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.     

Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes).

Gene constructs consisting of human growth hormone (hGH) gene driven by promoter/regulatory sequence of mouse metallothionein (mMT), viral thymidine kinase (vTK), rat cholecystokinin (rCCK), or chicken beta-actin (cBA) gene were injected into the cytoplasm of fertilized medaka eggs via the micropyle. More than 49% of the injected embryos survived at hatching. Up to 26% of the survivors showed integration of the introduced gene construct, as determined by polymerase chain reaction analysis and subsequent confirmation by Southern blot hybridization of the genomic DNA. A significant fraction of F1 progeny, derived from crosses between transgenic founders and the nontransgenic individuals, inherited the transgene. Expression of hGH gene was also observed in some of the P1 founders and F1 transgenic progeny carrying mMT-hCG or cBA-hGH gene. Furthermore, the growth performance of the P1 mMT-hGH and cBA-hGH transgenic founders and F1 cBA-hGH F1 transgenic progeny was significantly greater than their full sibling, nontransgenic individuals. In addition to the microinjection experiment, a gene construct containing the long-terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) and rainbow trout (rt) GH2 cDNA was introduced into embryos of medaka by electroporation using an exponential decay electroporator. Approximately 70% of the electroporated embryos survived at hatching, and 20% of the survived individuals integrated RSVLTR-rtGH2 cDNA into their genomes. These two techniques will greatly enhance the ability to study regulation of gene expression in transgenic animals during differentiation and development.     

Fertility of transgenic female mice expressing bovine growth hormone or human growth hormone variant genes

Although growth hormone (GH) exerts various direct and indirect stimulatory effects on gonadal development and function, excessive levels of GH in acromegalic patients and in transgenic animals are often associated with reproductive disorders. We have examined reproductive performance of transgenic female mice expressing the following hybrid genes: mouse metallothionein-1 (MT)/human placental GH variant (hGH.V), MT/bovine GH(bGH), and phosphoenolpyruvate carboxykinase (PEPCK)/bGH. This allowed us to evaluate the effects of chronic GH excess in three animal models and to obtain some information on the significance of the lactogenic activity of the foreign GH (hGH.V vs. bGH) and on the developmental stage of transgene expression (MT vs. PEPCK). Transgenic animals from each line had elevated plasma insulin-like growth factor-I levels and greatly increased adult body weight. Plasma bGH levels were significantly higher in PEPCK/bGH than in MT/bGH transgenic mice. Approximately 20% of transgenic MT/hGH.V and MT/bGH females and over 60% of transgenic PEPCK/bGH females were infertile. Transgenic females that did reproduce ovulated either a normal or increased number of eggs but exhibited a variety of reproductive disorders including increased interval between pairing with a male and conception, increased interval between litters, reduced number of litters, reduced fetal growth, increased pre- and postnatal mortality, and alterations in sex ratio. Among adult offspring of these females, the proportion of transgenic animals was significantly less than the expected 50%. While some characteristics (e.g., fetal crown-rump length and weight on Day 14 of pregnancy) were affected to a comparable extent in transgenic females from all three lines, MT/hGH.V and PEPCK/bGH females were, in general, more severely affected than the MT/bGH animals. Sterility of PEPCK/bGH females appeared to be due to luteal failure since treatment with progesterone led to pregnancy. Greatly increased intervals between successive litters appeared to be due to failure to mate during postpartum estrus and to sterile matings during this period. Reduced fetal size and weight may have been due to chronic glucocorticoid excess because comparable changes could be induced in normal females by injections of dexamethasone during pregnancy, and plasma corticosterone levels were previously shown to be elevated in transgenic mice from each of these lines. Comparison of these results with data obtained from matings of normal female mice to transgenic males from the same lines suggests that reduced fetal growth is due primarily to maternal genotype, while reduced "transmission" of the hybrid genes is not, and presumably reflects increased mortality of transgenic progeny at various stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of insulin-like growth factor-I in neuroendocrine function and the consequent effects on sexual maturation: inferences from animal models

