HOXA1-stimulated oncogenicity is mediated by selective upregulation of components of the p44/42 MAP kinase pathway in human mammary carcinoma cells.

Expression of homeobox A1 (HOXA1) results in oncogenic transformation of immortalized human mammary epithelial cells with aggressive tumor formation in vivo. However, the mechanisms by which HOXA1 mediates oncogenic transformation is not well defined. To identify molecules that could potentially be involved in HOXA1-mediated oncogenic transformation, microarray analysis was utilized to characterize and compare the gene expression pattern in response to forced expression or depletion of HOXA1 in human mammary carcinoma cells. Gene expression profiling identified that genes involved in the p44/42 mitogen-activated protein (MAP) kinase activation pathway (GRB2, MAP kinase kinase (MEK1) and SDFR1) or p44/42 MAP kinase-regulated genes (IER3, EPAS1, PCNA and catalase) are downstream expression targets of HOXA1. Forced expression of HOXA1 increased GRB2 and MEK1 mRNA and protein expression and increased p44/42 MAP kinase phosphorylation, activity and Elk-1-mediated transcription. Use of a MEK1 inhibitor demonstrated that increased p44/42 MAP kinase activity is required for the HOXA1-mediated increase in cell proliferation, survival, oncogenicity and oncogenic transformation. Thus, modulation of the p44/42 MAP kinase pathway is one mechanism by which HOXA1 mediates oncogenic transformation of the human mammary epithelial cell.

Transcriptional activation of p53 by Pitx1.

Little is known about factors that stimulate transcription of the p53 tumor suppressor gene. Here, we report that the human pituitary homeobox 1 (hPitx1) transcription factor increases the expression of p53 at the mRNA and protein levels in human mammary carcinoma (MCF-7) cells. Increased p53 mRNA expression was due to activation of the p53 promoter by hPitx1. hPitx1 bound directly to the p53 promoter and functionally utilized two hPitx1 consensus elements. The predominant consensus element utilized by hPitx1 to stimulate p53 transcription was located within the first exon of the p53 gene. A hPitx1 mutant (hPitx1-R141P) acting as a dominant inhibitor repressed p53 transcription. Forced expression of hPitx1 resulted in cell-cycle arrest and p53-dependent apoptosis in p53-replete MCF-7 cells. Furthermore, hPitx1 stimulated the transcription of p53 target genes involved in cell-cycle arrest and apoptosis (p21 and PTGF-beta), again in a p53-dependent manner. Depletion of endogenous hPitx1 by small interfering RNA (siRNA) in MCF-7 cells resulted in decreased basal expression of p53 and consequently of p21 and placental transforming growth factor beta (PTGF-beta). Depletion of p53 by siRNA dramatically attenuated hPitx1-induced apoptosis in MCF-7 cells. Thus, p53 is a direct transcriptional target gene of hPitx1. This observation is concordant with the recent identification of hPitx1 as a tumor suppressor gene.

Signal transduction via the growth hormone receptor.

Rapid progress has been made recently in the definition of growth hormone (GH) receptor signal transduction pathways. It is now apparent that many cytokines, including GH, share identical or similar signalling components to exert their cellular effects. This review provides a brief discourse on the signal transduction pathways, which have been demonstrated to be utilized by GH. The identification of such pathways provides a basis for understanding the pleiotropic actions of GH. The mechanisms by which the specific cellular effects of GH are achieved remain to be elucidated.

Growth hormone receptor expression in the rat gastrointestinal tract.

We have used immunohistochemistry to define the cellular distribution of GH receptors in the gastrointestinal tract (GIT) and its derivatives. Immunohistochemistry was performed in the adult rat GIT with a panel of characterized monoclonal antibodies to the GH receptor. The most intense and heterogeneous immunoreactivity was observed in epithelial cell subpopulations of GIT mucosa. Mesenchymal elements of the GIT were homogenously and moderately immunoreactive. Intense immunoreactivity was observed in the ductal epithelium of the sublingual gland, scattered basal epidermal cells of the esophageal mucosa, zymogen cells of the gastric glands, scattered surface epithelial cells of the stomach, and scattered peripheral pancreatic acinar groups. Scattered enteroendocrine cells and parietal cells, crypt and villous columnar cells of the small intestine, surface columnar cells of the cecum/colon, crypt base columnar cells of the colon, and contiguous peripheral cords of pancreatic islet cells displayed strong immunoreactivity. No immunoreactivity was detectable in the mucous and serous acini of the sublingual and submandibular gland, respectively, mucous-secreting cells of the base of the cardiac and pyloric glands, surface epithelial cells of the fundus, paneth cells, goblet cells of cecum/colon, or mucous cells at the base of the cecal crypt. Other elements of the GIT were moderately or weakly immunoreactive. In support of our localization we can detect high affinity binding (Ka = 3 x 10(9] of [125I]human GH with ovine GH as displacing ligand to crude homogenates of adult rat stomach and intestine. We conclude that discrete epithelial cell subpopulations of the GIT and its derivatives are directly responsive to GH action. GH may, therefore, act independently of or synergistically with hepatic insulin-like growth factor-I in executing its physiological and/or growth-promoting role in the GIT.

