ABSTRACT
The biosynthesis of many of the peptides involved in homeostatic control requires peptidylglycine α-amidating monooxygenase (PAM), an ancient, highly conserved copper- and ascorbate-dependent enzyme. Using the production of amidated chromogranin A to monitor PAM function in tumor cells, physiologically relevant levels of hypoxia were shown to inhibit this monooxygenase. The ability of primary pituitary cells exposed to hypoxic conditions for 4 h to produce amidated chromogranin A was similarly inhibited. The affinity of the purified monooxygenase for oxygen (Km = 99 ± 19 µM) was consistent with this result. The ability of PAM to alter secretory pathway behavior under normoxic conditions required its monooxygenase activity. Under normoxic conditions, hypoxia-inducible factor 1a levels in dense cultures of corticotrope tumor cells expressing high levels of PAM exceeded those in control cells; expression of inactive monooxygenase did not have this effect. The effects of hypoxia on levels of two PAM-regulated genes (activating transcription factor 3 [Atf3] and FK506 binding protein 2 [Fkbp2]) differed in cells expressing high versus low levels of PAM. Putative hypoxia response elements occur in both human and mouse PAM, and hPAM has consistently been identified as one of the genes upregulated in response to hypoxia. Expression of PAM is also known to alter gene expression. A quarter of the genes consistently upregulated in response to hypoxia were downregulated following increased expression of PAM. Taken together, our data suggest roles for PAM and amidated peptide secretion in the coordination of tissue-specific responses to hypoxia.
Subject(s)
Chromogranin A/metabolism , Mixed Function Oxygenases/metabolism , Multienzyme Complexes/metabolism , Pituitary Gland, Anterior/enzymology , Pituitary Neoplasms/enzymology , Tumor Hypoxia , Activating Transcription Factor 3/genetics , Activating Transcription Factor 3/metabolism , Amidine-Lyases/genetics , Amidine-Lyases/metabolism , Animals , Cell Line, Tumor , Databases, Genetic , Female , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mixed Function Oxygenases/genetics , Multienzyme Complexes/genetics , Pituitary Gland, Anterior/pathology , Pituitary Neoplasms/genetics , Pituitary Neoplasms/pathology , Rats , Tacrolimus Binding Proteins/genetics , Tacrolimus Binding Proteins/metabolismABSTRACT
In animal models, estrogens are complete carcinogens in certain target sites. 4-Hydroxyestradiol (4-OH-E2), an endogenous metabolite of 17ß-estradiol (E2), is known to have prominent estrogenic activity plus potential genotoxicity and mutagenicity. We report here our finding that 4-OH-E2 does not induce pituitary tumors in ACI female rats, whereas E2 produces 100% pituitary tumor incidence. To probe the mechanism, we conducted a short-term animal experiment to compare the proliferative effect of 4-OH-E2 in several organs. We found that, whereas 4-OH-E2 had little ability to stimulate pituitary cell proliferation in ovariectomized female rats, it strongly stimulates cell proliferation in certain brain regions of these animals. Further, when we used in vitro cultured rat pituitary tumor cells as models, we found that 4-OH-E2 has similar efficacy as E2 in stimulating cell proliferation, but its potency is approximately 3 orders of magnitude lower than that of E2. Moreover, we found that the pituitary tumor cells have the ability to selectively metabolize 4-OH-E2 (but not E2) with ultrahigh efficiency. Additional analysis revealed that the rat pituitary expresses a membrane-bound catechol-O-methyltransferase that has an ultralow Km value (in nM range) for catechol estrogens. On the basis of these observations, it is concluded that rapid metabolic disposition of 4-OH-E2 through enzymatic O-methylation in rat anterior pituitary cells largely contributes to its apparent lack of cell proliferative and tumorigenic effects in this target site.
Subject(s)
Catechol O-Methyltransferase/metabolism , Estrogens, Catechol/pharmacology , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/metabolism , Animals , Biocatalysis , Carcinogenesis/drug effects , Cell Proliferation/drug effects , Estrogens, Catechol/chemistry , Female , Humans , Methylation , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/enzymology , Rats , Rats, Inbred ACI , Tumor Cells, CulturedABSTRACT
The murine double-stranded RNA-binding protein termed protein kinase R (PKR)-associated protein X (RAX) and the human homolog, protein activator of PKR (PACT), were originally characterized as activators of PKR. Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax(-/-) mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eukaryotic translation initiation factor 2 α subunit (eIF2α) (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax(-/-) mice. Generating rax(-/-) mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax(-/-) defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax(-/-) mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21(WAF1/CIP1). These results demonstrate that PKR kinase activity is required for onset of the rax(-/-) phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development.
