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TGF-ß1-induced PAI-1 gene expression requires MEK activity and cell-to-substrate adhesion

Center for Cell Biology & Cancer Research, Albany Medical College, Albany, New York 12208, USA

View larger version (103K): [in a new window]   Fig. 1. PAI-1 deposition in T2 cells after stimulation with serum or TGF-ß1. Quiescent (Q) cell cultures were stimulated by addition of FBS or TGF-ß1 (to final concentrations of 20% and 1 ng/ml, respectively). After 4 hours, cells were fixed and processed (see Materials and Methods) for visualization of microfilament organization (Actin) and PAI-1 immunolocalization (PAI-1).

View larger version (31K): [in a new window]   Fig. 2. Stimulation of cell motility by serum or TGF-ß1. Confluent cultures of T2 cells were incubated in serum-free DMEM for 3 days prior to scrape-wounding. Cells were maintained in the FBS-free medium to assess basal migration (A). The percent (%) wound closure was determined 24 hours later. Data plotted are the means±s.d. of 20 individual measurements made on each of triplicate cultures for each treatment condition. Asterisks indicate a statistically significant difference (Student’s t-test, P>0.01) in cell migration for TGF-ß1- and FBS-supplemented cultures compared with basal (FBS-free) motility. The effect of targeted PAI-1 ablation on basal (-TGF-ß1) as well as TGF-ß1-induced (+TGF-ß1) cell locomotion was assessed in EC-1 and 4HH cell cultures by evaluation of the extent (%) of wound closure in the absence and presence of growth factor (1 ng/ml) (B). Data plotted are the means±s.d. of 15 individual measurements on duplicate cultures/treatment group. Asterisk indicates a statistically significant difference (P<0.01) between motility under growth factor-free and supplemented conditions for EC-1 cells. By contrast, 4HH cells were unresponsive to TGF-ß1 in this assay.

View larger version (45K): [in a new window]   Fig. 3. Metabolic requirements for TGF-ß1-induced PAI-1 expression. To assess pathways underlying induced PAI-1 expression, quiescent (Q) T2 cells were stimulated with serum (20%) or TGF-ß1 (1 ng/ml) for 2 hours, in the presence or absence of a 30 minute pretreatment with the indicated inhibitors, prior to RNA isolation (A). Northern blots were hybridized with 32P-labeled cDNA probes for PAI-1 and A-50 simultaneously. The inability of puromycin to attenuate either serum or TGF-ß1-induced PAI-1 transcripts and the sensitivity of expression to actinomycin D indicated that PAI-1 induction by both stimuli was an immediate-early (i.e., primary) response. TGF-ß1-induced PAI-1 expression in T2 cells is MEK-dependent (B). Quiescent (Q) T2 cells were stimulated with TGF-ß1 (1 ng/ml) for 2 hours in the absence or presence of a 30 minute pretreatment with PD98059 (50 nM) prior to isolation of RNA. Northern blots were hybridized with 32P-labeled cDNA probes to PAI-1 and A-50.

View larger version (64K): [in a new window]   Fig. 4. Coupled ERK immunoprecipitation/MBP kinase assay for assessment of TGF-ß1-induced ERK activation. ERK1/2 were immunoprecipitated from lysates of quiescent (Q), FBS- and TGF-ß1-stimulated T2 cells. Exposure of quiescent cells to FBS (20%) was for 15 minutes and stimulation with TGF-ß1 (1 ng/ml) was for 30, 60 and 90 minutes prior to cell disruption and ERK1/2 immunoprecipitation. MBP phosphorylation reaction products (MBP-P) were separated by gel electrophoresis; equivalent loading of MBP and ERK per lane was confirmed by Ponceau S staining (not shown) and ERK2 western blotting, respectively (A). In contrast to the relatively rapid rate of ERK activation by serum (15 minutes), TGF-ß1-induced changes in ERK activity were not evident until 60 minutes after growth factor addition (A), remained elevated for approximately 2 hours and then rapidly declined (B). Coupled ERK immunoprecipitation/MBP phosphorylation (MBP-P) assays (C) confirmed that ERK activation in TGF-ß1-stimulated T2 cells is sensitive to the same pharmacologic inhibitors that attenuate growth factor-induced PAI-1 expression. The more pathway restrictive inhibitor herbimycin A (250 nM) (Fukazawa et al., 1994) attenuated MBP phosphorylation but not to the same extent as genistein or PD98059.

