Pathophysiological and clinical importance of insulin-like growth factor-I with respect to bone metabolism

The modern concept of causality of diseases emphasizes the study of natural defense functions of the organism and possibilities of influencing them, which will lead to effective prevention of these diseases. A great deal of information has been obtained on the system growth hormone (GH)/insulin-like growth factor (IGF)-I, which is of quite fundamental importance for the integrity of the organism. An imbalance of the system may be the cause of diseases of the neonatal period, as well as diseases associated with aging. In old age, the synthesis of the crucial peptide system, IGF-I, declines as well as the sensitivity of tissues to this hormone. At the same time the changes in the expression of IGF-binding proteins (IGFBP) occur. Systemic growth factors are present in measurable concentrations in the circulation, they are, however, taken up or synthesized by some tissues, where they act as local cellular regulators. IGF-I is produced by many tissues, including bones under the control of estrogens, growth hormone and the parathyroid hormone. A decline of bone IGF-I in the cortical portion of bones is one of the many mechanisms leading to the development of involutional osteoporosis. Correlation studies, which have provided evidence of a relationship between the IGF system and the building of peak bone mass and its subsequent loss contributed to the understanding of the pathogenesis of this disease. It may be foreseen that the results of intervention studies focused on the effects of the recombinant IGF-I will also influence therapeutic and preventive approaches. Modern antiresorption pharmacotherapy stabilizes or enhances bone density and reduces the risk of fractures. The addition of effective anabolics might increase the effectiveness of treatment by shifting the remodeling equilibrium in favor of formative processes. Because both recombinant GH and IGF-I have certain therapeutic limitations, it is considered to utilize substances which either stimulate endogenous IGF-I production directly in the bone or modulate synthesis and distribution of binding proteins for the peptide. Further new findings related to physiology and pathophysiology of this peptide will contribute to designing new strategies in the prevention of osteoporosis and other serious diseases of old age, such as diabetes, neoplasias or cardiovascular diseases.     

Regulation of breast cancer growth by insulin-like growth factors

The IGFs may be important autocrine, paracrine or endocrine growth factors for human breast cancer. IGF-I and II stimulate growth of cultured human breast cancer cells. IGF-I is slightly more potent, paralleling its higher affinity for the IGF-I receptor. Antibody blockade of the IGF-I receptor inhibits growth stimulation induced by both IGFs, suggesting that this receptor mediates the growth effects of both peptides. However, IGF-I receptor blockade does not inhibit estrogen (E₂)-induced growth suggesting that secreted IGFs are not the major mediators of E₂ action. Several breast cancer cell lines express IGF-II mRNA by both Northern analysis and RNase protection assay. IGF-II activity is found in conditioned medium by radioimmuno and radioreceptor assay, after removal of somatomedin binding proteins (BP) which are secreted in abundance. IGF-I is undetectable. BPs of 25 and 40 K predominate in ER-negative cell lines while BPs of 36 K predominate in ER-positive cells. Blockade of the IGF-I receptor inhibits anchorage-independent and monolayer growth in serum of a panel of breast cancer cell lines. Growth of one line (MDA-231) was also inhibited in vivo by receptor antibody treatment of nude mice. The antibody had no effect on growth of MCF-7 tumors. These data suggest the IGFs are important regulators of breast cancer cell proliferation and that antagonism of this pathway may offer a new treatment strategy.     

