Estradiol and testosterone concentrations increase with fasting in weaned pups of the northern elephant seal (Mirounga angustirostris).

Although neonatal development is generally associated with increased levels of circulating testosterone (T) and estradiol (E2), food deprivation may inhibit steroidogenesis. Therefore, these potentially conflicting stimuli were examined in fasting weaned northern elephant seal (Mirounga angustirostris) pups by measuring serum concentrations of T, E2, progesterone (P4), and luteinizing hormone (LH) by either radioimmunoassay (P4, LH) or enzymeimmunoassay (T, E2). Blood samples were obtained from 20 male and 20 female pups at both early (<1 wk postweaning) and late (6-8 wk postweaning) periods during their natural postweaning fast. T in males (early: 2.9 +/- 0.4 ng/mL; late: 16 +/- 2 ng/mL; P < 0.0001) and E2 in females (early: 42 +/- 6 pg/mL; late: 67 +/- 5 pg/mL; P < 0.01) increased between the two measurement periods, while P4 (early: 2.5 +/- 0.3 ng/mL; late: 2.1 +/- 0.3 ng/mL; P > 0.05) did not. LH increased (early: 46 +/- 4 pg/mL; late: 65 +/- 6 pg/mL; P < 0.05) in males but not in females (early: 69 +/- 9 pg/mL; late: 65 +/- 6 pg/mL; P > 0.05). Increases in LH and T suggest that LH may stimulate T secretion. Alternatively, relatively low concentrations of LH in both males and females may reflect negative feedback inhibition imposed by elevated T and E2 concentrations. Despite the inherent postweaning fast, concentrations of T and E2 increased, suggesting that they may be critical for the continued development of pups. Therefore, compensatory mechanisms may exist that alleviate the fasting-induced inhibition of gonadal steroidogenesis during neonatal development in elephant seal pups.

GH and ghrelin increase with fasting in a naturally adapted species, the northern elephant seal (Mirounga angustirostris).

After nursing, pups of the northern elephant seal (Mirounga angustirostris) are approximately 46% body fat and rely almost entirely on the oxidation of their large fat stores to sustain their metabolism for the ensuing 8-12 week postweaning fast, which is a natural component of their life history. Thus, fasting pups provide an ideal opportunity to examine the hormonal alterations associated with prolonged food deprivation in a naturally adapted model. Cortisol, ghrelin, glucagon, growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, blood urea nitrogen (BUN), glucose and non-esterified fatty acids (NEFA) were examined in 20 male and 20 female pups blood sampled early (<1 week postweaning) and late (6-8 weeks postweaning) during the fast. Mean cortisol, ghrelin, GH, and glucagon increased 1.8-, 1.8-, 1.4-, and 2.3-fold between early and late periods, while mean IGF-I and insulin decreased 97% and 38%, respectively. NEFA increased 2.3-fold, while BUN and glucose decreased 46% and 11%, respectively. NEFA was significantly and positively correlated with cortisol and GH; individually; however, when the relationship was examined as a multiple regression the correlation improved suggesting that cortisol and GH act synergistically to promote lipolysis during the fast. GH and BUN were negatively and significantly correlated between early and late fasting suggesting that GH may promote protein sparing as well. The decrease in glucose may be responsible for stimulating glucagon, resulting in the maintenance of relative hyperglycemia. The increases in cortisol, ghrelin, glucagon, and GH suggest that these hormones may be integral in mediating the metabolism of seal pups during prolonged fasting.

[Oligoastrocytoma with signet-ring cell differentiation. A morphological, ultrastructural and immunohistochemical study]. / Oligoastrocitoma con diferenciación en células en anillo de sello. Estudio morfológico, ultraestructrural e inmunohistoquímico.

We present a case of a mixed glial tumor (oligoastrocytoma) with signet-ring cells. This cellular feature is a rare differentiation in glial tumors of the central nervous system. Histological, immunohistochemical and ultrastructural findings have been analyzed. Signet-ring cells showed intense expression with GFAP, S-100 and vimentin. A differential diagnosis with other primary brain tumors and cerebral metástases with signet-ring cell differentiation was discussed.