View larger version (26K): [in a new window]   Fig. 5. The MEK inhibitor PD98059 attenuates both basal and TGF-ß1-stimulated T2 cell migration. Initial experiments were designed to determine the optimal concentration of TGF-ß1 on wound-induced motility (A). After scrape-injury, TGF-ß1 was added (in the concentrations indicated) and extent of migration determined 24 hours later. Data plotted is % increase in wound closure relative to non-supplemented (FBS-free) cultures. Asterisks indicate those concentrations for which motility was significantly different from basal migration (Student’s t-test, P>0.0005). To assess the requirement for MEK activity in stimulated cell movement, monolayer scrape wound-closure assays were carried out in TGF-ß1-supplemented (concentration range 0, 1, 2 and 5 ng/ml) serum-free medium in the presence (P) or absence of PD98059 (50 µM) (B). TGF-ß1 at 1 and 2 ng/ml significantly increased T2 cell directional motility (Student’s t-test, P>0.001; asterisks) relative to basal mobility (0 ng). In this series of experiments, cells exposed to 5 ng/ml of the growth factor actually had a decreased rate of locomotion relative to unsupplemented controls. At each concentration of TGF-ß1, PD98059 effectively reduced wound closure; there was no significant difference in the % closure rate among any of the treatment groups in the presence of PD98059.

View larger version (61K): [in a new window]   Fig. 6. TGF-ß1 concentration-dependent increase in relative PAI-1 mRNA transcripts. Quiescent T2 cells (Q) were stimulated with TGF-ß1 at the indicated concentrations and RNA isolated 2 hours later. Northern blots (insert for example) were scanned and the average PAI-1 transcript abundance, normalized to A-50 signal, calculated for 2 separate experiments.

View larger version (52K): [in a new window]   Fig. 7. Optimum response of the PAI-1 gene to TGF-ß1 stimulation in T2 cells requires substrate adhesion. TGF-ß1-induced PAI-1 transcripts requires adhesion (to a fibronectin (FN) substrate) since cells cultured in suspension (agarose, Ag) or stimulated with TGF-ß1 (1 ng/ml) in suspension (Ag+TGF-ß1) did not express PAI-1 mRNA (A). Cells plated onto FN from suspension culture (AgFN) did produce low levels of PAI-1 transcripts, whereas plating onto FN in the presence of TGF-ß1 (AgFN+TGF-ß1) yielded optimal induction. PAI-1 induction as a consequence of FN attachment alone was also attenuated by addition of PD98059 during the 2 hour period of adhesion suggesting that MEK activity was also required for adhesion-dependent expression under growth factor-free conditions (B).

View larger version (29K): [in a new window]   Fig. 8. Matrix-type dependency of basal and TGF-ß1-induced PAI-1 expression. Quiescent (Q) T2 cells were trypsinized and replated on plastic dishes coated with BSA, fibronectin (FN) or vitronectin (VN) in the presence or absence of TGF-ß1 (1 ng/ml) for a 2 hour period. While both FN and VN induced PAI-1 mRNA transcripts, the amplitude of induction was significantly greater on FN-coated surfaces; TGF-ß1 stimulated expression only on T2 cells adhering to FN.

View larger version (42K): [in a new window]   Fig. 9. Relative spreading of T2 cells on fibronectin and vitronectin. Suspended T2 cells were seeded in serum-free medium to dishes previously coated with fibronectin or vitronectin (10 µg/ml). After 4 hours, random fields were photographed (representative examples shown) and the percent spread cells (i.e. non-refractive) calculated. Data plotted are the means±s.d. for assessments on three separate dishes/substrate. There was no difference in either T2 cell attachment or spreading on fibronectin or vitronectin.