STAT3 upregulation in pituitary somatotroph adenomas induces growth hormone hypersecretion.

Pituitary somatotroph adenomas result in dysregulated growth hormone (GH) hypersecretion and acromegaly; however, regulatory mechanisms that promote GH hypersecretion remain elusive. Here, we provide evidence that STAT3 directly induces somatotroph tumor cell GH. Evaluation of pituitary tumors revealed that STAT3 expression was enhanced in human GH-secreting adenomas compared with that in nonsecreting pituitary tumors. Moreover, STAT3 and GH expression were concordant in a somatotroph adenoma tissue array. Promoter and expression analysis in a GH-secreting rat cell line (GH3) revealed that STAT3 specifically binds the Gh promoter and induces transcription. Stable expression of STAT3 in GH3 cells induced expression of endogenous GH, and expression of a constitutively active STAT3 further enhanced GH production. Conversely, expression of dominant-negative STAT3 abrogated GH expression. In primary human somatotroph adenoma-derived cell cultures, STAT3 suppression with the specific inhibitor S3I-201 attenuated GH transcription and reduced GH secretion in the majority of derivative cultures. In addition, S3I-201 attenuated somatotroph tumor growth and GH secretion in a rat xenograft model. GH induced STAT3 phosphorylation and nuclear translocation, indicating a positive feedback loop between STAT3 and GH in somatotroph tumor cells. Together, these results indicate that adenoma GH hypersecretion is the result of STAT3-dependent GH induction, which in turn promotes STAT3 expression, and suggest STAT3 as a potential therapeutic target for pituitary somatotroph adenomas.

Constitutive somatostatin receptor subtype-3 signaling suppresses growth hormone synthesis.

Somatostatin signals through somatostatin receptor subtypes (SSTR) 2 and 5 to attenuate GH secretion. Although expressed in normal pituitary glands and in GH-secreting pituitary tumors, SSTR3 function was unclear, and we have now determined the role of SSTR3 in somatotroph function. Stable rat pituitary tumor cell (GC) transfectants of human SSTR3 (GpSSTR3(WT)) showed suppression of rat (r) GH promoter activity, GH mRNA expression, and secreted GH concordant with suppressed cAMP/protein kinase A (PKA) signaling. In contrast, cAMP levels and GH expression were unchanged in cells expressing a mutant SSTR3 DRY motif (GpSSTR3(R141A)). GH expression was rescued by treatment of GpSSTR3(WT) with forskolin and 8-bromo-cAMP. GpSSTR3(WT) exhibited activation of glycogen synthase kinase3-ß (GSK3-ß), a PKA substrate, which was also reversed by 8-Bromo-cAMP treatment. Moreover, SSTR3-dependent GH transcriptional inhibition was rescued by inhibition of GSK3-ß. GpSSTR3(WT) exhibited elevated Pit-1 serine phosphorylation and decreased Pit-1 occupancy of the rGH promoter with sustained Pit-1 expression. GSK3-ß and Pit-1 physically interacted with each other, indicating that Pit-1 may be a GSK3-ß phosphorylation substrate. In conclusion, constitutive SSTR3 activity mediates transcriptional repression of GH through cAMP/PKA, leading to subsequent activation of GSK3-ß and increased Pit-1 phosphorylation and ultimately attenuating Pit-1 binding to the rGH promoter.

Sca1⁺ murine pituitary adenoma cells show tumor-growth advantage.

