metallothionein and Pituitary-Neoplasms

Hyperactivation of the GHRH receptor or downstream signaling components is associated with hyperplasia of the pituitary somatotrope population, in which adenomas form relatively late in life, with less than 100% penetrance. Hyperplastic and adenomatous pituitaries of metallothionein promoter-human GHRH transgenic (Tg) mice (4 and > 10 months, respectively) were used to identify mechanisms that may prevent or delay adenoma formation in the presence of excess GHRH. In hyperplastic pituitaries, expression of the late G(1)/G(2) marker Ki67 increased, whereas the proportion of 5-bromo-2'-deoxyuridine-labeled cells (S phase marker) did not differ from age-matched controls. These results indicate cell cycle progression is blocked, with further evidence suggesting that enhanced p27 activity may contribute to this process. For adenomas, formation was associated with loss of p27 activity (nuclear localization and mRNA). Increased endogenous somatostatin (SST) tone may also slow the conversion from hyperplastic to adenomatous state because mRNA levels for SST receptors, sst2 and sst5, were elevated in hyperplastic pituitaries, whereas adenomas were associated with a decline in sst1 and sst5 mRNA. Also, SST-knockout Tg pituitaries were larger and adenomas formed earlier compared with those of SST-intact Tg mice. Unexpectedly, these changes were independent of changes in proliferation rate within the hyperplastic tissue, suggesting that endogenous SST controls GHRH-induced adenoma formation primarily via modulation of apoptotic and/or cellular senescence pathways, consistent with the predicted function of some of the most differentially expressed genes (Casp1, MAP2K1, TNFR2) identified by membrane arrays and confirmed by quantitative real-time RT-PCR.

Topics: Adenoma; Aging; Animals; Cell Proliferation; Female; Growth Hormone-Releasing Hormone; Human Growth Hormone; Humans; Male; Metallothionein; Mice; Mice, Knockout; Organ Size; Pituitary Gland; Pituitary Neoplasms; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins; Receptors, Neuropeptide; Receptors, Pituitary Hormone-Regulating Hormone; RNA, Messenger; Somatostatin; Somatotrophs

Excessive GHRH stimulation leads to somatotrope hyperplasia and, ultimately, pituitary adenoma formation in the metallothionein promoter-driven human GHRH (hGHRH) transgenic mouse. This pituitary phenotype is similar to that observed in humans with ectopic production of GHRH. In both mice and man, GHRH hyperstimulation also results in dramatic increases in circulating GH and IGF-I. To determine whether GH/IGF-I modulates the development and growth rate of GHRH-induced pituitary tumors, pituitary growth and histology were evaluated in mice generated from cross-breeding metallothionein promoter-driven hGHRH transgenic mice with GH receptor binding protein (GHR) gene disrupted mice (GHR(-/-)). Expression of the hGHRH transgene in 2-month-old GHR intact (GHR(+)) mice resulted in the doubling of pituitary weight that was largely attributed to an increase in the number of GH-immunopositive cells. Pituitary weight of GHR(+) hGHRH mice did not significantly change between 2 and 6 months of age, whereas at 12 months, weights increased up to 100-fold those of GHR(+) pituitaries, and 70% of the glands contained grossly visible adenomas. All adenomas stained positively for GH, whereas some showed scattered PRL staining. Pituitaries of GHR(-/-) mice were half the size of those of GHR(+) mice. Although reduced in size, the histological features of GHR(-/-) mouse pituitaries were suggestive of somatotrope hyperplasia. Despite evidence of somatotrope hyperplasia, pituitaries from GHR(-/-) mice as old as 28 months of age were similar in size to those of 2-month-old mice and did not show signs of adenoma formation. Expression of the hGHRH transgene in GHR(-/-) mice did not significantly increase pituitary size between 2 and 6 months of age. However, at 12 months the majority of GHR(-/-), hGHRH pituitaries developed adenomas with mean pituitary weight and histological features similar to those of GHR(+), hGHRH mice. These observations demonstrate that intact GH signaling is not required for GHRH tumor formation. Although the majority of GHR(+), hGHRH and GHR(-/-), hGHRH pituitaries developed tumors by 12 months of age, a small subset remained morphologically indistinct from those at 2 months of age. These observations taken together with the fact that overt tumor formation is preceded by a static pituitary growth phase between 2 and 6 months, indicates that protective mechanisms are in place to maintain pituitary mass despite hGHRH hyperstimulation.

