Endometrial stromal cells undergoing decidualization down-regulate their properties to produce proinflammatory cytokines in response to interleukin-1 beta via reduced p38 mitogen-activated protein kinase phosphorylation

The decidualized endometrium plays a role in regulating trophoblast invasion for successful implantation and maintenance of pregnancy. IL-1 beta, a proinflammatory cytokine, has been suggested to play a role in this process. Recently, several lines of evidence indicate the importance of p38 MAPK in various inflammatory responses. We investigated whether endometrial stromal cells (ESC) change their inflammatory responses to IL-1 beta as related to p38 MAPK phosphorylation during the process of decidualization. ESC were decidualized by the treatment with progesterone for 9 d, as determined as such by an increase in the production of prolactin and cAMP by the cells. Whereas IL-1 beta increased the production of IL-6, IL-8, and monocyte chemotactic protein-1, and expression of cyclooxygenase-2 mRNA in ESC cultured without treatment, the stimulatory effects of IL-1 beta were reduced in the decidualized cells. Treatment with SB202190, a p38 MAPK inhibitor, also reduced the stimulatory effects of IL-1 beta in nondecidualized ESC. P38 MAPK phosphorylation was increased by IL-1 beta in nondecidualized ESC, whereas the IL-1 beta-induced increase was suppressed in the decidualized cells. Treatment with 8-bromo-cAMP reduced IL-1 beta-induced phosphorylation of p38 MAPK in nondecidualized ESC. In contrast, treatment with H89, a protein kinase A inhibitor, reversed a reduction in IL-1 beta-induced p38 MAPK phosphorylation in the decidualized cells. In summary, decidualization seems to be a process during which endometrial cells diminish their response to IL-1 beta, a known key factor for implantation, leading to the down-regulation of inflammation-like events, which may be relevant to controlled trophoblast invasion. The altered property of decidualized cells is thought to be caused by attenuation of IL-1 beta-induced p38 MAPK phosphorylation in a way that involves the activation of the cAMP/protein kinase A pathway.     

Activation of synovial cell p38 MAP kinase by macrophage migration inhibitory factor

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine important in animal models of rheumatoid arthritis (RA). We investigated the utilization by MIF of mitogen activated protein (MAP) kinase signalling pathways in the stimulation of fibroblast-like synoviocytes (FLS), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin 6 (IL-6) and IL-8 expression. METHODS: Cultured human RA FLS were treated with recombinant MIF. Activation of MAPK was measured by Western blotting and blocked using specific inhibitors. The expression of COX-2, PGE(2), IL-6, and IL-8 were measured using flow cytometry, ELISA, and real-time polymerase chain reaction. RESULTS: MIF induced the phosphorylation of FLS p38 and extracellular-signal regulated kinase (ERK) MAP kinase. MIF significantly induced COX-2 and IL-6 protein and mRNA expression as well as PGE(2) and IL-8 production. Antagonism of p38 MAP kinase inhibited MIF induction of COX-2, PGE(2), and IL-6. In contrast, antagonism of ERK had no effect on COX-2, PGE(2), or IL-6. Neither antagonist inhibited MIF-induced IL-8. CONCLUSION: MIF activates RA FLS COX-2 and IL-6 expression via p38 MAP kinase activation and induces IL-8 via p38 and ERK MAP kinase-independent pathways.     

Expression of mitogen-activated protein kinase phosphatase 1, a negative regulator of the mitogen-activated protein kinases, in rheumatoid arthritis: up-regulation by interleukin-1beta and glucocorticoids

OBJECTIVE: Mitogen-activated protein kinases (MAPKs) are activated by proinflammatory stimuli. MAPK phosphatases (MKPs), in particular MKP-1, have been identified as endogenous negative regulators of MAPK activation. Since MAPKs are known to be important in rheumatoid arthritis (RA) synoviocyte activation, this study assessed the expression, regulation, and function of MKP-1 in RA. METHODS: MKP-1 expression was measured by Western blotting (WB) and real-time polymerase chain reaction (PCR). RA fibroblast-like synoviocytes (FLS) were treated with interleukin-1beta (IL-1beta), tumor necrosis factor alpha, fetal calf serum, and dexamethasone. Expression of MAPKs in RA FLS was analyzed by WB using phosphospecific antibodies, while IL-6 expression was assessed by real-time PCR. RESULTS: MKP-1 protein and messenger RNA were detected in cultured RA FLS. IL-1beta rapidly up-regulated MKP-1, coinciding with reciprocal down-regulation of ERK, JNK, and p38 MAPK phosphorylation. Dexamethasone rapidly and sustainably up-regulated MKP-1, and this also coincided with down-regulation of ERK, JNK, and p38 MAPK phosphorylation. In addition, dexamethasone augmented IL-1beta-induced up-regulation of MKP-1, and this was associated with inhibition of ERK, JNK, and p38 MAPK phosphorylation and IL-6 expression. Dexamethasone had no effect on the phosphorylation of upstream kinases such as MEKK-3/6. In the presence of glucocorticoid (GC) receptor antagonist RU 486, the dexamethasone-mediated up-regulation of MKP-1 was impaired. Moreover, inhibition of MKP-1 expression impaired dexamethasone-mediated inhibition of MAPK phosphorylation. CONCLUSION: This study demonstrates the expression of MKP-1 in RA FLS. Cytokine and GC regulation of MKP-1 may be important in determining the magnitude of the inflammatory response in RA that is mediated via MAPKs. The effects of GCs in RA may be mediated, in part, via GC receptor-dependent up-regulation of MKP-1.     

Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis

OBJECTIVE: To investigate whether stress- and mitogen-activated protein kinases (SAPK/MAPK), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, are significantly activated in rheumatoid arthritis (RA) synovial tissue compared with their activation in degenerative joint disease; to assess the localization of SAPK/MAPK activation in rheumatoid synovial tissue; and to search for the factors leading to stress kinase activation in human synovial cells. METHODS: Immunoblotting and immunohistology by antibodies specific for the activated forms of SAPK/MAPK were performed on synovial tissue samples from patients with RA and osteoarthritis (OA). In addition, untreated and cytokine-treated human synovial cells were assessed for SAPK/MAPK activation and downstream signaling by various techniques. RESULTS: ERK, JNK, and p38 MAPK activation were almost exclusively found in synovial tissue from RA, but not OA, patients. ERK activation was localized around synovial microvessels, JNK activation was localized around and within mononuclear cell infiltrates, and p38 MAPK activation was observed in the synovial lining layer and in synovial endothelial cells. Tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6 were the major inducers of ERK, JNK, and p38 MAPK activation in cultured human synovial cells. CONCLUSION: Signaling through SAPK/MAPK pathways is a typical feature of chronic synovitis in RA, but not in degenerative joint disease. SAPK/MAPK signaling is found at distinct sites in the synovial tissue, is induced by proinflammatory cytokines, and could lead to the design of highly targeted therapies.     

The mitogen-activated protein kinase dependent expression of prostaglandin H synthase-2 and interleukin-8 messenger ribonucleic acid by myometrial cells: the differential effect of stretch and interleukin-1{beta}

Infection and uterine stretch are the common causes of preterm labor. IL-1beta plays a key role in infection-induced preterm labor and increases prostaglandin H synthase 2 (PGHS-2) and IL-8 expression. We have shown that mechanical stretch of uterine myocytes in vitro up-regulates the expression of PGHS-2 and IL-8. In this study, we tested the hypotheses that both IL-1beta and mechanical stretch increase the myometrial expression of PGHS-2 and IL-8 via MAPK activation and that their effects are synergistic. MAPK activation was assessed in myocytes obtained from pregnant women undergoing cesarean section before the onset of labor after exposure to IL-1beta and stretch either alone or in combination. Specific inhibitors of ERK, p38, and c-Jun N-terminal kinase were used to define the role of each in the increased expression of PGHS-2 and IL-8 mRNA. We found that both IL-1beta and stretch activated all three MAPK subtypes but that they had no synergistic effect. The inhibitor studies showed that stretch-induced increases in both PGHS-2 and IL-8 mRNA expression were ERK1/2 and p38 dependent and that IL-1beta-induced increases of PGHS-2 mRNA expression were also ERK1/2 and p38 dependent, but those of IL-8 were dependent only on ERK1/2 activation. These data show that exposure of human uterine myocytes to both stretch and IL-1beta activates the MAPK system, which is responsible for the increase in PGHS-2 and IL-8 mRNA expression. We found no evidence of a synergistic effect of IL-1beta and stretch on myometrial expression of PGHS-2 and IL-8 mRNA.     

Effects of RWJ 67657, a p38 mitogen activated protein kinase (MAPK) inhibitor, on the production of inflammatory mediators by rheumatoid synovial fibroblasts

OBJECTIVE: To investigate the effect of the p38 mitogen activated protein kinase (MAPK) inhibitor RWJ 67657 on inflammatory mediator production by rheumatoid synovial fibroblasts (RSF). METHODS: RSF were pretreated with RWJ 67657 and stimulated with TNF alpha and/or IL-1 beta. Protein levels and mRNA expression of MMP-1, MMP-3, TIMP-1, IL-6, and IL-8 were determined, as was mRNA expression of COX-2 and ADAMTS-4. RESULTS: MMP-3 production was significantly inhibited at 1 microM RWJ 67657 and MMP-1 production at 10 microM, while TIMP-1 production was not inhibited. Inhibition of IL-6 and IL-8 protein production was seen at 0.1 microM RWJ 67657. Expression profiles of mRNA were in accordance with protein production. Inhibition of COX-2 mRNA expression occurred at 0.01 microM RWJ 67657. CONCLUSIONS: RWJ 67657 inhibits major proinflammatory mediator production in stimulated RSF at pharmacologically relevant concentrations. These findings could have important relevance for the treatment of rheumatoid arthritis.     

Interleukin-1β induces cyclo-oxygenase-2 expression in gastric cancer cells by the p38 and p44/42 mitogen-activated protein kinase signaling pathways

BACKGROUND AND AIMS: Cyclo-oxygenase-2 (COX-2) is the inducible enzyme in the gastric mucosa responsible for prostaglandin production during inflammation and ulcer healing. The regulation of COX-2 gene expression in gastric epithelial cells is not well understood. In this study, we investigated the effect of interleukin (IL)-1beta on COX-2 expression in the human gastric cancer cell, and explored the signaling pathways involved. METHODS: Gastric cancer cell line AGS was treated with IL-1beta or the inhibitors of mitogen-activated protein-Erk kinase (MEK) and p38 mitogen-activated protein (MAP) kinase prior to the addition of IL-1beta. The COX-2 mRNA or protein levels were measured by using RT-PCR or western blot analysis, respectively. Prostaglandin E2 (PGE2) production/secretion was determined by using the prostaglandin E2 EIA assay. The phosphorylation/activation of p44/42 and p38 MAP kinases were determined by using western blot analysis and using phospho-specific antibodies. RESULTS: Interleukin-1beta treatment dose- and time-dependently increased COX-2 mRNA and protein expression levels, and enhanced PGE2 production/secretion in AGS cells. In contrast, IL-1beta had no effect on the level of the constitutively expressed COX-1. In parallel to the increase of COX-2, we showed that p44/42 and p38 MAP kinase activities were also upregulated by IL-1beta treatment. To demonstrate the cause-effect relationship, we showed that inhibition of MEK and p38 MAP kinase with specific inhibitors suppressed IL-1beta-mediated increases in COX-2 mRNA and protein levels, and the PGE2 production. CONCLUSIONS: Our results demonstrated that in human gastric cancer cells, IL-1beta upregulates the COX-2 gene expression through the activation of MEK/p44/42 and p38 MAP kinases pathway.     

p38 Inhibition attenuates the pro-inflammatory response to C-reactive protein by human peripheral blood mononuclear cells

An active role for C-reactive protein (CRP) in inflammatory vascular diseases has been recently suggested. Monocytes play an important role in vascular pathology and are activated by p38 mitogen activated protein kinase (MAPK) dependent mechanisms in many inflammatory settings. Therefore, we investigated whether CRP directly promotes a pro-inflammatory phenotype in human peripheral blood mononuclear cells (HPBMC) via p38 MAPK signaling. CRP exposure leads to a rapid phosphorylation of p38 MAPK in HPBMC. CRP-induced p38 kinase activity in HPBMC was blocked by treatment with an inhibitor of p38 kinase, SD-282. CRP-induced the expression of tissue factor protein and the secretion of IL-6, IL-8, IL-1beta, TNFalpha and PGE(2). Co-exposure to CRP and SD-282 blocked the secretion of these pro-inflammatory and pro-thrombotic mediators. CRP treatment elevated IL-6, IL-8, IL-1beta, TNFalpha, COX-2 and TF mRNA expression. These effects of CRP also required p38 activity, since SD-282 blocked mRNA induction of each. Taken together these data suggest a mechanistic relationship between p38 MAPK signaling and CRP-induced pro-inflammatory and pro-thrombotic activities in HPBMC. Thus, p38 inhibition may represent a novel approach to attenuate inflammation and its consequences in cardiovascular disease.     

Involvement of p38 MAPK,JNK, p42/p44 ERK and NF-kappaB in IL-1beta-induced chemokine release in human airway smooth muscle cells

Asthma is an inflammatory disease, in which eotaxin, MCP-1 and MCP-3 play a crucial role. These chemokines have been shown to be expressed and produced by IL-1beta-stimulated human airway smooth muscle cells (HASMC) in culture. In the present study we were interested to unravel the IL-1beta-induced signal transduction leading to chemokine production. Using Western blot, we observed an activation of p38 MAPK, JNK kinase and p42/p44 ERK when HASMC were stimulated with IL-1beta. We also observed a significant decrease in the expression and the release of eotaxin, MCP-1 and MCP-3 in the presence of SB203580, an inhibitor of p38 MAPK (71 +/- 6%, P < 0.05, n = 8 and 39 +/- 10% P < 0.01, n = 10 respectively), curcumin, an inhibitor of JNK kinase (83 +/- 4.9% and 88 +/- 3.4% respectively, P < 0.01, n = 4). U0126, an inhibitor of p42/p44 ERK, also produced a significant decrease in chemokine production (46.3 +/- 9%, P < 0.01 n = 10 and 67.8 +/- 12%, P < 0.01, n = 12). Pyrrolydine dithiocarbamate, an inhibitor of NF-kappaB was also able to reduce the eotaxin, MCP-1 and MCP-3 expression and production (50 +/- 13%, P < 0.05, n = 10 and 23 +/- 7%, P < 0.05, n = 12). We conclude that p38 MAPK, JNK kinase, ERK and NF-kappaB are involved in the IL-1beta-induced eotaxin, MCP-1, and MCP-3 expression and release in HASMC.