The role of tumor stem cells in benign tumors such as pituitary adenomas remains unclear. In this study, we investigated whether the cells within pituitary adenomas that spontaneously develop in Rb+/- mice are hierarchically distributed with a subset being responsible for tumor growth. Cells derived directly from such tumors grew as spheres in serum-free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor. Some cells within growing pituitary tumor spheres (PTS) expressed common stem cell markers (Sca1, Sox2, Nestin, and CD133), but were devoid of hormone-positive differentiated cells. Under subsequent differentiating conditions (matrigel-coated growth surface), PTS expressed all six pituitary hormones. We next searched for specific markers of the stem cell population and isolated a Sca1(+) cell population that showed increased sphere formation potential, lower mRNA hormone expression, higher expression of stem cell markers (Notch1, Sox2, and Nestin), and increased proliferation rates. When transplanted into non-obese diabetic-severe combined immunodeficiency gamma mice brains, Sca1(+) pituitary tumor cells exhibited higher rates of tumor formation (brain tumors observed in 11/11 (100%) vs 7/12 (54%) of mice transplanted with Sca1(+) and Sca1(-) cells respectively). Magnetic resonance imaging and histological analysis of brain tumors showed that tumors derived from Sca1(+) pituitary tumor cells were also larger and plurihormonal. Our findings show that Sca1(+) cells derived from benign pituitary tumors exhibit an undifferentiated expression profile and tumor-proliferative advantages, and we propose that they could represent putative pituitary tumor stem/progenitor cells.

Growth hormone is a cellular senescence target in pituitary and nonpituitary cells.

Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins, chromosomal instability, or DNA damage is associated with p53/p21 activation, culminating in either senescence or apoptosis, depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH, in contrast to circulating pituitary-derived endocrine GH, has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway, with subsequent marked induction of intracellular pituitary GH in vitro. In contrast, GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo, treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland, as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence, likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary, in nonpituitary cells GH exerts antiapoptotic properties. Thus, the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence.

Constitutive somatostatin receptor subtype 2 activity attenuates GH synthesis.

Somatostatin signals predominantly through somatostatin receptor (SSTR) subtype 2 to attenuate GH release. However, the independent role of the receptor in regulating GH synthesis is unclear. Because we had previously demonstrated constitutive SSTR2 activity in mouse corticotrophs, we now analyzed GH regulation in rat pituitary somatotroph (GC) tumor cells, which express SSTR2 exclusively and are devoid of endogenous somatostatin ligand. We demonstrate that moderately stable SSTR2 overexpression (GpSSTR2(WT) cells) was associated with decreased GH promoter activity, GH mRNA, and hormone levels compared with those of control transfectants (GpCon cells). In contrast, levels of GH mRNA and peptide and GH promoter activity were unchanged in GpSSTR2(DRY) stable transfectants moderately expressing DRY motif mutated SSTR2 (R140A). GpSSTR(2DRY) did not exhibit an enhanced octreotide response as did GpSSTR2(WT) cells; however, both SSTR2(WT)-enhanced yellow fluorescent protein (eYFP) and SSTR2(DRY)-eYFP internalized on octreotide treatment. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, increased GH synthesis in wild-type GC cells and primary pituitary cultures. GpSSTR2(WT) cells induced GH synthesis more strongly on SAHA treatment, evident by both higher GH peptide and mRNA levels compared with the moderate but similar GH increase observed in GpCon and GpSSTR2(DRY) cells. In vivo SAHA also increased GH release from GpSSTR2(WT) but not from control xenografts. Endogenous rat GH promoter chromatin immunoprecipitation showed decreased baseline acetylation of the GH promoter with exacerbated acetylation after SAHA treatment in GpSSTR2(WT) compared with that of either GpSSTR(2DRY) or control cells, the latter 2 transfectants exhibiting similar GH promoter acetylation levels. In conclusion, modestly increased SSTR2 expression constitutively decreases GH synthesis, an effect partially mediated by GH promoter histone deacetylation.

Glucagon receptor is required for long-term survival: A natural history study of the Mahvash disease in a murine model

Background and aim We have described a novel Mahvash disease of hyperglucagonemia and pancreatic neuroendocrine tumors (PNETs) associated with an inactivating glucagon receptor mutation, and identified the glucagon receptor-deficient (..) (AU)

Antecedentes y objetivo: Hemos descrito la nueva enfermedad de Mahvash en la que existen hiperglucagonemia y tumores neuroendocrinos pancreáticos (TNEP) asociados con una mutación inactivante del receptor de glucagón, e (..) (AU)

Glucagon receptor is required for long-term survival: a natural history study of the Mahvash disease in a murine model.

