Evidence for a direct effect of growth hormone on osteoblasts

In order to determine whether growth hormone (GH) exerts a direct effect on osteoblasts, in vitro and in vivo immunocytological studies were carried out on newborn rat calvaria and a clonal osteoblast-like cell line (MC3T3-E1) isolated from newborn mouse calvaria. After exposure to human growth hormone (hGH) or 1,25 dihydroxyvitamin D3 (1,25(OH)₂D₃), a significant increase in alkaline phosphatase activity was observed in MC3T3-E1 cells. Simultaneous exposure of MC3T3-E1 cells to hGH and 10 nM 1,25(OH)₂D₃ showed a synergistic effect of the two hormones on this activity. The optimal dose of hGH was 0.1 nM. An immunocytological procedure was performed on ultrathin frozen sections from 7-day-old rat calvaria and MC3T3-E1 cells cultured with hGH. GH-like immunoreactivity was observed in both cases. In calvaria, endogenous GH-like immunoreactivity was localized at the same ultrastructural level (plasma membrane, cytoplasmic and nuclear matrices) as exogenous GH-like immunoreactivity in MC3T3-E1 cells. Following the initial step of binding to the plasma membrane, GH may be internalized in the cytoplasmic matrix and nucleus. In situ hybridization revealed the presence of mRNA coding for GH receptor in calvaria cells. The density of these receptors seemed to be lower in osteoblasts than in hepatocytes. In MC3T3-E1 cells, hGH induced a dose-dependent secretion of insulin-like growth factor 1. In conclusion, these results indicate that GH may act directly on osteoblasts.     

Specific binding sites for growth hormone in cultured mouse thymic epithelial cells

Growth Hormones bound specifically to murine Thymic epithelial cells, which represent the major component of thymic micro-environment and can be modulated by pituitary hormones. The Kds found with human growth hormone and bovine growth hormone were 0.14 and 0.27 nM with a Bmax 0.56 and 0.35 fmol/10(6) cells respectively. Competition experiment analysis showed ED50 of 0.24 nM for hGH, 0.46 nM for rGH, 0.71 nM for bGH, 11.8 nM for hPRL and 11.2 nM for oPRL. No specific binding of [125I]-oPRL was observed under the same conditions. Both hPRL and bGH showed a negative regulatory effect on the number of the hGH binding sites when incubated with the culture for three days. The presence of GH receptors on Thymic epithelial cells provides biochemical evidence for the effect of GH on thymic function.     

活化素抑制大鼠垂体GH_3细胞中人生长激素基因启动子的活性

本研究旨在探讨活化素(activin)对大鼠垂体GH3细胞中人生长激素(hGH)基因启动子活性的影响及其可能的调节机制。采用荧光素酶报告基因方法。首先建立含hGH基因启动子(-484～+30bp)和荧光素酶融合基因的稳定转染GH3细胞株,然后加入活化素或同时加入活化素与相关信号转导途径的激动剂,通过检测细胞培养液和细胞裂解液中GH的含量,以及GH3细胞内荧光素酶的变化,反映活化素对GH分泌、合成和hGH基因启动子活性的影响。将含不同长度hGH基因启动子序列的荧光素酶表达质粒分别转染GH3细胞,观察它们对活化素的反应,寻找活化素影响hGH基因启动子活性的关键DNA序列。结果表明,活化素(5,50nmol/L)能抑制大鼠垂体GH3细胞中GH的分泌和合成,活化素(5,50nmol/L)还能够抑制GH3细胞中hGH基因启动子的活性,使之仅达对照组的77%和69%;在胞内信号转导激动剂中,丝裂原活化蛋白激酶激酶(MAPKK/MEK)特异性激动剂C6ceramide(1μmol/L)完全取消了活化素对hGH基因启动子活性的抑制作用;活化素发挥抑制作用所需要的hGH基因启动子关键序列位于-132～-66bp之间。上述研究表明,活化素能抑制大鼠垂体GH3中hGH基因启动子的活性,它可能是通过抑制细胞内依赖MAPK的信号转导途径来完成的,同时hGH启动子上-132～-66bp的序列在其中发挥重要的作用。     

Biochemical and cytochemical studies of preadipocyte differentiation in serum-free culture of porcine stromal-vascular cells: interaction of dexamethasone and growth hormone.

