CCK2 (CCK(B)/gastrin) receptor mediates rapid protein kinase D (PKD) activation through a protein kinase C-dependent pathway

Addition of gastrin or cholecystokinin octapeptide (CCK-8) to cultures of Rat-1 cells stably transfected with the CCK2 (CCK(B)/gastrin) receptor induced protein kinase D (PKD) activation that was detectable within 1 min and reached a maximum ( approximately 10-fold) after 2.5 min of hormonal stimulation. Half-maximal PKD activation for both CCK-8 and gastrin was achieved at 10 nM. Treatment with various concentrations of the selective PKC inhibitors Ro 31-8220 or GF-I potently blocked PKD activation induced by subsequent addition of CCK-8 in a concentration-dependent fashion. Our results indicate that PKC-dependent PKD activation is a novel early event in the action of gastrin and CCK-8 via CCK2 receptors.     

Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway.

Interferon-gamma (IFN-gamma) induced intercellular adhesion molecule-1 (ICAM-1) expression in human NCI-H292 epithelial cells, as shown by enzyme-linked immunosorbent assay and immunofluorescence staining. The enhanced ICAM-1 expression resulted in increased adhesion of U937 cells to NCI-H292 cells. Tyrosine kinase inhibitors (genistein or herbimycin), Src family inhibitor (PP2), or a phosphatidylinositol-phospholipase C inhibitor (U73122) attenuated the IFN-gamma-induced ICAM-1 expression. Protein kinase C (PKC) inhibitors (staurosporine or Ro 31-8220) also inhibited IFN-gamma-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression; this effect was inhibited by tyrosine kinase or Src inhibitor. ICAM-1 promoter activity was enhanced by IFN-gamma and TPA in cells transfected with pIC339-Luc, containing the downstream NF-kappaB and gamma-activated site (GAS) sites, but not in cells transfected with GAS-deletion mutant, pIC135 (DeltaAP2). Electrophoretic gel mobility shift assay demonstrated that GAS-binding complexes in IFN-gamma-stimulated cells contained STAT1alpha. The IFN-gamma-induced ICAM-1 promoter activity was inhibited by tyrosine kinase inhibitors, a phosphatidylinositol-phospholipase C inhibitor, or PKC inhibitors, and the TPA-induced ICAM-1 promoter activity was also inhibited by tyrosine kinase inhibitors. Cotransfection with a PLC-gamma2 mutant inhibited IFN-gamma- but not TPA-induced ICAM-1 promoter activity. However, cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited both IFN-gamma- and TPA-induced ICAM-1 promoter activity. The ICAM-1 promoter activity was stimulated by cotransfection with wild type PLC-gamma2, PKCalpha, c-Src, JAK1, or STAT1. An immunocomplex kinase assay showed that both IFN-gamma and TPA activated c-Src and Lyn activities and that these effects were inhibited by staurosporine and herbimycin. Thus, in NCI-H292 epithelial cells, IFN-gamma activates PLC-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and c-Src or Lyn, resulting in activation of STAT1alpha, and GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.     

Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCbetaI, PKCepsilon, and PKCzeta. PKCzeta is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCepsilon is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCbetaI, but only a partial down-regulation of PKCepsilon. Moreover, no translocation of PKCbetaI or PKCzeta from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCepsilon was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform epsilon, in the negative control of ERK-1/2 through the induction of their specific phosphatase.     

Prostaglandin F2alpha increases glucose transport in 3T3-L1 adipocytes through enhanced GLUT1 expression by a protein kinase C-dependent pathway

The effect of prostaglandin F2alpha (PGF2alpha) on glucose transport in differentiated 3T3-L1 adipocytes was examined. Whereas PGF2alpha had little influence on insulin-stimulated 2-deoxyglucose uptake, it increased basal glucose uptake in a time- and dose-dependent manner, reaching maximum at approximately 8 h. The long-term effect of PGF2alpha on glucose transport was inhibited by both cycloheximide and actinomycin D. In concord, while the content of GLUT4 protein was not altered, immunoblot and Northern blot analyses revealed that both GLUT1 protein and mRNA levels were increased by exposure of cells to PGF2alpha. The effect of PGF2alpha on glucose uptake was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA), the stimulatory effects of PGF2alpha on glucose transport and GLUT1 mRNA accumulation were both inhibited. In accord, PMA was shown to stimulate GLUT1 mRNA accumulation. To further investigate if PKC may be activated by PGF2alpha, we tested several diacylglycerol-sensitive PKC isozymes and found that PGF2alpha was able to activate PKCepsilon. Taken together, these results indicate that PGF2alpha may enhance glucose transport in 3T3-L1 adipocytes by stimulating GLUT1 expression via a PKC-dependent mechanism.     

Ultrasound induces cyclooxygenase-2 expression through integrin, integrin-linked kinase, Akt, NF-kappaB and p300 pathway in human chondrocytes

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. However, the precise molecular events generated by US in chondrocytes have not been clarified well. Here we found that US stimulation transiently increased the surface expression of alpha2, alpha5, beta1 or beta3 but not alpha3 or alpha4 integrins in human chondrocytes, as shown by flow cytometric analysis. US stimulation increased prostaglandin E(2) formation as well as the protein and mRNA levels of cyclooxygenase-2 (COX-2). At the mechanistic level, anti-integrin beta1 and beta3 antibodies or beta1 and beta3 integrin small interference RNA attenuated the US-induced COX-2 expression. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited the potentiating action of US. US stimulation promotes kinase activity of ILK, phosphorylation of Akt. In addition, US stimulation also induces IKKalpha/beta phosphorylation, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. The binding of p65 to the NF-kappaB element, as well as the recruitment of p300 and the enhancement of p50 acetylation on the COX-2 promoter was enhanced by US. Taken together, our results provide evidence that US stimulation increases COX-2 expression in chondrocytes via the integrin/ILK/Akt/NF-kappaB and p300 signaling pathway.     

Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway.

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol esters, membrane-permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co-transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS-7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC-dependent signal transduction pathway.     

Rho and Rho-associated kinase modulate the tyrosine kinase PYK2 in T-cells through regulation of the activity of the integrin LFA-1

We have examined the role of the small GTPase Rho and its downstream effector, the Rho-associated kinase (ROCK), in the control of the adhesive and signaling function of the lymphocyte function-associated antigen-1 (LFA-1) integrin in human T-lymphocytes. Inhibition of Rho (either by treatment with C3-exoenzyme or transfection with a dominant-negative form of Rho (N19Rho)) or ROCK (by treatment with Y-27632) results in the following: (a) partial disorganization and aggregation of cortical filamentous actin (F-actin); (b) induction of LFA-1-mediated cellular adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) through a mechanism involving clustering of LFA-1 molecules, rather than alterations in the level of expression or in the affinity state of this integrin; and (c) induction of cellular polarization and activation of the tyrosine kinase PYK2. Transfection of T-cells with a constitutively active form of Rho (V14Rho) blocks the clustering of LFA-1 on the membrane and the LFA-1-mediated activation of PYK2. Importantly, the activation of PYK2 caused by inhibition of Rho or ROCK takes place only when the T-cells are plated onto ICAM-1 but not when they are either prevented from interacting with ICAM-1 with anti-LFA-1 blocking antibodies or when they are plated on the nonspecific poly-l-lysine substrate. These results indicate that the small GTPase Rho regulates the tyrosine kinase PYK2 in T-cells through the F-actin-mediated control of the activity of the integrin LFA-1. These findings represent a novel paradigm for the regulation of the activity of a cytoplasmic tyrosine kinase by the small GTPase Rho.     

Down-regulation by antisense oligonucleotides establishes a role for the proline-rich tyrosine kinase PYK2 in angiotensin ii-induced signaling in vascular smooth muscle

Abnormal vascular smooth muscle cell (VSMC) growth plays a key role in the pathogenesis of hypertension and atherosclerosis. Angiotensin II (Ang II) elicits a hypertrophic growth response characterized by an increase in protein synthesis in the absence of DNA synthesis and cell proliferation. Intracellular signaling mechanisms linking angiotensin type I receptor activation to protein synthesis in VSMC have not been fully characterized. The present study investigates the role of the nonreceptor proline-rich tyrosine kinase 2 (PYK2) in Ang II-induced VSMC protein synthesis and in the regulation of two signaling pathways that have been implicated in the control of protein synthesis, the extracellular signal-regulated kinase (ERK1/2) and the phosphatidylinositol 3-kinase/Akt pathways. PYK2 antisense oligonucleotides were used to down-regulate PYK2 expression in cultured VSMC. An 80% down-regulation in PYK2 expression resulted in an approximately 80% inhibition of ERK1/2 (3.8 +/- 1.3 versus 16.6 +/- 1.8), p70S6 kinase (1.03 +/- 0.03 versus 3.8 +/- 0.5), and Akt activation (3.0 +/- 0.8 versus 16.0 +/- 1.0) by Ang II. Furthermore, PYK2 down-regulation resulted in a complete inhibition of Ang II-induced VSMC protein synthesis. These data conclusively identify PYK2 as an upstream regulator of both the ERK1/2 and the phosphatidylinositol 3-kinase/Akt pathways that are involved in Ang II-induced VSMC protein synthesis.     

Protein kinase C-dependent activation of a myelin basic protein kinase by gastrin-releasing peptide in Swiss 3T3 fibroblasts

Addition of gastrin releasing peptide to serum-starved Swiss 3T3 mouse fibroblasts results in a transient appearance of a myelin basic protein-kinase activity in cytosolic extracts. Increased kinase activity is also observed upon stimulation of cells with bradykinin, epidermal growth factor or 4 beta-phorbol dibutyrate. Chromatographic analysis of the cytosolic extracts show that both gastrin-releasing peptide and 4 beta-phorbol dibutyrate induce the appearance of a kinase activity similar to that induced by epidermal growth factor. The response to gastrin-releasing peptide is abolished by down-regulation of protein kinase C and attenuated by acute inhibition of protein kinase C using staurosporine. The effect of epidermal growth factor was also suppressed under these conditions, albeit to a lesser extent. The results indicate (1) that activation of myelin basic protein kinase(s) may be common to different growth factors, and (2) that protein kinase C may participate in this response, at least in the case of gastrin-releasing peptide.     

Cholecystokinin activates PYK2/CAKbeta by a phospholipase C-dependent mechanism and its association with the mitogen-activated protein kinase signaling pathway in pancreatic acinar cells.

PYK2/CAKbeta is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125(FAK)) that can be activated by a number of stimuli including growth factors, lipids, and some G protein-coupled receptors. Studies suggest PYK2/CAKbeta may be important for coupling various G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin (CCK) is known to activate both phospholipase C-dependent cascades and MAPK signaling pathways; however, the relationship between these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and activation of PYK2/CAKbeta by both activation of high affinity and low affinity CCK(A) receptor states. Blockage of CCK-stimulated increases in protein kinase C activity or CCK-stimulated increases in [Ca(2+)](i), inhibited by 40-50% PYK2/CAKbeta but not p125(FAK) tyrosine phosphorylation. Simultaneous blockage of both phospholipase C cascades inhibited PYK2/CAKbeta tyrosine phosphorylation completely and p125(FAK) tyrosine phosphorylation by 50%. CCK-8 stimulated a rapid increase in PYK2/CAKbeta kinase activity assessed by both an in vitro kinase assay and autophosphorylation. Total PYK2/CAKbeta under basal conditions was largely localized (77 +/- 7%) in the membrane fraction, whereas total p125(FAK) was largely localized (86 +/- 3%) in the cytosolic fraction. With CCK stimulation, both p125(FAK) and PYK2/CAKbeta translocated to the plasma membrane. Moreover CCK stimulation causes a rapid formation of both PYK2/CAKbeta-Grb2 and PYK2/CAKbeta-Crk complexes. These results demonstrate that PYK2/CAKbeta and p125(FAK) are regulated differently by CCK(A) receptor stimulation and that PYK2/CAKbeta is probably an important mediator of downstream signals by CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and neoplastic tissues.     

