Stress-activated Protein kinase/c-Jun N-terminal Kinase Research Articles

Utility and Limitations of SP600125, an Inhibitor of Stress-Responsive c-Jun N-Terminal Kinase

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) belongs to the mitogen-activated protein kinase (MAPK) family and plays an important role in many biological contexts. JNK is deeply involved in several serious human disorders, including inflammation, obesity, diabetes, neuronal disease and cancer. Accordingly, JNK has been recognized as an appropriate target for the treatment of these diseases, and much effort has been expended over the past 15 years to isolate JNK inhibitors that can inactivate this kinase. In 2001, the compound SP600125 was reported as the first JNK-specific inhibitor. Many researchers have subsequently employed SP600125 in in vitro and in vivo models to evaluate whether certain disease-associated events are JNK-dependent. Indeed, more than 1300 studies citing the use of SP600125 as a JNK inhibitor in cell cultures and animal models have been reported. However, although SP600125 has been employed successfully to inhibit JNK in several situations, there have been questions about its specificity for JNK. SP600125 can bind to a broad range of protein kinases and inhibit some of them with similar or greater potency than JNK, confirming that many additional kinases may be targets of SP600125. In this article, we review both the usefulness of SP60125 as a JNK inhibitor and the limitations to its specificity.

Rho-kinase limits FGF-2-stimulated VEGF release in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is involved in FGF-2-stimulated VEGF release in MC3T3-E1 cells. FGF-2 induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly enhanced the FGF-2-stimulated VEGF release. Fasudil, another Rho-kinase inhibitor, also amplified the VEGF release. FGF-2 significantly stimulated VEGF accumulation and fasudil enhanced FGF-2-stimulated VEGF accumulation also in whole cell lysates. Neither Y27632 nor fasudil affected the phosphorylation levels of p44/p42 MAP kinase or p38 MAP kinase. Y27632 and fasudil markedly strengthened the FGF-2-induced phosphorylation of SAPK/JNK. Y27632 as well as fasudil enhanced FGF-2-stimulated VEGF release and Y27632 enhanced the FGF-2-induced phosphorylation levels of SAPK/JNK also in human osteoblasts. These results strongly suggest that Rho-kinase negatively regulates FGF-2-stimulated VEGF release in osteoblasts.

Oxidation of Cell Surface Thiol Groups by Contact Sensitizers Triggers the Maturation of Dendritic Cells

p38 mitogen-activated protein kinase (MAPK) has a crucial role in the maturation of dendritic cells (DCs) by sensitizers. Recently, it has been reported that the oxidation of cell surface thiols by an exogenous impermeant thiol oxidizer can phosphorylate p38 MAPK. In this study, we examined whether sensitizers oxidize cell surface thiols of monocyte-derived DCs (MoDCs). When cell surface thiols were quantified by flow cytometry using Alexa fluor maleimide, all the sensitizers that we examined decreased cell surface thiols on MoDCs. To examine the effects of decreased cell surface thiols by sensitizers on DC maturation, we analyzed the effects of an impermeant thiol oxidizer, o-phenanthroline copper complex (CuPhen). The treatment of MoDCs with CuPhen decreased cell surface thiols, phosphorylated p38 MAPK, and induced MoDC maturation, that is, the augmentation of CD83, CD86, HLA-DR, and IL-8 mRNA, as well as the downregulation of aquaporin-3 mRNA. The augmentation of CD86 was significantly suppressed when MoDCs were pretreated with N-acetyl-L-cystein or treated with SB203580. Finally, we showed that epicutaneous application of 2,4-dinitrochlorobenzene on mouse skin significantly decreased cell surface thiols of Langerhans cells in vivo. These data suggest that the oxidation of cell surface thiols has some role in triggering DC maturation by sensitizers.

Rho-kinase regulates prostaglandin D2-stimulated heat shock protein 27 induction in osteoblasts

We previously reported that prostaglandin D(2) (PGD(2)) stimulates heat shock protein 27 (HSP27) induction through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In addition, we recently showed that PGD(2) activates Rho-kinase, resulting in the regulation of interleukin-6 synthesis via activation of p38 MAP kinase but not p44/p42 MAP kinase in these cells. In the present study, in order to investigate whether Rho-kinase is involved in the PGD(2)-stimulated HSP27 induction in MC3T3-E1 cells, we examined the effects of Rho-kinase inhibitors on HSP27 induction. Y27632 and fasudil, Rho-kinase inhibitors, markedly suppressed the HSP27 induction stimulated by PGD(2) in a dose-dependent manner without affecting levels of HSP70 in the presence of PGD(2). Immunofluorescence microscopy studies also revealed that Y27632 and fasudil markedly suppressed the induction of HSP27. Y27632 and fasudil attenuated the PGD(2)-induced phosphorylation levels of SAPK/JNK. In conclusion, Rho-kinase inhibitors regulate PGD(2)-stimulated HSP27 induction via activation of both SAPK/JNK and p38 MAP kinase in osteoblasts.

Inhibition by minodronate of basic fibroblast growth factor-stimulated vascular endothelial growth factor synthesis in osteoblast-like cells

In our previous study, we showed that basic fibroblast growth factor (bFGF) stimulates the synthesis of vascular endothelial growth factor (VEGF) via the activation of p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of minodronate, a newly developed bisphosphonate, on bFGF-induced VEGF synthesis in MC3T3-E1 cells. Minodronate significantly reduced the synthesis of VEGF induced by bFGF in a dose-dependent manner in a range between 3 and 100 µM. The bFGF-stimulated phosphorylation of p44/p42 MAP kinase and SAPK/JNK was reduced by minodronate. These results strongly suggest that minodronate suppresses bFGF-stimulated VEGF synthesis via the inhibition of p44/p42 MAP kinase and SAPK/JNK in osteoblasts.

Cross‐regulation between β1‐ and β3‐adrenoceptors following chronic β‐adrenergic stimulation in neonatal rat cardiomyocytes

We have previously shown that beta-adrenoceptors continuously stimulated with noradrenaline induces an increase in beta(3)-adrenoceptors (G alpha(i)PCRs) and a decrease in beta(1)-adrenoceptors (G alpha(s)PCRs) at functional, genomic and protein levels. This compensatory modification induced by noradrenaline is probably one of the consequences of cardiac depression observed in heart disease. Therefore, we investigated further the interaction between beta(1)- and beta(3)-adrenoceptors in neonatal rat cardiomyocytes. Functional studies were performed by cyclic adenosine monophosphate (cAMP) accumulation assays in cells untreated or treated with dobutamine and ICI 118551 (beta(1)-adrenoceptor) or CL-3162436243 (beta(3)-adrenoceptor) for 24 h in the presence or absence of protein kinase inhibitors. Beta-adrenoceptor and protein kinase expression was monitored by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by Western blotting, respectively. Chronic beta(1)- or beta(3)-adrenoceptor stimulation reduced beta(1)-adrenoceptor-mediated cAMP accumulation in association with a decrease in beta(1)-adrenoceptor mRNA and protein levels through protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38MAPK) activation. In contrast, both treatments induced an increase in beta(3)-adrenoceptor expression and beta(3)-adrenoceptor-inhibited forskolin response through PKC, extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and p38MAPK phosphorylation, although no beta(3)-adrenoceptor response was observed in untreated cells. ERK1/2 and p38MAPK were activated by both treatments. The modulation of beta(1)- or beta(3)-adrenoceptor function did not require stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) although chronic beta(1)-adrenoceptor stimulation activated SAPK/JNK. Beta(3)-adrenoceptor treatment activated Akt although PI3K was not involved in beta(3)-adrenoceptor up-regulation. We show for the first time that chronic beta(1)- or beta(3)-adrenoceptor stimulation leads to the modulation of beta(1)- and beta(3)-adrenoceptors by a cross-regulation involving PKC, PI3K p38MAPK and MEK/ERK1/2 pathway, and through protein kinase A when beta(1)-adrenoceptors are chronically activated.

Involvement of Rho-kinase in TGF-β-stimulated heat shock protein 27 induction in osteoblasts

We previously reported that transforming growth factor-β (TGF-β) stimulates heat shock protein 27 (HSP27) induction through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In addition, we recently reported that the Rho-kinase inhibitors Y27632 and fasudil suppressed the TGF-β-induced phosphorylation of SAPK/JNK, but not p44/p42 MAP kinase, p38 MAP kinase or Smad2. In the present study, to investigate whether Rho-kinase is involved in TGF-β-stimulated HSP27 induction in MC3T3-E1 cells, we examined the effects of Rho-kinase inhibitors on HSP27 induction. Y27632 and fasudil significantly suppressed the HSP27 induction stimulated by TGF-β in a dose-dependent manner, without affecting the protein levels of HSP70 or HSP90. Immunofluorescence microscopy also revealed that TGF-β clearly stimulated, while Y27632 and fasudil markedly suppressed, HSP27 induction in the cytosol of these cells. Taken together, these findings indicate that Rho-kinase regulates TGF-β-stimulated HSP27 induction via SAPK/JNK activation in osteoblasts.

Cytokine-induced activation of mixed lineage kinase 3 requires TRAF2 and TRAF6

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple mitogen-activated protein kinase (MAPK) pathways in response to growth factors, stresses and the pro-inflammatory cytokine, tumor necrosis factor (TNF). MLK3 is required for optimal activation of stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling by TNF, however, the mechanism by which MLK3 is recruited and activated by the TNF receptor remains poorly understood. Here we report that both TNF and interleukin-1β (IL-1β) stimulation rapidly activate MLK3 kinase activity. We observed that TNF stimulates an interaction between MLK3 and TNF receptor associated factor (TRAF) 2 and IL-1β stimulates an interaction between MLK3 and TRAF6. RNA interference (RNAi) of traf2 or traf6 dramatically impairs MLK3 activation by TNF indicating that TRAF2 and TRAF6 are critically required for MLK3 activation. We show that TNF also stimulates ubiquitination of MLK3 and MLK3 can be conjugated with lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitin chains. Our results suggest that K48-linked ubiquitination directs MLK3 for proteosomal degradation while K63-linked ubiquitination is important for MLK3 kinase activity. These results reveal a novel mechanism for MLK3 activation by the pro-inflammatory cytokines TNF and IL-1β.

Involvement of Rho-kinase in prostaglandin E 1-stimulated VEGF synthesis through stress-activated protein kinase/c- Jun N-terminal kinase in osteoblast-like MC3T3-E1 cells

We have previously shown that prostaglandin E 1 (PGE 1) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the PGE 1-stimulated VEGF synthesis in these cells. PGE 1 induced within 3 min the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632 and fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly suppressed the PGE 1-stimulated VEGF synthesis. Y27632 and fasudil markedly reduced the PGE 1-induced phosphorylation of SAPK/JNK without affecting the phosphorylation levels of p38 MAP kinase or p44/p42 MAP kinase. These results strongly suggest that Rho-kinase functions at a point upstream of SAPK/JNK and regulates PGE 1-stimulated VEGF synthesis in osteoblasts.

Mechanisms of interleukin-1β-induced GDNF release from rat glioma cells

Glial cell line-derived neurotrophic factor (GDNF) is highly expressed both in neurons and astrocytes in injured tissues. Astrocytes support neurons by releasing neurotrophic factors including GDNF. It has been reported that various agents including cytokines such as interleukin (IL)-1β induce GDNF mRNA expression and the release in astrocytes. However, the mechanism behind the GDNF synthesis and release remains unclear. Herein, we investigated the mechanisms of the IL-1β-induced GDNF release from rat C6 glioma cells. IL-1β time dependently stimulated GDNF release from C6 cells. IL-1β induced the phosphorylation of inhibitor kappa B (IκB), p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase, stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) and signal transducer and activator of transcription (STAT) 3. The IL-1β-stimulated levels of GDNF were suppressed by wedelolactone, an inhibitor of IκB kinase, SB203580, an inhibitor of p38 MAP kinase, PD98059, an inhibitor of MAP kinase kinase 1/2 or Janus family of tyrosine kinase (JAK) inhibitor I, an inhibitor of upstream kinase of STAT3. On the contrary, SP600125, an inhibitor of SAPK/JNK, failed to reduce the IL-1β-effect. These results strongly suggest that IL-1β stimulates GDNF release through the pathways of IκB-nuclear factor kappa B, p38 MAP kinase, p44/p42 MAP kinase and JAK-STAT3, but not through the SAPK/JNK pathway in glioma cells.

Structural characteristics and in vitro macrophage activation of acetyl fucoidan from Cladosiphon okamuranus.

We investigated a structural characteristics of acetyl fucoidan (CAF) isolated from commercially cultured Cladosiphon okamuranus. The CAF-induced macrophage activation and its signaling pathways in murine macrophage cell line, RAW 264.7 were also investigated. From the results of methylation analysis, CAF consisted of alpha-1-->3 linked L: -fucosyl residues and substituted sulfate and acetyl groups at C-4 on the main chain. CAF induced production of nitric oxide (NO), tumor necrosis factor-alpha and interleukin-6 in RAW 264.7 cells. Sulfate and acetyl groups of CAF involved in CAF-induced NO production. Neutralizing anti-Toll-like receptor 4 (TLR4), anti-CD14 and anti-scavenger receptor class A (SRA) but not anti-complement receptor type 3 monoclonal antibodies decreased CAF-induced NO production. The results of immunoblot analysis indicated that CAF activated mitogen-activated protein kinases (MAPKs) such as p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). SB203580 (p38 MAPK inhibitor) and SP600125 (SAPK/JNK inhibitor), but not U0126 (MAPK/ERK kinase 1/2 inhibitor) decreased CAF-induced NO production. The results suggested that CAF induced macrophage activation through membrane receptors TLR4, CD14 and SRA, and MAPK signaling pathways.

P38 MAP kinase controls EGF receptor downregulation via phosphorylation at Ser1046/1047

The desensitization mechanism of the EGF receptor (EGFR) is important for the regulation of cancer cells. Although the phosphorylation of EGFR at Tyr1045 and Ser1046/1047 (Ser1046/7) reportedly accounts for such desensitization, the precise mechanism still remains unknown. Therefore, the present study investigated the upstream signals of these phosphorylations in SW480 colon cancer cells. Anisomycin, a potent kinase activator, induced the activation of both p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), but not p44/p42 MAPK. Anisomycin caused EGFR degradation and this was abolished by a specific p38 MAPK inhibitor, SB203580. Surprisingly, whereas EGF induced phosphorylation at Tyr1045, but not Ser1046/7, anisomycin induced the phosphorylation of EGFR at Ser1046/7, but not Tyr1045. In addition, though both EGF and anisomycin caused EGFR internalization, the EGFR internalized by anisomycin was not associated with an ubiquitin ligase, c-Cbl. Furthermore, SB203580 or gene silencing using p38 MAPK-siRNA suppressed anisomycin-induced phosphorylation of EGFR at Ser1046/7. These results strongly suggest that p38 MAPK directs EGFR toward desensitization via its phosphorylation at Ser1046/7.

Oxidative stress resistance through blocking Hsp60 translocation followed by SAPK/JNK inhibition in aged human diploid fibroblasts

The stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway is a well-known senescence-related stress activated protein kinase. Multiple environmental stresses induce programmed cell death, such as apoptosis. Normal human diploid fibroblast (HDF) cells have a limited life span in vitro, halting proliferation after a fixed number of cell divisions. Aged passage HDF showed resistance to oxidative stress involving heat shock proteins (Hsp60) through a mechanism involving the translocation of Hsp60 from the mitochondria to the cytosol. The present study showed that the translocation of Hsp60 from the mitochondria to the cytosol followed by high levels of p-SAPK/JNK activation as a result of oxidative stress was observed in the young cells only. The inhibition of SAPK/JNK activation by SP600125 under oxidative stress almost completely blocked the translocation of Hsp60 in both young and aged cells. This suggests that aged HDF cells are resistant to oxidative stress by blocking the translocation of Hsp60 from the mitochondria to the cytosol followed by SAPK/JNK inhibition. Overall, the mechanism of resistance by oxidative stress in aged cells is induced by blocked of the translocation of Hsp60 followed by SAPK/JNK inactivation.

Rho-kinase inhibitors decrease TGF-β-stimulated VEGF synthesis through stress-activated protein kinase/c- Jun N-terminal kinase in osteoblasts

We have previously reported that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In order to investigate whether Rho-kinase is involved in the TGF-β-stimulated VEGF synthesis in these cells we examined the effects of Rho-kinase inhibitors on the VEGF synthesis. TGF-β time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1) which is a well known substrate of Rho-kinase. Y27632 and fasudil, Rho-kinase inhibitors, significantly reduced the TGF-β-stimulated VEGF synthesis as well as the MYPT-1 phosphorylation. Y27632 and fasudil failed to affect the TGF-β-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or Smad2. On the contrary, Y27632 as well as fasudil markedly suppressed the TGF-β-induced phosphorylation of SAPK/JNK. Taken together, our results strongly suggest that Rho-kinase regulates TGF-β-stimulated VEGF synthesis via SAPK/JNK activation in osteoblasts.

D-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance

In this paper, the role of D-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous D-Asp were detected, along with aspartate racemases that convert D-Asp to L-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered D-Asp. D-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since D-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that D-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.

The HMG-CoA inhibitor, simvastatin, triggers in vitro anti-tumour effect and decreases IgM secretion in Waldenstrom macroglobulinaemia.

Waldenstrom macroglobulinaemia (WM) is an incurable lymphoplasmacytic lymphoma with secretion of serum monoclonal immunoglobulin M (IgM). We previously showed that patients receiving cholesterol-lowering statins, had the lowest IgM value in a large cohort of patients with WM. Simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19(+) WM cells. Interestingly, those effects were reversed by addition of mevalonate and geranylgeranylpyrophosphate, demonstrating that simvastatin inhibited cell growth, survival and IgM secretion on BCWM.1 WM cells by inhibition of geranylgeranylated proteins. Furthermore, simvastatin overcame tumour cell growth induced by co-culture of WM cells with bone-marrow stromal cells. Simvastatin also decreased IgM secretion by BCWM.1 cells at an early time-point that had not affected cell survival. Simvastatin-induced cytotoxicity was preceded by a decrease in Akt (protein kinase B, PKB) and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways at 18 h. In addition, simvastatin induced an increase in stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) MAPK followed by caspase-8, -9, -3 and poly(ADP-ribose) polymerase (PARP) cleavages at 18 h, leading to apoptosis. Furthermore, simvastatin enhanced the cytotoxicity induced by bortezomib, fludarabine and dexamethasone. Our studies therefore support our earlier observation of statin-mediated anti-WM activity and provide the framework for future clinical trials testing simvastatin in WM.

Potentiation by platelet-derived growth factor-BB of FGF-2-stimulated VEGF release in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of platelet-derived growth factor-BB (PDGF-BB) on FGF-2-induced VEGF release in MC3T3-E1 cells. PDGF-BB significantly enhanced the FGF-2-stimulated VEGF release. The amplifying effect of PDGF-BB was dose dependent in the range between 0.1 and 30 ng/ml. AG1295, a selective inhibitor of PDGF receptor kinase, which reduced the autophosphorylation of PDGF receptor-(R), suppressed the enhancement by PDGF-BB without affecting the FGF-2 effect. PDGF-BB failed to strengthen the FGF-2-induced phosphorylation of p44/p42 MAP kinase or SAPK/JNK. The amplification by PDGF-BB of FGF-2-stimulated VEGF release was reduced by PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK. These results strongly suggest that PDGF-BB potentiates FGF-2-stimulated VEGF release at a point downstream from p44/p42 MAP kinase and SAPK/JNK in osteoblasts.

Function of Rho-kinase in prostaglandin D 2-induced interleukin-6 synthesis in osteoblasts

We have previously reported that prostaglandin D 2 (PGD 2) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGD 2-stimulated IL-6 synthesis in MC3T3-E1 cells. PGD 2 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGD 2-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGD 2-stimulated IL-6 synthesis. The PGD 2-stimulated IL-6 synthesis was reduced by PD98059, a MEK inhibitor, and SB203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, but not SP600125, an inhibitor of stress-activated protein kinase/c – Jun N-terminal kinase (SAPK/JNK). However, Y27632 and fasudil failed to affect the PGD 2-induced phosphorylation of p44/p42 MAP kinase. On the other hand, Y27632 as well as fasudil markedly attenuated the PGD 2-induced phosphorylation of p38 MAP kinase. In addition, PGD 2 additively induced IL-6 synthesis in combination with endothelin-1 which induces IL-6 synthesis through p38 MAP kinase regulated by Rho-kinase. These results strongly suggest that Rho-kinase regulates PGD 2-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.

Involvement of Rho-kinase in prostaglandin F2α-stimulated interleukin-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGF(2alpha)-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF(2alpha) time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGF(2alpha)-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGF(2alpha)-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect the PGF(2alpha)-induced phosphorylation of p44/p42 MAP kinase. SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by PGF(2alpha). While SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), failed to reduce the synthesis. Y27632 as well as fasudil attenuated the PGF(2alpha)-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates PGF(2alpha)-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.

The role of the novel Th17 cytokine IL-26 in intestinal inflammation

Background and aims:Interleukin 26 (IL-26), a novel IL-10-like cytokine without a murine homologue, is expressed in T helper 1 (Th1) and Th17 cells. Currently, its function in human disease is...