Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β-Slug signaling

Abstract

Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial-mesenchymal transition (EMT), TGF-β1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM(-/-)) wounds. Correspondingly, VIM(-/-) wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM(-/-) wounds. Vimentin reconstitution in VIM(-/-) fibroblasts restored both their proliferation and TGF-β1 production. Similarly, restoring paracrine TGF-β-Slug-EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-β1-Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation.

Keywords: epithelial–mesenchymal transition; fibroblast proliferation; keratinocyte migration; vimentin intermediate filaments; wound healing.

Fig. 1.

VIM^−/− mice display wound-healing defect in a burn wound model. (A) Representative pictures showing immunohistochemical labeling of pan-keratin in WT and VIM^−/− wounds on days 9 (D9) and 15 (D15) postinjury. (Scale bar, 100 μm.) (B) Quantification of the percentage of wound reepithelialization at different time points after wounding in VIM^−/− and WT wounds. Data are shown as means ± SEM; n = 6. (C) Representative wound pictures from VIM^−/− and WT mice during the 15-d wound-healing period. (D) Quantification of the remaining wound area at different time points after wounding in WT and VIM^−/− groups. Data are shown as means ± SEM; n = 6–12. (E) Comparison of the healing times (scab falling off) in the days after wounding. Data are shown as means ± SEM; n = 12. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S1.

VIM^−/− mice have slower wound healing in an excisional wound model (related to Fig. 1). (A) Representative pictures of immunohistochemical labeling of pan-keratin in wounds in VIM^−/− and WT mice on days 9 (D9) and 15 (D15) postinjury. (B) Representative pictures of wounds in WT and VIM^−/− mice 15 d after excisional injury. (C) Quantification of the wound area remaining at different time points after wounding in the WT and VIM^−/− groups. Data are shown as means ± SEM; n = 4. *P < 0.05; **P < 0.01; ns, not significant.

Fig. S2.

Vimentin control mice have normal skin composition and collagen accumulation (related to Figs. 2 and 5). (A) Representative pictures showing immunohistochemical labeling of pan-keratin in uninjured skin from control mice on E14 and from 18-d-old, 10-wk-old, and 10-mo-old mice. (B) Representative confocal pictures of immunofluorescent labeling of N-cadherin (red) and keratin 5 (K5, green) in skins of uninjured control mice. (C and D) Quantitation of K5⁺DAPI⁺ cells (C) and N-cadherin⁺ DAPI⁺ cells (D) in the epidermal and dermal region. (E) Representative pictures of Picro-Sirius Red staining of collagen in the corresponding sections of skins from VIM^−/− and WT control mice. (F) The quantitation of collagen accumulation (Picro-Sirius Red-positive areas) in the mesenchymal/dermal regions of wounds. In A, B, and E, D, dermis region; E, epidermis region. (Scale bars, 100 μm.) In C, D, and F, data are shown as means ± SD; n = 2–4.

Fig. 2.

Compromised reepithelialization and EMT differentiation in VIM^−/− wounds. (A and B) Representative pictures of confocal images of keratin 6 (green) and DAPI (red) (A) and keratin 14 (green) and DAPI (red) (B) in VIM^−/− and WT wounds on day 9 and 15 after skin burn injury. D, dermis region; S, scab region. (Scale bars, 200 μm.) (C) Representative confocal images of keratin expression as visualized by a pan-keratin antibody (green), vimentin (red), and DAPI (blue) in VIM^−/− and WT wounds on day 15 postinjury. White arrows indicate the region of thin and poor keratinization in VIM^−/− wounds. (D) Quantification of average epidermis thickness on day 15 after burn injury. Bars indicate the mean fold changes relative to WT ± SEM; n = 3. (E) qRT-PCR analysis of mRNA transcripts for Slug, N-cadherin (Cdh2), FSP-1 (S100a4), and fibronectin (Fn1) in isolated epidermal regions of VIM^−/− and WT wounds on days 0, 3, 9, and 15 after burn injury. Bars indicate the mean fold changes ± SEM relative to day 0 WT; **P < 0.01; n = 3.

Fig. S3.