BACKGROUND AND AIM: We have described a novel Mahvash disease of hyperglucagonemia and pancreatic neuroendocrine tumors (PNETs) associated with an inactivating glucagon receptor mutation, and identified the glucagon receptor-deficient (Gcgr(-/-)) mice as its murine model. We aim to elucidate the natural history of the rare Mahvash disease by long-term observation of the Gcgr(-/-) mice. MATERIALS AND METHOD: Wild type (WT) (n=52), heterozygous (n=127), and Gcgr(-/-) (n=56) mice living under standard vivarium conditions were observed without specific treatments over 22 months. Autopsy was performed on dead animals. RESULTS: The WT and heterozygous mice did not exhibit any measurable differences. The Gcgr(-/-) mice became progressively lethargic and cachexic after 12 months. Random glucose levels were stable in WT and heterozygous mice but decreased with age in the Gcgr(-/-) mice. At the end of observation, 28/56 Gcgr(-/-), 7/52 WT, and 24/127 heterozygous mice died. The survival curve of Gcgr(-/-) mice began to separate from those of WT and heterozygous mice at 12 months and the survival difference widened with age. At 18 months, survival probability was 17% for Gcgr(-/-) mice but 77% for WT and 81% for heterozygous mice. Autopsy revealed numerous PNETs up to 15 mm in diameter in most well-preserved Gcgr(-/-) pancreata (17/20) but none in WT or heterozygous ones. Four Gcgr(-/-) mice developed liver or subcutaneous metastasis. CONCLUSION: The untreated Mahvash disease may cause cachexia, severe hypoglycemia, and early death. Patients with Mahvash disease need to undergo life-long surveillance for PNETs. Functional glucagon receptor is thus required for long-term survival.

Estradiol partially recapitulates murine pituitary cell cycle response to pregnancy.

Because pregnancy and estrogens both induce pituitary lactotroph hyperplasia, we assessed the expression of pituitary cell cycle regulators in two models of murine pituitary hyperplasia. Female mice were assessed during nonpregnancy, pregnancy, day of delivery, and postpartum. We also implanted estradiol (E(2)) pellets in female mice and studied them for 2.5 months. Pituitary weight in female mice increased 2-fold after E(2) administration and 1.4-fold at day of delivery, compared with placebo-treated or nonpregnant females. Pituitary proliferation, as assessed by proliferating cell nuclear antigen and/or Ki-67 staining, increased dramatically during both mid-late pregnancy and E(2) administration, and lactotroph hyperplasia was also observed. Pregnancy induced pituitary cell cycle proliferative and inhibitory responses at the G(1)/S checkpoint. Differential cell cycle regulator expression included cyclin-dependent kinase inhibitors, p21(Cip1), p27(Kip1), and cyclin D1. Pituitary cell cycle responses to E(2) administration partially recapitulated those effects observed at mid-late pregnancy, coincident with elevated circulating mouse E(2), including increased expression of proliferating cell nuclear antigen, Ki-67, p15(INK4b), and p21(Cip1). Nuclear localization of pituitary p21(Cip1) was demonstrated at mid-late pregnancy but not during E(2) administration, suggesting a cell cycle inhibitory role for p21(Cip1) in pregnancy, yet a possible proproliferative role during E(2) administration. Most observed cell cycle protein alterations were reversed postpartum. Murine pituitary meets the demand for prolactin during lactation associated with induction of both cell proliferative and inhibitory pathways, mediated, at least partially, by estradiol.

EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas.

