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Published as doi: 10.1096/fj.05-4838fje.
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(The FASEB Journal. 2006;20:1006-1008.)
© 2006 FASEB

FAK-dependent regulation of myofibroblast differentiation

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

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

{dagger} Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York

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

ABSTRACT

Fibroblasts and myofibroblasts both participate in wound healing. Transforming growth factor beta (TGFß) induces fibroblasts to differentiate into myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to "de-differentiate" into fibroblasts. TGFß induces expression of smooth muscle alpha actin (SM{alpha}A) and incorporation into in stress fibers, a phenotype of differentiated myofibroblasts. Additionally, TGFß induces the expression of fibronectin and fibronectin integrins. Fibronectin-generated signals contribute to the TGFß-mediated myofibroblast differentiation. Because fibronectin signals are transmitted through focal adhesion kinase (FAK), it was predicted that FAK would be essential to TGFß-mediated myofibroblast differentiation. To determine whether the FAK signaling pathway is required for myofibroblast differentiation, we used two approaches to decrease FAK in mouse embryo fibroblasts (MEFs): 1) FAK +/+ MEFs, in which FAK protein expression was greatly decreased by short hairpin RNA (shRNA), and 2) FAK –/– MEFs, which lack FAK. In both cases, the majority of cells were myofibroblasts, expressing SM{alpha}A in stress fibers even after treatment with FGF/h. Furthermore, both the surface expression of FGFRs and FGF signaling were greatly reduced in FAK–/– MEFs. We conclude that FAK does not contribute to TGFß-dependent myofibroblast differentiation. Instead, FAK was necessary for FGF/h signaling in down-regulating expression of SM{alpha}A, which is synonymous with myofibroblast differentiation. FAK activation could contribute to regulating myofibroblast differentiation, thereby ameliorating fibrosis.—Greenberg, R. S., Bernstein, A. M., Benezra, M., Gelman, I. H., Taliana, L., Masur, S. K. FAK-DEPENDENT REGULATION OF MYOFIBROBLAST DIFFERENTIATION.


Key Words: FGF • TGFß • fibrosis

DURING WOUND REPAIR, fibroblasts at the wound margin differentiate into myofibroblasts, exemplified by their SM{alpha}A containing stress fibers (1 2 3 4 5 6) . The force generated by integrin-mediated interaction between SM{alpha}A stress fiber terminals and the extracellular matrix (ECM) contributes to wound closure (4 , 7 8 9 10 11 12) . In a properly healed wound, few myofibroblasts remain and the persistence of myofibroblasts correlates with fibrosis (1 , 5 , 13 14 15 16 17) . Thus, understanding the pathways that influence myofibroblast differentiation in a wound will contribute to promoting healthy repair.

Previous studies show that in fibroblasts TGFß induces expression and incorporation into stress fibers of SM{alpha}A, which is synonymous with myofibroblast differentiation (1 , 3 , 18 19 20 21 22 23) . TGFß also induces fibronectin synthesis, secretion, and incorporation into the ECM, as well as expression of fibronectin integrins (17 , 18 , 24 25 26 27 28 29 30 31) . Signals from both TGFß and fibronectin are required to promote myofibroblast differentiation in many tissues (3 , 21 , 26 , 30 , 32 33 34 35 36 37) . Integrin-mediated fibronectin signals are transmitted by FAK, a 120-kDa nonreceptor protein tyrosine kinase. Focal adhesion kinase F-actin (FAK) is localized to focal adhesions where it is activated by phosphorylation after integrin-mediated cell attachment (38 39 40 41 42) . Thus, it was hypothesized that FAK would transduce the integrin-mediated fibronectin signals that promoted SM{alpha}A expression and stress fiber organization, and thus myofibroblast differentiation. Given that 1) TGFß stimulates expression of SM{alpha}A, fibronectin and fibronectin integrins and that 2) FAK transmits signals from fibronectin integrins, we reasoned that myofibroblast differentiation induced by TGFß and fibronectin would be FAK dependent.