Growth hormone specifically stimulates the expression of low density lipoprotein receptors in human hepatoma cells.

Human GH (hGH) has been shown to stimulate hepatic low density lipoprotein (LDL) receptor expression in man in vivo. To further characterize this effect in vitro, we determined the expression of LDL receptors in cultured human hepatoma (HepG2) cells exposed to hGH. After incubation with hGH, stimulation of LDL receptors appeared at a concentration of 0.25 nM hGH. The presence of hGH receptors on HepG2 cells could be demonstrated by immunocytochemistry using a hGH receptor-specific monoclonal antibody. Binding studies, using 125I-labeled hGH, revealed high affinity binding with the appropriate somatogenic specificity. The LDL receptor induction was specific for hGH, as both bovine GH and recombinant human PRL were without effect. The LDL receptor stimulation occurred in parallel with increased levels of LDL receptor messenger RNA. Inclusion of dexamethasone and thyroid hormone in the incubation medium enhanced the LDL receptor stimulation by hGH. Although incubation with insulin-like growth factor-I (IGF-I) stimulated LDL receptor expression, the hGH-induced stimulation was unaltered after preincubation of cells with a monoclonal mouse anti-IGF-I antibody, suggesting that the release of IGF-I is not involved in LDL receptor stimulation by hGH. We conclude that hGH specifically induces the LDL receptor in cultured HepG2 cells at both the protein and the messenger RNA level, and that the induction is independent of IGF-I release.

Growth hormone (GH) regulation of gastric structure and function in the GH-deficient rat: up-regulation of intrinsic factor.

In a recent study we identified GH receptor/binding protein in cells of the gastric mucosa. In order to define the role of the GH receptor/binding protein in gastric function, we have investigated the effect of GH on gastric structure and function in the GH-deficient Lewis (dwarf) rat. Bovine GH, 65 micrograms/100 g body wt, was administered twice daily to adult male dwarf rats for 6 days (DW+) while control animals received vehicle only (DW-). Administration of GH produced a significant increase in body wt (P less than 0.001), stomach wt (P less than 0.01), and stomach to body wt ratio (P less than 0.05). GH administration also resulted in increased total gastric DNA, RNA, and protein content but did not produce significant differences in DNA, RNA, or protein content when normalized to stomach wt. Morphometric analysis of the gastric mucosa revealed a significantly (P less than 0.05) increased gastric epithelial height and mucosal surface area along with an increase in the proportion of nuclei with multiple nucleoli (P less than 0.01). The number of gastric mucosal cells in S-phase was determined by immunohistochemical detection of nuclear 5'-bromo-2'-deoxyuridine (BrdU) incorporated during a 2 h pulse of BrdU. GH treatment resulted in a 74% increase (P less than 0.05) in the number of BrdU-labeled nuclei/mm2 mucosa relative to vehicle-injected control animals. A modification of Zimmerman's method for the differential staining of gastric mucosa was used to delineate cell type for morphometric analysis. This showed that the density of differentiated (parietal and chief) cell types was not significantly different between DW- and to DW+ animals. Soluble extracts of gastric mucosa were prepared for estimation of pepsinogen content and [57Co]cyanocobalamin (vitamin B12) binding. GH administration produced no significant change in pepsinogen content per mg protein and did not affect the relative levels of pepsinogen isoenzymes as determined by polyacrylamide gel electrophoresis. GH administration did however result in an 86% increase (P less than 0.01) in [57Co]cyanocobalamin binding per mg protein. The increase in binding was totally displaceable by 1 microgram/ml unlabeled cyanocobalamin but not by 1 microgram/ml cobinamide dicyanide indicating it was the result of increased intrinsic factor rather than R protein. Sephadex S-300 gel filtration of mucosal extracts revealed an elution profile for [57Co]cyanocobalamin identical to that of purified porcine intrinsic factor and different from that of human salivary R protein. In conclusion, we have demonstrated that GH stimulates proliferation and enlargement of the gastric mucosa without significant alteration in cellular composition.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone promotion of tubulin polymerization stabilizes the microtubule network and protects against colchicine-induced apoptosis.

