PKC刺激与抑制对HeLa细胞G_1/S期进程的影响

蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）是一类分布广泛的丝氨酸／苏氨酸蛋白激酶,在真核细胞的跨膜信号传递中发挥重要的作用［１］．而细胞周期进程是一个复杂的调控过程,受控于多种磷酸酶与磷酸激酶作用下的中心调控体系［２］．大量报道表明ＰＫＣ信号...     

Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms

We reported previously that the protein kinase C (PKC) inhibitor GF109203X stimulated the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. PKCalpha, -epsilon, and -zeta were detected in hamster adrenal zona glomerulosa and NCI-H295 cells, and PKCtheta in NCI-H295 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibited basal and stimulated cytochrome P450 aldosterone synthase mRNA expression by angiotensin (AII), dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP), or KCl in NCI-H295 cells. Basal CYP11B2 promoter activity was inhibited in cells cotransfected with constitutively active (CA) PKCalpha, -epsilon, and -theta mutants, whereas it was increased with CA-PKCzeta. Dominant negative (DN) PKCalpha, -theta, -epsilon, and -zeta mutants stimulated the promoter activity. AII-, KCl-, and Bt2cAMP-stimulatory effects were abolished in cells cotransfected with CA-PKCalpha, -epsilon, or -theta. The effect of Bt2cAMP was abolished by CA-PKCzeta but AII and KCl were still able to enhance the promoter activity. DN-PKCalpha, -epsilon, -theta, or -zeta did not inhibit these effects. G?6976 enhanced promoter activity, providing further evidence that PKCalpha was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKCalpha, -epsilon, or -theta abolished the effects of AII, KCl, and Bt2cAMP on the activity of -102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (alpha) and atypical (zeta) PKC.     

Regulation of matrix Gla protein by parathyroid hormone in MC3T3-E1 osteoblast-like cells involves protein kinase A and extracellular signal-regulated kinase pathways

Inhibition of osteoblast-mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix gamma-carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH-mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3-E1 cells. Following transient transfection of these cells with a -748 bp murine MGP promoter-luciferase construct (pMGP-luc), PTH (10 (-7) M) induced promoter activity in a time-dependent manner with a maximal four- to six fold induction seen 6 h after PTH treatment. Both H-89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co-transfection of MC3T3-E1 cells with pMGP-luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose-dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12-myristate 13-acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal-regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA- and ERK-dependent pathways.     

Stimulation of Cell Elongation in Teleost Rod Photoreceptors by Distinct Protein Kinase Inhibitors

Abstract: The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

Reactive oxygen species mediate shear stress-induced fluid-phase endocytosis in vascular endothelial cells

To elucidate the role of shear stress in fluid-phase endocytosis of vascular endothelial cells (EC), we used a rotating-disk shearing apparatus to investigate the effects of shear stress on the uptake of lucifer yellow (LY) by cultured bovine aortic endothelial cells (BAEC). Exposure of EC to shear stress (area-mean value of 10 dynes/cm2) caused an increase in LY uptake that was abrogated by the antioxidant, N-acetyl-L-cysteine (NAC), the NADPH oxidase inhibitor, acetovanillone, and two inhibitors of protein kinase C (PKC), calphostin C and GF109203X. These results suggest that fluid-phase endocytosis is regulated by both reactive oxygen species (ROS) and PKC. Shear stress increased both ROS production and PKC activity in EC, and the increase in ROS was unaffected by calphostin C or GF109203X, whereas the activation of PKC was reduced by NAC and acetovanillone. We conclude that shear stress-induced increase in fluid-phase endocytosis is mediated via ROS generation followed by PKC activation in EC.     

4-Hydroxynonenal-induced MEL cell differentiation involves PKC activity translocation

4-Hydroxynonenal (HNE) is a highly reactive aldehyde, produced by cellular lipid peroxidation, able to inhibit proliferation and to induce differentiation in MEL cells at concentrations similar to those detected in several normal tissues. Inducer-mediated differentiation of murine erythroleukemia (MEL) cells is a multiple step process characterized by modulation of several genes as well as by a transient increase in the amount of membrane-associated protein kinase C (PKC) activity. Here we demonstrate that a rapid translocation of PKC activity from cytosol to the membranes occurs during the differentiation induced by HNE. When PKC is completely translocated by phorbol-12-myristate-13-acetate (TPA), the degree of HNE-induced MEL cells differentiation is highly decreased. However, if TPA is washed out from the culture medium before the exposition to the aldehyde, HNE gradually resumes its differentiative ability. The incubation of cells with a selective inhibitor of PKC activity, bisindolylmaleimide GF 109203X, partially prevents the HNE-induced differentiation in MEL cells. In conclusion, our results demonstrate that HNE-induced MEL cell differentiation is preceded by a rapid translocation of PKC activity, and that the inhibition of this phenomenon prevents the onset of terminal differentiation.     