We took advantage of the availability of MEFs lacking FAK to explore the role of FAK in regulating the differentiation of fibroblasts to myofibroblasts. On the basis of the previous studies described above implicating fibronectin signaling and FAK in myofibroblast differentiation, we predicted that in the absence of FAK, MEFs would remain fibroblasts even after TGFß treatment. In contrast to this prediction, in the absence of FAK, MEFs strongly expressed SM{alpha}A in stress fibers even without treatment with exogenous TGFß. However, FAK was necessary for FGF/h signaling and down-regulation of SM{alpha}A expression. These results were confirmed in FAK+/+ MEFs where FAK was greatly decreased by expression of shRNA specific for FAK. In the absence of FAK, FGFR surface expression and signals downstream of FGF were decreased. This is the first report to establish a role for FAK in promoting FGF signaling. Our data demonstrate that FAK plays a role in down-regulating SM{alpha}A. FAK’s regulation of myofibroblast differentiation may significantly contribute to preventing fibrosis.

MATERIALS AND METHODS

Cell culture
MEFs obtained and grown as described were FAK +/+ and FAK –/– MEFs (43) , FAK –/– (hygro), DA2 (FAK re-expressing) (44) , and Src/Fyn/Yes –/– (45) . FAK –/– (hygro) and DA2 MEFs, were grown on 0.1% gelatin-coated coverslips or cell culture dishes. All MEFs were passaged by addition of 0.5 x trypsin, EDTA (Invitrogen, Carlsbad, CA) and were split 1:3 to 1:10. For growth factor treatment MEFs were grown in supplemented serum-fee medium (SSFM): DMEM/F-12 (Invitrogen) with antibiotic-antimycotic, 50 µg/ml gentamicin solution, RPMI 1640 Vitamin Mix, ITS Liquid media supplement (5 µg/ml each of insulin, transferrin, and 0.05 µg/ml sodium selenite), 1 µg/ml glutathione, (Sigma, St. Louis, Mo); 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids. To promote the fibroblast phenotype in cultures of Src/Fyn/Yes –/–, FAK +/+ and DA2 MEFs, we added 1–20 ng/ml FGF-2 (Invitrogen, Carlsbad, CA) and 5 µg/ml heparin (Sigma) to SSFM. To induce the myofibroblast phenotype, we added 1 ng/ml TGFß (BD Biosciences Transduction, San Diego, CA) to SSFM. For serum starvation conditions, MEFs were incubated in serum free media with 0.2% BSA (Serologicals Corp., Norcross, GA) for 3 h (46) , and treated with growth factors for times indicated.

FAK shRNA and electroporation
The FAK shRNA expression construct, pSHAG/mFAK24, was designed and created as described (47) . The construct contains FAK shRNA sequences (29-mer) oligonucleotides ligated into the vector pSHAG, and was shown to decrease FAK protein expression in FAK +/+ MEFs (47) . To introduce FAK shRNA into FAK +/+ MEFs, electroporation of pSHAG/mFAK24 was performed on the Nucleofector device (Amaxa Biosystems, Köln, Germany). We used the MEF2 Nucleofector Kit (Amaxa Biosystems) according to the manufacturer’s protocol. 6.6 µg of pSHAG-mFAK24 or empty vector (pSHAG), and 4.4 µg enhanced GFP (EGFP)-C1 (BD Clontech, Palo Alto, CA) was electroporated per 2 x 106 FAK +/+ MEFs. By including EGFP-C1 in the electroporation, we determined by fluorescence microscopy that 80% of MEFs contained the plasmids.

Antibodies
All antibodies were purchased from commercial sources; Mouse anti-ERK (MK12), (BD Transduction, San Diego, CA); mouse antiphosphorylated ERK 1/2 (E4), rabbit-anti FRS2 (H-91) and rabbit-anti FGFR1 (flg; C-15) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse and rabbit anti-FAK (neither cross-reacts with Pyk2), and mouse antiphosphotyrosine (4G10) (Upstate Biotechnology, Lake Placid, NY); mouse anti-tubulin (B512), mouse anti-SM{alpha}A (1A4), and mouse anti-SM{alpha}A cy3 or FITC conjugate (Sigma); goat antimouse and goat anti-rabbit conjugated to either Alexa 488, Alexa 568, or Alexa 647 (Molecular Probes, Eugene, OR); rabbit gamma globulin, mouse gamma globulin, goat antimouse and goat anti-rabbit conjugated to HRP (Jackson ImmunoResearch Laboratories, West Grove, PA).