Cushing disease is a condition in which the pituitary gland releases excessive adrenocorticotropic hormone (ACTH) as a result of an adenoma arising from the ACTH-secreting cells in the anterior pituitary. ACTH-secreting pituitary adenomas lead to hypercortisolemia and cause significant morbidity and mortality. Pituitary-directed medications are mostly ineffective, and new treatment options are needed. As these tumors express EGFR, we tested whether EGFR might provide a therapeutic target for Cushing disease. Here, we show that in surgically resected human and canine corticotroph cultured tumors, blocking EGFR suppressed expression of proopiomelanocortin (POMC), the ACTH precursor. In mouse corticotroph EGFR transfectants, ACTH secretion was enhanced, and EGF increased Pomc promoter activity, an effect that was dependent on MAPK. Blocking EGFR activity with gefitinib, an EGFR tyrosine kinase inhibitor, attenuated Pomc expression, inhibited corticotroph tumor cell proliferation, and induced apoptosis. As predominantly nuclear EGFR expression was observed in canine and human corticotroph tumors, we preferentially targeted EGFR to mouse corticotroph cell nuclei, which resulted in higher Pomc expression and ACTH secretion, both of which were inhibited by gefitinib. In athymic nude mice, EGFR overexpression enhanced the growth of explanted ACTH-secreting tumors and further elevated serum corticosterone levels. Gefitinib treatment decreased both tumor size and corticosterone levels; it also reversed signs of hypercortisolemia, including elevated glucose levels and excess omental fat. These results indicate that inhibiting EGFR signaling may be a novel strategy for treating Cushing disease.

CEBPD suppresses prolactin expression and prolactinoma cell proliferation.

Hyperprolactinemia, usually caused by a pituitary lactotroph tumor, leads to galactorrhea and infertility. Increased prolactin (PRL) levels may be due to enhanced PRL expression or proliferation of PRL-secreting cells. We hypothesize that PRL expression and PRL-secreting cell proliferation are linked. Using microarray-based gene expression profiling, we identified CCAAT-enhancer-binding protein δ (CEBPD) transcription factor as a critical gene that regulates both PRL expression and lactotroph cell proliferation. CEBPD expression levels are decreased approximately 7-fold in experimental rat prolactinoma cells. Forced expression of this transcription factor in PRL-secreting cells (GH3 and MMQ) inhibited PRL expression and cellular proliferation, and CEBPD knockdown by small interfering RNA leads to increased PRL expression in both cell lines. To determine mechanisms underlying this observation, we determined binding of CEBPD to the PRL promoter and also showed marked suppression (96%) of PRL promoter activity. CEBPD and Pit1 interact and attenuate each other's binding to the PRL promoter. CEBPD also suppresses expression of proliferation-related genes, including c-Myc, survivin, as well as cyclins B1, B2, and D1. These results show that PRL expression and cell proliferation are controlled in part by CEBPD.

Pancreatic neuroendocrine tumors in glucagon receptor-deficient mice.

Inhibition of glucagon signaling causes hyperglucagonemia and pancreatic α cell hyperplasia in mice. We have recently demonstrated that a patient with an inactivating glucagon receptor mutation (P86S) also exhibits hyperglucagonemia and pancreatic α cell hyperplasia but further develops pancreatic neuroendocrine tumors (PNETs). To test the hypothesis that defective glucagon signaling causes PNETs, we studied the pancreata of mice deficient in glucagon receptor (Gcgr(-/-)) from 2 to 12 months, using WT and heterozygous mice as controls. At 2-3 months, Gcgr(-/-) mice exhibited normal islet morphology but the islets were mostly composed of α cells. At 5-7 months, dysplastic islets were evident in Gcgr(-/-) mice but absent in WT or heterozygous controls. At 10-12 months, gross PNETs (≥1 mm) were detected in most Gcgr(-/-) pancreata and micro-PNETs (<1 mm) were found in all (n = 14), whereas the islet morphology remained normal and no PNETs were found in any WT (n = 10) or heterozygous (n = 25) pancreata. Most PNETs in Gcgr(-/-) mice were glucagonomas, but some were non-functioning. No tumors predominantly expressed insulin, pancreatic polypeptide, or somatostatin, although some harbored focal aggregates of tumor cells expressing one of those hormones. The PNETs in Gcgr(-/-) mice were well differentiated and occasionally metastasized to the liver. Menin expression was aberrant in most dysplatic islets and PNETs. Vascular endothelial growth factor (VEGF) was overexpressed in PNET cells and its receptor Flk-1 was found in the abundant blood vessels or blood islands inside the tumors. We conclude that defective glucagon signaling causes PNETs in the Gcgr(-/-) mice, which may be used as a model of human PNETs. Our results further suggest that completely inhibiting glucagon signaling may not be a safe approach to treat diabetes.

