Paradoxical actions of endogenous and exogenous insulin-like growth factor-binding protein-5 revealed by RNA interference analysis.
Insulin-like growth factor-binding protein-5 (IGFBP-5) is abundantly expressed in bone cells. To determine the physiological role(s) of endogenous IGFBP-5 in regulating bone cell growth, differentiation, and survival, we used short double-stranded RNA (siRNA) to trigger RNA interference of IGFBP-5 in human osteosarcoma cells. The IGFBP-5 siRNA, targeting against a sequence unique to the IGFBP-5 middle domain, efficiently reduced IGFBP-5 mRNA and protein levels. The IGFBP-5 siRNA did not change the levels of IGFBP-4, a structurally related protein, or glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene. Knock-down of IGFBP-5 resulted in a significant increase in the number of transferase-mediated dUTP nick end labeling-positive cells and a decrease in a bone differentiation parameter (alkaline phosphatase activity) but had little effect on basal or insulin-like growth factor I-induced proliferation. Overexpression of a siRNA-resistant IGFBP-5 mutant in the IGFBP-5 knock-down cells restored the levels of survival to the control level; overexpression of IGFBP-4 or wild type IGFBP-5 had no such effect. Paradoxically, the addition of exogenous IGFBP-5 not only failed to rescue IGFBP-5 knock-down-induced apoptosis, it caused a further increase in apoptosis. Furthermore, the addition of exogenous IGFBP-5 alone increased apoptosis. This pro-apoptotic action of exogenous IGFBP-5 was abolished when IGF-I was added in excess, suggesting that exogenous IGFBP-5 increases apoptosis by binding to and inhibiting the activities of insulin-like growth factors. These results indicate that endogenous and exogenous IGFBP-5 exhibits opposing biological actions on cell survival and underscore the necessity and utility of studying IGFBP functions through loss-of-function approaches.
Yin
Ping
P
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, 48109, USA.
Xu
Qijin
Q
Duan
Cunming
C
D000469:Alkaline Phosphatase / Q000378:metabolism; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D002455:Cell Division; D045744:Cell Line, Tumor; D002470:Cell Survival; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection
D018975:Insulin-Like Growth Factor Binding Protein 5; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000469:Alkaline Phosphatase