We have investigated the effect of GH on microtubular physiology in Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)). We show here that after 30 min of human GH (hGH) treatment of CHO-GHR(1-638) cells, there was a significant increase in the level of polymerization of all four tubulin isoforms (alpha-, beta-, gamma-, and tyrosinated alpha-tubulin) compared with the serum-deprived state. However, this transient increase in the levels of polymerized tubulin after hGH treatment was particularly pronounced for beta- and tyr alpha-tubulin. For alpha- and gamma-tubulin, the hGH-induced increase in polymerization state lasted to approximately 3 h and then declined by 7 h, whereas for beta- and tyr alpha-tubulin there was a decrease in the polymerization state at 1-2 h after hGH treatment compared with the level at 30 min (but still greater than the serum-deprived state) followed by a second but lesser wave of increased polymerization lasting to 7 h. The changes in the polymerization state of the tubulins were not accompanied by comparative changes in the level of total cellular tubulin. The proline rich box 1 region of the GH receptor was required for hGH to stimulate tubulin polymerization indicative that this event is JAK dependent. Increased tubulin polymerization still occurred in response to hGH in a receptor truncation lacking the carboxyl terminal half of the intracellular domain of the GH receptor indicative that hGH induced changes in intracellular calcium concentration is not required for tubulin polymerization. Prior treatment of CHO-GHR(1-638) cells with hGH retarded colchicine induced microtubule depolymerization and also prevented colchicine induced apoptotic cell death. The integrity of the microtubule network was not required for GH-induced STAT5 mediated transcription as treatment of cells with colchicine, vincristine, or vinblastine did not alter the fold stimulation of the STAT5 mediated transcriptional response to GH. Thus one consequence of cellular treatment with GH is alteration in microtubule physiology.

Cellular localization of the growth hormone receptor/binding protein in the male and female reproductive systems.

We have used immunohistochemistry to localize GH receptor/binding protein (BP) in the male and female reproductive systems of adult rats. Testes and ovaries from neonatal animals were also examined to determine if GH receptor/BP expression in these tissues is developmentally regulated. Two monoclonal antibodies (MAb 43 and 263) were immunoreactive in identical locations whereas no immunoreactivity was evident when control monoclonal antibodies 7 and 50.8 were used. Localization of the receptor/BP was observed in both the nucleus and cytoplasm of immunopositive cells confirming our recent report of a nuclear GH receptor. Intense GH receptor/BP immunoreactivity in the male reproductive system was evident in the epithelium of the vas deferens and coagulating gland, the prostatic epithelium during the secretory phase, and the ductular epithelium of the coagulating and bulbourethral glands, respectively. Strong immunoreactivity was detectable in the Leydig and Sertoli cells, the epithelium of the ductus epididymis and seminal vesicles and smooth muscle of the tunica muscularis of the vas deferens, septae of the seminal vesicles, and in prostatic fibromuscular stroma. Cells of the seminiferous tubules (spermatogonia, primary and secondary spermatocytes, and spermatids) were moderately immunoreactive. No immunoreactivity was detectable in spermatozoa in the ductus epididymis or vas deferens, in scattered epithelial cells of the ductus epididymis, the prostatic epithelium in the nonsecretory phase, and mucous secreting cells of the bulbourethral glands. Leydig cells of 10-day postnatal rat testis were intensely immunoreactive whereas seminiferous tubular cells displayed homogenous immunoreactivity from moderate to strong. Intense GH receptor/BP immunoreactivity in the female reproductive system was evident in the germinal epithelium, the vascular endothelium of the myometrium, the epithelial lining of the fimbriae and oviduct, the endometrial epithelium and scattered endometrial glands, the mesothelium of the perimetrium, and the vascular endothelium of the endometrium. Strong immunoreactivity was exhibited by scattered oocytes, lutein cells of the corpus luteum, scattered endometrial glands, and the vascular endothelium of the endometrium. Moderate immunoreactivity was evident in scattered oocytes, granulosa cells, theca interna and externa, smooth muscle of the oviduct and myometrium, scattered endometrial glands, and luminally placed endometrial stroma cells. Ovarian granulosa cells from 10-day postnatal rats displayed strong immunoreactivity in contrast to moderate immunoreactivity in adult granulosa cells. In conclusion, we report a widespread distribution of the GH receptor/BP in the reproductive system of the rat by which GH may exert a direct action on reproductive function. The distribution is concordant with a role for GH in epithelial function and/or maintenance and also with a possible role for GH in the integrity of the endometrial vasculature.

