Articles de revues : « Kindline-2 »

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Mi, Yingchang, Wenbin Wu, Qing Zhang, Yan Li, Xiaoyan Li, Zheng Tian et Min Wang. « Expression of Kindlins in Acute Myeloid Leukemia ». Blood 118, n^o 21 (18 novembre 2011) : 4910. http://dx.doi.org/10.1182/blood.v118.21.4910.4910.

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Abstract Abstract 4910 The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. They comprise of three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, they share considerable sequence and structural similarities. A few of study revealed that Kindlin-2 influences solid tumor cell invasion and resistance. With regard to AML, the influence of Kindlins is still unknown. To evaluate the clinical significance of Kindlin-2 in acute myeloid leukemia (AML), we investigated the expression of Kindlin-2, kindling-3 in AML cells. 1. Materials and methods K562, KG-1a, HL60, U937, Jurkat cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified atmosphere of 5% CO2. Bone marrow (BM) samples were obtained from 88 patients with de novo AML from Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Samples of 9 normal donors and ITP were used as the control group. Bone marrow mononuclear cells (BMMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. Expressions of Kindlin-2, Kindlin-3 were detected by RQ-PCR. The following primers for real-time PCR were used: (a) Kindlin-2 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (b) Kindlin-2 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (c) Kindlin-3 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (d) Kindlin-3 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (e) GAPDH sense primer, 5'-GAAGGTGAAGGTCGGAGTC-3'; (f) GAPDH antisense primer, 5'-GAAGATGGTGATGGGATTTC-3'. Analysis was performed using ABI 7500 Sequence Detection software (Applied Biosystems). The expression of Kindlin-2 and Kindlin-3 were showed as RQ value calculated through ΔΔCt method [ΔΔCt = (CtKindlin □ CtGAPDH)sample □ (CtKindlin □ CtGAPDH)calibrator]. The ΔCt (CtKindlin □ CtGAPDH) of K562 was defined as calibrator, and the RQ of calibrator was 1.000. Relationships between Kindlin-2, Kindlin-3 and the patients' clinical data were analyzed. 2. Results Expression of Kindlins in newly diagnosis AML The level of Kindlin-2 in AML (0.163±1.665) was significantly lower than that in non-AML (1.683±1.395) controls (p=0.010). No significant difference was found between the AML and controls in levels of Kindlin-3 (p=0.216). Out of the 79 patients who accepted treatment, 61 patients achieved complete remission (CR) and 18 patients were NR. Patients with higher expression of Kindlin-2 had a higher CR rate (86.8% vs 68.3%) (p=0.050). Expression of kindling 3 was unrelated to CR rate. Both of kindling-2 and kindling-3 increased after CR. This finding implicates Kindlin-2 as a potential prognostic factor of AML. Disclosures: No relevant conflicts of interest to declare.

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Kadry, Yasmin A., Eesha M. Maisuria, Clotilde Huet-Calderwood et David A. Calderwood. « Differences in self-association between kindlin-2 and kindlin-3 are associated with differential integrin binding ». Journal of Biological Chemistry 295, n^o 32 (16 juin 2020) : 11161–73. http://dx.doi.org/10.1074/jbc.ra120.013618.

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The integrin family of transmembrane adhesion receptors coordinates complex signaling networks that control the ability of cells to sense and communicate with the extracellular environment. Kindlin proteins are a central cytoplasmic component of these networks, directly binding integrin cytoplasmic domains and mediating interactions with cytoskeletal and signaling proteins. The physiological importance of kindlins is well established, but how the scaffolding functions of kindlins are regulated at the molecular level is still unclear. Here, using a combination of GFP nanotrap association assays, pulldown and integrin-binding assays, and live-cell imaging, we demonstrate that full-length kindlins can oligomerize (self-associate) in mammalian cells, and we propose that this self-association inhibits integrin binding and kindlin localization to focal adhesions. We show that both kindlin-2 and kindlin-3 can self-associate and that kindlin-3 self-association is more robust. Using chimeric mapping, we demonstrate that the F2PH and F3 subdomains are important for kindlin self-association. Through comparative sequence analysis of kindlin-2 and kindlin-3, we identify kindlin-3 point mutations that decrease self-association and enhance integrin binding, affording mutant kindlin-3 the ability to localize to focal adhesions. Our results support the notion that kindlin self-association negatively regulates integrin binding.

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McDowall, Alison, Lena Svensson, Paula Stanley, Irene Patzak, Probir Chakravarty, Kimberley Howarth, Himalee Sabnis, Michael Briones et Nancy Hogg. « Two mutations in the KINDLIN3 gene of a new leukocyte adhesion deficiency III patient reveal distinct effects on leukocyte function in vitro ». Blood 115, n^o 23 (10 juin 2010) : 4834–42. http://dx.doi.org/10.1182/blood-2009-08-238709.

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Abstract In the disorder leukocyte adhesion deficiency III (LAD-III), integrins on platelets and leukocytes are expressed but fail to function and this leads to severe bleeding and infections at an early age. Mutation in the KINDLIN3 (FERMT3) gene is the cause of LAD-III in patients from the Middle East, Malta, and Turkey. We describe 2 novel homozygous mutations in the KINDLIN3 gene of a new African-American patient that destabilize KINDLIN3 mRNA leading to loss of kindlin-3 protein. Transfection of wild-type (WT) KINDLIN3 cDNA restored integrin-related adhesion and migration in the LAD-III patient's T and B lymphocytes. We analyzed the individual mutations separately in vitro to learn more about the function of the kindlin-3 protein. The first G>A mutation gives rise to a Gly308Arg change at the end of FERM (protein 4.1, ezrin, radixin, moesin) subdomain 2, and the second mutation is a base deletion causing early termination within the pleckstrin homology (PH) domain. This second mutation prevented membrane association of kindlin-3 and did not restore either adhesion or migration, whereas the FERM subdomain 2 mutation affected only migration. Thus, these LAD-III patient mutations have highlighted functionally important regions of kindlin-3 that alter leukocyte integrin-dependent function in 2 distinct ways.

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Malinin, Nikolay L., Edward F. Plow et Tatiana V. Byzova. « Kindlins in FERM adhesion ». Blood 115, n^o 20 (20 mai 2010) : 4011–17. http://dx.doi.org/10.1182/blood-2009-10-239269.

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The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. The 3 members of this family, Kindlin-1, -2, and -3, perform an essential role in activation of integrin adhesion receptors, and expression of at least 1 Kindlin paralog is required to enable integrin activation in physiologically relevant settings. In humans, deficiencies in Kindlin-3 lead to a number of abnormalities affecting hemostasis, the immune system, and bone function, whereas the lack of Kindlin-1 causes profound skin defects. The importance of Kindlins is underscored by the results of animal knockout studies, which clearly show the indispensable and nonredundant functions of all 3 Kindlins in development and normal physiology. This review discusses recent progress in the studies of Kindlin protein family, emphasizing newly identified functions and potential mechanisms underlying differential activities of the family members.

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Meller, Julia, Igor B. Rogozin, Eugenia Poliakov, Nahum Meller, Mark Bedanov-Pack, Edward F. Plow, Jun Qin, Eugene A. Podrez et Tatiana V. Byzova. « Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness ». Molecular Biology of the Cell 26, n^o 4 (15 février 2015) : 786–96. http://dx.doi.org/10.1091/mbc.e14-08-1294.

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Kindlins are integrin-interacting proteins essential for integrin-mediated cell adhesiveness. In this study, we focused on the evolutionary origin and functional specialization of kindlins as a part of the evolutionary adaptation of cell adhesive machinery. Database searches revealed that many members of the integrin machinery (including talin and integrins) existed before kindlin emergence in evolution. Among the analyzed species, all metazoan lineages—but none of the premetazoans—had at least one kindlin-encoding gene, whereas talin was present in several premetazoan lineages. Kindlin appears to originate from a duplication of the sequence encoding the N-terminal fragment of talin (the talin head domain) with a subsequent insertion of the PH domain of separate origin. Sequence analysis identified a member of the actin filament–associated protein 1 (AFAP1) superfamily as the most likely origin of the kindlin PH domain. The functional divergence between kindlin paralogues was assessed using the sequence swap (chimera) approach. Comparison of kindlin 2 (K2)/kindlin 3 (K3) chimeras revealed that the F2 subdomain, in particular its C-terminal part, is crucial for the differential functional properties of K2 and K3. The presence of this segment enables K2 but not K3 to localize to focal adhesions. Sequence analysis of the C-terminal part of the F2 subdomain of K3 suggests that insertion of a variable glycine-rich sequence in vertebrates contributed to the loss of constitutive K3 targeting to focal adhesions. Thus emergence and subsequent functional specialization of kindlins allowed multicellular organisms to develop additional tissue-specific adaptations of cell adhesiveness.

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Arnold, M., S. Rehart, M. Sauerbier, C. Biehl, C. Heck, U. Müller-Ladner et E. Neumann. « POS1035 FOCAL ADHESION PROTEINS KINDLIN-1, -2 AND TALIN-1 ARE REGULATED IN IL-1Β-STIMULATED RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS ». Annals of the Rheumatic Diseases 82, Suppl 1 (30 mai 2023) : 835.1–835. http://dx.doi.org/10.1136/annrheumdis-2023-eular.3477.

