IGF binding protein 3 exerts its ligand-independent action by antagonizing BMP in zebrafish embryos

Abstract

SummaryIGFBP3 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 IGFindependent actions by antagonizing Bmp signaling and that this mechanism is conserved.

40 YEARS OF IGF1: IGF-binding proteins

“…Martin et al 2009;Naspi, et 353 al. 2017;Zhong, et al 2011). In many situations, IGFBP-3 was a tumour suppressor, but it may also 354 have pro-tumourigenic actions; modulation of sphingolipids was proposed to underlie these 355 apparently opposing effects (Baxter 2014).…”

Dynamic transcriptional regulation of autocrine/paracrine igfbp1, 2, 3, 4, 5, and 6 in the skeletal muscle of the fine flounder during different nutritional statuses

“…Previous studies on the biology of Igfbps in fish have shown that Igfbps regulate embryonic development (Duan et al 1999, Kajimura et al 2005) and exert ligand-dependent and -independent mechanisms (Dai et al 2010, Zhong et al 2011, as well as showing that Igfbps are modulated by Gh treatment (Duan et al 1999, Cheng et al 2002, Chen et al 2004, Pedroso et al 2009b, oxygen availability (Kajimura et al 2005, Rahman & Thomas 2011, Sun et al 2011, reproduction , Chen et al 2010, and nutrition , Bower et al 2008, Pedroso et al 2009a, Peterson & Waldbieser 2009, Amaral & Johnston 2011, Macqueen et al 2011. Nevertheless, studies on local muscle-derived igfbps (autocrine/ paracrine) have been scarce and have recently shown contrasting results, reflecting a complex physiology of Igfbps and suggesting that intricate patterns of regulation have evolved between and within teleost lineages , Bower et al 2008, Amaral & Johnston 2011, Macqueen et al 2011.…”

Papp-a2 modulates development of cranial cartilage and angiogenesis in zebrafish embryos

“…Broadening of the axial mesoderm has previously been observed in zebrafish upon Igfbp-3 overexpression (Zhong et al, 2011), indicating that Papp-a2 might modulate BMP signaling by proteolytic cleavage of Igfbp-3. In accordance with such mechanism, human IGFBP-3 was recently reported to bind BMP2 and BMP4 thereby antagonizing their signaling activity Zhong et al, 2011). Furthermore, zebrafish Bmp1 cleaves Igfbp-3, and proteolytic cleavage of human IGFBP-3 by BMP1 is known to reduce its affinity for BMP4 .…”

“…In addition, broadening of the noto-expression domain has been reported in zebrafish embryos overexpressing igfbp-3 (Zhong et al, 2011), indicating a functional link between Pappa2 and Igfbp-3. For biochemical characterization, we transfected HEK293T cells with zebrafish papp-a2 cDNA and confirmed its expression by western blotting (Fig.…”