Biology of insulin-like growth factors in development

Insulin-like growth factors (IGFs) provide essential signals for the control of embryonic and postnatal development in vertebrate species. In mammals, IGFs act through and are regulated by a system of receptors, binding proteins, and related proteases. In each of the many tissues dependent on this family of growth factors, this system generates a complex interaction specific to the tissue concerned. Studies carried out over the last decade, mostly with transgenic and gene knockout mouse models, have demonstrated considerable variety in the cell type-specific and developmental stage-specific functions of IGF signals. Brain, muscle, bone, cartilage, pancreas, ovary, skin, and fat tissue have been identified as major in vivo targets for IGFs. Concentrating on several of these organ systems, we review here phenotypic analyses of mice with genetically modified IGF systems. Much progress has also been made in understanding the specific intracellular signaling cascades initiated by the binding of circulating IGFs to their cognate receptor. We also summarize the most relevant aspects of this research. Considerable efforts are currently focused on deciphering the functional specificities of intracellular pathways, particularly the molecular mechanisms by which cells distinguish growth-stimulating insulin-like signals from metabolic insulin signals. Finally, there is a growing body of evidence implicating IGF signaling in lifespan control, and it has recently been shown that this function has been conserved throughout evolution. Very rapid progress in this domain seems to indicate that longevity may be subject to IGF-dependent neuroendocrine regulation and that certain periods of the life cycle may be particularly important in the determination of individual lifespan.     

The insulin-like growth factors and their binding proteins

1. This review provides a brief overview of the structure of the insulin-like growth factors (IGFs or somatomedins), their mRNA and genes; the regulation and sites of production of these peptides; their binding and actions in target tissues; and the structure and biological role of their binding proteins. 2. Molecular cloning techniques have allowed the prediction of precursor forms of IGF-I and IGF-II, have provided tools to study the regulation of the synthesis and translation of IGF mRNAs, and have recently yielded the primary sequence of the IGF-I receptor, supplementing other rapidly-accumulating structural data. 3. Several of the IGF binding proteins have also been purified, and initial structural studies performed. 4. The increased knowledge of the structures of the IGFs, their receptors and binding proteins should now permit rapid progress in understanding the physiology and functions of these proteins.     

Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3.

In the circulation, most of the insulin-like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF-binding protein (IGFBP)-3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF-binding fraction separated from human plasma. IGF ligand blotting, cross-linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP-3 showed a 5-fold higher affinity for IGF-II than IGFBP-3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP-3 functions.     

Mosaic evolution of the insulin-like growth factors. Organization, sequence, and expression of the rat insulin-like growth factor I gene

Insulin-like growth factor I (IGF-I) plays a major role in mammalian growth and regenerative processes as a mediator of many of the biological effects of growth hormone. We have demonstrated recently that the human IGF-I gene is transcribed and processed into distinct messenger RNA molecules, each of which directs the synthesis of unique IGF-I-containing peptides. As a means to determine whether a similar model of IGF-I gene organization and expression is the paradigm in mammals and as an initial step in devising experimental approaches to the study of regulation of IGF-I biogenesis, we have isolated and characterized the rat IGF-I gene. The rat gene, like its human counterpart, is very large, extending over at least 73 kilobases, and is composed of five exons subdivided by four introns. As in the human example, the rat IGF-I gene hybridizes to several messenger RNAs: 0.8-1.2, 1.6-2.1, and 7.8 kilobases. There is extensive nucleotide and amino acid sequence conservation between the two genes. The predicted mature rat IGF-I protein is identical to the human peptide in 67 of 70 residues. A comparably high degree of amino acid sequence identity is also found for both the amino- and carboxyl-terminal extension peptides, suggesting that, like mature IGF-I, the extension molecules may have physiological function.     

Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells

Estrogen sensitizes the MCF-7 estrogen-responsive breast cancer cell line to the mitogenic effect of insulin and the insulin-like growth factors (IGFs). This sensitization is specific for estrogen and occurs at physiological concentrations of estradiol. Dose-response experiments with insulin, IGF-I, and IGF-II suggested that the sensitization is mediated through the type I IGF receptor. Binding experiments with 125I-IGF-I and hybridization of a type I IGF receptor probe to RNA showed that the levels of the type I IGF receptor and its mRNA are increased 7- and 6.5-fold, respectively, by estradiol. IGF-I and estradiol had similar synergistic effects on other estrogen-responsive breast cancer cell lines, but IGF-I alone increased the proliferation of the MDA MB-231 cell line which is not responsive to estrogens. These experiments suggest that an important mechanism by which estrogens stimulate the proliferation of hormone-dependent breast cancer cells involves sensitization to the proliferative effects of IGFs and that this may involve regulation of the type I IGF receptor.     