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BackgroundRheumatoid arthritis (RA) is a progressive chronic inflammatory autoimmune disease characterized by synovial hyperplasia, articular inflammation and excessive joint and bone erosion. Activated RA synovial fibroblasts (SF) are key players of cartilage erosion by attaching and invading into cartilage. The kindlin family of focal adhesion proteins consists of three members, whose function is to activate integrins and thereby enable focal adhesion. Integrins play a central role in regulating cell-matrix and cell-cell adhesion. The effects of integrin activation are cell adhesion and migration, which are known to be pathologically increased in RASF. It is well described that dysfunction of integrins and kindlins and their co-factor talin-1 are involved in the pathogenesis of certain diseases characterized by inflammation and malignant transformation. However, the role of kindlins and talin-1 in RA, and specifically RASF, remain unclear.ObjectivesTo investigate the compartmental and cellular localisation of kindlin-1, -2, -3 and talin-1 in RA synovial tissue and RASF, and the impact of pro-inflammatory activation on their expression on mRNA and protein level.MethodsSynovial fibroblasts were isolated from synovial tissue after joint replacement surgery (RA, osteoarthritis (OA)) or synovectomy of hand joints (non-arthritic controls). RASF were stimulated with 10 ng/ml IL-1β for 6, 17, 24 and 48 h. In a second approach, RASF were repetitively stimulated three times with 0.05 ng/ml IL-1β every 24 h. RNA was isolated, reverse-transcribed and qPCR performed for kindlin-1, -2, -3 and talin-1. Whole RNA sequencing was performed for the repetitive stimulation approach and evaluated using matrix combining pairwise contrasts (DESeq2 and DESeq) normalized counts and thresholds for significance of differential expression per contrast. Immunocytochemistry on RASF seeded on chamber slides was performed. Immunohistochemistry on synovial tissue was performed on 5 µm acetone-fixed cryosections. Biological replicates and Kruskal-Wallis-test with Dunn’s multiple comparison were used for statistics.ResultsIn RA synovial tissues, kindlin-2 and talin-1 protein were detectable adjacent to blood vessels, in the synovial lining layer and in the sublining. Kindlin-2 and talin-1 could be detected in cultured RASF with a consistent signal of the cell body without a nuclear signal. Kindlin-1 and -3 were neither detectable in synovial tissue nor in synovial fibroblasts. Relative mRNA levels (arbitrary units) of kindlins and talin-1 in RASF differed, with the strongest signal for talin-1 (663 ± 186), followed by kindlin-2 (88 ± 23), kindlin-1 (2.15 ± 2.06) and kindlin-3 (0.12 ± 0.08). In comparison to unstimulated controls, stimulation of RASF with IL-1β led to a temporary downregulation of kindlin-2 (-4.1-fold, p=0.0456) and talin-1 (-1.6-fold) after 17 h and returned to the baseline levels after 48 h. This time-dependent effect could also be observed by immunocytochemistry of IL-1β-stimulated RASF. Kindlin-1 mRNA expression was significantly upregulated after 6 h (2.9-fold, p=0.0178) of stimulation and downregulated after 17 h (-1.5-fold, p<0.0001), 24 h (-0.7-fold, p=0.0004) and 48 h (-1.2-fold, p<0.0001) compared to 6 h. After repetitive activation of RASF, kindlins and talin-1 were constantly regulated compared to the first stimulation (kindlin-1: 1.03-fold; kindlin-2: -1.06-fold; talin-1: 1.00-fold). In contrast, IL-6 showed an inflammatory adjustment to IL-1β leading to lower induction of IL-6 (49.1%) after repetitive IL-1β exposure.ConclusionFocal adhesion proteins kindlin-2 and talin-1, factors involved in integrin activation of cells, are present on RA synovial tissue and in RASF. As a time-dependent regulation of kindlin-2 and talin-1 after stimulation was observed, this effect may lead to a temporary and repetitive reproducible activation of RASF and activation of integrins, subsequently contributing to altered adhesion and migration of RASF under inflammatory arthritic conditions.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

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Tan, Hui-Foon, et Suet-Mien Tan. « The focal adhesion protein kindlin-2 controls mitotic spindle assembly by inhibiting histone deacetylase 6 and maintaining α-tubulin acetylation ». Journal of Biological Chemistry 295, n^o 18 (13 mars 2020) : 5928–43. http://dx.doi.org/10.1074/jbc.ra120.012954.

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Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.

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Sun, Jiaojiao, Desheng Xiao, Yuan Ni, Tianlong Zhang, Zhongyuan Cao, Zhou Xu, Huong Nguyen et al. « Structure basis of the FERM domain of kindlin-3 in supporting integrin αIIbβ3 activation in platelets ». Blood Advances 4, n^o 13 (10 juillet 2020) : 3128–35. http://dx.doi.org/10.1182/bloodadvances.2020001575.

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Abstract Kindlin-3, a protein 4.1, ezrin, radixin, and moesin (FERM) domain–containing adaptor in hematopoietic cells, is essentially required for supporting the bidirectional integrin αIIbβ3 signaling in platelets by binding to the integrin β3 cytoplasmic tail. However, the structural details of kindlin-3’s FERM domain remain unknown. In this study, we crystalized the kindlin-3’s FERM domain protein and successfully solved its 3-dimensional structure. The structure shows that the 3 kindlin-3’s FERM subdomains (F1, F2, and F3) compact together and form a cloverleaf-shaped conformation, which is stabilized by the binding interface between the F1 and F3 subdomains. Interestingly, the FERM domain of kindlin-3 exists as a monomer in both crystal and solution, which is different from its counterpart in kindlin-2 that is able to form a F2 subdomain-swapped dimer; nonetheless, dimerization is required for kindlin-3 to support integrin αIIbβ3 activation, indicating that kindlin-3 may use alternative mechanisms for formation of a functional dimer in cells. To evaluate the functional importance of the cloverleaf-like FERM structure in kindlin-3, structure-based mutations were introduced into kindlin-3 to disrupt the F1/F3 interface. The results show that integrin αIIbβ3 activation is significantly suppressed in platelets expressing the kindlin-3 mutant compared with those expressing wild-type kindlin-3. In addition, introduction of equivalent mutations into kindlin-1 and kindlin-2 also significantly compromises their ability to support integrin αIIbβ3 activation in CHO cells. Together, our findings suggest that the cloverleaf-like FERM domain in kindlins is structurally important for supporting integrin αIIbβ3 activation.

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Pluskota, Elzbieta, James J. Dowling, Natalie Gordon, Jeffrey A. Golden, Dorota Szpak, XiaoXia Z. West, Carla Nestor et al. « The integrin coactivator Kindlin-2 plays a critical role in angiogenesis in mice and zebrafish ». Blood 117, n^o 18 (5 mai 2011) : 4978–87. http://dx.doi.org/10.1182/blood-2010-11-321182.

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Abstract Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2+/− mice. RM1 prostate tumors grown in kindlin-2+/− mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2+/− mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2+/− mice. Vessels in the kindlin-2+/− mice were leaky, and BM transplantation from kindlin-2+/− to WT mice did not correct this defect. Endothelial cells derived from kindlin-2+/− mice had integrin expression levels similar to WT mice but reduced αVβ3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVβ3.

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Godbout, Elena, Dong Ok Son, Stephanie Hume, Stellar Boo, Vincent Sarrazy, Sophie Clément, Andras Kapus et al. « Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts ». Cells 9, n^o 12 (17 décembre 2020) : 2702. http://dx.doi.org/10.3390/cells9122702.

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We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-β1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

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Pluskota, Elzbieta, Dorota Szpak, Katarzyna Bialkowska, Kamila Bledzka et Edward F. Plow. « Kindlin-2 Maintains Vascular Barrier Integrity By Stabilizing Endothelial Adherens Junctions ». Blood 126, n^o 23 (3 décembre 2015) : 3452. http://dx.doi.org/10.1182/blood.v126.23.3452.3452.

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Abstract Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, angiogenesis, thrombosis and hemostasis. Its absence is embryonically lethal in mice, emphasizing its biological importance. In the present study, we examined the role of Kindlin-2 in regulation of vascular integrity. In vivo, Kindlin-2+/- mice showed enhanced (70%) permeability of tracheal vasculature to fluorescent beads as compared to wild-type (WT) littermates under both basal conditions or when stimulated with Platelet Activating Factor (PAF). Consistent with these in vivo observations, confluent monolayers of aortic endothelial cells (ECs) isolated from Kindlin-2+/- mice had increased (2-3-fold) baseline, PAF- and thrombin-induced permeability for Alexa-488-labeled bovine serum albumin (BSA) or FITC-dextran-10000 as compared to WT cells. Also, reduction of Kindlin-2 expression by 60-70% in human umbilical vein endothelial cell (HUVEC) monolayers with Kindlin-2-specific siRNA resulted in 3-fold increase in their baseline or thrombin/(PAF)-induced permeability of these markers. Mechanistically, Kindlin-2 co-localized with VE-cadherin and actin within adherens junctions in resting, confluent HUVEC monolayers as well as co-immunoprecipitated with these proteins and other components of adherens junctions, including α-, β- and γ-catenin. In contrast, Kindlin-2 did not co-localize or co-immunoprecipitate with any components of GAP or tight junctions. VE-cadherin-based complexes had less associated Kindlin-2 and actin in Kindlin-2+/- ECs and in HUVECs treated with Kindlin-2-specifc siRNA. Also, upon stimulation of HUVECs with PAF or thrombin, Kindlin-2 along with actin dissociated from VE-cadherin-based complexes, suggesting that Kindlin-2 stabilizes adherens junctions. In direct binding studies with recombinant proteins the SPR sensograms revealed direct interaction of Kindlin-2 with β- and γ-catenin, but not with α-catenin nor the VE-cadherin cytoplasmic tail. In addition, using actin spin-down assays we demonstrated that Kindlin-2 directly interacted with actin filaments and linked them to β- or γ-catenin. Using WT and deletion mutants of Kindlin-2 we mapped the β- and γ-catenin binding site to the F1 and F3 domains in Kindlin-2, while actin binding is very dependent on the F0 domain. Taken together, these data identify a previously unappreciated function of Kindlin-2. It plays a crucial role in maintaining vascular integrity by linking adherens junctions to actin filaments. Disclosures No relevant conflicts of interest to declare.

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Pluskota, Elzbieta, James J. Dowling, Natalie Gordon, Jeffrey A. Golden, Dorota Szpak, XiaoXia Zhang, Carla Nestor et al. « The Integrin Co-Activator Kindlin-2 Plays a Critical Role In Angiogenesis and Blood Vessel Integrity ». Blood 116, n^o 21 (19 novembre 2010) : 4. http://dx.doi.org/10.1182/blood.v116.21.4.4.

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Abstract Abstract 4 Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Our previous study demonstrated that reduction of kindlin-2 levels in human endothelial cells results in defective adhesive and migratory responses, suggesting that kindlin-2 may be implicated in angiogenesis. We tested this hypothesis in the kindlin-2+/− mice utilizing murine RM1 prostate tumor and Matrigel implant models. Staining of tumor sections for EC with CD31 showed shorter and thinner blood vessels and reduced vascular area by 3.5-fold in tumors grown in kindlin-2+/− mice compared to WT mice (P=0.0186, n=6). The vessels that did form in the kindlin-2+/− mice were immature as they lacked smooth muscle cells and pericytes, had thinner basement membrane, and were leaky as evidenced by increased by 2-fold area for plasma-derived fibrin in tumor sections (P=0.0006, n=5). Consistent with the blunted angiogenic response and vascular leakiness in the kindlin-2+/− mice, the tumors grown in kindlin-2+/− animals had 2-fold larger necrotic core and were 2.5-fold smaller than those derived in WT mice (P=0.042, n=7). Also, the permeability of preexisting blood vessels in ear and dorsal skin of WT and kindlin-2+/− mice was compared after injection of Evans blue dye. Baseline permeability of vasculature in ear and dorsal skin was enhanced by ∼70–100 % in kindlin-2+/− mice as compared WT mice (P<0.01, n=6). Application of mustard oil, a proinflammatory stimulus, on ear skin or VEGF on dorsal skin, enhanced permeability in WT mice by ∼75–80 % (P<0.05, n=6) while it had a negligible effect in the kindlin-2+/− mice (P=0.159, n=6). The impaired angiogenic response in the kindlin-2+/− mice was confirmed in a Matrigel model where VEGF-induced angiogenesis was abnormal. CD31 staining showed that in contrast to the matrigel plugs derived from WT mice, those obtained from the kindlin-2+/− mice contained shorter and thinner vessels, and the few thicker CD31+ structures that did form in the kindlin-2+/− mice were disorganized. As a consequence of blood leakage from abnormal vessels the Matrigel implants collected from the kindlin-2+/− mice exhibited a 3.5-fold increase in hemoglobin content as compared to WT mice (P=0.0149, n=5).Bone marrow transplant experiments were performed and showed that bone marrow-derived cells did not significantly contribute to abnormal angiogenesis and enhanced vascular permeability observed in the kindlin-2+/− mice. Tumor vascular area, plasma-derived fibrin content, tumor growth and vascular permeability in ear and dorsal skin were similar in WT recipients, which received bone marrow from kindlin-2+/− or WT donors. Aortic endothelial cells isolated from WT and kindlin-2+/− mice exhibited similar expression levels of the β1 and β3 integrins as well as VEGFR2. However, FACS analysis of soluble ligand binding showed significantly reduced activation of the β3 integrins in kindlin-2+/− ECs as compared to WT cells, whereas the β1 integrin activation was similar in both genotypes. Additionally, PMA or VEGF enhanced integrin activation in WT ECs, but failed to do so in the kindlin-2+/− cells. Accordingly, integrin-dependent cellular functions: adhesion, migration and spreading of the EC derived from the kindlin-2+/− mice were significantly blunted on the β3 integrin substrates but not on the β1 substrates. In control experiments, overexpression of kindlin-2 in the kindlin-2+/− EC restored defective spreading on vitronectin while a kindlin-2 that does not bind integrins, did not, indicating specificity. Also, tube formation by kindlin-2+/− ECs was significantly impaired compared to those developed by WT ECs. Taken together, kindlin-2 plays an important role in angiogenesis as well as in maintenance of vascular integrity. Disclosures: No relevant conflicts of interest to declare.

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Chen, Weiguo, Yulia Epshtein, Christen Vagts, Anne E. Cress et Jeffrey R. Jacobson. « Increased Kindlin-2 via SMURF1 Inhibition Attenuates Endothelial Permeability and Acute Lung Injury ». International Journal of Molecular Sciences 26, n^o 5 (22 février 2025) : 1880. https://doi.org/10.3390/ijms26051880.

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Integrin β4 (ITGB4) mediates lung endothelial cell (EC) inflammation attenuated by simvastatin, an HMG CoA-reductase inhibitor. The cytoplasmic domain of ITGB4 is predicted to bind kindlin-2. Kindlin-2 expression is mediated by SMURF1, an E3 ubiquitin ligase that promotes kindlin-2 ubiquitination and degradation. We hypothesized that increased kindlin-2 expression via the inhibition of SMURF1 mediates EC inflammatory responses relevant to acute lung injury (ALI). To investigate this, human lung ECs were treated with simvastatin (5 µM, 16 h) prior to the immunoprecipitation of kindlin-2 and Western blotting for ITGB4. Next, ECs were treated with a SMURF1 inhibitor, A01, and increased kindlin-2 expression was confirmed. In assays of barrier function, kindlin-2 was silenced (siRNA) in ECs prior to thrombin and measurements of transendothelial resistance (TER) and FITC-dextran transwell flux. Repeat assessments of barrier function were performed in A01-treated ECs. Finally, mice were pretreated with A01 prior to LPS; bronchoalveolar lavage (BAL) fluid was collected, and their lungs were used for histology. Simvastatin increased ITGB4:kindlin-2 association, while A01 increased kindlin-2 expression. Thrombin-induced EC barrier disruption was both increased after kindlin-2 silencing and decreased by A01. Finally, murine ALI was significantly attenuated by A01. Our findings suggest that the augmentation of kindlin-2 may serve as a novel ALI therapeutic strategy.

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Wei, Xiaofan, Xiang Wang, Yang Xia, Yan Tang, Feng Li, Weigang Fang et Hongquan Zhang. « Kindlin-2 regulates renal tubular cell plasticity by activation of Ras and its downstream signaling ». American Journal of Physiology-Renal Physiology 306, n^o 2 (15 janvier 2014) : F271—F278. http://dx.doi.org/10.1152/ajprenal.00499.2013.

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Kindlin-2 is an adaptor protein that contributes to renal tubulointerstitial fibrosis (TIF). Epithelial-to-mesenchymal transition (EMT) in tubular epithelial cells was regarded as one of the key events in TIF. To determine whether kindlin-2 is involved in the EMT process, we investigated its regulation of EMT in human kidney tubular epithelial cells (TECs) and explored the underlying mechanism. In this study, we found that overexpression of kindlin-2 suppressed epithelial marker E-cadherin and increased the expression of fibronectin and the myofibroblast marker α-smooth muscle actin (SMA). Kindlin-2 significantly activated ERK1/2 and Akt, and inhibition of ERK1/2 or Akt reversed kindlin-2-induced EMT in human kidney TECs. Mechanistically, kindlin-2 interacted with Ras and son of sevenless (Sos)-1. Furthermore, overexpression of kindlin-2 increased Ras activation through recruiting Sos-1. Treatment with a Ras inhibitor markedly repressed kindlin-2-induced ERK1/2 and Akt activation, leading to restraint of EMT. We further demonstrated that knockdown of kindlin-2 inhibited EGF-induced Ras-Sos-1 interaction, resulting in reduction of Ras activation and suppression of EMT stimulated by EGF. Importantly, we found that depletion of kindlin-2 significantly inhibited activation of ERK1/2 and Akt signaling in mice with unilateral ureteral obstruction. We conclude that kindlin-2, through activating Ras and the downstream ERK1/2 and Akt signaling pathways, plays an important role in regulating renal tubular EMT and could be a potential therapeutic target for the treatment of fibrotic kidney diseases.

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Huang, Shaobin, Wuguo Deng, Peng Wang, Yue Yan, Chuanbo Xie, Xiaoling Cao, Miao Chen et al. « Fermitin family member 2 promotes melanoma progression by enhancing the binding of p-α-Pix to Rac1 to activate the MAPK pathway ». Oncogene 40, n^o 37 (28 juillet 2021) : 5626–38. http://dx.doi.org/10.1038/s41388-021-01954-8.

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AbstractWe identified fermitin family member 2 (FERMT2, also known as kindlin-2) as a potential target in A375 cell line by siRNA library screening. Drugs that target mutant BRAF kinase lack durable efficacy in the treatment of melanoma because of acquired resistance, thus the identification of novel therapeutic targets is needed. Immunohistochemistry was used to identify kindlin-2 expression in melanoma samples. The interaction between kindlin-2 and Rac1 or p-Rac/Cdc42 guanine nucleotide exchange factor 6 (α-Pix) was investigated. Finally, the tumor suppressive role of kindlin-2 was validated in vitro and in vivo. Analysis of clinical samples and Oncomine data showed that higher levels of kindlin-2 predicted a more advanced T stage and M stage and facilitated metastasis and recurrence. Kindlin-2 knockdown significantly inhibited melanoma growth and migration, whereas kindlin-2 overexpression had the inverse effects. Further study showed that kindlin-2 could specifically bind to p-α-Pix(S13) and Rac1 to induce a switch from the inactive Rac1-GDP conformation to the active Rac1-GTP conformation and then stimulate the downstream MAPK pathway. Moreover, we revealed that a Rac1 inhibitor suppressed melanoma growth and metastasis and the combination of the Rac1 inhibitor and vemurafenib resulted in a better therapeutic outcome than monotherapy in melanoma with high kindlin-2 expression and BRAF mutation. Our results demonstrated that kindlin-2 promoted melanoma progression, which was attributed to specific binding to p-α-Pix(S13) and Rac1 to stimulate the downstream MAPK pathway. Thus, kindlin-2 could be a potential therapeutic target for treating melanoma.

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Bledzka, Kamila, Katarzyna Bialkowska, Khalid Sossey-Alaoui, Julia Vaynberg, Elzbieta Pluskota, Jun Qin et Edward F. Plow. « Kindlin-2 directly binds actin and regulates integrin outside-in signaling ». Journal of Cell Biology 213, n^o 1 (4 avril 2016) : 97–108. http://dx.doi.org/10.1083/jcb.201501006.

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Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK47/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK47/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK47/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses.

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Wei, Xiaofan, Xiang Wang, Jun Zhan, Yuhan Chen, Weigang Fang, Lingqiang Zhang et Hongquan Zhang. « Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation ». Journal of Cell Biology 216, n^o 5 (13 avril 2017) : 1455–71. http://dx.doi.org/10.1083/jcb.201609073.

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Integrin activation is an indispensable step for various integrin-mediated biological functions. Kindlin-2 is known to coactivate integrins with Talin; however, molecules that restrict integrin activation are elusive. Here, we demonstrate that the E3 ubiquitin ligase Smurf1 controls the amount of Kindlin-2 protein in cells and hinders integrin activation. Smurf1 interacts with and promotes Kindlin-2 ubiquitination and degradation. Smurf1 selectively mediates degradation of Kindlin-2 but not Talin, leading to inhibition of αIIbβ3 integrin activation in Chinese hamster ovary cells and β1 integrin activation in fibroblasts. Enhanced activation of β1 integrin was found in Smurf1-knockout mouse embryonic fibroblasts, which correlates with an increase in Kindlin-2 protein levels. Similarly, a reciprocal relationship between Smurf1 and Kindlin-2 protein levels is found in tissues from colon cancer patients, suggesting that Smurf1 mediates Kindlin-2 degradation in vivo. Collectively, we demonstrate that Smurf1 acts as a brake for integrin activation by controlling Kindlin-2 protein levels, a new mechanism that permits precise modulation of integrin-mediated cellular functions.

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Nelson, Colin, Daniel Hernandez-Cortes, Kendra D. Marr, Jaime MC Gard, Allan I. Paxson, William L. Harryman, Natalya K. Seppanen, John M. Ryniawec et Anne E. Cress. « Abstract 5418 : Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells ». Cancer Research 84, n^o 6_Supplement (22 mars 2024) : 5418. http://dx.doi.org/10.1158/1538-7445.am2024-5418.

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Abstract Hypoxia is a physiologically relevant feature of the prostate microenvironment that promotes migration and invasion resulting in extracapsular extension, the first step toward metastatic progression. Prostate cancer invasion depends on integrins as mechanosensing membrane receptors during creation of membrane lamellipodial protrusions and focal adhesions (FAs). The objective of this study was to determine the molecular events that promote membrane protrusions under hypoxia and whether this was dependent upon kindlin-2, an essential integrin adapter that marks activated β1 integrin heterodimers. DU145 cells depleted of one copy of FERMT2+/- (50% kindlin-2 expression) by CRISPR/Cas9 or DU145 FERMT2wt (normal kindlin-2 expression) were grown under acute exposure to hypoxia (1% O2) and compared to cells grown under normal tissue culture conditions. Immunofluorescence microscopy experiments were performed to analyze the spatial temporal expression of kindlin-2 complexes. Kindlin-2 complexes were confirmed by immunoprecipitation using an anti-kindlin-2 3A3-antibody from Sigma-Aldrich. Colocalization was determined by obtaining 2D immunofluorescence microscopy images analyzed using ImageJ 2.1.0/1.53c and Nikon NIS-Elements 5.30.04. Under hypoxic conditions, analysis over four time points (4h, 8h, 12h & 16h) increased the number and area of FAs (marked by paxillin (PXN)) containing kindlin-2 in a time-dependent manner by 2-fold and 1.5-fold, respectively, but not in DU145 FERMT2+/- cells. Additionally, hypoxia increased membrane area, perimeter, and the plasma membrane intensity of kindlin-2 exclusive of FAs in DU145 FERMT2wt cells which was maximal after 8 hours of hypoxia. Interestingly, limiting the kindlin-2 expression in FERMT2+/- cells resulted in a loss of hypoxia-induced lamellipodial protrusions (marked by lamellipodin (RAPH1)) containing kindlin-2 while hypoxia-induced kindlin-2 FA changes were preserved. The current working hypothesis is that lamellipodial protrusions are dependent upon kindlin-2 expression whereas the established FAs are stable under conditions of reduced kindlin-2 expression in hypoxia. This data suggests that an early stage of migration, lamellipodial extensions are sensitive to kindlin-2 availability. Further studies are required to determine the dynamic interplay between protrusive events and focal adhesions in relation to kindlin-2 for prostate cancer cells to migrate and invade. Citation Format: Colin Nelson, Daniel Hernandez-Cortes, Kendra D. Marr, Jaime MC Gard, Allan I. Paxson, William L. Harryman, Natalya K. Seppanen, John M. Ryniawec, Anne E. Cress. Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5418.

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Wang, Wei, Priyanka S. Rana, Akram Alkrekshi, Katarzyna Bialkowska, Vesna Markovic, William P. Schiemann, Edward F. Plow, Elzbieta Pluskota et Khalid Sossey-Alaoui. « Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion, and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway ». Cancers 14, n^o 3 (27 janvier 2022) : 639. http://dx.doi.org/10.3390/cancers14030639.

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Breast cancer (BC) is one of the leading causes of cancer-related deaths due in part to its invasive and metastatic properties. Kindlin-2 (FERMT2) is associated with the pathogenesis of several cancers. Although the role of Kindlin-2 in regulating the invasion-metastasis cascade in BC is widely documented, its function in BC initiation and progression remains to be fully elucidated. Accordingly, we generated a floxed mouse strain by targeting the Fermt2 (K2lox/lox) locus, followed by tissue-specific deletion of Kindlin-2 in the myoepithelial compartment of the mammary glands by crossing the K2lox/lox mice with K14-Cre mice. Loss of Kindlin-2 in mammary epithelial cells (MECs) showed no deleterious effects on mammary gland development, fertility, and lactation in mice bearing Kindlin-2-deletion. However, in a syngeneic mouse model of BC, mammary gland, specific knockout of Kindlin-2 inhibited the growth and metastasis of murine E0771 BC cells inoculated into the mammary fat pads. However, injecting the E0771 cells into the lateral tail vein of Kindlin-2-deleted mice had no effect on tumor colonization in the lungs, thereby establishing a critical role of MEC Kindlin-2 in supporting BC tumor growth and metastasis. Mechanistically, we found the MEC Kindlin-2-mediated inhibition of tumor growth and metastasis is accomplished through its regulation of the TGF-β/ERK MAP kinase signaling axis. Thus, Kindlin-2 within the mammary gland microenvironment facilitates the progression and metastasis of BC.

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Moslem, Mohsen, Reto Eggenschwiler, Christian Wichmann, Raymund Buhmann, Tobias Cantz et Reinhard Henschler. « Kindlin-2 Modulates the Survival, Differentiation, and Migration of Induced Pluripotent Cell-Derived Mesenchymal Stromal Cells ». Stem Cells International 2017 (2017) : 1–13. http://dx.doi.org/10.1155/2017/7316354.

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Kindlin-2 is a multidomain intracellular protein that can be recruited to β-integrin domains to activate signaling, initiate transcriptional programs, and bind to E-cadherin. To explore its involvement in cell fate decisions in mesenchymal cells, we studied the effects of Kindlin-2 modification (overexpression/knockdown) in induced pluripotent cell-derived mesenchymal stromal cells (iPSC-MSCs). Kindlin-2 overexpression resulted in increased proliferation and reduced apoptosis of iPSC-MSCs, as well as inhibition of their differentiation towards osteocytes, adipocytes, and chondrocytes. In contrast, siRNA-mediated Kindlin-2 knockdown induced increased apoptosis and increased differentiation response in iPSC-MSCs. The ability of iPSC-MSCs to adhere to VCAM-1/SDF-1α under shear stress and to migrate in a wound scratch assay was significantly increased after Kindlin-2 overexpression. In contrast, inhibition of mixed lymphocyte reaction (MLR) was generally independent of Kindlin-2 modulation in iPSC-MSCs, except for decreased production of interleukin-2 (IL-2) after Kindlin-2 overexpression in iPS-MSCs. Thus, Kindlin-2 upregulates survival, proliferation, stemness, and migration potential in iPSC-MSCs and may therefore be beneficial in optimizing performance of iPSC-MSC in therapies.

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Hernandez-Cortes, Daniel, Jaime M. C. Gard, Beatrice S. Knudsen, Noel A. Warfel et Anne E. Cress. « Abstract 3835 : Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 3835. http://dx.doi.org/10.1158/1538-7445.am2022-3835.

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Abstract The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) migration, invasion, metastasis, and decreased cancer-specific survival. Kindlin-2 (FERMT2) is a β1 integrin adaptor and mechanosensory focal adhesion (FA) protein that activates and clusters integrins in response to structural ECM alterations in the tumor microenvironment. Our goal was to determine if integrin-kindlin-2 adhesion complexes (kindlin-2:α6β1) were responsive to hypoxia, a physiologically relevant and altered microenvironment in PCa progression. Five different endpoints were tested including the biochemical analysis of kindlin-2 complexes, qRT-PCR, immunoblotting, immunocytochemistry, and electric cell impedance sensing (ECIS). Using DU145 prostate cancer cells grown under hypoxia (1% O2) for up to 16 hours, the results showed a reversible increase in kindlin-2:α6β1 complexes with maximal assembly within 4 hours and disassembly starting by 8 hours. Notably, kindlin-2:α6β1 complexes were found exclusively within membrane projections and were not observed within hypoxia-inducible paxillin (PXN)-containing FAs. The hypoxia induced kindlin-2:α6β1 complexes and classical FAs were dependent on kindlin-2 as determined by CRISPR-Cas9 heterozygous deletion of FERMT2. Protein co-localization of α6 integrin and PXN with kindlin-2 within membrane projections and FAs, respectively, was also induced under hypoxia. Further, non-invasive ECIS measurements in live cells confirmed functional cell-cell and cell-ECM dynamics driven by hypoxia and requiring kindlin-2. Our results indicate that the kindlin-2:α6β1 complexes are uniquely associated with FA-independent membrane projections induced by hypoxia, a tumor microenvironment associated with aggressive prostate cancer. The novel kindlin-2:α6β1 complexes may represent an actionable pharmacological target for blocking escape of organ confined disease and metastasis promoting steps of human prostate cancer. (Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226). Citation Format: Daniel Hernandez-Cortes, Jaime M.C. Gard, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3835.

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Yeon, Minjeong, Irene Bertolini et Dario C. Altieri. « Abstract 1276 : Kindlin-2 : a novel target of Parkin regulating cancer metastasis ». Cancer Research 84, n^o 6_Supplement (22 mars 2024) : 1276. http://dx.doi.org/10.1158/1538-7445.am2024-1276.

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Abstract Although targeted therapies in prostate cancer have clinically shown successful outcomes, resistance to these therapies and acquisition of lineage plasticity pose challenges for fully treating prostate cancer. Among several microscopic perspectives to solve these obstacles, much evidence has emerged indicating that mitochondria dynamics are closely associated with tumor metastasis and tumor progression in various types of cancer. The E3 ubiquitin ligase Parkin is associated with PINK1-mediated mitophagy, allowing cells to control mitochondria damages and generate the needed energy to survive in harsh physiological conditions. Interestingly, we found that Parkin potentially ubiquitinates proteins independently of PINK1-associated mitophagy. By SALIC analysis, we found that K681 and K582 of Kindlin-2 is one of the potential ubiquitination sites by Parkin in prostate cancer cells. Kindlin-2 is a cytoskeletal membrane protein and is a component of the focal adhesion complex. Computational modeling suggested that N-terminal region of Kindlin-2 binds to Parkin, with K581 and K582 of Kindlin-2 exposed outside for ubiquitination. Our studies showed that Kindlin-2 is ubiquitinated and degraded by wild-type Parkin but not by the catalytic site, C431S, mutant Parkin. Furthermore, K581A and K582A mutant Kindlin-2 did not exhibit poly-ubiquitination under conditions of Parkin overexpression. Surprisingly, in our immunofluorescence study, Kindlin-2 was recruited to the mitochondria and co-localized with Parkin. From a mitochondrial functional perspective, wild-type Kindlin-2 reduced mitochondria dynamics, whereas mutant Kindlin-2 didn’t in the presence of Parkin. This suggests that the cytoskeletal protein Kindlin-2 might play a role in mitochondrial dynamics that mediates tumor metastasis. Wild-type Kindlin-2 reduced both focal adhesion generation and decay, unlike mutant Kindlin-2 under Parkin-induced condition. Additionally, this pattern was observed in the single cell lamellipodia dynamics through live cell imaging. 2D and 3D cell invasive assays supported the evidence that Parkin decreases tumor metastatic potential by ubiquitinating Kindlin-2 and regulating mitochondria and focal adhesion dynamics. Our findings indicate that Parkin reduces tumor metastatic potential by ubiquitinating Kindlin-2 and demonstrate the possibility as a therapeutic agent for advanced and immune suppressive tumors. Furthermore, Parkin could be a promising agent for Parkin suppressed tumor patients in line with personalized cancer therapy development. Citation Format: Minjeong Yeon, Irene Bertolini, Dario C. Altieri. Kindlin-2: a novel target of Parkin regulating cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1276.

