Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation.     

P38 MAPK, but not p42/p44 MAPK mediated inducible nitric oxide synthase expression in C6 glioma cells

Recently mitogen-activated protein kinase (MAPK) has been reported to play an important role in phosphorylation cascades governing cell growth and protein expression in numerous cell types. In order to explore the signaling mechanism by which inducible nitric oxide synthase (iNOS) is regulated in C6 glioma cells, we investigated the role of MAPK in iNOS expression by using the specific MAPK inhibitors. First the induction of nitric oxide by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), alone or their combination, was studied in C6 glioma cells. Administration of LPS, TNFalpha, or IFNgamma alone had no detectable stimulatory effect on the production of nitric oxide (NO). However, combination of the three factors elicited a significant elevation of NO level in C6 cell culture medium. Subsequently pretreatment of C6 cells with a specific inhibitor of p38 MAPK, SB202190, resulted in a dose-dependent inhibition of NO production and iNOS expression, but PD98059, an inhibitor of p42/p44 MAPK activation, had no effect. These data suggest that p38 MAPK mediates iNOS expression in C6 glioma cells, but p42/p44 MAPK is not involved in this process.     

PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 muM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.     

Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB in IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.     

Mitogen-activated protein kinase phosphatase-1 (MKP-1) preferentially dephosphorylates p42/44MAPK but not p38MAPK in rat pinealocytes

We recently reported a diurnal and norepinephrine (NE) -induced expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the rat pineal gland and postulated that this MKP-1 expression might impact adrenergic-regulated arylalkylamine- N -acetyltransferase (AA-NAT) activity via modulation of MAPKs. In this study, we investigated the effect of depletion of MKP-1 expression by using doxorubicin, a topoisomerase inhibitor that suppresses the expression of MKP-1 in other cell types and small interfering RNA targeted against Mkp1 in NE-stimulated pinealocytes. We found that both treatments were effective in inhibiting NE induction of MKP-1 expression. Moreover, both treatments also resulted in a prolonged activation of p42/44MAPK and an increase in AA-NAT induction by NE. In contrast, treatment of pinealocytes with PD98059, an inhibitor of MAPK kinase, reduced NE-stimulated AA-NAT activity. Interestingly, suppressing MKP-1 expression had no effect on the time profile of NE-stimulated p38MAPK activation. These results indicate that MKP-1 modulates the profile of AA-NAT activity by selectively shaping the activation profile of p42/44MAPK but not that of p38MAPK.     

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.     

Signaling through the p38 and p42/44 mitogen-activated families of protein kinases in pancreatic beta-cell proliferation

The present study has focused on the role of the 42- and 44-kDa mitogen-activated protein kinases (p42/44 MAPKs) and the 38-kDa mitogen-activated protein kinase (p38 MAPK) in the proliferation of the pancreatic beta-cell line MIN6. MIN6 beta-cell proliferation was assessed by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA. Inhibition of both the p42/44 MAPK pathway using the MEK inhibitor PD098059 (PD) and the p38 MAPK pathway using the p38 inhibitor SB203580 (SB) caused a marked, concentration-dependent reduction in the BrdU immunostaining observed in the presence of 15% FCS when assessed using fluorescence immunocytochemistry. These data provide direct evidence of a role for p42/44 MAPKs in the mitogenic response of MIN6 beta-cells to FCS. Furthermore, these data also suggest a novel role for the p38 MAPK pathway in MIN6 beta-cell proliferation.     

p38 and p42/44 MAPKs differentially regulate progesterone receptor A and B isoform stabilization

Progesterone receptor (PR) isoforms (PRA and PRB) are implicated in the progression of breast cancers frequently associated with imbalanced PRA/PRB expression ratio. Antiprogestins represent potential antitumorigenic agents for such hormone-dependent cancers. To investigate the mechanism(s) controlling PR isoforms degradation/stability in the context of agonist and antagonist ligands, we used endometrial and mammary cancer cells stably expressing PRA and/or PRB. We found that the antiprogestin RU486 inhibited the agonist-induced turnover of PR isoforms through active mechanism(s) involving distinct MAPK-dependent phosphorylations. p42/44 MAPK activity inhibited proteasome-mediated degradation of RU486-bound PRB but not PRA in both cell lines. Ligand-induced PRB turnover required neosynthesis of a mandatory down-regulating partner whose interaction/function is negatively controlled by p42/44 MAPK. Such regulation strongly influenced expression of various endogenous PRB target genes in a selective manner, supporting functional relevance of the mechanism. Interestingly, in contrast to PRB, PRA stability was specifically increased by MAPK kinase kinase 1-induced p38 MAPK activation. Selective inhibition of p42/p44 or p38 activity resulted in opposite variations of the PRA/PRB expression ratio. Moreover, MAPK-dependent PR isoforms stability was independent of PR serine-294 phosphorylation previously proposed as a major sensor of PR down-regulation. In sum, we demonstrate that MAPK-mediated cell signaling differentially controls PRA/PRB expression ratio at posttranslational level through ligand-sensitive processes. Imbalance in PRA/PRB ratio frequently associated with carcinogenesis might be a direct consequence of disorders in MAPK signaling that might switch cellular responses to hormonal stimuli and contribute towards pathogenesis.     

Quantitative role of p42/44 and p38 in the production and regulation of cytokines TNF-alpha, IL-1beta and IL-12 by murine peritoneal macrophages in vitro by concanavalin A

In the present study the quantitative role of p42/44 and p38 in the production of TNF-alpha, IL-1beta and IL-12 by murine peritoneal macrophages, in vitro, on treatment with Concanavalin A (ConA) has been investigated. Maximum expression/production of cytokines TNF-alpha, IL-1beta and IL-12 was observed after 16 h by RT-PCR and 24 h by ELISA, on in vitro treatment with ConA. To investigate the role of MAP kinases in the production of cytokines, pharmacological inhibitors of MAP kinases--PD98059, SB202190 and SP600125, were used. The expression of TNF-alpha, IL-1beta and IL-12 was down regulated in the presence of PD98059 and SB202190 in a dose dependent manner, suggesting the involvement of p42/44 and p38 in ConA induced production of TNF-alpha, IL-1beta and IL-12 by macrophages. It was observed that SP600125 did not have any effect on the expression of TNF-alpha, IL-1beta and IL-12. Using different combinations of MAPK inhibitors, it was found that 45% signal is conveyed via p42/44 and 25% via p38 in the production of these cytokines, by ConA treated macrophages while 30% signal passes through unidentified pathways.     

Cyclic AMP inhibits agonist-induced heparin-binding EGF gene expression independently of effects on p42/p44 MAPK activation

Heparin-binding EGF-like growth factor (HB-EGF) mRNA levels are increased up to 20-fold in RIE-1 cells by two agonists that act through distinct receptor types. We demonstrated a common requirement for p42/p44 mitogen-activated protein kinase (MAPK) in this response using the selective MAPK kinase (MEK) inhibitor, PD 098059. Agonist-mediated induction of HB-EGF mRNA was markedly suppressed in cells that had been treated with cyclic AMP-elevating agents. In contrast, the activation of p42 MAPK in response to agonists was not affected by raising cellular cyclic AMP levels. We conclude that cyclic AMP negatively regulates the HB-EGF gene, but that the inhibitory action is either independent of the p42/p44 MAPK pathway or the site of action is distal to MAPK activation.