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First published online November 21, 2007
doi: 10.1242/10.1242/jcs.03492

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MAP-kinase activity necessary for TGF1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397

Tomoko Hayashida^1,3,*, Ming-Hua Wu², Amy Pierce^1,, Anne-Christine Poncelet⁴, John Varga² and H. William Schnaper^1,3

¹ Division of Kidney Diseases, Department of Pediatrics
² Division of Rheumatology, Department of Medicine, The Feinberg School of Medicine, Northwestern University, 303 E Chicago Ave, Chicago IL 60611, USA
³ Children's Memorial Research Center, Chicago IL 60614, USA
⁴ University of Washington, School of Medicine, Seattle, WA, USA

View larger version (85K): [in this window] [in a new window]   Fig. 1. TGF1 modulation of FAK phosphorylation. (A) Whole lysates of TGF1-treated cells were analyzed for FAK expression (top panel) and specific phosphorylation on tyrosine residues 397 or 925 (bottom panels) by immunoblotting. (B) Mesangial cells serum-deprived for 24 hours were treated with TGF1 (right panels) or vehicle (left panels) for 30 minutes, then fixed and stained for phospho-Y397 FAK (pY397, top) or phospho-Y925 FAK (pY925, bottom). Bar, 20 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 2. FAK phosphorylation and stress fiber formation in cells plated on pLL. (A) Attachment and spreading after 3 hours. Mesangial cells were serum deprived for 24 hours and then non-enzymatically lifted and re-plated on either gelatin-coated (left) or pLL-coated (right) glass slides. After 3 hours, the cells were fixed and stained for F-actin (red, top panels) and vinculin (green, middle panels). Merged images are shown at the bottom. (B) Effect of TGF1 on FA and cytoskeletal structures. (a) Representative merged images of immunostaining for F-actin (red) and phospho-Y397 FAK (green) with cells re-plated on gelatin (top panels) or pLL (bottom panels) for 6 hours, followed by treatment with TGF1 (right panels) or vehicle (left panels) for 30 minutes. Bar, 20 µm. (b) Zoomed-in images of the FAs, shown next to their respective images, were obtained, using Photoshop 7.0 software (Adobe), to demonstrate colocalization (yellow). Bar, 2 µm. (c) Colocalizing pixels were selected by CoLocalization Express software (shown as dots in the image plots). The pixels of colocalization from four separate images were summed and are shown as a graph below the images. White bars, vehicle; black bars, TGF1. *P<0.05, compared to values in control conditions with vehicle. (C) Cells plated on gelatin or pLL for 4 hours were treated with TGF1 (1.0 ng/ml) or vehicle for 30 minutes and FAK phosphorylation at Y397 (top) or Y925 (middle) was analyzed by immunoblotting. (Graph, below) A representative set of blots and densitometoric analysis of four experiments for phospho-Y397-FAK (left) and phospho-Y925-FAK (right) over FAK expression are shown. White bars, vehicle; black bars, TGF1 treated. *P<0.05, compared to values on gelatin without TGF1; P<0.05, compared to values on gelatin without TGF1.

