Deficiency in the LIM-only protein Fhl2 impairs skin wound healing

Abstract

After skin wounding, the repair process is initiated by the release of growth factors, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as fibroblast invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. 19:359-369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. 21:736-748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of alpha-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

Figure 1.

Expression and nuclear translocation of Fhl2 in myofibroblasts within human skin wounds. (A) Immunostaining of human wound tissue reveals strong up-regulation of Fhl2 in α-SMA–positive myofibroblast-like cells present in dermal granulation tissue 5 d after wounding. (B) Double immunostaining of human wound tissue indicates that Fhl2 immunosignals (arrows, red AEC stain) label α-SMA– and SM22-positive myofibroblasts. Bars: (A) 50 μm; (B) 25 μm.

Figure 2.

Delayed wound healing in Fhl2^−/− mice. Up-regulation of Fhl2 mRNA (A) and Fhl2 protein (B) in skin wounds 5 and 12 d after applying punch biopsies in Northern and Western blots, respectively. Fhl2^+/+ mice, Fhl2 ^−/− mice (Fhl2 ^−/−), and the rescue mouse strain carrying a SM22-promoter Fhl2 transgene in an Fhl2 ^−/−genetic background (Fhl2 ^−/− tgSM22Fhl2) were used. Gapdh and β-actin served as loading controls. (C) Percentage of entirely closed wounds in Fhl2^+/+, Fhl2 ^−/−, Fhl2 ^−/−-rescue, and Fhl2^+/+ tgSM22Fhl2 mice after 5 and 12 d, respectively. 38–42 wounds for each genotype and time point were monitored by macroscopic inspection, and punch biopsies were verified histologically. Error bars represent the SD.

Figure 3.

Fhl2 regulates fibroblast contractility and coactivates SRF-mediated α-SMA transcription in wound healing. (A) Defective collagen contraction in Fhl2 ^−/− embryonic fibroblasts (top). Collagen contraction and stimulation in response to treatment with S1P were restored after retransfecting Fhl2 cDNA into Fhl2 ^−/− fibroblasts (bottom). Transfections were done in triplicate, with either empty vector (pcDNA3) or Fhl2 expression plasmid (Fhl2). SDs were <2% in all cases. (B) HEK293, Fhl2 ^−/− fibroblasts (MEFs), and Fhl2 ^−/− mesenchymal stem cells (MSCs) were transfected with an α-SMA promoter-driven luciferase reporter construct and expression plasmids for SRF and Fhl2. Bars indicate the fold induction of transfecting SRF, Fhl2, and both expression vectors versus the luciferase activity of the reporter plasmid. n = 3. Error bars represent the SD. (C) Cutaneous lesions 5 d after applying skin punch wounds to Fhl2^+/+ and Fhl2 ^−/− mice. Images show hematoxylin and eosin staining (HE, top) and immunohistochemical stainings for α-SMA expression (bottom). There is strong α-SMA reactivity in the granulation tissue of Fhl2^+/+, but not of Fhl2 ^−/−, mice below the reepithelializing keratinocytes on top. Bar, 100 μm.

Figure 4.

Reduced motility of Fhl2^−/− mesenchymal stem cells. (A) Fhl2^+/+, Fhl2 ^−/−, and Fhl2 ^−/− -rescue stem cells were examined regarding shape (top), actin cytoskeleton organization, and distribution of Fhl2 protein (bottom). F-actin was visualized with Alexa Fluor 488–coupled phalloidin (green). Fhl2^+/+ and Fhl2 ^−/− cells were immunostained for Fhl2 with the F4B2 monoclonal antibody, Fhl2 ^−/− rescued cells with the anti-myc 9E10 monoclonal antibody. Bars, ∼50 μm. (B) Migration of Fhl2^+/+, Fhl2 ^−/−, and Fhl2 ^−/− -rescue mesenchymal stem cells. Cell migration on uncoated dishes into cell-free areas was photographed at 0 and 42 h.

Figure 5.

Reduction of p130Cas expression in Fhl2^−/− cells. (A) p130Cas, Src, or FAK proteins were immunoprecipitated from lysates of fibronectin-stimulated Fhl2^+/+ or Fhl2 ^−/− cells and analyzed by immunoprobing for the proteins indicated. (B) Recombinant expression of myc-Fhl2 in Fhl2 ^−/− cells reverses p130Cas expression. Immunoblot with anti-ERK1 antibody served as loading controls. 10 μg of total cell lysates were analyzed. (C) Expression control of p130Cas in Fhl2 ^−/− stem cells. Cells were infected with retroviruses containing the GFP vector or GFP+p130Cas. 48 h later the cells were harvested and one part was used for analysis of infection efficiency by FACSscan (top) or by immunoblotting (bottom). Black curve of the FACSscan profile, noninfected cells; blue curve, GFP-vector–infected cells; red curve, GFP+p130Cas–infected cells. For immunoblotting, 7.5 μg of total cell lysates were separated on 10% SDS-PAGE, and p130Cas and ERK1/2 (as loading controls) were detected with specific antibodies. (D) The second part of infected cells, along with noninfected Fhl2^+/+ and Fhl2 ^−/− cells, was used for migration assays. Migration of cells on noncoated or on fibronectin-precoated dishes was studied. The assays were performed twice with similar results. Only cell motility on noncoated dishes is shown. Error bars represent the SD. (E) Reduction of Rac activation in Fhl2 ^−/− cells (top) and increased Rac activation in p130Cas-overexpressing Fhl2 ^−/− cells (bottom). The cells were serum-starved overnight, trypsinized, and plated for 15, 30, and 60 min on cell culture dishes precoated with 20 μg/ml fibronectin. For precipitation of GTP-loaded Rac, cells were lysed in Triton X-100 lysis buffer, and 400 μg of protein lysates were rotated with GST-PAK3–coated glutathione beads. The precipitates and the lysates were analyzed for the presence of Rac1 by SDS-PAGE and Western Blotting. The fold of Rac activation was estimated densitometrically as the relative intensity of the GTP-Rac bands to the loading controls. Values at time point 0 were taken as unity.