Compromised reepithelialization and keratinocyte transdifferentiation in VIM^−/− wounds (related to Fig. 2). (A) Representative confocal images of the expression of pan-keratin (green) and DAPI (blue) in wounds of VIM^−/− and WT mice on day 9 postinjury (D9). The white arrowheads indicate examples of migrating pan-keratin⁺ cell colonies locating in the dermis–epidermis interface. (Scale bar, 100 μm.) (B) Quantitation of pan-keratin⁺ cell colony numbers in dermis–epidermis interface regions on day 9 postinjury. (C) Representative confocal images of the expression of α-SMA (red) and pan-keratin (green) in VIM^−/− and WT wounds on day 9 postinjury. The white arrowheads indicate examples of pan-keratin⁺α-SMA⁺ cells in the dermal region of WT wounds on day 9 postinjury. (Scale bars, 20 μm.) (D) Quantitation of pan-keratin⁺α-SMA⁺ cells in wounds in VIM^−/− and WT mice on day 9 postinjury. (E) Representative pictures of immunofluorescent labeling of keratin 5 (green) and N-cadherin (red) in wounds of VIM^−/− and WT mice on day 15 postinjury (D15). (Scale bar, 100 μm.) (F) Quantitation of N-cadherin⁺ K5⁺ cells in epidermis in day 15 wounds. In A and E, E: epidermis region; D: dermis region. In B, D, and F, data are shown as means ± SEM; n = 3. **P < 0.01.

Fig. 3.

TGF-β1–Slug signaling promotes keratinocyte differentiation and migration. (A) qRT-PCR analysis of transcripts for TGF-β1 (Tgfb1) in VIM^−/− and WT wounds on days 3, 9, and 15 after wounding. Bars show mean fold changes ± SEM relative to WT; n = 3. (B) Mouse keratinocytes were stimulated with 5–10 ng/mL TGF-β1 for 0, 3, or 5 d. Cell lysates were collected and blotted with antibodies against desmoplakin, E-cadherin, N-cadherin, vimentin, and Hsc-70 as loading control. (C and D) Quantification of Slug (C) and N-cadherin (D) intensity in B equalized to Hsc-70. Bars show mean fold changes ± SEM relative to day 3 control mice; n = 3. (E) Mouse keratinocytes were transfected with scramble siRNA (si Scra) or Slug siRNA (si Slug) oligos for 2 d and then were stimulated with 5 ng/mL TGF-β1 for 3 d. Cell lysates were collected for Western blotting analysis of Slug, N-cadherin, vimentin, desmoplakin, and loading control Hsc-70. (F and G) Quantification of Slug (F) and N-cadherin (G) intensity in E equalized to Hsc-70. Bars show the mean fold changes ± SEM relative to mock transfections (Ctrl); n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 4.

Vimentin promotes TGF-β production from fibroblasts driving EMT and migration of keratinocytes. (A) qRT-PCR analysis of transcripts for TGF-β1 (Tgfb1) in VIM^−/− and WT MDFs. Bars show mean fold changes ± SEM relative to WT; n = 6. (B) Level of active and latent forms of TGF-β1 in the supernatants of 6-d MDF cell cultures were analyzed by ELISA. Data are shown as mean ± SEM; n = 3. (C) VIM^−/− and WT MDF cell-culture media were extracted on 0, 3, and 6 d after cell growth. The growth medium of mouse keratinocytes was replaced with the MDF-conditioned medium for 5 d. Cell lysates were collected and blotted with antibodies against desmoplakin, E-cadherin, N-cadherin, vimentin, Slug, and loading control GAPDH. (D and E) In vitro wound-healing assay of mouse keratinocytes grown in 6-d conditioned medium from VIM^−/− MDFs and WT MDFs. The cell gap was monitored over 24 h, and the wound areas were measured and plotted against the time point. At least four wound scratches were analyzed per experiment. Data are shown as means ± SEM; n = 3. (F) Mouse keratinocytes grown in 6-d conditioned medium from the VIM^−/− and WT MDFs were treated with control IgG (Ctrl IgG, 10 μg/mL), pan–TGF-β neutralizing antibody 1D11 (1D1 Ab, 10 μg/mL), two chemical inhibitors of TGF-β receptors [LY2109761 (2 μM) and SB431542 (2 μM)] or were grown in the corresponding DMSO control (DMSO, 2 μM) for 3 d. The cell lysates from this experiment were blotted with antibodies against pSmad2/3, total Smad2/3, Slug, N-cadherin, and loading control GAPDH. (G and H) Quantification of Slug and N-cadherin intensity in F equalized to GAPDH; bars show the mean fold changes relative to WT Ctrl IgG ± SEM; n = 3. (I) In vitro wound-healing assay of mouse keratinocytes (at 16 h of wound healing) in the treatments in F. (J) The y axis shows the percentage of area covered (at 16 h of wound healing) by keratinocytes transfected with mock, scramble siRNA (si Scra), or Slug siRNA (si Slug) oligos for 2 d and incubated in 6-d WT or VIM^−/− MDF-conditioned medium. In I and J, at least four wound scratches were analyzed per experiment. Data are shown as means ± SEM; n = 3; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S4.