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Guo, Ling, Ting Cai, Keng Chen, Rong Wang, Jiaxin Wang, Chunhong Cui, Jifan Yuan et al. « Kindlin-2 regulates mesenchymal stem cell differentiation through control of YAP1/TAZ ». Journal of Cell Biology 217, n^o 4 (1 mars 2018) : 1431–51. http://dx.doi.org/10.1083/jcb.201612177.

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Precise control of mesenchymal stem cell (MSC) differentiation is critical for tissue development and regeneration. We show here that kindlin-2 is a key determinant of MSC fate decision. Depletion of kindlin-2 in MSCs is sufficient to induce adipogenesis and inhibit osteogenesis in vitro and in vivo. Mechanistically, kindlin-2 regulates MSC differentiation through controlling YAP1/TAZ at both the transcript and protein levels. Kindlin-2 physically associates with myosin light-chain kinase in response to mechanical cues of cell microenvironment and intracellular signaling events and promotes myosin light-chain phosphorylation. Loss of kindlin-2 inhibits RhoA activation and reduces myosin light-chain phosphorylation, stress fiber formation, and focal adhesion assembly, resulting in increased Ser127 phosphorylation, nuclear exclusion, and ubiquitin ligase atrophin-1 interacting protein 4–mediated degradation of YAP1/TAZ. Our findings reveal a novel kindlin-2 signaling axis that senses the mechanical cues of cell microenvironment and controls MSC fate decision, and they suggest a new strategy to regulate MSC differentiation, tissue repair, and regeneration.

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Rana, Priyanka, Wei Wang, Akram Alkrekchi, Katarzyna Bialkowska, Vesna Markovic, Edward F. Plow, Elzbieta Pluskota et Khalid Sossey-Alaoui. « Abstract P1-06-02 : Targeted deletion of Kindlin-2 in mouse mammary glands inhibits tumor growth, invasion and metastasis downstream of TGF-β/EGF oncogenic signaling pathway ». Cancer Research 82, n^o 4_Supplement (15 février 2022) : P1–06–02—P1–06–02. http://dx.doi.org/10.1158/1538-7445.sabcs21-p1-06-02.

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Abstract Cancer metastasis is a complex process by which cancer cells migrate through the blood and the lymphatic systems to lodge and proliferate in distant sites and organs in the body. Metastasis is the main cause of death in patients suffering from cancer, including those patients with breast cancer. Breast cancer (BC) is the most frequently diagnosed malignancy in women and is one of the leading causes of death due to cancer invasion, metastasis, and resistance to therapies. Among its variants, triple-negative breast cancer (TNBC) is considered the most aggressive due to its early invasive and metastatic properties with poor prognosis. Kindlin-2, which is encoded by Fermitin family homolog 2 gene (FERMT2) has been associated with pathogenesis of several types of cancers of epithelial origin. Our previous studies have addressed the role of Kindlin-2 as a major regulator of the invasion-metastasis cascade in breast cancer by controlling several hallmarks of cancer in tumor cells. The contribution of mammary epithelial cell Kindlin-2 in the mammary glands to the process of tumor progression and metastasis has, however, not been investigated. Accordingly, we generated a floxed mouse strain by targeting the FREMT2 (K2lox/lox) locus using a CRISPR/Cas9-based editing strategy, followed by tissue-specific deletion of the Kindlin-2 in the basal subtype of the mammary epithelial cells (MECs) in the mouse mammary glands by crossing the K2lox/lox mice with K14-cre mice. Loss of Kindlin-2 in the basal MECs had no deleterious effects on mammary glands development, mouse development and fertility and lactation in mice bearing the Kindlin 2-deletetion phenotype and their progeny. However, in a syngeneic mouse model of BC, the loss of Kindlin-2 in MECs inhibited tumor growth and metastasis in mice inoculated with the aggressive murine TNBC E0771 cells when implanted directly in their mammary fat pads. Injecting the E0771 cells via the tail vein of Kindlin-2-deleted mice had, however, no effect on tumor colonization in the lungs, when compared to wild-type mice, clearly supporting a critical role of MECs Kindlin-2 in BC tumor growth and metastasis. Mechanistically, we found the MECs Kindlin-2-mediated inhibition of tumor growth and metastasis is through the regulation of the TGF-β/ERK MAP. kinase signaling axis, in a similar manner that our published studies showed that Kindlin-2 regulates this oncogenic pathway in the BC cells. Thus, our findings strongly suggest that Kindlin-2 supports BC oncogenesis in both the tumor cells and the MECs in the mammary glands, through the regulation of the TGF-β/EGF oncogenic signaling pathway. Therefore, therapeutic strategies targeting Kindlin-2 in both the cancer cells and the mammary glands may be necessary for a successful inhibition of BC tumors. Citation Format: Priyanka Rana, Wei Wang, Akram Alkrekchi, Katarzyna Bialkowska, Vesna Markovic, Edward F Plow, Elzbieta Pluskota, Khalid Sossey-Alaoui. Targeted deletion of Kindlin-2 in mouse mammary glands inhibits tumor growth, invasion and metastasis downstream of TGF-β/EGF oncogenic signaling pathway [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-02.

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Bialkowska, Katarzyna, Eugene Podrez, Tatiana V. Byzova et Edward F. Plow. « Agonist-Induced Kindlin-3 Phsphorylation Regulates αIIbβ3 Integrin Activation In HEL Cells and Platelets ». Blood 122, n^o 21 (15 novembre 2013) : 22. http://dx.doi.org/10.1182/blood.v122.21.22.22.

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Abstract The contributions of integrins to platelet responses depend upon the dynamic regulation of their activation status, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin function. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin functions in hematopoietic cells, including platelets, where kindlin-3 deficiency can lead to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Despite this evidence, little is known about kindlin-3 structure-function relationship. Here, we used human platelets and human erythroleukemic HEL cell line that expresses integrin αIIbβ3 to investigate whether posttranslational modification(s) of kindlin-3 occurs and can influence its integrin activity. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen or bind soluble fibrinogen and PAC-1 antibody (specific for activated αIIbβ3) readily. Thrombopoietin or PMA stimulation activated αIIbβ3 such that the cells adhered and spread on fibrinogen and increased their binding of PAC-1 and soluble fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions in the adherent cells, and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. Since, as we have shown previously, β3 integrin phosphorylation regulates kindlin and integrin interaction, we sought to determine whether kindlin-3 is also phosphorylated. Human platelets were stimulated with thrombin and HEL cells with PMA, and kindlin-3 was immunoprecipitated from lysates of control and stimulated cells. A kindlin-3 peptide showing significant increase in phosphorylation upon agonist stimulation was identified in both platelets and HEL cells by mass spectrometry. T482 or S484 were identified as phosphorylation sites in sequence that resides in the kindlin-3 variable region, which is not present either in kindlin-1 or kindlin-2 but is conserved across all species in which kindlin-3 has been sequenced. When expressed in HEL cells, TS/AA kindlin-3 mutant displayed decreased soluble fibrinogen binding and cell spreading on immobilized fibrinogen when compared to wild-type kindlin-3. Membrane-permeable, poly-arginine tagged kindlin-3 peptide containing the candidate phosphorylation sites kindlin-3 was introduced into HEL cells and platelets. HEL cell adhesion and spreading was blunted by the kindlin-3 peptide when compared to a scramble poly-arginine control peptide. Moreover, thrombin-induced platelet aggregation was inhibited by kindlin-3 peptide but not by the scramble peptide. Thus, our data emphasizes a role of previously unknown, agonist-induced kindlin-3 phosphorylation, in integrin αIIbβ3 activation in HEL cells and platelets and provides a basis for functional differences between kindlin-3 and its other two paralogs, kindlin-1 and kindlin-2. Disclosures: No relevant conflicts of interest to declare.

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Böttcher, Ralph T., Maik Veelders, Pascaline Rombaut, Jan Faix, Marina Theodosiou, Theresa E. Stradal, Klemens Rottner, Roy Zent, Franz Herzog et Reinhard Fässler. « Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading ». Journal of Cell Biology 216, n^o 11 (14 septembre 2017) : 3785–98. http://dx.doi.org/10.1083/jcb.201701176.

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Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions. Consistently, abrogation of kindlin-2 binding to Arp2/3 impairs lamellipodia formation and cell spreading. Our findings identify kindlin-2 as a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Rac1 and supplying Rac1 with the Arp2/3 complex.

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Pluskota, Elzbieta, Yi Ma, Kamila M. Bledzka, Katarzyna Bialkowska, Dmitry A. Soloviev, Dorota Szpak, Eugene A. Podrez et al. « Kindlin-2 regulates hemostasis by controlling endothelial cell–surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism ». Blood 122, n^o 14 (3 octobre 2013) : 2491–99. http://dx.doi.org/10.1182/blood-2013-04-497669.

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Key Points Kindlin-2 regulates hemostasis in vivo by limiting CD39 and CD73 expression on the surface of endothelial cells. Kindlin-2 interacts directly with CHC and controls clathrin-dependent CD39 and CD73 endocytosis/recycling in endothelial cells.

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Bandyopadhyay, A., G. Rothschild, S. Kim, D. A. Calderwood et S. Raghavan. « Functional differences between kindlin-1 and kindlin-2 in keratinocytes ». Journal of Cell Science 125, n^o 9 (10 février 2012) : 2172–84. http://dx.doi.org/10.1242/jcs.096214.

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Liao, Zhongji, Hisashi Kato, Manjula Pandey, Joseph M. Cantor, Ararat J. Ablooglu, Mark H. Ginsberg et Sanford J. Shattil. « Interaction of kindlin-2 with integrin β3 promotes outside-in signaling responses by the αVβ3 vitronectin receptor ». Blood 125, n^o 12 (19 mars 2015) : 1995–2004. http://dx.doi.org/10.1182/blood-2014-09-603035.

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Key Points Interaction of the integrin β3 cytoplasmic tail with kindlin-2 selectively promotes outside-in signaling through αVβ3. Disruption of the kindlin-2/αVβ3 interaction impairs outside-in signaling and endothelial cell functions, both in vitro and in vivo.

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Mody, I., P. K. Stanton et U. Heinemann. « Activation of N-methyl-D-aspartate receptors parallels changes in cellular and synaptic properties of dentate gyrus granule cells after kindling ». Journal of Neurophysiology 59, n^o 3 (1 mars 1988) : 1033–54. http://dx.doi.org/10.1152/jn.1988.59.3.1033.

