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.     

Compound K protects MIN6N8 pancreatic β‐cells against palmitate‐induced apoptosis through modulating SAPK/JNK activation

Chronic elevation of NEFAs (non‐esterified fatty acids) due to insulin resistance and obesity has been shown to be associated with increased β‐cell apoptosis and with the aetiology of the reduced β‐cell mass of Type 2 diabetes. SAPK (stress‐activated protein kinase)/JNK (c‐Jun N‐terminal kinase) have been implicated in the control of apoptosis. C‐K [compound K; 20‐O‐β‐d ‐glucopyranosyl‐20(S)‐protopanaxadiol] is the main intestinal bacterial metabolite of protopanaxadiol ginsenosides. Currently, little is known about the effects of C‐K on β‐cells with the presence of NEFAs. The aim of the present study was to investigate the in vitro protective effect of C‐K on MIN6N8 mouse insulinoma β‐cells against NEFA‐induced apoptosis, as well as the modulating effect on SAPK/JNK activation. Our results have shown that C‐K inhibited the palmitate‐induced apoptosis through modulating SAPK/JNK activation. We conclude that C‐K protects against β‐cell death and that, by anti‐apoptotic activity, C‐K may contribute to the previously reported anti‐diabetic actions of ginseng.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Expression change of β-1,4 galactosyltransferase I, V mRNAs and Galβ1,4GlcNAc group in rat sciatic nerve after crush

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β-1,4-galactosylation is performed by a family of β-1,4-galactosyltransferases (β-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. In the present study, Real-time PCR revealed that the β-1,4-GalT I and V mRNAs reached peaks at 2 w after sciatic nerve crush. In situ hybridization showed that at 1 d after sciatic nerve crush, the expression levels of β-1,4-GalT I and V mRNAs were strong at the crush site, and decreased gradually from crush site to the distal segments. In addition, combined in situ hybridization for β1,4-GalT I and V mRNAs and immunohistochemistry for S100 showed that β1,4-GalT I and V mRNAs were mainly located in Schwann cells. Lectin blot showed that the expression of Galβ1,4GlcNAc group increased at 6 h immediately, reached a peak at 12 h and remained elevated up to 4 w after sciatic nerve crush. In conclusion, β1,4-GalT I and V might play important roles in the regeneration of the injuried sciatic nerve, and upregulation of Galβ1,4GlcNAc group might be correlated with the process of the sciatic nerve injury.     

Structural and functional analysis of tomato β‐galactosidase 4: insight into the substrate specificity of the fruit softening‐related enzyme

Plant β‐galactosidases hydrolyze cell wall β‐(1,4)‐galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato β‐galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of β‐(1,4)‐galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1–7 that belongs to the β‐galactosidase/exo‐β‐(1,4)‐galactanase subfamily. The enzyme can hydrolyze a wide range of plant‐derived (1,4)‐ or 4‐linked polysaccharides, and shows a strong ability to attack β‐(1,4)‐galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with β‐d ‐galactose. TBG4 comprises a catalytic TIM barrel domain followed by three β‐sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 β‐galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards β‐(1,6)‐galactobiose, and ~0.6‐fold activity towards β‐(1,4)‐galactobiose, compared with wild‐type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant β‐galactosidases towards β‐1,4 and β‐1,6 linkages.     

The role of hair follicle nestin‐expressing stem cells during whisker sensory‐nerve growth in long‐term 3D culture

We have previously reported that nestin‐expressing hair follicle stem cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, vibrissa hair follicles, including their sensory nerve stump, were excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND‐GFP mice), and were placed in 3D histoculture supported by Gelfoam®. β‐III tubulin‐positive fibers, consisting of ND‐GFP‐expressing cells, extended up to 500 µm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F‐actin. These findings indicate that β‐III tubulin‐positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND‐GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results of the present report suggest a major function of the nestin‐expressing stem cells in the hair follicle is for growth of the follicle sensory nerve. J. Cell. Biochem. 114: 1674–1684, 2013. © 2013 Wiley Periodicals, Inc.     