Inflammatory cytokines and infiltrated inflammatory cells in wounds in WT and VIM^−/− mice (related to Fig. 4). (A) qRT-PCR analysis of transcripts for IL-6 (Il6), IL-1α (Il1a), IL-1β (Il1b), IL-23 (Il23), and TNF-α (Tnf) in wounds in VIM^−/− and WT mice on days 3 (D3), 9 (D9), and 15 (D15) after wounding. Data are shown as means ± SEM; n = 3. (B) Representative pictures of immunohistochemical labeling of mouse neutrophils in wounds in VIM^−/− and WT mice on days 3 and 9 postinjury. The inflamed tissue is indicated by black dashed lines. (Scale bars, 20 mm.) (C) Comparison of the temporal regulation of inflammation (the wound-edge regions infiltrated with neutrophils) in wounds in VIM^−/− and WT mice. Data are shown as means ± SEM; n = 3. (D and E) Representative pictures (D) and quantitative comparison (E) of the myeloperoxidase activity of neutrophils (MPO), monocyte/macrophage marker CD11b, and T-cell marker Zap-70 in the wound-edge regions of wounds in VIM^−/− and WT mice 9 d after injury. (Scale bar, 100 μm.) Data in E are shown as mean ± SEM; n = 3. (F) Level of active and latent forms of TGF-β1 in the supernatants after 6 d of WT and VIM^−/− macrophage cell culture were analyzed by ELISA. Data are shown as means ± SEM; n = 5. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 5.

Vimentin promotes mesenchymal cell proliferation and collagen accumulation in vivo. (A) Representative confocal images of the expression of Ki67 (punctate green signal in the nucleus) and vimentin (red) in VIM^−/− and WT wounds on days 9 (D9) and 15 (D15) after burn wounding. Nuclei were counterstained with DAPI (blue). (Scale bars, 20 μm.) The white arrowheads indicate examples of ki67⁺ cells in dermal regions. D, dermis region; E, epidermis region. (B) Quantitation of ki67⁺ cells in mesenchymal/dermal regions of wounds. (C) Representative pictures of Picro-Sirius Red staining of collagen (Upper) and the immunohistochemical labeling of pan-keratin (Lower) in the corresponding sections of VIM^−/− and WT wounds on day 15 postinjury. The right lower corner of each panel shows an enlarged image of the area in the white box. (Scale bars, 100 μm.) (D) The quantitation of collagen accumulation (Picro-Sirius Red-positive areas) in mesenchymal/dermal regions of wounds. In B and D data are shown as means ± SEM; n = 3; *P < 0.05; **P < 0.01.

Fig. 6.

Vimentin promotes mesenchymal cell proliferation and paracrine EMT signaling in vitro. (A) The growth curve of VIM^−/− and WT MDF cells after transfected with different amounts of vectors encoding full-length vimentin (the total DNA per transfection was equalized with the control vector). Data are shown as mean ± SEM; n = 3. (B) Immunoblotting of vimentin, pERK1/2, total ERK1/2, and GAPDH expression of the cell lysates in A. (C and D) Western blotting of individual markers (C) and migration (D) of mouse keratinocytes growing in 6-d conditioned medium (D6) from VIM^−/− and WT MDFs transfected with the indicated amount of vimentin plasmids. Data are shown as means ± SEM; n = 3. (E) Scheme showing the working model. Vimentin has a profound effect on fibroblast proliferation, which activates both collagen production and TGF-β secretion. The active fibroblast TGF-β induces Slug–EMT signaling in keratinocytes, promoting EMT-like transdifferentiation and keratinocyte migration.

Fig. S5.

Vimentin does not influence fibroblast survival (related to Fig. 6). (A) The cell-survival curve of VIM^−/− and WT MDFs after transfection with different amounts of vectors encoding full-length vimentin (the total DNA per transfection was equalized with the control vector). Data are shown as means ± SEM; n = 3. (B) Representative confocal pictures of immunofluorescent labeling of DAPI (blue), keratin 5 (green), and vimentin (red) (Left) or keratin 14 (green) and N-cadherin (red) (Right) from isolated mouse keratinocytes. (Scale bars, 20 μm.)