Retinoid X receptor agonist inhibits TGF-β1-induced collagen synthesis in cardiac fibroblasts by repressing Smad2 activation

AIM: To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms. METHODS: CFs were cultured using myocardial tissue with dry method. Hypoxic environment was established for CFs by continuous nitrogen supplement. Type I and type III collagens in supernatants were detected by ELISA. Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents. The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining. RESULTS: Under hypoxic condition, TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-dependent manner in the CFs. At the concentration of 5 μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01). RXR agonist 9-cis-retinoic acid (9-cis-RA; 10^-9~10^-6mol/L) decreased TGF-β1 (5 μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic condition. The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01). Smad2 inhibitor (20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF-β1 under hypoxic condition. Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly increased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05). CONCLUSION: Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition.