Inhibition of latent transforming growth factor-beta1 activation by lentivirus-mediated short hairpin RNA targeting the CD36 gene in NR8383 cells

Abstract

CD36, a cell surface receptor for thrombospondin-1 (TSP-1), is believed to interact with latent transforming growth factor-beta1 (L-TGF-beta1) thereby activating its fibrogenic bioactivity. In this study, a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the rat CD36 gene (Lv-shCD36) is developed and tested. To observe the inhibitory effect of Lv-shCD36 on the activation of L-TGF-beta1, a rat alveolar macrophage cell line (NR8383), infected with either Lv-shCD36 or Lv-shCD36-NC (non-silenced control lentivirus), was treated with 0.1 microg/ml bleomycin, which is known to stimulate alveolar macrophages to release increasing amounts of TGF-beta1. The results show that Lv-shCD36 can suppress expression of CD36 mRNA and protein in bleomycin-treated NR8383 cells. By quantifying active and total TGF-beta1 in the supernatant, it was discovered that the quantity of total TGF-beta1 is not significantly different between the three groups, while the quantity and percent of active TGF-beta1 in the Lv-shCD36 group was significantly lower than in either the bleomycin-treated group or the Lv-shCD36-NC group, respectively (P < 0.05). These results suggest that Lv-shCD36 can inhibit activation of L-TGF-beta1 secreted in bleomycin-treated NR8383 cells by decreasing the expression of CD36 on the cell membrane, thereby reducing binding of CD36 to TSP-1.