RESUMO
Recent studies have revealed roles for immunoproteasome in regulating cell processes essential for maintaining homeostasis and in responding to stress and injury. The current study investigates how the absence of immunoproteasome affects the corneal epithelium under normal and stressed conditions by comparing corneas from wildtype (WT) mice and those deficient in two immunoproteasome catalytic subunits (lmp7(-/-)/mecl-1(-/-), L7M1). Immunoproteasome expression was confirmed in WT epithelial cells and in cells of the immune system that were present in the cornea. More apoptotic cells were found in both corneal explant cultures and uninjured corneas of L7M1 compared to WT mice. Following mechanical debridement, L7M1 corneas displayed delayed wound healing, including delayed re-epithelialization and re-establishment of the epithelial barrier, as well as altered inflammatory cytokine production compared to WT mice. These results suggest that immunoproteasome plays an important role in corneal homeostasis and wound healing.
Assuntos
Córnea/imunologia , Cisteína Endopeptidases/deficiência , Células Epiteliais/imunologia , Epitélio Corneano/imunologia , Complexo de Endopeptidases do Proteassoma/deficiência , Reepitelização/fisiologia , Animais , Apoptose/imunologia , Córnea/metabolismo , Córnea/patologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Desbridamento , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Expressão Gênica , Homeostase/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologiaRESUMO
PURPOSE: Due to its ability to disrupt transforming growth factor beta (TGF-ß) signaling, halofuginone has been successfully used to treat various fibrotic disorders. Here we investigated the antifibrotic potential of halofuginone in human corneal fibroblasts. METHODS: Human corneal fibroblasts were isolated from human donor corneas for in vitro experiments. TGF-ß was used to stimulate pro-fibrotic responses from corneal fibroblasts under halofuginone treatment. The expression of alpha smooth muscle actin (α-SMA) and fibronectin was analyzed by western blots. Phalloidin toxin was used to stain cultures for stress fiber assemblies. Quantitative reverse transcription PCR (qRT-PCR) and immunostaining were used to analyze the expression of type I collagen mRNA and protein, respectively. The expression of Smad2, Smad3, phospho-Smad2, and phospho-Smad3 was determined by western blots. RESULTS: Halofuginone was well tolerated by human corneal fibroblasts up to 10 ng/ml as demonstrated by a cell viability assay. At this concentration, TGF-ß-induced expression of the fibrotic markers α-SMA, fibronectin, and type I collagen was significantly reduced. Interestingly, under our experimental conditions, halofuginone treatment led to reduced protein expression of Smad3, which was both dose- and time-dependent. CONCLUSIONS: Our results suggest that halofuginone may exert its antifibrotic effects in the cornea via a novel molecular mechanism and may be used as an antifibrotic agent for corneal fibrosis treatment.
