Zhong, Y., Lu, L., Zhou, J., Li, Y., Liu, Y., Clemmons, D.R., and Duan, C. (2011) IGF binding protein 3 exerts its ligand-independent action by antagonizing BMP in zebrafish embryos. Journal of Cell Science. 124(11):1925-1935.
IGFBP3 is a multi-functional protein that has IGF-dependent and IGF-independent actions in cultured cells. Here we show that
the IGF binding domain (IBD), nuclear localization signal (NLS) and transactivation domain (TA) are conserved and functional
in zebrafish Igfbp3. The in vivo roles of these domains were investigated by expression of Igfbp3 and its mutants in zebrafish
embryos. Igfbp3, and its NLS and TA mutants had equally strong dorsalizing effects. Human IGFBP3 had similar dorsalizing effects
in zebrafish embryos. The activities of IBD and IBD+NLS mutants were lower, but they still caused dorsalization. Thus, the
IGF-independent action of Igfbp3 is not related to NLS or TA in this in vivo model. We next tested the hypothesis that Igfbp3
exerts its IGF-independent action by affecting Bmp signaling. Co-expression of Igfbp3 with Bmp2b abolished Bmp2b-induced gene
expression and inhibited its ventralizing activity. Biochemical assays and in vitro experiments revealed that IGFBP3 bound
BMP2 and inhibited BMP2-induced Smad signaling in cultured human cells. In vivo expression of Igfbp3 increased chordin expression in zebrafish embryos by alleviating the negative regulation of Bmp2. The elevated level of Chordin acted together
with Igfbp3 to inhibit the actions of Bmp2. Knockdown of Igfbp3 enhanced the ventralized phenotype caused by chordin knockdown. These results suggest that Igfbp3 exerts its IGF-independent actions by antagonizing Bmp signaling and that this
mechanism is conserved.