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. 2007 Jun;18(6):2169-78.
doi: 10.1091/mbc.e06-12-1121. Epub 2007 Apr 4.

FAK is required for TGFbeta-induced JNK phosphorylation in fibroblasts: implications for acquisition of a matrix-remodeling phenotype

Shangxi Liu  1 , Shi-wen Xu, Laura Kennedy, Daphne Pala, Yunliang Chen, Mark Eastwood, David E Carter, Carol M Black, David J Abraham, Andrew Leask
Affiliations

FAK is required for TGFbeta-induced JNK phosphorylation in fibroblasts: implications for acquisition of a matrix-remodeling phenotype

Shangxi Liu et al. Mol Biol Cell. 2007 Jun.

Abstract

Transforming growth factor beta (TGFbeta) plays a critical role in connective tissue remodeling by fibroblasts during development, tissue repair, and fibrosis. We investigated the molecular pathways in the transmission of TGFbeta signals that lead to features of connective tissue remodeling, namely formation of an alpha-smooth muscle actin (alpha-SMA) cytoskeleton, matrix contraction, and expression of profibrotic genes. TGFbeta causes the activation of focal adhesion kinase (FAK), leading to JNK phosphorylation. TGFbeta induces JNK-dependent actin stress fiber formation, matrix contraction, and expression of profibrotic genes in fak+/+, but not fak-/-, fibroblasts. Overexpression of MEKK1, a kinase acting upstream of JNK, rescues TGFbeta responsiveness of JNK-dependent transcripts and actin stress fiber formation in FAK-deficient fibroblasts. Thus we propose a FAK-MEKK1-JNK pathway in the transmission of TGFbeta signals leading to the control of alpha-SMA cytoskeleton reorganization, matrix contraction, and profibrotic gene expression and hence to the physiological and pathological effects of TGFbeta on connective tissue remodeling by fibroblasts.