[Endogenous neuregulin-1 expression in the anterior pituitary of female Wistar-Furth rats during the estrous cycle].

OBJECTIVE: To study the changes in endogenous neuregulin-1 (Nrg1) expression in the anterior pituitary of female Wistar-Furth rats in different phases of the estrous cycle. METHODS: Female Wistar-Furth rats during estrous cycles were used. RT-PCR was employed to study the changes in the expression of Nrg1 isoforms and their cognate receptors ErbB-2 and ErbB-4 in the anterior pituitary in different phases of the estrous cycle. Western blotting was used to detect Nrg1 expression at the protein level. Immunofluorescence staining was used to identify hypophyseal cells expressing Nrg1 and observe the localization and distribution of Nrg1 and functional phosphorylation of ErbB-4. The co-expression of Nrg1 and ErbB-4 in the anterior pituitary of Rhesus monkey was also investigated. RESULTS: Some of the Nrg1 isoforms, especially type III Nrg1s, were expressed at a higher level during the estrous cycle I (E1) and estrous cycle II (E2), a result consistent with that of Western blotting for samples of the anterior pituitaries collected at these phases. Immunofluorescence staining identified the gonadotrophs as the main source of Nrg1, and showed an extensive distribution of Nrg1 in the anterior pituitary in E1 and E2 phases accompanied by apparent phosphorylated activation of ErbB-4. Adjacent distribution of Nrg1- and ErbB-4-positive cells was also observed in the anterior pituitary of male Rhesus monkeys. CONCLUSION: Our results provide evidence for the expression of multiple Nrg1 isoforms and the presence of Nrg1/ErbB-4 signaling in the anterior pituitary of female Wistar-Furth rats. This signaling demonstrates an estrous cycle phase-related pattern. Additionally, Nrg1/ErbB-4-based juxtacrine signaling may exist in the anterior pituitary of male non-human primate.

HER2/ErbB2 receptor signaling in rat and human prolactinoma cells: strategy for targeted prolactinoma therapy.

Dopamine agonist resistance or intolerance is encountered in approximately 20% of prolactinoma patients. Because human epidermal growth factor receptor 2 (HER2)/ErbB2 is overexpressed in prolactinomas and ErbB receptor ligands regulate prolactin (PRL) gene expression, we tested the role of HER2/ErbB2 in prolactinoma hormone regulation and adenoma cell proliferation to assess the rationale for targeting this receptor for prolactinoma therapy. As we showed prolactinoma HER2 overexpression, we generated constitutively active HER2-stable GH3 cell transfectants (HER2CA). PRL mRNA levels were induced approximately 250-fold and PRL secretion was enhanced 100-fold in HER2CA cells, which also exhibited increased proliferation. Lapatinib, a dual tyrosine kinase inhibitor (TKI) of both epidermal growth factor receptor (EGFR)/ErbB1 and HER2, blocked receptor signaling, and suppressed PRL expression more than gefitinib, a TKI of EGFR/ErbB1. Lapatinib also suppressed colony formation in soft agar more than gefitinib. Oral lapatinib treatment caused tumor shrinkage and serum PRL suppression both in HER2CA transfectant-inoculated Wistar-Furth rats and in estrogen-induced Fischer344 rat prolactinomas. In cultured human cells derived from resected prolactinoma tissue, lapatinib suppressed both PRL mRNA expression and secretion. These results demonstrate that prolactinoma HER2 potently induces PRL and regulates experimental prolactinoma cell proliferation. Because pituitary HER2 signaling is abrogated by TKIs, this receptor could be an effective target for prolactinoma therapy.

Heregulin regulates prolactinoma gene expression.

