Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C.

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO(-)) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts. ONOO(-)-mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO(-) treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO(-), implying that activation of ERK by ONOO(-) depends on activation of PKC. Indeed, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha and ERK phosphorylation by ONOO(-). Indeed, ONOO(-) exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using G?6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO(-)-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.     

Bradykinin-mediated cell proliferation depends on transactivation of EGF receptor in corneal fibroblasts

In previous studies, bradykinin (BK) has been shown to induce cell proliferation through BK B2 receptor (B2R) via p42/p44 MAPK in Statens Seruminstitut Rabbit Corneal Cells (SIRCs). In addition to this pathway, EGFR transactivation pathway has been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we further investigate whether these transactivation mechanisms participating in BK-induced cell proliferation in SIRCs. Using an immunofluorescence staining and RT-PCR, we initially characterize that SIRCs were corneal fibroblasts and predominantly expressed B2R by BK. Inhibition of p42/p44 MAPK by the inhibitors of Src, EGFR, and Akt or transfection with respective siRNAs prevents BK-induced DNA synthesis in SIRCs. The mechanisms underlying these responses were mediated through phosphorylation of Src and EGFR via the formation of Src/EGFR complex which was attenuated by PP1 and AG1478. Moreover, BK-induced p42/p44 MAPK and Akt activation was mediated through EGFR transactivation, which was diminished by the inhibitors of MMP-2/9 and heparin-binding EGF-like factor (HB-EGF). Finally, increased nuclear translocation of Akt and p42/p44 MAPK turns on early gene expression leading to cell proliferation. These results suggest that BK-induced cell proliferation is mediated through c-Src-dependent transactivation of EGFR via MMP2/9-dependent pro-HB-EGF shedding linking to activation of Akt and p42/p44 MAPK in corneal fibroblasts.     

Phosphorylation of p42/44(MAPK) by various signal transduction pathways activates cytosolic phospholipase A(2) to variable degrees

Arachidonic acid has been implicated to play a role in physiological and pathophysiological processes and is selectively released by the 85-kDa cytosolic phospholipase A(2) (cPLA(2)). The activity of cPLA(2) is regulated by calcium, translocating the enzyme to its substrate, and by phosphorylation by a mitogen-activated protein kinase (MAPK) family member and a MAPK-activated protein kinase. In this study, the signal transduction pathways in growth factor-induced phosphorylation of p42/44(MAPK) and cPLA(2) activation were investigated in Her14 fibroblasts. p42/44(MAPK) in response to epidermal growth factor was not only phosphorylated via the Raf-MEK pathway but mainly through protein kinase C (PKC) or a related or unrelated kinase in which the phosphorylated p42/44(MAPK) corresponded with cPLA(2) activity. Serum-induced phosphorylation of p42/44(MAPK) also corresponded with cPLA(2) activity but is predominantly mediated via Raf-MEK and partly through PKC or a related or unrelated kinase. In contrast, activation of PKC by phorbol ester did not result in increased cPLA(2) activity, while p42/44(MAPK) is phosphorylated, mainly via Raf-MEK and through MEK. Moreover, p42/44(MAPK) phosphorylation is present in quiescent and proliferating cells, and p42/44(MAPK) is entirely phosphorylated via Raf-MEK, but it only corresponds to cPLA(2) activity in the former cells. Collectively, these data show that p42/44(MAPK) in proliferating, quiescent, and stimulated cells is phosphorylated by various signal transduction pathways, suggesting the activation of different populations of p42/44(MAPK) and cPLA(2).     

Stimulation of Na+/H+ antiport and pyruvate kinase activities by high glucose concentration in human erythrocytes

This study aims to demonstrate the effect of high glucose concentrations on NHE-1 and PK activities and investigate the implicated signal transduction pathways. Erythrocytes drawn from healthy volunteers were incubated in the presence of 5 or 50 mM of glucose, fructose, galactose or mannitol. When appropriate, specific inhibitors of NHE-1, PKC or p42/44 MAPK were used. Erythrocyte NHE-1 activity has been estimated by fluorometrical determination of the intracellular pH and quantification of sodium uptake using 22Na. Pyruvate kinase activity was measured by a NADH-lactate dehydrogenase enzymatic assay. p42/44 MAPK activity was assessed with a specific enzyme linked immunosorbent assay (ELISA). Increased concentrations of glucose but not galactose, fructose or mannitol enhanced erythrocyte NHE-1, PK and p42/44 MAPK activity. Inhibition of PKC, counteracted these effects of glucose. Similarly, inhibition of NHE 1 abolished the effect of high glucose on PK and p42/44 MAPK as well. Finally, inhibition of p42/44 MAPK also hindered the effect of glucose on NHE-1 and PK activities. The data of the present study indicate an acute effect of glucose on signal transduction pathways in human erythrocytes. This pathway involves NHE-1, PKC, and p42/44 MAPK. A positive feedback between NHE 1 and p42/44 MAPK is suggested.     

Bile acids stimulate PKCalpha autophosphorylation and activation: role in the attenuation of prostaglandin E1-induced cAMP production in human dermal fibroblasts

The aim was to identify the specific PKC isoform(s) and their mechanism of activation responsible for the modulation of cAMP production by bile acids in human dermal fibroblasts. Stimulation of fibroblasts with 25-100 microM of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) led to YFP-PKCalpha and YFP-PKCdelta translocation in 30-60 min followed by a transient 24- to 48-h downregulation of the total PKCalpha, PKCdelta, and PKCepsilon protein expression by 30-50%, without affecting that of PKCzeta. Increased plasma membrane translocation of PKCalpha was associated with an increased PKCalpha phosphorylation, whereas increased PKCdelta translocation to the perinuclear domain was associated with an increased accumulation of phospho-PKCdelta Thr505 and Tyr311 in the nucleus. The PKCalpha specificity on the attenuation of cAMP production by CDCA was demonstrated with PKC downregulation or inhibition, as well as PKC isoform dominant-negative mutants. Under these same conditions, neither phosphatidylinositol 3-kinase, p38 MAP kinase, p42/44 MAP kinase, nor PKA inhibitors had any significant effect on the CDCA-induced cAMP production attenuation. CDCA concentrations as low as 10 microM stimulated PKCalpha autophosphorylation in vitro. This bile acid effect required phosphatidylserine and was completely abolished by the presence of G?6976. CDCA at concentrations less than 50 microM enhanced the PKCalpha activation induced by PMA, whereas greater CDCA concentrations reduced the PMA-induced PKCalpha activation. CDCA alone did not affect PKCalpha activity in vitro. In conclusion, although CDCA and UDCA activate different PKC isoforms, PKCalpha plays a major role in the bile acid-induced inhibition of cAMP synthesis in fibroblasts. This study emphasizes potential consequences of increased systemic bile acid concentrations and cellular bile acid accumulation in extrahepatic tissues during cholestatic liver diseases.     

Thrombin Mediates Migration of Rat Brain Astrocytes via PLC, Ca2+, CaMKII, PKCα, and AP-1-Dependent Matrix Metalloproteinase-9 Expression

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain unclear. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 and migration of RBA-1 cells, which were inhibited by pretreatment with the inhibitor of Gq-coupled receptor (GPAnt2A), Gi/o-coupled receptor (GPAnt2), PC-PLC (D609), PI-PLC (U73122), Ca²⁺-ATPase (thapsigargin, TG), calmodulin (CaMI), CaMKII (KN62), PKC (Gö6976 or GF109203X), MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) or the intracellular calcium chelator (BAPTA/AM) and transfection with siRNA of PKCα, Erk2, JNK1, p38 MAPK, c-Jun, or c-Fos. In addition, thrombin-induced elevation of intracellular Ca²⁺ concentration was attenuated by PPACK (a thrombin inhibitor). Thrombin further induced CaMKII phosphorylation and PKCα translocation, which were inhibited by U73122, D609, KN62, TG, or BAPTA/AM. Thrombin also induced PKCα-dependent p42/p44 MAPK and JNK1/2, but not p38 MAPK activation. Finally, we showed that thrombin enhanced c-Fos expression and c-Jun phosphorylation. c-Fos mRNA levels induced by thrombin were reduced by PD98059, SP600125, and Gö6976, but not SB202190. Thrombin stimulated in vivo binding of c-Fos to the MMP-9 promoter, which was reduced by pretreatment with SP600125 or PD98059, but not SB202190. These results concluded that thrombin activated a PLC/Ca²⁺/CaMKII/PKCα/p42/p44 MAPK and JNK1/2 pathway, which in turn triggered AP-1 activation and ultimately induced MMP-9 expression in RBA-1 cells.     

Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.     

Activation of mitogen-activated protein kinases is required for alpha1-adrenergic agonist-induced cell scattering in transfected HepG2 cells

Activation of alpha1B-adrenergic receptors ((alpha1B)AR) by phenylephrine (PE) induces scattering of HepG2 cells stably transfected with the (alpha1B)AR (TFG2 cells). Scattering was also observed after stimulation of TFG2 cells with phorbol myristate acetate (PMA) but not with hepatocyte growth factor/scatter factor, epidermal growth factor, or insulin. PMA but not phenylephrine rapidly activated PKCalpha in TFG2 cells, and the highly selective PKC inhibitor bisindolylmaleimide (GFX) completely abolished PMA-induced but not PE-induced scattering. PE rapidly activated p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), and AP1 (c-fos/c-jun). Selective blockade of p42/44 MAPK activity by PD98059 or by transfection of a MEK1 dominant negative adenovirus significantly inhibited the PE-induced scattering of TFG2 cells. Selective inhibition of p38 MAPK by SB203850 or SB202190 also blocked PE-induced scattering, whereas treatment of TFG2 cells with the PI3 kinase inhibitors LY294002 or wortmannin did not inhibit PE-induced scattering. Blocking JNK activation with a dominant negative mutant of JNK or blocking AP1 activation with a dominant negative mutant of c-jun (TAM67) significantly inhibited PE-induced cell scattering. These data indicate that PE-induced scattering of TFG2 cells is mediated by complex mechanisms, including activation of p42/44 MAPK, p38 MAPK, and JNK. Cell spreading has been reported to play important roles in wound repair, tumor invasion, and metastasis. Therefore, catecholamines acting via the (alpha1)AR may modulate these physiological and pathological processes.     

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: A hypothesis for calcium-dependent and calcium-independent events activated by flow

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical fórces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolárity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP₂ levels0 and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.     

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.     

Induction of multiple matrix metalloproteinases in human dermal and synovial fibroblasts by Staphylococcus aureus: implications in the pathogenesis of septic arthritis and other soft tissue infections

Infections of body tissue by Staphylococcus aureus are quickly followed by degradation of connective tissue. Patients with rheumatoid arthritis are more prone to S. aureus-mediated septic arthritis. Various types of collagen form the major structural matrix of different connective tissues of the body. These different collagens are degraded by specific matrix metalloproteinases (MMPs) produced by fibroblasts, other connective tissue cells, and inflammatory cells that are induced by interleukin-1 (IL-1) and tumor necrosis factor (TNF). To determine the host's contribution in the joint destruction of S. aureus-mediated septic arthritis, we analyzed the MMP expression profile in human dermal and synovial fibroblasts upon exposure to culture supernatant and whole cell lysates of S. aureus. Human dermal and synovial fibroblasts treated with cell lysate and filtered culture supernatants had significantly enhanced expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, and MMP-11 compared with the untreated controls (p < 0.05). In the S. aureus culture supernatant, the MMP induction activity was identified to be within the molecular-weight range of 30 to >50 kDa. The MMP expression profile was similar in fibroblasts exposed to a combination of IL-1/TNF. mRNA levels of several genes of the mitogen-activated protein kinase (MAPK) signal transduction pathway were significantly elevated in fibroblasts treated with S. aureus cell lysate and culture supernatant. Also, tyrosine phosphorylation was significantly higher in fibroblasts treated with S. aureus components. Tyrosine phosphorylation and MAPK gene expression patterns were similar in fibroblasts treated with a combination of IL-1/TNF and S. aureus. Mutants lacking staphylococcal accessory regulator (Sar) and accessory gene regulator (Agr), which cause significantly less severe septic arthritis in murine models, were able to induce expression of several MMP mRNA comparable with that of their isogenic parent strain but induced notably higher levels of tissue inhibitors of metalloproteinases (TIMPs). To our knowledge, this is the first report of induction of multiple MMP/TIMP expression from human dermal and synovial fibroblasts upon S. aureus treatment. We propose that host-derived MMPs contribute to the progressive joint destruction observed in S. aureus-mediated septic arthritis.     

Selective secretion of protein kinase C isozymes by thrombin-stimulated human platelets

The protein kinase C (PKC) was secreted from thrombin-stimulated human platelets in a time- and dose-dependent manner. The PKC specific inhibitors Ro31-8220 (0.05 microM) and GF 109203X (0.5 microM) totally inhibited the secreted kinase activity. Western blot analysis of the secretory components showed reactivity to PKCalpha, PKCbetaII, and PKCdelta antibodies, but not to PKCbetaI, and p42/44 MAPK, although they were present in lysed platelets. The fractionation of platelets secreted components showed that PKC activity increased in both soluble and microparticle fractions after thrombin treatments. This is the first report demonstrating that activated human platelets selectively secrete protein kinase C isozymes. Protein kinase C secreted by platelets in this unique manner may have an extracellular role in the plasma, and may regulate cellular functions, including remodeling of vascular endothelial cells.     

Thyroid hormones regulate DNA-synthesis and cell-cycle proteins by activation of PKCalpha and p42/44 MAPK in chick embryo hepatocytes

The molecular mechanism by which thyroid hormones exert their effects on cell growth is still unknown. In this study, we used chick embryo hepatocytes at different stages of development as a model to investigate the effect of the two thyroid hormones, T3 and T4, and of their metabolite T2, on the control of cell proliferation. We observed that T2 provokes increase of DNA-synthesis as well as T3 and T4, independently of developmental stage. We found that this stimulatory effect on the S phase is reverted by specific inhibitors of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (p42/44 MAPK), Ro 31-8220 or PD 98059. Furthermore, the treatment with thyroid hormones induces the activation of PKCalpha and p42/44 MAPK, suggesting their role as possible downstream mediators of cell response mediated by thyroid hormones. The increase of DNA-synthesis is well correlated with the increased levels of cyclin D1 and cdk4 that control the G1 phase, and also with the activities of cell-cycle proteins involved in the G1 to S phase progression, such as cyclin E/A-cdk2 complexes. Interestingly, the activity of cyclin-cdk2 complexes is strongly repressed in the presence of PKC and p42/44 MAPK inhibitors. In conclusion, we demonstrated that the thyroid hormones could modulate different signaling pathways that are able to control cell-cycle progression, mainly during G1/S transition.