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MEK/ERK pathway mediates cell-shape-dependent plasminogen activator inhibitor type 1 gene expression upon drug-induced disruption of the microfilament and microtubule networks

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

View larger version (39K): [in a new window]   Fig. 1. CD induces PAI-1 expression in growth-arrested R22 cells as a function of concentration and time of exposure. CD (in final concentrations of 2, 5 or 10 µM) or DMSO vehicle alone (0 µM) was added to serum-deprived R22 cell cultures. Total RNA was isolated 4 hours later and northern blots hybridized with 32P-labeled PAI-1 and A50 cDNAs (A). PAI-1 mRNA levels increased as a function of CD concentration; maximal expression was attained with 10 µM CD (Hawks and Higgins, 1998). The histogram in (B) represents a quantitative analysis of PAI-1 mRNA abundance (mean±s.d.) from three different experiments normalized to the A50 signal. Immunoblot detection of PAI-1 protein on transfers of electrophoretic separations of R22 cell extracts (20 µg protein/lane) at various times after CD treatment (C). PAI-1 increased as early as 1 hour post-stimulation and reached maximal levels by approximately 4 hours.

View larger version (67K): [in a new window]   Fig. 2. In situ distribution of CD-induced PAI-1 protein in quiescent R22 cells. The fractions of PAI-1 protein-expressing cells and the relative intensity of immunoreactive PAI-1 staining were assessed by fluorescence microscopy; both increased as a function of the time of CD exposure. Nuclei were visualized by DAPI staining. Although microfilament structure was progressively disrupted in 0.1 and 1.0 µM CD-treated cells, PAI-1 expression was only evident in 10 µM CD-stimulated cultures. Induction was more closely associated with changes in cell shape (Fig. 3) than actin skeleton disorganization. The apparent nuclear region accumulation of PAI-1 is probably the collective result of CD-induced cellular arborization and Golgi collapse around the nucleus.

View larger version (81K): [in a new window]   Fig. 3. Inverse correlation between extent of morphological response of quiescent R22 cells to cytoskeletal disruption and PAI-1 transcript abundance. Cells maintained under serum-free culture conditions for 3 days were generally well spread with little membrane ruffling (A). A 4 hour exposure to 10 µM CD resulted in an arborized phenotype (B), although the cellular periphery remained well demarcated and easily visible for footprint imaging. Colchicine exposure (10 µM, 4 hours) (C) did not produce the same arborized effect as CD, although the cellular footprint area was similarly reduced (by 35%) in CD- and colchicine-treated cultures. Computerized imaging was done on 75 randomly selected individual cells per culture condition in three sets of 25 cells each. The footprint area was significantly reduced by treatment with either drug at the maximal PAI-1 transcript-inducing concentration of 10 µM (Fig. 4). Comparison between DMSO and CD-treated cells indicated an approximate reciprocal relationship between cellular footprint area and PAI-1 expression (D). The histogram in (D) illustrates the mean±s.d. (in arbitrary units) for triplicate determinations of footprint area and PAI-1 levels.

View larger version (55K): [in a new window]   Fig. 4. Colchicine stimulates PAI-1 synthesis in quiescent R22 cells. Growth-arrested cells were treated with DMSO (solvent control) or colchicine (10 µM, final concentration) for 4 hours. The cellular microtubule network and PAI-1 protein were visualized in fixed and permeabilized cells by indirect immunocytochemistry with antibodies to tubulin and PAI-1, respectively (A). Cells exposed to DMSO were well spread with a highly branched microtubule network. Colchicine treatment disrupted the microtubule skeleton and significantly reduced the cell-spread area (Fig. 3, legend). PAI-1 protein was virtually undetectable by immunocytochemistry (A) and resolved at only extremely low levels by western blotting of extracts of control cells (B). Abundant PAI-1 protein was easily identified, by contrast, by both techniques in 4 hour colchicine-stimulated cells (A,B). Nuclei were stained with DAPI.

View larger version (66K): [in a new window]   Fig. 5. Metabolic requirements for CD-induced PAI-1 mRNA expression. Serum-deprived cells were pretreated for 30 minutes with actinomycin D (Act D; 5 µg/ml) or puromycin (Puro; 100 µg/ml) prior to a 4 hour exposure to CD (final concentration 10 µM). Optimal concentrations of inhibitors were determined previously (e.g. Ryan et al., 1996; Hawks and Higgins, 1998). Northern blots were probed with 32P-labeled cDNAs to PAI-1 and A50 (A). CD-induced PAI-1 transcription appeared to utilize a secondary (i.e. protein-synthesis-dependent) response mechanism. To evaluate the potential role of autocrine TGF-ß as a secondary response intermediate, quiescent R22 cell cultures were pretreated for 30 minutes with TGF-ß neutralizing antibodies prior to addition of either CD or TGF-ß1. RNA was isolated, and blots were probed for PAI-1 and A50 transcripts (B). Representative blots are shown.