Oligoastrocitoma con diferenciación en células en anillo de sello. Estudio morfológico, ultraestructural e inmunohistoquímico / Oligoastrocytoma with signet-ring cell differentiation. A morphological, ultrastructural and immunohistoche-mical study

Presentamos un caso de tumor glial mixto (oli-goastrocitoma) con células en anillo de sello. Esta diferenciación celular es rara en tumores gliales del sistema nervioso central. En este estudio analizamos las características morfológicas, ultraestructurales e inmunohistoquímicas del tumor. Las células neoplásicas con características morfológicas en anillo de sello mostraban expresión de GFAP, S-100 y vimentina. En la discusión consideramos el diagnóstico diferencial con otros tumores primarios del sistema nervioso central, así como con metástasis cerebrales de neoplasias con diferenciación en células en anillo de sello (AU)

Structure and regulation of expression of the mouse GH receptor.

GH-binding protein (GHBP) in the mouse consists of a ligand-binding domain, which is identical to the extracellular portion of the GH receptor (GHR), and a hydrophilic C-terminal domain, in place of the transmembrane and intracellular domains of the GHR. The two proteins are encoded by separate mRNAs which are derived from a single gene by alternative splicing. Determination of the gestational profiles of GHR and GHBP mRNA expression in mouse liver and placenta shows that in the liver, the 1.4 kb mRNA corresponding to the mouse GHBP increases approximately 20-fold between non-pregnant and late pregnant mice, whereas the relative increase in the expression of the 4.2 kb mouse GHR was 8-fold. The rise in the steady-state levels of both mRNAs began on day 9 of gestation. Mouse GHBP mRNA levels continue to rise until day 15 of pregnancy, while GHR mRNA abundance reaches a plateau by day 13. By elucidating the temporal changes in GHR and GHBP mRNA abundance during pregnancy and lactation in multiple maternal tissues and by assessing the ontogeny of these mRNAs in fetal and early postnatal mouse liver, our studies have demonstrated that the alternative splicing of mouse GHR/GHBP mRNA precursor is regulated in a tissue-, developmental stage- and physiological state-specific manner. In vitro studies using hepatocytes in culture have begun to elucidate the hormonal factor(s) involved in the gestation control of the expression of GHR and GHBP. Treatment of hepatocytes with GH or estradiol (E2) alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR mRNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2- and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. Our studies in the mouse suggest that GHBP is an important cell-surface receptor for GH in the liver. These studies postulate that an arginine-glycine-aspartic acid sequence found on mouse GHBP but absent in other species is responsible for the association of GHBP with the plasma membrane by binding to one or more integrins on the surface of liver cells.

[Idiopathic granulomatous hypophysitis. Morphological and immunohistochemical study of a case]. / Hipofisitis granulomatosa idiopática. Estudio morfológico e inmunohistoquímico de una observación.

Inflammatory diseases of the pituitary gland constitute a group of interest because of their scarce frequency, because the disorder presents with symptoms of hypopituitarism and expanding sellar mass and because of their therapeutics implications. We present one case of idiopathic granulomatous hypophysitis, in a 55-years-old patient with daily headaches, panhypopituitarism and a sellar mass lesion. Granulomatous hypophysitis is characterized by granulomas with epithelioid histiocytes and multinucleated giant cells but also shows lymphocyte collections. With respect to immunohistochemistry our results show histiocytes (CD68+) and an heterogeneous inflammatory infiltrate (CD45RO+ y CD20+). We analyze the differential diagnosis with another granulomatous processes, infectious or not infectious, and with the histiocytosis. We examine the possible relation with the lymphocytic hypophysitis.

Structurally distinct membrane-associated and soluble forms of GH-binding protein in the mouse.

It has previously been shown that the large increase in GH-binding capacity of mouse liver microsomes during pregnancy is due largely to an increase in the amount of GH-binding protein (GHBP), with a more modest increase in GH receptor (GHR). Here we show that mouse liver GHBP is predominantly present as a membrane-associated protein structurally distinct from the soluble form of GHBP present in serum. Liver GHBP is associated with both intracellular membranes and the plasma membrane. Membrane-associated GHBP and soluble GHBP appear to be identical polypeptides distinguished by the addition of different N-glycans to asparagine residues. The pattern of release of GHBP from membranes by various treatments indicates that GHBP associates with membranes through noncovalent interactions with one or more membrane protein, but not with GHR. Covalent crosslinking provides evidence for several GHBP-associated membrane polypeptides, with molecular masses ranging from 58 kDa to over 200 kDa. These studies in the mouse and similar studies in the rat suggest that GHBP is an important cell-surface receptor for GH in the liver of these species. We postulate that an arginine-glycine-aspartic acid sequence found on rat and mouse GHBP but absent in other species is responsible for the association of GHBP with the plasma membrane by binding to one or more integrins on the surface of liver cells.

Hipofisitis granulomatosa idiopática. Estudio morfológico e inmunohistoquímico de una observación / Hypophysitis granulomatous idiopathic. Morphological and Immunohistochemical study. Case report

Los procesos inflamatorios de la glándula hipofisaria constituyen un grupo de interés por su escasa frecuencia. Con frecuencia se presentan como lesiones ocupantes de espacio y cursan con cuadros de panhipopituitarismo. Ello platea problemas de diagnostico diferencial con implicaciones terapéuticas importantes En este trabajo presentamos un caso de hipofisitis granulomatosa idiopática, en una paciente de 55 años con crisis diarias de cefalea y panhipopituitarismo y un aumento de tamaño de la glándula hipofisaria. La morfología destaca una lesión inflamatoria granulomatosa no necrotizante, con células multinucleadas gigantes y un infiltrado linfoplasmocitario. El estudio inmunohistoquímico muestra la presencia de macrófagos (CD68+) y un infiltrado inflamatorio heterogéneo (CD45RO y CD20+).Se analiza el diagnóstico diferencial con otros procesos granulomatosos infecciosos o no infecciosos y con la histiocitosis.En la discusión examinamos la posible relación con la hipofisitis linfocitaria (AU)

Growth and characterization of N-methyl-N-nitrosourea-induced mammary tumors in intact and ovariectomized rats.

It is well established that 85-90% of chemically induced mammary tumors in rats will disappear or diminish significantly in size after the ovaries are removed from the animal. However, it is less well established whether a high percentage of these mammary tumors will grow back with prolonged time after ovariectomy. It is also not known what changes in gene expression take place in the tumors as they develop an independence from hormones for growth. This study was carried out to investigate this. Virgin, 50-day-old female Sprague-Dawley rats were injected with N-methyl-N-nitrosourea (MNU) at the dose of 50 mg MNU/kg body wt. When at least one mammary tumor had grown to 1.0-1.5 cm in one dimension, the animal was bilaterally ovariectomized and reduction and then re-growth of the tumors monitored. Control animals were treated identically except they were not ovariectomized when tumors appeared. Re-growths and new tumors and tumors that developed in the control rats were removed when they reached 1.0-1.5 cm in diameter and all animals were killed 25 weeks after the MNU injection. All the animals in the study (100%) developed mammary tumors after MNU injection with an average latency of 56.5 days. After ovariectomy, 93% of the tumors showed 50% or more reduction in size and 76% of the tumors could not be detected by palpation. However, in 96% of the animals where tumor reduction or disappearance occurred, a re-growth or new mammary tumor development took place with an average latency period of 52.8 days from the day of ovariectomy. Of these post-ovariectomy tumors, 36% occurred at a location where tumors had developed prior to ovariectomy, but 64% appeared at new locations. The circulating levels of 17beta-estradiol (E2) was undetectable in the ovariectomized (OVX) rats and significant reduction was seen in the serum concentrations of progesterone (P4), prolactin (PRL), growth hormone (GH) and insulin-like growth factor-I (IGF-I). The tumors from the OVX rats showed indications of progression as evident from loss of differentiation and invasive characteristics. Comparison between tumors from OVX and intact rats revealed a significantly increased expression of P450 aromatase and elevated activation of extracellular signal-regulated kinase 1 and 2, but reduced levels of the progesterone receptor and cyclin D1 in OVX rats. However, the estrogen receptor (ER) content remained similar in tumors from both groups, at least at the protein level, and so did the expression of IGF-I, IGF-II, insulin receptor substrate-1 (IRS1), IRS-2 and epidermal growth factor receptor. IGF-I receptor (IGF-IR) and ErbB-2 were expressed, respectively, in 50 and 70% of the tumors from the OVX animals, whereas these genes were expressed in 100% of the tumors from the intact rats. It is concluded that chemically induced rat mammary tumors may still depend on the ER and local syntheses of E2 and growth factors for growth initially after ovariectomy. However, as these tumors progress, they develop a more aggressive phenotype and lose their dependency on the ER and possibly growth factors.

