Enhancement of fibroblast collagenase-1 (MMP-1) gene expression by tumor promoter okadaic acid is mediated by stress-activated protein kinases jun N-terminal kinase and p38

Collagenase-1 (matrix metalloproteinase-1, MMP-1) is expressed by several types of cells, including fibroblasts, and apparently plays an important role in the remodeling of collagenous extracellular matrix in various physiologic and pathologic situations. Here, we have examined the molecular mechanisms of the activation of fibroblast MMP-1 gene expression by a naturally occurring non-phorbol ester type tumor promoter okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatase 2A. We show that in fibroblasts OA activates three distinct subgroups of mitogen activated protein kinases (MAPKs): extracellular signal-regulated kinase 1,2 (ERK 1,2), c-Jun N-terminal-kinase/stress-activated protein kinase (JNK/SAPK) and p38. Activation of MMP-1 promoter by OA is entirely blocked by overexpression of dual-specificity MAPK phosphatase CL100. In addition, expression of kinase-deficient forms of ERK 1,2, SAPKβ, p38, or JNK/SAPK kinase SEK1 strongly inhibited OA-elicited activation of MMP-1 promoter. OA-elicited enhancement of MMP-1 mRNA abundance was also strongly prevented by two chemical MAPK inhibitors: PD 98059, a specific inhibitor of the activation of ERK1,2 kinases MEK1,2; and SB 203580, a selective inhibitor of p38 activity. Results of this study show that MMP-1 gene expression in fibroblasts is coordinately regulated by ERK1,2, JNK/SAPK, and p38 MAPKs and suggest an important role for the stress-activated MAPKs JNK/SAPK and p38 in the activation of MMP-1 gene expression. Based on these observations, it is conceivable that specific inhibition of stress-activated MAPK pathways may serve as a novel therapeutic target for inhibiting degradation of collagenous extracellular matrix.     

p38 MAPK信号传导通路

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Bradykinin B<Subscript>2</Subscript> receptor-mediated proliferation via activation of the Ras/Raf/MEK/MAPK pathway in rat vascular smooth muscle cells

It has been suggested that bradykinin (BK) plays an important role in regulating neointimal formation after vascular injury. However, implication of BK in the growth of rat vascular smooth muscle cells (VSMCs) is controversial. Therefore, we examined the mitogenic effect of BK on VSMCs associated with activation of mitogen-activated protein kinase (MAPK). Both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were activated by BK in time- and concentration-dependent manners. Pretreatment of these cells with neither pertussis toxin nor cholera toxin attenuated the BK-induced responses. Pretreatment of VSMCs with Hoe 140 (a selective B(2) receptor antagonist), U73122 (an inhibitor of phospholipase C), and BAPTA/AM (an intracellular Ca(2+) chelator) inhibited both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to BK. BK-induced [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were inhibited by pretreatment of VSMCs with tyrosine kinase inhibitors (genistein and herbimycin A), protein kinase C (PKC) inhibitors (staurosporine, Go-6976, and Ro-318220), an MAPK kinase inhibitor (PD98059), and a p38 MAPK inhibitor (SB203580). Overexpression of the dominant negative mutants, H-Ras-15A and Raf-N4, suppressed p42/p44 MAPK activation induced by BK and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. From these results, we concluded that the mitogenic effect of BK is mediated through activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF-BB. BK-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase all of which are associated with cell proliferation in rat cultured VSMCs.     

Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells

Interleukin 1beta (IL-1beta) induces expression of the inducible nitric-oxide synthase (iNOS) with concomitant release of nitric oxide (NO) from glomerular mesangial cells. These events are preceded by activation of the c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38(MAPK). Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKbeta/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1beta. Similarly, overexpression of the kinase-dead mutant form of p38alpha(MAPK) also inhibits IL-1beta-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1beta can activate MKK4/SEK1, MKK3, and MKK6 in renal mesangial cells; therefore, we examined the role of these MAPK kinases in the modulation of iNOS induced by IL-1beta. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38(MAPK) phosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1beta-induced p38(MAPK) (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38(MAPK); however, in the absence of IL-1beta, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38alpha(MAPK) signaling cascades are necessary for the IL-1beta-induced expression of iNOS and production of NO in renal mesangial cells.     

