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.     

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.     

Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5.

A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.     

AMP-activated protein kinase positively regulates FGF-2-stimulated VEGF synthesis in osteoblasts

AMP-activated protein kinase (AMPK) is recognized as a regulator of energy homeostasis. We have previously reported that basic fibroblast growth factor (FGF-2) stimulates vascular endothelial growth factor (VEGF) release through 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 involvement of AMPK in FGF-2-stimulated VEGF release in these cells. FGF-2 time-dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an AMPK inhibitor, which suppressed the FGF-2-induced phosphorylation of AMPK, significantly inhibited the VEGF release stimulated by FGF-2. The AMPK inhibitor also reduced the mRNA expression of VEGF induced by FGF-2. The FGF-2-induced phosphorylation of both p44/p42 MAP kinase and SAPK/JNK was attenuated by compound C. These results strongly suggest that AMPK positively regulates the FGF-2-stimulated VEGF synthesis via p44/p42 MAP kinase and SAPK/JNK in osteoblasts.     

SAPK/JNK plays a role in transforming growth factor-beta-induced VEGF synthesis in osteoblasts.

We previously reported that transforming growth factor-beta (TGF-beta) activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase, resulting in the stimulation of vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK), another member of the MAP kinase superfamily, in TGF-beta-induced VEGF synthesis in these cells. TGF-beta markedly induced SAPK/JNK phosphorylation. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced TGF-beta-induced VEGF synthesis. SP600125 suppressed TGF-beta-induced SAPK/JNK phosphorylation. PD98059, an inhibitor of upstream kinase of p44/p42 MAP kinase and SB203580, an inhibitor of p38 MAP kinase, each failed to reduce TGF-beta-induced SAPK/JNK phosphorylation. A combination of SP600125 and PD98059 or SP600125 and SB203580 suppressed TGF-beta-stimulated VEGF synthesis in an additive manner. These results strongly suggest that TGF-beta activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a role in addition to p42/p44 MAP kinase and p38 MAP kinase in TGF-beta-induced VEGF synthesis.     

c-Jun N-Terminal Kinase Mediated VEGFR2 Sustained Phosphorylation is Critical for VEGFA-Induced Angiogenesis In Vitro and In Vivo

c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is involved in the regulation of various cellular functions including cell cycle, proliferation, apoptosis. However, whether JNK/SAPK directly regulates the angiogenesis of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor A (VEGFA) has not yet been fully elucidated. Our present study firstly demonstrated VEGFA-induced angiogenic responses including the increase of cell viability, migration, and tube formation with a concentration-dependent manner in HUVECs. Further results showed that VEGFA induced the activation of JNK/SAPK, p38 kinase and extracellular signal-regulated kinases 1 and 2 (ERK1/2), while JNK/SAPK inhibitor SP600125 and specific siRNA both blocked all those angiogenic effects induced by VEGFA. Furthermore, VEGFA induced the phosphorylation of ASK1, SEK1/MKK4, MKK7, and c-Jun, which are upstream or downstream signals of JNK/SAPK. In addition, in vivo matrigel plug assay further showed that SP600125 inhibited VEGFA-induced angiogenesis. Further results showed that SP600125 and JNK/SAPK siRNA decreased VEGFA-induced VEGFR2 (Flk-1/KDR) sustained phosphorylation in HUVECs. Taken together, all these results demonstrate that JNK/SAPK regulates VEGFA-induced VEGFR2 sustained phosphorylation, which plays important roles in VEGFA-induced angiogenesis in HUVECs.     

Purinergic inhibition of Na+,K+,Cl− cotransport in C11-MDCK cells: Role of stress-activated protein kinases

Previously, we observed that sustained activation of P2Y₁ leads to inhibition of Na⁺,K⁺,Cl⁻ cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y₁ agonists.     

Nocodazole-induced p53-dependent c-Jun N-terminal kinase activation reduces apoptosis in human colon carcinoma HCT116 cells

