Progress in growth factor research最新文献

Insulin-like growth factor (IGF) bioavailability is modulated by specific IGFBPs, six of which are known (IGFBPs 1–6). Since IGFBPs have equal or higher affinity for IGFs than do IGF receptors, it is believed that degradation of IGFBPs by IGFBP-degrading proteinases is an important step in regulating IGF bioactivity. Recent studies from our laboratory have demonstrated that at least two IGFBPs, i.e. IGFBP-3 and -5, are degraded under physiologic conditions by matrix metalloproteinases (MMPs). In vitro, we have demonstrated that IGFBP-3 is degraded in human dermal fibroblast cultures by MMPs using a variety of techniques, including proteinase inhibition with a specific inhibitor of MMPs, i.e. tissue inhibitor of metalloproteinases (TIMP-1), immunoabsorption with specific antisera to human MMPs and a unique method developed in our laboratory, IGFBP-3 substrate zymography. Using similar methods, we have also demonstrated that MMPs, along with an unidentified 97-kDa proteinase, degrade IGFBP-5 in murine osteoblast cultures. In rat pregnancy serum, we have shown that degradation of IGFBP-3 is associated with MMP activity present in the serum, which likely arises from the placental compartment. Analysis of the cleavage products of IGFBP-3 produced by MMPs 1, 2 and 3 reveals that MMPs cleave exclusively within the non-homologous, mid-region of the molecule. Together, these studies suggest that MMPs, beyond their previously described roles as extracellular matrix degrading enzymes, may also exert effects on cellular growth and differentiation via degradation of IGFBPs.

The migratory behaviour of cells is fundamental to diverse biologic processes such as tumor metastasis, development of atherosclerotic plaques, embryonic development and wound healing. We have examined the effects of IGF-I and IGFBPs on the migration of Chinese Hamster ovary (CHO) cells, smooth muscle cells (SMC) and human breast cancer cells (HBC) and have studied the involvement of integrin receptors in migration induced by IGF-I and by IGFBPs.

Using a monolayer wounding assay, we determined the effect of IGFBP-1 on SMC to be qualitatively similar to its effect we reported earlier on CHO cells, in that there is a direct stimulation of migration mediated by the α5β1 integrin. IGFBP-2 has no direct effect on SMC migration, and although it also contains the ArgGlyAsp sequence, we can detect no integrin binding. Unlike CHO cells, SMC are stimulated to migrate by IGF-I. IGFBP-2 and IGFBP-1 both inhibit this IGF-I receptor-mediated stimulation. We have also studied the migration of HBC using a Boyden chamber apparatus and have shown a potent chemotactic effect of IGF-I.

We have investigated the mechanisms for IGF-I stimulation of SMC and HBC migration. IGF-I stimulation of SMC migration requires the presence of either 0.2% serum or vitronectin, because of a requirement for ligand binding by the αVβ3 integrin (vitronectin receptor). MCF-7 HBC migrate toward a concentration gradient of IGF-I, the only growth factor that was able to stimulate these cells to migrate. Integrin ligand binding was also necessary for MCF-7 cells to migrate in response to IGF-I; αVβ5 integrin was required for migration on vitronectin and α2β1 was required on collagen.

These studies demonstrate that the stimulation of cell migration by IGFBP-1 and IGF-I involves signaling by members of the integrin family of receptors. The mechanisms by which the IGF-I receptor and integrin receptors interact are not yet known.

In normal subjects, the major form of circulating IGF is the GH-dependent 150 K complex. As demonstrated by gel-permeation chromatography, the acid-labile subunit (ALS) purified from human serum, incubated for 2 h at 20°C with [¹²⁵I]IGF-I and rIGFBP-3, is able to increase not only the molecular weight (mol. wt.) of the IGF-IGFBP-3 complex, but also the amount of IGF-I bound. In both charcoal and polyethylene glycol ligand binding assays, competitive binding curves for the displacement of [¹²⁵I]IGF-I from rIGFBP-3 by increasing concentrations of unlabeled IGF-I showed an increased binding activity of rIGFBP-3 in the presence of ALS. The effect of ALS on rIGFBP-3 binding activity was dose dependent. In addition, ligand and immunoblot revealed that ALS and rIGFBP-3 are able to form a high mol. wt. complex in the absence of IGF peptide. On the basis of these data, ALS seems to have a more complex function than that of simply increasing the mol. wt of the IGF-IGFBP-3 complex.

The regulation of ALS synthesis by rat hepatocytes in primary culture has also been evaluated by immunoblot. In agreement with the in vivo finding of an inhibitory effect of octreotide (a somatostatin analog) on the formation of the 150 K complex in acromegalic subjects, we could observed in vitro that octreotide produces a dose-dependent inhibition of ALS secretion into the hepatocyte conditioned medium. TGF-β1 was also inhibitory at high doses. On the contrary, we could not evidence any effect of IGF-I or IGF-II, while a small increase has been noted after incubation with T3.

