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FAK-dependent regulation of myofibroblast differentiation

Roseanne S. Greenberg^*,1, Audrey M. Bernstein^*, Miriam Benezra^*, Irwin H. Gelman, Lavinia Taliana^* and Sandra K. Masur^*

^* Department of Ophthalmology, Mount Sinai School of Medicine, New York New York, USA; and

Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, USA

¹Correspondence: Department of Ophthalmology, Box 1183, Mt. Sinai School of Medicine, 1 Gustave Levy Pl., New York, NY 10029-6574, USA. E-mail: roseanne.greenberg{at}mssm.edu

SPECIFIC AIMS

The aim of this study was to determine the role of focal adhesion kinase (FAK) in the phenotypic shift between fibroblasts and myofibroblasts, two cell types important in wound healing. Signals from transforming growth factor beta (TGFßbeta;) and fibronectin induce fibroblasts to differentiate into smooth muscle - actin (SMA)-expressing myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to loose SMA expression and dedifferentiate into fibroblasts. Because we found that FAK was not necessary for TGFßbeta;-mediated myofibroblast differentiation, but FAK was necessary for FGF/h-mediated inhibition of myofibroblast differentiation, we embarked on the elucidation of the role of FAK role in FGF signaling.

PRINCIPAL FINDINGS

1. The absence of FAK leads to unregulated myofibroblast differentiation
Earlier reports implicated TGFßbeta;-induced fibronectin signaling and FAK in myofibroblast differentiation. This led to the prediction that in the absence of FAK, cells would remain fibroblasts even after TGFßbeta; treatment. To evaluate this, we used mouse embryonic fibroblasts (MEFs) lacking or expressing FAK (FAK –/– and FAK +/+) and counted the number of cells in which SMA was expressed and incorporated into stress fibers, a phenotype of differentiated myofibroblasts (Fig. 1 ). We studied the cells in serum-free medium in the absence of added growth factors, or with TGFßbeta; to promote the myofibroblast phenotype (SMA expression) or with FGF/h to promote the fibroblast phenotype (lack of SMA expression). In contrast to the prediction, FAK –/– MEFs were myofibroblasts under all three growth conditions. Most notably after treatment with FGF/h, FAK –/– MEFs remained as myofibroblasts, (Fig. 1C, D , FAK –/–: 95% myofibroblasts), whereas FAK +/+ MEFs (Fig. 1A, B ), and DA2 (FAK re-expressing) MEFs, (Fig. 1E, F ), were predominately fibroblasts (FAK +/+: 2%, and DA2: 3% myofibroblasts). Thus, FAK was not only dispensable for myofibroblast formation, but the absence of FAK promoted the myofibroblast phenotype.

View larger version (95K): [in this window] [in a new window]   Figure 1. FAK –/– MEFs were predominately myofibroblasts even after treatment with FGF-2/heparin. FAK +/+, FAK –/–, and DA2 MEFs were treated with 1–20 ng/ml FGF-2 and 5 µg/ml heparin in SSFM for at least 3 days. FAK, (arrows in A, E), was immunodetected in fixed cells with monoclonal anti-FAK followed by secondary goat anti-mouse conjugated to Alexa 488, and SMA, (arrow heads in D), with mouse anti SMA conjugated to cy3. FAK was immunodetected in FAK +/+ (A) and DA2 MEFs (E), but not in FAK –/– MEFs (C). In contrast, SMA stress fibers were not detected in FAK +/+ MEFs (B) nor DA2 MEFs (F), which express FAK, but were detected in FAK –/– MEFs (D). Scale bars = 10 µm. Percent myofibroblasts: FAK +/+ MEFs: 2% ± 3%; FAK –/– MEFs: 95% ± 7%; DA2: 3% ± 5%. Values are means from 4 independent experiments ± SD P < 0.05 FAK –/– compared with FAK +/+, and P < 0.05 FAK –/– compared with DA2.

