Activation of extracellular signal-regulated kinase 1/2 and Sp1 may contribute to the expression of tissue inhibitor of metalloproteinases-1 induced by transforming growth factor-β1 in human pulmonary arterial smooth muscle cells.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) plays an important role in the development of pulmonary arterial hypertension. However, the molecular regulatory mechanisms of TIMP-1 in the pulmonary arteries are not fully understood, especially in human pulmonary arterial smooth muscle cells (HPASMCs). We investigated the signaling pathway involved in the regulation of TIMP-1 in HPASMCs induced by transforming growth factor (TGF)-β1. Cultured HPASMCs were incubated with different concentrations of TGF-β1 (0-40 ng/mL) for 24 h or with 10 ng/mL TGF-β1 for different times (1-48 h). Western blot, real-time polymerase chain reaction and enzyme-linked immunosorbent assay analyses showed that TGF-β1 enhanced the expression and secretion of TIMP-1 in a time-dependent and dose-dependent fashion. TGF-β1 could phosphorylate two of the three mitogen-activated protein kinases-extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, but not c-Jun NH2-terminal kinase. Of these kinases, only the inhibition of ERK1/2 by U0126, which was a specific inhibitor of mitogen-activated protein kinase/ERK1/2, effectively blocked the TGF-β1-induced expression of TIMP-1. Mithramycin, an inhibitor of Sp1 transcription factor, also significantly inhibited the expression of TIMP-1. Additionally, electrophoretic mobility shift assay showed that TGF-β1 could up-regulate the DNA-binding activity of Sp1 and that U0126 and mithramycin could effectively inhibit these events. TGF-β1 could stimulate the expression and secretion of TIMP-1 in HPASMCs in a time-dependent and dose-dependent fashion, and ERK1/2 and Sp1 signaling pathways might be involved in these activities.

Tang W ，Yang J ，Zhang F ，Guo H ，Peng F ，Wang X ... -  《-》

Transforming growth factor-beta1 induces tissue inhibitor of metalloproteinase-1 expression via activation of extracellular signal-regulated kinase and Sp1 in human fibrosarcoma cells.

Kwak HJ ，Park MJ ，Cho H ，Park CM ，Moon SI ，Lee HC ，Park IC ，Kim MS ，Rhee CH ，Hong SI ... -  《MOLECULAR CANCER RESEARCH》

TGF-beta-induced expression of tissue inhibitor of metalloproteinases-3 gene in chondrocytes is mediated by extracellular signal-regulated kinase pathway and Sp1 transcription factor.

Transforming growth factor (TGF-beta1) is a potent inducer of chondrogenesis and stimulant of cartilage extracellular matrix (ECM) synthesis. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is located in ECM and is the major inhibitor of matrix metalloproteinases (MMPs) and aggrecanase, the principal enzymes implicated in collagen and aggrecan degradation in arthritis. We investigated the role of extracellular-signal-regulated kinase (ERK)-mitogen-activated protein kinases (MAPK) and Sp1 transcription factor in TGF-beta-induced TIMP-3 gene in chondrocytes and chondrosarcoma cells. TGF-beta time-dependently induced a sustained phosphorylation of ERK-MAPKs in primary human or bovine chondrocytes. Inhibitors of this pathway, PD98059 and U0126, downregulated TGF-beta-induced expression of TIMP-3 RNA and protein. Since the ERKs can phosphorylate Sp1, and the promoter of human TIMP-3 gene contains four Sp1-binding sites, we investigated whether Sp1 is a downstream target of this pathway. Mithramycin and WP631, the agents that prevent binding of Sp1 to its consensus site, downregulated TGF-beta-inducible TIMP-3 expression. Indeed, mithramycin blocked TGF-beta-stimulated Sp1 binding activity. Transfection of cytomegalovirus (CMV) promoter-Sp1 plasmid increased TIMP-3 promoter (-940 to +376)-driven luciferase activity. Depletion of Sp1 by transfection of an antisense phosphorothioate oligonucleotide suppressed TGF-beta-induced TIMP-3 protein expression, while its sense homolog had no effect. These results suggest that activation of ERK-MAPK pathway and Sp1 transcription factor play a pivotal role in the induction of TIMP-3 by TGF-beta in chondrocytes.