Prolactin receptor expression in the epithelia and stroma of the rat mammary gland.

The importance of prolactin (PRL) in regulating growth and differentiation of the mammary gland is well known. However, it is not well established whether PRL acts solely on the mammary epithelia or if it can also directly affect the mammary stroma. To determine where PRL could exert its effects within the mammary gland, we investigated the levels of expression and the localization of the PRL receptor (PRLR) in the epithelia and stroma of the rat mammary gland at different physiological stages. For these studies, we isolated parenchymal-free 'cleared' fat pads and intact mammary glands from virgin, 18-day-pregnant and 6-day-lactating rats. In addition, intact mammary tissues were enzymatically digested to obtain epithelial cells, free of stroma. The mammary tissues, intact gland, stroma and isolated epithelia, were then used for immunocytochemistry, protein extraction and isolation of total RNA. PRLR protein was detected in tissues using specific polyclonal antisera (PRLR-l) by immunocytochemistry and Western blot analysis. Messenger RNA for PRLR was measured by ribonuclease protection assay. Immunocytochemistry and Western blots with the PRLR-1 antisera detected PRLR in wild-type rat and mouse tissues, whereas the receptor protein was absent in tissues from PRLR gene-deficient mice. PRLR was found to be present both in the epithelia and stroma of mammary glands from virgin, pregnant and lactating rats, as determined by immunocytochemistry and Western blotting. Western blots revealed the predominance of three bands migrating at 88, 90 and 92 kDa in each of the rat mammary samples. These represent the long form of the PRLR. During pregnancy and lactation, PRLR protein increased in the epithelial compartment of the mammary gland but did not change within the stromal compartment at any physiological stage examined. We also found PRLR mRNA in both the epithelia and stroma of the mammary gland. Again, the stroma contained lower levels of PRLR mRNA compared with the epithelia at all physiological stages examined. Also, the PRLR mRNA levels within the stroma did not change significantly during pregnancy or lactation, whereas PRLR mRNA within the epithelia increased twofold during pregnancy and fourfold during lactation when compared with virgin rats. We conclude from this study that PRLR is expressed both in the stromal and epithelial compartment of the mammary gland. This finding suggests PRL may have a direct affect on the mammary stroma and by that route affect mammary gland development.

PRL, placental lactogen, and GH induce NA(+)/taurocholate-cotransporting polypeptide gene expression by activating signal transducer and activator of transcription-5 in liver cells.

We investigated the transcriptional regulation of the Na(+)/taurocholate cotransporting polypeptide gene by PRL, placental lactogen, and GH. In primary hepatocytes, ovine PRL induced a dose-dependent phosphorylation and nuclear translocation of signal transducers and activators of transcription-5a and -5b, but not -1 or -3, whereas mouse placental lactogen I and rat GH activated -5a, -5b, and -1. In EMSAs, ovine PRL, mouse placental lactogen I, and rat GH increased the specific DNA binding of nuclear signal transducer and activator of transcription-5 to its consensus element in both transfected HepG2 cells and primary hepatocytes. PRL, placental lactogen I, and GH also increased Na(+)/taurocholate cotransporting polypeptide mRNA expression in hepatocytes from control and pregnant (mouse placental lactogen I) rats. Genistein, a phosphotyrosine kinase inhibitor, inhibited PRL-induced signal transducer and activator of transcription-5 activation and Na(+)/taurocholate-cotransporting polypeptide mRNA. In HepG2 cells transiently cotransfected with either the long form of the rat PRL receptor or rat GH receptor, signal transducer and activator of transcription-5a and a -5-responsive luciferase expression vector containing the Na(+)/taurocholate-cotransporting polypeptide promoter, mouse placental lactogen I, like ovine PRL, activated -5a via the long form of the rat PRL receptor; whereas rat GH activated -5a via rat GH receptor, leading to transactivation of the Na(+)/taurocholate-cotransporting polypeptide promoter. These data establish that PRL and placental lactogen I induce Na(+)/taurocholate-cotransporting polypeptide gene expression via signal transducer and activator of transcription-5 proteins in liver, and indicate that these hormones play an important role in regulating liver metabolic function.

