FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro

Abstract

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis-ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-β1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-β3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-β1 + FAKi attenuated TGF-β1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-β1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

Keywords: 3D cell culture; corneal scarring; extracellular matrix (ECM); focal adhesion kinase (FAK); α-smooth muscle actin (αSMA).

Figure 1

Identification of differentially expressed fibrotic and wound-healing genes following TGF-β1 or -β3 treatment in hCF constructs. A RT² Profiler PCR array analysis of human fibrosis and wound-healing gene expression was performed. Total RNA was extracted from human corneal fibroblast (hCF) 3D constructs that were untreated or treated continuously with TGF-β1 or -β3 for 4 weeks, subjected to cDNA synthesis, and analyzed with Human Fibrosis and Wound Healing PCR Array. (A) Pie chart showing distribution of targeted 86 genes and their relevant biological processes (labeled 1–8) within CR array. (B) Heatmap of targeted genes comparing RNA profile derived from hCF + TGF-β1 or hCF + TGF-β3 relative to untreated hCF constructs with a * p < 0.05: ACTA2, Alpha smooth muscle actin; AGT, Angiotensinogen; GREM1, Gremlin 1; ITGAV, Integrin Subunit Alpha V; ITGB1, Integrin Subunit Beta 1; JUN, Jun Proto-Oncogene, AP-1 Transcription Factor Subunit; MAPK14, Mitogen-Activated Protein Kinase 14; MMP14, Matrix Metallopeptidase 14; PDGFRB, Platelet Derived Growth Factor Receptor Beta; SRC, SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase. Fold change values: blue (−2) to yellow (+2) through grey.

Figure 2

TGF-β1 treatment induced ACTA2/αSMA and FAK expression in hCF 3D constructs. Characterization of ACTA2/αSMA and FAK expression in 4-week human corneal fibroblast (hCF) 3D constructs. mRNA and protein were isolated from 4-week hCF constructs that were either untreated (control) or treated continuously with TGF-β1 or TGF-β3. (A,C) Extracted mRNA from constructs per experimental condition was examined by qRT-PCR analysis for ACTA2 and FAK. (B,D) Cell lysates were prepared from constructs and analyzed for relevant target proteins (αSMA and FAK) and β-Actin (loading control). Bands were measured by densitometry analysis, and average fold change of targeted proteins are shown relative to control ± SEM; n = 3 per group. * p < 0.05, **** p < 0.0001. ACTA2, Alpha smooth muscle actin; FAK, Focal Adhesion Kinase.

Figure 3

FAK inhibition decreased expression of αSMA in 3D hCF constructs. (A) In 2D cell culture (2D), hCF were growth arrested 48 h prior to stimulation with either no growth factors (control), TGF-β3, TGF-β1, or TGF-β1 + FAK inhibitor (FAKi). After a further 24 h, the 2D cultures were examined for αSMA localization. Green = αSMA, Blue = TO-PRO-3. (B) In 3D constructs, hCF constructs were generated and stimulated with vitamin C to secrete their own extracellular matrix for 2 weeks. 3D hCF constructs were treated with either no growth factors (control), TGF-β3, TGF-β1, or TGF-β1 + FAKi. After a further 24 h, the 3D constructs were examined for αSMA localization. Red = αSMA, Blue = TO-PRO-3. Images of (C) 2D cell culture and (D) 3D constructs were captured, and fluorescent intensity of αSMA was quantified using Image-J software. Average fold change of fluorescent intensity relative to control is shown for all experimental groups ±SEM. All images were taken at 20× magnification. Scale bar: 50μm. Representative western blot images of αSMA and FAK via different treatment groups are shown. n = 3 per group. ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001. ACTA2, Alpha smooth muscle actin; FAK, Focal Adhesion Kinase.

Figure 4

FAK inhibition decreased αSMA expression at mRNA and protein level in 2D and 3D culture. (A,B) In 2D culture, hCFs were growth arrested 48 h prior to stimulation with either no growth factors (control), TGF-β3, TGF-β1, or TGF-β1 + FAK inhibitor (FAKi). (C–F) In 3D culture, hCF constructs were generated and stimulated with vitamin C to secrete their own extracellular matrix for 2 weeks. 3D hCF constructs were treated with no growth factors (control), TGF-β3, TGF-β1, or TGF-β1 + FAKi. Isolated mRNA from each experimental condition was examined by qRT-PCR analysis for levels of (A,C) ACTA2 and (E) FAK. Cell lysates were prepared from each experimental condition and average fold change of (B,D) αSMA and (F) FAK with respect to β-Actin and relative to control was measured by densitometry analysis. Representative western blot images of αSMA and FAK via different treatment groups are shown. Data are shown as mean ± SEM; n = 3 per group. ns: non-significant, * p < 0.05, ** p < 0.01, *** p< 0.001, **** p < 0.0001. ACTA2, Alpha smooth muscle actin gene; αSMA, Alpha smooth muscle actin protein; FAK, Focal Adhesion Kinase.