IGF-I analogues that bind poorly to IGFBPs are substantially more potent than IGF-I at stimulating growth in rats. However, rodents differ from other mammals because they contain only minimal circulating levels of IGF-II and they are poorly responsive to GH. In this report we review a series of experiments carried out in pigs, a species that is both GH responsive and has high blood concentrations of IGF-II. Intravenous bolus administration of IGFs to 55 kg pigs depressed blood glucose with the potency greatest for analogues such as des(1–3)IGF-I, R³IGF-I and LongR³IGF-I that showed the weakest binding to pig IGFBP-3, a similar efficacy pattern to that reported in the rat. Chronic subcutaneous administration of LongR³IGF-I, however, reduced growth rates, led to a depression in food intake and lowered concentrations of IGF-I, IGF-II and IGFBP-3. IGF-I itself depressed IGF-II concentrations and did not stimulate growth. Subcutaneous infusion of IGFs over a 3-day period, also in 55 kg pigs, demonstrated that analogues that bound least well to IGFBP-3 were the most effective at reducing the concentration of this binding protein, suggesting that the inhibition of growth was related to the depression of IGFBP-3. On the other hand, IGF-I and LongR³IGF-I increased growth rats in neonatal pigs, especially under conditions of reduced food intake. As these anabolic effects occur at a developmental stage where the animals are insensitive to GH in a manner analogous to the situation in rats, it is plausible that the feed-back inhibition of GH secretion explains the catabolic response to IGFs in older pigs.

This paper reviews actions of antiestrogens on IGF physiology, and discusses the potential significance of the recent observations that (i) effects of antiestrogens on the uterus are correlated with their effects on uterine IGF-I and IGFBP-3 gene expression, and that (ii) the potent antiestrogen and growth inhibitor ICI 182780 induces autocrine production of IGFBP-3 by estrogen receptor-positive breast cancer cells, while the growth stimulatory action of estradiol is associated with suppression of IGFBP-3 expression.

To delinate regions of IGFBP-3 involved in ligand and cell-surface binding, DNAs encoding human IGFBP-3[1–264] and several variants were transfected into CHO cells. Of three deletion (Δ) mutants, IGFBP-3[1–88], [1–184], and [Δ89–184], none bound IGF-I tracer by ligand blotting, although all were detectable by immunoblotting. No ALS binding was detectable, as predicted by the lack of IGF binding. Normal-sequence IGFBP-3 associated with the CHO cells and was partly displaceable by IGF-I. Whereas IGFBP-3[1–88] and [1–184] failed to cell-associate, the non-IGF-binding central deletion variant [Δ89–184] did associate with CHO cells but was not displaced by IGF-I. To further examine the role of the carboxy-terminal domain in cell-association, the basic sequence IGFBP-3[228–232] (KGRKR) was altered to the corresponding IGFBP-1 residues MDGEA, a major charge reversal. This variant showed reduced IGF-I binding, and bound ALS with decreased affinity as determined by Scatchard analysis. It showed no cell binding, implicating the basic domain in cell-association. We conclude that, whereas the central and carboxy-terminal domain deletions fail to bind IGF-I, the ability to cell associate requires the carboxy-terminal but not the central domain. Specifically, the basic region [228–232] is essential for cell binding, and also affects IGF-I binding, and independently, ALS affinity.

The insulin-like growth factor binding proteins (IGFBPs) have conserved characteristics of their genomic organization, including similar locations of exon borders relative to nucleotides encoding conserved cysteine residues. Furthermore, the human IGFBP genes, as well as the human homeobox (HOX) genes, are localized to chromosomes 2, 7, 12, and 17. Although little is known about the evolution of the IGFBP genes, the association of human IGFBP and homeobox (HOX) genes at four chromosomal loci may indicate that their ancestral genes were linked prior to the first duplication of chromosomal DNA containing the ancestral HOX cluster. The hypothesis that IGFBPs are ancient proteins is supported by the reported detection of IGFBP activity in serum from the Agnathan species, Geotria australis, a primitive vertebrate.

Further studies of IGFBPs in different species are needed to understand the evolution of this protein/gene family. Chicken provides a good intermediate model, since birds diverged from mammals ∼300 million years ago. A complementary DNA (cDNA) clone encoding chicken insulin-like growth factor binding protein-5 (cIGFBP-5) was isolated. The deduced amino acid sequence is 83% identical to human IGFBP-5 and encodes a mature polypeptide of 251 amino acids. The conservation of IGFBP-5 primary structure across vertebrate species suggests maintenance of important functions during evolution.

Insulin-like growth factor (IGF) bioavailability is modulated by a family of six IGF binding proteins (IGFBPs) that binds IGF with affinities similar to that of the type 1 IGF receptor. Proteolytic degradation of IGFBP-3, the major serum IGFBP, was first reported in pregnancy serum and suggested to be an additional mechanism involved in the regulation of IGF bioavailability. In this paper, the presence of serum IGFBP-3 proteolysis and its potential role in the regulation of IGF bioavailability is discussed partly in view of our recent finding of IGFBP-3 proteolysis by the tissue plasminogen activator (tPA)-plasminogen-plasmin system in human serum.

In this review paper, three pieces of clinical information in childhood are presented: (1) IGFBP-3 may replace GH provocation tests in the diagnosis of GH deficiency (GHD); (2) IGFBP-3 levels are regulated by IGF-I levels in a short period, and (3) ratio of free IGF-I to total IGF-I is high in serum of early infancy, similarly to serum of pregnancy, only partially owing to the presence of IGFBP-3 proteolytic activity. Each paper will be published soon.