Immunofluorescence
MEFs were grown on 12-mm glass coverslips, and fixed by 3% paraformaldehyde (Fisher Scientific, www.Fishersci.com) in PBS, for 15 min at 25°C. Immunodetection was performed as described (29) . All cells were viewed with a Zeiss Axioskop, or Zeiss Axiovert 200 microscope equipped with CCD cameras. In Fig. 1A and B , optical sections were generated by inserting an ApoTome slider (Carl Zeiss, Thornwood, NY) into the illumination path on a Zeiss Axiovert 200 equipped with a x63 objective lens. The ApoTome slider projected a series of three shifted images of a grid of lines onto the focal plane (i.e., grid projection or "structured illumination"). The images were subsequently combined, and the out-of-focus haze removed using AxioVision software (Carl Zeiss) and images collected on an Axiocam MRm CCD camera. All images were processed by Adobe PhotoShop software. All experiments were performed at least 3 times with similar results obtained, and representative images are shown.


Figure 1
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  		Figure 1. FAK –/– MEFs were predominantly 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 antimouse conjugated to Alexa 488, and SM{alpha}A, (arrowheads in D), with mouse anti SM{alpha}A conjugated to cy3. FAK was immunodetected in FAK +/+ (A) and DA2 MEFs (E), but not in FAK –/– MEFs (C). In contrast, SM{alpha}A stress fibers were not detected in FAK +/+ MEFs (B) nor DA2 MEFs (F), which express FAK, but were detected in FAK –/– MEFs (D). 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.

Immunoprecipitation, SDS-PAGE and immunoblotting
MEFs were lysed in modified radio-immunoprecipitation assay (RIPA) buffer: (150 mM NaCl, 2 mM EDTA, 1% DOC, 0.1% SDS, 1% Triton X-100, 10% glycerol, 50 mM HEPES (pH 7.5), 100 mM NaF, 3 mM sodium vanadate, 10 mM sodium pyrophosphate, CompleteTM EDTA-free protease inhibitors (Roche Diagnostics, Munich, Germany), and 1 mM PMSF), or 1% SDS lysis buffer (1% SDS, 10 mM Tris pH, 7.6, CompleteTM EDTA-free protease inhibitors, and 1:2,000 dilution of Benzonase (Sigma) added after lysis. For immunoprecipitation, lysates were precleared with Protein A-sepharose beads (Sigma) for 20 min, 4°C. Precleared lysates were mixed with primary antibodies and incubated overnight at 4°C. To each lysate, 40 µl protein A-sepharose beads were added and incubated for 60 min at 4°C. The beads were then collected by centrifugation, 3,000 rpm, and washed 4 times in lysis buffer followed by 1x PBS wash. The immune-bead-associated complexes were eluted with 1x SDS sample buffer in PBS with boiling for 10 min. Lysates or immunoprecipitated bead supernatants were separated by 7.5% or 10% (Fig. 3 only) SDS-PAGE, transferred to ProtranTM nitrocellulose (Schleicher and Schuell, Keene, NH) or PVDF (Millipore, Bedford, MA) membranes and immunodetection performed as described (29) . Chemiluminescence was visualized on a Kodak Image Station 440CF (Kodak) or on Kodak BioMax MS film (Fisher Scientific). Densitometry was performed using Kodak 1D software. Where indicated, blots were reprobed after stripping using Restore Western blot Stripping buffer (Pierce, Rockford, IL).


Figure 3
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  		Figure 3. FAK shRNA inhibited FAK expression and increased SM{alpha}A expression. FAK +/+ MEFs were electroporated with pSHAG-shRNA24 containing FAK-shRNA or with empty vector. By including EGFP-C1 in the electroporation, we determined by fluorescence microscopy that 80% of MEFs contained the plasmids. After electroporation, MEFs were incubated with or without 5 ng/ml FGF-2 and 5 µg/ml heparin in SSFM for 3 days. A) In immunoblots of FAK-shRNA expressing FAK +/+ MEFs, 1, FAK expression was decreased, and 3, SM{alpha}A expression was increased. Cell lysates were analyzed for 1, FAK, and 3, SM{alpha}A. After detection, blots were stripped, and reprobed for, 2, tubulin, as a loading control. 4, Bar graph for normalization of SM{alpha}A band intensity. The ratio of SM{alpha}A/tubulin band intensities for untreated empty vector was made equal to 1. Values are means from two independent experiments ± range. *P < 0.05, empty vector without FGF/h compared to empty vector with FGF/h. ** P < 0.05, empty vector with FGF/h compared to shRNA with FGF/h. B) MEFs were incubated with 5 ng/ml FGF-2 and 5 µg/ml heparin in SSFM for 3 days. FAK was detected in 1, MEFs nucleofected with empty vector, which did not express 2, SM{alpha}A. In contrast, FAK was not detected in 3, FAK-shRNA expressing MEFs, which 4, had increased SM{alpha}A expression in stress fibers, as compared to 2, empty vector controls. MEFs were fixed and immunodetected for both FAK and SM{alpha}A with 1, 3, monoclonal anti-FAK, followed by secondary goat antimouse conjugated to Alexa 647 and 2,4, SM{alpha}A-cy3. Scale bar = 10 µm. Percent myofibroblasts: empty vector: 18% ± 11%; FAK-shRNA: 64% ± 2%. Values are means from two independent experiments ± range. P < 0.05, FAK-shRNA compared to empty vector.

TGFß assay of MEF conditioned media
MEFs were plated at 3 x 104 cells per well in 24-well dishes in DMEM/F-12 containing 10% FBS. After cells attached, the medium was replaced by SSFM with or without 20 ng/ml FGF-2 and 5 µg/ml heparin. Conditioned media was collected after 1, 2, and 3 days of growth in SSFM. To assay the amount of TGFß in media, we used a well-documented, standard assay: Mink lung epithelial cells (MLECs) stably transfected with the promoter fragment of the human PAI-1 promoter, which is specifically regulated by TGFß (48 49 50 51) . The MLECs were plated at 1 x 104 cells/well in 96-well dishes in 10% FBS containing DMEM with 200 µg/ml G418 (Sigma). After cell attachment, the MLEC medium was removed, and MEF conditioned medium was added to the MLECs for 14 h, followed by lysis of MLECs with Passive Lysis Buffer (Promega, Madison, WI). 20 µl of cell lysate for each condition was assayed for luciferase activity as an index of TGFß activity (Dual Luciferase, Promega) (52) .

FGF binding
MEFs were plated at 2 x 104 cells per well in gelatin-coated 24-well dishes with DMEM/F-12 containing 10% FBS. 18 h later, cells were washed twice and incubated for 20 min with binding buffer (DMEM containing 1 mg/ml BSA, 10 µg/ml heparin, 50 mM HEPES pH 7.5) at 4°C. Next, cells were incubated in 0.4 ml of binding buffer containing 2 ng (0.175 µCi) [125I] FGF-2 (Amersham, Piscataway, NJ) with or without 200 ng/ml nonradioactive FGF-2. After 4 h, cells were washed on ice 3 times with 1 ml PBS to wash off nonbound FGF-2, twice with 1 ml of 2 M NaCl in 20 mM HEPES pH 7.5 to dissociate low-affinity binding, and twice with 1 ml of 2 M NaCl in 20 mM NaAc pH 4.0 to dissociate FGF-2 bound to FGFRs (53 and references therein). The last two high salt-acid washes were pooled, and counted in a Cobra II (Packard) gamma counter. Radioactive counts from samples containing unlabeled and [125I] FGF-2 were subtracted from samples containing only [125I] FGF-2. To determine the number of cells and thus the amount of [125I] FGF-2 bound per cell, MEFs were plated on gelatin-coated coverslips in 24-well dishes, grown in parallel to MEFs used for the binding assay, fixed, stained with Hoechst nuclear stain, and counted. The amount of [125I] FGF-2 bound by FAK –/– MEFs is expressed as a percentage, where FAK +/+ and DA2 were set at 100%.

Statistical analysis
Statistical differences between experimental groups were determined by ANOVA and values of P < 0.05 were considered significant (54) . All the analysis was performed by using Microsoft Excel® 2004 for Macintosh®.

RESULTS

The absence of FAK leads to unregulated myofibroblast differentiation
On the basis of the studies implicating FAK in fibronectin signaling and in myofibroblast differentiation, we predicted that in the absence of FAK, (FAK –/–) MEFs would remain fibroblasts even after TGFß treatment. However, we found that FAK –/– MEFs had SM{alpha}A stress fibers without addition of exogenous TGFß. Furthermore, whereas FGF/h-treated FAK +/+ (Fig. 1 A, B), and DA2 (FAK re-expressing) MEFs (Fig. 1E, F ), expressed little SM{alpha}A protein and thus were predominantly fibroblasts, (FAK +/+: 2%, and DA2: 3% myofibroblasts), FAK –/– MEFs had SM{alpha}A stress fibers (Fig. 1C, D , FAK –/–: 95% myofibroblasts). Increasing FGF-2 concentration to 50 ng/ml did not cause FAK –/– MEFs to loose their SM{alpha}A stress fibers and become fibroblasts (data not shown). Thus, FAK was not only dispensable for myofibroblast differentiation, but the absence of FAK promoted the myofibroblast phenotype. Next, we tested whether FAK was necessary for TGFß-induced-myofibroblasts to "de-differentiate" to fibroblasts in response to FGF/h. We found that FGF/h treatment reversed the TGFß-induced myofibroblast phenotype in FAK +/+ MEFs but not in FAK –/– MEFs (compare Fig. 2 with Fig. 1D ). On the basis of the inability of FGF to induce the fibroblast phenotype in FAK –/– MEFs, we concluded that FAK was necessary for FGF-mediated negative regulation of SM{alpha}A expression.


Figure 2
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  		Figure 2. FGF/heparin treatment reversed the TGFß-induced myofibroblast phenotype in FAK +/+ MEFs. FAK was immunodetected in FAK +/+ MEFs (A). B) SM{alpha}A was immunodetected in stress fibers of FAK +/+ MEFs treated with 1 ng/ml TGFß1 in SSFM for 3 days before fixation. C) SM{alpha}A was not detected in FAK +/+ myofibroblasts grown in parallel to those cells in A and B, and replated and treated with 20 ng/ml FGF-2 and 5 µg/ml heparin in SSFM for 3 days before fixation. D) Hoechst nuclear stain of cells in C. Scale bar = 10 µm.

FAK shRNA decreases FAK and increases SM{alpha}A protein expression
To confirm that the absence of FAK promoted the myofibroblast phenotype, we used FAK shRNA to decrease FAK protein expression. As determined by immunoblotting (Fig. 3 A) and immunofluorescence (Fig. 3B ), FAK shRNA decreased FAK protein expression in FAK +/+ MEFs as compared to MEFs containing the empty vector. Furthermore, knockdown of FAK inhibited FGF-mediated decrease in SM{alpha}A, (Fig. 3A, B ). After FGF/h treatment, 64% of FAK +/+ MEFs expressing the FAK-shRNA were myofibroblasts as compared to 18% of FAK +/+ MEFs nucleofected with empty vector (Fig. 3B ). Thus knockdown of FAK protein, not only the knockout of FAK, decreased FGF-mediated inhibition of myofibroblast differentiation.

FAK –/– MEFs and FAK +/+ MEFs secrete comparable amounts of active TGFß
Increased expression of SM{alpha}A in FAK –/– MEFs could reflect more secretion of active TGFß by FAK –/– MEFs as compared to FAK +/+ MEFs. To test this hypothesis, we measured active TGFß in conditioned media of MEFs using the standard MLEC luciferase assay (48) , (see Materials and Methods). We found that FAK +/+ MEFs and FAK –/– MEFs secreted comparable amounts of active TGFß (Fig. 4 ). Thus, it was the FAK –/– MEFs inability to respond to the FGF/h signal, and not a differential increase in TGFß, which caused FAK –/– MEFs to have increased SM{alpha}A expression (Figs. 1 2 3) . On the basis of these results, we explored FAK’s role in the FGF signaling pathway.


Figure 4
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  		Figure 4. FAK +/+ and FAK –/– MEFs secrete active TGFß. TGFß activity was comparable in conditioned media from FAK +/+ and FAK –/– MEFs. Media were collected from FAK +/+ and FAK –/– MEFs grown for 1, 2, and 3 days in SSFM. The conditioned media were added to MLECs, expressing the firefly luciferase gene under control of the TGFß-inducible PAI-1 promoter. After 14 h, MLEC cell extracts were assayed for luciferase activity. There was no significant difference between the amount of active TGFß in FAK +/+ as compared to FAK –/– conditioned media. Values are means from 2 independent experiments done in triplicate ± SD

Src/Fyn/Yes –/– MEFs respond to growth factor-mediated regulation of phenotypes
Because Src family tyrosine kinases are involved in both FGF and FAK signaling, we posed the question of whether Src family tyrosine kinases mediated FAK’s negative regulation of the SM{alpha}A expression. We used MEFs null for the ubiquitously expressed Src family kinases Src, Fyn, and Yes. Src/Fyn/Yes –/– MEFs responded to FGF/h by decreasing SM{alpha}A expression (Fig. 5 A, B) and to TGFß by increasing SM{alpha}A expression (Fig. 5C, D ). Therefore, we concluded that these Src family kinases do not play a role in this phenotype modulation.


Figure 5
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  		Figure 5. Src/Fyn/Yes tyrosine kinases were not required for the FGF/heparin-induced fibroblast phenotype. A, B) Src/Fyn/Yes triple –/– MEFs treated with 20 ng/ml FGF-2 and 5 µg/ml heparin in SSFM, were fibroblasts. C, D) When treated with 1ng/ml TGFß, Src/Fyn/Yes –/– MEFs were myofibroblasts. MEFs were treated with growth factors for 3 days and detected for SM{alpha}A (A, C) and (B, D) nuclei as in Figs. 1 and 2 . Scale bar = 10 µm.

FAK affects FGF receptor signaling
Having determined that FAK –/– MEFs were defective in FGF/h-dependent inhibition of SM{alpha}A expression, we sought to identify the mechanism of FAK’s regulation of FGF signaling. To evaluate whether FAK was required for FGF receptor (FGFR) expression, we measured the cell surface expression of FGFRs by incubation with [125I]FGF-2. We found that FAK –/– MEFs bound 50% less [125I]FGF-2 as compared to FAK +/+ and DA2 MEFs (Fig. 6 A, B). Furthermore, FGFR phosphorylation in FGF/h-treated FAK –/– MEFs was significantly decreased compared to FAK +/+ MEFs (Fig. 6C ).


Figure 6
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  		Figure 6. 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: (A) FAK +/+, P < 0.05 or (B) DA2 (hygro), P < 0.05. 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 F-actin +/+ 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.

FAK is necessary for FGF signaling via FRS2 and ERK
FRS2 is immediately downstream from FGFRs and links FGF signaling to ERK activation. We asked whether FGF signaling through FRS2 and ERK was decreased in FAK –/– MEFs. Treatment of FAK +/+ MEFs with FGF/h induced the phosphorylation of FRS2 (Fig. 6D ) and ERK (Fig. 7 ). In contrast, treatment of FAK –/– MEFs with FGF/h induced little or no phosphorylation of FRS2 (Fig. 6D ), nor ERK (Fig. 7) . Thus, FAK was necessary for efficient FGF signaling to ERK. This is the first report to establish a role for FAK in regulating signaling through the FGF/FRS2/ERK pathway. In summary, in the absence of FAK, there was reduced FGFR surface expression and activity, and decreased FGF signaling via the FGFR/FRS2/ERK pathway.


Figure 7
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  		Figure 7. FAK was required for FGF-2-dependent ERK phosphorylation. In FAK +/+ MEFs, treatment with FGF/h increased phosphorylated ERK 1/2. In FAK –/– MEFs, there was little p-ERK, and FGF/h treatment did not significantly change p-ERK. FAK +/+, and FAK –/– MEFs were serum-starved, treated with 20 ng/ml FGF-2 and 5 µg/ml heparin or untreated for 5 min. Chemiluminescence was visualized on a Kodak Image Station 440 CF. The ratio of phosphorylated ERK/total ERK band intensities (determined using bands for both ERK-2 and ERK-1) for untreated FAK +/+ was made equal to 1. Values are means from 2 independent experiments ± range. *P < 0.05, FAK +/+ MEFs without FGF/h compared to FAK +/+ MEFs with FGF/h. **P < 0.05, FAK +/+ MEFs with FGF/h compared to FAK –/– MEFs with FGF/h.