We found that both FAK +/+ MEFs and FAK –/– MEFs secreted comparable amounts of active TGFßbeta;. We posit that this secreted TGFßbeta; stimulates SMA expression and thus myofibroblast differentiation. We determined that both cell types have the ability to respond to exogenous TGFßbeta; with SMA synthesis. However, we found that FGF/h treatment decreases SMA expression only in cells expressing FAK. Thus FAK was necessary for FGF-mediated negative regulation of SMA expression.

2. FAK shRNA decreases FAK and increases SMA protein expression
Using an alternative approach to decrease FAK protein expression in the MEFs, we nucleofected FAK+/+ MEFs with FAK short hairpin RNA (shRNA). As determined by immunoblotting and immunofluorescence expression of FAK shRNA in FAK +/+ MEFs greatly decreased FAK protein expression. Concomitantly with the decrease in FAK concentration, there was a strongly decreased response to FGF, which was reflected in its inability to inhibit myofibroblast differentiation. After FGF/h treatment, only 18% of FAK +/+ MEFs (nucleofected with empty vector) were myofibroblasts, whereas 64% of MEFs, in which FAK was knocked down, remained as myofibroblasts.

3. FGFR cell-surface expression and downstream signaling are decreased in FAK –/– MEFs
Our results indicated that FAK was integral to the FGF-dependent inhibition of SMA expression. FGF mediates its effect by binding to its receptor (FGFR) and activation of a signaling cascade. To evaluate whether FAK was required for FGFR expression, we measured the cell surface expression of FGFRs by incubation of cells with [¹²⁵I] FGF-2. We found that FAK –/– MEFs, bound 50% less [¹²⁵I] FGF-2, as compared to FAK +/+ and DA2 MEFs (Fig. 2 A, B). Furthermore, FGFR phosphorylation in FGF/h-treated FAK –/– MEFs was significantly decreased as compared to FAK +/+ MEFs (Fig. 2C ). The difference in phosphorylation is greater than the 50% difference in FGFR expression. This may be because FGFRs signal synergistically, or FAK may directly contribute to FGFR phosphorylation.

View larger version (23K): [in this window] [in a new window]   Figure 2. FAK was required for maximal surface expression of FGFRs and for phosphorylation of FGFRs, and FRS2. A, B) FGFR surface expression was reduced in the absence of FAK. FGFR surface expression was determined by [125I]FGF-2 binding assay as described in Material and Methods. Percent [125I]FGF-2 bound per cell of FAK –/– MEFs was compared to FAK-expressing MEFs: FAK +/+, P < 0.05 (A) or DA2 (hygro), P < 0.05 (B). FAK –/– MEFs bound 50% less [125I]FGF-2 compared to MEFs containing FAK (FAK +/+ and DA2). Values are means from 3 independent experiments ± SD. C) FGFR1 phosphorylation was decreased in the absence of FAK. FGFR tyrosine phosphorylation was determined in FAK +/+ and FAK –/– MEFs that were serum-starved and incubated with or without 50 ng/ml FGF-2 and 5 µg/ml heparin for 10 min. Cell lysates were immunoprecipitated with 1, rabbit anti FGFR1 or 2, nonspecific rabbit IgG. Chemiluminescence was visualized on Kodak Biomax MS film. 1: In response to FGF/h, FAK +/+ MEFs had significantly more FGFR1 phosphorylation than FAK –/– MEFs. Experiment was performed 2 times with similar results. D) FRS2 phosphorylation was absent in FAK –/– MEFs. FAK +/+ and FAK –/– MEFs were serum-starved and incubated with or without 20 ng/ml FGF-2 and 5 µg/ml heparin for 10 min. Cell lysates were immunoprecipitated with 1, rabbit anti-FRS2 or 2, nonspecific rabbit IgG. Chemiluminescence was visualized on a Kodak Image Station 440 CF. Experiment was performed 3 times with similar results.