Qureshi HY ，Sylvester J ，El Mabrouk M ，Zafarullah M ... -  《JOURNAL OF CELLULAR PHYSIOLOGY》

Platelet-derived growth factor and transforming growth factor-beta modulate the expression of matrix metalloproteinases and migratory function of human airway smooth muscle cells.

Ito I ，Fixman ED ，Asai K ，Yoshida M ，Gounni AS ，Martin JG ，Hamid Q ... -  《-》

Plasminogen activator inhibitor type-1 gene expression and induced migration in TGF-beta1-stimulated smooth muscle cells is pp60(c-src)/MEK-dependent.

Transforming growth factor-beta1 (TGF-beta1) stimulates expression of plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor (SERPIN) important in the control of stromal barrier proteolysis and cell-to-matrix adhesion. Pharmacologic agents that target MEK (PD98059, U0126) or src family (PP1) kinases attenuated TGF-beta1-dependent PAI-1 transcription in R22 aortic smooth muscle cells. Pretreatment with PP1 at concentrations that inhibited TGF-beta1-dependent PAI-1 expression also blocked ERK1/2 activation/nuclear accumulation suggesting that the required src kinase activity is upstream of ERK1/2 in the TGF-beta1-initiated signaling cascade. The IC(50) of the PP1-sensitive kinase, furthermore, specifically implied involvement of pp60(c-src) in PAI-1 induction. Indeed, addition of TGF-beta1 to quiescent R22 cells resulted in a 3-fold increase in pp60(c-src) autophosphorylation and kinase activity. Transfection of a dominant-negative pp60(c-src) construct, moreover, reduced TGF-beta1-induced PAI-1 expression levels to that of unstimulated controls or PP1-pretreated cells. A >/=170 kDa protein that co-immunoprecipitated with TGF-beta1-activated pp60(c-src) was also phosphorylated transiently in response to TGF-beta1. TGF-beta1 is known to transactivate the 170 kDa EGF receptor (EGFR) by autocrine HB-EGF or TGF-alpha mechanisms suggesting involvement of EGFR activation in certain TGF-beta1-initiated responses. Incubation of quiescent R22 cells with the EGFR-specific inhibitor AG1478 prior to growth factor (EGF or TGF-beta1) addition effectively blocked EGFR activation as determined by direct visualization of receptor internalization. AG1478 suppressed (in a dose-dependent fashion) EGF-induced PAI-1 protein levels and, at a final concentration of 2.5 muM, virtually eliminated EGF-dependent PAI-1 synthesis. More importantly, AG1478 similarly repressed inducible PAI-1 levels in TGF-beta1-stimulated R22 cultures. PP1, PD98059, and U0126 also inhibited TGF-beta1-dependent cell motility at concentrations that significantly attenuated PAI-1 expression. Consistent with the AG1478-associated reductions in EGF- and TGF-beta1-stimulated PAI-1 expression, pretreatment of R22 cell cultures with AG1478 effectively suppressed growth factor-stimulated cell motility. These data indicate that two major phenotypic characteristics of TGF-beta1-exposure (i.e., transcription of specific target genes [e.g., PAI-1], increased cell motility) are linked in the R22 vascular smooth muscle cell system, require pp60(c-src) kinase activity and MEK signaling and involve activation of an AG1478-sensitive (likely EGFR-dependent) pathway.

Samarakoon R ，Higgins CE ，Higgins SP ，Kutz SM ，Higgins PJ ... -  《JOURNAL OF CELLULAR PHYSIOLOGY》