miR-218 regulates focal adhesion kinase-dependent TGFβ signaling in fibroblasts

Abstract

Scarring, which occurs in essentially all adult tissue, is characterized by the excessive production and remodeling of extracellular matrix by α-smooth muscle actin (SMA)-expressing myofibroblasts located within connective tissue. Excessive scarring can cause organ failure and death. Oral gingivae do not scar. Compared to dermal fibroblasts, gingival fibroblasts are less responsive to transforming growth factor β (TGFβ) due to the reduced expression, due to the reduced expression and activity of focal adhesion kinase (FAK) by this cell type. Here we show that, compared with dermal fibroblasts, gingival fibroblasts show reduced expression of miR-218. Introduction of pre-miR-218 into gingival fibroblasts elevates FAK expression and, via a FAK/src-dependent mechanism, results in the ability of TGFβ to induce α-SMA. The deubiquitinase cezanne is a direct target of miR-218 and has increased expression in gingival fibroblasts compared with dermal fibroblasts. Knockdown of cezanne in gingival fibroblasts increases FAK expression and causes TGFβ to induce α-smooth muscle actin (α-SMA). These results suggest that miR-218 regulates the ability of TGFβ to induce myofibroblast differentiation in fibroblasts via cezanne/FAK.

FIGURE 1:

Expression levels of miR-218 are higher in human dermal fibroblasts than in human gingival fibroblasts. As described in Materials and Methods, RNA was prepared from human dermal fibroblasts (HDFs) and human gingival fibroblasts (HGFs) and subjected to real-time PCR analysis to detect miR-218. Relative expression compared with HGFs (N = 3, *p < 0.05, Student's t test).

FIGURE 2:

miR-218 modulates profibrotic gene expression in dermal and gingival fibroblasts. As described in Materials and Methods, HDFs and HGFs were transfected with anti–miR-218 or anti-miRNA negative control and pre–miR-218 or pre-miRNA negative control, respectively. Forty-eight hours posttransfection, RNA was harvested and subjected to real-time PCR analysis to detect (A) miR-218, FAK, endothelin-1, α-SMA, and CCN2 mRNAs. Average relative expression compared with HGFs ± SD (N = 3, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA test). (B) Protein was assessed by Western blot analysis to detect anti-FAK and anti–β-actin antibodies or enzyme-linked immunosorbent assay (ELISA) to detect ET-1.

FIGURE 3:

Transfection of pre–miR-218 into human gingival fibroblasts results in increased cell adhesion to fibronectin. As described in Materials and Methods, HGFs were transfected with pre–miR-218 or pre-miRNA negative control and subjected to a cell adhesion assay on fibronectin. Adherent cells were detected using MTT assay. Average ± SD (N = 3, *p < 0.05, Student's t test).

FIGURE 4:

miR-218 enhances TGFβ responses in gingival fibroblasts. (A) miR-218 promotes TGFβ-induced α-SMA expression in HGFs: RNA analysis. HGFs were treated with or without pre-miRNA negative control or pre-miR218 miRNA and treated with or without TGFβ1 for 6 h. Simultaneously, HDFs were treated with or without TGFβ1 for 6 h. RNA was harvested and subjected to real-time PCR analysis to detect α-SMA mRNA. Average ± SD (N = 3, **p < 0.01, one-way ANOVA test). (B) miR-218 promotes TGFβ-induced α-SMA expression in HGFs: protein analysis. HGFs were treated with or without pre-miRNA negative control or pre-miR218 miRNA and treated with or without TGFβ1 for 24 h. Protein was extracted and subjected to Western blot analysis with anti–α-SMA and anti–β-actin antibodies. (C, D) Effect of the ALK5 receptor inhibitor SB431542, the FAK/src inhibitor PP2, and the ET receptor antagonist PD145065 on the ability of pre–miR-218 to enhance TGFβ responses in gingival fibroblasts. Cells were treated and transfected as in A but were pretreated for 45 min before addition of TGFβ with or without DMSO, SB431542, PP2, or PD145065, as indicated. RNA was harvested and subjected to real-time PCR analysis to detect (C) α-SMA or (D) ET-1 mRNA. Average ± SD (N = 3, **p < 0.01, ***p < 0.001, one-way ANOVA test).

FIGURE 5:

Cezanne siRNA increases FAK and TGFβ-induced α-SMA expression in gingival fibroblasts. (A) HGFs were treated with or without control siRNA or siRNA recognizing cezanne. Simultaneously, HDFs were cultured as a control. Forty-eight hours later, protein was harvested and subjected to Western blot analysis with antibodies detecting cezanne, FAK, or β-actin protein levels, Alternatively, HGFs treated as in A were subjected to (B) real-time PCR analysis to detect cezanne or FAK or (C) real-time PCR analysis or ELISA to detect ET-1 levels. (N = 3, *p < 0.05, ***p < 0.001, Student's t test). (D) Cezanne siRNA enhances TGFβ-induced α-SMA expression in HGFs. HGFs were treated with or without control siRNA or cezanne siRNA and treated with or without TGFβ1 for 6 h (for RNA analysis) or 24 h (for protein analysis). RNA was harvested and subjected to real-time PCR analysis to detect α-SMA mRNA. Average ± SD (N = 3, **p < 0.01, one-way ANOVA test). Protein was subjected to Western blot analysis to detect α-SMA or β-actin protein.

FIGURE 6:

Cezanne expression is a direct target of miR-218. (A) Promoter-reporter constructs containing two predicted miR-218 recognition sequences or with the putative sequences mutated were transfected into cells with pre-miRNA negative control or pre–miR-218. Cells were cotransfected with control vector expressing Renilla luciferase. Adjusted average expression values ± SD (N = 3, **p < 0.01, ***p < 0.001, Student's t test). (B). HGFs were transfected with pre-miRNA negative control or pre–miR-218. In addition, HDFs were treated with anti-miRNA negative control or anti–miR-218. Forty-eight hours later, cells were harvested for RNA and subjected to real-time PCR analysis to detect cezanne (N = 3, **p < 0.01, ***p < 0.001, Student's t test). (C) HGFs were transfected with pre-miRNA negative control or pre–miR-218. As a control, HDFs were cultured in parallel. Forty-eight hours later, protein was harvested and subjected to Western blot analysis with anti-cezanne or anti–β-actin protein.

FIGURE 7:

Summary of the findings. Compared to HGFs, in HDFs there is elevated miR-218 expression. miR-218 suppresses the deubiquitinase cezanne, leading to elevated FAK expression. Thus HDFs show elevated FAK-dependent transcriptional responses to TGFβ1 compared with HGF.