Progress in growth factor research最新文献

Identification of sites of expression of IGF, IGF receptors and IGFBPs in the olfactory bulb of the rat brain suggested the presence of a paracrine IGF system. Since cell association of IGFBPs has been suggested as an important factor in their modulation of IGF action, we investigated whether IGFBPs are cell associated in olfactory bulb (OB). This was supported by des(1–3)IGF-I only partially competing for [¹²⁵I]IGF-I binding to rat OB membrane, suggesting the presence of a cell associated IGFBP. Affinity cross-linking of [¹²⁵I]IGF-I to rat OB membrane demonstrated a 39-kDa complex which was reduced by IGF-I and IGF-II, but not by des(1–3)IGF-I or insulin. Western ligand blotting of solubilised membrane showed a 38-kDa IGFBP which was immunoprecipitated by anti-IGFBP-2 antiserum but not by anti-IGFBP-5 antiserum. We conclude that in the rat IGFBP-2 is associated with membranes from OB. Whether the cell membrane association is due to integrin binding via its RGD sequence or glycosaminoglycan binding is currently under investigation. Cell associated IGFBP-2 may modulate IGF action in the neonatal rat OB.

The insulin-like growth factor binding proteins have been shown to modify IGF actions. IGFBP-5 binds to extracellular matrix (ECM) and its ability to potentiate IGF activity is dependent upon the amount that is ECM associated. To determine the specific regions of IGFBP-5 that are required for ECM association, site directed mutagenesis has been used to prepare several forms of IGFBP-5. Mutants that have had the amino acids between positions 201 and 218 altered have been useful. Mutation of the lysine 211 resulted in no change in the affinity of IGFBP-5 for ECM or heparin Sepharose; however, it resulted in a major reduction in affinity for IGF-I following heparin binding. Other mutations which disrupted heparin binding also resulted in loss of this affinity shift. Most disruptive were mutations of amino acids 211, 214, 217 and 218 and 202, 206 and 207. Mutation of residues 201 plus 292 had some effect, but substitution for 207, 211, 217 and 218 had no effect. When binding to intact ECM was analyzed, similar results were obtained. This suggests that amino acids 202, 206 and 214 are definitely involved in heparin and ECM binding. When binding to proteoglycans such as tenascin and heparin sulfate proteoglycan was analyzed, similar results were obtained. IGFBP-5 also binds to other proteins in ECM, including type IV collagen and plasminogen activator inhibitor-I. Specific antisera for plasminogen activator inhibitor-1 can coprecipitate IGFBP-5. IGFBPs are degraded by specific proteases. Three proteases that degrade IGFBP-2, -4 and -5 have been characterized. They are serine proteases that cleave these proteins at basic residues. Although several well characterized serine proteases cleave IGFBP-4 or -5, the proteases in cell conditioned media appear to be distinct.

Osteoporosis develops because of an age-dependent imbalance between the rates of bone formation and bone resorption (i.e. bone formation rate is inadequate compared with bone resorption rate to maintain bone volume). With regard to the mechanism for the deficiency in bone formation, we propose that age-associated changes in the IGF system components contribute to an age-related decrease in the skeletal capacity for osteoblast cell proliferation. As a means of testing this hypothesis, we have measured serum levels of IGFBP-4 and IGFBP-5 since our studies have shown that the mitogenic actions of IGFs in bone cells are modulated by inhibitory IGFBP-4 and stimulatory IGFBP-5. By using newly developed and validated radioimmunoassays for measurement of IGFBP-4 and IGFBP-5, we found that the circulating level of IGFBP-4 increases with age while that of IGFBP-5 declines with age. In subjects from 23–87 years, serum IGFBP-4 concentrations showed a significant positive correlation with serum PTH while serum IGFBP-5 concentrations showed a significant positive correlation with IGF-I. These age-related changes in the serum levels of IGF system components are consistent with our previous findings of age-related decreases in the femoral cortical contents of IGF-I, IGF-II and IGFBP-5. Although the biological implications of the sequestration of IGFs in bone unknown, we have hypothesized that the level of the IGFs in bone is a reflection of their integrated local secretion by osteoblasts. Based on our data, we now propose a model in which (a) underproduction of the stimulatory components and overproduction of an inhibitory component of the IGF system occur as a consequence of aging, and (b) these changes lead to an age-related decrease in the local (autocrine/paracrine) as well as the hormonal (endocrine) actions of the IGFs, which in aggregate could contribute to the decrease in osteoblast proliferation and the deficiency in bone formation. In conclusion, although our findings provide indirect evidence that age associated changes in IGF system components could lead to a deficit in bone formation, further studies are needed to demonstrate a cause and effect relationship between changes in bone cell production of IGF system components and the age-related uncoupling of bone formation from resorption.

