Gotowa bibliografia na temat „PKC-Beta2”

We investigated the protein expression of different protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta1, PKC-beta2, PKC-gamma, PKC-delta, PKC-epsilon, PKC-eta and PKC-zeta) in the hippocampus of normal control mice and progressive changes in PKC isoforms expression during and after pilocarpine induced status epilepticus (PISE). We showed the reduced expression of PKC-delta, PKC-eta and PKC-zeta in interneurons in the CA1 area and in the hilus of the dentate gyrus during or after PISE. Increased expression of PKC-alpha and PKC-beta1 was demonstrated in the stratum pyramidale of CA3 area, and PKC-epsilon was up-regulated in the stratum lucidum of the CA3 area during or after PISE. Our results suggest that hippocampal PKC isoforms may play different roles in seizure generation, and be targets for development of anti-convulsive drugs.

Although activation of adenosine 3',5'-cyclic monophosphate by histamine and of Ca2+-dependent signaling pathways by cholinergic agonists is a generally recognized mechanism for increasing parietal cell HCl secretion, the role of protein kinase C (PKC) in this process is controversial. In this study, acid-secretory responses of gastric glands from rabbits [measured as accumulation of aminopyrine (AP)] were found to be relatively resistant to the PKC inhibitors calphostin C, chelerythrine chloride, staurosporine, and the bisindolylmaleimide-like inhibitors Ro 31-8220, Go 6976, and bisindolylmaleimide I hydrochloride. Western analyses of the PKC isozyme profile in highly enriched parietal cells (98% purity) indicated that this cell type expresses abundant levels of the novel isoforms PKC-epsilon and PKC-mu and abundant levels of the atypical isoforms PKC-iota, PKC-lambda, and PKC-zeta. In contrast, there appeared to be low to undetectable expression of the classical isoforms PKC-alpha and PKC-beta1/beta2, respectively. Relatively high concentrations of Ro 31-8220 potentiated both carbachol- and histamine-stimulated AP accumulation (IC50 857 +/- 100 and 910 +/- 98 nM, respectively). There was a similar dose dependence for Ro 31-8220 inhibition of in situ phosphorylation of a parietal cell phosphoprotein, pp66 (IC50 750 +/- 120 nM). Similar concentrations of Ro 31-8220 also inhibited phosphorylation of the cytoskeletal, actin membrane cross-linking phosphoprotein ezrin, but not other phosphoproteins. Ezrin phosphorylation was increased by carbachol and 12-O-tetradecanoylphorbol 13-acetate (TPA). Because carbachol and TPA stimulate pp66 phosphorylation in a Ca2+-independent manner, our results suggest that one or more novel PKC isoforms may be involved in negative regulation of HCl secretion. In related experiments, PKC-epsilon, but not PKC-mu, was immunolocalized by confocal microscopy to a parietal cell compartment that bore a striking resemblance to that containing filamentous actin. Moreover, pp66 was enriched in a Triton X-100-insoluble parietal cell fraction, suggesting a potential cytoskeletal localization for this unknown protein. Given their location and sensitivity to Ro 31-8220, it is possible that pp66 and ezrin interact in a PKC-dependent manner to regulate the well-known morphological changes that occur in concert with agonist-dependent activation of parietal cell HCl secretion.

