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Published as doi: 10.1096/fj.07-095521.
 (The FASEB Journal. 2008;22:2508-2520.)
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Deficiency in the LIM-only protein FHL2 impairs assembly of extracellular matrix proteins

Jung Park{dagger},1, Carola Will*,1, Bernd Martin{ddagger}, Lucia Gullotti§,2, Nicolaus Friedrichs§, Reinhard Buettner§, Holm Schneider||, Stephan Ludwig* and Viktor Wixler*,3

* Institute of Molecular Virology, Münster University Hospital Medical School, Münster, Germany;

{dagger} Department of Experimental Medicine I, University of Erlangen-Nürnberg, Erlangen, Germany;

{ddagger} Department of Gynecology and Obstetrics, Medical School, J. W. Goethe-University, Frankfurt, Germany;

§ Institute of Pathology, University Hospital Medical School, Bonn, Germany;

|| Experimental Neonatology, Department of Pediatrics, Medical University of Innsbruck, Innsbruck, Austria

3Correspondence: Institute of Molecular Virology, Münster University Hospital Medical School, Von-Esmarch-Str. 56, D-48149 Münster, Germany. E-mail: vwixler{at}uni-muenster.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have described the scaffolding protein FHL2 as a component of focal adhesion structures, to which it is recruited via binding to both {alpha}- or β-integrin subunits. Using mesenchymal stem cells from wild-type and FHL2-knockout mice, we show here that inactivation of FHL2 leads to impaired assembly of extracellular matrix proteins on the cell surface and to impaired bundling of focal adhesions. Both altered properties can be restored by reexpression of recombinant FHL2 protein in FHL2-null cells. Molecular analysis of integrin-mediated signaling revealed a higher phosphorylation of FAK at tyrosine 925 in FHL2-knockout cells compared to their wild-type counterpart. Consequently, the activation of the mitogenic kinase ERK was more pronounced in knockout cells on cell adhesion. The growth factor-induced activation of ERK, however, was not altered. The perturbed organization of extracellular matrix on FHL2-null cells was improved when the increased activation of MAPK was inhibited. Our findings point to a role of FHL2 in bundling of focal adhesion structures, in integrin-mediated ERK activation, and subsequently in proper allocation of matrix proteins on the cell surface.—Park, J., Will, C., Martin, B., Gullotti, L., Friedrichs, N., Buettner, R., Schneider, H., Ludwig, S., Wixler, V. Deficiency in the LIM-only protein FHL2 impairs assembly of extracellular matrix proteins.


Key Words: fibronectin • integrin-mediated ERK activation • focal adhesions • cytoskeleton


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE EXTRACELLULAR MATRIX (ECM) is a highly organized structural framework that provides mechanical support for organs, serves as a store of many biologically active substances and regulates such fundamental processes as tissue development and regeneration (1 2 3 4) . Formation of regular ECM networks takes place on cell surfaces and, despite the high self-assembly capacity of many ECM proteins, it is more efficient when matrix proteins are bound to cell surface receptors (5 6 7) . Several studies have demonstrated the dependence of ECM assembly on integrin activity and downstream intracellular signaling (4 , 8 9 10 11 12) . Binding of integrins to matrix proteins leads to activation of the focal adhesion kinase (FAK) and formation of focal adhesion (FA) structures. These multiprotein complexes connect the ECM to the cytoskeleton of cells and serve as initiation sites for activation of many intracellular signaling pathways regulating the shape of cells and their migration, proliferation, and gene transcription. They also act as sites of matrix assembly (13 14 15) . The molecular mechanisms by which the matrix assembly is regulated are still poorly understood, but evidence suggests involvement of FA and the classical mitogenic Ras-Raf-MEK-ERK pathway (2 , 4 , 10 , 16 17 18) . Inactivation of FAK, the key enzyme of FA complexes, leads not only to changes of FA and microfilament structures and retarded cell migration but also to impaired fibronectin (FN) assembly (11 , 19) . The MAP kinase ERK, activated by growth factors as well as by integrins via FAK, regulates in turn the recycling of FA structures and the actin-myosin contraction by phosphorylation of FAK, paxillin, calpain, or myosin light chain kinase (20 , 21) . However, overexpression or constitutive activation of ERK or its upstream activators, Ras or Raf, results in disruption of the actin cytoskeleton and impairment of ECM assembly. This finding has been well described in tumors with an activated Ras-ERK pathway, and high or prolonged ERK activity is assumed to cause inactivation of integrin receptors, which leads to modulation of stress fibers and changes in cell surface tension (8 , 10 , 22) , which are critical for appropriate assembly of ECM proteins on the cell surface (4) .

Scaffolding proteins are essential components of FA complexes. They bridge integrins with enzymes and structural proteins and ensure their coordinated action. Previously, we described the LIM-only protein FHL2 (four-and-a-half LIM domain protein 2) as an adaptor protein recruited to focal adhesion sites by binding directly to different {alpha}- and β-integrin subunits (23 , 24) . Other authors identified FHL2 also as interacting with FAK and the phosphorylated form of ERK (25 , 26) , two additional components of FA. While the functional consequence of the first interaction is still unknown, binding to ERK has been shown to prevent the nuclear translocation of the MAPK in cardiomyocytes but not to alter its kinase activity. Finally, we recently demonstrated that FHL2-deficient cells develop low contractile forces and have less-developed actin stress fibers and decreased cell motility (27) . Therefore, we studied here the role of FHL2 in the assembly of ECM proteins and could show that its loss results in impaired matrix assembly and increased integrin-mediated ERK activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA constructs
For stable expression of FHL2 in mesenchymal stem cells, the myc-tagged human FHL2 was removed from the pCS2+MT plasmids and recloned in the pBabe-neo vector as described previously (28) . The myc-tagged C4 and BXB Raf constructs were obtained by cloning the appropriate inserts from the pKRSPA (29) in the pCS2+MT vector. The Raf-C4 construct includes the N-terminal part of c-Raf kinase containing the Ras-binding domain but lacking the C-terminal kinase domain, whereas the Raf-BXB carries the kinase-active C-terminal half (30) . Correct inframe cloning of the constructs was verified by DNA sequencing.