Endogenous IGFBP-3 has been examined in the circulation and in four different extravascular fluids in normal healthy adults and in patients with psoriasis or arthritis. In all of these cases there was no apparent increase of IGFBP-3 protease activity in the circulation. In contrast, endogenous IGFBP-3 from normal skin interstititial fluid and synovial fluid from healthy adults was found to be predominantly in the 29 kDa proteolytically modified form. This indicated that in these extravascular fluids in normal healthy adults a protease was active which was similar, if not identical, to that found in the circulation in pregnancy and other conditions. This was confirmed by the fragmentation of recombinant IGFBP-3 when incubated with these fluids. When the skin interstitial fluid or synovial fluid were taken from abnormal tissues (psoriasis in the former and osteoarthritis or rheumatoid arthritis in the latter) there was a considerable reduction in the amount of endogenous IGFBP-3 in the ‘clipped’ form and a reduction in the protease activity. In psoriatic lesions, this reduction in IGFBP-3 protease activity was shown to be due to the presence of an inhibitor in the interstitial fluid but not in the circulation. In both peritoneal and follicular fluid, the ratio of intact to fragmented IGFBP-3 appeared to relate to the oestrogen status. In peritoneal fluid there was a decrease in intact IGFBP-3 during the late proliferative/early secretory phase of the endometrial cycle. In the ovary there was an increase in the amount of fragmented IGFBP-3 in the follicular fluid from the dominant follicle in comparison with atretic follicles from the same ovary. There is normally little proteo-lysis of IGFBP-3 in the circulation but this increases in many conditions where there is increased metabolic activity. The same enzyme(s) appear to be active in many extravascular fluids but under very different regulation. The activity in these extravascular fluids is normally high but can be decreased with local tissue inflammation; this decrease appears to be mediated by the induction of a local inhibitor.

We have examined the polarity of the IGF system in differentiated HT29-D4 colonic epithelial cells cultured on permeable supports. Type I IGF receptors (∼30,000 per cell; Kd∼1 nM) are highly polarized (>97%) in the basolateral membrane, and this figure does not change whatever the stage of post-confluent differentiation. In early differentiated cells, i.e., up to day 7 post-confluence, IGF-II, IGFBP-2, IGFBP-4 (>96%) and IGFBP-6 (∼85%) are recovered in the basolateral medium. In contrast, in well differentiated cells, e.g. at day 23, a differential distribution of the IGFBPs secretory pathways is observed: IGFBP-2 and IGFBP-4 continue to be predominantly secreted from the basolateral surface whereas IGFBP-6 is almost all (>96%) targeted towards the apical surface. As a result, IGF-II is secreted in equal quantities in both apical and basolateral compartments. Since the constitutive secretory pathway in intestine epithelial cells is known to be basolateral only, it is suggested that the IGFBP-6 apical release results from an active sorting. In addition, IGFBP-6 secretory level is down-regulated (∼60% decrease) whereas those of IGFBP-2 and IGFBP-4 remain constant during the differentiation process. Although speculative, we suggest that this IGFBPs differential secretory sorting could regulate the IGFBPs basolateral secretory profile, that in turn could ensure a fine tuning of IGF-II autocrine bioavailability towards the IGF-responsive basolateral membrane of the colonic epithelial cells.

The role of the insulin-like growth factor system on mammary gland development and involution following pregnancy and lactation was studied. Transgenic mice expressing rat IGF-I and human IGFBP-3 specifically in the mammary gland tissue, were created using the whey acidic protein gene. Mammary gland development was normal in transgenic animals expressing either rIGF-I or hIGFBP-3. In contrast, involution of the gland was delayed in both groups of transgenic mice. Specifically, the number of apoptotic cells was less in the involuting glands of transgenic mice compared with control animals. These results confirm the hypothesis that the IGF system regulates mammary gland function.

IGFBP-1 secretion in humans is regulated by insulin and the counter-regulatory hormones with a high production rate and rapid turnover. The IGFBP-1 fasting levels are both genetically and environmentally determined. Serum IGFBP-1 levels may be regulated by so far unknown factors during certain conditions. The fasting IGFBP-1 level can be used as a marker of diurnal IGFBP-1 and insulin secretion.

From a small beginning 21 years ago, the investigation of the insulin-like growth factor binding proteins (IGFBPs) has grown to occupy a major part of the research on the insulin-like growth factors (IGFs). A single abstract at the Endocrine Society in 1974 has grown into a torrent of more than 1400 papers published in the past two decades, and more than 200 in the past year alone. Instead of one possible binding protein for somatomedin we now have seven members of a homologous protein family that was completely uncharacterized only a decade ago. The Third International Symposium on IGF Binding Proteins clearly marked a milestone in the development of this field of research. Concentrated on the three days of this meeting was a rich collection of 50 oral presentations and 89 posters. The quality and breadth of the presentations proved that not only is research on the IGFBPs vigorous and healthy, but it is clearly dealing with important scientific issues. The publication of this collection of papers from the Third International Symposium on IGF Binding Proteins serves as a roadmap for where this field of research is now, and the likely direction for developments as we approach the third millennium.

An insulin response element (IRE) has been identified ∼100 base pairs (bp) 5′ to the transcription start site of the human insulin-like growth factor binding protein-1 (hIGFBP-1) gene. This cis element appears crucial to the multihormonal regulation of hIGFBP-1 expression in liver, since (i) an intact IRE is required for maximal stimulation of hIGFBP-1 promoter activity by dexamethasone, and (ii) the IRE confers activity. Further progress in understanding how the IRE confers insulin and glucocorticoid effects requires identification of transcription factors confering effects of these hormones. D-site binding protein (DBP), and members of the hepatic nuclear factor 3 (HNF 3) and high mobility group I/Y (HMG I/Y) protein families, each known to bind DNA elements similar in sequence to the IRE, were tested for IRE binding. DBP, HMGI and HNF 3β each protected the hIGFBP-1 IRE from DN AseI digestion. Additional studies are required to establish whether binding of any of these proteins to the IRE is important to the regulation of hIGFBP-1 expression by insulin and/or glucocorticoids.