Inhibition of latent transforming growth factor-beta1 activation by lentivirus-mediated short hairpin RNA targeting the CD36 gene in NR8383 cells.
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.
Wang
Xin
X
Division of Pneumoconiosis, School of Public Health, China Medical University, Shenyang, People's Republic of China.
Chen
Ying
Y
Lv
Lina
L
Chen
Jie
J
D000704:Analysis of Variance; D000818:Animals; D001761:Bleomycin; D015153:Blotting, Western; D018955:CD36 Antigens / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D055029:Inverted Repeat Sequences / Q000235:genetics; D016086:Lentivirus; D009841:Oligonucleotides / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
D018955:CD36 Antigens; D009841:Oligonucleotides; C508962:Tgfb1 protein, rat; D053773:Transforming Growth Factor beta1; D001761:Bleomycin; D012313:RNA