Progress in growth factor research最新文献

The IGFBP proteases were first described in pregnancy serum as a proteolytic activity against IGFBP-3. Since then, IGFBP proteases have been described in many other clinical situations, in various body fluids, and have been shown to cleave IGFBP-2 to -6 with varying specificity. The molecular nature of some of these proteases is being unraveled and three classes of IGFBP proteases have been recognized. These include kallikreins, cathepsins and matrix metalloproteinases (MMPs). We utilized two cellular systems to demonstrate the significance of IGFBP proteases in cellular growth regulation. In primary cultures of prostatic cells, we have shown that prostate-specific antigen (PSA) has the ability to enhance IGF mitogenic action by reducing the effects of IGFBPs. Similar kallikreins such as gamma nerve growth factor (NGF) share this activity. Within the prostatic milieu, we have also demonstrated epithelial production of the acid-activated IGFBP protease, cathepsin D, and its secretion into seminal plasma, as well as the serum of patients with prostate malignancy. We have also identified MMPs in prostatic cells and fluids. Using cultured airway smooth muscle (ASM) cells, we have demonstrated the synergism between IGFs and inflammatory agents in mediating ASM cell proliferation. Examination of this phenomenon revealed that these agents (e.g. leukotriene D₄ and interleukin1-β) induce the secretion of an IGFBP protease which cleaves the IGFBPs secreted by ASM cells, allowing IGFs to stimulate proliferation. Using several methods, including immunoblotting and immunodepletion techniques, we have identified this protease as MMP-1. These two pathophysiological systems demonstrate the importance of IGFBP proteases as autocrine paracrine growth regulators. Furthermore, IGFBP proteases may be critical elements in malignant and benign proliferative diseases, including prostate cancer and the ASM hyperplasia of long-standing asthma.

In mammals IGF-I is part of a 150-kDa binding protein complex, which also contains a glycosylated acid-labile protein (ALS) and a glycosylated acid-stable IGF binding subunit IGFBP-3. Administration of free IGF-I in vivo induces not only acute insulin-like effects but also growth stimulation. Since co-injection with IGFBP-3 only partially blocked the hypoglycemic response of free IGF-I in hypophysectomized rats, we were interested in the growth stimulating activity of the IGFI-IGFBP-3 complex in pituitary-deficient mice compared to that obtained by IGF-I alone. Therefore, the effects of subcutaneously administered IGF-I, IGFBP-3 and the IGF-I-IGFBP-3 complex on somatic growth and organ growth of pituitary-deficient Snell dwarf mice were studied after 4 weeks of treatment.

Treatment with IGF-I alone induced a significant increase in body length and weight, as well as in weights of the submandibular salivary glands, kidneys and quadriceps femooris muscles as compared to buffer treated controls. No significant changes were found in liver, brain, heart and thymus. IGFBP-3 alond had no effect. However, the stimulating effects of IGF-I alone on body length and weight, as well as on the weight of the kidneys, were fully neutralized by co-injection with IGFBP-3. In contrast, the weights of submandibular salivary glands and m. quadriceps femoris were increased by treatment with the complex compared to controls and not significantly different from animals treated with IGF-I alone.

Our data show that in GH-deficient mice administration of IGFBP-3 differentially inhibits the IGF-I induced body and organ growth. This calls for extra vigilance when exploring presumed adbantages of administering an IGF-I-IGFBP-3 complex to GH-deficient individuals in order to obtain stimulation of growth.