Isolation and culture of bone marrow-derived mesenchymal progenitor cells
Fresh bone marrows from femurs of 4 wk-old C57BL/6 wild-type or FHL2–/– mice were centrifuged at 350 g for 10 min. The resuspended cells were filtered through a 70 µm nylon filter and plated on FN-coated plates (100 ng/ml) with 1 x 105 mononuclear cells/cm2. After 3 days the nonadherent cells were removed, and the cell population left on the dish was further expanded in medium containing 60% Dulbecco modified Eagle medium with low glucose (DMEM-LG; Gibco BRL, Grand Island, NY, USA), 40% MCDB-201 (Sigma, St. Louis, MO, USA), supplemented with 1x insulin-transferrin-selenium (Sigma), 1x linoleic acid-bovine serum albumin (Sigma), 10–9M dexamethasone (Sigma), 10–4 M ascorbic acid 2-phosphate (Sigma), 100 U penicillin, 10 µg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD, USA), 10 ng/mL epidermal growth factor (EGF; Sigma), 10 ng/mL platelet-derived growth factor-BB (PDGF-BB; R&D Systems, Minneapolis, MN, USA), 1000 U/mL mouse LIF (Chemicon, Temecula, CA, USA), and 2% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT, USA), as described previously (31) . After 3 wk, the cultures were depleted of CD45+ cells using a FACS (Moflo; Dako, Copenhagen, Denmark), and the sorted cells were plated into FN-coated 96-well plates with a density of 10 cells/well. The expanded single clones were transferred into new dishes and cultured further for over 40 population doublings, with replating every 3 days and cells kept at a density of 1 x 103/cm2. Well-growing clones with a doubling time of roughly 35 h were selected and expanded for further experiments. The expanded cells were positive for the markers CD34, c-kit, sca1, Thy1, and CD13 and negative for CD45, CD10, and CD31 markers. According to this marker profile and to their potency to differentiate into osteogenic, chondrogenic, and adipogenic lineages, we identified these as mesenchymal cells. To rescue the FHL2-deficiency, the FHL2–/– cells were infected with retroviruses containing a myc-tagged FHL2 construct and a neomycin resistance gene, as described previously (28) . After selection of cells for G418 resistance, they were subcloned; clones with high myc-FHL2 expression were further expanded. These cells were described previously (27) .

Isolation and culture of embryonal fibroblasts
Murine embryonal fibroblasts were derived from 14.5-day-old embryos of C57BL/6 wild-type or FHL2-deficient mice. The embryo carcass without head and internal organs was minced using fine scissors and digested with trypsin. After centrifugation and removal of debris, the cells were filtered through a 70 µm nylon filter and cultured on plastic dishes in DMEM supplemented with penicillin (100 U/ml), streptomycin (10 µg/ml), and 10% FCS (Invitrogen, Karlsruhe, Germany). The cells were passaged three times to obtain a pure population of fibroblasts.

Analysis of matrix assembly
Cells were plated onto uncoated glass coverslips and incubated for 3 days without medium change either in stem cell cultivation medium, containing 10 ng/ml EGF and 10 ng/ml PDGF, or in DMEM:MCDB-201 medium mix without growth factors but with 2% FCS. When cells were incubated with the U0126 MEK inhibitor, the medium was replaced daily and dimethyl sulfoxide (DMSO) served as a control, as the U0126 stock compound was dissolved in DMSO. Cells were fixed with 2% paraformaldehyde and stained for ECM proteins without being permeabilized. For immunoblot analysis of proteins incorporated into polymerized ECM meshwork, two different procedures were used: 5 x 104 cells were cultivated in 6-well plates in DMEM:MCDB-201 medium mix without growth factors but with 2% FCS. In the first approach, the cells were then washed two times with PBS and exposed for 30 min at 37°C to 0.5% Triton X-100 in PBS as described (32) . The lysed cells were removed by washing several times with PBS and water. Proteins remaining on the dishes were scraped off and dissolved in 2x sample buffer [2% sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol, 10% glycerol, 0.002% bromphenol blue, 62 mM Tris-HCl, pH 6.8]. In the second approach, cells were lysed by scraping them off in deoxycholate (DOC) lysis buffer (2% DOC; 20 mM Tris-HCl, pH 8.9; 2 mM EDTA; 2 mM iodoacetic acid; 2 mM N-ethylmaleimide; 2 mM Pefablock). The samples were centrifuged at 13,000 g for 10 min, and the DOC-insoluble pellets were solubilized in a buffer containing 1% SDS, 25 mM Tris-HCl (pH 8.0), 2 mM EDTA, 2 mM iodoacetic acid, 2 mM N-ethylmaleimide, and 2 mM Pefablock. Aliquots of the samples were separated on an 8% SDS-polyacrylamide gel (reduced) and electroblotted to nitrocellulose for immunodetection. All experiments were repeated 2 to 4 times.

Mouse wound analysis
Punch wounds (0.6 cm) including the skin and cutaneous muscle were cut in the back of 6-wk-old anesthetized mice and left to heal by secondary intention, essentially as described previously (33) . At days 0, 5, and 12, wounds were dissected and paraffin-embedded for histology or snap-frozen in liquid nitrogen for protein extraction. All experiments were performed in compliance with animal welfare regulations (permission 50.203.2-BN12, 12/02 by the Regierungspräsidium Cologne, Germany). Gordon and Sweet’s method for silver staining of reticulin fibers as well as FN immunostainings were performed as described previously (34) .

Luciferase assays
Transfections of HeLa or FHL2-deficient stem cells were performed with Fugene 6 (Roche, Mannheim, Germany) as recommended by the manufacturer. The reporter plasmid pGL3/FN-luc (1 µg) was cotransfected with 100 ng of expression plasmids coding for FHL2 (pCMX-FHL2) or Lef1 (pCG-Lef1) as indicated. Relative light units were normalized to protein concentrations determined with the Bradford dye assay (Bio-Rad, Hercules, CA, USA). The FHL2 and Lef1 constructs were as described previously (28) . The pGL3/FN-luc contained –499/+20 bp of the Xenopus FN gene promoter (35) .