Phosphorylation of the mitochondrial protein Sab by stress-activated protein kinase 3

Mitogen-activated protein kinases (MAPKs) transduce extracellular signals into responses such as growth, differentiation, and death through their phosphorylation of specific substrate proteins. Early studies showed the consensus sequence (Pro/X)-X-(Ser/Thr)-Pro to be phosphorylated by MAPKs. Docking domains such as the "kinase interaction motif" (KIM) also appear to be crucial for efficient substrate phosphorylation. Here, we show that stress-activated protein kinase-3 (SAPK3), a p38 MAPK subfamily member, localizes to the mitochondria. Activated SAPK3 phosphorylates the mitochondrial protein Sab, an in vitro substrate of c-Jun N-terminal kinase (JNK). Sab phosphorylation by SAPK3 was dependent on the most N-terminal KIM (KIM1) of Sab and occurred primarily on Ser321. This appeared to be dependent on the position of Ser321 within Sab and the sequence immediately surrounding it. Our results suggest that SAPK3 and JNK may share a common target at the mitochondria and provide new insights into the substrate recognition by SAPK3.     

In the cellular garden of forking paths: how p38 MAPKs signal for downstream assistance

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes which connect cell-surface receptors to regulatory targets within cells and convert receptor signals into various outputs. In mammalian cells, four distinct MAPKs have been identified: the extracellular signal-related kinases (ERK)-1/2, the c-jun N-terminal kinases or stress-activated protein kinases 1 (JNK1/2/3, or SAPK1s), the p38 MAPKs (p38 alpha/beta/gamma/delta, or SAPK2s), and the ERK5 or big MAP kinase 1 (BMK1). The p38 MAPK cascade is activated by stress or cytokines and leads to phosphorylation of its central elements, the p38 MAPKs. Downstream of p38 MAPKs there is a diversification and extensive branching of signalling pathways. For that reason, we will focus in this review on the different signalling events that are triggered by p38 activity, and analyse how these events contribute to specific gene expression and cellular responses.     

Oncostatin M-induced activation of stress-activated MAP kinases depends on tyrosine 861 in the OSM receptor and requires Jak1 but not Src kinases

We have investigated the molecular mechanisms involved in the activation process of the stress-activated protein kinases (SAPK) p38 and JNK in response to the interleukin-6-type cytokine oncostatin M (OSM). Interestingly, activation of p38 and JNK originates from tyrosine residue 861 in the OSMR; the same tyrosine residue which we identified before to be involved in the activation of the mitogen-activated kinases Erk1/2 [Hermanns, H. M., Radtke, S., Schaper, F., Heinrich, P. C., and Behrmann, I. (2000) J. Biol. Chem. 275, 40742-40748]. Therefore, activation of members belonging to all three MAPK families is mediated by one tyrosine motif in the cytoplasmic region of the human OSMR. Concomitantly, point mutation of this residue abrogates the phosphorylation of these kinases. The Janus kinase Jak1 is absolutely essential for the activation of p38 in response to OSM, while Src kinase family members appear to be generally dispensable. Finally, we demonstrate that mutation of tyrosine 861 abrogates OSMR-mediated cell proliferation and identify Erk1/2 as mainly responsible for the proliferative effect. Erk1/2 activation is negatively influenced by p38 activation and inhibition of p38 significantly prolongs the half-life of OSM-induced Egr-1.     

Impaired synergistic activation of stress-activated protein kinase SAPK/JNK in mouse embryonic stem cells lacking SEK1/MKK4: different contribution of SEK2/MKK7 isoforms to the synergistic activation.