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1. The cellular and synaptic properties of rat dentate gyrus granule cells (GCs) were examined using intra-/extracellular and Ca2+-sensitive microelectrode recordings following epilepsy induced by kindling of the hippocampal commissures or amygdala. 2. The recordings were made in hippocampal slices prepared from sham-stimulated controls and animals that have received daily stimuli to reach stage IV-V of kindling. The average number of stimulation trials (60 Hz/1 s, 100-150 microA) required to reach full motor seizures (stage V) was 23 +/- 2 for commissural kindling and 14 +/- 1 for amygdala kindling. 3. The resting membrane potential of GCs following kindling (RMP; -72 +/- 3 mV) was not significantly different from the RMP of control GCs (-70 +/- 2 mV). Similarly, action potential height and threshold were unaffected by kindling. However, kindling altered other cellular properties of GCs regardless of the site of stimulation (hippocampal commissures or amygdala), the stage of kindling reached (IV or V), or the time elapsed between the last kindling stimulus and preparation of the hippocampal slices (24 h-6 wk). The input resistance of kindled GCs (55 +/- 4 M omega) was significantly higher than that of controls (40 +/- 3 M omega). In contrast to most control GCs, the slope conductance (GS) of kindled neurons, measured with constant-amplitude current injections at various membrane potentials, generally increased at membrane potentials more negative than rest. Furthermore, other voltage-dependent ionic conductances (see below), that were not normally encountered in control GCs, were present in kindled neurons. 4. The intracellularly recorded monosynaptic excitatory postsynaptic potentials (EPSPs) of kindled GCs, evoked through the stimulation of the lateral perforant pathway, differed significantly from the EPSPs of control GCs. The amplitudes of control EPSPs increased upon hyperpolarizations and decreased following depolarizations of the membrane, as expected for conventional EPSPs without contribution from voltage-dependent conductances. In contrast, the EPSPs of kindled GCs invariably increased in amplitude and duration at membrane potentials 5-20 mV depolarized from rest, indicating the presence of a characteristic voltage-dependent component. Frequently, following the synaptically triggered action potentials, kindled GCs displayed depolarizing afterpotentials. 5. Perfusion of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV; 30 microM) had no effect on the EPSPs of control GCs, but consistently reduced the amplitude and duration of EPSPs in kindled GCs.(ABSTRACT TRUNCATED AT 400 WORDS)

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Ma, Yan-Qing, Jun Qin, Chuanyue Wu et Edward F. Plow. « Kindlin-2 (Mig-2) : a co-activator of β3 integrins ». Journal of Cell Biology 181, n^o 3 (5 mai 2008) : 439–46. http://dx.doi.org/10.1083/jcb.200710196.

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Integrin activation is essential for dynamically linking the extracellular environment and cytoskeletal/signaling networks. Activation is controlled by integrins' short cytoplasmic tails (CTs). It is widely accepted that the head domain of talin (talin-H) can mediate integrin activation by binding to two sites in integrin β's CT; in integrin β3 this is an NPLY747 motif and the membrane-proximal region. Here, we show that the C-terminal region of integrin β3 CT, composed of a conserved TS752T region and NITY759 motif, supports integrin activation by binding to a cytosolic binding partner, kindlin-2, a widely distributed PTB domain protein. Co-transfection of kindlin-2 with talin-H results in a synergistic enhancement of integrin αIIbβ3 activation. Furthermore, siRNA knockdown of endogenous kindlin-2 impairs talin-induced αIIbβ3 activation in transfected CHO cells and blunts αvβ3-mediated adhesion and migration of endothelial cells. Our results thus identify kindlin-2 as a novel regulator of integrin activation; it functions as a coactivator.

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Attwaters, Michael. « Kindlin-2 reduces IVD inflammation ». Nature Reviews Rheumatology 18, n^o 3 (31 janvier 2022) : 125. http://dx.doi.org/10.1038/s41584-022-00753-z.

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Ye, Xi, et Kim A. Dora. « The kindlin-2 double act ». Journal of Physiology 595, n^o 20 (13 septembre 2017) : 6371. http://dx.doi.org/10.1113/jp275082.

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Shan, Ping, Jilong Zhang, Yulan Gou, Lijun Luo et Suiqiang Zhu. « Chaihu plus Longgu Muli Decoction Alleviated Brain Injury in Pentylenetetrazole-Kindled Epileptic Mice by Regulating Cyclooxygenase-2/Prostaglandin E2/Multidrug Transporter Pathway ». BioMed Research International 2021 (29 mars 2021) : 1–10. http://dx.doi.org/10.1155/2021/6652195.

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Objective. To evaluate the effect of CLMD administration on epileptic seizures and brain injury in pentylenetetrazole- (PZT-) kindled mice. Methods. The effect of pretreatment with CLMD (5, 10, and 20 ml/kg (mg/kg) by gavage) for seven days on PTZ-induced kindling, duration and grade of kindling-induced seizures, and pathological injury in the cortex and hippocampus was evaluated. Male BALB/c mice with adenosine A1 receptor knockout were subjected to intraperitoneal injection of PTZ (35 mg/kg) once every day until kindling was successfully induced. Quantitative reverse transcription polymerase chain reaction, immunofluorescence, and western blot were performed to assess the mRNA and protein levels of p-glycoprotein (PGP), multidrug resistance-associated protein 1 (MRP1), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and adenylate kinase (ADK) in the cortex and hippocampus. Results. PTZ successfully induced kindling in mice after 21 days, wherein CLMD showed an obvious dose-dependent antiepileptic effect. High-dose CLMD significantly increased the latency of epileptic seizures, decreased the sustained time of epileptic seizures and the seizure grade, and ameliorated the histopathological changes in the cortex and hippocampus. Furthermore, PTZ kindling induced significantly higher levels of PGP, MRP1, COX-2, PGE2, and ADK, but this effect was inhibited by pretreatment with CLMD in a dose-dependent manner. Conclusion. Pretreatment with CLMD attenuates PTZ-kindled convulsions and brain injury in mice. The mechanism may be related to the cyclooxygenase-2/prostaglandin E2/multidrug transporter pathway.

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Manevich-Mendelson, Eugenia, Sara W. Feigelson, Ronit Pasvolsky, Memet Aker, Valentin Grabovsky, Ziv Shulman, Sara Sebnem Kilic et al. « Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions ». Blood 114, n^o 11 (10 septembre 2009) : 2344–53. http://dx.doi.org/10.1182/blood-2009-04-218636.

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Abstract Leukocyte adhesion deficiency (LAD)–III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor–stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3–null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3–null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative β1 Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4–adhesive functions in human lymphocytes.

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Zhan, Jun, Xiang Zhu, Yongqing Guo, Yunling Wang, Yuxiang Wang, Guangliang Qiang, Miaomiao Niu et al. « Opposite Role of Kindlin-1 and Kindlin-2 in Lung Cancers ». PLoS ONE 7, n^o 11 (29 novembre 2012) : e50313. http://dx.doi.org/10.1371/journal.pone.0050313.

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Xiccato, G., A. Trocino, C. Boiti et G. Brecchia. « Reproductive rhythm and litter weaning age as they affect rabbit doe performance and body energy balance ». Animal Science 81, n^o 2 (octobre 2005) : 289–96. http://dx.doi.org/10.1079/asc50270289.

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Abstract One hundred and twenty multiparous does were synchronized to give birth the same day (initial kindling). The trial lasted until the successive (final) kindling. Immediately following initial kindling, 22 does were selected for comparative slaughter. The remaining does were assigned to three reproductive rhythms and mated 2 (R2), 11 (R11) or 26 (R26) days post partum. Within each rhythm, the does were further divided into two groups with litters weaned at 21 (W21) or 25 (W25) days of age. A total of 54 does were pregnant and were slaughtered soon after final kindling. By increasing the kindling-to-mating interval from 2 to 26 days, total milk production was increased (5590 to 6065 g for R2 and R26, respectively;P< 0·05); voluntary food intake during lactation was not affected (356 g/day on average), but during the dry period was reduced (182 to 169 g/day;P< 0·05) in this way accounting for a decrease during the experimental period on the whole (299 to 249 g/day;P< 0·01). At the final kindling, the number of kits born per litter was lower in does submitted to the R11 than to the R26 rhythm (P< 0·01). By increasing the kindling-to-mating interval, doe body water concentration decreased, while fat and energy increased (P< 0·01) and higher empty body gain was recorded (from −123 to −4, and to +97 g, in R2, R11 and R26 does, respectively;P< 0·001). As a result, body protein, fat and energy balances changed from negative values to approach equilibrium as reproductive rhythm became extensive (energy balance: −0·14, −0·02 and +0·01 of the initial body content in R2, R11 and R26 does, respectively;P< 0·001). At 28 days after kindling, blood leptin concentration was higher (P< 0·01) and IGF-1 lower (P< 0·05) in R26 does. Daily food intake throughout the experiment was lower (P< 0·05) in W21 does due to the longer dry period. Increasing weaning age from 21 to 25 days increased both number of kits born alive per litter (from 7·4 to 9·6;P< 0·05) and doe body water concentration, while body energy tended to decrease (P< 0·1). At 28 days after kindling lower blood leptin concentration was recorded in W21 than W25 does (1·87 v. 2·76 μg/l,P< 0·05).

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Jalilifar, Mostafa, Ali Yadollahpour, Ahmad Ali Moazedi et Zohreh Ghotbeddin. « Low Frequency Electrical Stimulation Either Prior to Or after Rapid Kindling Stimulation Inhibits the Kindling-Induced Epileptogenesis ». BioMed Research International 2017 (2017) : 1–8. http://dx.doi.org/10.1155/2017/8623743.

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Objective. Studies are ongoing to find appropriate low frequency stimulation (LFS) protocol for treatment of epilepsy. The present study aimed at assessing the antiepileptogenesis effects of LFS with the same protocol applied either just before or immediately after kindling stimulations.Method. This experimental animal study was conducted on adult Wistar rats (200 ± 20 g) randomly divided into kindle (n=7), LFS + Kindle (n=6), and Kindle + LFS groups (n=6). All animals underwent rapid kindling procedure and four packages of LFS (1 Hz) with 5 min interval were applied either immediately before (LFS-K) or after kindling stimulation (K-LFS). The after discharge duration (ADD), daily stages of kindling, and kindling seizure stage and number of stimulations required to reach each stage were compared between the three groups using two-way analysis of variance (ANOVA) followed by Tukey post hoc and one-way ANOVA, and Kruskal-Wallis test, respectively.Results. LFS in both protocols significantly decreased the ADD (p<0.05) and daily seizure stages (p<0.05) and increased the number of stimulations required to achieve stage 3 and stages 4 and 5 of kindling compared with the kindle group (stage 2:p>0.05, stages 3 to 5:p<0.05).Conclusion. Although LFS-K showed more inhibiting effect than K-LFS, the difference was not statistically significant.

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Matsuura, S., K. Hirayama et R. Murata. « Enhancement of synaptic facilitation during the progression of kindling epilepsy by amygdala stimulations ». Journal of Neurophysiology 70, n^o 2 (1 août 1993) : 602–9. http://dx.doi.org/10.1152/jn.1993.70.2.602.

