TGF-β1-induced PAI-1 gene expression requires MEK activity and cell-to-substrate adhesion

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Abstract
The type-1 inhibitor of plasminogen activator (PAI-1) is an important physiological regulator of extracellular matrix (ECM) homeostasis and cell motility. Various growth factors mediate temporal changes in the expression and/or focalization of PAI-1 and its protease target PAs, thereby influencing cell migration by barrier proteolysis and/or ECM adhesion modulation. TGF-β1, in particular, is an effective inducer of matrix deposition/turnover, cell locomotion and PAI-1 expression. Therefore, the relationship between motility and PAI-1 induction was assessed in TGF-β1-sensitive T2 renal epithelial cells. PAI-1 synthesis and its matrix deposition in response to TGF-β1 correlated with a significant increase in cell motility. PAI-1 expression was an important aspect in cellular movement as PAI-1-deficient cells had significantly impaired basal locomotion and were unresponsive to TGF-β1. However, the induced migratory response to this growth factor was complex. TGF-β1 concentrations of 1-2 ng/ml were significantly promigratory, whereas lower levels (0.2-0.6 ng/ml) were ineffective and final concentrations ≥5 ng/ml inhibited T2 cell motility. This same growth factor range progressively increased PAI-1 transcript levels in T2 cells consistent with a bifunctional role for PAI-1 in cell migration. TGF-β1 induced PAI-1 mRNA transcripts in quiescent T2 cells via an immediate-early response mechanism. Full TGF-β1-stimulated expression required tyrosine kinase activity and involved MAPK/ERK kinase (MEK). MEK appeared to be a major mediator of TGF-β1-dependent PAI-1 expression and T2 cell motility since PD98059 effectively attenuated both TGF-β1-induced ERK1/2 activation and PAI-1 transcription as well as basal and growth factor-stimulated planar migration. Since MEK activation in response to growth factors is adhesion-dependent, it was important to determine whether cellular adhesive state influenced TGF-β1-mediated PAI-1 expression in the T2 cell system. Cells maintained in suspension culture (i.e., over agarose underlays) in growth factor-free medium or treated with TGF-β1 in suspension expressed relatively low levels of PAI-1 transcripts compared with the significant induction of PAI-1 mRNA evident in T2 cells upon stimulation with TGF-β1 during adhesion to a fibronectin-coated substrate. Attachment to fibronectin alone (i.e., in the absence of added growth factor) was sufficient to initiate PAI-1 transcription, albeit at levels considerably lower than that induced by the combination of cell adhesion in the presence of TGF-β1. T2 cells allowed to attach to vitronectin-coated surfaces also expressed PAI-1 transcripts but to a significantly reduced extent relative to cells adherent to fibronectin. Moreover, newly vitronectin-attached cells did not exhibit a PAI-1 inductive response to TGF-β1, at least during the short 2 hour period of combined treatment. PAI-1 mRNA synthesis in response to substrate attachment, like TGF-β1-mediated induction in adherent cultures, also required MEK activity as fibronectin-stimulated PAI-1 expression was effectively attenuated by the MEK inhibitor PD98059. These data indicate that cellular adhesive state modulates TGF-β1 signaling to particular target genes (i.e., PAI-1) and that MEK is a critical mediator of the PAI-1+/promigratory phenotype switch induced by TGF-β1 in T2 cells.

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