Short-term exposure to pregnancy levels of estrogen prevents mammary carcinogenesis.

It is well established that pregnancy early in life reduces the risk of breast cancer in women and that this effect is universal. This phenomenon of parity protection against mammary cancer is also observed in rodents. Earlier studies have demonstrated that short-term administration of estradiol (E) in combination with progesterone mimics the protective effect of parity in rats. In this study, the lowest effective E dosage for preventing mammary cancer was determined. Rats were injected with N-methyl-N-nitrosourea at 7 weeks of age; 2 weeks later, the rats were subjected to sustained treatment with 20 microg, 100 microg, 200 microg, or 30 mg of E in silastic capsules for 3 weeks. Treatments with 100 microg, 200 microg, and 30 mg of E resulted in serum levels of E equivalent to those of pregnancy and were highly effective in preventing mammary cancer. E treatment (20 microg) did not result in pregnancy levels of E and was not effective in reducing the mammary cancer incidence. In another set of experiments, we determined the effect of different durations of E with or without progesterone treatments on mammary carcinogenesis. These experiments indicate that a period as short as one-third the period of gestation is sufficient to induce protection against mammary carcinogenesis. The pioneering aspect of our study in contrast to long-term estrogen exposure, which is thought to increase the risk of breast cancer, is that short-term sustained treatments with pregnancy levels of E can induce protection against frank mammary cancer.

Parous rats regain high susceptibility to chemically induced mammary cancer after treatment with various mammotropic hormones.

Parity in humans and rats provides significant protection against mammary tumor development. This study was carried out to investigate whether treatment of parous rats with mammotropic hormones would affect methyl-nitrosourea (MNU)-induced mammary carcinogenesis. Parous rats were treated with 17beta-estradiol (E2), progesterone (P4) and thyroxine (T4) alone or in combination. E2 (20 microg/60 days) and P4 (20 mg/60 days) were administered by silastic tubing and T4 in the drinking water (3 microg T4/ml). Hormonal treatments commenced 7 days before MNU injection and continued for 33 weeks. Animals were palpated weekly for tumor detection. The effects of the hormonal treatments on the circulating concentrations of E2, P4, growth hormone (GH), prolactin (PRL), T4 and insulin-like growth factor-I (IGF-I) after 7 days of treatment, the time of MNU injection, was assessed. Animals treated with E2 had significantly elevated circulation concentrations of GH, PRL and P4, and serum levels of E2 were more consistent in this group than in the other animal groups. P4 treatment caused elevation in P4 concentration in serum but did not affect the circulating levels of other hormones. The proliferation of the mammary gland at the time of MNU injection was elevated in animal groups treated with E2 either alone or with P4 and T4 and in animals treated with P4 alone, but the mammary gland was most differentiated in untreated parous rats and least in animals treated with E2 either alone or with P4 and T4. Mammary tumor incidence was 10% in parous rats that did not receive any hormonal treatment. Treatments with E2 or P4 alone significantly increased the susceptibility of parous animals to 67 and 50.0%, respectively; a tumor incidence similar to that of untreated AMV rats (64%). Parous rats treated with E2 plus P4 had tumor incidence higher than 90%. T4 administered did not affect mammary carcinogenesis.

Alternative 5'-untranslated regions of mouse GH receptor/binding protein messenger RNA are derived from sequences adjacent to the major L2 promoter.

Heterogeneity of 5' untranslated region (5'UTR) sequences is a common feature of growth hormone receptor/binding protein (GHR/BP) mRNA from a number of species. Two major 5'UTR sequences (designated L1 and L2 in the mouse) have been cloned from rodents, ruminants and primates, and are known to correspond to transcripts generated from independently regulated promoters. A variable number of other 5'UTRs with diverse sequences have been cloned from rat, human and bovine tissues. To characterize alternative 5'UTR usage in mouse GHR/BP mRNA, we carried out 5' rapid amplification of cDNA ends using RNA from non-pregnant mouse liver and adipose tissue. Three novel 5'UTR sequences were obtained. Sequencing of genomic DNA revealed that exons corresponding to these three sequences are clustered within 1 kb downstream of the exon encoding 5'UTR L2, and the associated L2 promoter. The novel 5'UTRs are present at very low levels relative to the total pool of GHR/BP mRNA in liver, fat, kidney, and mammary gland as determined by ribonuclease protection assays. On the basis of these data, we propose that these 5'UTR sequences may result from the use of cryptic transcription start sites and splice donor sites under the influence of the adjacent L2 promoter/enhancer region.

Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia.

The factors that regulate pancreatic beta cell proliferation are not well defined. In order to explore the role of murine placental lactogen (PL)-I (mPL-I) in islet mass regulation in vivo, we developed transgenic mice in which mPL-I is targeted to the beta cell using the rat insulin II promoter. Rat insulin II-mPL-I mice displayed both fasting and postprandial hypoglycemia (71 and 105 mg/dl, respectively) as compared with normal mice (92 and 129 mg/dl; p < 0.00005 for both). Plasma insulin concentrations were inappropriately elevated, and insulin content in the pancreas was increased 2-fold. Glucose-stimulated insulin secretion by perifused islets was indistinguishable from controls at 7.5, 15, and 20 mm glucose. Beta cell proliferation rates were twice normal (p = 0. 0005). This hyperplasia, together with a 20% increase in beta cell size, resulted in a 2-fold increase in islet mass (p = 0.0005) and a 1.45-fold increase in islet number (p = 0.0012). In mice, murine PL-I is a potent islet mitogen, is capable of increasing islet mass, and is associated with hypoglycemia over the long term. It can be targeted to the beta cell using standard gene targeting techniques. Potential exists for beta cell engineering using this strategy.

Sensitivity of the cervical transformation zone to estrogen-induced squamous carcinogenesis.

Regions where one type of epithelium replaces another (metaplasia) have a predilection for cancer formation. Environmental factors are closely linked to metaplastic carcinogenesis. In particular, cervical cancers associated with human papillomavirus (HPV) infection develop primarily at the transformation zone, a region where metaplastic squamous cells are detected in otherwise columnar epithelial-lined endocervical glands. Previously, we reported estrogen-induced multistage vaginal and cervical carcinogenesis in transgenic mice expressing HPV16 oncogenes in basal squamous epithelial cells. In the present study to investigate the threshold neoplastic response to exogenous estrogen, we treated groups of transgenic mice with lower hormone doses. A 5-fold reduction in estrogen dose induced squamous carcinogenesis solely at the cervical transformation zone compared with other reproductive tract sites. Further study delineated stages of transformation zone carcinogenesis, including formation of hyperplastic lower uterine glands and emergence of multiple foci of squamous metaplasia from individual stem-like glandular reserve cells, followed by neoplastic progression of metaplasia to dysplasia and squamous cancer. We propose that a combination of low-dose estrogen and low-level HPV oncogene expression biases transformation zone glandular reserve cells toward squamous rather than columnar epithelial fate decisions. Synergistic activation of proliferation by viral oncoprotein cell cycle dysregulation and estrogen receptor signaling, together with altered paracrine stromal-epithelial interactions, may conspire to support and promote neoplastic progression and cancer formation.

Growth hormone (GH) and 17beta-estradiol regulation of the expression of mouse GH receptor and GH-binding protein in cultured mouse hepatocytes.

In the present study, primary mouse hepatocytes from 8- to 10-week-old virgin female Swiss-Webster mice were perfused with collagenase (100 U/ml) using the two-step method. Isolated hepatocytes were plated in a rat tail type I collagen sandwich configuration to examine the regulation of GH receptor (GHR) and GH-binding protein (GHBP) expression by GH and 17beta-estradiol (E2). After 48 h of initial plating, hepatocytes were divided into groups of five replicates and treated for 24 h with medium containing no hormones (controls), GH (100 ng/ml), E2 (10(-9) M), E2 (10(-9) M) plus GH (100 ng/ml), or E2 plus GH and ICI 182-780 at different concentrations. Treatment of hepatocytes with GH or E2 alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR messenger RNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2 and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. In the present study we have shown that in cultured hepatocytes from virgin female mice, GH or E2 alone did not affect the concentrations of GHBP and GHR. However, E2 and GH together significantly up-regulated GHR and GHBP expression.

Structure and expression of the mouse growth hormone receptor/growth hormone binding protein gene.