Stromal-vascular cells from adipose tissue of pigs 5-7 days of age were grown in serum for 2-3 days and switched to serum-free (insulin, transferrin and selenium) conditions +/- test hormones for 6-7 days. The interaction of dexamethasone (DEX) and human growth hormone (hGH) was evaluated since glucocorticoids augment and hGH antagonizes the effect of insulin. Low levels (1-10 nM) of DEX with insulin doubled (p less than 0.05) specific activity of glycerol phosphate dehydrogenase (GPDH) and doubled (p less than 0.05) the number of detectable fat cells relative to insulin alone. DEX with insulin enhanced the morphological differentiation of preadipocytes and markedly increased fat cell cluster numbers in the presence of hGH. Furthermore, 1-10 nM of DEX partially blocked (p greater than 0.05) the inhibitory effect of 10 nM hGH on GPDH activity, but 1-100 nM DEX had no effect (p greater than 0.05) on the ability of hGH to compromise lipid deposition. DEX alone (no insulin or hGH) induced the appearance of esterase-reactive but lipid-free cells. Cells with these characteristics were increased in number by DEX in the presence of hGH but were nearly absent in the presence of insulin and DEX. Therefore, transient exposure to GH in vivo may have no permanent effect on adipose tissue development in the continued presence of glucocorticoids.     

Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3',5'-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids

We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.     

Ontogenesis of hepatic growth hormone receptors in the rat

Detailed ontogenic studies of the binding of human (hGH) and bovine growth hormone (bGH) have been performed in liver preparations from male and female rats during the neonatal, weanling, pre- and post-pubertal periods. Specific binding of both hormones was readily detected at all ages, with no apparent interference due to occupancy by endogenous hormones. No sex difference in binding was observed prior to weaning (22 days) for hGH, which binds to both somatotrophic and lactogenic sites. However, after weaning a marked sex-related dissociation in the pattern of binding did occur, with female rats binding 3-4 times more hGH than in the pre-weaning period and male rats binding hGH to only half their pre-weaning levels. A very similar pattern was seen for binding of bGH (which binds only to somatotrophic sites) except that in male rats, the post-weaning levels did not fall. Binding patterns for either hGH or bGH prior to weaning did not mirror the known age-related pattern of circulating rat GH levels, suggesting the absence of a definitive auto-regulation system for the GH-GH receptor system under normal circumstances in vivo. The possible role of the weaning process per se in the post-weaning changes of GH binding seen in male and female rats still requires elucidation.     

Growth hormone (GH) stimulates protein synthesis in cells transfected with GH receptor complementary DNA

An expression vector containing a rat GH receptor cDNA was transfected into Chinese hamster ovary (CHO) cells, and stable cell lines expressing GH receptors were established. In contrast to nontransfected CHO cells, expression of GH receptors in transfected cells resulted in the appearance of high affinity (Kd = 1.53 nM) specific binding of GH. Cross-linking of [125I]hGH to the receptors and subsequent sodium dodecyl sulfate (SDS)-electrophoresis gave an estimated receptor mol wt of 84,000. GH treatment stimulated protein synthesis 60% over basal levels in GH receptor-expressing CHO cells, but not in the receptor-negative parental cells. The effect was observed only under serum-free conditions and was time and dose dependent. These results show that heterologous expression of the rat GH receptor results in the appearance of specific binding of GH and the acquisition of a functional GH response.     