Cellular localization of the growth hormone binding protein in the rat.

In the rat a GH-binding protein (GHBP) exists that is derived from the GH receptor gene by an alternative messenger RNA splicing mechanism such that the transmembrane and intracellular domains of the GH receptor are replaced by a hydrophilic carboxy terminus. Previous immunohistochemical studies detailing the localization of the GH receptor binding protein (BP) have used monoclonal antibodies that recognize extracellular region-specific epitopes common to both the GH receptor and GHBP. In this study we have used a monoclonal antibody (MAb 4.3) specific for the carboxy terminus of the rat GHBP to map its somatic distribution in the rat and have compared this distribution with that of a MAb recognizing both the BP and the GH receptor. A variety of tissues including the skeletal and muscular systems, the gastrointestinal tract and derivatives, the male and female reproductive systems, skin, central and peripheral nervous systems, and the 18 day gestation fetus were investigated. The distribution of GHBP immunoreactivity (MAb 4.3) was widespread and identical to that previously reported for the extracellular region of the GH receptor (MAbs 263 and 43). Immunoreactivity was both cytoplasmic and nuclear, indicating a possible role for the GHBP in intracellular function. GHBP immunoreactivity was predominantly associated with epithelial/endothelial cell subtypes and with mesenchymal elements such as muscle, chondrocytes, and osteoblasts, as previously described for the GH receptor extracellular region. We also report here the distribution of the GH receptor/GHBP in the kidney, cardiovascular, and respiratory systems. The most prominent immunoreactivity (MAbs 4.3 and 263) was associated with the distal convoluted tubules and collecting ducts of the kidney, with the epithelium and smooth muscle of the broncho-alveolar tree (including type I and II pneumocytes), with the Purkinje and myocardial fibers of the heart and with the endothelium and smooth muscle of blood vessels. Thus we have identified sites of direct GH action in the cardiovascular, renal, and respiratory systems. In conclusion, the extensive cellular distribution of the GHBP in the rat indicates physiological function(s) other than the binding of GH in plasma. Since GHBP mRNA has also been reported in a number of tissues, it may be that the GHBP is synthesized locally to mediate intracellular transport of GH and/or transcriptional regulation by GH in a variety of target tissues.

Growth hormone-induced reorganization of the actin cytoskeleton is not required for STAT5 (signal transducer and activator of transcription-5)-mediated transcription.

We have investigated the effect of GH on the organization of the actin cytoskeleton within the cell. Human GH (hGH) treatment (50 nM) of Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)) resulted in a reorganization of actin filaments in the cell that was not observed upon GH treatment of the untransfected parental CHO cell line. hGH initially induced depolymerization of actin stress fibers similar in magnitude to that induced by treatment of the cells with 100 nM human insulin-like growth factor I. This loss of stress fibers was observed as early as 30 sec after addition of hGH to the medium, and maximal depolymerization of stress fibers was observed between 1-4 min after addition of hGH. This was followed by a slow, but submaximal, repolymerization of the stress fibers and the formation of localized focal filamentous actin containing complexes. Similar cytoskeletal changes were observed after hGH treatment in Swiss 3T3 fibroblasts and BRL cells stably transfected with rat GH receptor complementary DNA (BRL-GHR(1-6381)). Pretreatment of CHO-GHR(1-638) cells with wortmannin (a phosphatidylinositol 3-kinase inhibitor) and verapamil (a calcium channel antagonist) both inhibited the hGH-induced actin reorganization. The integrity of the actin cytoskeleton was not required for GH-induced STAT5 (signal transducer and activator of transcription-5)-mediated transcription, as treatment of cells with cytochalasins B and D did not alter the fold stimulation of the STAT5-mediated transcriptional response to GH. We conclude that GH induces a rapid reorganization of the actin cytoskeleton by a process requiring phosphatidylinositol 3-kinase activation and calcium influx, but this cytoskeletal reorganization is not required for the STAT5-mediated transcriptional response to GH.

Autocrine human growth hormone enhancement of human mammary carcinoma cell spreading is Jak2 dependent.

We investigated the role of autocrine production of human (h) GH in the attachment and spreading of mammary carcinoma cells in vitro. We used a previously described model system for the study of the autocrine/paracrine role of GH in which the hGH gene (MCF-hGH) or a translation-deficient hGH gene (MCF-MUT) was stably transfected into MCF-7 cells. No differences in attachment to a collagen matrix between MCF-hGH and MCF-MUT cells were observed in either serum-free medium (SFM) or medium containing exogenous hGH, 5% serum, or 10% serum. In contrast, MCF-hGH cells spread more rapidly on a collagen matrix than did MCF-MUT cells. Exogenous hGH and 10% serum interacted with autocrine production of hGH in an additive manner to increase cell spreading. MCF-hGH cells formed filipodia and stress fibers earlier than MCF-MUT cells during the process of cell spreading and possessed marked differences in morphology after spreading. MCF-MUT cells displayed uniform and symmetrical formation of stress fibers, whereas MCF-hGH cells displayed irregular and elongated stress fiber formation. The level of cytoplasmic phosphotyrosine was increased in MCF-hGH compared with MCF-MUT cells during spreading and displayed colocalization with Janus kinase 2 (JAK2). Basal JAK2 tyrosine phosphorylation was increased, and it increased further on spreading in MCF-hGH cells compared with MCF-MUT cells. Transient transfection of JAK2 complementary DNA resulted in interaction with autocrine hGH to increase the rate of cell spreading in MCF-hGH cells compared with MCF-MUT cells. Treatment with a selective JAK2 tyrosine kinase inhibitor (AG 490) reduced the rate of MCF-hGH cell spreading to the rate of MCF-MUT cell spreading. Thus, we conclude that autocrine production of hGH enhances the rate of mammary carcinoma cell spreading in a JAK2-dependent manner.

Constitutive nuclear localization of Janus kinases 1 and 2.

Both GH and the GH receptor have been reported to undergo rapid nuclear translocation. Janus kinases (JAK) 1 and 2 have been implicated in GH receptor signaling, and both of these kinases are phosphorylated by GH stimulation. In this report, we have investigated the subcellular distribution of JAK1 and JAK2. Both JAK1 and JAK2 exhibit a nucleocytoplasmic distribution by immunocytochemistry in unstimulated serum deprived CHO cells stably transfected with rat GH receptor complementary DNA (cDNA). The nucleocytoplasmic localization of JAK2 was verified by immunogold electron microscopy in both rat liver hepatocytes and CHO cells stably transfected with rat GH receptor cDNA. Nucleocytoplasmic localization of JAK2 was also verified by transient tranfection of CHO cells with a Haemophilus influenzae haemagglutinin (HA) epitope tagged JAK2 expression plasmid and subsequent localization of HA immunoreactivity. Western blot analysis of purified nuclear extracts revealed the presence of immunoreactive JAK1 at 130 kDa and immunoreactive JAK2 at 128 kDa. No change in the nuclear content of JAK1 or JAK2 was observed upon ligand stimulation of GH receptor cDNA transfected cells with 100 nM human GH for 5, 10, 15, 30, or 60 min. GH stimulation caused, however, the appearance of tyrosine phosphorylated 42- and 44-kDa proteins as well as tyrosine phosphorylated JAK2 in the nucleus. The constitutive nuclear localization of the Janus Kinases is suggestive of a novel nuclear role for JAK family members, in addition to their described cytosolic function and presents an interesting challenge to the subcellular site of hormone action.

The effects of autocrine human growth hormone (hGH) on human mammary carcinoma cell behavior are mediated via the hGH receptor.

The human GH (hGH) antagonist B2036 combines a single amino acid substitution impairing receptor binding site 2 (G120K) with eight additional amino acid substitutions that improve binding site 1 affinity. B2036 does not bind, activate, or antagonize the human PRL receptor and therefore is suitable to determine cellular effects mediated specifically through the hGH receptor. We have used this hGH receptor specific antagonist in MCF-7 cells stably transfected with either the hGH gene (MCF-hGH) or a translation deficient hGH gene (MCF-MUT) to determine whether the effects of autocrine hGH on mammary carcinoma cell behavior are mediated via the hGH receptor. Enhanced JAK2 tyrosine phosphorylation observed in MCF-hGH cells compared with MCF-MUT cells is abrogated by B2036 as is the autocrine hGH stimulated increase in total cell number and DNA synthesis. Interestingly, autocrine hGH functions as a potent inhibitor of apoptosis induced by serum withdrawal compared with exogenously added hGH, and the protection against apoptosis afforded by autocrine hGH is abrogated by B2036. B2036 also inhibited autocrine hGH stimulated transcriptional activation mediated by either STAT5, CHOP (p38 MAP kinase specific) or Elk-1 (p44/42 MAP kinase specific). Finally, B2036 inhibited the autocrine hGH-dependent enhancement of the rate of mammary carcinoma cell spreading on a collagen matrix. Thus, the effects of autocrine hGH on human mammary carcinoma cell behavior are mediated via the hGH receptor.

The mechanism of effect of growth hormone on preadipocyte and adipocyte function.

Growth hormone (GH) is not only the major regulator of postnatal somatic growth but also exerts profound effects on body composition through a combination of anabolic, lipolytic and antinatriuretic actions. GH enhancement of the lipolytic activity of adipose tissue in combination with a reduction of triglyceride accumulation via inhibition of lipoprotein lipase activity appears to be the major mechanism by which GH results in a reduction of the total fat mass. Recently, much progress has been made in understanding the molecular mechanism by which GH affects cellular function. This review provides a brief discourse and summary of the mechanism of effects of GH on preadipocyte/adipocyte function. It is intended to provide a functional understanding of the mechanism of action of GH as it relates to adipogenesis and adipocyte function.

Prenatal and adult growth hormone gene expression in rat lymphoid organs.

Growth hormone (GH) exerts its immune effects on mature lymphocytes through an autocrine/paracrine mechanism. We investigated the prenatal synthesis of GH mRNA in rat lymphoid organs using the sensitive in situ RT-PCR methodology. We show that GH transcripts are detectable in the thymus and liver of the 18-day fetus. At this stage, all thymocytes are immature and express the GH gene. In fetal liver, GH gene expression was localized in circulating lymphocytes and in hematopoietic cells surrounding GH mRNA-negative hepatocytes. In situ GH gene expression in fetal lymphoid organs was confirmed by in vitro RT-PCR showing that the amplified product from fetal lymphoid tissues was similar to the product obtained from the pituitary. Moreover, GH gene expression was detected in the thymus, spleen, and ileum Peyer's patches of adult rat, with a localization restricted to the lymphocytes and endothelial and smooth muscle cells of blood vessels. The autocrine/paracrine expression of the GH gene by lymphoid and hematopoietic cells during fetal growth might influence the generation of regulatory cells involved in immunity and hematopoiesis.

Growth hormone (GH) regulation of submandibular gland structure and function in the GH-deficient rat: upregulation of haptocorrin.

Based on localization studies of the GH receptor/binding protein (BP) in the gastrointestinal tract, we have recently demonstrated growth hormone regulation of gastric intrinsic factor. In order to define the role of GH in the submandibular gland (SMG) we have investigated the effect of GH on SMG structure and function with particular reference to haptocorrin. Bovine GH (65 micrograms/100 g body weight) was administered twice daily to adult male dwarf rats for 6 days (DW+) while control animals received vehicle (DW-). Administration of GH produced a significant increase in body weight (P < 0.001) and allometric increase in SMG weight (P = 0). There was no change in RNA or protein content per g SMG and GH administration produced a small decrease in DNA content normalized to SMG weight. Morphometric analysis of the SMG revealed a significant increase in the percentage area of the gland occupied by tubular (GH receptor/BP expressing) structures and a significant increase in the diameter of both the intralobular striated and granular convoluted tubules. The effect of GH on cellular proliferation in the ductular and acinar components was determined by the immunohistochemical detection of nuclear 5'-bromo-2'-deoxyuridine (BrdU) incorporated during a 2-h pulse of BrdU. GH treatment induced a 5.5-fold increase in the labelling index of tubular cells whereas the acinar cell labelling index increased only 3.3-fold. Soluble extracts of SMG were prepared for estimation of 57Co-cyanocobalamin (vitamin B12) binding. GH administration resulted in an increase in total 57Co-cyanocobalamin (CBL) binding per mg SMG protein. To determine the contribution of haptocorrin (R-protein) the amount of cobinamide dicyanide (CD) displaceable binding was calculated. GH administration produced a 70% increase in CD displaceable CBL binding per mg SMG indicating GH regulation of haptocorrin. A comparison of total SMG CBL binding and CD displaceable CBL binding between male and female rats detected no sex difference. Therefore sex-specific GH secretory profiles are unlikely to be of importance in the regulation of haptocorrin. In conclusion we have demonstrated that GH stimulates hypertrophy and hyperplasia of components of the SMG in the dwarf rat. The observed upregulation of haptocorrin may synergize with the GH-stimulated increase in intrinsic factor to facilitate absorption of CBL during the anabolic state.