Topics: Adenoma; Aging; Animals; Gene Expression; Growth Hormone; Growth Hormone-Releasing Hormone; Humans; Hyperplasia; Male; Metallothionein; Mice; Mice, Inbred Strains; Mice, Knockout; Phenotype; Pituitary Gland; Pituitary Gland, Anterior; Pituitary Neoplasms; Promoter Regions, Genetic; Receptors, Somatotropin; Reference Values; Signal Transduction; Transgenes

To study the neurophysiological functions of metallothioneins (MTs), localization of MT-I and -II was examined immunohistochemically in a variety of brain lesions in dogs, including infarct, laminar cortical necrosis, hemorrhage, invasive growth of tumour, inflammatory lesions in granulomatous meningoencephalitis and distemper encephalitis. MT-I and -II were demonstrated in both nucleus and cytoplasm of hypertrophic astrocytes in most brain lesions examined regardless of the type, size, localization and duration of the lesions. In addition, MT expression was stronger in a population of hypertrophic astrocytes localizing inside of the surviving brain tissue rather than those localizing at the boundary between the surviving brain tissue and necrotic area, where severe inflammatory changes were developing. These results suggest that MT-I and -II may play roles not only in protection of neurons from metals and free radicals ubiquitous in the inflammatory lesions but also in repair of injured neural tissues.

Topics: Adenocarcinoma; Animals; Astrocytes; Brain Diseases; Brain Neoplasms; Cerebral Hemorrhage; Cerebral Infarction; Distemper; Dog Diseases; Dogs; Encephalitis, Viral; Female; Glial Fibrillary Acidic Protein; Hypertrophy; Inflammation; Male; Meningoencephalitis; Metallothionein; Necrosis; Neoplasm Invasiveness; Pituitary Neoplasms

Low molecular weight (LMW) GTP-binding proteins are hypothesized to play a role in the vectorial transport of intracellular vesicles. Mutational studies in yeast and subcellular localization in mammalian cells suggest that a family of LMW GTP-binding proteins, termed rab, target intracellular vesicles to their appropriate acceptor compartment. In this report, we demonstrate that an elasmobranch homologue of rab3A, o-rab3, plays a significant role in the sequestration of regulated secretory vesicles. When transfected into the murine endocrine cell line AtT-20, the wild-type o-rab3 protein is localized exclusively to the tips of the processes, a region of the cell known to accumulate proteins associated with regulated secretory vesicles. Two mutations, Gln81 to Leu (Q81L) and Asn135 Ile (N135I), which alter GTP binding or rate of hydrolysis, blocked the localization of the o-rab3 protein to the tips of cell processes. These mutations also hindered the sequestration of ACTH-containing secretory vesicles to the process tips but did not affect the basal or stimulated release of ACTH. Moreover, the sequestration of the protein VAMP to the process tip was also hindered by the mutation. The results demonstrate a role for the rab3 proteins in localization, sequestration, and storage of secretory vesicles near their release site.

Topics: Adrenocorticotropic Hormone; Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Cadmium; Cell Line; Cytoplasmic Granules; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Intracellular Membranes; Metallothionein; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Pituitary Neoplasms; Promoter Regions, Genetic; rab3 GTP-Binding Proteins; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants.

Topics: Animals; Base Sequence; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Gene Expression Regulation, Neoplastic; Genes, fos; Humans; Isoproterenol; Kinetics; Metallothionein; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Pituitary Neoplasms; Pro-Opiomelanocortin; Promoter Regions, Genetic; Protein Kinases; Proto-Oncogene Proteins c-fos; Receptors, Adrenergic, beta; RNA, Messenger; Sequence Deletion; Transfection; Tumor Cells, Cultured

Morphologic changes in the pituitary, liver, and pancreas of mice with the metallothionein-human growth hormone--releasing hormone (MT-hGRH) transgene were analyzed by in situ hybridization histochemistry (ISH). There was progression from somatotroph hyperplasia to neoplasia in pituitaries of transgenic mice. Pituitary neoplasms were present between 9 to 12 months of age in some mice. Magnetic resonance imaging (MRI) readily identified enlarged pituitaries in MT-hGRH transgenic mice. Serum mouse GH and hGRH levels were marked elevated in MT-hGRH transgenic mice. In situ hybridization histochemistry showed mRNA for hGRH in liver, pituitary, pancreas, spleen, and in most other tissues examined. Combined ISH and immunohistochemistry in the pituitary gland showed that some of the GH cells also produced hGRH, and ultrastructural immunohistochemical analysis of pituitaries showed that GH and hGRH were localized in the same cell and within the same secretory granules. Liver cells of MT-hGRH transgenic mice showed evidence of hypertrophy, and the pancreatic islets were hyperplastic with significant increases in the islet cell areas. The morphologic changes in the liver were distinctive enough to separate control littermates from MT-hGRH transgenic mice in all cases. The enlarged pancreatic islets had increased numbers of insulin-producing cells. Immunoreactive hGRH and hGRH mRNA were both localized in islet cells, and an intense hybridization signal of hGRH mRNA, but only weak staining for hGRH protein, were detected in the liver of transgenic mice. These results indicate that excessive hGRH production leads to distinct morphologic changes in various organs in MT-hGRH transgenic mice and that there is temporal progression from hyperplasia to adenomatous somatotrophs in pituitaries with chronic stimulation by hGRH that involves paracrine, endocrine, and autocrine mechanisms.