To investigate the role of p185(her2/neu)/ErbB3 signaling in pituitary tumor function, we examined these receptors in human prolactinomas. Immunofluorescent p185(her2/neu) was detected in almost all (seven of eight), and ErbB3 expression in a subset (four of eight) of tumors (seven adenomas and one carcinoma). Quantitative PCR also showed abundant ErbB3 mRNA in tumor specimens derived from a rarely encountered prolactin-cell carcinoma. Activation of p185(c-neu)/ErbB3 signaling with heregulin, the ErbB3 ligand, in rat lacto-somatotroph (GH4C1) tumor cells specifically induced prolactin (PRL) mRNA expression approximately 5-fold and PRL secretion approximately 4-fold, whereas growth hormone expression was unchanged. Heregulin (6 nmol/L) induced tyrosine phosphorylation and ErbB3 and p185(c-neu) heterodimerization, with subsequent activation of intracellular ERK and Akt. The Akt signal was specific to ErbB3 activation by heregulin, and was not observed in response to epidermal growth factor activation of epidermal growth factor receptor. Gefitinib, the tyrosine kinase inhibitor, suppressed heregulin-mediated p185(c-neu)/ErbB3 signaling to PRL. Heregulin induction of PRL was also abrogated by transfecting cells with short interfering RNA directed against ErbB3. Pharmacologic inhibition of heregulin-induced phosphoinositide-3-kinase/Akt (with LY294002) and ERK (with U0126) signaling, as well as short interfering RNA-mediated mitogen-activated protein kinase-1 down-regulation, showed ERK signaling as the primary transducer of heregulin signaling to PRL. These results show ErbB3 expression in human prolactinomas and a novel ErbB3-mediated mechanism for PRL regulation in experimental lactotroph tumors. Targeted inhibition of up-regulated p185(c-neu)/ErbB3 activity could be useful for the treatment of aggressive prolactinomas resistant to conventional therapy.

Fibroblast growth factor-2 autofeedback regulation in pituitary folliculostellate TtT/GF cells.

To investigate paracrine regulation of pituitary cell growth, we tested fibroblast growth factor (FGF) regulation of TtT/GF folliculostellate (FS) cells. FGF-2, and FGF-4 markedly induced cell proliferation, evidenced by induction of pituitary tumor transforming gene-1 (Pttg1) mRNA expression and percentage of cells in S phase. Signaling for FGF-2-induced FS cell proliferation was explored by specific pharmacological inhibition. A potent inhibitory effect on FGF-2 action was observed by blocking of Src tyrosine kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (>or=0.1 microm), followed by protein kinase C (PKC) inhibition with GF109203X. Treatment with FGF-2 (30 ng/ml; 10 min) activated phosphorylation of signal transducer and activator of transcription-3, ERK, stress-activated protein kinase/c-Jun N-terminal kinase, Akt, and focal adhesion kinase. Src inhibition with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine suppressed FGF-2-induced Akt and focal adhesion kinase, indicating effects downstream of FGF-2-induced Src activation. FGF-2 also markedly induced its own mRNA expression, peaking at 2-4 h, and this effect was suppressed by Src tyrosine kinase inhibition. The PKC inhibitor GF109203X abolished FGF-2 autoinduction, indicating PKC as the primary pathway involved in FGF-2 autoregulation in these cells. In addition to pituitary FGF-2 paracrine activity on hormonally active cells, these results show an autofeedback mechanism for FGF-2 in non-hormone-secreting pituitary FS cells, inducing cell growth and its own gene expression, and mediated by Src/PKC signaling.

Diminished pancreatic beta-cell mass in securin-null mice is caused by beta-cell apoptosis and senescence.