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1. A quantitative analysis of facilitation during the kindling stimulation to the amygdala was conducted by measuring the area between the excitatory potential and the baseline in the averaged tetanic response recorded at the entorhinal cortex. The changes in facilitation were then compared with the development of electrographic afterdischarges (AD) and behavioral seizures in response to successive kindling stimulations. 2. Kindling train pulses (n = 99 or 100; duration: 0.5 ms; frequency: 10 Hz; intensity: AD threshold) were applied to conscious rats until at least one generalized seizure occurred or until 13 stimuli were delivered. 3. Facilitation of the entorhinal responses by kindling stimulation first occurred in the monosynaptic excitatory component and was then followed by a progressive increase in the polysynaptic component that was manifested as the later negative peaks. A clear progressive enhancement was observed in the facilitation by successive kindling stimulations, which also induced prolongation of the AD duration and progression of the seizure stages, indicating that activity-dependent enhancement of facilitation (EF) occurred during the progression of kindling epilepsy. 4. Quantitative analysis revealed that the EF that occurred with the progression of seizure stages was statistically significant (P < 0.001, Friedman test). The AD duration (r = 0.89) and the long-term potentiation (r = 0.85) of the entorhinal responses by single test amygdala stimuli showed a very good linear relation to the EF.(ABSTRACT TRUNCATED AT 250 WORDS)

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Mia, Md Saimon, Yagna Jarajapu, Reena Rao et Sijo Mathew. « Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2 ». International Journal of Molecular Sciences 22, n^o 19 (30 septembre 2021) : 10599. http://dx.doi.org/10.3390/ijms221910599.

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The tumor microenvironment plays a critical role in defining the growth and malignancy of solid tumors. Extracellular matrix (ECM) proteins such as collagen, vitronectin, and fibronectin are major components of the tumor microenvironment. Tumor growth-promoting reciprocal interaction between ECM and cytoplasmic proteins is regulated by the cell surface receptors called integrins. This study investigated the mechanism by which integrin β1 promotes pancreatic tumor growth. In MIA PaCa-2 pancreatic cancer cell line, the loss of integrin β1 protein reduced the ability of cells to proliferate in a 3D matrix and compromised the ability to form a focal adhesion complex. Decreased expression of integrin α5 was observed in KO cells, which resulted in impaired cell spreading and adhesion on vitronectin and fibronectin. Reduced expression of the integrin-associated protein, kindlin-2 was also recorded. The downregulation of kindlin-2 decreased the phosphorylation of Smad2/3 by reducing the expression of TGF-β receptor 2. These results unravel a new mechanism of integrin β1 in tumor growth by modifying the expression of kindlin-2 and TGF-β receptor 2 signaling.

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Brunner, Molly, Angélique Millon-Frémillon, Genevieve Chevalier, Inaam A. Nakchbandi, Deane Mosher, Marc R. Block, Corinne Albigès-Rizo et Daniel Bouvard. « Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition ». Journal of Cell Biology 194, n^o 2 (18 juillet 2011) : 307–22. http://dx.doi.org/10.1083/jcb.201007108.

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The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization.

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Zai, Neelum A. Yousaf, Lamyae El Khalki, Wei Wang, Justin Szpendyk et Khalid Sossey-Alaoui. « Abstract 4381 : Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC ». Cancer Research 84, n^o 6_Supplement (22 mars 2024) : 4381. http://dx.doi.org/10.1158/1538-7445.am2024-4381.

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Abstract Background: Kindlins are a small gene family of FERM domain-containing adaptor proteins that function as essential drivers of integrin activation. Kindlin-2 (K2) is the most widely expressed member of the Kindlin family; its homozygous deletion in mice is embryonically lethal, K2 expression is also dysregulated in several human cancers, including the breast. We established K2 as a major driver of TNBC tumors progression and metastasis through the regulation of several hallmarks of cancer. A well-established pathway whereby K2 regulates TNBC oncogenic behavior is through the regulation of integrin inside-out signaling by directly binding to the cytoplasmic tail of integrin β subunit. Interestingly, our recent studies found K2 to play a major role in activating the TGF-β-mediated regulation of the CSF1/EGF signaling through macrophage polarization to the M2 tumorigenic state and their increased tumor infiltration, as well as inhibiting tumor infiltration of cytotoxic lymphocytes, therefore, driving tumor immune evasion. Here we present novel findings involving K2 in the stabilization of the β1-Integrin:TβRI complexes by establishing a physical bridge that links β1-Integrin to TβRI. Loss of K2 results in the degradation of this protein complex and therefore, the inhibition of the oncogenic pathways downstream of these two major regulators of several hallmarks of cancer. Methods: We applied a combination of in vitro assays; CRISPR/Cas9 gene editing, cell migration, 3D tumor-sphere, solid binding, co-immunoprecipitation, cell adhesion and spreading, as well as western blot and flow cytometry analyses with MDA-MB-231 and 4T1 TNBC cell lines. We also used preclinical in vivo mouse models of TNBC tumor progression and metastasis to support or investigations. Results: We found that the K2/β1-Integrin direct interaction is mediated through the C-terminal F3 domain of K2, while the K2/TβRI direct interaction is mediated through the F2 domain of K2. Disruption of this bridge, via CRISPR/Cas9-mediated knockout of K2, leads to β1-Integrin and TβRI degradation, and inhibition of the oncogenic pathways downstream of both β1-Integrin and TβRI, and inhibited tumor growth and metastasis in levels comparable to loss of expression of either β1-Integrin and TβRI. Treatment of the K2-deficent cells with the proteasome inhibitor MG-132 restored expression of both β1-Integrin and TβRI, confirming that K2 is required for the stabilization of the β1-integrin/TβR1 complexes at the protein level. Rescue of K2 expression in the K2-KO cells restored their oncogenic activities both in vitro and in vivo. Conclusion: We identified K2 to play a novel and major in the stabilization of the β1-integrin/TβR1 complexes and the regulation of their downstream oncogenic signaling. These studies have a significant translational impact as they may identify new therapeutically targeted pathways that are desperately needed for the treatment of TNBC tumors. Citation Format: Neelum A. Yousaf Zai, Lamyae El Khalki, Wei Wang, Justin Szpendyk, Khalid Sossey-Alaoui. Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4381.

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Andreeva-Gateva, P., D. Bakalov, Z. Sabit et B. Tenchov. « Effects of Ketogenic Diet on Corneal Kindling Mouse Model ». Acta Medica Bulgarica 47, n^o 2 (1 juillet 2020) : 7–11. http://dx.doi.org/10.2478/amb-2020-0015.

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AbstractAim/objective: Corneal kindling mouse test is a model of decreasing the seizure threshold after repetitive subchronical electrical stimuli. Ketogenic diet (KD) is used for the treatment of children with pharmacoresistant epilepsy since more than 100 years. Surprisingly, very few studies testing the effect of the KD in corneal kindling test were published. The aim of this study was to evaluate the effect of the KD on the seizure activity in corneal kindling mouse model.Methods: 50 adult male ICR mice (25-35 g) were randomly distributed in four groups, as follows: group 1 – standard diet (SD) treated controls (n = 10); group 2 – KD treated (n = 10), group 3 – kindled mice on SD treatment (n = 15); group 4 – kindled mice on KD treatment (n = 15). The diet was started at day one, one week before the start of the kindling and it continued for four weeks. At the end of the experiment, kindled mice were challenged with 6-Hz test and their behavior was assessed.Results: In kindled mice on SD the seizure latency time significantly decreased at days 14, 21 and 28. Mice on KD displayed relatively constant seizure latency during the experiment. At day 28 the duration of provoked seizures was statistically higher as compared with mice on KD (median values 101 vs 2 sec, p < 0.05). Blood ketone levels were statistically higher (p < 0.05), and blood glucose level was statistically lower (p < 0.05) in the KD treated group, as compared with SD treated mice.Conclusion: KD effectively suppressed the seizure activity in corneal kindling test. Further studies are needed for elucidating the molecular mechanisms which can explain this effect.

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Mishchenko, Mariia, Sergiy Shtrygol’, Andrii Lozynskyi, Mykhailo Hoidyk, Dmytro Khyluk, Tatyana Gorbach et Roman Lesyk. « Evaluation of 5-[(Z)-(4-nitrobenzylidene)]-2-(thiazol-2-ylimino)-4-thiazolidinone (Les-6222) as Potential Anticonvulsant Agent ». Scientia Pharmaceutica 90, n^o 3 (19 septembre 2022) : 56. http://dx.doi.org/10.3390/scipharm90030056.

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It was determined that the studied 5-[(Z)-(4-nitrobenzylidene)]-2-(thiazol-2-ylimino)-4-thiazolidinone (Les-6222) affects the cyclooxygenase pathway of the arachidonic acid cascade, the markers of damage to neurons on models of PTZ kindling. In the model of chronic epileptogenesis in mice (pentylenetetrazole kindling), a 4-thiazolidinone derivative showed high anticonvulsant activity, which is weaker than the effect of sodium valproate and higher than Celecoxib. The mentioned compound has a pronounced anti-inflammatory effect in the brain on the background of the PTZ kindling, reliably inhibiting COX-1 and COX-2. The predominant inhibition of COX-2 by 44.5% indicates this enzyme’s high selectivity of Les-6222. According to the molecular docking study results, the studied compound revealed the properties of COX-1/COX-2 inhibitor and especially 5-LOX/FLAP. The decreasing content of 8-isoprostane in the brain of mice of the Les-6222 group indicates a beneficial effect on cell membranes in the background of oxidative stress during the long-term administration of PTZ. In addition, Les-6222 significantly decreased the content of neuron-specific enolase, indicating neuroprotective properties in the background of chronic epileptogenesis. The obtained results experimentally substantiate the feasibility of further developing Les-6222 as a promising anticonvulsant agent.

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Chen, Zhefeng, Kai Shen, Ziyang Zheng, Jinchun Zhou, Shujie Zhao, Huanghe Song, Jiuxiang Liu, Xuan Zhao, Feng Liu et Qiang Zuo. « Kindlin-2 Promotes Chondrogenesis and Ameliorates IL-1beta-Induced Inflammation in Chondrocytes Cocultured with BMSCs in the Direct Contact Coculture System ». Oxidative Medicine and Cellular Longevity 2022 (12 avril 2022) : 1–13. http://dx.doi.org/10.1155/2022/3156245.