Investigating the binding of β‐1,4‐galactan to Bacillus licheniformis β‐1,4‐galactanase by crystallography and computational modeling

Microbial β‐1,4‐galactanases are glycoside hydrolases belonging to family 53, which degrade galactan and arabinogalactan side chains in the hairy regions of pectin, a major plant cell wall component. They belong to the larger clan GH‐A of glycoside hydrolases, which cover many different poly‐ and oligosaccharidase specificities. Crystallographic complexes of Bacillus licheniformis β‐1,4‐galactanase and its inactive nucleophile mutant have been obtained with methyl‐β(1→4)‐galactotetraoside, providing, for the first time, information on substrate binding to the aglycone side of the β‐1,4‐galactanase substrate binding groove. Using the experimentally determined subsites as a starting point, a β(1→4)‐galactononaose was built into the structure and subjected to molecular dynamics simulations giving further insight into the residues involved in the binding of the polysaccharide from subsite ?4 to +5. In particular, this analysis newly identified a conserved β‐turn, which contributes to subsites ?2 to +3. This β‐turn is unique to family 53 β‐1,4‐galactanases among all clan GH‐A families that have been structurally characterized and thus might be a structural signature for endo‐β‐1,4‐galactanase specificity. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Induction of TNF-α by LPS in Schwann Cell is Regulated by MAPK Activation Signals

Mitogen-activated protein kinases (MAPKs) are important mediators of cytokine expression and are critically involved in the immune response. The lipopolysaccharide (LPS) of gram-negative bacteria induces the expression of cytokines and proinflammatory genes via the toll-like receptor 4 (TLR4) signaling pathway in diverse cell types. In vivo, Schwann cells (SCs) at the site of injury may also produce tumor necrosis factor-- α (TNF-α). However, the precise mechanisms of TNF-α synthesis are still not clear. The purpose of the present study was to elucidate the underlying molecular mechanisms in the cultured SCs for its ability to activate the MAPKs and TNF-α gene, in response to LPS. Using enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment with LPS stimulated the synthesis of TNF-α in a concentration- and time-dependent manner. Intracellular location of TNF-α was detected under confocal microscope. Moreover, LPS activated extracellular signal-regulated kinase (ERK1/2), P38 and stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and induced their phosphorylation. LPS-elicited SCs TNF-α production was also drastically suppressed by PD98059 (ERK inhibitor), SB202190 (P38 inhibitor), or SP600125 (SAPK/JNK inhibitor). Additionally, the expression of CD14 and TLR4 was examined by RT–PCR. It was demonstrated that the expression of CD14, TLR4 was crucial for the SCs responses to LPS. In conclusion, the results provide novel mechanisms for the response of SCs to LPS stimulation, through MAPKs signaling pathways. Chun Cheng and Yongwei Qin contributed equally to this work.     

Proteomic analysis of SP600125‐controlled TrkA‐dependent targets in SK‐N‐MC neuroblastoma cells: Inhibition of TrkA activity by SP600125

The c‐Jun N‐terminal kinase (JNK) is well known to play an important role in cell death signaling of the p75 neurotrophin receptor. However, little has been studied about a role of JNK in the signaling pathways of the tropomyosin‐related kinase A (TrkA) neurotrophin receptor. In this study, we investigated JNK inhibitor SP600125‐controlled TrkA‐dependent targets by proteomic analysis to better understand an involvement of JNK in TrkA‐mediated signaling pathways. PDQuest image analysis and protein identification results showed that hnRNP C1/C2, α‐tubulin, β‐tubulin homolog, actin homolog, and eIF‐5A‐1 protein spots were upregulated by ectopic expression of TrkA, whereas α‐enolase, peroxiredoxin‐6, PROS‐27, HSP70, PP1‐gamma, and PDH E1‐alpha were downregulated by TrkA, and these TrkA‐dependent upregulation and downregulation were significantly suppressed by SP600125. Notably, TrkA largely affected certain PTM(s) but not total protein amounts of the SP600125‐controlled TrkA‐dependent targets. Moreover, SP600125 strongly suppressed TrkA‐mediated tyrosine phosphorylation signaling pathways as well as JNK signaling, indicating that SP600125 could function as a TrkA inhibitor. Taken together, our results suggest that TrkA could play an important role in the cytoskeleton, cell death, cellular processing, and glucose metabolism through activation or inactivation of the SP600125‐controlled TrkA‐dependent targets.     

Arachidonic acid induces an increase of β-1,4-galactosyltransferase I expression in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n‐6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, and it might be present in the extracellular microenvironment. AA and its metabolites are implicated in FAK activation and cell migration in MDA‐MB‐231 breast cancer cells, and an epithelial‐to‐mesenchymal‐like transition process in mammary non‐tumorigenic epithelial cells MCF10A. During malignant transformation is present an altered expression of glycosiltransferases, which promote changes on the glycosilation of cell‐surface proteins. The β‐1,4‐galactosyltransferase I (GalT I) is an enzyme that participates in a variety of biological functions including cell growth, migration, and spreading. However, the participation of AA in the regulation of GalT I expression and the role of this enzyme in the cell adhesion process in breast cancer cells remains to be investigated. In the present study, we demonstrate that AA induces an increase of GalT I expression through a PLA2α, Src, ERK1/2, and LOXs activities‐dependent pathway in MDA‐MB‐231 breast cancer cells. Moreover, MDA‐MB‐231 cells adhere to laminin via GalT I expression and pretreatment of cells with AA induces an increase of cell adhesion to laminin. In conclusion, our findings demonstrate, for the first time, that AA promotes an increase of GalT I expression through an AA metabolism, Src and ERK1/2 activities‐dependent pathway, and that GalT I plays a pivotal role in cell adhesion to laminin in MDA‐MB‐231 breast cancer cells. J. Cell. Biochem. 113: 3330–3341, 2012. © 2012 Wiley Periodicals, Inc.     

Impact of integrin–matrix interaction and signaling on insulin gene expression and the mesenchymal transition of human β‐cells

A critical shortage of donor pancreata currently prevents the development of a universal cell‐based therapy for type I diabetes. The ex vivo expansion of insulin‐producing β‐cells offers a potential solution but is problematic due to the inherent tendency of these cells to transition into mesenchymal‐like cells that are devoid of function. Here, we demonstrate for the first time that exposure to elements of the extracellular matrix (ECM) directly potentiates the mesenchymal transition of cultured fetal β‐cells and causes associated declines in insulin gene expression. Individual ECM constituents varied in their ability to induce such responses, with collagen‐IV (C‐IV) and fibronectin inducing strong responses, whereas laminin‐1 had no significant effect. Mesenchymal transition and concomitant losses in insulin gene expression observed on C‐IV were found to be dependent on β₁‐integrin ligation and were augmented in the presence of hepatocyte growth factor. Importantly, selective inhibition of c‐Src, c‐Jun N‐terminal kinase (JNK), and extracellular signal‐regulated kinase (ERK) prior to exposure to C‐IV prevented mesenchymal transition and effectively preserved insulin expression. Fetal β‐cells undergoing mesenchymal transition were found to acquire α₁β₁ expression, and ligation of this integrin then promotes declines in insulin gene expression and a marked increase in β‐cell motility. Inhibition of Src‐, ERK‐, or JNK‐dependent signaling combined with the selective regulation of matrix exposure may ultimately facilitate the development of more effective β‐cell expansion protocols. J. Cell. Physiol. 224:101–111, 2010 © 2010 Wiley‐Liss, Inc.     

Expression of Src Suppressed C Kinase Substrate in Rat Neural Tissues During Inflammation

Src-suppressed C kinase substrate (SSeCKS), an in vivo and in vitro protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including brain, lung, heart, kidney etc., indicating a possible role of SSeCKS in inflammatory process. However, the expression and biological function of SSeCKS during neuronal inflammation remains to be elucidated, so we established an inflammatory model injected with LPS to investigate the gene expression patterns of SSeCKS in neural tissues by using TaqMan quantitative real-time PCR and immunohistochemistry in rat. Real-time PCR showed that LPS stimulated the expression of SSeCKS mRNA in a dose- and time-dependent manner in sciatic nerves, spinal cords and dorsal root ganglions. Immunohistochemistry showed that SSeCKS colocalized with nerve fibers in sciatic nerve after LPS administration, but there was no colocalization between SSeCKS and Schwann cells. In addition, SSeCKS colocalized with neurons which existed in dorsal root ganglions and spinal cords. These findings indicated that SSeCKS might play some important roles in sciatic nerve fibers and neurons in spinal cords and dorsal root ganglions after LPS injection.     