Angiopoietin-like protein 3 modulates barrier properties of human glomerular endothelial cells through a possible signaling pathway involving phosphatidylinositol-3 kinase/protein kinase B and integrin alphaVbeta3

Podocytes can influence glomerular endothelial cell (GEnC) barrier properties and take part in the development of proteinuria by some molecules. Angiopoietin-like protein 3 (Angptl3), secreted by podocytes, is a member of the angiopoietin-like protein family that has important biological functions in endothelial cells. In our previous studies, we showed that mRNA expression of Angptl3 increased significantly in kidneys of children with minimal change nephrotic syndrome. And the mRNA level of Angptl3 was increased in the glomerulus of adriamycin rats with the development of proteinuria. It was also found that Angptl3 was expressed in the cytoplasm of cultured podocytes. Thus, Angptl3 might influence the biological functions of GEnCs in a paracrine manner. In this study, we found that Angptl3 could increase the permeability of GEnCs and increase the level of protein kinase B phosphorylation in cultured GEnCs in vitro. LY294002, a phosphatidylinositol-3 kinase inhibitor, could prevent the increase of permeability of GEnCs induced by Angptl3. Our results also indicated that the integrin αVβ3 antibody (LM609) could block the Angptl3-induced protein kinase B phosphorylation.     

c-jun-NH2JNK mediates invasive potential and EGFR activation by regulating the expression of HB-EGF in a urokinase-stimulated pathway

In this study, we demonstrated that tyrosine phosphorylation of EGFR and the autocrine expression of uPA and HB-EGF depend on the activity of c-jun amino-terminal kinase (JNK) in human prostatic DU-145 cells. These cells overexpress EGFR and produce a high amount of uPA. Treatment with either SP600125, a specific chemical inhibitor of JNK, or the expression of a dominant-negative JNK form inhibited autocrine production of uPA and HB-EGF, which block EGFR phosphorylation and mitigates invasive capacity. Our data provided evidence that in DU-145 cells, the maintenance of the activation level of EGFR, which determines the cellular invasive potential, operates through an autocrine loop involving the JNK-dependent production of uPA and HB-EGF activity. Moreover, we found that exogenously added uPA stimulates autocrine production of HB-EGF, and that blocking HB-EGF activity curbed DU-145 cell invasive potential.     

Transforming growth factor-beta induces collagenase-3 expression by human gingival fibroblasts via p38 mitogen-activated protein kinase

Human collagenase-3 (matrix metalloproteinase 13 (MMP-13)) is characterized by exceptionally wide substrate specificity and restricted tissue specific expression. Human skin fibroblasts in culture express MMP-13 only when they are in three-dimensional collagen (Ravanti, L., Heino, J., López-Otín, C., and K?h?ri. V.-M. (1999) J. Biol. Chem. 274, 2446-2455). Here we show that MMP-13 is expressed by fibroblasts during normal human gingival wound repair. Expression of MMP-13 by human gingival fibroblasts cultured in monolayer or in collagen gel was induced by transforming growth factor-beta1 (TGF-beta1). Treatment of gingival fibroblasts with TGF-beta1 activated two distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2) in 15 min and p38 MAPK in 1 and 2 h. Induction of MMP-13 expression by TGF-beta1 was blocked by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a selective inhibitor of ERK1/2 activation. Adenovirus-mediated expression of dominant negative p38alpha and c-Jun potently inhibited induction of MMP-13 expression in gingival fibroblasts by TGF-beta1. Infection of gingival fibroblasts with adenovirus for constitutively active MEK1 resulted in activation of ERK1/2 and JNK1 and up-regulation of collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) production but did not induce MMP-13 expression. In addition, activation of p38 MAPK by constitutively active MKK6b or MKK3b was not sufficient to induce MMP-13 expression. These results show that TGF-beta-elicited induction of MMP-13 expression by gingival fibroblasts is dependent on the activity of p38 MAPK and the presence of functional AP-1 dimers. These observations demonstrate a fundamental difference in the regulation of collagenolytic capacity between gingival and dermal fibroblasts and suggest a role for MMP-13 in rapid turnover of collagenous matrix during repair of gingival wounds, which heal with minimal scarring.     

Bcl-xL blocks activation of related adhesion focal tyrosine kinase/proline-rich tyrosine kinase 2 and stress-activated protein kinase/c-Jun N-terminal protein kinase in the cellular response to methylmethane sulfonate

The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade.     

Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism

The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. Here we demonstrate that pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. We observe that the dominant-negative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, a membrane-permeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents.