Effect of recombinant porcine IGFBP-3 on IGF-I and long-R3-IGF-I-stimulated proliferation and differentiation of L6 myogenic cells.
ABSTRACT Insulin-like growth factor (IGF)-I stimulates both proliferation and differentiation of myogenic precursor cells. In vivo, IGFs are bound to one of the members of a family of six high-affinity IGF binding proteins (IGFBP 1-6) that regulate their biological activity. One of these binding proteins, IGFBP-3, affects cell proliferation via both IGF-dependent and IGF-independent mechanisms and it has generally been shown to suppress proliferation of cultured cells; however, it also may stimulate proliferation depending upon the cell type and the assay conditions. Cultured porcine embryonic myogenic cells (PEMCs) produce IGFBP-3 and its level drops significantly immediately prior to differentiation. Additionally, IGFBP-3 suppresses both IGF-I and Long-R3-IGF-I-stimulated proliferation of embryonic porcine myogenic cells. In this study, we have examined the effects of recombinant porcine IGFBP-3 (rpIGFBP-3) on IGF-I- and Long-R3-IGF-I-stimulated proliferation and differentiation of the L6 myogenic cell line. L6 cells potentially provide a good model for studying the actions of IGFBP-3 on muscle because they contain no non-muscle cells and they do not produce detectable levels of IGFBP-3. RpIGFBP-3 suppresses both IGF-I and Long-R3-IGF-I-stimulated proliferation of L6 cells, indicating that it suppresses proliferation via both IGF-dependent and IGF-independent mechanisms. Our data also show that rpIGFBP-3 causes IGF-independent suppression of proliferation without increasing the level of phosphosmad-2 in L6 cultures. Additionally, rpIGFBP-3 suppresses IGF-I-stimulated differentiation of L6 cells. In contrast, however, rpIGFBP-3 does not suppress Long-R3-IGF-I-stimulated differentiation. This suggests that rpIGFBP-3 does not have IGF-independent effects on L6 cell differentiation.
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ABSTRACT: It is very clear that the GH-IGF axis plays a major role in controlling the growth and differentiation of skeletal muscles, as it does virtually all of the tissues in the animal body. One aspect of this control is unquestioned: circulating GH acts on the liver to stimulate expression of the IGF-I and IGFBP3 genes, substantially increasing the levels of these proteins in the circulation. It also seems that GH stimulates expression of IGF-I genes in skeletal muscle, although there are a number of cases in which skeletal muscle IGF-I expression is elevated in the absence of GH. It is substantially less clear that GH acts directly on skeletal muscle to stimulate its growth; the presence of GH receptor mRNA in skeletal muscle is well established, but most investigators have been unsuccessful in demonstrating any specific binding of GH to skeletal muscle or to myoblasts in culture. It has been equally difficult to show direct actions of GH on cultured muscle cells; the only positive report concludes that the early insulin-like effects of GH can result from direct interactions between GH and isolated muscle cells. The effects of the IGFs on skeletal muscle are much clearer. It is well established by studies in a number of laboratories on a variety of systems that IGFs stimulate many anabolic responses in myoblasts, as they do in other cell types. IGFs have the unusual property of stimulating both proliferation and differentiation of myoblasts, responses that are generally believed to be mutually exclusive; in myoblasts, they are in fact temporally separated. The stimulation of differentiation by IGF-I is (at least in part) a result of substantially increased levels of the mRNA for myogenin, the member of the MyoD family most directly associated with terminal myogenesis. As levels of myogenin mRNA rise, those of myf-5 mRNA (the only other member of the MyoD family expressed significantly in L6 myoblasts) fall dramatically, although myf-5 expression is required for the initial elevation of myogenin. The effects of IGFs are significantly modulated by IGFBPs secreted by myoblasts in serum-free medium, inhibitory IG-FBPs-4 and -6 are expressed and secreted by L6A1 myoblasts, while expression of IGFBP-5 rises dramatically as differentiation proceeds. Other myoblasts also secrete IGFBP-2. Even if exogenous IGFs are not added to the low-serum "differentiation" medium, myoblasts express sufficient amounts of autocrine IGF-II to stimulate myogenesis after a period of time; some myogenic cell lines, (such as Sol 8) are so active in expressing the IGF-II gene that it is not possible to demonstrate effects of exogenous IGFs. This autocrine expression of IGFs is by no means unique to skeletal muscle cells; indeed, it is so widely seen in cells responding to mitogenic stimuli that we suggest that IGFs can be viewed as extracellular second messengers that mediate most, if not all, such actions of agents that stimulate cell proliferation. The component of serum that suppresses IGF-II gene expression under "growth" conditions appears to be the IGFs themselves, which exhibit a very high potency in the feedback inhibition of IGF-II expression. In addition, IGFs have effects on the expression of other genes related to differentiation. Treatment of L6A1 cell with IGFs suppresses their expression of the myogenesis-inhibiting TGF beta s with a time course consistent with an initial proliferative step followed by differentiation, i.e. expression is first increased and then very substantially decreased. It is not established that this plays a role in control of differentiation, but experiments with FGF antisense constructs suggests that this may well be the case. Until recently, IGFs were the only circulating agents known to stimulate myoblast differentiation, in contrast to the relatively large number of growth factors that inhibit the process. It is now clear that thyroid hormones and RA also stimulate myogenesis, and that IL-15 enhances the stimulatory effEndocrine Reviews 11/1996; 17(5):481-517. · 14.87 Impact Factor
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ABSTRACT: It is well known that IGFs-I and -II stimulate both the proliferation and differentiation of myoblasts, but the role of the IGF binding proteins (IGFBPs) during these processes has not been established. In this study we show that IGF-I analogs with greatly reduced affinity for IGFBPs exhibited about a 10-fold increase in potency in stimulating proliferation (as in other cell types), but up to a 100-fold greater potency than native IGF-I in stimulating L6A1c differentiation. Analysis of conditioned media revealed that L6 cells secrete significant levels of IGFBPs that react with antisera to IGFBP-4, -5 and -6. Steady-state levels of IGFBP-4 mRNA were highest in proliferating myoblasts, while IGFBP-5 mRNA could not be detected in myoblasts although its levels were dramatically increased during IGF- or insulin-stimulated differentiation of myoblasts into myotubes. Elevated IGFBP-6 mRNA levels were found in quiescent cells in serum-free medium. IGF-I and IGF-II treatment elevated IGFBP-5 in conditioned media, but longR3IGF-I and insulin, which do not bind to IGFBPs, had smaller effects. This complex regulation of expression of different IGFBPs not only during different stages of muscle growth and differentiation, but also upon stimulation by IGFs or insulin, suggests that the IGFBPs play a specific and significant role in modulating the actions of the IGFs during myogenesis.Journal of Endocrinology 04/1995; 144(3):539-53. · 4.06 Impact Factor
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ABSTRACT: Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.Journal of Cellular Physiology 03/1999; 178(2):227-34. · 4.22 Impact Factor