Pituitary tumor transforming gene (PTTG) encodes a securin protein critical in regulating chromosome separation. PTTG-null (PTTG(-/-)) mice exhibit pancreatic beta-cell hypoplasia and insulinopenic diabetes. We tested whether PTTG deletion causes beta-cell senescence, resulting in diminished beta-cell mass. We examined beta-cell mass, proliferation, apoptosis, neogenesis, cell size, and senescence in PTTG(-/-) and WT mice from embryo to young adulthood before diabetes is evident. The roles of cyclin-dependent kinase inhibitors and DNA damage in the pathogenesis of diabetes in PTTG(-/-) mice were also addressed. Relative beta-cell mass in PTTG(-/-) mice began to decrease at 2-3 wk, whereas beta-cell proliferation rate was initially normal but decreased in PTTG(-/-) mice beginning at 2 months. Apoptosis was also much more evident in PTTG(-/-) mice. At 1 month, beta-cell neogenesis was robust in wild-type mice but was absent in PTTG(-/-) mice. In addition, the size of beta-cells became larger and macronuclei were prominent in PTTG(-/-) animals. Senescence-associated beta-galactosidase was also active in PTTG(-/-) beta-cells at 1 month. Cyclin-dependent kinase inhibitor p21 was progressively up-regulated in PTTG(-/-) islets, and p21 deletion partially rescued PTTG(-/-) mice from development of diabetes. mRNA array showed that DNA damage-associated genes were activated in PTTG(-/-) islets. We conclude that beta-cell apoptosis and senescence contribute to the diminished beta-cell mass in PTTG(-/-) mice, likely secondary to DNA damage. Our results also suggest that ductal progenitor beta-cells are exhausted by excessive neogenesis induced by apoptosis in PTTG(-/-) mice.

Rat prolactinoma cell growth regulation by epidermal growth factor receptor ligands.

Epidermal growth factor (EGF) regulates pituitary development, hormone synthesis, and cell proliferation. Although ErbB receptor family members are expressed in pituitary tumors, the effects of EGF signaling on pituitary tumors are not known. Immunoprecipitation and Western blot confirmed EGF receptor (EGFR) and p185(c-neu) protein expression in GH3 lacto-somatotroph but not in adrenocorticotropic hormone-secreting AtT20 pituitary tumor cells. EGF (5 nmol/L) selectively enhanced baseline ( approximately 4-fold) and serum-induced (>6-fold) prolactin (PRL) mRNA levels, whereas gefitinib, an EGFR antagonist, suppressed serum-induced cell proliferation and Pttg1 expression, blocked PRL gene expression, and reversed EGF-mediated somatotroph-lactotroph phenotype switching. Downstream EGFR signaling by ERK, but not phosphoinositide-3-kinase or protein kinase C, mediated the gefitinib response. Tumors in athymic mice implanted s.c. with GH3 cells resulted in weight gain accompanied by increased serum PRL, growth hormone, and insulin growth factor 1. Gefitinib decreased tumor volumes and peripheral hormone levels by approximately 30% and restored normal mouse body weight patterns. Mice treated with gefitinib exhibited decreased tumor tissue ERK1/2 phosphorylation and down-regulated tumor PRL and Pttg1 mRNA abundance. These results show that EGFR inhibition controls tumor growth and PRL secretion in experimental lacto-somatotroph tumors. EGFR inhibitors could therefore be useful for the control of PRL secretion and tumor load in prolactinomas resistant to dopaminergic treatment, or for those prolactinomas undergoing rare malignant transformation.

Skeletal muscle adaptations to testosterone and resistance training in men with COPD.

We recently reported increased leg lean mass and strength in men with chronic obstructive pulmonary disease (COPD) receiving 10 wk of testosterone (T) and leg resistance training (R) (Casaburi R, Bhasin S, Cosentino L, Porszasz J, Somfay A, Lewis M, Fournier M, Storer T. Am J Respir Crit Care Med 170: 870-878, 2004). The present study evaluates the role of muscle IGF and related factors as potential mechanisms for our findings, using quadriceps muscle biopsies from the same cohort. Patient groups were 1) weekly placebo (P) injections + no R; 2) P and R; 3) weekly injections of T + no R; and 4) T + R (TR). Muscle fibers were classified histochemically, and their cross-sectional areas (CSAs) and fiber density (number of fibers per unit area) were determined. Gene transcripts were determined by real-time PCR and protein expression by RIA. While no significant changes in fiber CSAs were noted across groups, increased trends were observed after 10 wk, and significant decrements in muscle fiber density were noted in all treated groups. A global increase in all myosin heavy chain (MyHC) mRNA isoforms was observed in TR patients. Muscle IGF-IEa and IGF-IEc mRNAs were significantly increased with TR group. Muscle IGF-I protein was increased in all intervention groups (greatest in TR). While TR IGF-II mRNA was increased, protein levels were unaltered. IGF binding protein-4 mRNA was increased with TR. Myogenin mRNA was increased in both T groups, while MyoD and myostatin were unchanged. Muscle atrophy F-box mRNA tended to increase with TR. Our data suggest that the combined interventions produced an enhanced local anabolic milieu driven in large part by the muscle IGF system, despite potentially negative biochemical influences present in COPD patients.