Berberine suppresses neuroinflammatory responses through AMP‐activated protein kinase activation in BV‐2 microglia

The AMPK cascade is a sensor of cellular energy change, which monitors the AMP/ATP ratio to regulate cellular metabolism by restoring ATP levels, but its regulation of neuroinflammation mechanism remains unclear. Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been shown to improve several metabolic disorders, such as obesity and type II diabetes. However, the effect of berberine on neuroinflammatory responses in microglia are poorly understood. This study shows that berberine represses proinflammatory responses through AMP‐activated protein kinase (AMPK) activation in BV‐2 microglia. Our findings also demonstrate that berberine significantly down‐regulates LPS‐ or interferon (IFN)‐γ‐induced nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) expression in BV‐2 microglia cells. Berberine also inhibited LPS‐ or IFN‐γ‐induced nitric oxide production. In addition, berberine effectively inhibited proinflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 expression. On the other hand, upon various inflammatory stimulus including LPS and IFN‐γ, berberine suppressed the phosphorylated of ERK but not p38 and JNK in BV‐2 microglia. AMPK activation is catalyzed by upstream kinases such as LKB1 and Ca2+/calmodulin‐dependent protein kinase kinase‐II (CaMKK II). Moreover, berberine induced LKB1 (Ser428), CaMKII (Thr286), and AMPK (Thr172) phosphorylation, but not AMPK (Ser485). Furthermore, the inhibitory effect of berberine on iNOS and COX‐2 expression was abolished by AMPK inhibition via Compound C, an AMPK inhibitor. Berberine‐suppressed ERK phosphorylation was also reversed by Compound C treatment. Our data demonstrate that berberine significantly induces AMPK signaling pathways activation, which is involved in anti‐neuroinflammation. J. Cell. Biochem. 110: 697–705, 2010. © 2010 Wiley‐Liss, Inc.     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Upregulation of lipopolysaccharide-induced interleukin-10 by prostaglandin A1 in mouse peritoneal macrophages

The cyclopentenone prostaglandins (cyPGs) prostaglandin A1 (PGA1) and 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) have been reported to exhibit antiinflammatory activity in activated monocytes/macrophages. However, the effects of these two cyPGs on the expression of cytokine genes may differ. In this study, we investigated the mechanism of action of PGA1 in lipopolysaccharide (LPS)-induced expression of interleukin (IL)-10 mRNA in mouse peritoneal macrophages. 15d-PGJ2 inhibited expression of LPSinduced IL-10, whereas PGA1 increased LPS-induced IL-10 expression. This synergistic effect of PGA1 on LPS-induced IL-10 expression reached a maximum as early as 2 h after simultaneous PGA1 and LPS treatment (PGA1/LPS), and did not require new protein synthesis. The synergistic effect of PGA1 was inhibited by GW9662, a specific peroxisome proliferator-activated receptor (PPAR) antagonist, and Bay-11-7082, a NF-kappaB inhibitor. The extracellular signalregulated kinases (ERK) inhibitor PD98059 increased the expression of PGA1/LPS-induced IL-10 mRNA, rather than inhibiting the IL-10 expression. Moreover, PGA1 inhibited LPS-induced ERK phosphorylation. The synergistic effect of PGA1 on LPS-induced IL-10 mRNA and protein production was inhibited by p38 inhibitor PD169316, and PGA1 increased LPS-induced p38 phosphorylation. In the case of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), the SAPK/JNK inhibitor SP600125 did not inhibit IL-10 mRNA synthesis but inhibited the production of IL-10 protein remarkably. These results suggest that the synergistic effect of PGA1 on LPS-induced IL-10 expression is NF-kappaB-dependent and mediated by mitogen-activated protein (MAP) kinases, p38, and SAPK/ JNK signaling pathways, and also associated with the PPARgamma pathway. Our data may provide more insight into the diverse mechanisms of PGA1 effects on the expression of cytokine genes.     

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.