The kinase inhibitor iso-H7 stimulates rat satellite cell differentiation through a non-protein kinase C pathway by increasing myogenin expression level

We analysed the signaling pathways involved in myogenic differentiation of primary cultures of rat satellite cells using substances targeting the protein kinase C (PKC) and the cAMP protein kinase (PKA) pathways. We have previously shown that iso-H7, which putatively inhibits both PKC and PKA, strongly stimulates satellite cell differentiation, as well as the PKA inhibitor HA 1004. In the study reported here, the effects of iso-H7 on satellite cell differentation were compared to those observed in the presence of agents which reduce PKC activity. It was shown that treatments with the highly specific PKC inhibitor GF109203X or with 12-O-tetradecanoylphorbol 13-acetate (TPA) which induced a partial PKC downregulation, did not significantly alter myogenic differentiation. Northern blot analyses showed that iso-H7 activated the expression of myogenin but not that of MyoD mRNA. Concurrently, iso-H7 increased myosin light-chain mRNA expression. In contrast, TPA had no effect on these syntheses. Taken together, these results showed that iso-H7 did not act intracellularly as a PKC inhibitor but rather as a PKA inhibitor as previously suggested. Our results are compatible with the hypothesis that a reduction in PKA activity controls satellite cell myogenesis through an increased myogenin mRNA expression.Abbreviations PKC protein kinase C - PKA cAMP-dependent protein kinase - CK creatine kinase - iso-H7 1-(5-isoquinolinesulfonyl)-3-methylpiperazine - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - HA 1004 N-(3-hydroxyethyl)-1-piperazine ethanesulfonate - TPA 12-O-tetradecanoyl phorbol 13-acetate - MLC myosin light chain - GAPDH glyceraldehyde 3-phosphate deshydrogenase     

Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells

We investigated the mechanism of 8-Br-cAMP-mediated phospholipase D (PLD) activation using a primary cell culture system of human endometrial stromal cells (ES cells). PLD activity was increased by the treatment of ES cells with 8-Br-cAMP, maximally at 5 min. To determine whether the effects of 8-Br-cAMP on PLD occurred as a consequence of PKC activation, ES cells were preincubated for 15 min with RO320432 (1 microM) and GF109203X (1 microM), the PKC inhibitors, or they were pretreated for 24h with phorbol myristate acetate (100 nM) to downregulate PKC. However, these treatments had no effects on PLD activation induced by 8-Br-cAMP. Furthermore, 8-Br-cAMP had no effects on the subcellular distribution of PKC alpha and PKC betaI, confirming no involvement of PKC. 8-Br-cAMP activated ERK1/2, maximally at 5 min, and PD98059 (MEK inhibitor: 50 microM) and transfection of ES cells with dominant negative (DN)-MEK completely inhibited 8-Br-cAMP-induced PLD activation, suggesting that ERK1/2 mediates the PLD activation. To investigate the involvement of protein kinase A (PKA), Src, and Ras in 8-Br-cAMP-induced PLD activation, we used PKA inhibitor, H89 and Rp-cAMPs, and transfections of DN-Src and DN-Ras. H-89 and Rp-cAMPs completely blocked 8-Br-cAMP-mediated PLD and ERK activation, implying the involvement of PKA in this PLD activation. In addition, transfection of ES cells with DN-Src, or DN-Ras partially inhibited 8-Br-cAMP-induced ERK1/2 and consequently PLD activation, whereas cotransfection of DN-Src and DN-Ras completely inhibited ERK1/2 and PLD activation, suggesting that Src and Ras independently regulate ERK/PLD activation. Taken together, these results demonstrate a novel pathway in ES cells that 8-Br-cAMP activate PLD through PKA and ERK1/2 and this ERK/PLD activation by 8-Br-cAMP is mediated by Src and Ras, separately.     

