Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation

Previous studies have shown that exogenous insulin-like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF-II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF-II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF-II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF-II overproduction. Similar to observations with other muscle cell lines, IGF-II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low-serum differentiation medium. Accelerated differentiation of IGF-II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24–48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle-specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein-5 when compared with controls. These results confirm a role for IGF-II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation. © 1996 Wiley-Liss, Inc.     

"Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II.

Differentiation of muscle cells to form postmitotic myotubes is usually viewed as being negatively controlled by medium components, sometimes designated "mitogens." However, we have found that a family of mitogenic agents, the insulin-like growth factors (IGFs), are potent stimulators of differentiation in myoblasts which act by inducing expression of the myogenin gene. We show here that this action of the IGFs occurs even when these growth factors are not added to the cell medium; upon transfer to low-serum "differentiation medium," myoblasts begin active expression of the IGF-II gene, at both the mRNA and protein levels. Furthermore, autocrine secretion of IGF-II is essential for the process of terminal differentiation of the cells. These conclusions are based upon four lines of evidence. (1) The rate of spontaneous differentiation in several sublines of myogenic cells correlates with their level of expression of IGF-II. (2) C2 and Sol 8 cells, which secrete high levels of IGF-II, are relatively insensitive to exogenous IGFs, in contrast to L6 lines, which exhibit lower levels of IGF-II gene expression. (3) An antisense oligodeoxyribonucleotide complementary to the first five codons of IGF-II inhibits myogenic differentiation in the absence but not in the presence of exogenous IGF-II. (4) Spontaneous differentiation in response to autocrine IGF-II involves the same mechanism that occurs in cells stimulated by the IGFs, i.e. elevation of expression of the myogenin gene.     

La PI3 kinase,une activité critique pour la différenciation des cellules musculaires IGFs dépendante

Evidence has accumulated that suggests that insulin-like growth factors (IGFs) exert a positive influence on myoblast differentiation. We have undertaken to study the signalling events required for differentiation resulting from type-1 IGF receptor stimulation in C2 myoblasts, where autocrine production of IGF-II was abolished by means of antisense RNA. Exposure of the cells to IGFs leads to a rapid and sustained activation of phosphatidyl-inositol 3-kinase followed by the expression of Myod, myogenin and differentiation. The fungal metabolite, wortmannin, inhibits both PI 3-kinase and muscle differentiation with an IC 50 in the nanomolar range. IGFs are also known to cause a rapid activation of MAP kinase. However, the synthetic inhibitor of MEK, PD098059, which prevents MAP kinase activation, does not affect myoblast differentiation. These results provide evidence that PI3-kinase, but not MAP kinase, is required for insulin-like growth factor receptor-dependent differentiation of muscle cells.     

Paradoxical decrease in myf-5 messenger RNA levels during induction of myogenic differentiation by insulin-like growth factors.

Having previously demonstrated that the insulin-like growth factors (IGFs) induce expression of the myogenin gene, we have now extended our investigation of the induction of myogenesis by the IGFs to a second member of the MyoD family, myf-5. This is the only myogenesis gene other than myogenin expressed early in the differentiation of L6 myoblasts, so its regulation was of particular interest because of our observations on myogenin. In contrast to myogenin, myf-5 mRNA was detectable in proliferating myoblasts, but the steady state levels of myf-5 mRNA fell strikingly for 48 h after the cells were switched to low serum medium containing IGF-II in both murine cell lines and myoblasts cultured from human muscle. In spite of this decrease, translation of myf-5 mRNA appeared essential during the early stages of stimulation of myogenesis by the IGFs; an antisense oligodeoxynucleotide complementary to the first five codons of myf-5 blocked the increase in myogenin mRNA and inhibited morphological (cell fusion) and biochemical (creatine kinase elevation) aspects of myogenesis. We conclude that expression of myf-5 is essential for the initial induction of myogenin by the IGFs, but that subsequent elevation of myogenin expression is independent of myf-5, possibly resulting from autoinduction of the myogenin gene. The functional significance of the dramatic decrease in myf-5 mRNA levels during differentiation is not obvious.     

Activation of insulin-like growth factor II expression during skeletal muscle regeneration in the rat: correlation with myotube formation.

Insulin-like growth factors (IGFs) are important stimulators of proliferation and differentiation of cultured myoblasts. It has previously been shown that IGF-I is induced during muscle regeneration in rodents, however, little is known about the expression of IGF-II. Therefore, two in vivo models were used to analyze IGF-II mRNA expression during skeletal muscle regeneration in the rat: injection of the snake venom notexin and induction of ischemia. During the regeneration process the levels of both IGF-I and IGF-II mRNA were transiently induced, as analyzed by solution hybridization. Both IGF-I-like immunoreactivity and IGF-II-like immunoreactivity were found to be present during muscle regeneration. In a time course study, induction of IGF-II was preceded by IGF-I, both at the mRNA and protein levels. Using alpha- and beta-actin as markers for different stages of skeletal muscle differentiation, together with the immunohistochemistry data, it is concluded that the expression of IGF-I and IGF-II occurs at different differentiation stages, and that IGF-II appears concomitant to the formation of myotubes. These results suggest that each IGF has a distinct role during the differentiation of muscle cells.     

Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression.

Stimulation of myogenic differentiation by the insulin-like growth factors (IGFs) has been established for many years, but our attempts to elucidate the mechanism of that stimulation have been successful only in eliminating some likely possibilities. The recent discovery of a family of muscle determination genes has opened a new approach to this question, allowing specific focus on those genes that might play central roles in controlling myogenesis. We now report that IGF-I stimulates terminal myogenic differentiation in L6A1 cells by inducing a large increase in expression of the myogenin gene. This conclusion is supported by the following observations. 1) Myogenin mRNA is elevated by IGF-I, with a concentration dependency that parallels the stimulation of differentiation, including a decrease in stimulation at higher concentrations. 2) The time course of elevation of myogenin mRNA is consistent with its acting as an intermediate in the signalling pathway between occupancy of the IGF-I receptor and induction of expression of muscle-specific genes. 3) Inhibitors of myogenesis also inhibit elevation of myogenin mRNA in response to IGF-I. 4) An antisense oligonucleotide to the N-terminus of myogenin prevents the stimulation of differentiation by IGF-I and IGF-II, but has no effect on other actions of IGF-I on myoblasts. MyoD has been reported not to be expressed in L6 cells, and the expression of myf-5 and herculin/myf-6/MRF4 is reportedly low or undetectable. Thus, the stimulation of differentiation by IGF-I can be attributed largely, if not entirely, to increased expression of the myogenin gene. However, the relatively long time period between addition of the IGFs and elevation of myogenin mRNA as well as the inhibition of this process by several inhibitors indicate that increased myogenin mRNA levels are not a simple direct result of occupation of the IGF-I receptor.     

Insulin-like growth factors (IGF-I and IGF-II) inhibit C2 skeletal myoblast differentiation and enhance TNF alpha-induced apoptosis

IGF-I and IGF-II are thought to be unique in their ability to promote muscle cell differentiation. Murine C2 myoblasts differentiate when placed into low serum media (LSM), accompanied by increased IGF-II and IGF binding protein-5 (IGFBP-5) production. Addition of 20 ng/ml TNF alpha on transfer into LSM blocked differentiation, IGF-II and IGFBP-5 secretion and induced apoptosis. We, therefore, wished to assess whether IGFs could protect against the effects of TNF alpha. Neither inhibition of differentiation or induction of apoptosis was rescued by co-incubation with IGF-I or IGF-II. A lower dose of TNF alpha (1 ng/ml) while not inducing apoptosis still inhibited myoblast differentiation by 56% +/- 12, (P < 0.001), indicating that induction of apoptosis is not the sole mechanism by which TNF alpha inhibits myoblast differentiation. Addition of IGF-I or IGF-II alone reduced differentiation by 49% +/- 15 and 33% +/- 20, respectively, (P < 0.001), although neither induced apoptosis. For muscle cells to differentiate, they must arrest in G0. We established that addition of IGF-I, IGF-II or TNF alpha to the myoblasts promoted proliferation. The myoblasts could not exit the cell cycle as efficiently as controls and differentiation was thus reduced. Unexpectedly, co-incubation of IGF-I or IGF-II with 1 ng/ml TNF alpha enhanced the inhibition of differentiation and induced apoptosis. In the absence of apoptosis we show an association between IGF-induced inhibition of differentiation and increased IGFBP-5 secretion. These results indicate that the effects of the IGFs on muscle may depend on the cytokine environment. In the absence of TNF alpha, the IGFs delay differentiation and promote myoblast proliferation whereas in the presence of TNF alpha the IGFs induce apoptosis.     

The glycosylated IgII Extracellular domain of EMMPRIN is implicated in the induction of MMP-2

Insulin-like growth factor (IGF)-I and IGF-II play major roles in the regulation of skeletal muscle growth and differentiation, and both are locally expressed in muscle cells. Recent studies have demonstrated that IGF-II up-regulates its own gene expression during myogenesis and this auto-regulatory loop is critical for muscle differentiation. How local IGF-I is regulated in this process is unclear. Here, we report that while IGF-II up-regulated its own gene expression, it suppressed IGF-I gene expression during myogenesis. These opposite effects of IGF-II on IGF-I and IGF-II genes expression were time dependent and dose dependent. It has been shown that IGFs activate the PI3K-Akt-mTOR, p38 MAPK, and Erk1/2 MAPK pathways. In myoblasts, we examined their role(s) in mediating the opposite effects of IGF-II. Our results showed that both the PI3K-Akt-mTOR and p38 MAPK pathways played critical roles in increasing IGF-II mRNA expression. In contrast, mTOR was required for down-regulating the IGF-I gene expression by IGF-II. In addition, Akt, Erk1/2 MAPK, and p38 MAPK pathways were also involved in the regulation of basal levels of IGF-I and IGF-II genes during myogenesis. These findings reveal a previously unrecognized negative feedback mechanism and extend our knowledge of IGF-I and IGF-II gene expression and regulation during myogenesis.     

Investigating the prevalence of queuine in Escherichia coli RNA via incorporation of the tritium-labeled precursor, preQ(1)

Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.     

Insulin-like growth factors induce apoptosis as well as proliferation in LIM 1215 colon cancer cells

The insulin-like growth factor (IGF) system plays an important role in cell proliferation and survival. However, more recently, a small number of studies have shown that IGFs induce apoptosis in some cells. Our initial studies showed this occurred in LIM 1215 colon cancer cells but not RD rhabdomyosarcoma cells. IGFs induced both proliferation and apoptosis in LIM 1215 cells, and the induction of apoptosis was dose-dependent. [R54, R55]IGF-II, which binds to the IGF-I receptor with normal affinity but does not bind to the IGF-II receptor, induced apoptosis to the same extent as IGF-II, whereas [L27]IGF-II, which binds to the IGF-I receptor with 1000-fold reduced affinity, had no effect on apoptosis. These results suggest that the IGF-I receptor is involved in induction of apoptosis. Western blot analyses demonstrated that Akt and Erk1/2 were constitutively activated in RD cells. In contrast, phosphorylation of Akt and Erk1/2 were transient and basal expression of Akt protein was lower in LIM 1215 cells. Analysis of apoptosis-related proteins showed that IGFs decreased pro-caspase-3 levels and increased expression of pro-apoptotic Bad in LIM 1215 cells. IGFs co-activate proliferative and apoptotic pathways in LIM 1215 cells, which may contribute to increased cell turnover. Since high turnover correlates with poor prognosis in colorectal cancer, this study provides further evidence for the role of the IGF system in its progression.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis

During the last decade, involvement of growth hormone (GH), insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. This review provides an update on the GH, IGF system and their role in ovarian follicular development. In vitro studies and knockout experiments demonstrated an important role of GH in preantral follicle growth and differentiation through their binding with GH receptors, which are located both in the oocyte and follicular somatic tissues. Furthermore, GH stimulates the development of small antral follicles to gonadotrophin-dependent stages, as well as maturation of oocytes. With regard to the IGF system, IGF-I has no effects on primordial follicle development, but both IGF-I and IGF-II stimulate growth of secondary follicles. Depending on the species studies and method used, these proteins have been detected in oocytes and/or somatic cells. In antral follicles, these IGFs stimulate granulosa cell proliferation and steroidogenesis in most mammals. The bioavailability of IGFs is regulated by a family of intrafollicular expressed IGF binding proteins (IGFBPs). Facilitation of IGF can be increased through the activity of specific IGFBP proteases, which degrade the IGF/IGFBP complex, resulting in the production of IGFBP fragments and release of attached IGF.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.