Characterization of the slowly dissociable human growth hormone binding component of isolated rat hepatocytes

Human growth hormone (hGH) bound to specific sites on rat hepatocytes. The time course of hGH dissociation was comprised of more than one component. Dissociation was resolved into rapid (t1/2 = 10.5 min) and slow (t 1/2 = 6.4 h) fractions. The amount of slowly dissociable hormone increased for the first 75 min during which time cells and [125I]hGH associated. Subsequently, the amount of slowly dissociable hGH was constant. The time courses of hGH receptor binding and subsequent retention of slowly dissociable label were similar. The capacity of hepatocytes to accumulate slowly dissociable label was saturated by hGH over the same concentration range as the high-affinity binding site (KD approximately 2 nM). This suggested that a receptor-mediated process was responsible for the accumulation of slowly dissociable hGH. Rapidly dissociable label was intact [125I]hGH and fragments resulting from growth hormone degradation. Slowly dissociable hGH recovered from hepatocytes by acid extraction was intact and immunocompetent. There was a large increase in the extent of [125I]hGH degradation between 23 and 37 degrees C. Over this temperature range, the proportion of hGH not in rapid equilibrium with the medium decreased. High concentrations of hGH decreased the amount of slowly dissociable [125I]hGH retained by hepatocytes by competing for high-affinity sites. The interaction of [125I]hGH with low-affinity degradative systems was favored by the presence of hGH. The temperature and concentration dependencies of hGH retention and degradation distinguished these proceses.     

Induction of lactogenic receptors. I. In the liver of hypophysectomized female rats.

To identify the hormones which affect lactogenic receptors in the liver of chronically hypophysectomized female rats, hormones were injected s.c. for 7 days. Specific binding (%, SB) of labelled ovine prolactin (PRL) in liver membrane preparations (1000,000 X g pellet) of controls was 1%. Estradiol (E2), cortisone (Con), ACTH or bovine growth hormone (bGH) treatment did not induce hepatic binding sites for PRL. Human GH and a single dose of 2mg PRL (but not lower doses) increased SB of PRL. Treatment with oPRL plus ACTH was less effective than hGH plus ACTH (13 vs 28%); combinations of oPRL plus Con as well as administration of oPRL plus ACTH to hypophysectomized and adrenalectomized female rats did not induce SB for PRL. Therapy with oPRL plus hGH (26%) was more potent than oPRL plus bGH (2%). These studies suggest that PRL, GH, and ACTH induce and in concert with sex steroids, modulate the lactogenic receptors in the female rat liver. The effect of ACTH is not due to increased adrenal corticoid secretion.     

Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus carpio) and its enhancement by deoxycholate

Summary Recombinant human growth hormone was administered orally to carp and serum levels of absorbed bioactive hormone were investigated using a highly sensitive Nb2 rat lymphoma cell bioassay and radioimmumoassay. Serum levels of bioactive hGH reached maximum values 30 min after oral intubation and then gradually decreased. Co-administration of the hormone with deoxycholate to fasted carp resulted in up to a 1000-fold increase in absorption compared to aqueous solutions of the hormone, but had no effect on the kinetics of the absorption process. Absorption of the hormone in starved fish was significantly greater than in fed fish. A linear dose-response relationship was observed for hGH in starved fish and the level of absorption in fed fish was influenced by the time interval from the last meal. The ratio of bioactive to immunoactive hGH in fasted fish indicated little loss of bioactivity and also that deoxycholate may be protective against hGH degradation. The present study demonstrates for the first time that biologically active hGH is absorbed in the common carp after oral intubation. Furthermore, the use of a biological detergent dramatically increased the extent of hGH absorption. Additional studies are required to establish the approapriate conditions (diet composition, feeding level, and frequency, etc.) in which polypeptide hormones could be introduced orally to fish.Abbreviations hGH human growth hormone - HRP horseradish peroxidase - RIA radioimmunoassay     

Chimeric bovine-human growth hormone prepared by recombinant DNA technology: binding properties and biological activity

A chimeric bovine GH (amino acids Met-Asp-Gln-greater than 1-23) and human GH (hGH) (amino acids 24-191) plasmid was constructed and expressed in Escherichia coli. The purified protein (chimeric GH) exhibited a 2-3 order of magnitude lower affinity toward lactogenic receptors in Nb2 lymphoma cells, microsomal fractions from bovine mammary gland and male rat liver. The affinity towards somatogenic receptors in IM-9 human lymphocytes and male rat liver was decreased to a much lesser degree. This diminished affinity towards lactogenic receptors was accompanied by a parallel decrease in the ability of the chimeric GH to stimulate proliferation of Nb2-11C lymphoma cells and the lipogenesis in bovine mammary gland. This implies that occupation of the respective receptors by either chimeric GH or hGH leads to identical postreceptoral effects. The chimeric GH was also capable of down-regulating the lactogenic receptors in Nb2 lymphoma cells and was recognized by three anti-hGH monoclonal antibodies. These and previously published results indicate that the N-terminal part of hGH is essential for the high affinity binding to lactogenic receptors and subsequent biological effect. Removal or replacement by a corresponding part of bovine GH converts the hormone, respectively to weak antagonist or agonists. Analysis of our data, based on hydropathy index leads us to suggest that the high affinity binding site of the hGH towards lactogenic receptors is mainly confined to amino acids nos. 8-18.     

Hormonal regulation of expression of the endogenous and transfected human growth hormone gene

Expression of the endogenous human GH (hGH) gene in response to glucocorticoids, thyroid hormone, and insulin was studied in cultures of dispersed GH-secreting human pituitary adenomas. Results were compared to those obtained when the hGH gene was transfected into rat pituitary tumor cells (GC). In the human pituitary cells the glucocorticoid dexamethasone [(Dex) 10(-6) M] increased the release of GH and the levels of GH mRNA by 2 to 4-fold (P less than 0.05). T3 (10(-8) M) had no effect on GH mRNA but increased hGH release by 2- to 6-fold (P less than 0.01). Insulin (5 x 10(-9) M) alone had no significant effect on either hGH mRNA or protein, but blunted the effect of Dex. Among 11 of 18 GC cell clones transfected with the hGH gene with detectable hGH mRNA expression, Dex increased hGH mRNA levels in seven and T3 treatment reduced hGH mRNA levels in eight. Conversely, rat GH mRNA levels from the endogenous rat gene were increased by either Dex or T3 in all 18 clones. Insulin alone or in combination with T3 or Dex was found to increase hGH mRNA levels in some cell lines and to decrease hGH mRNA levels in others; these effects were correlated strongly (r = 0.88; P less than 0.001) with the influence of insulin on the endogenous rat GH gene, implying that individual cellular differences can simultaneously affect the insulin responsiveness of both genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration in the receptor binding specificity of human growth hormone by genomic exon exchange

The biological activities of the GH-PRL family of hormones are mediated by selective binding to two classes of cell membrane receptors, somatogen and lactogen. Primate GH such as human GH (hGH) are unusual in that they bind to both classes of receptors. Replacement of exons 3 or 4 of the hGH gene by the corresponding exons of the rat PRL or rat GH genes results in the synthesis of chimeric proteins which retain the ability to bind to lactogen receptors but can no longer bind to somatogen receptors. This selective loss of somatogen receptor binding in the chimeric proteins suggests that certain of the structural determinants of somatogen and lactogen receptor binding activities in hGH are distinct and can be separately modified by a limited number of amino acid substitutions.     

Increased site 1 affinity improves biopotency of porcine growth hormone. Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp104 and Trp169 in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys165), which in addition to His169, restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.     

