The ternary complex factor Net regulates cell migration through inhibition of PAI-1 expression

Abstract

Net, Elk-1, and Sap-1 are members of the ternary complex factor (TCF) subfamily of Ets transcription factors. They form ternary complexes with serum response factor (SRF) on serum response elements of immediate early genes such as c-fos and egr-1 and mediate responses to growth factors and mitogen-activated protein kinase signaling. Although the TCFs have been extensively studied as intermediates in signaling cascades, surprisingly little is known about their different target genes and physiological functions. We report that Net homozygous mutant mouse embryonic fibroblasts have a defect in cell migration. This defect results at least in part from increased expression of plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor (serpin) that controls extracellular proteolysis and cell matrix adhesion. The defect in cell migration can be reverted by the addition of a PAI-1 blocking antibody. Net represses PAI-1 promoter activity and binds to a specific region of the promoter containing Ets binding sites in the absence of SRF. We conclude that Net is a negative regulator of PAI-1 expression and is thereby involved in cell migration.

FIG. 1.

Decreased migration of Net mutant MEFs during in vitro wound healing. (A) Time-lapse microscopy. Wild-type and Net mutant MEFs were grown to confluence and scrape wounded (wound edges are delimited by the white dashed lines). The plates were placed in a chamber fixed to a robotized platform of an inverted microscope. Images (magnification, ×40; Hoffman contrast) were collected every 5 min for 30 h. Time after wounding is indicated above the panels (0, 6 h, 12 h, and 18 h). Representative pictures of triplicates of two pairs of wild-type and Net mutant MEFs are shown. (B) Kinetics of wound healing. Wound areas are the average of triplicates with each of two pairs of wild-type and Net mutant MEFs. (C) BrdU incorporation during wound healing. Aphidicolin (40 μM) was added where indicated (panels b in B and C). (D) Actin and focal adhesion staining of migrating MEFs. Wild-type and Net mutant MEFs grown on coverslips were scrape wounded and stained after 10 h for the actin cytoskeleton (phalloidin staining) (top) or focal adhesions (vimentin immunocytostaining) (bottom). Representative pictures of migrating cells are shown. Mut, Net mutant.

FIG. 2.

Migration and adhesion of Net mutant and wild-type MEFs on ECM substrates. (A) Boyden chamber migration assay. MEFs that had migrated through porous membranes coated with BSA, VN, or FN were quantified 8 h after seeding of 2.5 × 10⁵ cells. (B) Adhesion assay. MEFs that had adhered to BSA-, VN-, or FN-coated wells were quantified 1 h after seeding of 2 × 10⁴ cells. Two pairs of wild-type and Net mutant MEFs were used (A and B). The cells that had passed through the matrix barrier (A) or adhered to the substrate (B) were stained, and the eluent OD was measured at 570 nm. Mut, Net mutant. (C) Boyden chamber migration assays. Mouse SEND cells were transfected with 20 nM siRNA targeting Net (@Net) or control GL2-Luciferase (@Luc). At 48 h posttransfection, cells were harvested for Western blotting (a) or Boyden chamber migration assays (b). SEND cells that had migrated through porous membranes coated with BSA or VN were quantified 8 h after seeding of 2.5 × 10⁵ cells per chamber.

FIG. 3.

PAI-1 is overexpressed in Net mutant MEFs. (A) Atlas 588 gene arrays were hybridized with probes derived from subconfluent growing MEFs. The spots for PAI-1, uPA, tPA, and the housekeeping gene HGPRT are shown. Mut, Net mutant. (B) PAI-1 and uPA mRNA quantified by real-time quantitative RT-PCR. The values were normalized to 28S rRNA. (C) Net, mutant Net (Net δ), Elk, Sap-1a, SRF, and PAI-1 proteins detected by immunoblotting; n.s., nonspecific band. (D) Chromogenic uPA activity assay performed on wild-type and Net mutant MEF cell extracts. (E) Down-regulation of endogenous Net by antisense RNA (a) or siRNA (b) increases PAI-1 expression in mouse SEND cells. (a) Cells were transfected with pBSK (lane 1) or increasing amounts of p601D-anti-Net that produces antisense (AS) net RNA (lane 2, 1 μg; lane 3, 2 μg), and immunoblotted for Net, PAI-1, and TBP. (b) Cells were not transfected (NT; lanes 1 and 4) or transfected with 20 nM siRNAs targeting GL2-luciferase (lanes 3 and 5), Net (lane 2), and SRF (lane 6). Protein extracts were harvested 48 h posttransfection and immunoblotted for Net, SRF, Elk-1, PAI-1, and TBP.

