Progress in growth factor research最新文献

The pathomechanism of growth retardation and catabolism in children with chronic renal failure (CRF) is multifactorial. Recent evidence indicates that in particular disturbances of the somatotropic hormone axis play an important pathogenic role. In preterminal CRF serum insulin-like growth factor (IGF)-I and IGF-II levels are normal, while in end-stage renal disease (ESRD), IGF-I levels are slightly decreased and IGF-II levels slightly increased. In view of the prevailing elevated growth hormone levels in ESRD, these serum IGF-I levels appear as inadequately low. Indeed, there is both clinical and experimental evidence for a decreased hepatic IGF-I production rate in CRF. This hepatic insensitivity to the action of GH is partially owing to a reduced GH receptor expression. The action and metabolism of IGFs are modulated by specific high-affinity IGF binding proteins (IGFBPs), which bind ∼99% of circulating IGF. IGFBP-1, IGFBP-2, and low molecular weight IGFBP-3 fragments are increased in CRF serum in relation to the degree of renal dysfunction. Both decreased renal filtration, in particular of low molecular weight IGFBP-3 fragments, and increased hepatic production of IGFBP-1 and -2 contribute to high IGFBP serum levels. Experimental and clinical evidence suggests that these excessive high-affinity IGFBPs in CRF serum inhibit IGF action on target tissues by competition with the type 1 IGF receptor for IGF binding.

Affinity-purified lysosomal protease cathepsin D cleaved recombinant human IGFBP-1 to -5 in fragments of defined sizes, while IGFBP-6 was not degraded. To assess the role of cathepsin D for proteolytic processing of IGFBP in vivo, serum from cathepsin D-deficient mice and conditioned media from cathepsin D-deficient fibroblasts and organ explants were analyzed. No differences for the pattern and level of IGFBPs were detected. When conditioned media from fibroblasts were incubated at acid pH, proteolysis of IGFBP-1 and -4 was observed only in media derived from cathepsin D-expressing cells. Additional experiments showed that the proteolysis of IGFBP-4 is mediated by cathepsin D and not by a protease activated by cathepsin D. The IGFBP-4 degrading activities in media from organ explants from cathepsin D-deficient mice were found to be sensitive to inhibitors of aspartyl and cysteine proteases. The data indicate that different classes of acid pH-dependent proteases can contribute to the regulation of IGFBP-4 abundance.

Growth factor induction is a major component of the response to central nervous system trauma. The insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) are among the molecules induced by injury that have demonstrated neuroprotective actions. Induction of IGFBPs 2, 3, 4 and 5 have been documented following injury and are hypothesized to function in transport or localization of the IGFs to injured cells. It is unclear what factors lead to induction of these molecules following trauma, however, several cytokines including ciliary neurotrophic factor (CNTF) and interleukin-1β (IL-1β) have been described as major injury signals and can induce aspects of reactive gliosis. To establish whether these cytokines also are responsible for inducing the IGFBPs following CNS injury, we injected CNTF or IL-1β intracerebrally into the neocortex of adult rats and measured changes in mRNA expression for the IGFBPs. IGFBP-2 mRNA showed a dramatic increase by 24–48 h following either CNTF or IL-1β injection as compared with the contralateral side injected with heat-inactivated cytokine. Neither CNTF nor IL-1β caused alterations in BP3 or BP5. Levels of BP4 and BP6 mRNAs also were unchanged following CNTF injection. These results suggest that IGFBP2 is uniquely regulated among the IGFBPs in the CNS and is induced by cytokines that signal CNS injury.

Adult rat serum contains two types of 150-kDa IGFBP complexes: ternary complexes containing bound IGF-I, intact IGFBP-3 and the acid-labile subunit (ALS), and binary complexes that contain ALS and proteolytically-nicked IGFBP-3 but which lack bound IGF. We present evidence that the binary complexes containing proteolytically-nicked IGFBP-3 can be formed in two ways: by direct association of IGFBP-3 with ALS in the absence of IGF, and by proteolysis of IGFBP-3 within 150-kDa ternary complexes, resulting in increased dissociation of IGF-I. The relative contributions of the two mechanisms is unknown. Preliminary results indicate that binary complexes also can form in vivo. Proteolysis of IGFBP-3 in the 150-kDa ternary complex provides a regulatable mechanism by which IGF-I may be mobilized from the circulating reservoir of 150-kDa complexes to the tissues.

