Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.     

Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1

The objective of the present study was to investigate the effect of leptin, alone or in combination with IL-1, on nitric oxide synthase (NOS) type II activity in vitro in human primary chondrocytes, in the mouse chondrogenic ATDC5 cell line, and in mature and hypertrophic ATDC5 differentiated chondrocytes. For completeness, we also investigated the signalling pathway of the putative synergism between leptin and IL-1. For this purpose, nitric oxide production was evaluated using the Griess colorimetric reaction in culture medium of cells stimulated over 48 hours with leptin (800 nmol/l) and IL-1 (0.025 ng/ml), alone or combined. Specific pharmacological inhibitors of NOS type II (aminoguanidine [1 mmol/l]), janus kinase (JAK)2 (tyrphostin AG490 and Tkip), phosphatidylinositol 3-kinase (PI3K; wortmannin [1, 2.5, 5 and 10 μmol/l] and LY294002 [1, 2.5, 5 and 10 μmol/l]), mitogen-activated protein kinase kinase (MEK)1 (PD098059 [1, 5, 10, 20 and 30 μmol/l]) and p38 kinase (SB203580 [1, 5, 10, 20 and 30 μmol/l]) were added 1 hour before stimulation. Nitric oxide synthase type II mRNA expression in ATDC5 chondrocytes was investigated by real-time PCR and NOS II protein expression was analyzed by western blot. Our results indicate that stimulation of chondrocytes with IL-1 results in dose-dependent nitric oxide production. In contrast, leptin alone was unable to induce nitric oxide production or expression of NOS type II mRNA or its protein. However, co-stimulation with leptin and IL-1 resulted in a net increase in nitric oxide concentration over IL-1 challenge that was eliminated by pretreatment with the NOS II specific inhibitor aminoguanidine. Pretreatment with tyrphostin AG490 and Tkip (a SOCS-1 mimetic peptide that inhibits JAK2) blocked nitric oxide production induced by leptin/IL-1. Finally, wortmannin, LY294002, PD098059 and SB203580 significantly decreased nitric oxide production. These findings were confirmed in mature and hypertrophic ATDC5 chondrocytes, and in human primary chondrocytes. This study indicates that leptin plays a proinflammatory role, in synergy with IL-1, by inducing NOS type II through a signalling pathway that involves JAK2, PI3K, MEK-1 and p38 kinase.     

JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes

Interleukin (IL)-6 decreases cardiac contractility via a nitric oxide (NO)-dependent pathway. However, mechanisms underlying IL-6-induced NO production remain unclear. JAK2/STAT3 and ERK1/2 are two well known signaling pathways activated by IL-6 in non-cardiac cells. However, these IL-6-activated pathways have not been identified in adult cardiac myocytes. In this study, we identified activation of these two pathways during IL-6 stimulation and examined their roles in IL-6-induced NO production and decrease in contractility of adult ventricular myocytes. IL-6 increased phosphorylation of STAT3 (at Tyr(705)) and ERK1/2 (at Tyr(204)) within 5 min that peaked at 15-30 min and returned to basal levels at 2 h. Phosphorylation of STAT3 was blocked by genistein, a protein tyrosine kinase inhibitor, and AG490, a JAK2 inhibitor, but not PD98059, an ERK1/2 kinase inhibitor. The phosphorylation of ERK1/2 was blocked by PD98059 and genistein but not AG490. Furthermore, IL-6 enhanced de novo synthesis of iNOS protein, increased NO production, and decreased cardiac contractility after 2 h of incubation. These effects were blocked by genistein and AG490 but not PD98059. We conclude that IL-6 activated independently the JAK2/STAT3 and ERK1/2 pathways, but only JAK2/STAT3 signaling mediated the NO-associated decrease in contractility.     

Possible participation of a JAK2 signaling pathway in recombinant rat interleukin-5-induced prolongation of rat eosinophil survival

Recombinant rat interleukin (IL)-5-induced prolongation of rat eosinophil survival in culture was inhibited in a concentration-dependent manner by the protein synthesis inhibitor cycloheximide, the DNA-dependent RNA synthesis inhibitor actinomycin D, and the tyrosine kinase inhibitor herbimycin A when examined 96 h after incubation. The MEK-1 inhibitor PD98059 inhibited IL-5-induced phosphorylation of both p44 and p42 MAP kinases, but the IL-5-induced prolongation of eosinophil survival was not inhibited. In contrast, the JAK2 inhibitor AG490 inhibited the IL-5-induced prolongation of eosinophil survival. Treatment of eosinophils with IL-5 resulted in phosphorylation of STAT5 but not STAT1, and the IL-5-induced phosphorylation of STAT5 was inhibited by AG490. These findings suggest that the activation of JAK2 tyrosine kinase and protein synthesis are required for the prolongation of rat eosinophil survival induced by recombinant rat IL-5. STAT5 phosphorylation might also participate in the IL-5-induced survival of rat eosinophils.     

Oncostatin M regulates eotaxin expression in fibroblasts and eosinophilic inflammation in C57BL/6 mice

Oncostatin M (OSM) is a member of the IL-6/LIF (or gp130) cytokine family, and its potential role in inflammation is supported by a number of activities identified in vitro. In this study, we investigate the action of murine OSM on expression of the CC chemokine eotaxin by fibroblasts in vitro and on mouse lung tissue in vivo. Recombinant murine OSM stimulated eotaxin protein production and mRNA levels in the NIH 3T3 fibroblast cell line. IL-6 could regulate a small induction of eotaxin in NIH 3T3 cells, but other IL-6/LIF cytokines (LIF, cardiotrophin-1 (CT-1)) had no effect. Cell signaling studies showed that murine OSM, LIF, IL-6, and CT-1 stimulated the tyrosine phosphorylation of STAT-3, suggesting STAT-3 activation is not sufficient for eotaxin induction in NIH 3T3 cells. OSM induced ERK-1,2 and p38 mitogen-activated protein kinase phosphorylation in NIH 3T3 cells, and inhibitors of ERK (PD98059) or p38 (SB203580) could partially reduce OSM-induced eotaxin production, suggesting partial dependence on mitogen-activated protein kinase signaling. OSM (but not LIF, IL-6, or CT-1) also induced eotaxin release by mouse lung fibroblast cultures derived from C57BL/6 mice. Overexpression of murine OSM in lungs of C57BL/6 mice using an adenovirus vector encoding murine OSM resulted in a vigorous inflammatory response by day 7 after intranasal administration, including marked extracellular matrix accumulation and eosinophil infiltration. Elevated levels of eotaxin mRNA in whole lung were detected at days 4 and 5. These data strongly support a role of OSM in lung inflammatory responses that involve eosinophil infiltration.     

Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils.

Stimulation of rat peritoneal neutrophils with staurosporine (64 nM) induced production of macrophage inflammatory protein-2 (MIP-2) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase/MAP kinase (ERK/MAPK). The staurosporine-induced MIP-2 production at 4 h was inhibited by the highly specific p38 MAPK inhibitor SB 203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD 98059 in a concentration-dependent manner. By treatment with SB 203580 (1 microM) or PD 98059 (50 microM), the staurosporine-induced increase in the levels of mRNA for MIP-2 was only partially lowered, although the staurosporine-induced MIP-2 production was completely inhibited. Consistent with the inhibition by the protein synthesis inhibitor cycloheximide, SB 203580 and PD 98059 inhibited MIP-2 production at 4 h either when added simultaneously with staurosporine or 2 h after stimulation with staurosporine. In contrast, the DNA-dependent RNA polymerase inhibitor actinomycin D did not inhibit MIP-2 production at 4 h when it was added 2 h after staurosporine stimulation. Dot blot analysis demonstrated that treatment with SB 203580 or PD 98059 down-regulates the stability of MIP-2 mRNA. These results suggested that p38 MAPK and ERK/MAPK pathways are involved in translation of MIP-2 mRNA to protein and stabilization of MIP-2 mRNA.     

Signaling pathways of magnolol-induced adrenal steroidogensis

This study focused on identifying the signalling mediating the effect of magnolol on corticosterone production. Magnolol-induced corticosterone production was completely inhibited by mitogen-activated protein kinase kinase (MEK)-inhibitor PD98059, tyrosine kinase (TK)-inhibitor genistein or Janus tyrosine kinase 2 (JAK2)-inhibitor AG490, suggesting that extracellular signal-regulated kinase (ERK) and JAK2 are both involved in this signaling cascade. Further, magnolol induced the transient phosphorylation of MEK, ERK, cAMP response-element binding protein (CREB) and the expression of 32 and 30 kDa steroidogenic acute regulatory protein (StAR) in a time-dependent manner. Inhibition of TK or JAK2 activities blocked magnolol-induced phosphorylation of MEK and ERK, again supporting the upstream role of JAK2. The activation of JAK2 or MEK apparently mediated the magnolol-induced phosphorylation of CREB and the upregulation of StAR. These findings demonstrate a novel pathway for magnolol to induce the expression of StAR, which regulates the rate-limiting step in sterodiogenesis.     

Leptin induces IL-8 expression via leptin receptor, IRS-1, PI3K, Akt cascade and promotion of NF-kappaB/p300 binding in human synovial fibroblasts

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-8 production caused by leptin in both rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). RASF and OASF expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-8 production. Leptin-mediated IL-8 production was attenuated by OBRl receptor antisense oligonucleotide, JAK2 inhibitor or STAT3 small interference RNA (siRNA). Transfection with insulin receptor substrate (IRS)-1 siRNA or dominant-negative mutant of p85 and Akt or pretreatment with phosphatidylinositol 3-kinase inhibitor (Ly294002 and wortmannin), Akt inhibitor, NF-kappaB inhibitor (PDTC) and NF-kappaB inhibitor peptide also inhibited the potentiating action of leptin. Stimulation of RASF with leptin activated IkappaB kinase alpha/beta (IKK alpha/beta), p65 phosphorylation at Ser(276), p65 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked IL-8 expression. The binding of p65 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 acetylation on the IL-8 promoter was enhanced by leptin, which was inhibited by wortmannin, Akt inhibitor or IRS-1 siRNA. These results suggest that leptin increased IL-8 production in synovial fibroblast via the OBRl/JAK2/STAT3 pathway, as well as the activation of IRS1/PI3K/Akt/NF-kappaB-dependent pathway and the subsequent recruitment of p300.