Insulin-like growth factor-binding protein-5 inhibits osteoblast differentiation and skeletal growth by blocking insulin-like growth factor actions

Signaling through the IGF-I receptor by locally synthesized IGF-I or IGF-II is critical for normal skeletal development and for bone remodeling and repair throughout the lifespan. In most tissues, IGF actions are modulated by IGF-binding proteins (IGFBPs). IGFBP-5 is the most abundant IGFBP in bone, and previous studies have suggested that it may either enhance or inhibit osteoblast differentiation in culture and may facilitate or block bone growth in vivo. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5 in bone, we studied its effects in differentiating osteoblasts and in primary bone cultures. Purified wild-type (WT) mouse IGFBP-5 or a recombinant adenovirus expressing IGFBP-5WT each prevented osteogenic differentiation induced by the cytokine bone morphogenetic protein (BMP)-2 at its earliest stages without interfering with BMP-mediated signaling, whereas an analog with reduced IGF binding (N domain mutant) was ineffective. When added at later phases of bone cell maturation, IGFBP-5WT but not IGFBP-5N blocked mineralization, prevented longitudinal growth of mouse metatarsal bones in short-term primary culture, and inhibited their endochondral ossification. Because an IGF-I variant (R3IGF-I) with diminished affinity for IGFBPs promoted full osteogenic differentiation in the presence of IGFBP-5WT, our results show that IGFBP-5 interferes with IGF action in osteoblasts and provides a framework for discerning mechanisms of collaboration between signal transduction pathways activated by BMPs and IGFs in bone.     

IGFs, IGFBPs, IGF-binding sites and biochemical markers of bone metabolism during differentiation in human pulp fibroblasts

OBJECTIVE: To investigate the role of the insulin-like growth factors (IGF) system during the differentiation of human pulp-derived fibroblasts (HPF). METHODS: Primary HPF were cultured for 24 days in DMEM medium with IGF-I or IGF-II (50 ng/ml each). Cell growth and morphology, alkaline phosphatase (ALP) activity, the concentration of free deoxypyridinoline (DPD), IGF-I, -II, IGFBP-2 and -3 were studied. The number of (125)I-IGF-I binding sites was estimated by Scatchard analysis. RESULTS: Light-microscopically visible nodules emerged during differentiation. Simultaneously, the ALP activity increased steadily between days 8 and 24, while the DPD concentration decreased by about 50%. The HPF produced high concentrations of IGF-II (2.00-1.30 microg/10(6) cells) but low IGF-I, IGFBP-2. IGFBP-2 was not changed, IGFBP-3 increased by 65% during differentiation. The number of IGF binding sites increased from 8,500 +/- 55 per cell (day 8) up to 22,000 +/- 570 (day 24). CONCLUSION: The increasing number of IGF-binding sites accompanied by alterations in the biochemical bone markers during the differentiation of HPF suggests an autocrine/paracrine role for the IGFs in the formation of dentinal hard tissue.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

IGFBP-5 regulates muscle cell differentiation by binding to IGF-II and switching on the IGF-II auto-regulation loop

IGF-II stimulates both mitogenesis and myogenesis through its binding and activation of the IGF-I receptor (IGF-IR). How this growth factor pathway promotes these two opposite cellular responses is not well understood. We investigate whether local IGF binding protein-5 (IGFBP-5) promotes the myogenic action of IGF-II. IGFBP-5 is induced before the elevation of IGF-II expression during myogenesis. Knockdown of IGFBP-5 impairs myogenesis and suppresses IGF-II gene expression. IGF-II up-regulates its own gene expression via the PI3K-Akt signaling pathway. Adding IGF-II or constitutively activating Akt rescues the IGFBP-5 knockdown-caused defects. However, an IGF analogue that binds to the IGF-IR but not IGFBP has only a limited effect. When added with low concentrations of IGF-II, IGFBP-5 restores IGF-II expression and myogenic differentiation, whereas an IGF binding–deficient IGFBP-5 mutant has no effect. These findings suggest that IGFBP-5 promotes muscle cell differentiation by binding to and switching on the IGF-II auto-regulation loop.     

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.     

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.     

IGF binding proteins and their functions

The insulin-like growth factor (IGF) binding proteins (IGFBPs) have several functions, including transporting the IGFs in the circulation, mediating IGF transport out of the vascular compartment, localizing the IGFs to specific cell types, and modulating both IGF binding to receptors and growth-promoting actions. The functions of IGFBPs appear to be altered by posttranslational modifications. IGFBP-3, -4, -5, and -6 have been shown to be glycosylated. Likewise all the IGFBPs have a complex disulfide bond structure that is required for maintenance of normal IGF binding. IGFBP-2, -3, -4, and -5 are proteolytically cleaved, and specific proteases have been characterized for IGFBP-3, -4, and -5. Interestingly, attachment of IGF-I or II to IGFBP-4 results in enhancement of proteolysis, whereas attachment of either growth factor to IGFBP-5 results in inhibition of proteolytic cleavage. Cleavage of IGFBP-3 results in the appearance of a 31 kDa fragment that is 50-fold reduced in its affinity for the IGF-I or IGF-II. In spite of the reduction in its affinity, this fragment is capable of potentiating the effect of IGF-I on cell growth responses; therefore, proteolysis may be a specific mechanism that alters IGFBP modulation of IGF actions. Other processes that result in a reduction in IGF binding protein affinity are associated with potentiation of cellular responses to IGF-I and -II. Specifically, the binding of IGFBP-3 to cell surfaces is associated with its ability to enhance IGF action and with a ten- to 12-fold reduction in its affinity for IGF-I and IGF-II. Likewise, binding of IGFBP-5 to extracellular matrix (ECM) results in an eightfold reduction in its affinity and a 60% increase in cell growth in response to IGF-I. Another post-translational modification that modifies IGFBP activity is phosphorylation. IGFBP-1, -2, -3, and -5 have been shown to be phosphorylated. Phosphorylation of IGFBP-1 results in a sixfold enhancement in its affinity for IGF-I and -II. Following this enhancement of IGFBP-1 affinity, this binding protein loses its capacity to potentiate IGF-I growth-promoting activity. Future studies using site-directed mutagenesis to modify these proteins should enable us to determine the effect of these posttranslational modifications on the ability of IGFBPs to modulate IGF biologic activity. © 1993 Wiley-Liss, Inc.     

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.     

IGFBP-4 degradation by pregnancy-associated plasma protein-A in MC3T3 osteoblasts

Insulin-like growth factor (IGF) signaling is critical for osteoblast development and IGF binding protein (IGFBP)-4 is one of the principle IGFBPs expressed by osteoblasts. Release of bound IGF via proteolytic degradation of IGFBP-4 is likely to be critical for osteoblast development. We have investigated whether IGF-sensitive, IGFBP-4 degradation in mouse MC3T3-E1 osteoblasts is due to the metzincin pregnancy-associated plasma protein (PAPP)-A. Degradation of IGFBP-4 by PAPP-A or MC3T3-E1 conditioned medium was enhanced by IGF-II but inhibited by mutation of basic residues at or near the PAPP-A cleavage site in IGFBP-4. Furthermore, immunodepletion of PAPP-A from MC3T3-E1 conditioned medium abolished IGFBP-4 degradation. We also found that PAPP-A messenger RNA was expressed throughout differentiation of MC3T3-E1 cells. These results demonstrate for the first time that PAPP-A is the IGFBP-4 protease in MC3T3-E1 cells, a widely used model for osteoblast development, and that PAPP-A may regulate IGF release throughout osteoblast differentiation.     

Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities

The insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of six homologous proteins with high binding affinity for IGF-I and IGF-II. Information from NMR and mutagenesis studies is advancing knowledge of the key residues involved in these interactions. IGF binding may be modulated by IGFBP modifications, such as phosphorylation and proteolysis, and by cell or matrix association of the IGFBPs. All six IGFBPs have been shown to inhibit IGF action, but stimulatory effects have also been established for IGFBP-1, -3, and -5. These generally involve a decrease in IGFBP affinity and may require cell association of the IGFBP, but precise mechanisms are unknown. The same three IGFBPs have well established effects that are independent of type I IGF receptor signaling. IGFBP-1 exerts these effects by signaling through alpha(5)beta(1)-integrin, whereas IGFBP-3 and -5 may have specific cell-surface receptors with serine kinase activity. The regulation of cell sensitivity to inhibitory IGFBP signaling may play a role in the growth control of malignant cells.     

The insulin-like growth factor system: towards clinical applications

Insulin-like growth factors (IGF-I and II) are important endocrine, paracrine and autocrine mediators of physiological growth. They promote cellular proliferation, survival and differentiation. Their effects are mediated mainly through the IGF-I receptor, but IGFs also bind to the IGF-II/mannose 6-phosphate and insulin receptors. IGF activity is modulated by a family of six high-affinity IGF binding proteins (IGFBPs); in most situations, IGFBPs inhibit IGF actions but they may also enhance them. Assays are now available for IGF-I, IGF-II and individual IGFBPs. IGF-I and IGFBP-3 assays have some utility in the diagnosis and management of acromegaly and growth hormone deficiency. There is a large body of in vitro and in vivo evidence supporting a pathogenic role for alterations in the IGF system in many diseases, including diabetes, cancer, cardiovascular disease and neuromuscular disease. More recently, epidemiological studies have linked high IGF-I levels with some cancers and low IGF-I levels with ischaemic heart disease. Preliminary studies of recombinant IGF-I as a treatment for diabetes, osteoporosis and neuromuscular disease have been performed in humans. In contrast, there is considerable interest in developing IGF inhibitors for the treatment of cancer. This apparent paradox highlights the need to develop therapeutics beyond the natural ligands and inhibitors, with characteristics such as ligand and tissue specificity. This will only become possible as we increase our understanding of this complex system. Additionally, as IGF and IGFBP assays are becoming more readily available, their role in the diagnosis and monitoring of diseases should be more clearly defined in the near future.     

Normal growth spurt and final height despite low levels of all forms of circulating insulin-like growth factor-I in a patient with acid-labile subunit deficiency

BACKGROUND: In a recently described patient with acid-labile subunit (ALS) deficiency, the inability to form ternary complexes resulted in a marked reduction in circulating total insulin-like growth factor (IGF)-I, whereas skeletal growth was only marginally affected. To further study the role of circulating versus locally produced IGF-I in skeletal growth in this patient, we now describe in detail growth changes and their relationship with several components of the circulating IGF system. DESIGN AND METHODS: We followed growth and development up to the final height in a patient with complete ALS deficiency and determined both spontaneous and growth hormone (GH)-stimulated changes in the IGF system, including measurements of total, free and bioactive IGF-I, total IGF-II and insulin-like growth factor binding protein (IGFBP)-1, IGFBP-2 and IGFBP-3. RESULTS: The patient had a delayed growth and pubertal onset. Six months of GH treatment had no effect on growth. At the age of 19.3 years, he spontaneously completed puberty and had a normal growth spurt for a late adolescent (peak height velocity of 8.4 cm/year). A normal final height was attained at 21.3 years (167.5 cm; -0.78 SDS). During as well as after puberty, basal levels of total, free and bioactive IGF-I were low, as were total IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3. GH treatment for 6 months normalized free IGF-I and increased bioactive IGF-I, but had no effect on growth velocity. CONCLUSIONS: This case story shows that in the presence of complete ALS deficiency, a height within normal limits can be obtained despite low levels of all forms of circulating IGF-I. Furthermore, the patient presented a delayed but normal growth spurt without any marked increment of circulating IGF-I.     

Characterization of insulin-like growth factor binding proteins (IGFBPs) secreted by bovine adrenocortical cells in primary culture: regulation by insulin-like growth factors (IGFs) and adrenocorticotropin (ACTH).

In previous studies, we have shown that insulin-like growth factor II (IGF-II) stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I [1]. The steroidogenic effect of both IGFs is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we therefore characterized the IGFBPs secreted by bovine adrenocortical cells in primary culture, and investigated the effect of corticotropin (ACTH) and recombinant human IGF-I and IGF-II on the regulation of IGFBP synthesis. By Western ligand blotting, four different molecular forms of IGF-binding proteins were identified in conditioned medium of bovine adrenocortical cells with apparent molecular weights of 39-44 kDa, 34 kDA, 29-31 kDa, and 24 kDa. In accordance to their electrophoretic mobility, glycosylation status and binding affinity, these bands were identified by immunoprecipitation and immunoblotting as IGFBP-3, IGFBP-2, IGFBP-1, and deglycosilated IGFBP-4, respectively. Quantification of the specific bands by gamma counting revealed that, in unstimulated cells, IGFBP-3 accounts for approximately half of the detected IGFBP activity, followed by IGFBP-1, IGFBP-2 and IGFBP-4. ACTH treatment predominantly increased the abundance of IGFBP-1 and to a lesser extent IGFBP-3 in a time and dose-dependent fashion. In contrast, IGF-I or IGF-II (6.5 nM) preferentially induced the accumulation of IGFBP-3 (1.9-fold) and to a lesser extent of IGFBP-4, but did not show any effect on IGFBP-1. When ACTH and IGFs were combined, an additive stimulatory effect on the accumulation of IGFBP-3 and IGFBP-4 was observed. In contrast to their different steroidogenic potency, no significant difference in the stimulatory effect of IGF-I and IGF-II on IGFBP secretion was found. In conclusion, bovine adrenocortical cells synthesize IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4, and their secretion is regulated differentially by ACTH and IGFs. These results, together with earlier findings, suggest that IGF-binding proteins play a modulatory role in the regulation of differentiated adrenocortical functions. Therefore, bovine adult adrenocortical cells provide a useful tissue culture model in which the complex interactions between two IGF-ligands, at least four IGF binding proteins and two IGF-receptors can be evaluated.     