Protein kinase Calpha and sphingosine 1-phosphate-dependent signaling in endothelial cell

Protein kinase C (PKC)-mediated signal transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the role of various PKC isoforms in sphingosine 1-phosphate (S1P)-stimulated endothelial cells is not well understood. PKCalpha and PKCepsilon activity are increased in endothelial cell cultures, and S1P receptor transfection studies indicate S1P(3) stimulates PKCalpha and S1P1 leads to PKCepsilon activity. Infection of endothelial cells with dominant negative (DN)PKCalpha adenovirus reduces cell migration and greatly inhibits morphogenesis in cells stimulated with S1P. This effect is specific to PKCalpha, as infection with DN PKCepsilon does not alter either migration or morphogenesis. The PKC-specific chemical inhibitor GF109203X also inhibits these two responses. Infection of endothelial cells with dominant negative PKCalpha reduces S1P-induced calcium rise. This maximal rise requires calcium uptake, but it does not require enzymatic activity of the kinase. Pretreatment of these cells with the PKC-specific inhibitor GF109203X does not inhibit S1P-induced calcium rise. S1P-induced morphogenesis but not cell migration is critically dependent on extracellular calcium. Pretreatment of endothelial cells with phorbol 12-myristate 13-acetate for 5min abolishes S1P-stimulated rise in calcium but had little or no effect on migration. The PMA-inhibited calcium rise can be prevented by PKC inhibitor or infection with dominant negative PKCalpha.     

Modulation of apoptosis of proliferating and differentiating HL-60 cells by protein kinase inhibitors: suppression of PKC or PKA differently affects cell differentiation and apoptosis.

The relationship between RA- or dbcaMP-mediated differentiation and subsequent apoptosis in HL-60 cells was assessed by modulating the levels of differentiation suppressing the activity of PKC and PKA with calphostin C or GF 109203X and H89, respectively. Results demonstrated that (1) RA and dbcAMP caused a dose-dependent increase in apoptosis concomitant with progressive differentiation; (2) the suppression of PKC activity resulted in an increase of apoptosis unrelated to the modulated levels of differentiation; (3) the inhibition of PKA decreased granulocytic differentiation, but did not significantly affect apoptosis; (4) the pretreatment of cells with dbcAMP strongly potentiated RA-mediated differentiation without apparent changes in apoptosis; (5) cell differentiation and apoptosis were associated with cell cycle arrest in G1 phase and G2/M phases, respectively. Our findings indicate that the functional maturity of differentiating cells is not directly related to the apoptotic programme, and suggest that induction of cell differentiation and apoptosis are regulated by separate mechanisms in which PKC and PKA are involved.     

Differentiation-associated genes regulated by TPA-induced c-Jun expression via a PKC/JNK pathway in KYSE450 cells

A group of potential differentiation-associated genes had been identified by microarray analysis as c-Jun/AP-1 target genes essential for epithelial differentiation program. Our previous study showed that c-Jun/AP-1 could bind and activate these gene promoters in vivo using chromatin immunoprecipitation. To further understand how the mitogen-activated protein kinase signaling pathways regulate AP-1 activity and expression of c-Jun target genes, our strategy was based on the use of 12-o-tetradecanoylophorbol-13-acetate (TPA) and pharmacological reagents to induce or block c-Jun expression. The mRNA and protein expression of these genes increased in response to TPA-induced c-Jun/AP-1 expression. Inhibitors of JNK (SP600125) and PKC (GF109203X) mainly blocked expression and phosphorylation of c-Jun, while inhibition of MEK-ERK activity with PD98059 (an inhibitor of MEK) had little effect. Expression of involucrin and keratin 4 in response to TPA was attenuated by pretreatments with GF109203X and SP600125, but not PD98059, suggesting involvement of PKC and JNK in this response. Taken together, these results suggested that differentiation-associated genes were regulated by TPA-induced c-Jun/AP-1 mainly via a PKC/JNK pathway in esophageal cancer cell line KYSE450.     

Inducible expression of the megakaryocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase.

Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.     

Role of protein kinase C delta in reactivation of Kaposi's sarcoma-associated herpesvirus

TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCdelta isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCdelta mutant supported an essential role for the PKCdelta isoform in virus reactivation, yet overexpression of PKCdelta alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway.     

The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C

Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways.