Inhibition of protein tyrosine kinases or protein kinase C prevents nonspecific killer T lymphocyte-mediated tumoricidal activity

The signal transduction events which govern major histocompatibility complex-unrestricted tumour cell destruction by nonspecific killer T lymphocytes induced with anti-CD3 antibody have not yet been determined. In this study we used pharmacologic inhibitors to investigate the role of protein tyrosine kinases (PTK) and protein kinase C (PKC) in this process. The PTK-inhibitors herbimycin A, genistein, and methyl 2,5-dihydroxycinnamate blocked anti-CD3-activated killer T (AK-T) lymphocyte-mediated killing of tumour target cells. The PKC-inhibitors staurosporine, calphostin C, and myristoylated PKC pseudosubstrate peptide, as well as PKC desensitization by phorbol 12-myristate 13-acetate pretreatment, also suppressed the cytolytic effector function of AK-T lymphocytes. Lack of tumoricidal activity was not due to reduced AK-T lymphocyte binding to tumour target cells but was associated with the abrogation of granule exocytosis, indicating that PTK and PKC are involved in the postbinding process which results in delivery of the `lethal hit' by AK-T lymphocytes. © 1997 Elsevier Science B.V. All rights reserved.     

Arachidonic acid activates mitogen-activated protein (MAP) kinase-activated protein kinase 2 and mediates adhesion of a human breast carcinoma cell line to collagen type IV through a p38 MAP kinase-dependent pathway

Adhesion of metastatic human mammary carcinoma MDA-MB-435 cells to the basement membrane protein collagen type IV can be activated by treatment with arachidonic acid. We initially observed that this arachidonic acid-mediated adhesion was inhibited by the tyrosine kinase inhibitor genistein. Therefore, we examined the role of the mitogen-activated protein (MAP) kinase family tyrosine phosphorylation-regulated pathways in arachidonic acid-stimulated cell adhesion. Arachidonic acid stimulated the phosphorylation of p38, the activation of MAP kinase-activated protein kinase 2 (MAPKAPK2, a downstream substrate of p38), and the phosphorylation of heat shock protein 27 (a downstream substrate of MAP kinase-activated protein kinase 2). Treatment with the p38 inhibitor PD169316 completely and specifically inhibited arachidonic acid-mediated cell adhesion to collagen type IV. p38 activity was specifically associated with arachidonic acid-stimulated adhesion; this was demonstrated by the observation that 12-O-tetradecanoylphorbol 13-acetate-activated cell adhesion was not blocked by inhibiting p38 activity. Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were also activated by arachidonic acid; however, cell adhesion to collagen type IV was not highly sensitive to PD98059, an inhibitor of MAP kinase kinase/ERK kinase 1 (MEK1) that blocks activation of the ERKs. c-Jun NH(2)-terminal kinase was not activated by arachidonic acid treatment of these cells. Together, these data suggest a novel role for p38 MAP kinase in regulating adhesion of breast cancer cells to collagen type IV.     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways

During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5) and beta(1). All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase.     

P2Y receptor-mediated inhibition of tumor necrosis factor alpha -stimulated stress-activated protein kinase activity in EAhy926 endothelial cells

In the EAhy926 endothelial cell line, UTP, ATP, and forskolin, but not UDP and epidermal growth factor, inhibited tumor necrosis factor alpha (TNFalpha)- and sorbitol stimulation of the stress-activated protein kinases, JNK, and p38 mitogen-activated protein (MAP) kinase, and MAPKAP kinase-2, the downstream target of p38 MAP kinase. In NCT2544 keratinocytes, UTP and a proteinase-activated receptor-2 agonist caused similar inhibition, but in 13121N1 cells, transfected with the human P2Y(2) or P2Y(4) receptor, UTP stimulated JNK and p38 MAP kinase activities. This suggests that the effects mediated by P2Y receptors are cell-specific. The inhibitory effects of UTP were not due to induction of MAP kinase phosphatase-1, but were manifest upstream in the pathway at the level of MEK-4. The inhibitory effect of UTP was insensitive to the MEK-1 inhibitor PD 098059, changes in intracellular Ca(2+) levels, or pertussis toxin. Acute phorbol 12-myristate 13-acetate pretreatment also inhibited TNFalpha-stimulated SAP kinase activity, while chronic pretreatment reversed the effects of UTP. Furthermore, the protein kinase C inhibitors Ro318220 and Go6983 reversed the inhibitory action of UTP, but GF109203X was ineffective. These results indicate a novel mechanism of cross-talk regulation between P2Y receptors and TNFalpha-stimulated SAP kinase pathways in endothelial cells, mediated by Ca(2+)-independent isoforms of protein kinase C.     

Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.     

Differential regulation of protooncogenes c-jun and jun D expressions by protein tyrosine kinase, protein kinase C, and cyclic-AMP mitogenic pathways in dog primary thyrocytes: TSH and cyclic-AMP induce proliferation but downregulate C-jun expression.

The expressions of the protooncogenes c-jun and jun D have been investigated in dog thyrocytes in a primary culture whose proliferation is stimulated by three distinct intracellular signaling pathways (1) the thyrotropin (TSH) or forskolin-cyclic-AMP-mediated cascade; (2) the protein kinase C pathway activated by diacylglycerol (DAG) and phorbol esters (TPA); (3) a protein tyrosine kinase system activated by epidermal growth factor (EGF). While the first cascade is compatible with the differentiated state of the cell, the two latter pathways induce dedifferentiation. Following the stimulation by TPA or EGF, the expression of c-jun was increased and the expression of jun D was faintly increased. Both expressions are superinduced in the presence of cycloheximide as in mitogenically stimulated fibroblasts but, in the presence of cycloheximide alone, the expressions of c-jun and jun D are clearly unstable with time. This indicates that cycloheximide controls should be included at all time points examined in such experiments. Increasing intracellular concentrations of cyclic-AMP by forskolin or TSH was followed by an inhibition of the expression of c-jun. This inhibition was independent of protein synthesis. Similarly, the TPA or EGF stimulation of c-jun expression was also inhibited by TSH or forskolin, as in fibroblasts in which cyclic-AMP inhibits proliferation. Our results show that the expression of c-jun is not universally correlated with the stimulation of cell proliferation. The stimulation of c-jun expression is not common between the three mitogenic pathways. It thus represents another of the very different responses elicited by the cyclic-AMP cascade as compared to the more studied tyrosine kinase and protein kinase C mitogenic pathways.     

Pertussis toxin activates tyrosine kinase signaling cascade in myelomonocytic cells: a mechanism for cell adhesion

Pertussis toxin (PTX) has recently been shown to specifically bind to CD14 to promote myelomonocytic cell adhesion to serum. The present study investigated the signaling mechanisms responsible for PTX-induced differentiated U937 cell adhesion. PTX-induced myelomonocytic cell adhesion was blocked by genistein or tyrphostin-47 (two protein tyrosine kinase inhibitors), LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), or PD098059 (a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor). PTX induced a rapid tyrosine phosphorylation of several discrete cytoplasmic proteins, which could be inhibited by genistein or tyrphostin 47. In addition, PTX induced phosphorylation of Akt and of ERK2, which could be completely blocked by LY294002 and PD098059, respectively, and by genistein or tyrphostin 47 as well. All of these PTX-induced signaling events could be reproduced using purified PTX B-oligomer (PTX-B) alone. Our data show that PTX can activate tyrosine kinase signaling cascade, including the downstream PI3K and ERK/MAPK pathways, in myelomonocytic cells to induce cell adhesion to serum.     

Advanced glycosylation end products induce nitric oxide synthase expression in C6 glioma cells: involvement of a p38 MAP kinase-dependent mechanism.

The mitogen-activated protein kinase (MAPK) pathway is believed to function as an important mediator of inducible nitric oxide synthase (iNOS) expression. In the present study, we investigated the role of the p38 MAPK signaling pathway in advanced glycosylation end products (AGEs)-induced iNOS expression in C6 glioma cells. AGEs caused a dose-dependent increase of nitrite accumulation in C6 glioma cells. The AGEs-stimulated nitrite production from C6 glioma cells was inhibited by actinomycin D, cyclohexamide, and the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), suggesting that the increase of AGEs-induced nitrite release is due to iNOS up-regulation. Consistently, treatment of C6 glioma cells with AGEs induced iNOS protein expression. AGEs-stimulated nitrite production was inhibited by pretreatment of C6 glioma cells with anti-AGEs antibodies (1:100 or 1:50). The tyrosine kinase inhibitor (genistein and tyrphostin), the Ras-farnesyl transferase inhibitor (FPT inhibitor-II), or the p38 MAPK inhibitor (SB203580) suppressed AGEs-induced iNOS expression and nitrite release from C6 glioma cells. AGEs activated p38 MAPK in C6 glioma cells, and this effect was blocked by genistein (20 microM), tyrphostin (30 microM), FPT inhibitor-II (20 microM), and SB203580 (10 microM). Taken together, our data suggest that AGEs may activate the pathways of tyrosine kinase and Ras to induce p38 MAPK activation, which in turn induces iNOS expression and NO production in C6 glioma cells.     

Actin depolymerization via the beta-adrenoceptor in airway smooth muscle cells: a novel PKA-independent pathway

Actin is a majorfunctional and structural cytoskeletal protein that mediates suchdiverse processes as motility, cytokinesis, contraction, and control ofcell shape and polarity. While many extracellular signals are known tomediate actin filament polymerization, considerably less is known aboutsignals that mediate depolymerization of the actin cytoskeleton. Humanairway smooth muscle cells were briefly exposed to isoproterenol,forskolin, or the cAMP-dependent protein kinase A (PKA) agoniststimulatory diastereoisomer of adenosine 3',5'-cyclic monophosphate(Sp-cAMPS). Actin polymerization was measured by concomitantstaining of filamentous actin with FITC-phalloidin and globular actinwith Texas red DNase I. Isoproterenol, forskolin, or Sp-cAMPS inducedactin depolymerization, indicated by a decrease in the intensity offilamentous/globular fluorescent staining. The PKA inhibitor Rpdiastereomer of adenosine 3',5'-cyclic monophosphothioate(Rp-cAMPS) completely inhibited forskolin-stimulated depolymerization, whereas it only partially inhibitedisoproterenol-induced depolymerization. The protein tyrosine kinaseinhibitors genistein or tyrphostin A23 also partially inhibitedisoproterenol-induced actin depolymerization. In contrast, thecombination of Rp-cAMPS and either tyrosine kinase inhibitor had anadditive effect at inhibiting isoproterenol-induced actindepolymerization. These results suggest that both PKA-dependent and-independent pathways mediate actin depolymerization in human airwaysmooth muscle cells.

     

Granulocyte colony-stimulating factor-induced activation of protein kinase-C in myeloid cells

Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, differentiation, and activation of myeloid cells. It binds to a high affinity receptor (G-CSF-R) expressed on myeloid cells, for which the signal transduction mechanisms other than protein tyrosine kinase (PTK) activation have not been completely identified. We explored the potential involvement of protein kinase-C (PKC) in G-CSF-R signal transduction. In this report, we provide direct evidence of PKC activation by G-CSF-R. G-CSF treatment of peripheral blood neutrophils, granulocytic cell lines (HL-60, NFS-60, KG-1), and monocytic cell lines (WEHI-3B,U-937) resulted in PKC activation. Chelerythrine chloride and HA-100, an isoquinolinesulfonamide derivative, the specific inhibitors of PKC, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), a chelator of intracellular calcium, and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8), an inhibitor of intracellular calcium release, blocked G-CSF-induced PKC activation in HL-60 cells, and reduced CD11b upregulation in neutrophils, but did not affect ligand-binding or down-modulation of G-CSF-R. Methyl 2,5-dihydroxycinnamate (MDHC), a potent inhibitor of protein tyrosine kinases (PTK), also inhibited PKC activation in response to G-CSF treatment, suggesting that PKC activation may occur downstream of PTK activation. Our results demonstrate the involvement of PKC in G-CSF-R signal transduction, and suggest a common signaling pathway in myeloid cells of granulocytic and monocytic lineages. J. Cell. Biochem. 66:286–296, 1997. © 1997 Wiley-Liss, Inc.     

Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway

TNF-alpha induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-alpha-induced ICAM-1 expression. TNF-alpha produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-alpha-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-alpha resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-alpha-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C(2) ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by TNF-alpha and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-alpha- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCalpha or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-alpha activates PC-PLC to induce activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.     

Induction of cyclooxygenase-2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB pathways

Lipopolysaccharide (LPS) was found to induce inflammatory responses in the airways and exerted as a potent stimulus for PG synthesis. This study was to determine the mechanisms of LPS-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). LPS markedly increased the expression of COX-2 and release of PGE(2) in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Both the expression of COX-2 and the generation of PGE(2) in response to LPS were attenuated by a tyrosine kinase inhibitor genistein, a phosphatidylcholine-phospholipase C inhibitor D609, a phosphatidylinositol-phospholipase C inhibitor U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. Furthermore, LPS-induced NF-kappaB activation correlated with the degradation of IkappaB-alpha, COX-2 expression, and PGE(2) synthesis, was inhibited by transfection with dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. LPS-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK inhibitor), but these two inhibitors had no effect on LPS-induced NF-kappaB activation, indicating that NF-kappaB is activated by LPS independently of activation of p42/p44 MAPK and p38 MAPK pathways in TSMCs. Taken together, these findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from LPS-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways. LPS-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS

In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PGE(1) in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, cholera toxin, and PGE(1) induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. Pretreatment with the Src kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2) or geldanamycin or the PKA inhibitor Rp-cAMPS only partly inhibited isoproterenol- or PGE(1)-induced actin depolymerization. In contrast, PP2 and geldanamycin did not inhibit forskolin-induced actin depolymerization, and AG-213 (an EGF receptor tyrosine kinase inhibitor) did not inhibit isoproterenol- or PGE(1)-induced actin depolymerization. PI3 kinase or MAP kinase inhibition did not inhibit isoproterenol-induced actin depolymerization. Moreover, isoproterenol but not forskolin induced tyrosine phosphorylation of an Src family member at position 416. These results further confirm that both PKA-dependent and PKA-independent pathways mediate actin depolymerization in human airway smooth muscle cells and that the PKA-independent pathway by which isoproterenol induces actin depolymerization in human airway smooth muscle cells involves Src protein tyrosine kinases and the G(s) protein.     

Intracellular signal transduction for interleukin-1 beta-induced endothelin production in human umbilical vein endothelial cells.

The authors investigated the intracellular signal transduction for interleukin (IL)-1 beta-induced endothelin (ET) production by endothelial cells from cultured human umbilical vein (HUVEC). Cultured HUVEC released immunoreactive (iR)-ET into the media in a time-dependent manner and a significant increase of iR-ET production was observed by the addition of IL-1 beta. The stimulating effect of IL-1 beta on iR-ET production was respectively inhibited by protein kinase C (C kinase) inhibitor (H-7), Ca-calmodulin inhibitor (W-7), cyclic AMP-dependent protein kinase (A kinase) inhibitor (H-8) and tyrosine kinase inhibitor (genistain) in a dose-dependent fashion. The data suggested that intracellular signal transduction for IL-1 beta-induced iR-ET production were via such pathways as C kinase, A kinase, Ca-calmodulin and tyrosine kinase in combination or independently, though possible mediation by other pathways cannot be ruled out.