Induction of hepatocyte growth factor expression by maleic acid in human fibroblasts through MAPK activation

Carboxylic acids have various biological activities and play critical roles in cellular metabolic pathways such as the tricarboxylic acid (TCA) cycle. It has been shown that some carboxylic acids induce cell proliferation and production of cytokines or growth factors. However, there have been no reports on effects of carboxylic acids on hepatocyte growth factor (HGF) expression. In this study, we found that only maleic acid among various carboxylic acids examined markedly induced HGF production from human dermal fibroblasts. Maleic acid also induced HGF production from human lung fibroblasts and neuroblastoma cells. The stimulatory effect was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) but not in phosphorylation of p38 was observed from 6 h and up to 24 h after maleic acid addition. The ERK kinase inhibitor PD98059 and the JNK inhibitor SP600125 potently inhibited maleic acid-induced HGF production, while the p38 inhibitor SB203580 did not significantly inhibit the production. The protein synthesis inhibitor cycloheximide completely inhibited upregulation of HGF mRNA induced by maleic acid but superinduced HGF mRNA expression upregulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These results suggest that maleic acid indirectly induced HGF expression from human dermal fibroblasts through activation of ERK and JNK and that de novo protein synthesis is required for maleic acid-induced upregulation of HGF mRNA.     

Synergism between interferon-gamma and cAMP in induction of hepatocyte growth factor in human skin fibroblasts

Interferon (IFN)-gamma stimulates hepatocyte growth factor (HGF) production markedly in various human leukemia cell lines, but its positive effect in human skin fibroblasts is slight. We examined the combined effect of IFN-gamma and various HGF inducers on HGF production in human skin fibroblasts. IFN-gamma synergistically enhanced HGF production stimulated by 8-bromo-cAMP, one of the most effective inducers of HGF: HGF secreted from cells incubated with 1 mM of 8-bromo-cAMP, 1000 U/ml of IFN-gamma and both of these was approximately 8, 1.5 and 24 times, respectively, that secreted from untreated cells. The effect of IFN-gamma was dose-dependent and was nullified by an anti-IFN-gamma antibody. Neither IFN-alpha nor IFN-beta had such an enhancing effect, but both these IFNs inhibited the synergistic effect of IFN-gamma and 8-bromo-cAMP. IFN-gamma also synergistically augmented HGF production induced by interleukin-1beta and cAMP-increasing agents cholera toxin, forskolin and prostaglandin E(2). HGF gene expression upregulated by cholera toxin, forskolin and 8-bromo-cAMP was markedly enhanced by IFN-gamma, which was detected as early as 3 h after its addition. The synergy between HGF inducers and IFN-gamma is not common to all HGF inducers, because HGF production stimulated by epidermal growth factor and protein-kinase-C-activating phorbol esters was significantly inhibited by IFN-gamma. These results indicate that IFN-gamma synergistically stimulates cAMP-induced HGF production and inhibits HGF production induced by growth factors and protein kinase C activators in human skin fibroblasts.     

Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.     

Mitogen-activated protein kinases-dependent induction of hepatocyte growth factor production in human dermal fibroblasts by the antibiotic polymyxin B

Hepatocyte growth factor (HGF) stimulates migration and proliferation of keratinocytes and has been suggested to be involved in wound healing. The cationic antibiotic polymyxin B (PMB) is commonly used as a topical antibiotic for wound care. If PMB possesses an HGF-inducing activity, the antibiotic is potentially beneficial for wound healing in addition to minimizing chances of infection. In this study, we found that PMB markedly induced HGF production from various types of cells including human dermal fibroblasts. Its effect was stronger than the effects of epidermal growth factor and cholera toxin and was comparable to the effect of 8-bromo-cAMP. Among the polymyxin family and polymyxin derivatives, colistin was also effective, whereas colistin methanesulfonate had only a marginal effect and PMB nonapeptide was ineffective. The stimulatory effect of PMB was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) was observed from 0.25h to 6h after the addition of PMB, while increase in phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected from 24h to 60h after PMB addition. The MAPK/ERK kinase inhibitor PD98059, the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 all potently inhibited PMB-induced HGF production. Lastly, proliferation of human dermal fibroblasts was significantly stimulated by PMB. These results indicate that PMB-induced HGF production and proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the induction of HGF production.     

ERK1/2 and JNKs, but not p38 kinase, are involved in reactive oxygen species-mediated induction of osteopontin gene expression by angiotensin II and interleukin-1beta in adult rat cardiac fibroblasts

Osteopontin (OPN), also called cytokine Eta-1, expressed in the myocardium co-incident with heart failure plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Angiotensin II (Ang II) and inflammatory cytokines are increased in the heart following MI. We studied the involvement of mitogen-activated protein kinases (ERK1/2, JNKs, p38 kinase) and reactive oxygen species (ROS) in Ang II- and cytokine-induced OPN gene expression in adult rat cardiac fibroblasts. Ang II alone increased OPN mRNA (3.3 +/- 0.3-folds; P < 0.05; n = 7), while interleukin-1beta (IL-1beta), tumor necrosis factor (TNF-alpha), and interferon-gamma (IFN-gamma) had no effect. A combination of Ang II with IL-1beta or TNF-alpha, not IFN-gamma, increased OPN mRNA more than Ang II alone. Nitric oxide donor, S-nitrosoacetylpenicillamine (SNAP), alone or in combination with Ang II had no effect. Diphenylene iodonium (DPI), inhibitor of NAD(P)H oxidase, and tiron, superoxide scavenger, inhibited Ang II- and Ang II+ IL-1beta-stimulated increases in OPN mRNA. Ang II activated ERK1/2 within 5 min of treatment, not JNKs. IL-1beta activated ERK1/2 and JNKs within 15 min of treatment. A combination of Ang II and IL-1beta activated ERK1/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL-1beta-stimulated activation of ERK1/2, while partially inhibiting JNKs. PD98059, ERK1/2 pathway inhibitor, and SP600125, JNKs inhibitor, partially inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. A combination of PD98059 and SP600125 almost completely inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL-1beta and TNF-alpha act synergistically with Ang II to increase OPN mRNA possibly via NO independent mechanisms. The synergistic increase in OPN mRNA involves ROS-mediated activation of ERK1/2 and JNKs, not P38 kinase, pathways in cardiac fibroblasts.     

HGF synthesis in human lung fibroblasts is regulated by oncostatin M

Oncostatin M (OSM) is a IL-6 family cytokine locally produced in acute lung injury. Its profibrotic properties suggest a role in lung wound repair. Hepatocyte growth factor (HGF), produced by fibroblasts, is involved in pulmonary epithelial repair. We investigated the role of OSM in HGF synthesis by human lung fibroblasts. We showed that OSM upregulated HGF mRNA in MRC5 cells and in human lung fibroblasts, whereas IL-6 and leukemia inhibitory factor did not. OSM induced HGF secretion to a similar extent as IL-1beta in both a time- and dose-dependent manner. HGF was released in its cleaved mature form, and its secretion was completely inhibited in the presence of cycloheximide, indicating a de novo protein synthesis. OSM in combination with prostaglandin E(2), a powerful HGF inductor, led to an additive effect. OSM and indomethacin in combination further increased HGF secretion. This could be explained, at least in part, by a moderate upregulation of specific OSM receptor beta mRNA expression through cyclooxygenase inhibition. These results demonstrate that OSM-induced HGF synthesis did not involve a PGE(2) pathway. OSM-induced HGF secretion was inhibited by PD-98059 (a specific pharmacological inhibitor of ERK1/2), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor) by 70, 82, and 100%, respectively, whereas basal HGF secretion was only inhibited by SP-600125 by 30%. Our results demonstrate a specific upregulation of HGF synthesis by OSM, most likely through a MAPK pathway, and support the suggestion that OSM may participate in lung repair through HGF production.     

Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts

Interleukin-1 (IL-1) plays a crucial role in the immunopathological responses involved with tissue destruction in chronic inflammatory diseases, such as periodontal disease, as it stimulates host cells including fibroblasts to produce various inflammatory mediators and catabolic factors. We comprehensively investigated the involvement of mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) and IkappaB kinases (IKKs)/IkappaBs/nuclear factor-kappaB (NF-kappaB) in IL-1beta-stimulated IL-6, IL-8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase-1 (MMP-1) production by human gingival fibroblasts (HGF). Three MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), which were simultaneously activated by IL-1beta, mediated subsequent c-fos and c-jun mRNA expression and DNA binding of AP-1 at different magnitudes. IKKalpha/beta/IkappaB-alpha/NF-kappaB was also involved in the IL-1 signaling cascade. Further, IL-1beta stimulated HGF to produce IL-6, IL-8, PGE(2) and MMP-1 via activation of the 3 MAPKs and NF-kappaB, as inhibitors of each MAPK and NF-kappaB significantly suppressed the production of IL-1beta-stimulated factors, though these pathways might also play distinct roles in IL-1beta activities. Our results strongly suggest that the MAPKs/AP-1 and IKK/IkappaB/NF-kappaB cascades cooperatively mediate the IL-1beta-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in HGF.     

Involvement of tyrosine kinases on cyclooxygenase expression and prostaglandin E2 production in human gingival fibroblasts stimulated with interleukin-1beta and epidermal growth factor

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and tyrosine kinase on prostaglandin E2 (PGE2) production in human gingival fibroblasts stimulated by interleukin-1beta (IL-1beta) and/or epidermal growth factor (EGF). The cytokine IL-1beta and to a lesser extent EGF, enhanced COX-2 mRNA levels in gingival fibroblasts. Simultaneous treatment with EGF and IL-1beta resulted in enhanced COX-2 mRNA levels accompanied by a synergistic stimulation of PGE2 biosynthesis compared to the cells treated with IL-1beta or EGF alone. Neither IL-1beta EGF nor the combination of IL-1beta and EGF enhanced COX-1 mRNA levels in gingival fibroblasts. The tyrosine kinase inhibitors, Herbimycin A and PD 153035 hydrochloride, reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta or the combination of IL-1beta and EGF whereas COX-1 mRNA levels were not affected. Furthermore, the COX-2 specific inhibitor, NS-398, abolished the PGE2 production induced by IL-1beta, EGF, or the combination. These results indicate that the synergy between IL-1beta and EGF on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase(s) are involved in the signal transduction of COX-2 in gingival fibroblasts.     

Dextran sulfate sodium enhances interleukin-1 beta release via activation of p38 MAPK and ERK1/2 pathways in murine peritoneal macrophages

Interleukin (IL)-1 beta is a pro-inflammatory cytokine that has been shown to play a pivotal role in the onset of inflammatory bowel disease (IBD), however, the molecular mechanisms underlying the production of IL-1 beta in IBD are not fully understood. We investigated dextran sulfate sodium (DSS)-induced IL-1 beta production and caspase-1 activities in murine peritoneal macrophages (pM phi). Further, the activation status of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun NH(2)-terminal kinase (JNK1/2), as well as their upstream target kinases, were examined by Western blotting. In addition, mRNA expression was assessed by RT-PCR and CXC chemokine ligand 16 (CXCL16) protein was detected by immunocytochemistry. DSS-treated pM phi released IL-1 beta protein in a time-dependent manner without affecting mRNA levels during 3-24 h, and caspase-1 activity peaked at 5 min (29-fold). IL-1 beta release and caspase-1 activity induced by DSS were significantly inhibited by a MAPK kinase 1/2 inhibitor, a p38 MAPK inhibitor, and NAC, however, not by JNK1/2 or a protein kinase C inhibitor. In addition, DSS strikingly induced the phosphorylation of p38 MAPK and ERK1/2 within 2 and 10 min, respectively. DSS also induced intracellular generation of reactive oxygen species (ROS). Pre-treatment with anti-CXCL16 for 24 h, but not anti-scavenger receptor-A, anti-CD36, or anti-CD68 antibodies, significantly suppressed DSS-induced IL-1 beta production. Our results suggest that DSS triggers the release of IL-1 beta protein from murine pM phi at a post-translational level through binding with CXCL16, ROS generation, and resultant activation of both p38 MAPK and ERK1/2 pathways, and finally caspase-1 activation.     

Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.     

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.     

Anti-inflammatory effects of hepatocyte growth factor: induction of interleukin-1 receptor antagonist

Hepatocyte growth factor (HGF) prevents liver failure in various animal models including endotoxin-induced acute liver failure. We were interested to find out whether human HGF exerts anti-inflammatory effects by modulation of cytokine synthesis. Therefore, human HepG2 cells were cultured with increasing concentrations of HGF. HGF dose-dependently upregulated the production of interleukin-1 receptor antagonist (IL-1Ra). Incubation of HepG2 cells with interleukin-1beta (IL-1beta) caused an increase in IL-1Ra levels, while interleukin-6 (IL-6) had no effect on IL-1Ra synthesis. Co-stimulation of HepG2 cells with HGF + IL-1beta resulted in a synergistic effect on IL-1Ra mRNA and protein expression. Stimulation of freshly isolated mouse hepatocytes from male C57 BL/6 mice with HGF increased IL-1Ra mRNA and protein synthesis dose-dependently. A co-stimulation with HGF and IL-1beta had a synergistic effect on IL-1Ra mRNA expression but only a partially additive effect on IL-1Ra protein synthesis. HGF-induced IL-1Ra production was significantly decreased by the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Accordingly, HGF stimulation specifically increased MAPK-dependent signalling pathway (p42/44). In contrast, in preactivated PBMC mRNA expression and protein synthesis of IL-1Ra, interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) were unaffected after stimulation with HGF. In conclusion, our data suggest that HGF exerts anti-inflammatory effects by modulating the signal transduction cascade leading to increased expression of IL-1Ra, which might explain the protective and regenerative properties of this cytokine in animal models of liver failure.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

c-jun amino-terminal kinase and mitogen activated protein kinase 1/2 mediate hepatocyte growth factor-induced migration of brain endothelial cells

Hepatocyte growth factor (HGF) influences several components of the angiogenic response, including endothelial cell migration. While recent studies indicate a crucial role of HGF in brain angiogenesis, the signaling pathways that regulate brain endothelial cell migration by HGF remain uncharacterized. Herein, we report that HGF stimulated human brain microvascular endothelial cell (HBMEC) migration in a dose- and time-dependent manner. Challenge of HBMECs with HGF activated the c-jun amino-terminal kinase (JNK), increased phosphorylation of the proline-rich tyrosine kinase 2 (Pyk-2) at Tyr(402) and activated c-Src. Inhibition of JNK by SP600125 or expression of a dominant negative JNK1 construct abrogated the migratory response of HBMECs to HGF. Treatment of HBMECs with the Src inhibitor PP2 markedly decreased HGF-stimulated JNK activation and migration to HGF. Moreover, expression of a mutant Pyk-2 construct prevented HGF-induced Pyk-2 phosphorylation at Tyr(402) and stimulation of HBMEC migration. Next, we examined activation of the extracellular signal regulated kinase (ERK) pathway. Stimulation of HBMECs by HGF led to rapid activation of ERK1/2, phosphorylation of Raf-1 at Ser(338) and Tyr(340/341) and MEK1/2 at Ser(222). Moreover, inhibition of ERK activation by UO126 and PD98059 markedly decreased HGF-stimulated HBMEC migration. HGF also activated AKT, while inhibition of AKT by LY294002 induced a modest decrease of HGF-induced HBMEC migration. These results highlight a model whereby JNK and ERK play a critical role in regulation of brain endothelial cell migration by HGF.