FIG. 4.

PAI-1 expression after in vitro scrape wounding of wild-type and mutant MEFs. Wild-type and mutant MEFs were grown to confluence in 10-cm plates and scraped to form a criss-cross of wounds 1 cm apart. At 0, 2 h, 4 h, 8 h and 12 h after wounding, the cell culture medium was sampled and total RNA or cell lysates were prepared. (A) PAI-1 mRNA induction after wounding quantified by real-time quantitative RT-PCR (values normalized by 28S rRNA). Mut, Net mutant. (B) PAI-1 in cell extracts and secreted in the culture medium was detected by Western blotting. TBP was used as an internal control. n.s., nonspecific band. (C) Immunocytochemistry of PAI-1 protein expression at 0 and 4 h after wounding. The white dashed lines delimit the wound edges. Nuclei are stained blue (Hoechst), and PAI-1 is stained red.

FIG.5.

Effect of anti-PAI-1 blocking antibody on in vitro wound healing. Wild-type and Net mutant MEFs were grown to confluence and scrape wounded. The plates were placed in a chamber fixed to a robotized platform of an inverted microscope and examined by time-lapse video microscopy. The graphs show the kinetics of wound healing. Wound areas are the averages of duplicates with each of two pairs of wild-type and Net mutant MEFs. Wound areas of wild-type (wt; blue curve) and Net mutant (mut; red curve) fibroblasts in the absence of antibody (A) or in the presence of different concentrations of PAI-1-neutralizing monoclonal antibody in the medium of wild-type (Ba) or mutant (Ca) MEFs are shown. The histograms represent wound areas at 24 h postwounding with different concentrations of PAI-1-neutralizing monoclonal antibody for wild-type (Bb) and Net mutant MEFs (Cb). (*, P < 0.05; **, P < 0.01).

FIG. 6.

Endogenous Net binds to the PAI-1 promoter in vivo. Chromatin immunoprecipitation assays were performed with wild-type (WT, lanes 1 to 4) and Net mutant (Mut, lanes 5 to 8) MEFs. Formaldehyde cross-linked and sonicated cell lysates were used for clathrin (Ctrl, control; lanes 2 and 6), Net (lanes 3 and 7) and SRF (lanes 4 and 8) IPs. The Net antibody recognizes both wild-type (lane 3) and mutated (Net δ; lane 7) forms of Net. Coprecipitated DNA fragments were analyzed by PCR with specific primers for the c-fos SRE (positive control, promoter region −338 to −153), and PAI-1 proximal (−715 to −367) and distal (−2019 to −1540) promoter regions. PCR products were resolved in a 2% agarose gel.

FIG. 7.

Mapping of Net-responsive elements in the PAI-1 promoter. (A) Schematic representation of PAI-1 promoter 5′ deletion and point mutants linked to luciferase coding sequence (Luc) and relative luciferase activities of these constructs in wild-type (wt) and Net mutant (mut) MEFs. Each point was repeated in triplicate in two independent experiments (**, P < 0.005; *, P < 0.05). Luciferase activities were corrected for β-galactosidase activity expressed from the pCMV-LacZ internal control. For each construct, the values are relative to wild-type MEFs. (B) Sequence of the PAI-1 (−519; −319) promoter region and localization of the three putative EBSs mutated in pP(−1001, 3EBS mut)Luc.