Progress in growth factor research最新文献

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic growth factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and is thought to play an important role in the mesoderm induction. Although hematopoietic cells derive from the mesoderm, relatively few studies have, until recently, addressed the role of FGF-2 in hematopoiesis. FGF-2 is expressed in cells of the bone marrow including stromal cells, and possibly cells from several hematopoietic cell lineages. It is stored in the bone marrow extra-cellular matrix and released by enzymes such as heparanase, plasmin, or phospholipase C and D. FGF-receptors (FGF-Rs) are expressed in leukemic cell lines and in hematopoietic cells. FGF-2 positively regulates hematopoiesis, by acting on stromal cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. The action of FGF-2 is most likely indirect since its action, on megakaryocytopoiesis for example, is abrogated by anti-IL6 antibodies. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of TGF-β Taken together, these results demonstrate that FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is now recognized as a normal process in the regulation of insulin-like growth factor (IGF) activity, its major effect being to increase IGF bioavailability. In order to characterize the proteolytic fragments of IGFBP-3, we reproduced this proteolysis in vitro using plasmin which provokes cleavages that are similar to those induced in vivo by (unidentified) specific IGFBP-3 proteases.

Two major peaks were purified by RP-HPLC. One contained a 16 kDa fragment and the other comprised two fragments of 22 and 25 kDa. Competitive binding experiments showed that the 16 kDa material had no affinity for IGFs. The 22–25 kDa fragments had considerably reduced affinity, particularly for IGF-I. In a chick embryo fibroblast assay where DNA synthesis was stimulated by IGF-I or insulin, the 22–25 kDa fragments weakly inhibited IGF-I-induced cell proliferation and had no effect on stimulation by insulin. The 16 kDa fragment unexpectedly proved to be a potent inhibitor of both IGF- and insulin-induced cell growth. This proteolytic fragment of IGFBP-3 therefore exhibits intrinsic inhibitory activity.

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen : estradiol (A : E₂) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E₂ production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FF_a) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FF_e), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FF_e, but not in FF_a in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FF_e is higher than in FF_a, whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E₂- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ in A-dominant follicles in TSX ovaries, compared to A-dominant follicles from normally cycling women. Furthermore, TSX follicles do not contain IGFBP-4 protease, similar to A-dominant follicles from normally cycling women and those with PCOS. In summary, A-dominant follicles contain physiologic levels of IGF-I and IGF-II, high levels of inhibitory IGFBPs and low levels of IGFBP-4 protease, thereby favoring low levels of bioavailable IGFs in these follicles. In contrast, maximal IGF bioavailability is maintained in the E-dominant follicle by increased IGF-II production, decreased IGFBP production and increased IGFBP-degradation.

Maternal undernutrition inhibits fetal growth and alters circulating levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs). This study investigates whether the fetal IGF axis could be reprogrammed by maternal undernutrition and hence be a potential contributing factor to changes in fetal and postnatal metabolism. Ewes were either fed ad lib. or undernourished from day −60 to day 30 of gestation, and then both groups were fed ad lib. These groups were further divided at day 105, either being fed ad lib or undernourished until day 115. Fetal blood samples were obtained at day 105 and day 115. IGFBP-1 and IGFBP-3 levels were lower at day 105 in the periconceptually undernourished fetuses. Levels of IGFBP-1 were increased and IGFBP-3, IGFBP-4, IGF-1, glucose and insulin were reduced at day 115 after undernutrition. The degree of change in IGFBP-1, IGFBP-3 and IGF-I between day 105 and day 115 was greater in fetuses receiving low periconceptual nutrition. These results indicate that periconceptual undernutrition is able to reprogramme the fetal IGF axis such that the responses of IGF-I and the IGFBPs to undernutrition in late gestation are markedly altered.

Glucocorticoids stimulate and insulin inhibits hepatic production of IGFBP-1 at the level of gene transcription. We previously identified contiguous insulin and glucocorticoid response sequences in the proximal rat IGFBP-1 promoter. This insulin response sequence (IRS) is palindromic (CAAAACAAATTATTTTG) and each half resembles an IRS in the phosphoenolpyruvate carboxykinase (PEPCK) gene. We have reported that both the IGFBP-1 and PEPCK IRSs bind hepatocyte nuclear factor-3 (HNF-3) proteins [1]. We now report that IRSs from the IGFBP-1 and PEPCK, as well as an IRS which also binds HNF-3 in the rat tyrosine aminotransferase (TAT) gene, also interact with another DNA/protein complex in gel shift studies. Further, methylation interferences studies, gel shift and transient transfection studies with site-specific mutations identified a single base in the first half of the IRS that is critical both for interactions with proteins in this complex, and for maximal effects of insulin and glucocorticoids, on promoter function. Of note, a 250-fold excess of an oligo containing a C/EBP binding site (but not other AT-rich sequences) inhibits the formation of this complex in gel shift assays. Nevertheless, interactions with this C/EBP site are negligible at lower titers (≤ 100-fold excess), and antibodies against known C/EBP proteins do not react with this complex. Similarly, preincubation with CHOP, a truncated member of the C/EBP family which contains a β-leucine zipper domain, does not prevent or alter the mobility of this novel DNA/protein complex, indicating that components of this complex do not form heterodimers with β-ZIP proteins. We conclude that HNF-3 proteins and this novel C/EBP-related DNA/protein complex may play an important role in mediating interactions between glucocorticoids and insulin in the regulation of IGFBP-1 and perhaps multiple hepatic genes.