It is known that growth hormone (GH) plays an important role in growth and development.Additionally, emerging evidence suggest that it also influences hypothalamic-pituitary-gonadal function. We have found that GH from different species has different effects in mice. In rodents, human GH (hGH) binds to both GH and prolactin (PRL) receptors; it has both somatotrophic and lactotrophic effects. Since PRL has a profound effect on neuroendocrine function, the results obtained from hGH treatment or from transgenic animals expressing the hGH gene reflect PRL-like effects of this hormone. However, bovine GH (bGH) is purely somatogenic and therefore the effects of bGH represent the function of the natural GH produced in rodents. Furthermore, our studies in mice and rats have shown that not all effects of GH are stimulatory and the duration of exposure of the hypothalamo-hypophyseal-gonadal system to GH might influence the secretions of gonadotropins and gonadal steroids. In humans, excess productions of GH in acromegaly and GH resistance in Laron syndrome adversely affect reproduction. Similarly, it has been demonstrated that in transgenic mice expressing various GH genes, in insulin-like growth factor-I (IGF-I) gene-knockout mice, in GH receptor gene-disrupted (GHR-KO) mice, and in Ames dwarf mice the onset of puberty and/or fertility is altered. Therefore, excess or subnormal secretion of GH can affect reproduction. We have shown that the hypothalamic-pituitary functions are affected in transgenic mice expressing the GH genes, Ames dwarf mice and in GH receptor gene knockout mice. The majority of the GH effects are mediated via IGF-I and the aforementioned effects may be due to the GH-induced IGF-I secretion or due to the absence of this peptide production. It is important to realize that the syntheses and actions of IGF binding proteins are controlled by IGF-I. Furthermore, some IGF binding proteins can inhibit IGF-I action. Therefore, the concentrations of IGF binding proteins and the ratio of these binding proteins and IGF-I within the body might play a pivotal role in modulating IGF-I effects on the neuroendocrine-gonadal system.     

Thyroid hormone activates fibroblast growth factor receptor-1 in bone

Thyroid hormone (T3) and the T3 receptor (TR) alpha gene are essential for bone development whereas adult hyperthyroidism increases the risk of osteoporotic fracture. We isolated fibroblast growth factor receptor-1 (FGFR1) as a T3-target gene in osteoblasts by subtraction hybridization. FGFR1 mRNA was induced 2- to 3-fold in osteoblasts treated with T3 for 6-48 h, and FGFR1 protein was stimulated 2- to 4-fold. Induction of FGFR1 was independent of mRNA half-life and abolished by actinomycin D and cycloheximide, indicating the involvement of an intermediary protein. Fibroblast growth factor 2 (FGF2) stimulated MAPK in osteoblasts, and pretreatment with T3 for 6 h induced a more rapid response to FGF that was increased in magnitude by 2- to 3-fold. Similarly, T3 enhanced FGF2-activated autophosphorylation of FGFR1, but did not modify FGF2-induced phosphorylation of the docking protein FRS2. These effects were abolished by the FGFR-selective inhibitors PD166866 and PD161570. In situ hybridization analyses of TRalpha-knockout mice, which have impaired ossification and skeletal mineralization, revealed reduced FGFR1 mRNA expression in osteoblasts and osteocytes, whereas T3 failed to stimulate FGFR1 mRNA or enhance FGF2-activated MAPK signaling in TRalpha-null osteoblasts. These findings implicate FGFR1 signaling in T3-dependent bone development and the pathogenesis of skeletal disorders resulting from thyroid disease.     

Mode of growth hormone action in osteoblasts

Growth hormone (GH) affects bone size and mass in part through stimulating insulin-like growth factor type 1 (IGF-1) production in liver and bone. Whether GH acts independent of IGF-1 in bone remains unclear. To define the mode of GH action in bone, we have used a Cre/loxP system in which the type 1 IGF-1 receptor (Igf1r) has been disrupted specifically in osteoblasts in vitro and in vivo. Calvarial osteoblasts from mice homozygous for the floxed IGF-1R allele (IGF-1R(flox/flox)) were infected with adenoviral vectors expressing Cre. Disruption of IGF-1R mRNA (>90%) was accompanied by near elimination of IGF-1R protein but retention of GHR protein. GH-induced STAT5 activation was consistently greater in osteoblasts with an intact IGF-1R. Osteoblasts lacking IGF-1R retained GH-induced ERK and Akt phosphorylation and GH-stimulated IGF-1 and IGFBP-3 mRNA expression. GH-induced osteoblast proliferation was abolished by Cre-mediated disruption of the IGF-1R or co-incubation of cells with an IGF-1-neutralizing antibody. By contrast, GH inhibited apoptosis in osteoblasts lacking the IGF-1R. To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in similarly treated mice lacking the IGF-1R in osteoblasts were not significantly affected. These results indicate that although direct IGF-1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo.     