Subject(s)
Pituitary Gland, Anterior/metabolism , RNA-Binding Proteins/metabolism , eIF-2 Kinase/antagonists & inhibitors , Amino Acid Substitution , Animals , Cell Cycle , Cell Line , Cell Proliferation , Crosses, Genetic , Enzyme Activation , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Female , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , Mutation , Organ Size , Phosphorylation , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/enzymology , Pituitary Gland, Anterior/growth & development , Protein Processing, Post-Translational , RNA Interference , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolismABSTRACT
The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating various biological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly and restrictedly expressed in neural and reproductive tissues and plays significant roles in these organs. Although the expression of UCH-L1 in the anterior pituitary gland has been reported, the detailed localization and the role of UCH-L1 remain obscure. In the present study, we detected UCH-L1 protein exclusively in hormone-producing cells, but not non-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition, the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes and mammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed a comparative analysis using genetically UCH-L1-deficient gad mice. Significant decreases in the numbers of gonadotropes and mammotropes were observed in gad mice, suggesting a close involvement of UCH-L1 in these cells. Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Taken together, this is the first report to definitely demonstrate the presence of UCH-L1 in mouse anterior pituitary gland, and our results might provide a novel insight for better understanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in the reproduction.
Subject(s)
Gonadotrophs/enzymology , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/physiology , Animals , Cell Line , Gonads/physiology , Hypothalamus/physiology , Male , Mice , Mice, Inbred ICR , Pituitary Gland/physiology , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/enzymology , Reproduction/geneticsABSTRACT
Gonadotropin expression is precisely regulated within the hypothalamic-pituitary-gonadal axis through the complex interaction of neuropeptides, gonadal steroids. and both gonadal- and pituitary-derived peptides. In the anterior pituitary gland, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) modulates gonadotropin biosynthesis and secretion, acting both alone and in conjunction with GnRH. Steroid hormone feedback also influences gonadotropin expression via both direct and indirect mechanisms. Evidence from nonpituitary tissues suggests that PACAP may be a target for gonadal steroid regulation. In the present study, we show that androgen markedly stimulates rat (r) PACAP promoter-reporter activity in the LßT2 mature mouse gonadotrope cell line. 5'-Serial deletion analysis of reporter constructs identifies 2 regions of androgen responsiveness located at (-915 to -818) and (-308 to -242) of the rPACAP promoter. Androgen receptor (AR) binds directly to DNA cis-elements in each of these regions in vitro. Site-directed mutagenesis of 3 conserved hormone response element half-sites straddling the (-308 to -242) region dramatically blunts androgen-dependent PACAP promoter activity and prevents AR binding at the mutated promoter element. Chromatin immunoprecipitation demonstrates that endogenous AR binds the homologous region on mouse chromatin in LßT2 cells in both the presence and absence of androgen. These data demonstrate that androgen stimulates PACAP gene expression in the pituitary gonadotrope via direct binding of AR to a specific cluster of evolutionarily conserved hormone response elements in the proximal rPACAP gene promoter. Thus, androgen regulation of pituitary PACAP expression may provide an additional layer of control over gonadotropin expression within the hypothalamic-pituitary-gonadal axis.
Subject(s)
Androgens/metabolism , Gonadotrophs/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism , Pituitary Gland, Anterior/metabolism , Receptors, Androgen/metabolism , Animals , Cell Line , Chromatin Immunoprecipitation , Gonadotropins/biosynthesis , Gonadotropins/metabolism , Mice , Mice, Inbred C57BL , Mutagenesis, Site-Directed , Pituitary Adenylate Cyclase-Activating Polypeptide/biosynthesis , Pituitary Adenylate Cyclase-Activating Polypeptide/genetics , Pituitary Gland, Anterior/enzymology , Promoter Regions, Genetic , Protein Binding/genetics , Rats , Regulatory Sequences, Nucleic Acid , Response Elements/genetics , Signal Transduction/genetics , Transcription, GeneticABSTRACT
Folliculostellate (FS) cells of the anterior pituitary gland express a variety of regulatory molecules. Using transgenic rats that express green fluorescent protein specifically in FS cells, we recently demonstrated that FS cells in vitro showed marked changes in motility, proliferation, and that formation of cellular interconnections in the presence of laminin, a component of the extracellular matrix, closely resembled those observed in vivo. These findings suggested that FS cells express matrix metalloproteinase-9 (MMP-9), which assists their function on laminin. In the present study, we investigate MMP-9 expression in rat anterior pituitary gland and examine its role in motility and proliferation of FS cells on laminin. Immunohistochemistry, RT-PCR, immunoblotting, and gelatin zymography were performed to assess MMP-9 expression in the anterior pituitary gland and cultured FS cells. Real-time RT-PCR was used to quantify MMP-9 expression in cultured FS cells under different conditions and treatments. MMP-9 expression was inhibited by pharmacological inhibitor or downregulated by siRNA and time-lapse images were acquired. A 5-bromo-2'-deoxyuridine assay was performed to analyze the proliferation of FS cells. Our results showed that MMP-9 was expressed in FS cells, that this expression was upregulated by laminin, and that laminin induced MMP-9 secretion by FS cells. MMP-9 inhibition and downregulation did not impair FS motility; however, it did impair the capacity of FS cells to form interconnections and it significantly inhibited proliferation of FS cells on laminin. We conclude that MMP-9 is necessary in FS cell interconnection and proliferation in the presence of laminin.
