Identification of cadherin 11 as a mediator of dermal fibrosis and possible role in systemic sclerosis

Abstract

Objective: Systemic sclerosis (SSc) is a chronic autoimmune disease clinically manifesting as progressive fibrosis of the skin and internal organs. Recent microarray studies demonstrated that cadherin 11 (Cad-11) expression is increased in the affected skin of patients with SSc. The purpose of this study was to examine our hypothesis that Cad-11 is a mediator of dermal fibrosis.

Methods: Biopsy samples of skin from SSc patients and healthy control subjects were used for real-time quantitative polymerase chain reaction analysis to assess Cad-11 expression and for immunohistochemistry to determine the expression pattern of Cad-11. To determine whether Cad-11 is a mediator of dermal fibrosis, Cad-11-deficient mice and anti-Cad-11 monoclonal antibodies (mAb) were used in the bleomycin-induced dermal fibrosis model. In vitro studies with dermal fibroblasts and bone marrow-derived macrophages were used to determine the mechanisms by which Cad-11 contributes to the development of tissue fibrosis.

Results: Levels of messenger RNA for Cad-11 were increased in skin biopsy samples from patients with SSc and correlated with the modified Rodnan skin thickness scores. Cad-11 expression was localized to dermal fibroblasts and macrophages in SSc skin. Cad-11-knockout mice injected with bleomycin had markedly attenuated dermal fibrosis, as quantified by measurements of skin thickness, collagen levels, myofibroblast accumulation, and profibrotic gene expression, in lesional skin as compared to the skin of wild-type mice. In addition, anti-Cad-11 mAb decreased fibrosis at various time points in the bleomycin-induced dermal fibrosis model. In vitro studies demonstrated that Cad-11 regulated the production of transforming growth factor β (TGFβ) by macrophages and the migration of fibroblasts.

Conclusion: These data demonstrate that Cad-11 is a mediator of dermal fibrosis and TGFβ production and suggest that Cad-11 may be a therapeutic target in SSc.

Figure 1

Accumulation of elevated amounts of cadherin 11 (Cad-11) in the affected skin of patients with systemic sclerosis (SSc) and in the lesional skin of mice with bleomycin-induced dermal fibrosis. A, Elevated levels of Cad-11 mRNA in biopsy samples of affected skin from SSc patients (n = 6) relative to the levels in skin from healthy control subjects (n = 9). Values are the mean ± SEM. B, Correlation between Cad-11 mRNA levels, as assessed by microarray expression profiling, and modified Rodnan skin thickness scores (MRSS) in patients with diffuse SSc (Spearman’s r = 0.6301, P = 0.0006). C–G, Immunohistochemistry of skin biopsy samples from healthy control subjects and SSc patients, using anti–Cad-11 antibodies. Cad-11 expression is seen on fibroblasts (F) (spindle-shaped cells at arrows) and inflammatory cells (G) (round cells) in the dermis of SSc patients. Original magnification × 200 in C and E; × 400 in D, F, and G. H and I, Quantification of Cad-11–positive fibroblasts (H) and inflammatory cells (I) in skin biopsy samples from 9 SSc patients and 4 healthy controls. Values are the mean ± SD of at least 6 microscopic fields per sample. * = P < 0.05 versus controls.

Figure 2

Reduced fibrosis of lesional skin from cadherin 11 (Cad-11)–knockout (KO) mice. Cad-11–KO mice and wild-type (WT) mice received daily injections of phosphate buffered saline (PBS) or bleomycin (Bleo) for 28 days, and lesional skin was analyzed. A and B, Representative sections stained with hematoxylin and eosin (A) or Masson’s trichrome (B), showing a reduction in dermal thickness (arrows) in Cad-11–KO mice injected with bleomycin relative to that in WT mice. Original magnification × 100. C, Quantification of dermal thickness, demonstrating decreased dermal thickness in Cad-11–KO mice injected with bleomycin relative to that in WT mice. Values are the mean ± SEM of 15 mice per group. D, Quantification of soluble collagen levels by Sircol colorimetric assay, demonstrating decreased collagen in biopsy samples of lesional skin from Cad-11–KO mice injected with bleomycin relative to that in samples from WT mice. Values are the mean ± SEM of 15 mice per group.

Figure 3

Amelioration of bleomycin (Bleo)–induced skin fibrosis by treatment with anti–cadherin 11 (anti–Cad-11) antibody. A and B, Dermal thickness (A) and collagen accumulation (B) in the skin of C57BL/6 mice injected for 28 days with bleomycin or phosphate buffered saline (PBS). Starting on day 14, mice also received either PBS, isotype control, or anti–Cad-11 antibody (13C2 or 23C6) for 14 days. (Representative images are available upon request from the corresponding author.) C, Dermal thickness in the skin of C57BL/6 mice injected for 28 days with bleomycin or PBS. Starting on day 28, mice also received either no additional treatment or treatment with isotype control or anti–Cad-11 antibody 23C6 for 14 days. Anti–Cad-11 antibody treatment reduced dermal thickness. Values are the mean ± SEM of 15 mice per group in A and B and 5 mice per group in C. NS = not significant.

Figure 4

Expression of inflammatory and fibrotic genes in lesional skin from cadherin 11 (Cad-11)–knockout (KO) mice. Cad-11–KO and wild-type (WT) mice received daily injections of phosphate buffered saline (PBS) or bleomycin (Bleo) for 7 days. Total RNA in lesional skin samples was analyzed by real-time quantitative polymerase chain reaction for the relative expression of the proinflammatory genes CCL2 (A) and interleukin-6 (IL-6) (B) and the profibrotic genes Col1a2 (C), connective tissue growth factor (CTGF) (D), and α-smooth muscle actin (α-SMA) (E). Results were normalized to 18S RNA. Values are the mean ± SEM of 6 mice per group. * = P < 0.05. NS = not significant.

Figure 5

Transforming growth factor β (TGFβ) and its signaling pathway in patients with systemic sclerosis (SSc) as well as in cadherin 11 (Cad-11)–knockout (KO) versus wild-type (WT) mice. A and B, Correlation of Cad-11 mRNA levels with cartilage oligomeric matrix protein (COMP) levels (Spearman’s r = 0.9118, P < 0.0001) (A) and thrombospondin 1 (TSP-1) levels (Spearman’s r = 0.9186, P < 0.0001) (B) in skin biopsy samples from patients with SSc. C, Decreased expression of TGFβ mRNA in skin samples from Cad-11–KO mice as compared to WT mice, as determined by real-time quantitative polymerase chain reaction analysis. Results were normalized to 18S RNA. Mice had received daily injections of phosphate buffered saline (PBS) or bleomycin (Bleo). D, Decreased levels of TGFβ in bone marrow–derived macrophages from the femurs of Cad-11–KO mice as compared to WT mice, as determined by enzyme-linked immunosorbent assay. Macrophages were cultured for 72 hours in the presence of interleukin-4. E–H, Decreased numbers of fibroblasts and inflammatory cells expressing p-Smad2 (E and F) and p-ERK (G and H) in lesional skin from Cad-11–KO mice as compared to WT mice, as determined by immunohistochemistry using specific antibodies. Values in C–H are the mean ± SEM of triplicate determinations in at least 6 microscopic fields (n = 4 mice per group). * = P < 0.05. I, Similar expression of p-Smad2 and p-ERK in dermal fibroblasts and inflammatory cells from WT mice and Cad-11–KO mice stimulated in vitro with TGFβ, as determined by Western blotting (n = 3 mice per group).

Figure 6

Cadherin 11 (Cad-11)–dependent expression of β-catenin and migration in dermal fibroblasts. A, Decreased number of fibroblasts expressing β-catenin in bleomycin-injected skin from Cad-11–knockout (KO) mice as compared to wild-type (WT) mice, as determined by immunohistochemistry. Values are the mean ± SEM of 6 microscopic fields per mouse (n = 9 mice per group). B, Decreased levels of β-catenin in lysates of cultured dermal fibroblasts obtained from Cad-11–KO mice as compared to WT mice, as determined by Western blotting and densitometry. Values are the mean ± SEM (n = 9 WT mice and 8 Cad-11–KO mice). C, Decreased migratory capacity of dermal fibroblasts from Cad-11–KO mice as compared to WT mice, as determined by Boyden chamber analysis. The migration index is the number of cells migrating to 5% fetal bovine serum divided by the number of cells migrating to 0.1% bovine serum albumin. Values are the mean ± SEM (n = 4 WT mice and 4 Cad-11–KO mice). * = P < 0.05 versus WT mice. D, Representative images of Boyden chamber membranes, demonstrating decreased migratory capacity of dermal fibroblasts from Cad-11–KO mice relative to that of dermal fibroblasts from WT mice.