Prolactin receptor expression in the gastrointestinal tract: characterization of the prolactin receptor of gastric mucosa.

There is evidence that prolactin (PRL) influences gastrointestinal function. However, the sites at which prolactin exerts these effects are not known. A monoclonal antibody was therefore generated against the rabbit mammary gland prolactin receptor (MAb 218) and used to study the distribution of the prolactin receptor in the rabbit gastrointestinal tract (GIT) by immunohistochemistry. MAb 218 is an IgG1 kappa-precipitating antibody which precipitates major affinity cross-linked mammary gland prolactin receptor subunits of molecular masses 45 and 80 kDa. It has an affinity of 0.8 x 10(9) mol/l for the prolactin receptor and does not react with GH or insulin receptors in precipitation assays. MAb 218 immunoreactivity was observed in classical prolactin target cells such as mammary gland epithelium, and this immunoreactivity was abolished by preincubation of MAb 218 with purified prolactin receptor but not by preincubation with purified GH receptor. In the GIT, the most intense immunoreactivity was associated with the oesophageal epithelium, chief (zymogenic) cells of the fundic mucosa, pancreatic islets of Langerhans and surface epithelial cells of the duodenum and jejunum. Other specific elements of the GIT were immunoreactive at lower levels or were immunonegative. No immunoreactivity was observed in these locations with a control monoclonal antibody (MAb 50.8) of identical isotype to 218. To support the immunohistochemical findings, rabbit gastric mucosal membranes were used to show the presence of lactogen-specific binding. Scatchard analysis of 125I-labelled human GH binding to the gastric mucosal membranes with rat prolactin as displacing ligand yielded an affinity constant (Ka) of 1.0 +/- 0.2 x 10(9) mol/l with a capacity of 3.5 +/- 0.4 fmol/mg protein. Affinity cross-linking and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the gastric receptor revealed lactogenic hormone-binding subunits of molecular masses 43, 68 and 83 kDa. The 68 kDa subunit was not seen in rabbit mammary gland or ovarian tissue, and may be unique to gastric mucosa. In conclusion, we have demonstrated the presence of a high affinity lactogenic receptor in specific epithelial cell subpopulations of the GIT. This localization of the prolactin receptor in the GIT will assist in further functional assignment of prolactin to gastrointestinal physiology.

Localization of the growth hormone receptor/binding protein in skin.

Acromegaly is characterized by coarsening of facial features, acanthosis nigricans, hypertrichosis and oily skin. To determine the site through which GH exerts these effects, we have used immunohistochemistry to localize the GH receptor/binding protein (BP) in rat, rabbit and human skin. Three monoclonal antibodies (MAb 1, 43, 263) were immunoreactive in identical locations, whereas no immunoreactivity was evident when control monoclonal antibodies (MAb 50.8 and MAb 7 (rat] were used. Skin from neonatal and adult animals was used to determine whether GH receptor/BP expression was developmentally regulated. Immunoreactivity of the GH receptor/BP in the three species was consistently localized in the stratum basale and stratum spinosum. Intermittent staining was observed in the stratum granulosum. Scattered basal epidermal cells often displayed more intense immunoreactivity. This distribution was observed at all maturational stages examined. Intense GH receptor/BP immunoreactivity was observed in all histological layers of the lower one-third of hair follicles and in hair matrix cells of the dermal papillae. Immunoreactivity was also detected in the outer epithelial root sheath of the upper two-thirds of hair follicles, in sebaceous glands and in fibroblasts of the connective tissue sheath surrounding the follicle. GH receptor/BP immunoreactivity was also present in the secretory duct and myoepithelial cells of human eccrine sweat glands. Fibroblasts, Schwann cells of peripheral nerve fascicles, skeletal muscle cells and adipocytes of the dermis were also immunoreactive as were medial smooth muscle and endothelial cells of arteries. These results provide evidence that GH acts locally on the epidermis and epidermal appendages concordant with our recent localization of GH receptor/BP to epithelial cell types of the gastrointestinal and reproductive systems.

High stromal and epithelial human gh gene expression is associated with proliferative disorders of the mammary gland.

We have demonstrated and localized human GH (hGH) gene expression in surgical specimens of normal human mammary gland and in proliferative disorders of the mammary gland of increasing severity using sensitive in situ RT-PCR methodology. hGH mRNA identical to pituitary hGH mRNA was first detected by RT-PCR of RNA derived from samples of normal human mammary gland. Cellular localization of hGH gene expression in the normal mammary gland exhibited restriction to luminal epithelial and myoepithelial cells of the ducts and to scattered stromal fibroblasts. We subsequently examined the expression of the hgh gene in three progressive proliferative disorders of the human mammary gland, i.e. A benign lesion (fibroadenoma), a pre-invasive stage (intraductal carcinoma) and an invasive ductal carcinoma. hGH mRNA was readily detected in the tumoral and non-tumoral epithelial components and also in cells of the reactive stroma including fibroblasts, myofibroblastic and myoepithelial cells, inflammatory infiltrate lymphocytes and endothelial cells in areas of neovascularization. In all three proliferative disorders examined, the intensity of the cellular labeling observed in both the epithelial and stromal compartments was always stronger compared with that in adjacent normal tissue. hGH protein was also present in significantly higher concentration in extracts derived from proliferative disorders of the mammary gland compared with extracts derived from normal mammary gland. We also examined hGH gene expression in axillary lymph nodes not containing and containing metastatic mammary carcinoma. hGH gene expression was evidenced in metastatic mammary carcinoma cells and in reactive stromal cells by both in situ hybridization and in situ RT-PCR. In contrast, in lymph nodes not containing metastatic mammary carcinoma, hGH mRNA was detected only by use of in situ RT-PCR. Thus, increased expression of the hGH gene in the epithelial component and the de novo stromal expression in proliferative disorders of the mammary gland are suggestive of a pivotal role for autocrine hGH in neoplastic progression of the mammary gland.

Zonation of cytochrome P450 enzyme expression in rat liver. Isozyme-specific regulation by pituitary dependent hormones.

The effect of hypophysectomy and subsequent infusion of growth hormone (GH) or injections of triiodothyronine (T3) on the acinar expression pattern of four homonally regulated P450 isozymes was studied to elucidate the involvement of pituitary dependent hormones in regulating the characteristic centrilobular expression pattern of most members of the cytochrome P450 (CYP) gene family in rat liver. Hypophysectomy was previously observed to allow high expression of CYP2B1/2 and 3A1/2 in the normally silent periportal region. In the present study, it had much less effect on the zonation of the ethanol-inducible P450 2E1 form: only a moderate shift of 2E1 staining towards the periportal region was observed by immunohistochemistry. Subsequent injections with T3 moderately decreased CYP2E1 expression in the periportal region and no significant countereffect of GH was discerned. T3 treatment, previously observed to block only the periportal expression of CYP3A1/2, counteracted the increased CYP2B1/2 expression caused by hypophysectomy equally in the periportal and perivenous region. This was true both at the protein and mRNA level, as analysed from cell lysates obtained by in situ perfusion of livers by zone-restricted digitonin treatment. Thus, although hypophysectomy and subsequent GH and T3 treatment affect the total expression of CYP2B1/2, 2E1, and 3A1/2 similarly, the zonal effects were isozyme-specific. In contrast, the perivenous zonation normally seen for the dioxin-inducible P450 1A2 form was steepened rather than diminished by hypophysectomy, both in male and female rats. Administration of GH by the female-type continuous infusion had no effect in male rats, but partially counteracted the effect of hypophysectomy in females, suggesting an involvement of GH. In contrast to other CYP genes investigated, the female-characteristic expression of CYP2C12 was found to be completely non-zonated. Hypophysectomy and continuous GH administration dramatically affected the amount of mRNA of both P450 2C12 and the male-specific 2C11 form, but analysis of periportal and perivenous cell lysates indicated that these effects were not zone-specific. The distribution of the GH receptor was investigated to explain the zonal effects of GH. Immunohistochemically, a moderate perivenous dominance was observed, whereas the mRNA abundance of both GH receptor and GH binding protein was slightly higher in the periportal region. Thus, zonal regulation by GH does not appear to result from a GH receptor zonation; rather, a sinusoidal GH gradient may be involved. These data, combined with our previous results, indicate that pituitary-dependent hormones regulate the zone-specific expression of some P450 forms strongly (i.e. 2B1/2 and 3A1/2), and other forms are moderately regulated (i.e. 1A2 and 2E1), or are affected across the whole acinus (i.e. 2C11, 2C12).