A critical role for PKC zeta in endothelin-1-induced uterine contractions at the end of pregnancy

We have previously shown that protein kinase C (PKC) and/or PKC are necessary for endothelin-1 (ET-1)-induced human myometrial contraction at the end of pregnancy (Eude I, Paris P, Cabrol D, Ferré F, and Breuiller-Fouché M. Biol Reprod 63: 1567–1573, 2000). Here, we report that the selective inhibitor of PKC isoform, Rottlerin, does not prevent ET-1-induced contractions, whereas LY-294002, a phosphatidylinositol (PI) 3-kinase inhibitor, affects the contractile response. This study characterized the in vitro contractile response of cultured human pregnant myometrial cells to ET-1 known to induce in vitro contractions of intact uterine smooth muscle strips. Cultured myometrial cells incorporated into collagen lattices have the capacity to reduce the size of these lattices, referred to as lattice contraction. Neither the selective conventional PKC isoform inhibitor, Gö-6976, or rottlerin affected myometrial cell-mediated gel contraction by ET-1, whereas this effect was blocked by LY-294002. We found that treatment of myometrial cell lattices with an inhibitory peptide specific for PKC or with an antisense against PKC resulted in a significant loss of ET-1-induced contraction. Evidence is also presented by using confocal microscopy that ET-1 induced translocation of PKC to a structure coincident with the actin-rich microfilaments of the cytoskeleton. We have shown that PKC has a role in the actin organization in ET-1-stimulated cells. Accordingly, our results suggest that PKC plays a role in myometrial contraction in pregnant women. protein kinase C; uterine smooth muscle; parturition     

Multiple signaling pathways are involved in endothelin-1-induced brain endothelial cell migration

We have observed that the vasoactive peptide endothelin-1 is a potent inducer of migration of primary human brain-derived microvascular endothelial cells. By blocking signal transduction pathways with specific inhibitors, and using dominant negative mutant infections, we have demonstrated that multiple pathways are involved in endothelin-1-induced migration. Absolutely required for migration are protein tyrosine kinase Src, Ras, protein kinase C (PKC), phosphatidylinositol 3-kinase, ERK, and JNK; partial requirements were exhibited by cAMP-activated protein kinase and p38 kinase. Partial elucidation of the signal transduction sequences showed that the MAPKs ERK, JNK, and p38 are positioned downstream of both PKC and cAMP-activated protein kinase in the signal transduction scheme. The results show that human brain endothelial cell migration has distinct characteristics, different from cells derived from other vascular beds, or from other species, often used as model systems. Furthermore, the results indicate that endothelin-1, secreted by many tumors, is an important contributor to tumor-produced proangiogenic microenvironment. This growth factor has been associated with increased microvessel density in tumors and is responsible for endothelial cell proliferation, migration, invasion, and tubule formation. Because many signal transduction pathways investigated in this study are potential or current targets for anti-angiogenesis therapy, these results are of critical importance for designing physiological antiangiogenic protocols. signal transduction; angiogenesis; microvessels; vasoactive peptides     

Role of protein kinase C isoforms in the regulation of interleukin-13-induced 15-lipoxygenase gene expression in human monocytes

We reported previously that interleukin-13 (IL-13) induces tyrosine phosphorylation/activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6 in primary human monocytes. We recently revealed that p38 MAPK-mediated serine phosphorylation of both Stat1 and Stat3 is required for the induction of 15-lipoxygenase (15-LO) expression by IL-13. In this study, we present data indicating that another serine/threonine kinase, PKCdelta, is also required for IL-13-induced 15-LO expression. PKCdelta, a member of the novel protein kinase C (PKC) subclass, was rapidly phosphorylated and activated upon exposure to IL-13. Treatment of cells with rottlerin, a PKCdelta inhibitor, blocked IL-13-induced 15-LO mRNA and protein expression, whereas Go6976, an inhibitor of the conventional PKC subclass, had no inhibitory effects. Down-regulation of cellular PKCdelta protein levels by PKCdelta-specific antisense oligodeoxyribonucleotides also inhibited 15-LO expression markedly. IL-13-induced 15-LO expression resulted in significant inhibition of synthesis of the potent chemotactic factor leukotriene B4, and that process was reversed by rottlerin, presumably through the blockage of PKCdelta-dependent 15-LO expression. Furthermore, our data demonstrate that IL-13-mediated activation of PKCdelta and p38 MAPK are independent pathways, because inhibition of one kinase activity had no effect on the other, suggesting that the two pathways act in parallel to regulate the downstream targets necessary for 15-LO expression. Inhibition of PKCdelta activation by rottlerin also markedly attenuated IL-13-induced Stat3 DNA binding activity. Our findings indicate that PKCdelta plays an important role in regulating IL-13-induced 15-LO expression in human monocytes and subsequently modulates the inflammatory responses mediated by 15-LO products.     