There are three potential N-glycosylation sites (Asn-X-Ser/Thr) located in the non-conserved central region of the human IGFBP-3 sequence (Asn⁸⁹, Asn¹⁰⁹, Asn¹⁷²—sites 1, 2 and 3, respectively). Upon ligand blotting with IGFs, IGFBP-3 appears as two bands (40–45 kDa) representing different glycosylated forms. We have mutated the N-glycosylation sites in permutations of three single, three double and one triple mutations and expressed these variant cDNAs. Each mutant protein was detected by radioimmunoassay, indicating that glycosylation is not required for the secretion of the protein from CHO cells. Ligand blotting using [¹²⁵I]IGF-I indicated that all seven mutants retained IGF-I binding. Based on the molecular weights of the variant proteins, there are approximately 4, 5 and 6 kDa of carbohydrate on sites 1, 2 and 3, respectively. Furthermore, the two forms of IGFBP-3 represent the protein glycosylated either at all three sites or at Asn⁸⁹ and Asn¹⁰⁹ only. There appears to be no difference between the mutants and the fully-glycosylated rhIGFBP-3 in their acid-labile subunit (ALS) binding. Analysis of variance confirmed that the association constant for ALS was not significantly changed by any mutation [K_a (fully-glycosylated) = 12.5 ± 4.1 nM⁻¹; mean K_a (all mutants) = 22.1 ± 3.0 nM⁻¹]. While glycosylation does not appear to play a role in IGFBP-3 ligand binding, it may affect the turnover rate of the protein or be involved in rendering the protein resistant to proteolysis.

Circulating IGF-I exists primarily as part of a ternary 150 kDa complex comprising equimolar amounts of IGF-I, IGFBP-3 and acid labile subunit (ALS). It is also known that in contrast to IGF-I and IGFBP-3 there exists a substantial quantity of unbound ALS in the circulation. As part of our preclinical development program, we have investigated the pharmacokinetic properties of a complex of human recombinant IGF-I and IGFBP-3. Systematic administration of rhIGF-I/IGFBP-3 results in binding of this binary complex to endogenous free ALS and thus leads to increased circulating levels of 150 kD ternary complex (IGF-I/IGFBP-3/ALS). In contrast to the administration of free IGF-I, IGF-I/IGFBP-3 dosing leads to increased systemic IGF-I exposure (i.e. increased area under the time vs. serum concentration curve or AUC) and decreased clearance (CL). Upon administration of equimolar doses of IGF-I/IGFBP-3 to the rat and monkey, it was found that in the monkey AUC was increased and CL decreased when compared to the rat. In addition, the pharmacokinetic profile suggests that saturation of excess ALS occurs at considerably lower doses of rhIGF-I/IGFBP-3 in the monkey than in the rat. Finally, the bioavailability of rhIGF-I/IGFBP-3 was assessed in the rat and found to be approximately 85% and 50% after intramuscular and sub-cutaneous administration, respectively. It was concluded that the formation of the 150 kD ternary complex had a significant impact on increasing the systemic exposure to rhIGF-I when administered as the binary complex (rhIGF-I/IGFBP-3). In addition, IGFBP-3 increases the therapeutic index of rhIGF-I even at doses that significantly exceed the saturation of ALS.

We have previously shown that a polyclonal anti-IGF-I antiserum administered together with IGF-I potentiates IGF-I activity in vivo. The anti-IGF-I antiserum has a modest affinity for IGF-I, similar to that for enhancing IGFBPs, and treated animals have significantly higher circulating IGF-I concentrations than their controls. Our recent findings have demonstrated that the anti-IGF-I activity decreases the clearance of IGF-I by at least 2-fold and that it abolishes the acute hypoglycaemic action of a single subcutaneous dose of IGF-I. Interestingly, we have been unable to demonstrate potentiation of the growth-promoting activity of the potent non-IGFBP binding IGF-I analogue LR³IGF-I, even though the analogue binds to the antiserum in vitro; rather native IGF-I/antibody complexes perform even better than LR³IGF-I. In IGF-I/antibody-treated dwarf rats, most IGF-I may be found in an uncharacterised high molecular weight antibody complex which is probably responsible for improved IGF-I performance. Thus, the anti-IGF-I antibody may be behaving in a similar manner to a high molecular weight IGFBP and is effective in potentiating IGF-I action in vivo.

A number of lines of evidence suggest that IGFs are important mitogens in human breast cancer: (1) IGFs are the most potent growth factor in human breast cancer cells; (2) estrogen stimulates expression of IGF-II and the type 1 IGF receptor; and (3) stromal cells express IGFs, which may act in a paracrine manner. Numerous studies have demonstrated that IGFBPs modulate the mitogenic effects of IGFs in the local environment. In particular, we have recently demonstrated that IGFBP-3 inhibits the growth of Hs578T and MDA-MB-231 human breast cancer cells in an IGF-independent manner. Further studies revealed the existence of cell surface-associated IGFBP-3 receptors. Receptor binding and the subsequent antiproliferative action of IGFBP-3 was inhibited by IGFs, owing to the formation of an IGF-IGFBP-3 complex that prevents the binding of IGFBP-3 to its receptors. In addition, exogeneously added soluble heparin or heparan sulfate inhibited the binding of IGFBP-3 to the cell surface in a dose-dependent manner. However, when heparin and heparan sulfate linkages of glycosaminoglycans on the cell surface were enzymatically removed, IGFBP-3 binding was only minimally affected. These data suggest that soluble heparin or heparan sulfate forms a complex with IGFBP-3, thereby inhibiting receptor binding of IGFBP-3, rather than competing with cell-surface glycosaminoglycans for binding of IGFBP-3.

Additionally, the role of IGFBP-3 in the antiproliferative effects of transforming growth factor (TGF)-β and retinoic acid (RA) is supported by out observations that: (1) inhibition of IGFBP-3 gene expression using an IGFBP-3 antisense oligodeoxynucleotide not only blocks TGF-β and RA simulation of IGFBP-3 production by up to 90%, but also inhibits their antiproliferative effects by 40–60%; and (2) treatment with IGF-II and IGF-II analogs diminish TGF-β effects by blocking TGF-β induced binding of IGFBP-3 to the cell surface.

Taken together, our results support the hypothesis that IGFBP-3 is an important antiproliferative factor in human breast cancer, acting in an IGF-independent manner in addition to its ability to modulate the binding of IGF peptides to IGF receptors.

We examined the effects of GH and prolactin deficiency upon milk production, apoptosis and IGFBP production by the mammary gland. GH deficiency produced a 15% reduction in milk yield, prolactin a 50% reduction and combined prolactin- and GH-deficiency an 85% reduction in milk production. Litter removal led to complete inhibition of milk synthesis within 24 h owing in large part to milk accumulation. GH- and prolactin-deficiency also led to significant loss of mammary cells within 48 h and this was owing at least in part to apoptosis as judged by the appearance of characteristic DNA ladders. Prolactin replacement therapy could prevent all of these changes whilst GH was partially effective. The effects of GH are believed to be mediated via IGF-I, however, we were unable to mimic the effects of GH with IGF-I, IGF-II or a combination of IGF-I, IGF-II and IGFBP-3. We hypothesized that the cell-survival effects of exogenous IGFs might be blocked by an inhibitory IGFBP produced by the gland. Indeed the involuting mammary gland produces large concentrations of an IGFBP which Northern blotting identified as IGFBP-5. There was also a small increase in IGFBP-4 mRNA expression. Both appear to be produced by the secretory epithelial cells as judged by in situ hybridization. Preliminary studies using mouse “mammosphere” cultures suggest that they will be useful for investigating a potential causal relationship between IGFBP-5 synthesis and apoptosis.

The mouse ALS gene spans at least 6 kb. It contains 2 exons which encode a protein highly homologous to human and rat ALS. It was localized to mouse chromosome 17 by fluorescent in situ hybridization. The 5′ flanking region lacks a TATA box but contains GC boxes that may be recognised by transcription factors such as Sp1. Hepatic ALS mRNA is decreased in rats following hypophysectomy, and restored by systemic treatment with recombinant human GH. In transient transfection assays, GH stimulated ALS promoter activity in a rat hepatoma cell line, but not in 3T3-F442A mouse preadipocyte fibroblasts, suggesting that utilisation of the ALS promoter is cell-type specific. The rat hepatoma system is a promising system to study the regulation of ALS gene expression, and the signalling pathways of CH regulation.

Heparin affin regulatory peptide (HARP), also called Pleiotrophin (PTN), is a polypeptide that displays a high affinity for heparin and that shares approximately 50% sequence homology with Midkine (MK). According to this structural homology, these two molecules constitute a new family of heparin-binding proteins. The biological properties of HARP and MK remain largely a subject of debate. Both proteins have been described as neurite outgrowth promoting agents whereas until recently the mitogenic activity has been controversial. The aim of this review is to summarize the information on HARP with special focus on the recent data relating to its mitogenic properties.