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Figures

Figure 1.
Figure 1.
TGFβ induces JNK phosphorylation in fibroblasts: requirement for FAK. (A) TGFβ1 activates FAK and JNK phosphorylation in fak+/+ cells. Fibroblasts from fak+/+ and fak−/− mice were serum-starved and treated with or without TGFβ1 (4 ng/ml) for 30 min. Cell extracts were prepared and subjected to Western blot analysis with anti-phospho-JNK1, anti-total JNK1 antibodies, anti-phospho-FAK, and anti-FAK antibodies. (B) TGFβ1 results in redistribution of phospho-JNK epitopes in fak+/+ cells. Fibroblasts from fak+/+ and fak−/− mice were treated with TGFβ for 30 min, fixed in paraformaldehyde, and stained with anti-phospho-JNK antibody followed by FITC-conjugated secondary antibody (green stain). Cells were also stained with anti-vinculin antibody followed by Texas red–conjugated secondary antibody (to detect the cell periphery, red stain). Cells were counterstained with DAPI (to detect the nucleus, blue stain). Note that addition of TGFβ to fak+/+ cells resulted in the appearance of phospho-JNK epitopes throughout the cell, including at the cell periphery (arrow). Phospho-JNK staining remains perinuclear in fak−/− cells. Please also note that a 30-min treatment with TGFβ was insufficient to cause appearance of “supermature” FA in fak+/+ cells (C) The FAK/src inhibitor PP2 reduces TGFβ1-induced JNK phosphorylation in fak+/+ cells. Fibroblasts from fak+/+ were serum-starved and treated with or without TGFβ1 (4 ng/ml) for 30 min. Cells were pretreated with DMSO or PP2 (10 μM), as indicated. Cell extracts were prepared and subjected to Western blot analysis with anti-phospho-JNK1 and anti-total JNK1 antibodies or anti-phospho-FAK or anti-FAK antibodies as indicated.
Figure 2.
Figure 2.
MEFs deficient in FAK are smaller and less spread than fak +/+ MEFs. (A) Fibroblasts from wild-type (WT) and fak−/− (KO) mice were cultured and subjected to bright-field microscopy. Photographs were taken, and cell area (μm) was calculated using Northern Eclipse image analysis software. Average ± SD is shown (100 cells total/cell type). (B) A standard cell viability (MTT) assay was used to show that no significant difference in cell proliferation over 24 h was observed between WT and KO cells (n = 3; average ± SD is shown).
Figure 3.
Figure 3.
TGFβ induces α-SMA stress fiber formation in fibroblasts: requirement for JNK and FAK. (A) Indirect immunofluorescence analysis. Fibroblasts from wild-type (WT) and fak−/− (KO) mice were serum-starved and treated with or without TGFβ1 (4 ng/ml) for 24 h in the presence or absence of the JNK inhibitor SP600125 (SP). Cells were then fixed in paraformaldehyde and stained with α-SMA antibody followed by Texas red–conjugated secondary antibody. (B) Fluorescence intensity analysis. Cells obtained in A were analyzed using Northern Eclipse software by measuring fluorescence intensity across the cells, perpendicular to the α-SMA stress fibers. Intensity corresponds to individual α-SMA stress fibers. Note that JNK inhibition significantly impairs the formation of α-SMA stress fibers in normal fibroblasts and that the resultant cells are similar in appearance to fak−/− fibroblasts.
Figure 4.
Figure 4.
TGFβ induces collagen gel contraction in fibroblasts: requirement for JNK and FAK. Floating gel contraction assay. (A) Fibroblasts from wild-type (WT) and fak−/− (KO) mice were placed within collagen gel lattices. After polymerization, lattices were detached from tissue culture plates, treated with or without TGFβ1 (4 ng/ml) for 24 h in the presence or absence of the JNK inhibitor SP600125 (SP). Contraction was monitored by measuring gel weight. *Significantly different from untreated control WT cells (p < 0.05). (B) A standard cell viability (MTT) assay was used to show that no significant difference in cell proliferation within the collagen gels was observed between WT and KO cells in the presence or absence of TGFβ or the JNK inhibitor (n = 3; average ± SD is shown).
Figure 5.
Figure 5.
TGFβ induces collagen gel contraction in fibroblasts: requirement for JNK and FAK. FPCL assay. Fibroblasts from wild-type (WT) and fak−/− (KO) mice were placed within collagen gel lattices. After polymerization, lattices were detached from tissue culture plates and treated with or without TGFβ1 (4 ng/ml) for 24 h in the presence or absence of the JNK inhibitor SP600125 (SP). Contraction force generated across the gel was measured.
Figure 6.
Figure 6.
TGFβ is unable to induce “supermature” highly vinculin-positive focal adhesions (FA) in fak−/− fibroblasts. (A) Immunofluorescence analysis. Fibroblasts from wild-type (WT) and fak−/− (KO) mice were serum-starved and treated with or without TGFβ1 (4 ng/ml) for 24 h. Cells were then fixed in paraformaldehyde and stained with anti-vimentin antibody followed by Texas red–conjugated secondary antibody. Note appearance of intense vinculin-positive staining at the cell periphery of WT cells treated with TGFβ (arrow, corresponding to supermature FAs). (B) Quantification of supermature FA. Image analysis software was used to measure the intensity of vinculin staining per FA (average fluorescence intensity) in cells photographed for A. The average intensity ± SD for 100 FA is shown. *Significantly different from untreated control WT cells (p < 0.05). (C) Quantification of number of FAs per cell. WT cells possess more FAs (number of vinculin dots) per cell than fak−/− (KO) cells (100 cells, average number of FAs/cell ± SD) is shown. Note that TGF|gb treatment of either WT or KO cells did not significantly increase the number of FAs/cell.
Figure 7.
Figure 7.
TGFβ induces type I collagen and α-SMA proteins in fibroblasts: requirement for JNK and FAK. Fibroblasts from wild-type (WT) and fak−/− (KO) mice were serum-starved and treated with or without TGFβ1 (4 ng/ml) for 24 h in the presence and absence of SP600125. Proteins were subjected to Western blot analysis with anti-type I collagen, anti-α-SMA, and anti-GAPDH antibodies, as indicated. Note that JNK inhibition reduced α-SMA and type I collagen induction in response to TGFβ in WT cells. Also, please note that induction of α-SMA and type I collagen protein did not occur in fak−/− cells.
Figure 8.
Figure 8.
TGFβ is unable to induce expression of a cohort of profibrotic mRNAs in fak−/− fibroblasts. Fibroblasts from wild-type (WT) and fak−/− (KO) mice were serum-starved and treated with or without TGFβ1 (4 ng/ml) for 6 h. mRNAs were harvested and subjected to real-time PCR analysis with primers detecting type I collagen, α-SMA, thrombospondin-1, integrin α5, tenascin C, and vinculin. Samples were standardized to GAPHH. Fold-increase in response to TGFβ is shown. Note that vinculin was responsive to TGFβ in wild-type (WT) and fak−/− (KO) cells. Note that SD within samples was <10%. *Significantly different fold-induction in response to TGFβ in KO cells compared with WT cells (p < 0.05).
Figure 9.
Figure 9.
Transfection of MEKK1 expression vector restores the ability of TGFβ is to induce expression of a cohort of profibrotic mRNAs in fak−/− fibroblasts. Fibroblasts from fak−/− (KO) mice were transfected with empty expression vector or expression vector encoding activated MEKK1. Cells were serum-starved for 18 h after transfection and treated with or without TGFβ1 (4 ng/ml) for 6 h. mRNAs were harvested and subjected to real-time PCR analysis with primers detecting type I collagen, α-SMA, thrombospondin-1, integrin α5, tenascin C, or vinculin. Samples were standardized to GAPDH. Fold-increase in response to TGFβ is shown. Note that SD within samples was < 10%. *Significantly different fold-induction in response to TGFβ in KO cells transfected with activated MEKK1 compared with empty expression vector (p < 0.05).
Figure 10.
Figure 10.
Transfection of MEKK1 expression vector restores the ability of TGFβ is to induce α-SMA stress fibers in fak−/−fibroblasts. Fibroblasts from fak−/− (KO) mice were cotransfected with empty expression vector or expression vector encoding activated MEKK1 together with an expression vector encoding GFP. Cells were serum-starved for 18 h after transfection and treated with or without TGFβ1 (4 ng/ml) for 12 h. Cells were subjected to indirect immunofluorescence analysis using an anti-α-SMA antibody and an appropriate secondary antibody. Transfected cells were detected (using the GFP tag, arrow). Overexpression of MEKK1 restored the ability of TGFβ to induce α-SMA stress fibers.
Figure 11.
Figure 11.
Summary of the contribution of FAK and JNK to a matrix remodeling phenotype in fibroblasts. TGFβ activates FAK, which is required for JNK activation. JNK is, in return, required for the induction of α-SMA stress fiber organization, matrix contraction, and induction of expression of a cohort of profibrotic gene.
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References