Organization of the human genes for insulin-like growth factors I and II

Recently, we have reported the isolation of cDNAs encoding the precursors of insulin-like growth factors I and II (IGF-I and II) [(1983) Nature 306, 609-611; (1985) FEBS Lett. 179, 243-246. These cDNAs were employed as specific probes to detect and isolate the corresponding genes from human cosmid DNA libraries. Three cosmids were detected, together containing the entire cDNA sequence of IGF-I, and one cosmid containing the sequence of IGF-II cDNA. Southern blot hybridization, physical mapping and nucleotide sequence analysis of these cosmids revealed that the IGF-I and -II genes have a discontinous structure. The IGF-I gene contains at least four exons spanning a region of probably more that 45 kilobasepairs (kb), while the IGF-II gene consists of at least five exons, spanning a region of 16 kb.     

Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors.

BALB/c3T3 cells are exquisitely growth regulated and require both platelet-derived growth factor and insulin-like growth factor-1 (IGF-1) for optimal proliferation. BALB/c3T3 cells that constitutively express IGF-1 and elevated levels of IGF-1 receptor (IGF-1R) are capable of growth in serum-free medium without the addition of any exogenous growth factors. BALB/c3T3 cells overexpressing only the IGF-1R plasmid required IGF-1 or insulin for serum-free growth. Antisense oligodeoxynucleotides complementary to IGF-1R mRNA inhibited IGF-1-mediated cell growth. Under these conditions, neither the epidermal growth factor receptor nor phospholipase C gamma 1 was autophosphorylated. These findings indicate that constitutive expression of IGF-1 and IGF-1R allows 3T3 cells to grow in serum-free medium without addition of those exogenous growth factors that are required by the parent cell line.     

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 factors and their binding proteins in growth control and carcinogenesis

Interest in the role of the insulin-like growth factor (IGF) axis in growth control and carcinogenesis has recently been increased by the finding of elevated serum insulin-like growth factor I (IGF-I) levels in association with three of the most prevalent cancers in the United States: prostate cancer, colorectal cancer, and lung cancer. IGFs serve as endocrine, autocrine, and paracrine stimulators of mitogenesis, survival, and cellular transformation. These actions are mediated through the type 1 IGF-receptor (IGF-1R), a tyrosine kinase that resembles the insulin receptor. The availability of free IGF for interaction with the IGF-1R is modulated by the insulin-like growth factor-binding proteins (IGFBPs). IGFBPs, especially IGFBP-3, also have IGF-independent effects on cell growth. IGF-independent growth inhibition by IGFBP-3 is believed to occur through IGFBP-3-specific cell surface association proteins or receptors and involves nuclear translocation. IGFBP-3-mediated apoptosis is controlled by numerous cell cycle regulators in both normal and disease processes. IGFBP activity is also regulated by IGFBP proteases, which affect the relative affinities of IGFBPs, IGFs and IGF-1R. Perturbations in each level of the IGF axis have been implicated in cancer formation and progression in various cell types.     

Nutrition, insulin, insulin-like growth factors and cancer.

