Endothelin-1 induces expression of matrix-associated genes in lung fibroblasts through MEK/ERK

The endothelins are a family of endothelium-derived peptides that possess a variety of biological activities, including potent vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and pulmonary fibrosis. Here, we use genome-wide expression array analysis to show that the addition of ET-1 (100 nm, 4 h) to normal lung fibroblasts directly induces expression of matrix and matrix-associated genes, including the profibrotic protein CCN2 (connective tissue growth factor, or CTGF). ET-1 induces the MEK/ERK MAP kinase pathway in fibroblasts. Blockade of the MEK/ERK kinase pathway with U0126 abrogates the ability of ET-1 to induce expression of matrix and matrix-associated mRNAs and the CCN2 protein. The CCN2 promoter possesses an ET-1 response element, which maps to the previously identified basal control element-1 (BCE-1) site. Our results suggest that ET-1 induces a program of matrix synthesis in lung fibroblasts and that ET-1 may play a key role in connective tissue deposition during wound repair and in pulmonary fibrosis.     

Phospholipase D1 in caveolae: regulation by protein kinase Calpha and caveolin-1

Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Calpha (PKCalpha) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12-myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCalpha-stimulated PLD1 activity and the interaction between PLD1 and PKCalpha with an IC50 of 0.5 microM. PMA elicits translocation of PKCalpha to the CEMs, inducing PLD activation through the interaction of PKCalpha with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCalpha-dependent PLD activity through the molecular interaction between PLD1, PKCalpha, and caveolin-1 in caveolae.     

Suppression of urokinase expression and invasiveness by urinary trypsin inhibitor is mediated through inhibition of protein kinase C- and MEK/ERK/c-Jun-dependent signaling pathways

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) to evaluate the effect on expression of urokinase-type plasminogen activator (uPA), since the action of uPA has been implicated in matrix degradation and cell motility. Preincubation of the cells with UTI reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. UTI-induced down-regulation of PMA-stimulated uPA expression is irreversible and is independent of a cytotoxic effect. Down-regulation of uPA by UTI is mediated by its binding to the cells. We next asked whether the mechanism of inhibition of uPA expression by UTI was due to interference with the protein kinase C second messenger system. An assay for PKC activity demonstrated that UTI does not directly inhibit the catalytic activity of PKC and that PMA translocation of PKC from cytosol to membrane was inhibited by UTI, indicating that UTI inhibits the activation cascade of PKC. PMA could also activate a signaling pathway involving MEK1/ERK2/c-Jun-dependent uPA expression. When cells were preincubated with UTI, we could detect suppression of phosphorylation of these proteins. Like several types of PKC inhibitor, UTI inhibited PMA-stimulated invasiveness. We conclude that UTI markedly suppresses the cell motility possibly through negative regulation of PKC- and MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes.     

Ras-independent activation of ERK signaling via the torso receptor tyrosine kinase is mediated by Rap1

In Drosophila embryos, the Torso receptor tyrosine kinase (RTK) activates the small G protein Ras (D-Ras1) and the protein kinase Raf (D-Raf) to activate ERK to direct differentiation of terminal structures . However, genetic studies have demonstrated that Torso, and by extension other RTKs, can activate Raf and ERK independently of Ras . In mammalian cells, the small G protein Rap1 has been proposed to couple RTKs to ERKs. However, the ability of Rap1 to activate ERKs remains controversial, in part because direct genetic evidence supporting this hypothesis is lacking. Here, we present biochemical and genetic evidence that D-Rap1, the Drosophila homolog of Rap1, can activate D-Raf and ERK. We show that D-Rap1 binds D-Raf and activates ERKs in a GTP- and D-Raf-dependent manner. Targeted disruption of D-Rap1 expression decreased both Torso-dependent ERK activation and the ERK-dependent expression of the zygotic genes tailless and huckebein to levels similar to those achieved in D-Ras1 null embryos. Furthermore, combined deficiencies of D-Ras1 and D-Rap1 completely abolished expression of these genes, mimicking the phenotype observed in embryos lacking D-Raf. These studies provide the first direct genetic evidence of Rap1-mediated activation of the MAP kinase cascade in eukaryotic organisms.     