DISCUSSION

The data presented here can be organized into a working model of TGFß/FGF signaling in myofibroblast differentiation that alters the current paradigm. The established roles of TGFß in increasing fibronectin in myofibroblast differentiation, and FAK in modulating signals from fibronectin had suggested that FAK would contribute to TGFß-mediated myofibroblast differentiation. In contrast, we found that the absence of FAK curtailed the optimal functioning of FGF signaling in myofibroblast de-differentiation or maintaining the fibroblast phenotype. Where FAK was greatly decreased (by shRNA) or absent (FAK –/– MEFs), SM{alpha}A was expressed in stress fibers even after addition of FGF/h to cells. Furthermore, in the absence of FAK, FGFR surface expression was greatly reduced as was phosphorylation of FRS2 and ERK, which are downstream from FGFRs. Thus we postulate that FAK is necessary for FGF signaling via FRS2/ERK, and that these signals are involved in the negative regulation of SM{alpha}A (Fig. 8 ). This is the first report to establish a role for FAK in promoting FGF signaling and in FGF-mediated down-regulation of SM{alpha}A, and thus in opposing myofibroblast differentiation.


Figure 8
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  		Figure 8. FAK signals increase FGFR surface expression and inhibit SM{alpha}A expression. Numbers indicate sequential steps in the signaling scheme. TGFß 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 SM{alpha}A (step 5). TGFßR, TGFß receptor; FGFR, FGF receptor; HSPG, heparan sulfate proteoglycan; G, Grb2; S, Sos.

A major defect of FAK –/– MEFs is decreased migration because of decreased focal adhesion turnover (43 , 44 , 55 , 56) . These data are consistent with our finding that FAK –/– MEFs are myofibroblasts because myofibroblasts are less migratory than fibroblasts (57) . After the initial formation of focal adhesions, it is likely that the high expression of SM{alpha}A in FAK –/– MEFs stabilizes focal adhesions, increases mechanical tension, and contributes to decreased motility. This is consistent with the finding that inhibition of either SM{alpha}A-mediated contractile activity (18 , 58) or SM{alpha}A expression (18 , 57) increases focal adhesion and migration (18 , 57 , 58) . Matrix molecules like fibronectin may play a role in myofibroblast differentiation in a FAK-independent manner. This may be via p130Cas and/or phosphatidylinositol 3-kinase (PI3K), which can be activated downstream from ß1 integrins in FAK-independent pathways (59 60 61) . Extracellular signals are likely to be transduced by ß1 integrins since TGFß treatment of ß1 –/– MEFs did not induce SM{alpha}A expression (R. S. Greenberg, Thesis 2006).

FAK –/– MEFs bound 50% less FGF-2 on their cell surface than FAK-expressing FAK +/+, or DA2 MEFs (Fig. 6A, B ). The reduced expression of FGFRs on FAK –/– MEFs suggests that FAK contributes to reduced endocytosis and to enhanced retention of FGFRs in the cell membrane or to increased transcription of FGFRs genes. In fact, there is recent evidence for a positive role for FAK in regulating ß1 integrins’ cell surface residence. In FAK –/–, MEFs ß1 integrins were endocytosed at a higher rate than in FAK +/+ MEFs (62) . The additional role for FAK to provide signals that increase expression of FGFRs mRNA or protein is suggested by other data: E2F-mediated transcriptional activation of the FGFR1 and FGFR2 genes is induced by Cyclin D (63 64 65) , whose expression is increased by FAK (66 , 67) .

Although the reduced expression of FGFRs on FAK –/– MEFs leads to decreased FGFR phosphorylation, the difference in FGFR phosphorylation between FAK –/– and FAK +/+ MEFs is greater than the 50% difference in FGFR expression (Fig. 6C ). This may be because FGFRs signal synergistically, or FAK may directly contribute to FGFR phosphorylation.

Incorporating our data in a proposed model (Fig. 8) , places FAK upstream of FGFR in a pathway that regulates SM{alpha}A. The myofibroblast phenotype seems to be a default phenotype; input from FAK is required to maintain the fibroblast phenotype. In the absence of FAK, there is no FGF signaling via the FRS2/ERK pathway. This allows for unopposed TGFß signaling, and thus expression of SM{alpha}A. When FAK is present, FGF signaling is intact and can overcome TGFß-mediated myofibroblast differentiation. Another protein that is increased in TGFß-mediated myofibroblast differentiation is smooth muscle protein 22-alpha (SM-22{alpha}), (A.M. Bernstein and J. J. Tomasek unpublished observations). In cells treated simultaneously with FGF-2/h and TGFß, there was decreased expression of SM-22{alpha}. However, when an ERK inhibitor was added with these growth factors, the amount of SM-22{alpha} protein was not decreased (68) . This is consistent with our finding that ERK phosphorylation was absent in the FAK –/– myofibroblasts. These combined data suggest that ERK is involved in FGF/h-mediated inhibition of myofibroblast differentiation.

Our data indicate a role for FAK in positively contributing to FGF-dependent signaling, and down-regulating SM{alpha}A expression. FAK activity early in wound healing would promote FGF-mediated fibroblast motility and would delay myofibroblast differentiation. In contrast, myofibroblast formation before FGF/FAK-mediated proliferation and migration of fibroblasts could inhibit wound healing. These data can be integrated with previous studies that support a role for TGFß-mediated activation of FAK in myofibroblast differentiation (31 , 34) : TGFß increases fibronectin and integrin expression (17 , 18 , 24 25 26 27 28 29 30 31) , (Fig. 8 , step 1), which leads to FAK activation (Fig. 8 , step 2). So while TGFß induces SM{alpha}A expression by one pathway, its activation of FAK 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. 8 , step 3). Increased cell-surface expression of FGFRs transmits FGF signals and increases activation of the FGF pathway via FRS2/ERK (Fig. 8 , step 4), which negatively regulates the levels of SM{alpha}A (Fig. 8 , step 5). This is consistent with the data that show that when FAK was greatly decreased or absent, cells were refractory to FGF/h-mediated inhibition of SM{alpha}A expression (Figs. 1 2 3) . Our findings add FAK dependence to FGF’s inhibition of SM{alpha}A induction. We hypothesize that in vivo, FAK activation by TGFß may contribute to a negative feedback mechanism to prevent excessive myofibroblast differentiation, ameliorating fibrosis.

Although Src family tyrosine kinases are involved in both FGF and FAK signaling, our results with Src/Fyn/Yes –/– MEFs indicate that the Src family tyrosine kinases are not required for FGF/FAK-mediated negative regulation of SM{alpha}A expression (Fig. 5) . This is consistent with the finding that FGF-1 was able to induce phosphorylation of FRS2 in Src/Fyn/Yes –/– MEFs (69) . These data indicate that in Src/Fyn/Yes –/– MEFs there is sufficient FGF signaling to decrease SM{alpha}A in response to FGF/h.

Failure to down-regulate ECM deposition and/or SM{alpha}A stress fiber formation during wound closure has been linked to fibrosis (1 , 5 , 15 , 17) . Inhibition of TGFß-mediated SM{alpha}A production was shown to reduce fibrosis in the skin (70 , 71) and in the cornea (34 , 72) . Furthermore, exogenous application of FGF-2 has been found to promote healing in the skin (73) , heart (74 , 75) , and cornea (76 77 78 79) . Our data suggest that reduction of FAK activation would negatively affect the ability of FGF to reduce SM{alpha}A expression. Furthermore, activation of FAK and stimulation of FGF signaling could attenuate excess and irreversible SM{alpha}A expression that is found in fibrosis. These insights add to our knowledge of molecular mechanisms of fibrosis and could contribute to interventions that promote successful wound healing.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Eye Institute (NEI) Grant R01 EY09414 (SKM), an NEI Core Grant P30 EY001867, and Research to Prevent Blindness grants. AMB was supported by NEI Grant F32 EY07049. Microscopy was performed at the Mount Sinai School of Medicine Microscopy Shared Research Facility, supported in part, with funding from NIH-NCI shared resources Grant 1-R24-CA095823 and National Science Foundation Major Research Instrumentation Grant DBI-9724504. LT and MB each received a postdoctoral research fellowship from Fight for Sight, Prevent Blindness America.

We greatly appreciate the generosity of Dr. D. Ilic for FAK +/+ and FAK –/– MEFs, Dr. D. D. Schlaepfer for FAK –/– (hygro) and DA2 (FAK re-expressing) MEFs, Dr. P. Soriano for Src/Fyn/Yes –/– MEFs, S. Sachdev for pSHAG/FAKshRNA24, and Dr. D.L. Benson, and Dr. D. Sternberg for use of their Nucleofector devices. We thank Dr. S. C. Henderson and P. Carman for help with microscopy, and Dr. M. Goldfarb and Dr. L. Ossowski for discussions and for sharing their wisdom.

Received for publication October 10, 2005. Accepted for publication December 13, 2005.

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