The involvement of the IGF system in the growth regulation of hormone-dependent (e.g. endometrial and breast) cancer cells was studied. We chose two opposing effects of tamoxifen: the paradoxical stimulation of Ishikawa endometrial cancer cells growth and its inhibitory effects on MCF-7 mammary cancer cells. The results clearly confirm our working hypothesis that the IGF system is involved in growth regulation of these cancer cells irrespective of the direction of the drug effect. The following par-ameters of the IGFs system were studied: IGF-I receptors, IGF-I stimulated protein tyrosine phosphorylation, and membrane-associated and secreted IGF-binding proteins (IGFBPs). In Ishikawa cells, tamoxifen, similar to estradiol, increased IGF-I stimulated tyrosine phosphorylation of cellular substrates in accordance with its effect on cell growth. This effect of tamoxifen was inverted in MCF-7 cells. Tamoxifen did not affect the number or affinity of IGF-I receptors in both Ishikawa and MCF-7 cells, however, it caused a three-fold decrease in membrane-associated IGFBPs in the endometrial cells but an increase in these proteins in breast cancer cells. Similar but much less pronounced changes in soluble IGFBPs were observed. Our results indicate that the opposing growth effects of tamoxifen on endometrial and mammary cancer cells are associated with modulation of the IGF system components, mainly with reciprocal changes in membrane-associated IGFBPs.

Three hemizygous transgenic (Tg) mouse lines were generated with a fusion gene composed of the mouse metallothionein promoter (mMT-1) and a full length human insulin-like growth factor binding protein-1 (hIGFBP-1) cDNA that was truncated in its 3′ untranslated (3′UT) region. The transgene was ectopically expressed in the brain of each line and resulted in postnatal brain-growth retardation that was manifested by 2 weeks of age. Despite the expression of the transgene in multiple other tissues and high serum hIGFBP-1 concentrations in two of the three lines, studies designed to detect alterations in somatic growth, in reproduction and in glucose metabolism revealed few other abnormalities. Unexpectedly, however, we found that the regulation of the transgene shared characteristics with that of the native gene, despite the fact that it lacked the endogenous gene's 5′ regulatory region, as well as most of its 3′ UT region. Our studies suggest that factors controlling mRNA stability are important to regulation of both the native and transgene, and that an AU-rich element 17 base pairs (bp) from the end of coding sequence is responsible for the instability of the transgene and in part for instability of the endogenous gene.

The acid-labile subunit (ALS) of the ternary insulin-like growth factor binding protein (IGFBP) complex has a central role in regulating the bioavailability of circulating IGF. We have shown that gene expression of ALS in vivo and in vitro is regulated by a variety of factors, including growth hormone (GH). Our aim was to isolate and characterise the ALS gene as a step in defining the mechanism of its regulation. Southern analysis of rat genomic DNA suggests that the ALS gene exists as a single copy in the rat genome. In order to isolate this gene we screened 5×10⁵ clones and selected fragments of two genomic clones were sequenced. Comparison of this sequence with the cDNA identified two exons and a single ∼1.1 kb intron. Primer extension experiments suggest two major transcription initiation sites at −539 and −396 nts relative to the translational initiation codon, although there are no consensus TATA-boxes in this region. Analysis of 2.3 kb of 5′ flanking sequence identified two LF-A1 sites which may confer the liver-specific expression of the ALS gene. In addition, there are several elements that may be involved in regulation by growth hormone and cytokines.

It is generally accepted but not established that the insulin-like growth factor (IGF) binding site on the IGF binding proteins (IGFBPs) is in the N-terminal region. However, other workers have reported C-terminal fragments with IGF binding determinants. Therefore, we tested the hypothesis that both the N- and C-terminal regions of IGFBPs are involved in binding. Using a protein A gene fusion system, a cDNA encoding residues 1–147 (N147) was cloned into the plasmid pRIT2T and expressed in E. coli as a fusion protein. Since an Asn N-terminal to the gly of IGFBP-3 had been engineered into the cDNA construct, protein A was cleaved from N147 by hydroxylamine. Purified N147 was refolded in a DTT/cystamine redox system at pH 8.4 under nitrogen atmosphere. Both ligand binding Westerns and solution binding assays demonstrated that the recombinant derived N147 bound IGFs. The 147 and 176 residue N-terminal fragments, including a C-terminal fragment (residues 151–263) of IGFBP-3 were also expressed in pichia (yeast) as glycosylated proteins. Solution binding assays showed that they all bound labelled IGF-1. In conclusion, IGFBP-3 contains at least two binding determinants, one on the N- and one on the C-terminal domain. There may also be a possible contribution from the intermediate (I) domain. Our molecular genetic approach to mapping the binding region for IGFs on IGFBP-3 can now be tested on the other mutants we have prepared. Subsequently, site directed mutagenesis can be used to pinpoint key functional residues.

Insulin-like growth factor binding protein (IGFBP) proteases — their identification, regulation, and biological significance — are currently an area of ardent investigation. This has developed from the very recent realization that IGFBP availability and bioactivity is determined not only by gene expression, but also by the controlled proteolytic processing of the protein in the pertcellular environment. In each case identified so far, the modified IGFBP acts dramatically different from native or recombinant IGFBP in solution. This post-translational modification of IGFBP structure/function could have widespread significance since IGFBPs modulate the diverse growth-promoting activities of the IGFs. In fact, it may be argued that local IGF action is largely controlled by this mechanism. Therefore, knowledge of the form, function, and control of the various IGFBP proteases is likely to have major implications for our understanding of the physiology and pathophysiology of the IGFs.