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.

Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependent

it has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13-16 years old), young adults (18-35 years old), and adults (36-49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents.

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.

Dorsal and ventral medullary catecholamine cell groups contribute differentially to systemic interleukin-1 beta-induced hypothalamic pituitary adrenal axis responses

Medial parvocellular paraventricular corticotropin-releasing hormone (mPVN CRH) cells are critical in generating hypothalamic-pituitary-adrenal (HPA) axis responses to systemic interleukin-1 beta (IL-1 beta). However, although it is understood that catecholamine inputs are important in initiating mPVN CRH cell responses to IL-1 beta, the contributions of distinct brainstem catecholamine cell groups are not known. We examined the role of nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) catecholamine cells in the activation of mPVN CRH, hypothalamic oxytocin (OT) and central amygdala cells in response to IL-1 beta (1 mug/kg, i.a.). Immunolabelling for the expression of c-fos was used as a marker of neuronal activation in combination with appropriate cytoplasmic phenotypic markers. First we confirmed that PVN 6-hydroxydopamine lesions, which selectively depleted catecholaminergic terminals, significantly reduced IL-1 beta -induced mPVN CRH cell activation. The contribution of VLM (A1/C1 cells) versus NTS (A2 cells) catecholamine cells to mPVN CRH cell responses was then examined by placing ibotenic acid lesions in either the VLM or NTS. The precise positioning of these lesions was guided by prior retrograde tracing studies in which we mapped the location of IL-1 beta -activated VLM and NTS cells that project to the mPVN. Both VLM and NTS lesions reduced the mPVN CRH and OT cell responses to IL-1 beta. Unlike VLM lesions, NTS lesions also suppressed the recruitment of central amygdala neurons. These studies provide novel evidence that both the NTS and VLM catecholamine cells have important, but differential, contributions to the generation of IL-1 beta -induced HPA axis responses. Copyright (C) 2001 S. Karger AG, Basel.

Spatial distribution and developmental appearance of postjunctional P2X(1) receptors on smooth muscle cells of the mouse vas deferens

P2X(1)-type purinoceptors, have been shown to mediate fast transmission between sympathetic varicosities and smooth muscle cells in the mouse vas deferens but the spatial organization of these receptors on the smooth muscle cells remains inconclusive. Voltage clamp techniques were used to estimate the amplitudes of spontaneous excitatory junction currents (SEJCs) in cells of the vas deferens longitudinal smooth muscle layer. These currents involved the activation of about 6% of the P2X-type channels present on the cell, as compared to whole cell currents produced when isolated smooth muscle cells were exposed to maximal concentrations of either ATP or alpha,beta -MeATP. Immunofluorescence staining of the vas deferens with antibodies against P2X(1) receptor showed a diffuse, grainy distribution over the entire membrane of each smooth muscle cell. Anti-P2X(1) staining was not markedly clustered beneath anti-SV2-stained sympathetic varicosities. Similar results were obtained for cells in the urinary bladder. During development, P2X(1) mRNA was detected as early as embryonic day 15 (E15). Increasing intensities of diffuse immunostaining for P2X(1) were observed in the walls of the bladder, tail artery, and aorta from E15 until 6 weeks postnatal. The vas deferens showed increasing intensities of diffuse staining of its smooth muscle layers between 2 and 6 weeks postnatal, consistent with the time-course of development of fast purinergic transmission described previously. Together, the results suggest that the response of smooth muscle of the vas deferens to ATP released from sympathetic varicosities relies on rapidly desensitizing P2X(1) receptors, distributed diffusely across the smooth muscle cell surface. Synapse 42:1-11, 2001. (C) 2001 Wiley-Liss, Inc.

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the beta-amino acid agonist, taurine

The beta -amino acid, taurine, is a full agonist of the human glycine receptor al subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta -amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta -amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta -amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.

A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alpha beta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alpha beta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alpha beta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alpha beta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin las an allosterically acting 'competitive' antagonist) binds to this residue.

Estrogen receptors in human preadipocytes

Estrogen influences regional adipose tissue distribution and the accompanying cardiovascular disease risk. To elucidate the mechanisms of this link further, we assessed whether human preadipocytes (PAs) expressed estrogen receptors (ERs) and whether there were any regional or gender differences in ER complement. Human PAs expressed the ER alpha gene but not ERP by reverse transcriptase-polymerase chain reaction, possessed ER alpha protein on Western blotting, and displayed specific 17 beta -estradiol (E-2) binding with calculated dissociation constants of 0.78 nM, 0.96 nM, and 1.19 nM and maximal binding capacities of 9.3 fmol/mg, 14.6 fmol/ mg, and 18.2 fmol/mg from three whole cell binding assays. There were no regional differences in ER alpha complement for males or females. There were no gender differences in ER alpha complement for subcutaneous or visceral samples. We conclude that ER alpha but not ERP is present in human PAs. This suggests that the effect of estrogen on adipose tissue deposition has a contribution from the direct effect of estrogen on human PAs via ER alpha.

Peroxisome proliferator-activated receptor beta expression in human breast epithelial cell lines of tumorigenic and non-tumorigenic origin

Peroxisome proliferator-activated receptor beta (PPARbeta) is a member of the nuclear hormone receptor superfamily and is a ligand activated transcription factor. although the precise genes that it regulates and its physiological and pathophysiological role remain unclear. In view of the association of PPARbeta with colon cancer and increased mRNA levels of PPARbeta in colon tumours we sought in this study to examine the expression of PPARbeta in human breast epithelial cells of tumorigenic (MCF-7 and MDA-MB-231) and non-tumorigenic origin (MCF-10A). Using quantitative RT-PCR we measured PPARbeta mRNA levels in MCF-7. MDA-MB-231 and MCF-10A cells at various stages in culture. After serum-deprivation, MDA-MB-231 and MCF-10A cells had a 4.2- and 3.8-fold statistically greater expression of PPARbeta compared with MCF-7 cells. The tumorigenic cell lines also exhibited a significantly greater level of PPARbeta mRNA after serum deprivation compared with subconfluence whereas such an effect was not observed in non-tumorigenic MCF-10A cells. The expression of PPARbeta was inducible upon exposure to the PPARbeta ligand bezafibrate. Our results suggest that unlike colon cancer. PPARbeta overexpression is not an inherent property of breast cancer cell lines. However, the dynamic changes in PPARbeta mRNA expression and the ability of PPARbeta in the MCF-7 cells to respond to ligand indicates that PPARbeta may play a role in mammary gland carcinogenesis through activation of downstream genes via endogenous fatty acid ligands or exogenous agonists. (C) 2002 Elsevier Science Ltd. All rights reserved.

Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng, 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids. glucose and yeastolate ultrafiltrate) into Sf9 cell cultures grown in SF900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 mug/ml of NS1 at a peak cell density of 4.2 x 10(6) cells/ml. In contrast. optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 mug ml of NS1 produced at a peak cell density of 11.3 x 10(6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10(6) cells/ml, unless the cultures were stimulated by the addition of 4 mug/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 mug/ml. which was nearly 5-fold higher than optimised batch cultures. (C) 2002 Elsevier Science B.V. All rights reserved.

Systemic administration of interleukin-1 beta activates select populations of central amygdala afferents

The central nucleus of the amygdala (CeA) is activated robustly by an immune challenge such as the systemic administration of the proinflammatory cytokine interleukin-1beta (IL-1beta). Because IL-1beta is not believed to cross the blood-brain barrier in any significant amount, it is likely that IL-1beta elicits CeA cell recruitment by means of activation of afferents to the CeA. However, although many studies have investigated the origins of afferent inputs to the CeA, we do not know which of these also respond to IL-1beta. Therefore, to identify candidate neurons responsible for the recruitment of CeA cells by an immune challenge, we iontophoretically deposited a retrograde tracer, cholera toxin b-subunit (CTb), into the CeA of rats 7 days before systemic delivery of IL-1beta (1 mug/kg, i.a.). By using combined immunohistochemistry, we then quantified the number of Fos-positive CTb cells in six major regions known to innervate the CeA. These included the medial prefrontal cortex, paraventricular thalamus (PVT), ventral tegmental area, parabrachial nucleus (PB), nucleus tractus solitarius, and ventrolateral medulla. Our results show that after deposit of CTb into the CeA, the majority of double-labeled cells were located in the PB and the PVT, suggesting that CeA cell activation by systemic IL-1beta is likely to arise predominantly from cell bodies located in these regions. These findings may have significant implications in determining the central pathways involved in generating acute central responses to a systemic immune challenge. J. Comp. Neurol. 452:288-296, 2002. (C) 2002 Wiley-Liss, Inc.

Functional beta(1)- and beta(2)-adrenoceptors in the left and right atrium of pre-hypertensive rats

There is a small increase in the functional beta(2)-adrenoceptor response on the spontaneously hypertensive rat (SHR) left atrium in the early stages of hypertension. In the present study, the functional beta(1)- and beta(2)-adrenoceptors of the left and right atrium in SHR pre-hypertension and age-matched (5-week-old) Wistar Kyoto (WKY) rats were characterized. Contractility methods with isoprenaline, T-0509 (a selective beta(1)-adrenoceptor agonist) and procaterol (a selective beta(2)-adrenoceptor agonist) were used. At 5 weeks, the SHRs were pre-hypertensive. Isoprenaline was more potent on the left atrium of 5-week-old SHRs than WKY rats. Bisoprolol, a selective beta(1)-adrenoceptor antagonist, was more potent against isoprenaline and T-0509 on the SHR than WKY rat left atrium. ICI 118,551, a selective beta(2)-adrenoceptor antagonist, was more potent against procaterol and T-0509 on the SHR than WKY rat left atrium. The results with bisoprolol and ICI 118,551 suggest that there are more functional beta(1)- and beta(2)-adrenoceptors on the left atrium of 5-week-old SHRs than WKY rats. Isoprenaline, T-0509 and procaterol were equipotent on the right atrium of 5-week-old WKY rats and SHRs. Bisoprolol was more potent against isoprenaline, T-0509 and procaterol on the SHR than WKY rat right atrium. ICI 118,551 was more potent against T-0509, but not isoprenaline and procaterol, on the SHR than WKY rat left atrium. This suggests there are more functional beta(1)-adrenoceptors, and probably more functional beta(2)-adrenoceptors, on the right atrium of 5-week-old SHRs than WKY rats. These functional differences in beta(1)-and beta(2)-adrenoceptor-mediated responses of the left and right atria of pre-hypertensive SHRs cannot be caused by hypertension, and may be associated with the onset of hypertension.

Genes induced by growth hormone in a model of adipogenic differentiation

A substantial number of GH regulated genes have been reported in mature hepatocytes. but genes involved in GH-initiated cell differentiation have not yet been identified. Here we have studied a, ell-characterised model of GH-dependent differentiation, adipogenesis of 3T3-F442A preadipocytes, to identify genes rapidly induced by GH. Using the suppression subtractive hybridisation technique, we have identified eight genes induced within 60 min of GH treatment, and verified these by northern analysis. Six were identifiable as Stat 2. Stat 3, thrombospondin-1. oncostatin M receptor beta chain. a DEAD box RNA helicase. and muscleblind. a developmental transcription factor. Bioinformatic approaches assigned one of the two remaining unknown genes as a novel 436 residue serine,threonine kinase. As each of the identified genes hake important developmental roles. they may be important in initiating GH-induced adipogenesis. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells

Background: Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor Cell proliferation and, following clonal expansion of these cells. promotion of differentiation along the osteogenic lineage. Objectives: We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Method: The cell populations were assessed for mineralization potential after long-term culture in media containing beta-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogcnic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin. osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results: As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP. osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion: The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.