Rosiglitazone suppresses TGF-β1-induced myofibroblast activation and extra cellular matrix synthesis in pterygium fibroblasts at least partly through the modulation of the p38 MAPK pathway.

Citation: Kim SW, Kim H-I, Thapa B, Nuwromegbe S, Lee K. Critical role of mTORC2-Akt signaling in TGF-b1-induced myofibroblast differentiation of human pterygium fibroblasts. Invest Ophthalmol Vis Sci. 2019;60:82-92. https://doi.org/10.1167/iovs.18-25376PURPOSE. Profibrotic activation is essential for pterygium development. In this study, we investigated the role of the mechanistic target of rapamycin (mTOR) in regulating TGF-b1induced myofibroblastic responses in human pterygium fibroblasts (HPFs) and elucidated the relative contributions of mTOR signaling components. METHODS.HPFs were pretreated with the mTOR inhibitors rapamycin and Torin2, and TGF-b1induced expression of profibrotic markers, including a-smooth muscle actin (a-SMA) and fibronectin, was evaluated. RNA interference-based approaches targeting raptor and rictor, regulatory subunits of mTOR complex 1 (mTORC1) and 2 (mTORC2), respectively, were used to determine the impact of each mTOR complex on HPFs. The contractile phenotype of HPFs was assessed by a collagen gel contraction assay. RESULTS.The mTOR active-site inhibitor Torin2, which suppresses both mTORC1 and mTORC2 activity in HPFs, inhibited TGF-b1-induced expression of a-SMA and fibronectin. The allosteric inhibitor rapamycin only partially suppressed mTORC1 activity and exhibited a minimal effect on the induction of profibrotic markers. The induction of a-SMA and fibronectin in HPFs was abrogated by RNA interference-mediated knockdown of rictor but was only moderately affected by raptor knockdown. Akt inhibition mimicked the effect of Torin2 and rictor knockdown on myofibroblast differentiation of HPFs. mTOR inhibition potently reduced the contractile ability of HPFs in collagen gel contraction assays.CONCLUSIONS. This study found that mTOR signaling promoted profibrotic activation of HPFs and confirmed the importance of the mTORC2-Akt axis in TGF-b1-induced myofibroblast differentiation. Therefore, our study may open up new avenues for the development of novel therapeutic strategies involving targeting of mTOR signaling to treat pterygium.

Increased TGF-β1 synthesis after corneal alkali injury is implicated in corneal fibrosis, as it promotes transdifferentiation of keratocytes into myofibroblasts. The activation of 5-adenosine monophosphate-activated protein kinase (AMPK) by 5-amino-4imidazole carboxamide riboside-1-β-D-ribofuranoside (AICAR) inhibits TGF-β1-induced fibrosis in other cell types. We investigated the antifibrotic effect of AICAR in corneal fibroblasts after alkali injury. METHODS. Mouse models of corneal alkali burn, produced by placing 2-mm-diameter filter paper soaked in 0.1-N NaOH on the right cornea for 30 seconds, were treated with the test drugs 4× daily for 21 days. The central cornea was scanned by optical coherence tomography (OCT). Corneal tissues were obtained and processed for western blotting and immunohistochemistry. For in vitro analysis, primary human corneal fibroblasts were treated directly with TGF-β1 to induce fibrosis, with or without AICAR pretreatment. Myofibroblast activation and extracellular matrix (ECM) protein synthesis were detected by western blotting, real-time PCR, and collagen gel contraction assay. Signaling proteins were analyzed by western blotting. RESULTS. Alkali injury induced the upregulation of TGF-β1 expression, which led to increased α-smooth muscle actin (α-SMA) and fibronectin synthesis and myofibroblast differentiation. AMPK activation by AICAR significantly suppressed TGF-β1 and ECM protein expression. The antifibrotic effect of AICAR was AMPK dependent, as treatment with the AMPK inhibitor Compound C attenuated the antifibrotic response. CONCLUSIONS. AMPK activation by AICAR suppresses the myofibroblast differentiation and ECM synthesis that occur after alkali injury in corneal fibroblasts.

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