Keratoconus in vitro and the key players of the TGF-β pathway

Purpose: Keratoconus (KC) is a corneal thinning disease of unknown etiology whose pathophysiology is correlated with the presence of a thin corneal stroma and altered extracellular matrix (ECM). Transforming growth factor-β (TGF-β) signaling is a key regulator of ECM secretion and assembly in multiple tissues, including the anterior segment of the eye, and it has been linked to KC. We have previously shown that human keratoconus cells (HKCs) have a myofibroblast phenotype and altered ECM assembly compared to normal human corneal fibroblasts (HCFs). Moreover, TGF-β3 treatment promotes assembly of a more normal stromal ECM and modulates the fibrotic phenotype in HKCs. Herein, we identify alterations in TGF-β signaling that contribute to the observed fibrotic phenotype in HKCs. Methods: HCFs and HKCs were stimulated with TGF-β1, TGF-β2, or TGF-β3 isoforms (0.1 ng/mL) in the presence of a stable vitamin C derivative (0.5 mM) for 4 weeks. All samples were examined using RT–PCR and western blotting to quantify changes in the expressions of key TGF-β signaling molecules between HCFs and HKCs. Results: We found a significant downregulation in the SMAD6 and SMAD7 expressions by HKCs when compared to HCFs (p≤0.05). Moreover, stimulation of HKCs with any of the three TGF-β isoforms did not significantly alter the expressions of SMAD6 or SMAD7. HCFs also showed an upregulation in TGF-βRI, TGF-βRII, and TGF-βRIII following TGF-β3 treatment, whereas HKCs showed a significant two-fold downregulation. Conclusions: Overall, our data shows the decreased expressions of the regulatory SMADs SMAD6 and SMAD7 by HKCs contribute to the pathological ECM structure observed in KC, and TGF-β3 may attenuate this mechanism by downregulating the expression of the key profibrotic receptor, TGF-βRII. Our study suggests a significant role of altered regulation of TGF-β signaling in KC progression and that it may enable novel therapeutic developments targeting TGF-β receptor regulation.