View larger version (34K): [in this window] [in a new window]   Fig. 3. TGF1 induction of collagen expression on pLL. (A) Mesangial cells serum-deprived for 24 hours were trypsinized and re-plated on either gelatin-or pLL-coated plates. After 3 hours, these cells and two additional plates that were not trypsinized (retained on plate, two left-most lanes) were treated with TGF1 (1.0 ng/ml, 8 hours) and were analyzed for 1(I) collagen mRNA and 28S expression. Representative blots from one of six independent experiments are shown. (B) Cells transiently transfected with a luciferase construct of–0.42(I) collagen promoter and a plasmid expressing -galactosidase were trypsinized and re-plated on either gelatin-or pLL-coated plates. After 20 hours of treatment with TGF1 (1.0 ng/ml), cells were harvested and analyzed for luciferase activity. Relative luciferase activity (mean ± s.e.m., n=3) corrected for -galactosidase expression of a representative experiment out of five independent experiments is shown. White bars, vehicle; black bars, TGF1. *P<0.05 compared to values on gelatin without TGF1. Numbers in the graph represent fold induction by TGF1 over control.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effects of pLL on Smad and ERK signals. (A) Cells on either gelatin-or pLL-coated plates were prepared as described in Fig. 3 and total protein harvested after TGF1 treatment (1.0 ng/ml, 30 minutes) was subjected to immunoblotting with anti-linker-region phospho-Smad3 [207/212; pSmad3(L)], anti-C-terminal phospho-Smad3 [423/425; pSmad3(C)] and anti-Smad2/3 antibodies. Representative blots from one of five independent experiments are shown. WCL, whole-cell lysate. (Right) Intensity of the immunoreactive bands for C-terminal (triangles; white, gelatin; black, pLL) or linker-region (squares) phosphorylation of Smad3 were corrected for Smad3 expression levels (y axis) and plotted against phospho-Y397 FAK levels corrected for FAK expression (x axis) in each corresponding experiment (representative blots for FAK shown in Fig. 2C). (B) Cells were transiently transfected with a plasmid that expresses an activation domain of Elk linked to the DNA-binding region of yeast Gal4, along with a Gal4-luciferase reporter construct, then replated on either gelatin or pLL. After 20 hours of TGF1 treatment, cells were harvested and luciferase activity was analyzed to indicate Elk activation. Graph shows mean ± s.e.m. (n=3) of luciferase activity after correction for -galactosidase expression from five separate experiments. Numbers in the graph represent fold induction by TGF1 over control. White bars, vehicle; black bars, TGF1 (1.0 ng/ml 20 hours). *P<0.05, compared to values on gelatin without TGF1.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Overexpressed caMEK restores phosphorylation of the Smad3 linker region and–0.42(I) collagen promoter activity in cells cultured on pLL. (A) HKCs were transfected with a construct that expresses caMEK, along with an Elk-Gal transactivation system (left-hand graph) or the–0.42(I) collagen promoter (right-hand graph) and then the cells were re-plated on either gelatin-or pLL-coated six-well plates. TGF1-induced reporter activity was corrected for -galactosidase expression and representative results (mean ± s.e.m.) performed in triplicate from one of three experiments are shown as a graph. Numbers in the graph indicate relative increase of Gal4-Elk-luc activity over control in the absence of TGF1 (left-hand graph) or fold induction of 2(I) collagen promoter activity by TGF1 over each control (right-hand graph). White bars, vehicle; black bars, TGF1 (1.0 ng/ml, 12 hours). (B) HKCs transfected with constructs expressing Flag-Smad3 and caMEK were re-plated on either gelatin or pLL and cultured for another 3 hours followed by TGF1 treatment (1.0 ng/ml, 30 minutes). Expressed Smad3 was immunoprecipitated with anti-Flag affinity gel and phosphorylation was evaluated with phospho-specific antibodies (top panels). Y397-FAK phosphorylation and Flag expression using whole-cell lysate (WCL) are shown in the bottom panels. LB, lysis buffer control for immunoprecipitation.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Effects of FAK mutants on Elk-Gal-reporter and–0.42(I) collagen promoter activity induced by TGF1. (A) Mesangial cells were transfected with the Elk-Gal reporter system as described for Fig. 4C, along with an expression vector for either wild-type FAK (WT) or point mutants (Y397F or Y925F) of FAK, and TGF1 induction of ERK activity was evaluated as Elk-mediated luciferase activity. (B)–0.42(I) collagen promoter activity was stimulated by TGF1 in the presence of either a wild-type or point-mutant (Y397F or Y925F) FAK. A representative result (mean ± s.e.m.) performed in triplicate from one of four separate experiments is shown. Values were corrected for -galactosidase expression. Fold induction by TGF1 in each condition is shown in the graphs. White bars, vehicle; black bars, TGF1. *P<0.05, compared to each control by a post-hoc analysis.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Overexpressing caMEK bypasses inhibition of–0.42(I) collagen promoter activity by the Y397F FAK mutant. Collagen promoter activity in mesangial cells was analyzed in the presence of either wild type (wt) or Y397F FAK, along with an expression vector for caMEK or its control empty vector. Luciferase values were corrected for -galactosidase expression and representative results (mean ± s.e.m.) obtained in triplicate from one of four separate experiments is shown. Numbers in the graph indicate fold induction by TGF1. White bars, vehicle; gray or black bars, TGF1 (1.0 ng/ml, 24 hours). *P<0.05 compared to each control by a post-hoc analysis.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Effect of expressing a Y397F FAK mutant on TGF1-induced Smad3 phosphorylation. (A) Wild type (WT) or Y397F FAK was transfected along with a vector expressing Flag-tagged Smad3 in HKCs for 20 hours, followed by treatment with TGF1 (1.0 ng/ml) or vehicle for 30 minutes. Expressed Smad3 was immunoprecipitated with anti-Flag affinity gel and Smad phosphorylation was evaluated with phospho-specific antibodies. LB, lysis buffer control for immunoprecipitation. (Bottom) Intensities of the immunoreactive bands for phosphorylated Smad3 at either the C-terminus [423/425; pSmad3(C)] or linker region [207/212; pSmad3(L)] were corrected for expression levels of Smad3 construct detected as Flag expression and demonstrated graphically (mean ± s.e.m., n=6, *P<0.05 compared to those with wild-type FAK with TGF1). (B) Flag-tagged Smad3 wild-type or His-tagged Smad3 EPSM constructs were transfected. Expressed proteins were immunoprecipitated with appropriate anti-tag antibody and Smad3 phosphorylation levels either for linker-region (top panel) or C-terminal (second panel) serines were evaluated. Expression of the Smad3 constructs detected by the corresponding antibody to the tag is also shown (bottom panels).

View larger version (40K): [in this window] [in a new window]   Fig. 9. ECM accumulation and Smad phosphorylation and FAK phosphorylation in kidneys of bleomycin-treated mice. (A) Protein lysate from frozen kidney samples of bleomycin-treated (bleo) or control mice were analyzed for expression of type I collagen and Smad3 phosphorylation by immunoblotting. Representative results from two animals are shown. (B) Immunofluorescent staining of renal cortex sections for phospho-Y397 FAK (top panels) and FAK (bottom panels) is shown. DAPI staining (blue) for nuclei is also shown to locate glomeruli in the upper panels.

View larger version (18K): [in this window] [in a new window]   Fig. 10. Proposed scheme of interactions between the FAK/ERK and TGF/Smad pathways in mesangial cell collagen expression. The pathway described in the present study is shown in the box. The exact mechanism(s) by which phosphorylation of the Smad3 linker region and ERK activity affect type I collagen gene transcription are uncertain; therefore, the connections are indicated by broken lines. MEK, a constitutively active form of which we used to directly activate ERK in the absence of phospho-Y397 FAK in Figs 5 and 7, is directly upstream of ERK.

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