View larger version (28K): [in a new window]   Fig. 6. CD-induced PAI-1 expression is sensitive to tyrosine kinase inhibitors. Quiescent R22 cultures were pretreated for 30 minutes with progressively increasing concentrations of genistein (A) or PPI (B) prior to CD treatment. RNA was isolated, and northern blots were probed for PAI-1 and A50 transcripts. Representative blots are shown in A and B. —/— refers to DMSO vehicle only. The corresponding histograms illustrate PAI-1 mRNA abundance normalized to A50 signal for replicate experiments (mean±s.d.). Concentrations of genistine and PP1 (>25 µM and 10 nM, respectively) effectively ablated PAI-1 transcript expression.

View larger version (60K): [in a new window]   Fig. 7. CD activates pp60c-src kinase activity. Cells were incubated in 10 µM CD and extracts prepared at the times indicated for the immune complex assay of pp60c-src activity. Autophosphorylated pp60c-src kinase and phosphorylated IgG heavy chain were evident at 7.5 minutes after CD addition and maximal at 15 minutes post-stimulation (A). Kinase activity rapidly declined thereafter. Addition of PP1 to cultures prior to CD treatment effectively inhibited pp60c-src kinase activity at the optimal 15 minute time point. Equal gel loading was confirmed by visualization of the IgG heavy chain by staining with Ponceau S (B) and by western blotting for pp60c-src and IgG (C). The histogram indicates quantitative data (mean±s.d.) for three independent experiments.

View larger version (32K): [in a new window]   Fig. 8. CD-mediated PAI-1 expression and ERK1/2 activation is MEK dependent. Quiescent R22 cells were pretreated with PD98059 (at the indicated concentrations) prior to addition of CD. Northern blots of total cellular RNA were probed with 32P-labeled cDNA probes to PAI-1 and A50; PAI-1 mRNA abundance was normalized to A50 signal (A). CD-induced ERK1/2 activity was assessed by a coupled immunoprecipitation—in-vitro-kinase assay using myelin basic protein (MBP) as a phosphorylation substrate with or without PD98059 pretreatment (B). Western blotting and Ponceau S staining (B) served to confirm ERK1/2 and MBP levels, respectively, for normalization of MBP phosphorylation activity. The histograms (A,B) illustrate results of three separate experiments (mean±s.d.). Untreated groups in both panels refer to DMSO vehicle only.

View larger version (44K): [in a new window]   Fig. 9. ERK1 and ERK2 are activated by CD treatment. The upper panels in (A,B) illustrate MBP phosphorylation by activated ERK1 and ERK2, respectively, as determined by the coupled immunoprecipitation—in-vitro-kinase assay at various times (mins) following CD addition. PD98059 (20 µM) or serum (final concentration of 20%) was added where indicated. Western blotting and Ponceau S staining served to confirm ERK and MBP levels, respectively (middle and bottom panels in A,B), used for normalization of MBP phosphorylation activity. Data plotted are the means±s.d. for at least three different experiments.

View larger version (47K): [in a new window]   Fig. 10. CD-induced ERK1/2 activity is dependent on a src-like tyrosine kinase. Quiescent R22 cells were preincubated with the indicated inhibitors prior to a 2 hour treatment with CD (10 µM final concentration); control cultures were maintained without additives. Serum-stimulation (FBS to a final concentration of 20%) provided a positive control for ERK activation. The MAPK assay used myelin basic protein (MBP) as the phosphorylation substrate (pMBP; upper panel). Western blot probed with pan-ERK antibodies (middle panel) and Ponceau S staining of MBP (bottom panel) served to confirm equivalent protein loading as well as to confirm normalization of MBP phosphorylation (mean±s.d.) in replicate experiments (histogram).

View larger version (47K): [in a new window]   Fig. 11. Colchicine-induction of PAI-1 expression is sensitive to PP1, puromycin and PD98059 and associated with ERK activation. Pretreatment of quiescent R22 cultures with PP1 significantly attenuated and completely ablated colchicine-mediated PAI-1 expression at 170 and 340 nM, respectively (A, left panel). PAI-1 induction was also effectively inhibited by exposure to puromycin (100 µg/ml) (A, right panel). Similarly, PD98059 at a final concentration of 10 µM reduced cellular PAI-1 levels by 65% whereas use of the inhibitor at 25 µM effectively blocked CD-induced PAI-1 synthesis (B). Exposure of quiescent R22 cells to colchicine-stimulated ERK1/2 phosphorylation by 30 minutes and ERK remained activated for at least 4 hours after addition of the drug (C). ERK phosphorylation, like PAI-1 induction, was PD98059 sensitive. In all cases, colchicine was used at 10 µM.

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