IGFs stimulate zebrafish cell proliferation by activating MAP kinase and PI3-kinase-signaling pathways

Abstract

Insulin-like growth factor (IGF)-I and -II have been cloned from a number of teleost species, but their cellular actions in fish are poorly defined. In this study, we show that both IGF-I and -II stimulated zebrafish embryonic cell proliferation and DNA synthesis in a concentration-dependent manner, whereas insulin had little mitogenic activity. Affinity cross-linking and immunoblotting studies revealed the presence of IGF receptors with the characteristics of the mammalian type I IGF receptor. Competitive binding assay results indicated that the binding affinities of the zebrafish IGF-I receptors to IGF-I, IGF-II, and insulin are 1.9, 2.6, and >190 nM, indicating that IGF-I and -II bind to the IGF-I receptor(s) with approximately equal high affinity. To further investigate the cellular mechanism of IGF actions, we have studied the effects of IGFs on two major signal transduction pathways: mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase). IGFs activated MAPK in zebrafish embryonic cells in a dose-dependent manner. This activation occurred within 5 min of IGF-I stimulation and disappeared after 1 h. IGF-I also caused a concentration-dependent activation of protein kinase B, a downstream target of PI3 kinase, this activation being sustained for several hours. Inhibition of MAPK activation by the MAPK kinase inhibitor PD-98059 inhibited the IGF-I-stimulated DNA synthesis. Similarly, use of the PI3 kinase inhibitor LY-294002 also inhibited IGF-I-stimulated DNA synthesis. When both the MAPK and PI3 kinase pathways were inhibited using a combination of these compounds, the IGF-I-stimulated DNA synthesis was completely negated. These results indicate that both IGF-I and -II are potent mitogens for zebrafish embryonic cells and that activation of both the MAPK and PI3 kinase-signaling pathways is required for the mitogenic action of IGFs in zebrafish embryonic cells.

Characterization of GHRs, IGFs and MSTNs, and analysis of their expression relationships in blunt snout bream, Megalobrama amblycephala

“…But both of IGF-I and -II Table 3 Expression level of GHRs, IGFs and MSTNs in M. amblycephala adult different tissues (mean ± SE). were detected in zebrafish embryonic cells (Pozios et al, 2001), possibly related to the regulatory role for the IGF system in teleost oocyte maturation (Picha et al, 2012). In the present study, IGF-II seemed irrelevant to somitogenesis, according to increasing expression at somitesforming period (neurula period, 16 hpf); whereas IGF-I was not vigorous during embryonic development, and further research was needed to investigate about the absence of IGF-I from fertilized ovum to hatching.…”

Calcium deficiency-induced and TRP channel-regulated IGF1R-PI3K-Akt signaling regulates abnormal epithelial cell proliferation

“…As igfbp5a mRNA is specifically expressed in NaR cells, and IGFs are potent mitogens for zebrafish cells, 16 we tested the idea that low [Ca 2 þ ] stimulates NaR cell proliferation by activating IGF signaling in these cells. We examined phospho-Akt (pAkt) and phospho-Erk (pErk) because commercially available mammalian phospho-IGF1R antibodies did not yield specific signals in zebrafish larvae.…”

“…Signaling through the IGF1R is transduced primarily by the PI3K-Akt and the MEKErk cascades in zebrafish cells. 16 In extracellular fluids, IGFs are present in complexes with a family of high-affinity IGFBPs. These IGFBPs bind to IGFs with equal or even greater affinities than do the IGF1Rs and modulate the distribution, stability, and biological activities of IGFs.…”

Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss)

“…IGF-I has mitogenic and anabolic effects in fish myocytes in culture (Castillo et al 2004). This growth factor induces proliferation, measured as thymidine incorporation, in primary cell cultures of trout myocytes (Castillo et al 2004) as well as DNA synthesis in zebrafish embryonic cells (Pozios et al 2001). However, to our knowledge, the effects of IGF-I in fish adipocyte lineage have not been studied, although IGF-I binding has been characterized in brown trout (Salmo trutta) adipose tissue (Planas et al 2000).…”