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.     

Pituitary mammosomatotroph adenomas develop in old mice transgenic for growth hormone-releasing hormone

It has been shown that mice transgenic for human growth hormone-releasing hormone (GRH) develop hyperplasia of pituitary somatotrophs and mammosomatotrophs, cells capable of producing both growth hormone and prolactin, by 8 months of age. We now report for the first time that old GRH-transgenic mice, 16 to 24 months of age, develop pituitary mammosomatotroph adenomas. These findings provide conclusive evidence that protracted stimulation of secretory activity can cause proliferation, hyperplasia and adenoma of adenohypophysial cells.     

Unexpected thymic hyperplasia in transgenic mice harboring a neuronal promoter fused with simian virus 40 large T antigen.

The hypothalamic peptide growth hormone-releasing factor (GRF) regulates the secretion and production of growth hormone from the anterior pituitary (M. C. Gelato and G. R. Merriam, Annu. Rev. Physiol. 48:569-591). To study GRF gene regulation, transgenic mice were generated that harbor the human GRF promoter fused to the coding sequences from the simian virus 40 early region. These mice had normal hypothalamic functions but unexpectedly suffered from severe thymic hyperplasia. Immunohistochemical analysis revealed that large T antigen was expressed in the thymic epithelial cells. These cells have endocrine properties and are known to produce thymic hormones [corrected]. The thymic hyperplasia was the apparent consequence of inappropriate production of T-cell maturation factors by epithelial cells and could involve increased self renewal of apparently normal T stem cells in the thymus.     

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.     

Regulation of growth hormone-releasing hormone gene expression and biosynthesis

Growth hormone-releasing hormone (GRH) was initially isolated, characterized, sequenced, and cloned from human tumors and subsequently from the hypothalamus of humans and other animal species. Extensive structure-function studies have indicated the amino terminus to be most important for its biologic action, and the primary mechanism of its bioinactivation occurs by cleavage of an amino terminal dipeptide. The GRH gene is expressed primarily in the hypothalamic arcuate nucleus but also in the placenta. Expression of the GRH gene is regulated by growth hormone in a classical feedback manner, with hypophysectomy leading to increased expression that is reversed by growth hormone treatment. GRH gene overexpression in transgenic mice leads to a syndrome similar to that of ectopic GRH secretion with massive pituitary hyperplasia and markedly enhanced growth. The transgenic mouse has been used for studies of GRH biosynthesis and provides a suitable model for the study of precursor processing to the mature hormone.     

Cell-specific processing of preprosomatostatin in cultured neuroendocrine cells

We have previously found that preprosomatostatin is processed accurately to both somatostatin-14 and somatostatin-28 in pituitary gonadotrophs of transgenic mice. The foreign somatostatin peptides have been shown to enter the regulated secretory pathway of these cells. To determine whether accurate preprosomatostatin processing can occur in any neuroendocrine cell, we introduced preprosomatostatin cDNA expression vectors into several different neuroendocrine cell lines. We found that prosomatostatin was cleaved efficiently to somatostatin-14 and somatostatin-28 in RIN 5F and AtT20 cells, but not in GH4 or PC12 cells. The ability of a particular cell type to process prosomatostatin did not correlate with cellular storage capacity and was independent of the level of biosynthesis of the precursor. These data suggest that prosomatostatin processing requires specific pathways which are present in some neuroendocrine cells, but not in others.     

Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells

Summary Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 ± 160 vs. 387 ± 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10⁻¹² to 10⁻⁸ M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10⁻¹² to 10⁻⁸ M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.     

Effects of growth hormone-releasing factor and somatostatin on growth hormone secretion and cellular cyclic AMP levels. Cultured ovine and rat anterior pituitary cells show markedly different responses

Human pancreatic growth hormone-releasing factor (GRF-44-NH2) stimulated growth hormone (GH) secretion and intracellular cyclic AMP levels in cultured pituitary cells from both sheep and rat. Somatostatin (SRIF), over a wide range of doses and time, showed no significant effect on the elevated cyclic AMP levels in sheep cells, but did block the GH release in a dose-dependent manner. In rat cells, however, SRIF inhibited GRF-stimulated cyclic AMP levels by 75% maximum (still 8-fold greater than the basal levels) and GH release to almost half the basal value. We conclude that somatostatin inhibits GRF-elevated cyclic AMP levels in rat pituitary cells but not in sheep cells.     

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

Summary When the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [³H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.Abbreviations GH growth hormone - GHRF GH-releasing factor - PRL prolactin - SDS sodium dodecyl sulphate - SRIF somatostatin (somatropin release-inhibiting factor)     

Rat atrial natriuretic factor suppresses proopiomelanocortin-derived peptides secretion from both anterior and intermediate lobe cells and growth hormone release from anterior lobe cells of rat pituitary in vitro

Synthetic rat atrial natriuretic factor (ANF) was found to attenuate, in a dose-dependent manner, basal and corticotropin-releasing factor-induced secretion of proopiomelanocortin-derived peptides from cultured anterior and intermediate lobe cells of rat pituitary. ANF was also found to suppress basal and growth hormone-releasing factor-stimulated secretion of growth hormone from anterior lobe cells of rat pituitary. These results, together with reports of the existence of ANF-positive neurons in the hypothalamus and ANF-positive fibers in the median eminence, suggest that hypothalamic ANF is probably involved in the regulation of pituitary hormone secretion, especially that of proopiomelanocortin-derived peptides and growth hormone.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

Intracellular Routing and Release of Caseins and Growth Hormone Produced into Milk from Transgenic Mice

Growth hormone (GH) secretion, in mammary tissue from transgenic mice, containing a chimeric gene composed of the regulatory region of whey acidic protein gene and the structural region of GH gene, was compared to casein secretion. GH was expressed in milk and for a small percentage (1:1000) in blood as revealed by SDS-polyacrylamide gel electrophoresis and radio-immunoassay. As attested by immunofluorescence and immunogold electron microscopy, caseins and GH followed the same secretory pathway. However, contrary to caseins, which are essentially in micellar form, GH was detected in a nonaggregated form in secretory vesicles and in the lumen of the acini. Newly synthesized caseins and GH were carried simultaneously, mainly to the lumen of the acini, but also to the base of the cell. Secretion of newly synthesized proteins was increased by prolactin (PRL). As shown by immunoblotting, the proportion of GH versus other proteins, secreted in the presence of PRL was not modified, suggesting that GH secretion is subjected to the same hormonal regulation by PRL as other milk proteins. These results show that, in lactating mammary epithelial cells from transgenic mice, a recombinant GH and the caseins are carried simultaneously to the lumen and suggest that secretion of both proteins is increased by PRL during the same time course. Transport of these newly synthesized proteins occurs also to the base of the cell.     

Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones

Cells derived from rat islet tumor and grown in culture (parent cells-RIN-m) and two clones obtained from them were used to study the effect of various secretagogues on insulin, glucagon, and somatostatin secretion. Parent cells secreted all three hormones in various quantities, while clone 5F secreted predominantly insulin and clone 14B secreted predominantly somatostatin. The secretory behavior of these cells were compared to each other and to that of normal islets. In general, as in the case of normal islets, insulin secretion was stimulated by calcium, potassium, tolbutamide, theophylline, and glucagon. It was inhibited by somatostatin. Glucagon secretion was stimulated by calcium, arginine, and theophylline. Somatostatin secretion was stimulated in clone 14B by arginine, tolbutamide, theophylline, and insulin. These cells differ from normal islets, in that they do not respond to glucose or arginine with increased insulin secretion. Also somatostatin failed to inhibit glucagon secretion. The similarity in insulin secretory responses of parent cells and clone 5F suggests that local or paracrine islet hormone secretion plays only a negligible role in the control of other hormone secretion in these cells.     

Anatomy of the Hypophysiotropic Somatostatinergic and Growth Hormone-releasing Hormone System Minireview

The central control of growth hormone (GH) secretion from the pituitary gland is ultimately achieved by the interaction between two hypothalamic neurohormones, somatostatin which inhibits and growth hormone-releasing hormone (GHRH) which stimulates GH release. The regulation of the somatostatin and GHRH release from the hypothalamus is regulated by a range of other neuropeptides, neurotransmitters, neurohormones. In this mini review we attempt to provide a short summary covering the anatomy and chemical characteristics of the various cell populations regulating GH secretion as a tribute to Miklós Palkovits who pioneered the field of functional neuroanatomy of hypothalamic networks.Special Issue Dedicated to Miklós Palkovits.     

Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene

A transgenic animal model system was used to analyze the mitogenic effects of GRF on its target cell, the pituitary somatotroph. We have previously established a strain of mice that express a mouse metallothionein-I/human GRF (hGRF) fusion gene, and that grow to be abnormally large due to GH hypersecretion. We show here that chronic GRF production in these mice leads to the development of enormous pituitary glands. The increase in pituitary size appears to be largely the result of a selective proliferation (hyperplasia) of somatotrophs, the GH-producing cells. This observation provides direct evidence that a neuropeptide may act as a specific trophic factor for its target cell. In addition to this effect on pituitary development, we find that the pituitary is a major site of expression of mouse metallothionein-I/hGRF mRNA, and of hGRF peptide. This tissue specificity was unexpected in that neither component of the fusion gene is highly expressed in the normal pituitary. It suggests that pituitary somatotrophs might produce and respond to GRF in an essentially autocrine fashion in these transgenic animals.     

Peptidase inactivation of hypothalamic releasing hormones.

With the structural characterization of the hypothalamic hormones, luteinizing hormone-releasing hormone (LH-RH), thyrotrophin-releasing (TRH), melanocyte-stimulating hormone release-inhibiting hormine (MIH), and growth hormone release-inhibiting hormone, (GH-RIH or somatostatin), it has been possible to investigate their enzymic inactivation by peptidases which are present at various sites in the body. Enzymes may play an important part in the control of polypeptide hormone levels and the peptidases acting on these four hypothalamic hormones may regulate the amount of TRH, LH-RH, MIH and somatostatin released from the hypothalamus, or their action at the level of the pituitary and their removal from the circulation. By studying the peptidase enzymes, further information may be obtained on the physiological mechanisms controlling the secretion and actions of hypothalamic hormones, as well as on the design of analogues with increased or competitive activity.     

Effect of chronic hyperghrelinemia on ingestive action of ghrelin

The stomach hormone ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Systemic administration of ghrelin will cause elevations in growth hormone (GH) secretion, food intake, adiposity, and body growth. Ghrelin also affects insulin secretion, gastric acid secretion, and gastric motility. Several reports indicate that repeated or continuous activation of GHS-R by exogenous GHSs or ghrelin results in a diminished GH secretory response. The purpose of this study was to examine the extent to which the acute stimulation of food intake by exogenous ghrelin is altered by chronic hyperghrelinemia in transgenic mice that overexpress the human ghrelin gene. The present findings show that the orexigenic action of exogenous ghrelin is not diminished by a chronic hyperghrelinemia and indicate that the food ingestive pathway of the GHS-R is not susceptible to desensitization. In contrast, the epididymal fat pad growth response, like the GH response, to exogenous ghrelin is blunted in ghrelin transgenic mice with chronic hyperghrelinemia.     

Use of transgenic mice to study the routing of secretory proteins in intestinal epithelial cells: analysis of human growth hormone compartmentalization as a function of cell type and differentiation [published erratum appears in J Cell Biol 1990 Jan;110(1):following 227]

The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.     

Pituitary response to growth hormone-releasing factor in rats with functional or anatomical lesions of the central nervous system that inhibit endogenous growth hormone secretion

The pituitary growth hormone (GH) response to the growth hormone-releasing factor, hpGRF-44, was evaluated in male rats with various lesions of the central nervous system. These included an electrical lesion of the ventromedial hypothalamus, a chemical lesion of the arcuate nucleus induced by neonatal treatment with monosodium glutamate, a functional lesion of catecholamine synthesis with alpha-methyl-p-tyrosine or a functional lesion of catecholamine storage with reserpine. The first three lesions appear to partially inhibit normal somatostatin secretion since in every instance hpGRF-44 administration induced a significant increase in plasma GH concentrations. In contrast, reserpine blocked the GH response to hpGRF-44, presumably by stimulating somatostatin secretion. The pituitary GH response to hpGRF-44 in the above described models was enhanced by pretreatment of the rats with antibodies against somatostatin. The pituitary GH response to repeated injections of hpGRF-44 was also evaluated in rats with an anatomical lesion of the arcuate nucleus or a functional lesion of catecholamine synthesis. The maximum GH response did not vary over time to the repeated injections of hpGRF-44 in rats with lesions of the arcuate nucleus; however, interruption of catecholamine synthesis resulted in a significant decrease in the GH response to hpGRF-44 over time.