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The osteoarthritis caused by trauma or inflammation is associated with severe patient morbidity and economic burden. Accumulating studies are focusing on the repair of articular cartilage defects by constructing tissue-engineered cartilage. Recent evidence suggests that optimizing the source and quality of seed cells is one of the key points of cartilage tissue engineering. In this study, we demonstrated that Kindlin-2 and its activated PI3K/AKT signaling played an essential role in promoting extracellular matrix (ECM) secretion and ameliorating IL-1beta-induced inflammation in chondrocytes cocultured with bone marrow stem cells (BMSCs). In vivo experiments revealed that coculture significantly promoted hyaline cartilage regeneration. In vitro studies further uncovered that chondrocytes cocultured with BMSCs in the direct contact coculture system upregulated Kindlin-2 expression and subsequently activated the PI3K/AKT signaling pathway, which not only increases Sox9 and Col2 expression but also restores mitochondrial membrane potential and reduces ROS levels and apoptosis under inflammatory conditions. Overall, our findings indicated that direct contact BMSC-chondrocyte coculture system could promote chondrogenesis, and identified Kindlin-2 represents a key regulator in this process.

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Schloemer, Nathan J., Alex M. Abel, Monica S. Thakar, Yan-Qing Ma et Subramaniam Malarkannan. « Impact of Kindlin-3 in NK Cell-Mediated Anti-Tumor Cytotoxicity and Inflammatory Cytokine Production ». Blood 126, n^o 23 (3 décembre 2015) : 208. http://dx.doi.org/10.1182/blood.v126.23.208.208.

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Abstract Natural Killer (NK) cells are innate lymphocytes that play a central role in anti-viral and anti-tumor responses through direct cytotoxicity and production of inflammatory cytokines. Tumors can evade T-cell mediated immune-surveillance by down-regulation of the Class I Major Histocompatibility Complex (MHC-I) (Haworth et al, Ped Blood and Cancer, 2015). However, this lack of 'self' MHC-I serves as an activation stimulus for NK cells to recognize tumor cells. The molecular mechanism for 'self' recognition and destruction of 'missing self' are poorly understood. Integrins facilitate cell-to-cell interactions and are hypothesized to play a role in the 'self' versus 'missing self' recognition (Crozat et al, Blood, 2011). One of the critical regulators of integrin activation is Kindlin-3, which helps in their 'inside-out' signaling. Kindlin-3 binds to the cytoplasmic tail of β2-integrin and induces a conformational change increasing ligand affinity (Ye et al, Curr Biol, 2013). Consequently, Kindlin-3 is localized at the immunologic synapse and has been shown to interact with Adhesion and Degranulation Adaptor Protein (ADAP) (Kasirer-Friede et al, Blood 2014). As our group has shown, ADAP plays a central role in the signaling transduction for inflammatory cytokine production in NK cells (Rajasekaran et al, Nat Immunol, 2013). Clinically, defects in Kindlin-3 functions in humans are manifested by severe immune deficiency and bleeding disorder defined as Leukocyte Adhesion Deficiency-III (LAD-III). Based on these observations, we hypothesized Kindlin-3-dependent integrin function is critical for NK cell-mediated anti-tumor cytotoxicity and production of inflammatory cytokines. To define the role of Kindlin-3 in NK cell effector functions, we utilized a murine model. Kindlin-3 knock-in (K3KI) mice carry a double substitution mutation disrupting the binding of Kindlin-3 to β2-integrin (Xu et al, Arterioscler Thromb Vac Biol, 2014). NK cells from K3KI mice were evaluated for development and effector functions. Flow cytometry was utilized to identify maturation and developmental populations. Inflammatory cytokine production was assessed by Interferon-γ release following NK cell and tumor co-culture as well as plate-bound antibody activation. Cytotoxicity was assessed by 51Cr-release assay and the following tumor cells were used: cells representing, 1) 'self' (RMA and EL4 thymomas with autologous MHC-I); 2) 'missing-self' (RMA/S with decreased MHC expression relative to RMA); 3) 'induced self' (EL4 thymomas stably expressing H60, an activating ligand for NKG2D; 4) 'non-self' (YAC1 lymphoma with allogeneic MHC). Our results show an overall increase in the peripheral NK populations collected from spleens of K3KI mice, as seen in patients with LAD-III. However, no significant maturational defects were noted in the bone marrow of the K3KI mice. In vitro analyses reveal that K3KI NK cells have significantly impaired anti-tumor cytotoxicity relative to wild type controls (Figure 1). There was a significant reduction in the cytotoxic ability of K3KI NK cells towards 'induced self' or 'missing self' recognition (p<0.05). In contrast, K3KI NK cells have augmented inflammatory interferon-γ cytokine production compared to wild type controls when co cultured with the same tumor models in which cytotoxicity was significantly impaired (Figure 2). These data reveal the essential role of Kindlin-3 interaction with integrin for the effector functions of the NK cell. Currently, we are delineating the signaling mechanisms which mediate this divergence in NK cell functions dependent on Kindlin-3. Our studies reveal an undefined role for Kindlin-3 in NK cells and may help to identify novel therapeutic targets to modulate NK cell effector functions. Disclosures No relevant conflicts of interest to declare.

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Montanez, E., S. Ussar, M. Schifferer, M. Bosl, R. Zent, M. Moser et R. Fassler. « Kindlin-2 controls bidirectional signaling of integrins ». Genes & ; Development 22, n^o 10 (15 mai 2008) : 1325–30. http://dx.doi.org/10.1101/gad.469408.

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Neugebauer, Volker, Fatiha Zinebi, Rex Russell, Joel P. Gallagher et Patricia Shinnick-Gallagher. « Cocaine and Kindling Alter the Sensitivity of Group II and III Metabotropic Glutamate Receptors in the Central Amygdala ». Journal of Neurophysiology 84, n^o 2 (1 août 2000) : 759–70. http://dx.doi.org/10.1152/jn.2000.84.2.759.

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G-protein-coupled metabotropic glutamate receptors (mGluRs) are being implicated in various forms of neuroplasticity and CNS disorders. This study examined whether the sensitivities of mGluR agonists are modulated in a distinct fashion in different models of synaptic plasticity, specifically, kindling and chronic cocaine treatment. The influence of kindling and chronic cocaine exposure in vivo was examined in vitro on the modulation of synaptic transmission by group II and III metabotropic glutamate receptors using whole cell voltage-clamp recordings of central amygdala (CeA) neurons. Synaptic transmission was evoked by electrical stimulation of the basolateral amygdala (BLA) and ventral amygdaloid pathway (VAP) afferents in brain slices from control rats and from rats treated with cocaine or exposed to three to five stage-five kindled seizures. This study shows that after chemical stimulation with chronic cocaine exposure or after electrical stimulation with kindling the receptor sensitivities for mGluR agonists are altered in opposite ways. In slices from control rats, group II agonists, (2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (LCCG1) and (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), depressed neurotransmission more potently at the BLA-CeA than at the VAP-CeA synapse while group III agonist, L(+)-2-amino-4-phosphonobutyrate (LAP4), depressed neurotransmission more potently at the VAP-CeA synapse than at the BLA-CeA. These agonist actions were not seen (were absent) in amygdala neurons from chronic cocaine-treated animals. In contrast, after kindling, concentration response relationships for LCCG1 and LAP4 were shifted to the left, suggesting that sensitivity to these agonists is increased. Except at high concentrations, LCCG1, LY354740, and LAP4 neither induced membrane currents nor changed current-voltage relationships. Loss of mGluR inhibition with chronic cocaine treatment may contribute to counter-adaptive changes including anxiety and depression in cocaine withdrawal. Drugs that restore the inhibitory effects of group II and III mGluRs may be novel tools in the treatment of cocaine dependence. The enhanced sensitivity to group II and III mGluR agonists in kindling is similar to that recorded at the lateral to BLA synapse in the amygdala where they reduce epileptiform bursting. These findings suggest that drugs modifying mGluRs may prove useful in the treatment of cocaine withdrawal or epilepsy.

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Nayden, Naydenov, et Ivanov Andrei. « P-183 Kindlin 1and Kindlin 2 Regulate Adhesion and Migration of Colonic Epithelial Cells ». Inflammatory Bowel Diseases 20 (décembre 2014) : S100—S101. http://dx.doi.org/10.1097/01.mib.0000456902.63774.1b.

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Aleshin, Vasily A., Anastasia V. Graf, Artem V. Artiukhov, Alexander L. Ksenofontov, Lev G. Zavileyskiy, Maria V. Maslova et Victoria I. Bunik. « Pentylenetetrazole-Induced Seizures Are Increased after Kindling, Exhibiting Vitamin-Responsive Correlations to the Post-Seizures Behavior, Amino Acids Metabolism and Key Metabolic Regulators in the Rat Brain ». International Journal of Molecular Sciences 24, n^o 15 (3 août 2023) : 12405. http://dx.doi.org/10.3390/ijms241512405.

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Epilepsy is characterized by recurrent seizures due to a perturbed balance between glutamate and GABA neurotransmission. Our goal is to reveal the molecular mechanisms of the changes upon repeated challenges of this balance, suggesting knowledge-based neuroprotection. To address this goal, a set of metabolic indicators in the post-seizure rat brain cortex is compared before and after pharmacological kindling with pentylenetetrazole (PTZ). Vitamins B1 and B6 supporting energy and neurotransmitter metabolism are studied as neuroprotectors. PTZ kindling increases the seizure severity (1.3 fold, p < 0.01), elevating post-seizure rearings (1.5 fold, p = 0.03) and steps out of the walls (2 fold, p = 0.01). In the kindled vs. non-kindled rats, the post-seizure p53 level is increased 1.3 fold (p = 0.03), reciprocating a 1.4-fold (p = 0.02) decrease in the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) controlling the glutamate degradation. Further, decreased expression of deacylases SIRT3 (1.4 fold, p = 0.01) and SIRT5 (1.5 fold, p = 0.01) reciprocates increased acetylation of 15 kDa proteins 1.5 fold (p < 0.01). Finally, the kindling abrogates the stress response to multiple saline injections in the control animals, manifested in the increased activities of the pyruvate dehydrogenase complex, malic enzyme, glutamine synthetase and decreased malate dehydrogenase activity. Post-seizure animals demonstrate correlations of p53 expression to the levels of glutamate (r = 0.79, p = 0.05). The correlations of the seizure severity and duration to the levels of GABA (r = 0.59, p = 0.05) and glutamate dehydrogenase activity (r = 0.58, p = 0.02), respectively, are substituted by the correlation of the seizure latency with the OGDHC activity (r = 0.69, p < 0.01) after the vitamins administration, testifying to the vitamins-dependent impact of the kindling on glutamate/GABA metabolism. The vitamins also abrogate the correlations of behavioral parameters with seizure duration (r 0.53–0.59, p < 0.03). Thus, increased seizures and modified post-seizure behavior in rats after PTZ kindling are associated with multiple changes in the vitamin-dependent brain metabolism of amino acids, linked to key metabolic regulators: p53, OGDHC, SIRT3 and SIRT5.

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