Functional Mechanisms And Evolutional Study Of Insulin-like Growth Factor Binding Protein 3 (IGFBP-3)

IGF binding protein (IGFBP)-3 is a secreted protein that binds IGF and modulates its actions in extracellular environments. IGFBP-3 also has ligand-independent actions in cultured mammalian cells. In addition to its IGF binding domain (IBD), mammalian IGFBP-3 has a functional nuclear localization signal (NLS) and transactivation domain (TA). The specific roles of these structural domains of IGFBP-3 in mediating its in vivo actions are unclear. Here we show that these domains are conserved in zebrafish IGFBP-3. To study the functional roles of these domains, several IGFBP-3 mutants were engineered. The IBD mutant failed to bind IGF-I but had normal nuclear localization. The NLS mutant had greatly reduced nuclear localization but showed normal IGF binding. The IBD+NLS double mutant had impaired IGF binding and nuclear localization. We also determined critical residues in the TA domain in IGFBP-3 and engineered a TA dead mutant. When expressed in zebrafish embryos, wild-type IGFBP-3, NLS, and TA mutants had strong dorsalizing effects. The activities of the IBD mutant and the IBD+NLS mutant were significantly lower, suggesting that IGFBP-3 acts in IGF-dependent and -independent manners and that the IGF-independent action is not related to its nuclear localization or transactivation activity. Since the phenotypes caused by IGFBP-3 resemble those of bmp2b mutants, we co-expressed zebrafish Bmp2b with IGFBP-3 and the IBD mutant. Both IGFBP-3 and IBD reduced the ventralizing effect of Bmp2b and abolished the Bmp2b-induced direct target gene expression in vivo. Overexpression of IGFBP-3 and IBD mRNA in zebrafish increased chordin gene expression. Co-expression of IGFBP-3 and Chording mRNA has additive effect on dosalizing zebrafish embryos. In vitro ligand blotting assay revealed that IGFBP-3 binds to BMP-2 and reduces BMP-2 induced Smad1/5/8 phosphorylation. Furthermore, addition of human IGFBP-3 with BMP-2 into cultured C3H/10T1/2 cells inhibited BMP-2 induced cell osteoblast differentiation. These results suggest that IGFBP-3 binds to BMP-2 and inhibiting BMP-2 signaling.Two episodes of complete or whole genome duplication (WGD) occurred on the vertebrate lineage (2R hypothesis). Sea lamprey is one of the earliest vertebrate exist in the world, and is divergent after 1R and this is followed by 2R. An Igfbp-3 gene was cloned from an ancient Agnatha sea lamprey (Petromyzon marinus), which encodes 6 distinct ORFs. In these ORFs, IGFBP-3 ORF1 contains entire IGF-binding domain (IBD), nuclear localization signal (NLS), and transactivation activity (TA) domain. IGFBP-3 can bind IGFs, localize in the nuclear, and has transactivation activity in its N-domain. IGFBP-3 ORF1 dorsalizes zebrafish embryos when overexpressed in zebrafish embryos via microinjection its mRNA, and this action is partially IGF-dependent but independent of NLS or TA. IGFBP-3 inhibits angiogenesis of flk1:EGFP transgenic zebrafish embryos in an IGF-independent manner. Co-microinjection of IGFBP-3 ORF1 or its IBD mutant mRNA with Bmp2b mRNA reveals that IGFBP-3 ORF1 can antagonize BMP signaling in an IGF-independent manner. Moreover, IGFBP-3 ORF1 inhibits tp63, a BMP downstream direct target, mRNA expression in an IGF-independent manner. Intriguingly, other IGFBP-3 ORFs have not similar functions as ORF1 mentioned above. By sequence analysis, IGFBP-3 contains IBD, NLS, and TA in all sequenced animals. Furthermore, human IGFBP-3 can dorsalize zebrafish embryos and inhibits BMP signaling when overexpressed in zebrafish embryos.Taken together, IGFBP-3 has functional and conserved IBD, NLS, and TA. It has dorsalization activity and inhibits angiogenesis when overexpressed in zebrafish embryos. IGFBP-3 exerts its IGF-independent action via antagonize BMP signaling. The action of IGFBP-3 is evolutionary conserved in vertebrates.