Regulation of growth hormone and prolactin gene expression and secretion by chimeric somatostatin-dopamine molecules.

Dopamine (DA) regulates both prolactin (PRL) secretion and gene expression, whereas somatostatin (SRIF) inhibits GH secretion with unclear effects on GH gene expression. We therefore tested the effects of SRIF analogs and chimeric SRIF/DA compounds BIM 23A760 and BIM 23A761 on GH and PRL secretion and gene expression in primary rat pituitary cultures and pituitary tumor GH(3) and MMQ cells. Chimeric SRIF/DA molecules suppressed GH release with a similar efficacy to SRIF receptor subtype 2 agonists in rat pituitary and GH(3) cells. After 24 h, BIM 23A760 and BIM 23A761 did not exert additive effects on GH secretion, and after 48 h were less effective than the combination of respective mono-receptor agonists in GH(3) cells. Real-time PCR did not reveal changes in GH mRNA levels after treatment with SRIF analogs and SRIF/DA molecules. SRIF/DA compounds suppressed PRL and PRL mRNA in rat pituitary and MMQ cells with a similar efficacy to D(2)-DA receptor agonist. In GH(3) cells, they suppressed PRL and PRL mRNA levels with a similar efficacy to SRIF receptor subtype 2 agonists. SRIF/DA molecules did not exhibit additive effects on PRL secretion and mRNA levels as compared with cotreatment with mono-receptor ligands. The results show that SRIF analogs and SRIF/DA molecules inhibit GH and PRL secretion and suppress PRL but not GH gene expression.

Pyridoxal phosphate inhibits pituitary cell proliferation and hormone secretion.

Pyridoxal phosphate (PLP), a bioactive form of pyridoxine, dose-dependently (10-1000 microm) inhibited cell proliferation in rat pituitary MMQ and GH3 cells and in mouse AtT-20 cells. After 4 d, MMQ cell numbers were reduced by up to 81%, GH3 cell numbers were reduced by up to 64% (P < 0.05), and AtT-20 cell numbers were reduced by up to 90%. Cell proliferation rates recovered and dose-dependently reverted to control levels after PLP withdrawal. After 4 d, PLP (400 and 1000 microm) decreased [3H]thymidine incorporation by up to 71% (P < 0.05). PLP (400-1000 microm) reduced GH3 cell GH and prolactin secretion and AtT-20 cell ACTH secretion (adjusted for cell number) by approximately 70% after 2 d. The 100 microm PLP also inhibited prolactin secretion (65%, P < 0.05) in primary rat pituitary cells treated for 2 d. PLP decreased the percentage of AtT-20 and GH3 cells in S phase and increased those in G0-G1 phase. Furthermore, PLP induced AtT-20 and GH3 cell apoptosis (28 vs. 6, P < 0.05; 26 vs. 3, P < 0.05, respectively) and dose-dependently reduced content of the antiapoptosis gene Bcl-2. These results indicate that pharmacological doses of PLP inhibit pituitary cell proliferation and hormone secretion, in part mediated through PLP-induced cell-cycle arrest and apoptosis. Pyridoxine may therefore be appropriate for testing as a relatively safe drug for adjuvant treatment of hormone-secreting pituitary adenomas.