In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides

In vitro actions of purified plerocercoid growth factor (PGF) were compared with those of insulin and human growth hormone (hGH) in adipose tissue from normal male rats. Insulin-like effects were measured by the ability of PGF, insulin, or hGH to stimulate oxidation of [U-14C]glucose to 14CO2, to stimulate lipogenesis, and to inhibit epinephrine-induced lipolysis. PGF and insulin stimulated significant increases in glucose oxidation and lipogenesis in adipose tissue that had not been preincubated as well as in tissue that had been preincubated. hGH stimulated insulin-like effects only in tissue that had been preincubated for 3 hr. Insulin, hGH, and PGF inhibited epinephrine-induced lipolysis of preincubated (3 hr) adipose tissue. hGH produced a dramatic lipolytic response in tissue freshly removed from normal rats but no dose of PGF was lipolytic. PGF did not displace 125I-insulin from its receptors on adipocytes but did competitively inhibit 125I-hGH binding to adipocytes. These results suggest that PGF has direct insulin-like actions which are initiated by binding a GH receptor, but PGF had no anti-insulin action and the insulin-like activity of PGF was unaffected by refractoriness of adipose tissue to GH.     

Differential in vivo activities of bovine growth hormone analogues

In rodents, bovine (b) growth hormone (GH) binds only to GH receptors, while human (h) GH binds to both GH and PRL receptors. The phenotypic consequences of expression of bGH and hGH in transgenic mice are different and, in some cases, opposite. In the present study, site-directed in vitro mutagenesis of the bGH gene was used systematically to eliminate its differences from hGH at one, two, three or four sites suspected of conferring lactogenic activity: D11, H18, S57 and T60, respectively (corresponding to sites 12, 19, 57 and 60 of the bGH molecule). The resulting bGH analogues were expressed in cell lines and in transgenic mice. All of the seven bGH analogues produced retained their ability to bind to GH receptors and exhibited somatogenic activity in vitro and in vivo. However, none of them were able to bind to PRL receptors or to elicit detectable lactogenic response in vitro. Transgenic animals expressing any of the generated analogues were characterized by gigantism and splanchnomegaly. The effects of expression of each of the double, triple or quadruple mutants on the seminal vesicle weight resembled the effects of wild-type hGH and differed from the effects of expression of wild-type bGH. There were differences between the effects of the expression of different bGH analogues on plasma PRL levels and on the PRL response to pharmacological blockade of catecholamine synthesis. Plasma LH levels in ovariectomized females were suppressed by several of the analogues tested, an effect not seen in animals expressing wild-type bGH or hGH. Dopamine turnover in the median eminence of male mice was also altered in animals expressing different bGH analogues but not in those expressing wild-type bGH or hGH. In ovariectomized females, the effects of different bGH analogs on the turnover of dopamine and norepine phrine in the median eminence included changes resembling those detected in animals expressing hGH, as well as alterations differing from the effects of bot h bGH and hGH.The results indicate that biological actions of these bGH analogues cannot be characterized simply in terms of enhanced or reduced somatogenic or lactogenic activity and raise a possibility that different sites, domains or features of the tri-dimensional structure of GH are involved in its actions on different cellular targets     

Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.     

Effects of expression of human or bovine growth hormone genes on sperm production and male reproductive performance in four lines of transgenic mice.

Reproductive performance was studied in transgenic males from lines expressing and transmitting four hybrid genes: mouse metallothionein-I/human growth hormone (GH) (MT/hGH), MT/hGH placental variant (MT/hGH.V), MT/bovine GH (MT/bGH) and phosphoenolpyruvate carboxykinase/bGH (PEPCK/bGH). Each male was exposed to three normal females for 1 week and to three different normal females for another week. Females were examined for vaginal plugs and necropsied on day 14 of pregnancy. Males were killed for analysis of organ weights, numbers of testicular spermatids, numbers of epididymal sperm and measurements of plasma glucose concentration. Fertility of MT/hGH and MT/hGH.V transgenic males was significantly lower than in normal males, primarily because most males failed to impregnate any females. In females that became pregnant, the numbers of corpora lutea, total fetuses and live fetuses did not differ from those in females mated to normal (nontransgenic) males. Fetal crown-rump length on day 14 of pregnancy did not differ between litters sired by normal or by transgenic males. Weights of testes and seminal vesicles were significantly greater in all four types of transgenic male, but daily sperm production per unit weight (g-1) of testis was not affected and epididymal sperm reserves were either normal or slightly higher than normal. Plasma glucose concentrations were significantly higher in PEPCK/bGH mice than in other mice. Average or individual reproductive performance of transgenic males from the various lines did not correlate with any of the parameters examined except for significantly heavier seminal vesicles in MT/hGH and MT/hGH.V males than in normal males; these transgenic males exhibited a high incidence of infertility. Since hGH and hGH.V, but not bGH, are lactogenic in rodents, it was concluded that chronic stimulation of GH and prolactin receptors by ectopically produced human GHs in transgenic mice compromises male fertility by an unknown mechanism. Reduced fertility of transgenic males with MT/hGH or MT/hGH.V hybrid genes is due to failure to inseminate or impregnate females rather than to reduced numbers of spermatozoa or gross changes in the male reproductive system.     

Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes

The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated, by the use, of labelled ovine prolactin) was increased 2–3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.     

Dominant dwarfism in transgenic rats by targeting human growth hormone (GH) expression to hypothalamic GH-releasing factor neurons.

Expression of human growth hormone (hGH) was targeted to growth hormone-releasing (GRF) neurons in the hypothalamus of transgenic rats. This induced dominant dwarfism by local feedback inhibition of GRF. One line, bearing a single copy of a GRF-hGH transgene, has been characterized in detail, and has been termed Tgr (for Transgenic growth-retarded). hGH was detected by immunocytochemistry in the brain, restricted to the median eminence of the hypothalamus. Low levels were also detected in the anterior pituitary gland by radioimmunoassay. Transgene expression in these sites was confirmed by RT-PCR. Tgr rats had reduced hypothalamic GRF and mRNA, in contrast to the increased GRF expression which accompanies GH deficiency in other dwarf rats. Endogenous GH mRNA, GH content, pituitary size and somatotroph cell number were also reduced significantly in Tgr rats. Pituitary adrenocorticotrophic hormone (ACTH) and thyroid-stimulating hormone (TSH) levels were normal, but prolactin content, mRNA levels and lactotroph cell numbers were also slightly reduced, probably due to feedback inhibition of prolactin by the lactogenic properties of the hGH transgene. This is the first dominant dwarf rat strain to be reported and will provide a valuable model for evaluating the effects of transgene expression on endogenous GH secretion, as well as the use of GH secretagogues for the treatment of dwarfism.     

The human growth hormone gene cluster locus control region supports position-independent pituitary- and placenta-specific expression in the transgenic mouse

The human growth hormone (hGH) cluster contains five genes. The hGH-N gene is predominantly expressed in pituitary somatotropes, whereas the remaining four genes, the chorionic somatomammotropin genes (hCS-L, hCS-A, and hCS-B) and hGH-V, are expressed selectively in the placenta. In contrast, the mouse genome contains a single pituitary-specific GH gene and lacks any GH-related CS genes. Activation of the hGH transgene in the mouse is dependent on its linkage to a previously described locus control region (LCR) located -15 to -32 kilobases upstream of the hGH cluster. The sporadic, nonreproducible expression of hCS transgenes lacking the LCR suggests that they may be dependent on hGH LCR activity as well. To determine whether the hCS genes could be expressed with appropriate placental specificity, a series of five transgenic mouse lines carrying an 87-kilobase human genomic insert encompassing the majority of the hGH gene cluster and the entire contiguous LCR was established. All of the hGH cluster genes were appropriately expressed in each of these lines. High level expression of hGH was restricted to the pituitary and hCS to the labyrinthine layer of the placenta. The expression of the GH cluster genes in their respective tissues paralleled transgene copy numbers irrespective of the transgene insertion site in the host mouse genome. These studies have extended the utility of the transgenic mouse model for the analysis of the full spectrum of hGH gene cluster activation. Further, they support a role for the hGH LCR in placental hCS, as well as pituitary hGH gene activation, and expression.     

Acute effects of human growth hormone on liver cells in vitro: a comparison with livers of mice transgenic for human growth hormone.

We have examined the effects of human growth hormone (hGH), in concentrations comparable to those measured in plasma of transgenic mice expressing foreign GHs, on rat liver cells in culture. This treatment produced, within 24 and 48 hr, extreme heterogeneity in liver cell size, enlargement of nuclei, increase in the numbers of large nucleoli and nuclear protrusions, as well as appearance of numerous lipid droplets and accumulation of glycogen. These changes most likely indicate massive metabolic alterations and resemble changes present in vivo in the livers of mice transgenic for hGH and other foreign GHs. Since morphological alterations in vitro were apparent within 24 hr, we conclude that GH acutely and directly affects liver cell morphology and function in vitro and that the pathological lesions in vivo in the livers of transgenic mice are very likely a consequence of GH action.     