The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells

The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34(cdc2) levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1-3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9.     

Tetrodotoxin attenuates isoproterenol-induced hypertrophy in H9c2 rat cardiac myocytes

Thyroid stimulating hormone (TSH) is shown to have definite anabolic effects on skeletal metabolism. Previous studies have demonstrated that Insulin-like growth factors (IGF-I and IGF-II) and their six high affinity binding proteins (IGFBPs 1-6) regulate proliferation and differentiation of bone-forming osteoblasts. The current study was intended to determine whether the anabolic effects of TSH on human osteoblastic (SaOS2) cells are mediated through insulin-like growth factor system components. TSH given at 0.01 ng to 10 ng/ml dose levels for 24 and 48 h significantly increased human osteoblastic (SaOS2) cell proliferation and alkaline phosphatase activity, the differentiation marker. TSH significantly increased IGFs (IGF-I and IGF-II) mRNA expression after 6 and 24 h and their protein levels after 24 and 48 h of treatment, respectively. Unlike the IGFs, the IGFBPs responded differently to TSH treatment. Though there were some inconsistencies in the regulation of stimulatory IGF binding protein-3 and -5 by TSH treatment, there was an overall increase at the mRNA abundance and protein levels. Again, the inconsistency persisted at the regulation of the inhibitory IGFBPs 2, 4, and 6 especially at the level of mRNA expression due to TSH treatment, there is an overall decrease in the levels of IGFBP-2, 4, and 6 in the conditioned media (CM) of SaOS2 cell cultures. The IGFBP proteases which control the availability of IGFs are also regulated by hormones. Pregnancy-Associated Plasma Protein-A (PAPP-A) is responsible for the proteolysis of IGFBP-4. TSH treatment significantly unregulated the expression of PAPP-A both at mRNA and protein levels. In conclusion, TSH promotes human osteoblastic (SaOS2) cell proliferation and differentiation by upregulating IGFs and their stimulatory IGF binding proteins and down regulating the inhibitory IGF binding proteins.     

Expression of mRNAs encoding insulin-like growth factor (IGF) ligands, IGF receptors and IGF binding proteins during follicular growth and atresia in the ovine ovary throughout the oestrous cycle

The components of the insulin-like growth factor (IGF) system appear to be involved in the regulation of ovarian follicular growth and atresia in sheep. However, previous studies have only investigated a select few components of the system. The aim of the present study was to investigate the expression of mRNA encoding all of the components of the sheep IGF system among follicles of varying size and health status throughout the oestrous cycle using sheep-specific ribonucleotide probes and in situ hybridisation. For all IGF components, gene expression was unaffected by stage of oestrous cycle. IGF-I mRNA expression in all classes of follicle was generally low throughout the oestrous cycle, while IGFBP-1 mRNA expression could not be demonstrated at all. In contrast, there was relatively intense follicular expression of mRNAs encoding all remaining IGF system components. For IGF-II, both IGF receptors and IGFBP-2, -3, -4, -5, and -6, gene expression decreased as follicles increased in diameter (P < 0.01). IGF-II, type I IGF-R and IGFBP-2, -3, -4, and -6 mRNA expression significantly decreased as follicles progressed from healthy to atretic status (P < 0.01), whereas gene expression for type II IGF-R and IGFBP-5 was greater in atretic follicles (P < 0.01). This study demonstrates the spatial patterns of follicular gene expression for all of the IGF system components in cycling sheep for the first time. These results further highlight the potential functional role of IGF-II, in contrast to IGF-I, in the autocrine and/or paracrine regulation of follicle growth in sheep.     

Role for cyclic adenosine monophosphate in modulating insulin-like growth factor binding protein secretion by muscle cells

The modulation of insulin-like growth factor-binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP-4 and IGFBP-5 by L6 and BC₃H-1 muscle cells was stimulated by treatment with either insulin, IGF-I, or IGF-II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE₁]) increase secretion of IGFBP-4 and IGFBP-5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF-I, or dcAMP. The effects of dcAMP and either insulin or IGF-I were additive, but the effects of insulin and IGF-I were not additive. These results suggest that insulin/IGF-I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP-4 and IGFBP-5 secretion. IGFBP-5 secretion by BC₃H-1 cells was increased by either insulin or IGF-I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP-5 secretion. Additionally, these agents were not synergistic with either insulin or IGF-I. However, indomethacin and progesterone depressed IGFBP-5 secretion by BC₃H-1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP-4 and IGFBP-5 secretion by L6 and BC₃H-1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF-I and cAMP-dependent pathways, whereas IGFBP-5 secretion by BC₃H-1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation. J. Cell. Physiol. 174:293–300, 1998. © 1998 Wiley-Liss, Inc.