Abstract Background. The protein kinase C (PKC) inhibitor PKC412 (N-benzylstaurosporine) is a derivate of the naturally occurring alkaloid staurosproine and has been shown to inhibit the conventional isoforms of PKC (alfa, beta1, beta2 and gamma). PKC412 has been shown to have an antitumor effect on non-small cell lung cancer and acute leukemia with FLT3 mutations, but little is known about its effect on multiple myeloma up to date. Methods. Since PKC is also an inhibitor of a tyrosin kinase which is associated with VEGF, and inhibits the release of Interleukin-6, TNF alfa, and that of growth factor dependent C-FOS, we postulated that PKC412 might have also strong anti-myeloma features. Here we evaluated the anti-myeloma effect of PKC412 in the multiple myeloma cell lines INA-6, OPM-2 and RPMI 8226 by measuring its effect on their proliferation rate, the apoptosis rate and the Interleukin-6 mRNA expression. Results. PKC412 showed strong anti-myeloma effects in all three celllines. 50nM of PKC412 was enough to drop the proliferation rate in all three cell lines under 10% compared to untreated cells(p<0.01). The apoptosis rate increased in INA cell line up to 2,5 times and in RPMI cell line up to 3 times (p<0.05), whereas only a moderate increase was observed in the OPM2 cell line with 500nM of PKC412. As expected, the IL-6 mRNA expression decreased after PKC412 treatment in all three cell lines more than 50%. The addition of Bevacizumab to PKC412 in RPMI and OPM-2 cell lines did not increased the apoptosis rate significantly, whereas the addition of short-interference RNA (RNAi) against VEGF increased the apoptosis rate in RPMI 8226 cells about 20% (p<0.05) and in OPM-2 cells up to 30% (p<0.01) compared to PKC412 alone, which was also associated concordantly with a further reduction of the proliferation rate in RPMI and OPM-2 cells up to 30%. Conclusions. PKC412 shows strong anti-myeloma effects and might be effective also in the treatment of patients with multiple myeloma. These in-vitro studies might encourage to initiate clinical trials with PKC412 in patients with multiple myeloma.

Abstract Peripheral artery disease (PAD) is atherosclerotic occlusion of vessel outside the heart and most commonly affects the lower extremities. Diabetes (DM) accelerates the course and severity of PAD. Studies have shown that vascular endothelial cell NF-κB activity is required for post ischemic adaptation in experimental PAD. To better understand how DM contributes to PAD severity, we investigated the role of DM hyperglycemia in the activation of NF-κB under ischemic conditions. Induced ischemia in human vascular endothelial cell (HUVEC) cultures increased components of both canonical and non-canonical NF-κB pathways in the nucleus (p65 1.0 ± 0.1 vs 1.5 ± 0.2, p< 0.05, RelB 1.0 ± 0.1 vs 1.5 ± 0.2, p<0.01). Similarly, HUVEC acutely exposed to high glucose (HG, 25 mM) activated both canonical (IκB-α degradation, normal vs. HG 1.25 ± 0.02 vs 0.9 ± 0.0, p<0.05) and non-canonical NF-κB (p100 degradation, normal vs HG 0.021±0.001 vs 0.016±0.000, p<0.05) pathways. Prolonged exposure (3 days) of HUVEC to high glucose before ischemia resulted in impaired NF-κB activation as evident from decreased IκB phosphorylation (pIκB/IκB in normal glucose and ischemia 1.56 ± 0.22 vs 1.12 ± 0.35, p<0.01). To understand the signaling pathways underlying the ischemic activation of the NF-κB pathway, we used an array of antibodies to phosphoproteins involved in the inflammatory pathway. Compared to the lysates from cells grown in normal glucose, the lysates from cells grown in prolonged high glucose had dramatically increased phosphorylation of PKC-β2 (PKC-β2pSer661, 8-fold increase). To test whether this increase in PKC-β2pSer66 impairs NF-κB activation by ischemia, we treated HUVECS with prolonged high glucose exposure and ruboxystaurin (Rbx) (20 nM), an inhibitor of PKC-β2 phosphorylation, prior to ischemic exposure. Immunoblotting results confirmed that inhibition of PKC-β2 phosphorylation enhanced the ischemia induced NF-κB activation in HUVEC in this condition. We then tested the effect of Rbx on PKC-β2 phosphorylation and NF-κB activation in vivo in Akita mice, a model for type 1 diabetes. Consistent with our in vitro findings, in experimental PAD, NF-κB activity in the ischemic hind limb of Akita mice was significantly lower than those of the wild type (WT) mice as measured by IκB-α degradation (WT ischemic vs Akita ischemic; 0.04 ± 0.03 vs 0.10 ± 0.04 p<0.05). However, treatment of Akita mice with Rbx increased NF-κB activation in the ischemic hind limb (Akita ischemic 0.10 ± 0.04 vs ischemic+ Rbx 0.05 ± 0.02, p<0.05). Moreover, compared to the WT mice, the untreated Akita mice showed an impaired perfusion in the ischemic limbs (% perfusion recovery, WT vs Akita; 80.1 ± 10.3 vs 55.7 ± 10.1, p<0.05, n=5-8) that was improved in Rbx treated Akita mice (96.3 ± 2.3, p<0.01). Thus, hyperglycemic conditions increase PKC-β2pSer66 in endothelial cells attenuating salutary NF-κB activation contributing to poor PAD outcomes in DM.

