IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis

Abstract

Insulin-like growth-factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs) 1 . Although some of the actions of IGFBPs have been reported to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown 1,2 . Here we report a previously unknown function for IGFBP-4 as a cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation in vitro, and knockdown of Igfbp4 attenuated cardiomyogenesis both in vitro and in vivo. The cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signalling. IGFBP-4 physically interacted with a Wnt receptor, Frizzled 8 (Frz8), and a Wnt co-receptor, lowdensity lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Although IGF-independent, the cardiogenic effect of IGFBP-4 was attenuated by IGFs through IGFBP-4 sequestration. IGFBP-4 is therefore an inhibitor of the canonical Wnt signalling required for cardiogenesis and provides a molecular link between IGF signalling and Wnt signalling.The heart is the first organ to form during embryogenesis, and abnormalities in this process result in congenital heart diseases, the most common cause of birth defects in humans 3 . Molecules that mediate cardiogenesis are of particular interest because of their potential use for cardiac regeneration 4,5 . Previous studies have shown that soluble growth factors such as bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), Wnts and Wnt inhibitors mediate the tissue interactions that are crucial for cardiomyocyte specification 3,4 . We proposed that there might be additional soluble factors that modulate cardiac development and/or cardiomyocyte differentiation.P19CL6 cells differentiate into cardiomyocytes with high efficiency in the presence of 1% dimethylsulphoxide (DMSO) 6 . We cultured P19CL6 cells with culture media conditioned by various cell types in the absence of DMSO, and screened the cardiogenic activity of the conditioned media. The extent of cardiomyocyte differentiation was assessed by the immunostaining with MF20 monoclonal antibody that recognizes sarcomeric myosin heavy chain (MHC). Among the several cell types tested, culture media conditioned by a murine stromal cell line OP9 induced cardiomyocyte differentiation of P19CL6 cells without DMSO treatment (Fig. 1a, left and middle panels). Increased MF20-positive area was accompanied by the induction of cardiac marker genes such as aMHC, Nkx2.5 and GATA-4, and by the increased protein levels of cardiac troponin T (cTnT) (Fig. 1a, right panel). In contrast, culture media conditioned by COS7 cells, mouse embryonic fibroblasts, NIH3T3 cells, HeLa cells, END2 cells (visceral endoderm-like cells), neonatal rat cardiomyocytes and neonatal rat cardiac fibroblasts did not induce cardiomyocyte differentiation of P19CL6 cells in the absence of DMSO (Fig. 1a and data not shown). From these observations, we p...

Ex vivo expansion of hematopoietic stem cells

“…These results are accordant with the reported role of nephroblastoma overexpressed (CCN3/NOV), an IGFBP domain-containing protein, which supports expansion of human HSCs [149]. Multiple membrane proteins, including cell surface integrins [139,[142][143][144], Frizzle 8, and low-density lipoprotein (LDL) receptor-related protein 6 [150], were shown to mediate the IGF-independent effects of IGFBP2. Interestingly, extrinsic IGFBP2 can also be taken up into the cytosols of oxidative stressed cells [143,145].…”

Prohibitin-2 Binding Modulates Insulin-like Growth Factor-binding Protein-6 (IGFBP-6)-induced Rhabdomyosarcoma Cell Migration

“…PHB2 Is a Cell Surface Protein That Binds IGFBP-6-Although a number of IGF-independent actions of IGFBPs have been described via distinct mechanisms (34), there is limited information regarding cell surface proteins ("IGFBP receptors") that are responsible for these actions (35,36). IGFBP-1 and -2 interact with cell surface integrins to modulate cell migration and adhesion via Arg-Gly-Asp (RGD) sequences in their C-terminal domains (37,38).…”

Secreted and Transmembrane Wnt Inhibitors and Activators

“…Of the six IGFBP protein family members, IGFBP-1, -2, and -6, but not IGFBP-3 and -5, are also able to bind directly to LRP6 and Fz8 and to inhibit Wnt/b-catenin signaling, albeit with a lower efficiency compared with IGFBP-4 (Zhu et al 2008a). Thus, the lack of cardiac phenotypes in Igfb-4-null mice or Igfb-3, -4, and -5 triple-knockout mice (Ning et al 2006) may be caused by genetic redundancies between IGFBP-4 and other IGFBPs.…”

“…IGFBP-4 was identified in a screen for factors able to induce cardiomyocyte differentiation of P19CL6 cells (Zhu et al 2008a). It promotes cardiogenesis in an IGF-independent fashion, namely, by antagonizing Wnt/b-catenin signaling (Zhu et al 2008a). It binds directly to LRP6 and Fz8 via the carboxy-terminal thyroglobulin domain and blocks binding of Wnt3a to the receptors (Fig.…”