Topics: Adenoma; Animals; Growth Hormone-Releasing Hormone; Hormones, Ectopic; Hyperplasia; Immunohistochemistry; In Situ Hybridization; Liver; Metallothionein; Mice; Mice, Transgenic; Pancreas; Pituitary Gland; Pituitary Neoplasms; Recombinant Fusion Proteins

Topics: Animals; Cadmium; Cell Line; Chromosome Deletion; Gene Expression; Humans; Insulin; Metallothionein; Mice; Mutagenesis, Site-Directed; Pituitary Neoplasms; Proinsulin; Promoter Regions, Genetic; Protein Precursors; Transfection; Zinc

Pituitary GH3 cells were transfected with a human growth hormone-releasing hormone (hGRH) precursor minigene fused to the promoter region of either a cytomegalic immediate early gene (pCMV) or the mouse metallothionein-1 gene (mMT) to examine the molecular heterogeneity of the translation products. Expression of the hGRH message occurred following transfection of the cells with each fusion gene. Extracts of pCMV-hGRH-transfected GH3 cells as well as the culture medium contained detectable levels of immunoreactive (ir)-hGRH peptides. Analysis of molecular heterogeneity by reverse-phase high performance liquid chromatography and radioimmunoassay indicated that both mature forms of hGRH (hGRH(1-44)-NH2 and hGRH(1-40)-OH) were synthesized in the cells, although hGRH(1-44)-NH2 was the primary form secreted into the medium. A high molecular weight form of ir-hGRH, believed to represent the hGRH precursor (or a partially processed form of the precursor) was detected in cells and, in smaller amounts, in the medium. Several ir-hGRH peptides, presumed cleavage products of the mature forms of hGRH, were also found. The efficiency of processing of the hGRH precursor and metabolism of the mature hormonal forms in transfected cells grown in the presence of four different peptidase inhibitors varied with the inhibitor present. Transfected GH3 cells, therefore, possess all of the necessary enzymes for and are capable of processing the hGRH precursor to mature GRH and provide a model to study hGRH biosynthesis.

Topics: Animals; Blotting, Northern; Cells, Cultured; Chromatography, High Pressure Liquid; Cloning, Molecular; Cytomegalovirus; Gene Expression Regulation, Viral; Genes, Viral; Growth Hormone-Releasing Hormone; Humans; Metallothionein; Mice; Pituitary Gland; Pituitary Neoplasms; Plasmids; Promoter Regions, Genetic; Protease Inhibitors; Protein Precursors; Radioimmunoassay; Transfection; Tumor Cells, Cultured; Viral Fusion Proteins

Preprovasopressin-neurophysin II (prepro-AVP-Np), the precursor of the cyclic, amidated nonapeptide, arginine vasopressin (AVP), is present in the central and peripheral nervous systems, adrenal glands, and gonads of rats. To study cell-specific processing of prepro-AVP-Np, a fusion gene consisting of the heavy metal-inducible promoter of the mouse metallothionein I gene and the rat prepro-AVP-Np gene was introduced by cellular transfection into several defined cell phenotypes: a fibroblast line (BHK), a pituitary growth hormone and prolactin-producing cell line (GH4), a pituitary cell line that produces several amidated peptides (AtT-20), and an insulin-producing pancreatic islet line (RIN- 1046-38). Clonal cell lines were isolated and prepro-AVP-Np-specific transcripts were detected by Northern blot hybridization analyses. Fibroblast BHK and pituitary GH4 cells transfected with the fusion gene synthesized a polypeptide (Mr = 18,000) characteristic of the glycosylated precursor, pro-AVP-Np; in metal -treated cells, this protein was the major secreted cysteine-labeled polypeptide. Extracts of RIN-1046-38 and AtT-20 cells transfected with the fusion gene contained predominantly processed neurophysin and amidated arginine vasopressin, whereas extracts of BHK and GH4 cells contained mainly precursors of AVP and neurophysin. These observations indicate that the pathways involving specific post-translational processing of pro-AVP-Np are more efficiently utilized in the prohormone-producing AtT-20 and RIN-1046-38 cells than in GH4 and BHK cells that do not synthesize any recognized prohormones.

Topics: Animals; Arginine Vasopressin; Cell Line; Cloning, Molecular; Genes; Genes, Synthetic; Metallothionein; Mice; Neurophysins; Oxytocin; Pituitary Neoplasms; Protein Biosynthesis; Protein Precursors; Transcription, Genetic; Vasopressins