Characterization of the specific and sustained <Emphasis Type="Italic">GH1</Emphasis> expression induced by rAAV2/1 in normal adult male rats

Our aim was to investigate the long-term effects of intramuscular injection of rAAV2/1-CMV-GH1 viral particles on GH1 expression in normal adult male rats. We found that specific and sustained GH1 expression did not improve muscle exercise performance despite inducing local muscle hypertrophy. Injection of rAAV2/1-CMV-GH1 had some systemic effects on the liver and heart and on lipid metabolism in the healthy rats. Serum levels of hGH (human growth hormone), insulin, glucose and leptin increased significantly, which might induce insulin resistance. The serum concentration of IGF-1 (insulin-like growth factor 1), IGF-BP3 (insulin-like growth factor binding protein 3) and PIIINP (N-terminal propeptide of type III procollagen) markedly increased at 24 weeks after injection of GH1. In conclusion, GH1 expression driven by AAV2/1 in normal animals did not improve muscle strength but did increase muscle mass and may have systemic effects in healthy animals.     

Lack of a direct effect of homocysteic acid on growth hormone and prolactin receptors.

The effects of homoystine derivatives upon GH and prolactin receptors which are reported to stimulate growth in the rat were investigated. Several concentrations of homocysteic acid, homocysteine, homocysteinethiolactone, homoystine as well as other amino-acids were used in GH and prolactin receptor assay systems to determine their growth promoting or the lactogenic activities. None of the aminoacids used displaced labelled hGH or ovine PRL. It os concluded that derivatives of homoystine stimulate growth by an indirect mechanism and exhibit a lack of a direct stimulation of GH and prolactin receptors.     

Growth hormone regulation of growth hormone-releasing hormone gene expression

Slot-blot hybridization technique was used to evaluate growth hormone-releasing hormone (GHRH) mRNA levels in the hypothalamus of long-term (14 days) hypophysectomized (HPX) rats treated or not with 125 micrograms hGH/rat, twice daily IP, since the first day postsurgery. In addition, mRNA levels were determined in the hypothalamus of short-term (4 days) GH-treated (250 micrograms hGH/rat, twice daily IP) intact rats. GHRH mRNA levels were increased in HPX rats, and GH treatment partially counteracted this rise. Short-term administration of GH decreased GHRH mRNA levels in intact rats. These results, evaluated together with previous findings showing decreased hypothalamic GHRH-like immunoreactivity in both HPX rats and intact rats given GH (6, 7, 9), indicate that GH exerts a negative feedback action on the synthesis and release of GHRH.     

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)     

Human growth hormone stimulates proteinase activities of rabbit bone cells via IGF-I

Human growth hormone (hGH) and human insulin-like growth factor-I (hIGF-I) are known to have a marked influence on osteoclastic formation and bone resorption in an unfractionated rabbit bone cell model. This study investigated the effects of both of these factors on the induction of cysteine-proteinases and matrix metalloproteinase-2 (MMP-2) and MMP-9. After 4 days of rabbit bone cell culture, hGH and hIGF-I significantly modulated cathepsin, MMP-9 (latent form) and MMP-2 (active form) activities. Similar studies were performed in the presence of parathyroid hormone (hPTH). hPTH increased MMP-2 and MMP-9 activities whereas it had no effect on the production of cathepsins by bone cells. When neutralizing anti-hIGF-1 antiserum was added to the culture, the stimulatory effects of hGH were totally abolished, indicating that hGH-modulated cathepsin and metalloproteinase activities were partly mediated by local hIGF-I secretion. Cysteine-proteinase activities released by purified osteoclasts were very low and were not modulated by hGH and h-IGF-I. However, hIGF-I but not hGH increased MMP-2 and MMP-9 activities released by purified osteoclasts. It may be concluded that hGH markedly stimulates the expression of proteinases in total rabbit bone cells via local hIGF-I production by stromal cells. Cysteine-proteinase activities are mainly produced by non-osteoclastic cells, while MMP-2 and MMP-9 modulated by hIGF-I are mainly expressed by osteoclastic cells.