Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and platelet-derived growth factor in rat hepatic stellate cells

Proliferation of activated hepatic stellate cells (HSC) is an important event in the development of hepatic fibrosis. Insulin-like growth factor-1 (IGF-1) has been shown to be mitogenic for HSC, but the intracellular signaling pathways involved have not been fully characterized. Thus, the aims of the current study were to examine the roles of the extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (P13-K) and p70-S6 kinase (p70-S6-K) signaling pathways in IGF-1- and platelet-derived growth factor (PDGF)-induced mitogenic signaling of HSC and to examine the potential crosstalk between these pathways. Both IGF-1 and PDGF increased ERK, P13-K and p70-S6-K activity. When evaluating potential crosstalk between these signaling pathways, we observed that P13-K is required for p70-S6-K activation by IGF-1 and PDGF, and is partially responsible for PDGF-induced ERK activation. PDGF and IGF-1 also increased the levels of cyclin D1 and phospho-glycogen synthase kinase-30. Coordinate activation of ERK, P13-K and p70-S6-K is important for perpetuating the activated state of HSC during fibrogenesis.

Hepatocellular carcinoma

Detailed analyses of chromosomal damage in hepatocellular carcinoma have confirmed the results of previous studies that identified regions of significant loss. In addition, these studies examined the clinicopathological correlates of this damage, identified new sites for future investigation, and provided evidence of interactions between genes, The insulin-like growth factor II receptor gene is a target for inactivation through chromosomal loss and mutation, with loss also occurring in the cirrhotic liver. The insulin-like growth factor II receptor gene plays a central role in coordinating the competing actions of insulin-like growth factor and transforming growth factor-beta on cell proliferation. Our understanding of the changes in these growth factor pathways helps explain the apparent increase in risk of hepatocellular carcinoma in diabetic patients and the potential use of urinary transforming growth factor-beta in screening tests. Vaccination for hepatitis B in Taiwan has had a significant effect on the incidence of childhood hepatocellular carcinoma. Universal vaccination should result in a major reduction in the incidence of hepatocellular carcinoma worldwide.

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

Administration of growth hormone or IGF-I to pregnant rats on a reduced diet throughout pregnancy does not prevent fetal intrauterine growth retardation and elevated blood pressure in adult offspring

Increasing evidence from human epidemiological studies suggests that poor growth before birth is associated with postnatal growth retardation and the development of cardiovascular disease in adulthood. We have shown previously that nutritional deprivation in the pregnant rat leads to intrauterine growth retardation (IUGR), postnatal growth failure, changes in the endocrine parameters of the somatotrophic axis, and to increased blood pressure in later life. In the present study, we investigated whether administration of insulin-like growth factor-I (IGF-I) or bovine growth hormone (GH) during pregnancy could prevent IUGR and/or alter long-term outcome. Dams h-om day 1 of pregnancy throughout gestation received a diet of nd libitum available food or a restricted dietary intake of 30% of ad libitum fed dams. From day 10 of gestation, dams were treated for 10 days with three times daily subcutaneous injections of saline (100 mu l), IGF-I (2 mu g/g body weight) or GH (2 mu g/g body weight). Maternal weight gain was significantly increased (P

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.

Expression of growth factors in Dictyostelium discoideum

Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors. Many growth factors require posttranslational modifications making them unsuitable for production in Escherichia coli or other prokaryotes. Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins. Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring.

Super-CHO - A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6)cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6)cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Growth hormone-binding protein in normal and pathologic gestation: Correlations with maternal diabetes and fetal growth

To date, measurements of GH-binding protein (GHBP) during human pregnancy have been carried out using;assays susceptible to interference by the elevated levels of human placental GH typical of late gestation. We recruited a large cohort of pregnant women (n = 140) for serial measurements of GHBP and used the ligand immunofunctional assay for GHBP. For normal gravidas, GHBP levels fell throughout gestation. Mean levels were 1.07 nmol/L (SE = 0.18) in the first trimester, 0.90 nmol/L (SE = 0.08) at 18-20 weeks, 0.73 nmol/L (SE = 0.05) at 28-30 weeks, and 0.62 nmol/L (SE = 0.06) at 36-38 weeks. GHBP levels in the first trimester correlated significantly with maternal body mass index (r = 0.58; P < 0.01). GHBP levels in pregnancies complicated by noninsulin-dependent diabetes mellitus (NIDDM) were substantially elevated at all gestational ages. The mean value in the first quarter (2.29 nmol/L) was more than double the normal mean (P < 0.01). In contrast, patients with insulin-dependent diabetes mellitus (IDDM) showed reduced GHBP concentrations at 36-38 weeks. The correlation between body mass index and GHBP is consistent with a metabolic role for GHBP during pregnancy, as is the dramatic elevation in GHBP observed in cases of NIDDM. At 36 weeks gestation, GHBP was significantly elevated (P < 0.01) in those women whose neonates had low birth weight (

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.

Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimers

GH actions are dependent on receptor dimerization. The GH receptor antagonist, B2036-PEG, has been developed for treating acromegaly. B2036 has mutations in site 1 to enhance receptor binding and in site 2 to block receptor dimerization. Pegylation (B2036-PEG) increases half-life and lowers immunogenicity, but high concentrations are required to control insulin-like growth factor-I levels. We examined antagonist structure and function and the impact of pegylation on biological efficacy. Unpegylated B2036 had a 4.5-fold greater affinity for GH binding protein (GHBP) than GH but similar affinity for membrane receptor. Pegylation substantially reduced membrane binding affinity and receptor antagonism, as assessed by a transcription assay, by 39- and 20-fold, respectively. GHBP reduced antagonist activity of unpegylated B2036 but did not effect antagonism by B2036-PEG. B2036 down-regulated receptors, and membrane binding sites doubled in the presence of dimerization-blocking antibodies, suggesting that B2036 binds to a receptor dimer. It is concluded that the high concentration requirement of B2036-PEG for clinical efficacy relates to pegylation, which decreases binding to membrane receptor but has the advantages of reduced clearance, immunogenicity, and interactions with GHBP. Our studies suggest that B2036 binds to a receptor dimer and induces internalization but not signaling.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.