4. FAK is necessary for FGF signaling via FGF receptor substrate 2 (FRS2) and extracellular signal-regulated kinase (ERK)
FRS2 is the immediate downstream substrate of FGFRs, which links FGF signaling to ERK activation. We showed that in FAK –/– MEFs treatment with FGF/h induced little or no phosphorylation of FRS2 (Fig. 2D ), nor of ERK. In contrast, in FAK +/+ MEFs, treatment with FGF/h induced strong phosphorylation of FRS2 (Fig. 2D ), and ERK. This is the first report that establishes a role for FAK in regulating signaling through the FGF/FRS2/ERK pathway.

In summary, the absence of FAK coincided with reduced FGFR surface expression and activity, decreased FGF signaling via the FGFR/FRS2/ERK pathway, and increased expression of SMA. Thus, FAK plays a crucial role in FGF signaling, and FAK is necessary for FGF-mediated maintenance of the fibroblast phenotype and myofibroblast de-differentiation, but not for TGFßbeta;-induced differentiation to the myofibroblast phenotype.

CONCLUSIONS AND SIGNIFICANCE

This is the first report that establishes a role for FAK in promoting FGF signaling and in FGF-mediated down-regulation of SMA, a marker of myofibroblasts. In cells in which FAK protein was greatly reduced by shRNA or entirely absent (FAK –/– MEFs), treatment with FGF failed to decrease SMA expression. Furthermore, in the absence of FAK, FGFRs surface expression and signals downstream of FGF were decreased.

We propose a working model that implicates FAK in regulation of myofibroblast differentiation by integrating two different growth factor pathways (Fig. 3 ). Our model starts with previous data; as TGFßbeta; induces myofibroblast differentiation, it also increases expression of integrins and fibronectin (Fig. 3 , step 1). Integrin binding to fibronectin activates FAK in TGFßbeta;-mediated myofibroblast differentiation (Fig. 3 , step 2). FAK activation leads to increased cell-surface expression of FGFRs by increased retention of FGFR in the cell membrane and/or by increased FGFR gene expression (Fig. 3 , step 3). Increased cell-surface expression of FGFRs transmits FGF signals and increases activation of the FGF pathway via FRS2/ERK (Fig. 3 , step 4), which negatively regulates the levels of SMA (Fig. 3 , step 5). Our model includes a role for FAK in FGF inhibition of SMA induction due to the fact that the absence of FAK makes cells refractory to FGF/h-mediated inhibition of SMA expression. We hypothesize that in vivo, FAK activation by TGFßbeta; may contribute to a negative feedback mechanism that prevents excessive myofibroblast differentiation, which is linked to fibrosis. Failure to down-regulate extracellular matrix (ECM) deposition and/or SMA stress fiber formation on wound closure has been linked to fibrosis. Our results suggest that FAK activation could inhibit myofibroblast differentiation, reduce their proportion in a tissue, and ameliorate fibrosis. These insights add to our knowledge of molecular mechanisms of fibrosis and hopefully contribute to improved approaches to wound healing.

View larger version (36K): [in this window] [in a new window]   Figure 3. FAK signals increase FGFR surface expression and inhibit SMA expression. Numbers indicate sequential steps in the signaling scheme. TGFßbeta; induces myofibroblast differentiation and increases expression of integrins and fibronectin (step 1). Fibronectin binding to integrins activates FAK (step 2). FAK activation leads to increased cell-surface expression of FGFRs by increased retention of FGFR in the cell membrane and/or by increased FGFR gene expression (step 3). Increased cell-surface expression of FGFRs transmits FGF signals and increases activation of the FGF pathway via FRS2/ERK (step 4), which negatively regulate the levels of SMA (step 5). TGFßbeta;r, TGFßbeta; receptor; FGFR, FGF receptor; HSPG, heparin sulfate proteoglycan; G, Grb2; S, Sos.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4838fje

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