The mouse growth hormone receptor/growth hormone-binding protein (GHR/BP) gene produces several distinct mRNA forms through alternative splicing, including mRNAs encoding the membrane-bound growth hormone receptor (GHR) and the soluble growth hormone-binding protein (GHBP). Transcripts are also heterogeneous in their 5' regions due to alternative selection of two major 5' untranslated region (5'UTR) sequences, designated L1 and L2. Here we report the cloning of all mouse GHR/BP coding exons as well as the exon encoding 5'UTR L2, the most widely expressed 5'UTR. The mouse GHR/GHBP gene contains 11 coding exons, 9 of which are homologous in size and sequence to human GHR exons 2-10. The two mouse exons that do not have homologs in the human gene are designated exons 4B and 8A. Exon 4B, located between exons 4 and 5, encodes an 8-amino acid segment of the ligand binding domain that is unique to mouse GHR and GHBP. Analysis by reverse transcriptase-polymerase chain reaction indicated that exon 4B is constitutively present in mouse GHR and GHBP mRNA. Exon 8A encodes the GHBP hydrophilic tail and 3'UTR sequence. 5'UTR L2 is encoded by a single exon located at least 27 kb upstream of exon 2 and at least 12 kb upstream of the exon encoding 5'UTR L1. The transcription start sites of UTR L2 were mapped and the 5' flanking region sequenced. The exon and proximal promoter region are GC rich, and share a high level of conservation with the equivalent exons in the sheep, bovine and human GHR genes. A CCAAT motif and several putative Sp1 motifs are present, and there is no TATA box. Homology between the mouse sequence and other species is limited to a region of 450 bp upstream of the exon due to the insertion of a fragment of a LINE-1 element upstream of the mouse L2 exon. Ribonuclease protection assays were used to confirm that 5'UTR L2 is widely expressed in multiple tissues and is the predominant form of transcript except in the liver during pregnancy, in which 5'UTR L1 is the major form.

Transcriptional upregulation of hepatic GH receptor and GH-binding protein expression during pregnancy in the mouse.

In the mouse, GH-binding protein (GHBP) and GH receptor (GHR) are encoded by a single gene via alternative splicing. We previously demonstrated that the steady-state levels of the GHR and GHBP mRNAs are significantly elevated in mouse liver during pregnancy. Hepatic GHR and GHBP mRNAs are associated primarily with one of two different 5' untranslated regions (5' UTRs), designated 5' UTR Liver1 (L1) and Liver2 (L2). Distinct promoters associated with each of these 5' UTRs have recently been characterized. In the present study, we have investigated the role of transcriptional activation in the pregnancy-induced upregulation of GHR and GHBP mRNAs in liver. We also report on the relative contribution of the 5' UTR L1 and 5' UTR L2 promoters to the hepatic expression of the GHR/GHBP gene in the liver. Our approach was to compare, by ribonuclease protection assay (RPA), GHR/GHBP transcript levels in hepatic nuclear and total cellular RNA samples from virgin and late-pregnant mice. In these RPAs we utilized riboprobes that were complementary to the coding region of GHR/GHBP transcripts, as well as to the two noncoding, alternative first exons 5' UTR L1 and L2. When employing the coding region probe, RPAs revealed that the gestational increase in the levels of nuclear GHR/GHBP transcripts were statistically comparable with the increase in GHR/GHBP transcript levels in total cellular RNA. This finding suggests that enhanced transcriptional activity, rather than increased cytoplasmic half-life, is responsible for the upregulation of GHR/GHBP RNA in the pregnant liver. In RPAs utilizing the noncoding region probes, both nuclear and total cellular GHR/GHBP transcripts associated with 5' UTR L1 were significantly upregulated in late-pregnant as compared with virgin mice. In contrast, the levels of both nuclear and total GHR/GHBP transcripts associated with 5' UTR L2 were comparable between nonpregnant and pregnant animals. Moreover, 5' UTR L2-containing transcripts were present at levels that were only 3-5% of the 5' UTR L1-associated transcripts in the late-pregnant liver. Thus, we conclude that the gestational upregulation of the GHR/GHBP gene in the mouse liver can be ascribed to the significantly enhanced transcriptional activity of the 5' UTR L1 promoter.