    1. Chen Y., Blom I. E., Sa S., Goldschmeding R., Abraham D. J., Leask A. CTGF expression in mesangial cells: involvement of SMADs, MAP kinase, and PKC. Kidney Int. 2002;62:1149–1159. - PubMed
    1. Chen Y., et al. Matrix contraction by dermal fibroblasts requires transforming growth factor-beta/activin-linked kinase 5, heparan sulfate-containing proteoglycans, and MEK/ERK: insights into pathological scarring in chronic fibrotic disease. Am. J. Pathol. 2005;67:1699–1711. - PMC - PubMed
    1. Chen Y., Shi-wen X., Eastwood M., Black C. M., Denton C. P., Leask A., Abraham D. J., et al. Contribution of activin receptor-like kinase 5 (transforming growth factor beta receptor type I) signaling to the fibrotic phenotype of scleroderma fibroblasts. Arthritis. Rheum. 2006;54:1309–1316. - PubMed
    1. Cox B. D., Natarajan M., Stettner M. R., Gladson C. L. New concepts regarding focal adhesion kinase promotion of cell migration and proliferation. J. Cell Biochem. 2006;99:35–52. - PubMed
    1. Desmouliere A., Chaponnier C., Gabbiani G. Tissue repair, contraction, and the myofibroblast. Wound Repair Regen. 2005;13:7–12. - PubMed

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