Les polymorphonucléaires neutrophiles, sont des acteurs essentiels du système immunitaire inné, responsables de la phagocytose des agents pathogènes. Lors de la phagocytose, les neutrophiles produisent des quantités substantielles d'anion superoxyde, qui génèrent ensuite des espèces réactives de l'oxygène (ERO) telles que le peroxyde d'hydrogène, les radicaux hydroxyles et l'acide hypochloreux, indispensables à la destruction des microbes. L'enzyme responsable de la production de superoxyde est le complexe NADPH oxydase, composé de protéines membranaires (gp91phox/NOX2 et p22phox) et de protéines cytosoliques (p47phox, p67phox, p40phox et Rac1/2). Lors de l'activation, ces composants cytosoliques transloquent vers la membrane, entraînant l'assemblage et l'activation de l'enzyme. Une régulation adéquate de l'activité de la NADPH oxydase est essentielle pour équilibrer une élimination efficace des pathogènes et éviter les dommages tissulaires excessifs dus aux ERO. L' objectifs de ma thèse vise à examiner la phosphorylation de la p47phox dans les neutrophiles humains stimulés par le zymosan opsonisé par le sérum (OZ), un agent connu pour induire la phagocytose. Nous nous concentrons sur l'identification des sites de phosphorylation spécifiques et l'élucidation des voies de signalisation impliquées dans ce processus. Les neutrophiles humains ont été isolés du sang veineux de volontaires sains par sédimentation au Dextran et centrifugation sur Ficoll. Le zymosan a été opsonisé en utilisant du sérum autologue riche en immunoglobulines (IgG) et en protéines du complément (C3b et C3bi). Les neutrophiles ont ensuite été stimulés avec du zymosan non opsonisé ou opsonisé, et la phosphorylation de p47phox a été évaluée par SDS-PAGE et Western blot avec des anticorps spécifiques. La production de ROS a été mesurée en utilisant la chimioluminescence amplifiée par le luminol. La microscopie confocale a été utilisée pour visualiser les interactions des neutrophiles avec le zymosan opsonisé fluorescent. Divers inhibiteurs de kinases ont été utilisés pour disséquer les voies de signalisation conduisant à la phosphorylation de p47phox. Les résultats montrent que le zymosan opsonisé par le sérum (OZ) a induit une phosphorylation rapide et transitoire de p47phox aux sites Ser304, Ser315, Ser320 et Ser328, détectable en 20 secondes et atteignant un pic à 40-60 secondes. Cette phosphorylation diminue sur 10 minutes, tandis que la production de ROS est restée soutenue pendant plus de 30 minutes. Le zymosan non opsonisé n'a pas induit de phosphorylation significative ni de production de ROS. La phosphorylation a eu lieu lors du contact avec le OZ, avant la phagocytose, et a été principalement induite par les opsonines IgG et C3bi via leurs récepteurs respectifs, Fc-gamma R et CR3. Les études utilisant des inhibiteurs ont révélé que les kinases tyrosine Src et Syk, PI3K, PLC, PLD, le calcium et PKC-beta2 sont essentiels pour la phosphorylation de p47phox et l'activation subséquente de la NADPH oxydase. Cette étude élucide les événements de phosphorylation spécifiques et les voies de signalisation qui régulent l'activation de la NADPH oxydase dans les neutrophiles humains pendant la phagocytose. Le zymosan opsonisé par le sérum induit une phosphorylation rapide de p47phox aux sites Ser304, Ser315, Ser320 et Ser328, nécessaire pour initier mais non maintenir l'activité de la NADPH oxydase. Les IgG et C3bi sont les principales opsonines conduisant ce processus via les récepteurs Fc-gamma R et CR3. Les voies de signalisation clés impliquent les kinases tyrosine Src et Syk, PI3K, PLC, PLD, le calcium et PKC-beta2. Ces résultats améliorent notre compréhension de l'activation des neutrophiles et offrent des cibles thérapeutiques potentielles pour moduler les réponses immunitaires