Microtubule-interfering agents are widely used in cancer chemotherapy, and prognostic results vary significantly from tumor to tumor, depending on the p53 status. In preliminary experiments, we compared the expression and phosphorylation profiles of more than 100 protein kinases and protein phosphatases in human colorectal carcinoma cell line HCT116 between p53+/+ and p53-/- cells in response to short term nocodazole treatment through application of Kinetworks immunoblotting screens. Among the proteins tracked, the regulation of the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 at Thr-183/Tyr-185 was the major difference between p53+/+ and p53-/- cells. With the loss of the p53 gene, the levels of phosphorylation of Ser-63 of c-Jun and Thr-183/Tyr-185 of JNK1/2 in p53-/- cells did not increase as markedly as in p53+/+ cells in response to a 1-h treatment with nocodazole or other microtubule-disrupting drugs such as vinblastine and colchicine. Similar observations were also made in MCF-7 and A549 tumor cells, which were rendered p53-deficient by E6 oncoprotein expression. However, arsenate-induced JNK activation in p53-/- cells was preserved. Inhibition of p53 expression by its antisense oligonucleotide also attenuated nocodazole-induced JNK activation in p53+/+ cells. Surprisingly, cotransfection of p53+/+ cells with dominant negative mutants of JNK isoforms and treatment of p53+/+ cells with the JNK inhibitor SP600125 actually further enhanced apoptosis in p53+/+ cells by up to 2-fold in response to nocodazole. These findings indicate that inhibition of p53-mediated JNK1/2 activity in certain tumor cells could serve to enhance the apoptosis-inducing actions of cancer chemotherapeutic agents that disrupt mitotic spindle function.     

Prostaglandin D2 induces the phosphorylation of HSP27 in osteoblasts: function of the MAP kinase superfamily

We previously reported that prostaglandin D(2) (PGD(2)) stimulates the induction of heat shock protein 27 (HSP27) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether PGD(2) stimulates the phosphorylation of HSP27 in MC3T3-E1 cells exposed to heat shock. In the cultured MC3T3-E1 cells, PGD(2) markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85 in a time-dependent manner. Among the mitogen-activated protein (MAP) kinase superfamily, p44/p42 MAP kinase and p38 MAP kinase were phosphorylated by PGD(2) which had little effect on the phosphorylation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The PGD(2)-induced phosphorylation of HSP27 was attenuated by PD169316, an inhibitor of p38 MAP kinase or PD98059, a MEK inhibitor. SP600125, a SAPK/JNK inhibitor did not affect the HSP27 phosphorylation. In addition, PD169316 suppressed the PGD(2)-induced phosphorylation of MAPKAP kinase 2. These results strongly suggest that PGD(2) stimulates HSP27 phosphorylation via p44/p42 MAP kinase and p38 MAP kinase but not SAPK/JNK in osteoblasts.     

A mutant P53 can activate apoptosis through a mechanism distinct from those induced by wild type P53

Tau is a microtubule-associated protein that is abnormally hyperphosphorylated in the filamentous lesions that define a number of neurodegenerative diseases collectively referred to as tauopathies. We previously showed that stress-activated protein (SAP) kinases phosphorylate tau protein at many of the hyperphosphorylated sites in vitro. Here we have developed a system to study the effects of five SAP kinases (SAPK1c/JNK1, SAPK2a/p38alpha, SAPK2b/p38beta, SAPK3/p38gamma and SAPK4/p38delta) on tau phosphorylation in intact cells. All kinases phosphorylated tau, albeit at different efficiencies. Tau was a good substrate for SAPK3/p38gamma and SAPK4/p38delta, a reasonable substrate for SAPK2b/p38beta and a relatively poor substrate for SAPK2a/p38alpha and SAPK1c/JNK1. These findings indicate that the aberrant activation of SAP kinases, especially SAPK3/p38gamma and SAPK4/p38delta, could play an important role in the abnormal hyperphosphorylation of tau that is an invariant feature of the tauopathies.     

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.     

PPAR-gamma ligands up-regulate basic fibroblast growth factor-induced VEGF release through amplifying SAPK/JNK activation in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release. In the present study, we investigated the effects of ciglitazone and pioglitazone, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, on the VEGF release by FGF-2 in MC3T3-E1 cells. The FGF-2-induced VEGF release was significantly enhanced by ciglitazone. The amplifying effect of ciglitazone was dose-dependent between 0.1 and 10 microM. Pioglitazone had a similar effect on the VEGF release. GW9662, an antagonist of PPAR-gamma, reduced the effects of ciglitazone and pioglitazone. Ciglitazone or pioglitazone markedly enhanced the phosphorylation of SAPK/JNK induced by FGF-2 without affecting both the FGF-2-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. GW9662 markedly reduced the amplification by ciglitazone of the SAPK/JNK phosphorylation. Taken together, these results strongly suggest that PPAR-gamma ligands up-regulate FGF-2-stimulated VEGF release resulting from amplifying activation of SAPK/JNK in osteoblasts.