IGF-II is up-regulated and myofibres are hypertrophied in regenerating soleus of mice lacking FGF6

Important functions in myogenesis have been proposed for FGF6, a member of the fibroblast growth factor family accumulating almost exclusively in the myogenic lineage. However, the use of FGF6(-/-) mutant mice gave contradictory results and the role of FGF6 during myogenesis remains largely unclear. Using FGF6(-/-) mice, we first analysed the morphology of the regenerated soleus following cardiotoxin injection and showed hypertrophied myofibres in soleus of the mutant mice as compared to wild-type mice. Secondly, to examine the function of the IGF family in the hypertrophy process, we used semiquantitative and real-time RT-PCR assays and Western blots to monitor the expression of the insulin-like growth factors (IGF-I and IGF-II), their receptors [type I IGF receptor (IGF1R) and IGF-II receptor (IGF2R)], and of a binding protein IGFBP-5 in regenerating soleus muscles of FGF6(-/-) knockout mice vs. wild-type mice. In the mutant, both IGF-II and IGF2R, but not IGF-I and IGF1R, were strongly up-regulated, whereas IGFBP5 was down-regulated, strongly suggesting that, in the absence of FGF6, the mechanisms leading to myofibre hypertrophy were mediated specifically by an IGF-II/IGF2R signalling pathway distinct from the classic mechanism involving IGF-I and IGF1R previously described for skeletal muscle hypertrophy. The potential regulating role of IGFBP5 on IGF-II expression is also discussed. This report shows for the first time a specific role for FGF6 in the regulation of myofibre size during a process of in vivo myogenesis.     

Complementary expression of IGF-II and IGFBP-5 during anterior pituitary development

The specification of the five individual hormone-secreting cell types in the anterior pituitary requires a series of sequential cell fate decisions. We have immortalized cells at several stages along this pathway of pituitary differentiation. Here, we present analysis of differences in gene expression between an anterior pituitary precursor cell line, alphaT1-1, and an immature gonadotrope cell line, alphaT3-1, identified by using cDNA subtraction. Messenger RNA expression of members of the insulin-like growth factor signaling system, IGF-II and IGFBP-5, was found in the alphaT1-1 precursor cell line, but not in the more differentiated cell line, alphaT3-1. This inferred stage specificity was confirmed in the mouse embryo by using in situ hybridization on embryonic days e10.5 through e18.5. Expression of IGF-II and IGFBP-5 mRNAs was both temporally and spatially regulated during pituitary development. IGF-II was highly expressed in the epithelium surrounding Rathke's pouch at e10.5, while IGFBP-5 expression was restricted to the adjacent oral epithelium. At e11.5 (represented by alphaT1-1), IGF-II was expressed throughout the pouch, but was coexpressed with IGFBP-5 and alpha-subunit in the ventral portion of the pouch epithelium. On e12.5, the two mRNAs were expressed in opposing dorsoventral (IGF-II) and ventrodorsal (IGFBP-5) patterns, with IGF-II excluded from the rostral, alpha-subunit-expressing region. A decrease of both mRNAs was observed at e14.5 (equivalent to alphaT3-1), with IGF-II levels low and IGFBP-5 concentrated in the anterior pituitary rostral tip. These findings suggest that the timing of IGF-II expression and regulation of its accessibility by IGFBP-5 may play a role in anterior pituitary differentiation, survival, and/or proliferation.     

Alteration of the insulin-like growth factor axis during in vitro differentiation of the human osteosarcoma cell line HOS 58

The insulin-like growth factors I and II (IGF-I, IGF-II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low-density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF-I and -II; (ii) synthesized low levels of IGFBP-2, -3, -4, and -5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF-I and -II expressions increased continuously in parallel with an upregulation of IGFBP-2, -3, -4, and -5 whereas the IGF-I receptor and IGF-II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF-IR, but low concentrations of IGFBP-2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF-IR expression, but increase of mitoinhibitory IGFBP-2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells.     

Insulin-like growth factor binding protein-6: the "forgotten" binding protein?

Insulin-like growth factor binding protein-6 (IGFBP-6) differs from IGFBPs 1-5 in that it binds IGF-II with marked preferential affinity over IGF-I. Human and rat IGFBP-6 lack 2 and 4 N-terminal cysteines and therefore the Gly-Cys-Gly-Cys-Cys motif present in IGFBPs 1-5. IGFBP-6 is O-glycolsylated, and five serine/threonine glycosylation sites in the non-conserved mid-region of human IGFBP-6 have been identified. O-Glycosylation inhibits proteolysis of IGFBP-6 by chymotrypsin and trypsin, but has no effect on high affinity IGF binding. IGFBP-6 is a relatively specific inhibitor of IGF-II actions; it has not been shown to potentiate IGF actions. IGFBP-6 is only cell-associated to a very limited extent, if at all. IGFBP-6 is often expressed in non-proliferative, quiescent states in vitro and differentiating agents increase its expression. IGFBP-6 expression is associated with inhibition of growth of tumour cells in vitro and in vivo. Although many questions remain regarding the biological role of IGFBP-6, its major function appears to be the regulation of IGF-II actions. This could be especially significant since IGF-II has been implicated as an autocrine tumour growth factor.     

IGF-II and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren's disease

Dupuytren's disease (DD) is a common and heritable fibrosis of the palmar fascia that typically manifests as permanent finger contractures. The molecular interactions that induce the development of hyper-contractile fibroblasts, or myofibroblasts, in DD are poorly understood. We have identified IGF2 and IGFBP6, encoding insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-6 respectively, as reciprocally dysregulated genes and proteins in primary cells derived from contracture tissues (DD cells). Recombinant IGFBP-6 inhibited the proliferation of DD cells, patient-matched control (PF) cells and normal palmar fascia (CT) cells. Co-treatments with IGF-II, a high affinity IGFBP-6 ligand, were unable to rescue these effects. A non-IGF-II binding analog of IGFBP-6 also inhibited cellular proliferation, implicating IGF-II-independent roles for IGFBP-6 in this process. IGF-II enhanced the proliferation of CT cells, but not DD or PF cells, and significantly enhanced DD and PF cell contractility in stressed collagen lattices. While IGFBP-6 treatment did not affect cellular contractility, it abrogated the IGF-II-induced contractility of DD and PF cells in stressed collagen lattices. IGF-II also significantly increased the contraction of DD cells in relaxed lattices, however this effect was not evident in relaxed collagen lattices containing PF cells. The disparate effects of IGF-II on DD and PF cells in relaxed and stressed contraction models suggest that IGF-II can enhance lattice contractility through more than one mechanism. This is the first report to implicate IGFBP-6 as a suppressor of cellular proliferation and IGF-II as an inducer of cellular contractility in this connective tissue disease.     

Dibutyryl cyclic adenosine monophosphate differentially regulates cell proliferation in low and high alkaline phosphatase SaOS-2 human osteosarcoma cells: Evidence for mediation by the insulin-like growth factor-II system

In the present study, we have sought to determine whether a given signal transduction pathway can have diverse effects on subpopulations of cells of a lineage depending upon the stage of differentiation. To test this hypothesis, we selected the cyclic adenosine monophosphate (cAMP) signal transduction pathway because of its recognized importance in mediating the actions of many hormones, e.g., parathyroid hormone which acts on the bone-forming cells, the osteoblasts. Subpopulations of human osteosarcoma SaOS-2 cells with low (LSaOS) and high (HSaOS) alkaline phosphatase (ALP) content were chosen as model systems for preosteoblasts (pre-OB) and osteoblasts (OB), respectively. Dibutyryl cyclic AMP (DBcAMP) treatment of serum free cultures produced a differential effect on the proliferation of LSaOS cells (40 ± 5% of control at 1 mM DBcAMP, P < 0.001) compared with HSaOS cells (no statistically significant effect). The finding supports the hypothesis. Next, we sought evidence for mediation, at least in part, by the insulin-like growth factor (IGF)-II regulatory system. We report that the basal expression of IGF-II, IGF binding protein (IGFBP)-3, and IGFBP-4 was higher in LSaOS cells than in HSaOS cells with the opposite true for type I IGF receptor. DBcAMP treatment of LSaOS cells decreased IGF-II and IGFBP-3 but increased IGFBP-4 and type I IGF receptor; no effect was observed for the type II IGF receptors. DBcAMP treatment of HSaOS cells had no detectable effect on IGF-II; IGFBP-3, or type I and type II IGF receptor expression; only IGFBP-4 expression increased with DBcAMP. These observations suggest that the differential regulation of cell proliferation by the cAMP signal transduction pathway may be mediated, at least in part, by the IGF-II regulatory system. © 1993 Wiley-Liss, Inc.     

Interaction of insulin-like growth factor II (IGF-II) with multiple plasma proteins: high affinity binding of plasminogen to IGF-II and IGF-binding protein-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.     

An in situ hybridization study of the insulin-like growth factor system in developing condylar cartilage of the fetal mouse mandible

The objective of this study was to investigate the involvement of the insulin-like growth factor (IGF) system in the developing mandibular condylar cartilage and temporomandibular joint (TMJ). Fetal mice at embryonic day (E) 13.0-18.5 were used for in situ hybridization studies using [35S]-labeled RNA probes for IGF-I, IGF-II, IGF-I receptor (-IR), and IGF binding proteins (-BPs). At E13.0, IGF-I and IGF-II mRNA were expressed in the mesenchyme around the mandibular bone, but IGF-IR mRNA was not expressed within the bone. At E14.0, IGF-I and IGF-II mRNA were expressed in the outer layer of the condylar anlage, and IGF-IR mRNA was first detected within the condylar anlage, suggesting that the presence of IGF-IR mRNA in an IGF-rich environment triggers the initial formation of the condylar cartilage. IGFBP-4 mRNA was expressed in the anlagen of the articular disc and lower joint cavity from E15.0 to 18.5. When the upper joint cavity was formed at E18.5, IGFBP-4 mRNA expression was reduced in the fibrous mesenchymal tissue facing the upper joint cavity. Enhanced IGFBP-2 mRNA expression was first recognized in the anlagen of both the articular disc and lower joint cavity at E16.0 and continued expression in these tissues as well as in the fibrous mesenchymal tissue facing the upper joint cavity was observed at E18.5. IGFBP-5 mRNA was continuously expressed in the outer layer of the perichondrium/fibrous cell layer in the developing mandibular condyle. These findings suggest that the IGF system is involved in the formation of the condylar cartilage as well as in the TMJ.     

Insulin-like growth factor family and combined antisense approach in therapy of lung carcinoma

BACKGROUND: Perturbation in a level of any peptide from insulin-like growth factor (IGF) family (ligands, receptors, and binding proteins) seems to be implicated in lung cancer formation; IGF ligands and IGF-I receptor through their mitogenic and anti-apoptotic action, and the mannose 6-phosphate/insulin-like growth factor II receptor (M6-P/IGF-IIR) possibly as a tumor suppressor. MATERIALS AND METHODS: To determine the identity, role, and mutual relationship of IGFs in lung cancer growth and maintenance, we examined IGF's gene (by RT-PCR) and protein (by immunohistochemistry) expression in 69 human lung carcinoma tissues. We also examined IGF-I receptor numbers (Scatchard analysis) and IGF-II production and release (by Western blot) in IGF-II/IGF-IR mRNA positive and negative lung carcinomas. Finally, the potential role of IGF-IR and IGF-II as growth promoting factors in lung cancer was studied using antisense oligodeoxynucleotides that specifically inhibit IGF-IR and IGF-II mRNA. RESULTS: Thirty-two tumors were positive for IGF-I, 39 for IGF-II, 48 for IGF-IR, and 35 for IGFBP-4 mRNA. Seventeen tumors were concomitantly positive for all four IGFs, whereas 34 were positive for IGF-II, IGF-IR, and IGFBP-4 mRNA. An elevated amount of IGF-II peptide was secreted into the growth medium of cell cultures established from five different IGF-II/IGF-IR mRNA positive lung cancer tissues. The cells also expressed elevated numbers of IGF-IR. Nine IGF-II-negative and 19 IGF-II-positive lung cancers of different stages were selected, and M6-P/ IGF-II receptor was determined immunohistochemically. Most of the IGF-II-negative tumors were strongly positive for M6-P/IGF-IIR. IGF-II-positive tumors were mostly negative for M6-P/IGF-II receptors. Antisense oligodeoxynucleotides to IGF-II significantly inhibited, by 25-60%, the in vitro growth of all six lung cancer cell lines. However, the best results (growth inhibition of up to 80%) were achieved with concomitant antisense treatment (to IGF-IR and IGF-II). CONCLUSION: Our data suggest that lung cancer cells produce IGF-IR and IGF-II, which in turn stimulates their proliferation by autocrine mechanism. Cancer cell proliferation can be abrogated or alleviated by blocking the mRNA activity of these genes indicating that an antisense approach may represent an effective and practical cancer gene therapy strategy.     

IGF binding protein-2 gene expression and the location of IGF-I and IGF-II in fetal rat lung.

Binding proteins for the insulin-like growth factors (IGF-BPs) are important modulators of the biological actions of IGF-I and IGF-II. The generation of IGFBPs within developing organs, and their spatial arrangement, may similarly determine IGF action at specific microanatomical sites. In situ hybridization studies with late gestation (days 16, 18 and 20) fetal rat lung using a cDNA probe for IGFBP-2 showed strong gene expression in the fetal lung epithelial structures (alveoli and airways). The sites of IGFBP-2 gene expression were associated with immunoreactive IGF-II at the apical surface of the epithelium. By day 20, there was also some IGFBP-2 gene expression and immunoreactive IGF-II at discrete sites in the mesenchyme. In contrast, immunoreactive IGF-I was found predominantly distributed in a punctate pattern, consistent with its presence in the lumen or walls of small vessels or capillaries, and in a granular, intracellular form in both epithelial and mesenchymal cells. These studies suggest that endogenously generated IGFBP-2 may determine the distribution of IGF-II, principally at the apical surface of lung epithelia. IGF-I does not colocalise with IGF-II peptide or the sites of IGFBP-2 gene expression. We conclude that the spatial distributions of these two related growth factors are separately controlled, to some extent by endogenously generated binding proteins.     

IGF-II is regulated by microRNA-125b in skeletal myogenesis

MicroRNAs (miRNAs) have emerged as key regulators of skeletal myogenesis, but our knowledge of the identity of the myogenic miRNAs and their targets remains limited. In this study, we report the identification and characterization of a novel myogenic miRNA, miR-125b. We find that the levels of miR-125b decline during myogenesis and that miR-125b negatively modulates myoblast differentiation in culture and muscle regeneration in mice. Our results identify IGF-II (insulin-like growth factor 2), a critical regulator of skeletal myogenesis, as a direct and major target of miR-125b in both myocytes and regenerating muscles, revealing for the first time an miRNA mechanism controlling IGF-II expression. In addition, we provide evidence suggesting that miR-125b biogenesis is negatively controlled by kinase-independent mammalian target of rapamycin (mTOR) signaling both in vitro and in vivo as a part of a dual mechanism by which mTOR regulates the production of IGF-II, a master switch governing the initiation of skeletal myogenesis.     

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.     

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.     

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.     

O-glycosylation of insulin-like growth factor (IGF) binding protein-6 maintains high IGF-II binding affinity by decreasing binding to glycosaminoglycans and susceptibility to proteolysis.

Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glycoprotein which specifically inhibits insulin-like growth factor (IGF)-II actions. The effects of O-glycosylation of IGFBP-6 on binding to glycosaminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied. Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was approximately threefold greater than that of glycosylated (g) IGFBP-6. When bound to glycosaminoglycans, IGFBP-6 had approximately 10-fold reduced binding affinity for IGF-II. Exogenously added n-gIGFBP-6 but not gIGFBP-6 also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n-gIGFBP-6 was inhibited by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O-glycosylation also protected human IGFBP-6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affinity of IGFBP-6 for IGF-II, even with a relatively small reduction in apparent molecular mass as observed with chymotrypsin. Analysis by ESI-MS of IGFBP-6 following limited chymotryptic digestion showed that a 4.5-kDa C-terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding site were cleaved. The truncated, multiply cleaved IGFBP-6 remained held together by disulphide bonds. In contrast, trypsin cleaved IGFBP-6 in the mid-region of the molecule, resulting in a 16-kDa C-terminal peptide which did not bind IGF-II. These results indicate that O-glycosylation inhibits binding of IGFBP-6 to glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP-6 in a high-affinity, soluble form and so contributing to its inhibition of IGF-II actions.     

Co-expression of messenger ribonucleic acids encoding IGF-I, IGF-II, type I and II IGF receptors and IGF-binding proteins (IGFBP-1 to -6) during follicular development in the ovary of seasonally anoestrous ewes

In recent years, it has become apparent that components of the insulin-like growth factor (IGF) system are involved in the regulation of ovarian follicular development in sheep. The majority of previous studies have concentrated on investigating only a select few components and not the whole system. The aim of the present study was to use five seasonally anoestrous ewes to investigate the expression of mRNA encoding all 10 components of the sheep IGF system among various-sized follicles within the ovary, using sheep-specific ribonucleotide probes and in situ hybridisation. IGF-I mRNA expression was low and did not vary with follicle size. IGF-II mRNA expression was significantly higher (P < 0.05) in small follicles compared to large follicles. Both IGF receptors had significantly higher (P < 0.05) levels of mRNA expression in small follicles, with the type I receptor being expressed to a slightly greater extent than the type II receptor. IGFBP-2, -3, -4 and -5 gene expression followed a similar pattern to IGF-II and the IGF receptors, whereby expression decreased with increasing follicle size. Similar to IGF-I, IGFBP-6 mRNA expression showed little variation with follicle size. IGFBP-1 mRNA expression was observed at low and constant levels, albeit in small and medium-sized follicles only. These data demonstrate that all of the components of the IGF system are produced in the ovine follicle, and for some of the components, their gene expression varied with stage of follicle development. This study further emphasises the importance of IGF-II as the major IGF in the autocrine and paracrine regulation of follicle development in sheep.     

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.     

The expression pattern of an insulin-like growth factor (IGF)-binding protein gene is distinct from IGF-II in the midgestational rat embryo.

Insulin-like growth factor-II (IGF-II), the predominant form of IGF in fetal and neonatal serum and tissues, is found in vivo complexed with IGF-binding proteins. One of these binding proteins, IGFBP-2, is present at high levels in fetal rat plasma and binds both IGF-I and IGF-II with high affinity. We here have used in situ hybridization to compare the distribution of IGFBP-2 mRNA with that of IGF-II mRNA in embryonic day 13.5-15 rat embryos. The spatial patterns of IGF-II and IGFBP-2 expression in the fetal trunk were distinct and, in general, nonoverlapping. Most mesoderm derivatives that express IGF-II at high levels contained little, if any, IGFBP-2 mRNA. Instead, IGFBP-2 mRNA was expressed at high levels in many cell types derived from ectoderm and endoderm. The expression of IGFBP-2 mRNA in the central nervous system (CNS) during this developmental period was examined in particular detail. The three most prominent sites of IGFBP-2 expression in the CNS were comprised of cells with nonneuronal phenotypes: 1) the epithelium of the choroid plexus, a tissue that produces cerebrospinal fluid; 2) the floor plate, an area that can guide axonal outgrowth from commissural neurons of the spinal cord in vitro; and 3) the infundibulum, the progenitor of the posterior pituitary that is believed to influence differentiation of the adjacent intermediate pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)