The incidence of colon, pancreatic, and kidney cancers, as well as aggressive prostate cancer in men, and breast and endometrial cancer in women is invariably high in Western countries. Nutritional and related factors have been typically implicated. This review presents a model integrating nutrition, insulin and IGF-1 physiology ("bioactive" IGF-1), and carcinogenesis based on the following: (1) insulin and the IGF-1 axis function in an integrated fashion to promote cell growth and survival; (2) chronic exposure to these growth properties enhances carcinogenesis; (3) factors that influence bioactive IGF-1 will affect cancer risk. The model presented here summarizes the data that chronic exposure to high levels of insulin and IGF-1 may mediate many of the risk factors for some cancers that are high in Western populations. This hypothesis may help explain some of the epidemiologic patterns observed for these cancers, both from a cross-national perspective and within populations. Of particular importance is that some of relevant factors are modifiable through nutritional and lifestyle interventions. Out of a variety of perspectives presented, nutritional manipulation through the insulin pathway may be more feasible than attempting to influence total IGF-1 concentrations, which are determined largely by growth hormone. Further study is required to test these conclusions.     

A personal view on the early history of the insulin-like growth factors

Salmon and Daughaday, when trying to set up an in vitro assay for Growth Hormone (GH), failed to obtain a direct effect on sulphate uptake in cartilage of hypophysectomized (hypox) rats. They recognised that this was not the consequence of poor methodology or materials, but an encrypted message from the examined system. They decided to turn around and to try and decipher it. Treatment with GH appeared to render hypox rat serum active in stimulating sulphation in hypox rat cartilage. They proposed that GH induced an intermediary substance, responsible for this biological effect: sulphation factor (SF), later renamed to Somatomedin(s) (SM). This hypothesis met with great criticism and very few took on to study this hypothetical substance. Besides disbelief, slow progress was also due to initial lack of a practical assay and to the failure to find a tissue with enriched concentration from which to extract the activity. From experimental evidence, the concept gradually evolved that SF/SM was insulin-like and might be identical to NSILA (non-suppressible insulin-like activity). This again generated controversy. This characteristic was too far away from the known effects of GH to be readily acceptable as a physiological phenomenon. The subsequent recognition of the distinct characteristics of the receptors for SM/NSILA and insulin, the discovery of the SM/NSILA binding proteins and, much later, a beginning understanding of their interactions, modifications and breakdown, have gradually resolved this apparent contradiction. When the sequence of two NSILA molecules became known, they were named IGF-I and -II. Structural similarity with proinsulin and identity of IGF-I with SM-C and -A were established and it was found that Multiplication Stimulating Activity (MSA), a growth factor isolated from fetal calf serum and subsequently from conditioned media of a rat liver cell line, was the rat equivalent of IGF-II. Structure-function relations could be studied, a quest which is not yet brought to an end. Meanwhile, the endocrine profile of SF/SM had gradually emerged by measuring plasma levels with bioassays. The main determinants were found to be age, body size, GH and the nutritional state. Later, radioimmunoassays were developed, enabling consolidation and detailing of these early observations, and allowing explorations at the tissue level. As another aspect of the endocrine paradigm, in vivo effects of IGFs were studied. The initial demonstration of an effect of crude preparations on longitudinal growth in experimental animals raised heavy scepticism, since the effect might have been an artefact caused by contaminants. It took confirmation with highly purified preparations and biosynthetic IGF-I to ease this concern. Still, not until recent years it was demonstrated, by knocking out the genes, that a true physiological and not a pharmacological effect had been induced previously. When it was found that most tissues produce SMs and are sensitive to their actions, the concept emerged that IGFs may have para- and autocrine functions. Early experiments with combinations of growth factors in cell cultures had begun to define their specific roles in the cell cycle as competence or progression factors. SM-C fell in the latter category. Still, the awareness grew that, for obtaining physiologically meaningful results on the role of IGFs in complex, dynamic and tissue-specific environments, involving interactions of many hormones and growth factors, the intactness of tissue was a prerequisite. One result of this approach was the discovery of a direct interaction of GH with cartilage, leading, in concert with IGFs, to a clonal expansion of the cartilage cells of the growth plate. The isolation and sequencing of the IGF-I and -II genes, and later, of six IFG-BPs initiated the gradual elucidation of structure and function at the DNA and RNA level and the study of natural and synthetic IGF-variants. The generation of transgenic animals became feasible