MYPT1 protein isoforms are differentially phosphorylated by protein kinase G

Smooth muscle relaxation in response to NO signaling is due, in part, to a Ca(2+)-independent activation of myosin light chain (MLC) phosphatase by protein kinase G Iα (PKGIα). MLC phosphatase is a trimeric complex of a 20-kDa subunit, a 38-kDa catalytic subunit, and a 110-133-kDa myosin-targeting subunit (MYPT1). Alternative mRNA splicing produces four MYPT1 isoforms, differing by the presence or absence of a central insert and leucine zipper (LZ). The LZ domain of MYPT1 has been shown to be important for PKGIα-mediated activation of MLC phosphatase activity, and changes in LZ+ MYPT1 isoform expression result in changes in the sensitivity of smooth muscle to NO-mediated relaxation. Furthermore, PKGIα has been demonstrated to phosphorylate Ser-694 of MYPT1, but phosphorylation at this site does not always accompany cGMP-mediated smooth muscle relaxation. This study was designed to determine whether MYPT1 isoforms are differentially phosphorylated by PKGIα. The results demonstrate that purified LZ+ MYPT1 fragments are rapidly phosphorylated by PKGIα at Ser-667 and Ser-694, whereas fragments lacking the LZ domain are poor PKGIα substrates. Mutation of Ser-667 and Ser-694 to Ala and/or Asp showed that Ser-667 phosphorylation is more rapid than Ser-694 phosphorylation, suggesting that Ser-667 may play an important role in the activation of MLC phosphatase. These results demonstrate that MYPT1 isoform expression is important for determining the heterogeneous response of vascular beds to NO and NO-based vasodilators, thereby playing a central role in the regulation of vascular tone in health and disease.     

G蛋白,蛋白激酶C和Na^＋—H^＋交换在内皮素—1诱导培养心肌细胞肥大反应中的

内皮系-1（ET-1）是一种强的生长因子,并诱导心肌细胞肥大反应。在本实验中,我们探讨了G蛋白、蛋白激酶C（PKC）和Na＋－H＋交换在ET-1诱导的培养新生大鼠心肌细胞肥大反应中的作用。ET-1（10－10～10－7mol／L）促进3H－亮氨酸掺入,增加细胞蛋白质的含量和心肌细胞的表面积,且呈剂量依赖性,它们的EC50分别为5．2×10-10,5．2×10－10和7．3×10－10mol／L。用蛋白激酶C（PKC）抑制剂,Staurosporin（2nmol／L）预处理心肌细胞,可完全阻断ET-1诱导的心肌细胞的这些肥大反应,而蛋白激酶C激动剂,佛波酸酯（PMA）（10－8～10－6mol／L）呈剂量依赖性促进心肌细胞的肥大反应。用Na＋－H＋交换抑制剂,氨氯毗咪（10-4mol／L）预处理心肌细胞,可抑制ET－1诱导的心肌细胞肥大反应,但不影响PMA诱导的心肌细胞肥大反应。百日咳毒素（150ng／ml）预处理心肌细胞,可明显抑制ET－1诱导的心肌细胞肥大反应。这些结果提示,ET-1诱导的培养新生大鼠心肌细胞肥大反应是与百日咳毒素敏感的G蛋白相耦联,蛋白激酶C和Na＋．H＋交换可能在ET-1诱导的心肌细胞肥大反应中是重要的细胞内信使转导途径。     