The characteristics of hGH binding to the liver macrophages

Macrophages isolated from female rat liver as well as hepatocytes bind 125I-hGH. This study compares the effect of sex of the rat, hypophysectomy (hypox) and preincubation of the cells with oPrl on the binding of 125I-hGH to the cells. The percent of 125I-hGH to the hepatocytes was decreased in cells from hypox female and male rats, and hepatocytes preincubated with oPrl to 0.43, 0.21 and 0.39, respectively, of that observed in hepatocytes from normal female rats. In the hepatocytes from normal female, hypox female, and male rats, hGH was the most effective competitor for 125I-hGH binding with an ID50 of 0.73-0.99 nM. The concentration of oPrl, bGH and rGH that produced half-maximal inhibition (ID50) of 125I-hGH binding to hepatocytes from female rat liver was 6.3, 100, and 420 nM respectively. In hepatocytes from male and hypox female rats, and hepatocytes preincubated with oPrl, the ID50 for bGH and rGH varied from 2.1 to 15.9 nM. The percent of 125I-hGH bound by the macrophages from hypox female and male rats, and macrophages preincubated with oPrl was 0.06, 0.15 and 0.18, respectively, of that bound by macrophages from normal female rat liver. In contrast to hGH binding to the hepatocytes, the ID50 for hGH was 6 to 180-fold greater in macrophages from hypox female and male rats, and macrophages preincubated with oPrl compared to that observed in macrophages from normal female rats, Rat GH was the most effective competitor for 125I-hGH binding in the macrophages from the hypox female and male rat liver with ID50 of 5.5 and 85 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen antagonism on T3 and growth hormone control of the liver microsomal low-affinity glucocorticoid binding site (LAGS)

Male rat liver microsomes contain a low-affinity glucocorticoid binding site (LAGS) capable of binding all natural glucocorticoids and progesterone with a K_d from 20 to 100 nM. The LAGS level is under endocrine control by T₃, glucocorticoids and GH. These hormones act synergistically at physiological concentrations to increase the LAGS level. Since female rats show a LAGS level that is much lower than the males (0.15 vs 23 pmol/mg protein, respectively), here we investigated whether estradiol could decrease the LAGS in the male rat. Orchiectomized (OX) male rats showed a higher LAGS level than intact rats. This effect was reversed by implanting a Sylastic capsule containing testosterone. When the OX rats were implanted for 20 days with estrogen capsules that provided an estradiol level in serum of 40 pg/ml, their LAGS level decreased from 23 to 0.2 pmol/mg protein. This effect was not observed in intact male rats and can be partially reversed by testosterone implants into OX rats. Both hypophysectomized male rats and hypothyroid-orchiectomized male rats showed very low levels of LAGS. Administration of physiological doses of GH and/or T₃ to these rats greatly increased their LAGS level (from 0.3 to 15 and 16 pmol/mg protein, respectively). Implantation of estrogen capsules to these rats two weeks prior to starting treatment completely inhibited the increase in the LAGS level in response to T₃, and significantly decreased the response to hGH, and to a combination of hGH and T₃. These results suggest that physiological estradiol levels can antagonize the LAGS induction by T₃ and hGH in the male rat, and could be responsible for the low level of LAGS in the female rat. Moreover, estrogen capsules also inhibited the increase in the body and hepatic weights observed after hGH treatment, which suggests a powerful inhibitory effect of low estradiol levels on the male rat liver functions under regulation by T₃ and/or GH.     

Cloning and expression of ovine placental lactogen

Ovine placental lactogen (oPL) is active in a wide range of GH and PRL assays, a property that it shares with human GH (hGH). In addition, oPL is one of a small number of hormones that bind the human GH receptor with high affinity. In order to compare the sequence of oPL to the sequences of other members of the GH family, full-length cDNA clones have been isolated. These clones predict that the full sequence of oPL contains 198 amino acids preceded by a 38 amino acid signal sequence. The mature oPL sequence includes six cysteine and two tryptophan residues and shows substantially more identity to bovine PL (67%) and oPL (49%) than to mouse (31%) or human (25%) PL or to oGH (28%) or (26%) hGH. Like the natural hormone, oPL expressed in mammalian tissue cells binds with high affinity to a soluble form of the recombinant hGH receptor. Thus, oPL binds to the human receptor in spite of having a sequence that is considerably divergent from hGH. Interestingly, the sequence of oPL differs from hGH at most of the amino acids recently found by mutagenesis studies to be important residues in the binding of hGH to the human receptor.