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Atypical PKC (atypical + pkc)
Selected AbstractsUp-Regulation of Cell Surface Insulin Receptor by Protein Kinase C-, in Adrenal Chromaffin CellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2000Involvement of Transcriptional, Translational Events Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12- O -tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C-, (PKC-,) and novel PKC-, (but not atypical PKC-,) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-,. In the present study, chronic (,12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4,-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans -Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the ,-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor ,-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor ,-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-, up-regulates the density of the cell surface insulin receptor via transcriptional/translational events. [source] Involvement of protein kinase C, in interleukin-1, induction of ADAMTS-4 and type 2 nitric oxide synthase via NF-,B signaling in primary human osteoarthritic chondrocytesARTHRITIS & RHEUMATISM, Issue 12 2007Priya S. Chockalingam Objective Protein kinase C, (PKC,), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKC, in interleukin-1, (IL-1,),induced NF-,B signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. Methods Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKC,. Western blot analysis was used to evaluate phosphorylation of PKC, and NF-,B. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. Results Phosphorylation of PKC, and NF-,B was induced by IL-1, treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKC, suppressed IL-1,,induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1,,induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKC, and NO production. Furthermore, small interfering RNA, or short hairpin RNA,mediated knockdown of PKC, mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion Our results show that PKC, is involved in the regulation of IL-1,,induced NF-,B signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKC, could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA. [source] Possible role of the protein kinase C/CPI-17 pathway in the augmented contraction of human myometrium after gestationBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2003Hiroshi Ozaki Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (PDBu, 1 ,M) induced sustained contractions with no increase in [Ca2+]i in nonpregnant and pregnant human myometria. The contractile effects of PDBu in pregnant myometrium were much greater than those in nonpregnant myometrium, and the contractions in pregnant myometrium were accompanied by an increase in myosin light chain (MLC) phosphorylation at Ser19. The contraction induced by PDBu in pregnant myometrium was inhibited by the inhibitors of conventional PKC isoforms, bisindolylmaleimides and indolocarbazole, such as Go6976, Go6983, and Go6850 (1 ,M). LY333531 (1 ,M), a specific inhibitor of PKC,, also inhibited the PDBu-induced contraction in the pregnant myometrium. In the pregnant myometrium permeabilized with , -toxin, PDBu increased the contractions induced at fixed Ca2+ concentration (0.3 ,M) both in nonpregnant and pregnant myometria, indicating Ca2+ sensitization of contractile elements. Western immunoblot analysis indicated that pregnant myometrium contained PKC isozymes such as conventional PKC (,, ,, ,), novel PKC (,, ,, ,), and atypical PKC (, but not , and ,). RT-PCR and real-time RT-PCR analysis indicated that, among the conventional PKC, the levels of mRNA of , isoform in pregnant human myometrium were greater than those in nonpregnant myometrium. CPI-17 is a substrate for PKC, and the phosphorylated CPI-17 is considered to inhibit myosin phosphatase. The levels of CPI-17 mRNA and protein expression were also greater in the pregnant myometrium. These results suggest that the PKC-mediated contractile mechanism is augmented in human myometrium after gestation, and that this augmentation may be attributable to the increased activity of the , PKC isoform and CPI-17. British Journal of Pharmacology (2003) 140, 1303,1312. doi:10.1038/sj.bjp.0705552 [source] Role of atypical protein kinase C isozymes and NF-,B in IL-1,-induced expression of cyclooxygenase-2 in human myometrial smooth muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Sara V. Duggan Increased myometrial expression of cyclooxygenase-2 (Cox-2) at term results from elevated local levels of inflammatory cytokines, and its inhibition provides a potential route for intervention in human pre-term labor. We have identified a role for atypical protein kinase C (PKC) isozymes in IL-1,-induced Cox-2 expression in human myometrial smooth muscle cells (HMSMC). The PKC inhibitor GF109203X (10 µM) inhibited IL-1,-induced Cox-2 protein and RNA expression, which were also reduced by MAPK and nuclear factor ,B (NF-,B) inhibitors. GF109203X did not affect MAPK activities, and neither did it replicate the effect of p38 MAPK inhibition on Cox-2 mRNA stability, suggesting that PKC operates through an independent mechanism. The effect of GF109203X remained intact after depletion of conventional and novel PKC isozymes by phorbol ester pre-treatment. In contrast LY379196 (10 µM), which at micromolar concentrations inhibits all but atypical PKCs, did not affect Cox-2 expression. A peptide corresponding to the pseudosubstrate sequence of atypical PKCs blocked Cox-2 protein expression, whereas the sequence from conventional PKCs was ineffective. GF109203X did not affect NF-,B binding to nuclear proteins, but strongly reduced NF-,B-dependent transcription in luciferase reporter assays. Our findings indicate that IL-1,-induced Cox-2 expression in HMSMC in culture requires p38-MAPK-mediated mRNA stabilization and an independent activation of Cox-2 transcription which is dependent on the action of atypical PKCs, probably through direct stimulation of the transactivating activity of NF-,B. J. Cell. Physiol. 210: 637,643, 2007. © 2006 Wiley-Liss, Inc. [source] Involvement of protein kinase C, in interleukin-1, induction of ADAMTS-4 and type 2 nitric oxide synthase via NF-,B signaling in primary human osteoarthritic chondrocytesARTHRITIS & RHEUMATISM, Issue 12 2007Priya S. Chockalingam Objective Protein kinase C, (PKC,), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKC, in interleukin-1, (IL-1,),induced NF-,B signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. Methods Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKC,. Western blot analysis was used to evaluate phosphorylation of PKC, and NF-,B. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. Results Phosphorylation of PKC, and NF-,B was induced by IL-1, treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKC, suppressed IL-1,,induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1,,induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKC, and NO production. Furthermore, small interfering RNA, or short hairpin RNA,mediated knockdown of PKC, mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion Our results show that PKC, is involved in the regulation of IL-1,,induced NF-,B signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKC, could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA. [source] | |||||||||||||