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) family, plays an important role in a stress-induced signaling cascade. SAPK/JNK activation requires the phosphorylation of Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 (MKK4) and MKK7 (SEK2) have been identified as the upstream MAPK kinases. Here we examined the activation and phosphorylation sites of SAPK/JNK and differentiated the contribution of SEK1 and MKK7alpha1, -gamma1, and -gamma2 isoforms to the MAPK activation. In SEK1-deficient mouse embryonic stem cells, stress-induced SAPK/JNK activation was markedly impaired, and this defect was accompanied with a decreased level of the Tyr phosphorylation. Analysis in HeLa cells co-transfected with the two MAPK kinases revealed that the Thr and Tyr of SAPK/JNK were independently phosphorylated in response to heat shock by MKK7gamma1 and SEK1, respectively. However, MKK7alpha1 failed to phosphorylate the Thr of SAPK/JNK unless its Tyr residue was phosphorylated by SEK1. In contrast, MKK7gamma2 had the ability to phosphorylate both Thr and Tyr residues. In all cases, the dual phosphorylation of the Thr and Tyr residues was essentially required for the full activation of SAPK/JNK. These data provide the first evidence that synergistic activation of SAPK/JNK requires both phosphorylation at the Thr and Tyr residues in living cells and that the preference for the Thr and Tyr phosphorylation was different among the members of MAPK kinases.     

Histamine activates tyrosine hydroxylase in bovine adrenal chromaffin cells through a pathway that involves ERK1/2 but not p38 or JNK

In bovine adrenal chromaffin cells (BACC) histamine promotes a rapid increase in the intracellular levels of Ca2+ together with the release of catecholamines and the phosphorylation of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). In this study we investigated the role of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK1/2), stress activated protein kinase (p38) and Jun N-terminal kinases (JNK) on the histamine-induced activation and phosphorylation of TH. We found that in BACC histamine produced a rapid, long lasting and histamine type-1 (H1) receptor-dependent increase in the phosphorylation levels of ERK1/2, p38 and JNK which was accompanied by a H1 receptor-dependent increase in TH activity. This increase in TH activity was partially blocked by the MEK1/2 inhibitor U0126 but was unaffected by the p38 antagonist SB203580 or the JNK blocker JNKI1. To study the effect of MAPK inhibition on histamine-induced TH phosphorylation, we generated phospho-specific antibodies against the different phosphorylated forms of TH. Treatment with U0126 totally inhibited the histamine-induced phosphorylation of TH at Ser31, without affecting the phosphorylation of either Ser40 or Ser19. Neither SB203580 nor JNKI1 treatments produced any significant modification of the histamine-induced TH phosphorylation. Our data support the hypothesis that the up-regulation of the ERK1/2 pathway, but not that of p38 or JNK, promoted by histamine is involved in the phosphorylation of TH at Ser31 and that this phosphorylation event is required for the full activation of this enzyme.     

LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.     

Involvement of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase 1/2 in EGF‐induced angiogenesis

Angiogenesis is a process during which endothelial cells divide and migrate to form new capillaries from the preexisting blood vessels. The present study was designed to investigate whether MAPKs (mitogen‐activated protein kinases) play crucial roles in regulating EGF (epidermal growth factor)‐induced endothelial cell angiogenesis. Our results showed that EGF stimulated HUVEC (human umbilical vein endothelial cells) proliferation in a concentration‐dependent manner, of which the maximum effective concentration of EGF was 10 ng/ml. Western blot analysis showed that EGF at 10 ng/ml significantly induced the phosphorylation of ERK1/2 (extracellular signal‐regulated kinase 1 and 2) and p38 kinase at 5 min, while it induced the phosphorylation of JNK/SAPK (c‐Jun N‐terminal kinase/stress‐activated protein kinase) at 15 min. Further results showed that a JNK/SAPK inhibitor, SP600125, and a specific siRNA JNK/SAPK could both significantly inhibit EGF‐induced tube formation in HUVEC cells, and an ERK1/2 inhibitor PD098059 could also block the tube formation in some content, while a p38 inhibitor SB203580 failed to do so. Furthermore, only SP600125 significantly inhibited EGF‐induced HUVEC cell proliferation under no cytotoxic concentration, so did JNK/SAPK siRNA. In conclusion, JNK/SAPK and ERK1/2 signals therefore play critical roles in EGF‐mediated HUVEC cell angiogenesis.