ERK1 kinase assay
Kinase assays were performed as described previously (36) . ERK1 kinase was immunoprecipitated from cell lysates using protein G agarose beads and specific antibodies. The immune complexes were washed twice with lysis buffer and once with kinase buffer and then resuspended in 30 µl of kinase buffer (25 mM HEPES, pH 7.5; 25 mM sodium glycerophosphate; 10 mM MgCl2; 1 mM DTT; 100 µM ATP; 5 µCi {gamma}-32P-ATP) containing 5 µg of myelin basic protein (MBP). After incubation for 15 min at 30°C, the kinase mix was resolved by 12.5% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The phosphorylation of MBP was then quantified by phosphoimager scanning.

Western blotting
Cells were washed twice with PBS and lysed in RIPA buffer supplemented with 1 mM sodium vanadate, 1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin for 10 min. The lysates were cleared by centrifugation at 10,000 g for 10 min at 4°C. Supernatants were resolved by 10% SDS-PAGE, and, after electroblotting onto a nitrocellulose membrane, proteins were detected with appropriate antibodies using the ECL detection system (Bio-Rad Inc.). The antibodies used were: mAb anti-FHL2, clone F4B2-B11 (23) ; mAb anti-phospho-ERK (Cell Signaling, Danvers, MA, USA); rabbit polyclonal anti-ERK1 and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal antiphospho-FAK (Tyr925) (Cell Signaling); mAb anti-phospho-FAK (Tyr397); and mAb anti-FAK (Transduction Laboratory, Lexington, KY, USA). All Western blotting experiments were repeated 2 to 4 times.

Immunofluorescence
Indirect immunofluorescence staining was performed as described previously (23) . Briefly, cells grown on coverslips were rinsed with PBS and fixed with 2% PFA in PBS at room temperature for 10 min. After fixation, cells were permeabilized where indicated by incubation with 0.2% Triton X-100 in PBS for 2 min, washed with PBS, and incubated with the relevant antibodies diluted in PBS for 1 h. Primary antibodies were: anti-FHL2 mAb, clone F4B2, or rabbit polyclonal serum (23) ; anti-myc mAb, clone 9E10, derived from American Type Culture Collection (Manassas, VA, USA); mAb anti-paxillin (Transduction Laboratory); rabbit polyclonal anti-{alpha}5-integrin subunit (Chemicon); mAb anti-{alpha}-actinin, MAB1682 (Chemicon); rabbit polyclonal anti-FN (Sigma); and rabbit polyclonal anti-laminin (LN) {gamma}1 chain and anti-nidogen (Nd) 1 (both gifts of N. Smyth, University of Southampton, Southhampton, UK). The F-actin was visualized with Alexa488-labeled phalloidin (Invitrogen-BioSource, Karlsruhe, Germany). Secondary antibodies labeled with Cy3 or Alexa488 were also from BioSource. Cell images were taken using an Axiovert 2000 ApoTome microscope with an AxioCam digital camera and AxioVision software (Zeiss, Jena, Germany). Multiple trials of all immunofluorescence analysis were undertaken.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inactivation of FHL2 impairs ECM assembly
As the assembly of extracellular matrix proteins is regulated by integrins and depends on the integrity of FA structures, we investigated whether the absence of the integrin-binding protein FHL2 influences ECM organization. For these studies we used mesenchymal stem cell lines derived from bone marrow of FHL2 wild-type and knockout mice. The cells were incubated for 3 days on uncoated glass coverslips and stained with antibodies recognizing different ECM proteins. To reduce the possible influence of mitogenic stimuli but rather focus on the integrin contribution to matrix assembly, cells were grown in medium without growth factors and at low serum concentrations. Under these conditions, knockout cells were almost unable to form an ECM network, and the secreted proteins were deposited on cell surfaces as sparse large protein clusters (Fig. 1 A). In the presence of growth factors and FCS, they developed a network with only short and thin fibers and small meshes (Supplemental Fig. 1). The wild-type cells, in contrast, produced thick and long fibers spanning the whole cell surface and often neighboring cells. The fibers crosslinked and formed regular thick networks with wide meshes, independently of the cultivation conditions (Fig. 1A and Supplemental Fig. 1). Thus, FHL2–/– cells showed a defect in the organization of secreted matrix proteins on their surface compared with FHL2 wild-type cells. Furthermore, in accordance with the fact that FHL2 interacts with different integrin receptors that bind diverse ECM ligands (23) , a disordered matrix assembly was seen for different ECM proteins, including FN, LN {gamma}1, and Nd1.


Figure 1
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  		Figure 1. Inactivation of FHL2 impairs the assembly of ECM proteins. A) 5 x 103 FHL2+/+, FHL2–/–, or FHL2–/– rescue mesenchymal stem cells were plated in 0.5 ml medium on noncoated glass coverslips that were placed into 24-well plates and incubated for 3 days in DMEM:MCDB-201 medium mix with 2% FCS but without growth factors. The cells were fixed with 2% paraformaldehyde, not permeabilized, and stained with specific antibodies for FN, LN {gamma}1 chain, and Nd1 (in red), as described in Materials and Methods. To visualize the cells, the nuclei were counterstained with DAPI (blue). B) Primary embryonal fibroblasts obtained from FHL2 wild-type or FHL2-deficient mice were incubated and stained as described in A. Scale bars = 50 µm. C) Analysis of protein incorporation into ECM meshwork. Left panels: after incubation of cells in medium containing 2% FCS but no growth factors for 8 or 24 h, the cells were lysed with 0.5% Triton X-100 in PBS. After intensive washing of the plates, the remaining proteins were scrapped off in sample buffer. Right panels: conditioned media and DOC-soluble and -insoluble material were isolated from FHL2+/+ and FHL2–/– cells. Equal amounts of collected protein samples were separated by 8% SDS-PAGE, and after electroblotting the ECM proteins were analyzed by immunoblotting with antibodies indicated on the right of each panel. β-Actin blot shows the amount of F-actin left on dishes after lysis of cells with Triton. ERK1/2 proteins served as loading controls. D) FHL2-deficient mesenchymal stem cells (MSCs) and HeLa cells were transfected with an FN promoter-driven luciferase reporter construct and expression plasmids for FHL2 and Lef1. Bars indicate the fold induction of the luciferase activity of the reporter plasmid in the presence or absence of FHL2 or Lef1 (n=3).

To check that the organization of matrix proteins is indeed FHL2-dependent, we restored the FHL2 expression in these cells with retroviruses encoding a myc-tagged human FHL2. And in fact, expression of recombinant FHL2 rescued the defective matrix assembly in the knockout cells (Fig. 1A and Supplemental Fig. 1). The amount of recombinant myc-FHL2 protein in FHL2–/– rescue cells was lower than that of the endogenous protein in wild-type cells (Supplemental Fig. 2), and so the ECM assembly was not completely reverted to the level of FHL2+/+ cells. However, the network pattern of the matrix made by rescued cells was similar, though less well organized, to that of wild-type cells and significantly different from knockout cells. To ensure further that the disturbed matrix allocation in FHL2–/– cells is not a particular feature of the cell line used, possibly acquired during prolonged in vitro cultivation of mesenchymal stem cells, we analyzed the organization of FN and LNs in freshly isolated embryonal fibroblasts. Similarly to the stem cells, the assembly of both matrix proteins in FHL2–/– fibroblasts was less efficient compared to FHL2+/+ embryonal fibroblasts (Fig. 1B ).

Analysis of matrix proteins left on Petri dishes after removing adherent cells with 0.5% Triton X-100 according to the method of Gospodarowicz et al. (32) supported the conclusion of the immunofluorescence studies. FHL2–/– cells polymerized secreted proteins into matrix networks less efficiently than FHL2+/+ cells (Fig. 1C , left panels). Previous studies showed that incorporation of FN into DOC-insoluble matrix is the most adequate measure of its polymerization within the ECM fibrils (37) . To ensure that ECM assembly by knockout cells is indeed less efficient than in wild-type cells, we compared DOC-insoluble fractions of these cells after their cultivation for 8 or 24 h in medium with low serum content. Analysis of these fractions underlined the lower matrix assembly capacity of FHL2-deficient cells (Fig. 1C , right panels). The level of FN detected in conditioned medium of FHL2–/– cells was, however, slightly above that of FHL2+/+ cells. Because FHL2 can be translocated into the nucleus and serve as a transcriptional coregulator modifying the expression of different proteins (38) , we wondered whether the transcriptional activity of the FN gene might be regulated by FHL2. To test this hypothesis, we transiently transfected FHL2–/– cells with a luciferase reporter gene construct containing the Xenopus FN gene promoter (35) , either alone or together with the FHL2 transgene. The transcription factor Lef1 served as a positive control. As Fig. 1D (left panel) shows, expression of FHL2 in FHL2–/– mesenchymal stem cells did not change the FN promoter activity. Neither did FHL2 alter the reporter gene activity in another cell type when it was overexpressed there (Fig. 1D , right panel). Thus, FHL2 does not, at least not directly, regulate the transcription of the FN gene.

In summary, our data show that the integrin-binding protein FHL2 is involved in the regulation of ECM assembly. Cells lacking FHL2 organize ECM proteins less efficiently, and this is not due to decreased expression of ECM proteins. Importantly, the ECM organization can be restored by reintroduction of FHL2.

In the absence of FHL2, mice show a delay in wound healing (27) . To assess the role of FHL2 in matrix organization in vivo, we studied FN allocation and expression in skin during healing of acute wounds of wild-type and FHL2–/– mice. As expected, an elevated level of FN was seen in the granulation tissue of wild-type mice at day 5 after injury, while at day 12, the time point at which wounds of all wild-type mice were already closed, its expression was reduced (Fig. 2 A). Similar to wild-type mice, FN staining was hardly detected in the intact skin of FHL2-deficient mice, and FN was also up-regulated in wounded tissue at day 5. However, its distribution was more diffuse compared to wounds of wild-type mice and it did not decrease as fast as in wild-type mice. Immunoblot analysis of FN expression in the skin during wound healing confirmed the delayed down-regulation of the protein in FHL2-null mice (Fig. 2B ). To analyze the fibrillar deposition of the extracellular matrix during wound healing in FHL2 wild-type vs. knockout mice, Gordon staining of paraffin-embedded tissues was performed. In scar tissue of wild-type mice, a dense meshwork of fibers indicated normal wound healing (Fig. 2C1-3 ). In contrast, wounded tissue of FHL2-deficient mice consisted of faint, fragmented fibers in the scar area (Fig. 2C4-6 ). The fragility of this network was reflected by fresh bleeding into the scar tissue (Fig. 2C5 ).


Figure 2
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  		Figure 2. Impaired FN allocation in acute skin wounds of FHL2-deficient mice. A) Cutaneous lesions after applying skin punch wounds to FHL2 wild-type (+/+) and FHL2-deficient (–/–) mice. Hematoxylin and eosin staining (HE, left panels) and immunohistochemical stainings for FN expression (right panels) are shown. B) Immunoblot analysis of FN expression in wounds of wild-type and FHL2-deficient mice. Total RIPA lysates of wounded tissues (10 µg/lane) were separated on an 8% SDS-PAGE, and FN was detected by specific antiserum. ERK blot serves as a loading control. C) Impairment of the extracellular matrix in skin wounds visualized by silver staining. Top panels (1–3) illustrate wound healing in FHL2 wild-type mice; bottom panels (4–6) in FHL2-deficient mice. Panels 1 and 4 (x50) show the transition of squamous epithelium to scar tissue; the latter is marked with an asterisk. A dense meshwork of fibers within the scar (2, x200) of wild-type tissue occurs with a distinct basal membrane (arrow) in the surrounding skin (3, x200), while the scar field of the FHL2–/– tissue (4, x50; 5, x200) shows fragmented fibers and microbleeding in the tissue. Panel 6 (x100) shows the transition of normal tissue to the wound; note the fragmentation of the basal membrane (arrow).

Thus, our finding of poor fibrillary FN organization and its delayed down-regulation in wounded tissue in vivo suggests a possible feedback-regulation in knockout mice to compensate the allocation defect of dermal cells by overexpression of FN. In summary, these data together with the in vitro results clearly demonstrate that the FHL2 protein is important for correct organization of the extracellular matrix.

Inactivation of FHL2 enhances integrin-mediated stimulation of ERK and phosphorylation of FAK at Tyr925
While signaling via the MAPK-ERK pathway is necessary for functional modulation of focal adhesions, stress fibers, and cell shape and thus also for matrix assembly, its constitutive or prolonged activity impairs the organization of ECM proteins (4 , 8 , 10) . To test whether the absence of the scaffolding protein FHL2 might influence the activation of the mitogenic MAP kinase, we investigated ERK stimulation in FHL2-positive and FHL2-negative cells. To distinguish between integrin- and growth factor-mediated ERK stimulation, we analyzed the activation kinetics of ERK in two different ways: after adhesion of cells to FN in medium without growth factors and after stimulation of adherent cells with growth factors. For the first approach, FHL2 wild-type or knockout mesenchymal stem cells were starved overnight and then plated onto FN-coated dishes in medium containing no growth factors. As expected, phosphorylation of both ERK1 and ERK2 as well as their kinase activity were stimulated in both cell types during adhesion to FN. However, the phosphorylation of ERKs was much higher in FHL2-deficient than in FHL2 wild-type cells (Fig. 3 A). Consequently, the kinase activity of ERK1 precipitated from FHL2–/– cells was also higher (Fig. 3B ). The difference in ERK1 activation was reproducible and varied from 2- to 5-fold between different experiments. Importantly, FHL2 reexpression in FHL2–/– cells prevented the integrin-mediated prolonged enhanced phosphorylation of ERK1 and ERK2 as well as the increased kinase activation (Fig. 3B, C ). In the second approach, ERK activation was studied after stimulation of the cells by growth factors. Therefore, starved but still adherent cells were stimulated with a mixture of EGF and PDGF for different times, and RIPA cell lysates were analyzed for ERK activation with specific phospho-ERK antibodies. As Fig. 3D shows, ERK1 and ERK2 were rapidly activated in both cell types and, no significant differences in the degree or the kinetic of ERK phosphorylation between wild-type and knockout cells were detected.


Figure 3
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  		Figure 3. FHL2 deficiency alters integrin-mediated phosphorylation of ERK and FAK. FHL2+/+, FHL2–/–, or FHL2–/– rescue cells were starved overnight in medium with 0.25% FCS, trypsinized, washed, and resuspended in medium with 0.25% FCS. After recovery for 30 min in suspension, the cells were plated on FN-coated plates (20 µg/ml) for the times indicated. A) RIPA cell lysates were separated by 10% SDS-PAGE, and ERK1/2 proteins were analyzed by immunoblotting with antibodies as indicated. Numbers above the panels indicate relative ERK phosphorylation, estimated densitometrically as the relative intensity of the p-ERK bands to the loading controls. Values at time point 0 were taken as unity. Median values of 4 repeated experiments are shown. B) ERK1 was immunoprecipitated from cell lysates and assayed for its ability to phosphorylate MBP (top panel) in a kinase assay. Numbers above the panels indicate relative ERK activity, measured by phosphoimager scanning of pMBP bands. C) Total RIPA lysates of FHL2-negative and rescued cells were studied for ERK1/2 activation using phospho-specific antibodies. The fold of ERK activation was estimated as in A. Median values of 4 repeated experiments are shown. D) Adherent wild-type or FHL2-deficient cells were starved overnight in medium with 0.25% FCS and stimulated with 10 ng/ml EGF and 10 ng/ml PDGF for different times; RIPA lysates were analyzed by immunoblotting with specific antibodies. E) Phosphorylation of FAK at Tyr925 (top panel) and FAK at Tyr397 (bottom panel) after plating of starved cells on FN.

Integrin-mediated ERK activation requires phosphorylation of FAK at Tyr925. This creates an SH2-binding site for the growth factor receptor-bound protein 2 (Grb2) leading to activation of Ras, the upstream effector of ERK (20) . The enhanced integrin-triggered activation of ERK in FHL2-negative cells suggests a possible change in phosphorylation of FAK at Tyr925 in these cells. To prove this assumption, the phosphorylation of FAK at this residue was analyzed using phospho-specific antibodies. To ensure that the major stimulation of the kinase results from integrin activation, starved cells were trypsinized and kept for 30 min in suspension, followed by plating onto FN-coated dishes for different periods of time in medium without growth factors. Indeed, phosphorylation of the Tyr925 residue was enhanced in FHL2-deficient compared to FHL2 wild-type cells (Fig. 3E , top panels). The autophosphorylation activity of FAK in these cells, however, did not show significant differences (Fig. 3E , bottom panels, and ref. 27 ), suggesting that the initiation of FAK activation is rather not changed in FHL2-deficient cells. Taken together, our data show that only the integrin-mediated but not the growth factor-induced MAP kinase stimulation was affected in the absence of FHL2. This is in good agreement with the increased phosphorylation of FAK at Tyr925 and suggests that the enhanced ERK activation in FHL2–/– cells is mainly due to modified integrin/FAK signaling in these cells.

Inactivation of FHL2 alters clustering of focal adhesions
The formation of ECM frameworks depends on enzymatic activity and structural organization of focal adhesions. We have previously shown that FHL2 is present in FA at the termini of actin stress fibers where it is colocalized with integrins (24) and that absence of FHL2 alters actin stress fiber formation (27) . To address further the question of whether FHL2 influences the organization of focal adhesions, we analyzed focal adhesions in FHL2+/+, FHL2–/– and FHL2–/– rescued cells by immunofluorescence staining. FA structures were visualized with antibodies detecting different focal adhesion proteins (integrins, paxillin, and p-Y; Fig. 4 A) and talin, vinculin, tensin, and {alpha}-actinin (data not shown), and their formation was independent of FHL2 expression. However, the clustering pattern of FA proteins in FHL2-deficient cells was altered (Fig. 4A, B ). While in spread wild-type cells such proteins were present as thick and abundant clusters, located mostly at the cell periphery, they assembled in knockout cells into numerous and less-bundled complexes that were smaller in size and had a broader distribution. Counting of color pixels representing the paxillin or p-Y staining did not reveal a significant difference between wild-type and knockout cells. The numbers of FA pixels per cell were roughly the same, suggesting that only clustering of FA but not the total amount of cell-matrix contacts is changed in cells without FHL2. This fact is in accordance with data showing no difference in FAK autophosphorylation (Fig. 3E ) or cell attachment (Supplemental Fig. 4A). Investigation of focal contacts in embryonic fibroblasts confirmed the less-efficient bundling capacity of these structures in FHL2-deficient cells, further highlighting the fact that, in the presence of the scaffolding protein FHL2, the formation of integrin-dependent protein clusters proceeds more efficiently (Supplemental Fig. 3). The FHL2–/– rescue cells showed an intermediate pattern (Fig. 4A, B ), consistent with their lower expression of FHL2 protein when compared to wild-type cells (Supplemental Fig. 2). The altered organization of focal adhesions in FHL2–/– cells was not due to altered expression of integrins (27) or other focal adhesion proteins (Fig. 4C ) or due to altered adhesion properties (Supplemental Fig. 4A). Neither did expression of FHL2 alter the affinity state of integrin receptors, when this was measured by flow cytometry using the activation state-specific PAC1 antibody and the {alpha}IIb{alpha}7Aβ3 chimeric receptor (Supplemental Fig. 4B). The cytosolic part of the {alpha}IIb chain was exchanged for the cytodomain of integrin {alpha}7A, to which FHL2 shows a strong binding (23) .


Figure 4
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  		Figure 4. Absence of FHL2 alters clustering of focal adhesions. A) Cells were incubated on FN-coated coverslips for 3 h in full stem cell medium and stained for integrin {alpha}5, paxillin, or p-Tyr. B) Images represent a higher magnification of the corresponding frames in A. Scale bars = 20 µm. C) RIPA total cell lysates (5 µg/lane) were separated by 10% SDS-PAGE and immunoblotted with antibodies indicated. The ERK1/2 blot serves as a loading control.

Inhibition of the ERK pathway in FHL2-deficient cells restores ECM assembly
Finally, we tested whether down-regulation of the enhanced ERK activity in FHL2 knockout cells might restore the impaired assembly of ECM proteins. We analyzed matrix assembly on FHL2–/– cells under conditions where the Ras-ERK signaling pathway was inhibited. Two different experimental approaches were applied: inhibition of ERK by a synthetic kinase inhibitor or by expression of a dominant-negative Raf mutant. First, the U0126 compound was tested for its efficiency in integrin-induced ERK suppression. Starved cells were trypsinized and preincubated for 30 min in suspension with U0126 (39) and then allowed to adhere to FN-coated dishes in medium containing U0126 but no growth factors or serum. As shown in Fig. 5 A, the integrin-mediated up-regulation of ERK activity was efficiently blocked both in wild-type and knockout cells. We next incubated FHL2–/– cells for 3 days in the presence of the MEK inhibitor U0126 and studied its effect on FN assembly by immunofluorescence microscopy. Indeed, down-regulation of ERK activation significantly improved the assembly of FN in FHL2-deficient cells (Fig. 5B ). During incubation with the U0126 compound, cells did not proliferate. However, the inhibitor was not toxic, except at the highest concentration tested, when effectively no matrix assembly occurred. Not only FN assembly was altered in FHL2–/– cells on inhibition of ERK activation; laminins and nidogen were also better incorporated into ECM networks (Fig. 5C ). Consistent with the immunofluorescence data, increased accumulation of polymerized ECM proteins in U0126-treated cells was also shown by immunoblot analysis of Triton-insoluble cell-free fractions (Fig. 5D ). Treatment of wild-type cells with the U0126 MEK inhibitor did not significantly improve the already well organized ECM network of these cells (data not shown).


Figure 5
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  		Figure 5. Inhibition of ERK activity in FHL2–/– cells restores the deficiency in ECM assembly. A) FHL2+/+ or FHL2–/– cells were starved overnight in medium with 0.25% FCS, trypsinized, preincubated in suspension with indicated amounts of U0126 compound, and plated in starvation medium on FN-coated dishes. After 1 h, cells were lysed with RIPA, and 10 µg of cell lysates was separated by PAGE and analyzed by immunoblotting for phosphorylation of ERK1/2. The fold of ERK activation was estimated as in Fig. 3 A. Median values of 2 repeated experiments are shown. B, C) FHL2–/– cells were incubated on noncoated glass coverslips in DMEM:MCDB-201 medium mix with 2% FCS but without growth factors in the presence of indicated amounts of the U0126 compound for 3 days, fixed, and analyzed for ECM protein assembly by immunofluorescence microscopy. To visualize the cells, the F-actin or nuclei were counterstained with phalloidin (green) and DAPI (blue). Cells were not permeabilized. Scale bars = 50 µm. D) FHL2 knockout cells were incubated in medium without growth factors for 1 or 2 days in the presence of the U0126 compound. The cells were removed by 0.5% Triton X-100, and ECM proteins remaining on the plates were analyzed by immunoblotting. β-Actin blot shows the amount of actin left on dishes after cell bodies had been removed with Triton X-100. It roughly reflects the F-actin polymerization state after treatment of cells with U0126 shown in B.

In the second approach, we used myc-tagged dominant-negative or constitutively active mutants of the c-Raf kinase. The first mutant, Raf-C4, contained the Ras binding domain but lacked the C-terminal kinase domain. It competes with endogenous Raf for binding to Ras, thus blocking the stimulation of Raf and consequently that of MEK and ERK. The second mutant, Raf-BXB, lacked the N-terminal regulatory domain but possessed the kinase domain (29 , 30) . As mouse stem cells are difficult to transfect in amounts sufficient for kinase analysis of recombinant proteins, we tested the functional activity of the Raf mutants in HEK293 cells. As expected, transfection of the Raf-BXB construct prevented down-regulation of ERK1/2 phosphorylation during overnight starvation; conversely, the dominant-negative Raf-C4 mutant inhibited ERK1/2 phosphorylation after stimulation of the cells with growth factors (Fig. 6 A). We then transfected FHL2+/+ and FHL2–/– stem cells with Raf-C4 or Raf-BXB and analyzed the few transfected cells for their matrix assembly ability. The cells were cultivated on uncoated glass coverslips for 3 days, fixed, and costained with antibodies to FN and myc-tag. Immunofluorescence analysis showed that expression of the constitutively active Raf-BXB further disturbed the FN assembly in FHL2–/– cells and, the higher the expression of the Raf-BXB, the poorer the FN organization (Fig. 6B ). In contrast, cells expressing the dominant-negative Raf mutant (Raf-C4) organized FN molecules on their surface much better than nontransfected cells (Fig. 6B, C ), which confirmed the results obtained with the MEK inhibitor. Consistent with the fact that strong inhibition of the mitogenic Ras-ERK pathway has an antisurvival effect (40) , the most improved FN assembly was seen in cells with intermediate Raf-C4 expression (Fig. 6B, C ). Expression of Raf-BXB in wild-type cells efficiently blocked FN assembly (Fig. 6D ). Furthermore, it impaired the clustering of focal adhesions, as visualized by staining of these cells with antibodies to paxillin or FHL2 (Fig. 6E ). Taken together, these data support the hypothesis that the enhanced integrin-mediated ERK activity in FHL2–/– cells leads to the disturbed assembly of ECM proteins.


Figure 6
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  		Figure 6. Inhibition of ERK in FHL2–/– cells restores ECM assembly, and activation of ERK in FHL2+/+ cells decreases ECM polymerization. A) HEK 293 cells were transfected with indicated constructs. After 24 h, they were starved overnight in medium with 0.5% FCS and stimulated with 10 ng/ml EGF and 10 ng/ml PDGF for 15 min. RIPA lysates were analyzed by immunoblotting with antibodies as indicated. The fold of ERK activation was estimated as in Fig. 3 A. Median values of 2 repeated experiments are shown. B) Relation between expression of dominant negative Raf-C4 or constitutive-active Raf-BXB mutants and FN assembly. FHL2–/– stem cells were transfected with myc-tagged Raf-C4 or Raf-BXB constructs and after incubation for 3 days, they were fixed, permeabilized, and costained with rabbit polyclonal anti-FN antibodies for FN and with anti-myc mAb for Raf mutants. The coverslips with stained cells were coded, and immunosignals of transfected cells were simultaneously scored according to a semiquantitative scale from + (weak staining) to ++++ (strong staining) for Raf (red) and FN (green). The total amount of scored cells is shown on the right. Note that the degree of FN assembly varies depending on the expression of both Raf-C4 and Raf-BXB but in a different manner. The shadowed boxes describe the distribution trend of FN depending on Raf expression or activity. Numbers in the boxes represent the counted cells. C) FHL2–/– stem cells were transfected with myc-tagged Raf-C4 construct. After incubation for 3 days, cells were fixed, permeabilized, and costained with rabbit polyclonal anti-FN antibodies (green) and with anti-myc mAb, clone 9E10, for C4-Raf (red). Arrowheads show nontransfected cells. D, E) FHL2+/+ stem cells were transfected with myc-tagged Raf-BXB, cultured for 3 days and costained with antibodies to myc (green) and to FN, paxillin or FHL2 (in red). Scale bars = 50 µm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we show that loss of FHL2 causes altered bundling of focal adhesion structures and enhanced phosphorylation of FAK at Tyr925. As a result, the integrin-mediated activation of the mitogenic ERK pathway is increased in FHL2-deficient cells, leading to impaired assembly of secreted ECM proteins.

Focal adhesions are sites where ECM proteins are linked via integrins to the actin cytoskeleton. The assembly of these clusters is initiated by binding of integrins to ECM proteins and is further catalyzed by FAK and Src (20 , 21) . These kinases phosphorylate numerous substrates creating binding sites for other proteins, hence ensuring the formation of functional focal adhesion clusters. Although FHL2 directly associates with integrins and FAK (23 , 25) , focal adhesions were also formed in the absence of FHL2. This finding suggests that FHL2 is unlikely to be involved in recruiting FAK to integrins or in initiation of focal adhesions and indeed, the autophosphorylation of FAK at Tyr397 was not changed in FHL2-negative cells (Fig. 3E and ref. 27 ). Nevertheless, in FHL2-expressing cells, FHL2 was present in these structures as soon as they were formed during cell attachment. However, throughout further spreading and formation of actin stress fibers, the distribution of FHL2 changed, being present mostly along the actin cables (Supplemental Fig. 5A). Here, FHL2 was colocalized with {alpha}-actinin (Supplemental Fig. 5B), indicating that it is involved as a scaffolding protein in maintenance of stress fibers and formation of mechanical forces. In fact, inactivation of FHL2 results in poorer organization of actin stress fibers and reduced contractility (Supplemental Fig. 5C and ref. 27 ). Modulation of {alpha}-actinin-induced actin bundling has recently been described for FHL3 (41) , another FHL protein with high similarity to FHL2.

Although the scaffolding protein FHL2 was not necessary for the formation of focal adhesions per se, its inactivation impaired the maturation of these structures, suggesting that it plays a role in their bundling and stabilization. It seems that no other particular structural protein is missing in FA clusters of FHL2–/– cells, but rather their ability to form large stable protein clusters is impaired. Again, this is in good agreement with the better-organized cytoskeleton in FHL2 expressing cells, as formation of large focal adhesion clusters facilitates development of actin stress fibers and mechanical tension (42 43 44) . Although the phenotype of FHL2–/– cells is in many features similar to that of FAK–/– cells (11 , 19 , 45 , 46) , it is not identical. Like FAK-null cells, FHL2–/– cells show impaired focal adhesion clustering, a less-organized cytoskeleton, reduced Rac1 activation, lowered cell motility, and impaired matrix assembly. Further, these changes were restored in FHL2–/– cells by expression of the missing protein. However, in the absence of FHL2, the FAK phosphorylation was not reduced but rather enhanced, at least at Tyr925 and Tyr861 (Fig. 3E and ref. 27 ).

FHL2 is a scaffolding protein, and its overexpression does not necessarily change the composition or amount of formed protein clusters, which would alter cellular activities. Therefore, we could not measure any alterations of cell motility after overexpression of FHL2 in C2C12 myoblasts, which naturally express high amounts of endogenous FHL2 (23) . Similarly, overexpression of FHL2 in wild-type stem cells or NIH3T3 fibroblasts did not lead to significant changes in FN assembly on these cells. Lai et al. (47) , however, reported an increase of osteoblast migration through transwell membranes after overexpression of FHL2. The different cellular background or the amount of endogenously expressed FHL2 protein may be a reason for these differences. However, the delayed migration of our FHL2-negative stem cells was readily detectable and could be restored by expression of recombinant FHL2 protein and was based on decreased Rac1 activity (27) .

The impaired matrix assembly in FHL2 knockout cells is consistent with our observations that FHL2-negative cells have fewer bundled FA and stress fibers, reduced contraction of collagen matrices, and enhanced integrin-dependent activation of ERK (present study and ref. 27 ). Interestingly, only the integrin-mediated but not the growth factor-induced ERK activation was changed in FHL2–/– cells. The latter result is consistent with the higher phosphorylation of FAK at Tyr925 after cell adhesion to FN. It has previously been shown that prolonged or constitutive activation of the mitogenic Ras-ERK pathway reduces the ligand-binding affinity of integrins (8 , 10 , 22) , disrupting formation of focal adhesions and actin stress fibers (10 , 16) . This in turn reduces the mechanical tension on the surface of spread cells, which is of importance for proper association of matrix proteins and fibrillogenesis (2 3 4) . Therefore, we hypothesized that the increased ERK activation in FHL2–/– cells caused the decreased matrix assembly. Indeed, the disturbed matrix organization in FHL2–/– cells improved when the ERK activation was down-regulated either by a synthetic MEK inhibitor or a dominant-negative Raf mutant. Expression of a constitutively active Raf kinase had the opposite effect, substantially decreasing the matrix assembly in wild-type cells and reducing it still further in FHL2 knockout cells. Furthermore, not only was matrix assembly disturbed after expression of the active Raf-BXB, but formation of F-actin fibers and focal adhesion clustering was also impaired. Taken together, these data are in good agreement with previous observations that activation of the Ras-ERK pathway by oncogenic Ras or Raf inhibits integrin-mediated FN assembly (8 , 10) . Additionally, they show that a decreased matrix assembly may also occur in the presence of FHL2 protein under sustained ERK activity and that FHL2 acts upstream of ERK. These results indicate further that an FHL2/p-ERK interaction is not required for the regulation of matrix assembly, as might be expected given the direct binding ability of FHL2 to phosphorylated ERK. The FHL2/p-ERK association was shown in cardiomyocytes, where it had no influence on the kinase activity of ERK but prevented its nuclear translocation (26) . The enhanced activation of the integrin-mediated Ras-Raf-MEK-ERK pathway in FHL2-deficient cells results in the down-regulation of ECM protein polymerization efficiency. However, additional signaling cascades may also change in the absence of FHL2 and might also influence ECM assembly. Indeed, inhibition of ERK by the U0126 compound did not correct the impaired ECM organization in FHL2-negative cells to the level of wild-type cells, indicating the possible participation of other pathways. Alteration of the Rho-Rho-kinase-myosin pathway seems particularly plausible considering the impaired formation of actin stress fibers and cell contractility as well as decreased Rac activation in FHL2-negative cells (27) .

FHL2 has no intrinsic enzymatic activity but rather functions as a scaffold that can bring together different proteins, forming or modulating functional protein clusters, the efficiency of which will be attenuated in the absence of FHL2. Depending on the content of these clusters, FHL2 might induce different functional consequences. For example, the function of FHL2 as an enhancing or suppressing coregulator of transcription factors is well documented (38) . Hence, it is perhaps not surprising that the FHL2 knockout mouse reveal only a mild phenotype (47 48 49) . The function of this protein obviously can be substituted by other proteins. Further formation of functional protein clusters may also occur less efficiently without FHL2, and, therefore, any functional deficit would become visible only under certain conditions, such as the healing of acute skin wounds, where we saw a delay in wound closure. The reasons for this are complex as our studies show. First, FHL2 is an early response gene (50) and, while not expressed in intact dermis, it is rapidly up-regulated in fibroblasts on injury (27) . Second, FHL2 promotes migration of mesenchymal cells into the wounded area (27) . Third, FHL2–/– cells have a defect in matrix assembly (this study), which results in accumulation of poorly organized FN in wounds and impaired regeneration of the basement membrane in injured tissue. Fourth, FHL2 enters the nucleus on stimulation of cells with bioactive lipids, a large amount of which is released from blood cells into the injured tissue (51) . In the nucleus, FHL2 interacts with the SRF transcription factor and regulates the expression of {alpha}-SMA (52) . Incorporation of SMA into stress fibers stabilizes focal adhesions and increases the intracellular mechanical stress (43 , 53) . Indeed, expression of {alpha}-SMA in wounds of FHL2-deficient mice was reduced and, correspondingly, the fibroblasts had a contractility defect (27) . Contraction of granulation tissue being important for wound closure. Our findings point to an important role of FHL2 in the organization of focal adhesion structures and in integrin-mediated regulation of ERK activity and, as a consequence, in the assembly of ECM proteins.


   ACKNOWLEDGMENTS
 
We thank Dr. Neil Smyth for critical reading of the manuscript and Dr. Mirko Schmolke for help with flow cytometry experiments. This work was supported by grants from the Sander-Stiftung to V. W. and B. M. and by a grant from the Deutsche Forschungsgemeinschaft to R. B.


   FOOTNOTES
 
1 These authors contributed equally to this work.

2 Current address: Department of Biopathology and Biomedical Methodology, University of Palermo, Palermo, Italy

Received for publication July 30, 2007. Accepted for publication February 21, 2008.


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