Neutrophils, also known as polymorphonuclear leukocytes (PMNs), are essential players in the innate immune system, responsible for the phagocytosis of pathogens. During phagocytosis, neutrophils produce substantial amounts of superoxide anion, which subsequently generates reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals, and hypochlorous acid, crucial for microbial killing. The enzyme responsible for superoxide production is the NADPH oxidase complex, composed of membrane-bound proteins (gp91phox/NOX2 and p22phox) and cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). Upon activation, these cytosolic components translocate to the membrane, leading to the assembly and activation of the enzyme. Proper regulation of NADPH oxidase activity is essential to balance effective pathogen clearance and avoid excessive tissue damage due to ROS.The objectives of my thesis aims to investigate the phosphorylation of p47phox in human neutrophils stimulated by serum-opsonized zymosan (OZ), an agent known to induce phagocytosis. We focus on identifying the specific phosphorylation sites and elucidating the signaling pathways involved in this process. Human neutrophils were isolated from the venous blood of healthy volunteers using Dextran sedimentation and Ficoll centrifugation. Zymosan was opsonized using autologous serum rich in immunoglobulins (IgG) and complement proteins (C3b and C3bi). Neutrophils were then stimulated with either non-opsonized or opsonized zymosan, and p47phox phosphorylation was assessed using SDS-PAGE and Western blotting with specific antibodies. ROS production was measured using luminol-enhanced chemiluminescence. Confocal microscopy was employed to visualize neutrophil interactions with fluorescently labeled opsonized zymosan. Various kinase inhibitors were used to dissect the signaling pathways leading to p47phox phosphorylation. The results showed that serum-opsonized zymosan (OZ) induced rapid and transient phosphorylation of p47phox at Ser304, Ser315, Ser320, and Ser328, detectable within 20 seconds and peaking at 40-60 seconds. This phosphorylation declined over 10 minutes, while ROS production remained sustained for over 30 minutes. Non-opsonized zymosan did not induce significant phosphorylation or ROS production. Phosphorylation occurred upon contact with OZ, prior to phagocytosis, and was primarily induced by IgG and C3bi opsonins through their respective receptors, Fc-gamma R and CR3. Inhibitor studies revealed that Src and Syk tyrosine kinases, PI3K, PLC, PLD, calcium, and PKC-beta2 are crucial for p47phox phosphorylation and subsequent NADPH oxidase activation. This study elucidates the specific phosphorylation events and signaling pathways that regulate NADPH oxidase activation in human neutrophils during phagocytosis. Serum-opsonized zymosan induces rapid phosphorylation of p47phox at Ser304, Ser315, Ser320, and Ser328, which is necessary to initiate but not sustain NADPH oxidase activity. IgG and C3bi are the primary opsonins driving this process through Fc-gamma R and CR3 receptors. Key signaling pathways involve Src and Syk tyrosine kinases, PI3K, PLC, PLD, calcium, and PKC-beta2. These findings enhance our understanding of neutrophil activation and provide potential therapeutic targets for modulating immune responses