Using an improved procedure for transient transfection of H4-II-E rat hepatoma cells, we characterized the cis elements in the proximal promoter of the rat insulin-like growth factor binding protein-1 (rat IGFBP-1) gene that are required for basal (unstimulated) and dexamethasone-stimulated promoter activity. Three sites are required for optimal basal promoter activity: an AP-2 site (nt −286 to −293), the M4 region of the insulin response element (nt −108 to −99), and a hepatocyte nuclear factor-1 (HNF-1) site (nt −62 to −50). In addition to the glucocorticoid response element (nt −91 to −77), participation of two of three accessory sites is required for optimal stimulation by dexamethasone: the M4 and HNF-1 sites, and a third site located between nt −252 and −236. Further study will focus on how the interactions of tissue-specific and hormonally-responsive transcription factors are integrated.

Gene targeting allows mutations to be introduced selectively into any mouse locus of interest. This approach has already been used to demonstrate that insulin-like growth factor (IGF) peptides and receptors are required in vivo for normal prenatal growth. One of the IGFBP genes, IGFBP-2, has also been disrupted using gene targeting, and homozygous null BP-2 mice are characterized by a decreased spleen size most apparent during early postnatal stages and increased adult circulating levels of several other IGFBPs. These alterations are considered less dramatic than the phenotypes initially predicted based on the fetal IGFBP-2 expression pattern, although several physiological paradigms can be envisioned that will provide additional tests for specific aspects of IGFBP function. Since all six IGFBP genes are expressed during prenatal rodent development, as well as in adult tissues, the IGFBP-2 null phenotype must also be compared with genetic ablations involving members of other gene families and in the context of the other IGFBP expression patterns in rodent embryonic, extraembryonic, and uterine tissues.

To provide further insight into the function of the IGFBPs, transgenic (Tg) mice which overexpressed IGFBP-1 and IGFBP-3 were generated. In this report we have compared the phenotypic manifestations observed in these Tg mice. The IGFBP-1 Tg mice were significantly smaller at birth, birth weight and gained less weight in the postnatal period. Organ weight was proportionately reduced relative to body weight in most organs. However the brain was markedly smaller in IGFBP-1 Tg mice. Mean plasma levels of Tg-derived IGFBP-1 ranged from 8 to 80 ng ml⁻¹ in the different groups of IGFBP-1 Tg mice. In addition homozygous mice also demonstrated fasting hyperglycemia, impaired glucose tolerance and reduced fecundity. Two of the seven IGFBP-3 founders had measurable levels of hlGFBP-3 in the circulation and were bred to homozygosity. Maximal plasma levels of transgene-derived IGFBP-3 were 72–198 ng ml⁻¹. Transgene expression was detected in the kidney, small intestine and colon by Northern blot analysis. The birth weight, litter size and body weight of IGFBP-3 Tg mice were not significantly different from wild-type mice. However, the spleen, liver and heart of IGFBP-3 Tg mice derived from both founders were significantly heavier compared with organs from wild-type mice. The relative weight of other organs such as the brain, kidney and lungs were similar to wild-type mice. From these data, we conclude that over expression of IGFBP-1 results in inhibition of IGF action and in profound impairment of brain development, modest inhibition of fetal and postnatal growth and inhibition of the metabolic effects of the IGFs. In contrast, modest overexpression of hlGFBP-3 has little effect other than some selective organomegaly.

In the circulation, most of the IGFs are bound to a high molecular weight binding protein complex of 150 kDa that consists of IGF-I (or IGF-II), IGFBP-3 and the acid-labile subunit (ALS). Within rat liver, individual components of the 150 kDa complex are synthesized in different cellular compartments: ALS expression in localized in hepatocytes, but not in non-parenchymal cells. IGFBP-3 mRNA, however, is exclusively expressed in non-parenchymal and among them in endothelial and Kupffer cells.

Co-cultures of hepatocytes and Kupffer cells were used as a model to study the hormonal regulation of biosynthesis of the components of the 150 kDa complex. Although expressed in different liver cell populations IGFBP-3 and ALS were regulated synergistically. Insulin stimulated both the expression of ALS and IGFBP-3 in co-cultures in a dose-dependent manner, while expression of IGFBP-1 was decreased. Regulation of IGFBP-3 synthesis of Kupffer cells required a mediator that is secreted by hepatocytes, since IGFBP-3 expression in cultures of pure Kupffer cells did not respond to the stimulating effect of insulin.