Role of Cl- current in endothelin-1-induced contraction in rabbit basilar artery

Cl- efflux induces depolarization and contraction of smooth muscle cells. This study was undertaken to explore the role of Cl- channels in endothelin-1 (ET-1)-induced contraction in rabbit basilar artery. Male New Zealand White rabbits (n = 26), weighing 1.8-2.5 kg, were euthanized by an overdose of pentobarbital. The basilar arteries were removed for isometric tension recording. ET-1 produced a concentration-dependent contraction of the rabbit basilar artery in the normal Cl- Krebs-Henseleit bicarbonate buffer (123 mM Cl-). The ET-1-induced contraction was reduced by the following manipulations: 1) inhibition of Na+-K+-2Cl- cotransporter with bumetanide (3 x 10(-5) and 10(-4) M), 2) bicarbonate-free solution to disable Cl-/HCO exchanger, and 3) preincubation of rings with the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and indanyloxyacetic acid 94. The ET-1-induced contraction was enhanced by substitution of extracellular Cl- (10 mM) with methanesulfonic acid (113 mM). Cl- channels are involved in ET-1-induced contraction in the rabbit basilar artery.     

Characterization of protein kinase C and its isoforms in human T lymphocytes

Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function.     

Analysis of protein kinase C isoforms involved in the activation of laminin receptor in Raw264.7 macrophages

Adherence of hematopoietic macrophages to a laminin (LM) substratum requires protein kinase C (PKC)-dependent activation of LM receptor. This study was performed to analyze PKC isoform(s) leading to the activation of LM receptor during Raw264.7 macrophage-like cell adhesion to a LM substratum. Raw264.7 cells expressed multiple PKC isoforms, including alpha, beta I, delta, epsilon, zeta, lambda/iota, and mu. Among the PKC isoforms expressed, selective activation of PKC delta and epsilon was sufficient to induce cell adhesion to LM. PKC-dependent cell adherence was blocked by the selective inhibition of PKC delta, suggesting that PKC delta was the responsible PKC isoform leading to activation of LM receptor. PKC delta appeared to activate LM receptor in an intact microfilament-dependent pathway, because disruption of microfilament inhibited cell adhesion to LM without affecting PKC delta activation.     

Mechanisms of endothelin-1-induced contraction in pulmonary arteries from chronically hypoxic rats

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.     

Alkyl cinnamates as regulator for the C1 domain of protein kinase C isoforms

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Natural product curcumin is known to interact with PKC isoforms through the C1 domain and modulate PKC activity. The reported results demonstrate that the symmetric curcumin molecule might act as two separate units during its recognition of C1 domains. To understand the importance of the two halves of curcumin in PKC binding and to develop effective PKC regulators, we synthesized a series of alkyl cinnamates (1-8), characterized absorption and fluorescence properties and measured binding affinities with the C1b subdomains of PKC isoforms. The binding parameters of the monomeric compounds and liposomes containing compounds confirmed their interaction with the C1b subdomains of PKCδ and PKCθ. The molecular docking analysis with PKCδ and PKCθ C1b subdomains revealed that the alkyl cinnamates form hydrogen bond with the backbone of the protein at the same binding site as that of diacylglycerol and phorbol esters. The results show that the alkyl cinnamates bind to the activator binding site of PKCs and both methoxy and hydroxyl groups play important roles in the binding process.     

Signalling by protein kinase C isoforms in the heart

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)     

Selective phosphorylation of human DNA methyltransferase by protein kinase C

Human DNA methyltransferase, the enzyme thought to be responsible for the somatic inheritance of patterns of DNA methylation, is an effective substrate for phosphorylation by protein kinase C. This provides a plausible mechanistic link between the action of tumor promoting phorbol esters, which stimulate protein kinase C, and abnormal patterns of DNA methylation often observed in transformed cells.     

The two isoforms of the cAMP-dependent protein kinase catalytic subunit are involved in the control of dimorphism in the human fungal pathogen Candida albicans

We have cloned the Candida albicans TPK2 gene encoding a cAMP-dependent protein kinase (PKA) catalytic subunit and generated a tpk2 homozygous null mutant to assess its ability to germinate in liquid media. N-acetylglucosamine (GlcNAc)-induced germ-tube formation was attenuated in the tpk2 strain and enhanced by compounds that are known to increase the PKA activity in situ. Germination was completely blocked in the presence of the myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI). These results indicate that TPK1 acts positively in regulating the morphogenetic transition in C. albicans in the absence of the TPK2 gene. We were able to identify an mRNA from this second form of PKA in both wild-type and tpk2 null mutant cells. We found that PKA activity measured in the mutant lacking the TPK2 gene was about 10% of that displayed by the wild-type. The finding that the germinative response of tpk2 null mutant to serum was severely diminished at low serum concentrations indicates that the level of PKA is an important determinant of filamentous growth at low serum concentrations. The extent of germination attained at higher serum concentrations (5%) was similar in the wild-type and in the tpk2 null mutant strains suggesting that under these conditions germination was triggered through a PKA-independent pathway.     

Protein kinase Calpha is required for endothelin-1-induced proliferation of human myometrial cells.

The role of protein kinase C (PKC)-alpha in endothelin-1 (ET-1)-induced proliferation of human myometrial cells was investigated. Inhibition of conventional PKC with G? 6976 eliminated the proliferative effect of ET-1. Treatment of myometrial cells with an antisense oligonucleotide against PKCalpha efficiently reduced PKCalpha protein expression without effect on other PKC isoforms and resulted in the loss of ET-1-induced cell growth. Immunocytochemistry using an antibody against PKCalpha revealed that there was no PKCalpha immunoreactivity in the nuclei of quiescent nonconfluent untreated cells, whereas it is evenly distributed throughout the cytoplasm. Exposure of myometrial cells to ET-1 for 15 min caused the PKCalpha to shift towards the perinuclear area, and incubation for 60 min caused a shift towards the nucleus. These results reveal that PKCalpha is required for ET-1-induced human myometrial cell growth and suggest that targeting of PKCalpha by antisense nucleotides might be an important approach for the development of anticancer treatments.     

Regulation of human involucrin promoter activity by novel protein kinase C isoforms

Human involucrin (hINV) mRNA level and promoter activity increase when keratinocytes are treated with the differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). This response is mediated via a p38 mitogen-activated protein kinase-dependent pathway that targets activator protein 1 (Efimova, T., LaCelle, P. T. , Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395). In the present study we examine the role of various PKC isoforms in this regulation. Transfection of expression plasmids encoding the novel PKC isoforms delta, epsilon, and eta increase hINV promoter activity. In contrast, neither conventional PKC isoforms (alpha, beta, and gamma) nor the atypical isoform (zeta) regulate promoter activity. Consistent with these observations, promoter activity is inhibited by the PKCdelta-selective inhibitor, rottlerin, but not by Go-6976, an inhibitor of conventional PKC isoforms, and novel PKC isoform-dependent promoter activation is inhibited by dominant-negative PKCdelta. This regulation appears to be physiologically important, as transfection of keratinocytes with PKCdelta, -epsilon, or -eta increases expression of the endogenous hINV gene. Synergistic promoter activation (>/=100-fold) is observed when PKCepsilon- or -eta-transfected cells are treated with TPA. In contrast, the PKCdelta-dependent response is more complex as either activation or inhibition is observed, depending upon PKCdelta concentration.     

Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca²⁺‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.     

Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii

Protein kinase C (PKC) plays an important role in the control of proliferation and differentiation of a wide range of cell types, and fungi are no exception. Previous results reported by us on the effects of the phorbol ester, 12-myristate-13-acetate phorbol (PMA) and other PKC effector molecules, on dimorphism in Sporothrix schenckii suggested the presence of this enzyme in the fungus and its involvement in the control of morphogenetic transitions. The work summarized here confirms the presence of PKC in yeast and mycelium extracts of S. schenckii. Different isoforms of this enzyme were found to be present in the yeast and mycelium forms of the fungus and were identified by Western blot analysis using affinity purified anti-PKC isoforms specific antibodies: the γ and ζ isoforms were detected in both the yeast and mycelium forms of the fungus, while the β isoform was only detected in the yeast form. The presence of PKC was confirmed biochemically by measuring total enzyme activity in both forms of the fungus. No significant differences were observed for the PKC activity level recorded for both the mycelium and yeast forms of the fungus (p ≤ 0.05). These data confirm the presence of PKC activity in Sporothrix schenckii and constitutes the first evidence concerning the differential expression of PKC isoforms in the mycelium and yeast forms of a dimorphic fungus, supporting the possible involvement of this important signal transduction enzyme in the control of morphogenesis in this fungus. This revised version was published online in June 2006 with corrections to the Cover Date.