17β-estradiol inhibits TGF-β-induced collagen gel contraction mediated by human Tenon fibroblasts via Smads and MAPK signaling pathways

AIM: To investigate the impact of 17 beta-estradiol on the collagen gels contraction (CGC) and inflammation induced by transforming growth factor (TGF)-beta in human Tenon fibroblasts (HTFs). METHODS: HTFs were three-dimensionally cultivated in type I collagen- generated gels with or without TGF-beta (5 ng/mL), 17 beta-estradiol (12.5 to 100 mu mol/L), or progesterone (12.5 to 100 mu mol/L). Then, the collagen gel diameter was determined to assess the contraction, and the development of stress fibers was analyzed using immunofluorescence staining. Immunoblot and gelatin zymography assays were used to analyze matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases ( TIMPs) being released into culture supernatants. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription- quantitative polymerase chain reaction (RT- PCR) were used to detect interleukin ( IL)-6, monocyte chemoattractant proteins (MCP)-1, and vascular endothelial growth factor (VEGF) in HTFs at the translational and transcriptional levels. The phosphorylation levels of Sma- and Mad-related proteins (Smads), mitogen-activated protein kinases (MAPKs), and protein kinase B (AKT) were measured by immunoblotting. Statistical analysis was performed using either the Tukey-Kramer test or Student's unpaired t-test to compare the various treatments. RESULTS: The CGC caused by TGF-beta in HTFs was significantly inhibited by 17 beta-estradiol (25 to 100 mu mol/L), and a statistically significant difference was observed when comparing the normal control group with 17 beta-estradiol concentrations exceeding 25 mu mol/ L (P<0.05). The suppressive impact of 17 beta-estradiol became evident 24h after administration and peaked at 72h (P<0.05), whereas progesterone had no impact. Moreover, 17 beta-estradiol attenuated the formation of stress fibers, and the production of MMP-3 and MMP-1 in HTFs stimulated by TGF-beta. The expression of MCP-1, IL-6, and VEGF mRNA and protein in HTFs were suppressed by 100 mu mol/L 17 beta-estradiol (P<0.01). Additionally, the phosphorylation of Smad2 Smad3, p38, and extracellular signal-regulated kinase (ERK) were downregulated (P <0.01). CONCLUSION: 17 beta-estradiol significantly inhibits the CGC and inflammation caused by TGF-beta in HTFs. This inhibition is likely related to the suppression of stress fibers, inhibition of MMPs, and attenuation of Smads and MAPK (ERK and p38) signaling. 17 beta-estradiol may have potential clinical benefits in preventing scar development and inflammation in the conjunctiva.