Opposing roles of Ras/Raf oncogenes and the MEK1/ERK signaling module in regulation of expression and adhesive function of surface transglutaminase

Tissue transglutaminase (tTG) serves as a potent and ubiquitous integrin-associated adhesion co-receptor for fibronectin on the cell surface and affects several key integrin functions. Here we report that in fibroblasts, activated H-Ras and Raf-1 oncogenes decrease biosynthesis, association with beta1 integrins, and surface expression of tTG because of down-regulation of tTG mRNA. In turn, the reduction of surface tTG inhibits adhesion of H-Ras- and Raf-1-transformed cells on fibronectin and, in particular, on its tTG-binding fragment I(6)II(1,2)I(7-9), which does not interact directly with integrins. Analysis of Ras/Raf downstream signaling with specific pharmacological inhibitors reveals that the decrease in tTG expression is mediated by the p38 MAPK, c-Jun NH2-terminal kinase, and phosphatidylinositol 3-kinase pathways. In contrast, increased activation of the ERK pathway by constitutively active MEK1 stimulates tTG mRNA expression, biosynthesis, and surface expression of tTG, whereas MEK inhibitors or dominant negative MEK1 exert an opposite effect. This modulation of surface tTG by ERK signaling alters adhesion of cells on fibronectin and its fragment that binds tTG. Furthermore, transient stimulation of ERK signaling in untransformed fibroblasts by adhesion on fibronectin or growth factors elevates tTG biosynthesis, increases complex formation with beta1 integrins, and raises surface expression of tTG. Finally, ERK activation is required for growth factor-induced redistribution of tTG on the surface of adherent fibroblasts and co-clustering of beta1 integrins and tTG at cell-matrix adhesion contacts. Together, our data indicate that down-regulation of surface tTG by Ras and Raf oncogenes contributes to adhesive deficiency of transformed fibroblasts, whereas stimulation of biosynthesis and surface expression of tTG by the MEK1/ERK module promotes and sustains cell-matrix adhesion of untransformed cells. Contrasting effects of Ras/Raf oncogenes and their immediate downstream signaling module, MEK1/ERK, on tTG expression are consistent with adhesive function of surface tTG.     

Neutrophil elastase-initiated EGFR/MEK/ERK signaling counteracts stabilizing effect of autocrine TGF-beta on tropoelastin mRNA in lung fibroblasts

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938-18946, 2002). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-alpha-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-beta signaling, because TGF-beta type I receptor kinase inhibitor or TGF-beta neutralizing antibody dramatically decreases tropoelastin mRNA and protein levels. Half-life of tropoelastin mRNA in RFL-6 cells is >24 h, but it is decreased to approximately 8 h by addition of TGF-beta neutralizing antibody, EGF, TGF-alpha, or NE. Tropoelastin mRNA destabilization by NE, EGF, or TGF-alpha is abolished by AG1478 or U0126. EGF-dependent tropoelastin mRNA downregulation is reversed upon ligand withdrawal, whereas chronic EGF treatment leads to persistent downregulation of tropoelastin mRNA and protein levels and decreases insoluble elastin deposition. We conclude that NE-initiated EGFR/MEK/ERK signaling cascade overrides the autocrine TGF-beta signaling on tropoelastin mRNA stability and, therefore, decreases the elastogenic response in RFL-6 fibroblasts. We hypothesize that persistent EGFR/MEK/ERK signaling could impede the TGF-beta-induced elastogenesis/elastin repair in the chronically inflamed, elastase/anti-elastase imbalanced lung in emphysema.     

Ascochlorin suppresses oxLDL-induced MMP-9 expression by inhibiting the MEK/ERK signaling pathway in human THP-1 macrophages

The critical initiating event in atherogenesis involves the invasion of monocytes through the endothelial walls of arteries and the transformation of monocytes from macrophages into foam cells. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) and can then be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). Also, during a chronic inflammatory response, monocytes/macrophages produce the 92-kDa matrix metalloproteinase-9 (MMP-9) that may contribute to the extravasation, migration, and tissue remolding capacities of the phagocytic cells. Here, we investigate the effect of ascochlorin (ASC), a prenylphenol antiviral compound from the fungus Ascochyta viciae, on oxLDL-induced MMP-9 expression and activity in human THP-1 macrophages. ASC reduced oxLDL-induced MMP-9 expression and activity in a time-dependent and dose-dependent manner. Also, an analysis of MMP-9 activity using pharmacologic inhibitors showed that ASC inhibits MMP-9 activity via the extracellular signal-regulated kinase 1 and kinase 2 pathways. Our results suggest that ASC may be useful as a potent clinical antiatherogenic agent, a topic of considerable interest in the biological chemistry of chemotherapeutic agents.     

PDGF-BB-induced MT1-MMP expression regulates proliferation and invasion of mesenchymal stem cells in 3-dimensional collagen via MEK/ERK1/2 and PI3K/AKT signaling

Mesenchymal stem cells (MSCs) mobilize membrane type-1 matrix metalloproteinase (MT1-MMP) to traffic through both 3-dimensional (3D) collagen as well as basement membrane barriers, but factors capable of regulating the expression and activity of the protease remain unidentified. Herein, we report that the MT1-MMP-dependent invasive activities of rat MSCs are controlled by PDGF-BB. Furthermore, PDGF-BB also stimulates MSC proliferation in 3D type I collagen via an MT1-MMP-dependent process that is linked to pericellular collagen degradation. PDGF-BB stimulates MT1-MMP expression at both the mRNA and protein levels in concert with ERK1/2 and PI3K/AKT activation. Inhibition of ERK1/2 or PI3K/AKT activity potently suppresses both MT1-MMP-dependent invasive and proliferative activities. Basement membrane invasion is likewise stimulated by PDGF-BB in an MT1-MMP-dependent manner via ERK1/2 and PI3K/AKT signaling. Taken together, these data serve to identify PDGF-BB as an important MSC agonist that controls invasive and proliferative activities via MT1-MMP-dependent processes that are regulated by the ERK1/2 and PI3K/AKT signaling pathways.     

Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway

In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.     

Activation of extracellular signal-regulated kinase 1/2 inhibits type I collagen expression by human skin fibroblasts

Treatment with the lipid second messenger, ceramide, activates extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase, and p38 in human skin fibroblasts and induces their collagenase-1 expression (Reunanen, N., Westermarck, J., H?kkinen, L., Holmstr?m, T. H., Elo, I., Eriksson, J. E., and K?h?ri, V.-M. (1998) J. Biol. Chem. 273, 5137-5145). Here we show that C(2)-ceramide inhibits expression of type I and III collagen mRNAs in dermal fibroblasts, suppresses proalpha2(I) collagen promoter activity, and reduces stability of type I collagen mRNAs. The down-regulatory effect of C(2)-ceramide on type I collagen mRNA levels was abrogated by protein kinase C inhibitors H7, staurosporine, and Ro-31-8220 and potently inhibited by a combination of MEK1,2 inhibitor PD98059 and p38 inhibitor SB203580. Activation of ERK1/2 by adenovirus-mediated expression of constitutively active MEK1 resulted in marked down-regulation of type I collagen mRNA levels and production in fibroblasts, whereas activation of p38 by constitutively active MAPK kinase-3b and MAPK kinase-6b slightly up-regulated type I collagen expression. These results identify the ERK1/2 signaling cascade as a potent negative regulatory pathway with respect to type I collagen expression in fibroblasts, suggesting that it mediates inhibition of collagen production in response to mitogenic stimulation and transformation.     

Alpha2(I) collagen gene regulation by protein kinase C signaling in human dermal fibroblasts

We investigated the mechanisms by which protein kinase C (PKC) regulates the expression of the α2(I) collagen gene in normal dermal fibroblasts. Reduction of PKC-α activity by treatment with Gö697-6 or by overexpression of a dominant negative (DN) mutant form decreased α2(I) collagen gene expression. This decrease required a sequence element in the collagen promoter that contains Sp1/Sp3 binding sites. Reduction of PKC-δ activity by rottlerin or overexpression of DN PKC-δ also decreased α2(I) collagen gene expression. This effect required a separate sequence element containing Sp1/Sp3-binding sites and an Ets-binding site. In both cases, point mutations within the response elements abrogated the response to PKC inhibition. Forced overexpression of Sp1 rescued the PKC inhibitor-mediated reduction in collagen protein expression. A DNA affinity precipitation assay revealed that inhibition of PKC-δ by rottlerin increased the binding activity of endogenous Fli1 and decreased that of Ets1. On the other hand, TGF-β1, which increased the expression of PKC-δ, had the opposite effect, increasing the binding activity of Ets1 and decreasing that of Fli1. Our results suggest that PKC-δ is involved in the regulation of the α2(I) collagen gene in the presence or absence of TGF-β. Alteration of the balance of Ets1 and Fli1 may be a novel mechanism regulating α2(I) collagen expression.     

Integrins regulate microtubule nucleating activity of centrosome through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling

Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin β1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.     

Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.