Subject(s)
Matrix Metalloproteinase 9/genetics , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/enzymology , Animals , Animals, Genetically Modified , Cell Communication/physiology , Cell Proliferation , Cells, Cultured , Gene Expression , Green Fluorescent Proteins/genetics , Laminin/physiology , Male , Nerve Growth Factors/genetics , Rats , S100 Calcium Binding Protein beta Subunit , S100 Proteins/geneticsABSTRACT
The hypophysial pars tuberalis (PT) acts as an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis. Recently, we have identified an endocannabinoid system in the PT of hamsters and provided evidence that 2-arachidonoylglycerol is a messenger molecule that appears to play an essential role in seasonal reproduction and prolactin release by acting on the cannabinoid receptors in the PD. We now demonstrate the enzymes involved in endocannabinoid synthesis and degradation, namely sn-1-selective diacylglycerol lipase α, N-acylphosphatidylethanolamine-specific phospholipase D, and monoacylglycerol lipase, in the PT of man by means of immunohistochemistry. High-performance liquid chromatography coupled with tandem mass spectrometry revealed 2-arachidonoylglycerol and other endocannabinoids in the human PT. Furthermore, we detected the expression of the cannabinoid receptor 1 (CB1), a primary receptor for endocannabinoids, in the PD. Double-immunofluorescence staining for CB1 and various hypophysial hormones or S-100, a marker for folliculostellate (FS) cells, revealed that CB1 immunoreactivity was mainly localized to corticotrophs and FS-cells. A limited number of lactotrophs and somatotrophs also showed CB1 immunoreactivity, which was however absent from gonadotrophs and thyrotrophs. Our data thus indicate that the human PT comprises an endocannabinoid system, and that corticotrophs and FS-cells are the main target cells for endocannabinoids. The functional significance of this newly discovered pathway remains to be elucidated in man; it might be related to the control of stress responses and/or reflect a remnant seasonal control of hypophysial hormonal secretion.
Subject(s)
Arachidonic Acids/metabolism , Cannabinoid Receptor Modulators/metabolism , Endocannabinoids , Glycerides/metabolism , Neurotransmitter Agents/metabolism , Pituitary Gland, Anterior/enzymology , Receptor, Cannabinoid, CB1/metabolism , Adult , Aged , Aged, 80 and over , Arachidonic Acids/analysis , Cannabinoid Receptor Modulators/analysis , Chromatography, High Pressure Liquid , Female , Glycerides/analysis , Humans , Immunohistochemistry , Male , Middle Aged , Neurotransmitter Agents/analysis , Pituitary Gland, Anterior/chemistry , Tandem Mass SpectrometryABSTRACT
Regulated intramembrane proteolysis, a highly conserved process employed by diverse regulatory pathways, can release soluble fragments that directly or indirectly modulate gene expression. In this study we used pharmacological tools to identify peptidylglycine α-amidating monooxygenase (PAM), a type I secretory granule membrane protein, as a γ-secretase substrate. PAM, an essential enzyme, catalyzes the final step in the synthesis of the majority of neuropeptides that control metabolic homeostasis. Mass spectroscopy was most consistent with the presence of multiple closely spaced NH(2) termini, suggesting that cleavage occurred near the middle of the PAM transmembrane domain. The luminal domains of PAM must undergo a series of prohormone convertase or α-secretase-mediated cleavages before the remaining transmembrane domain/cytosolic domain fragment can undergo a γ-secretase-like cleavage. Cleavage by γ-secretase generates a soluble fragment of the cytosolic domain (sf-CD) that is known to localize to the nucleus. Although PAM sf-CD is unstable in AtT-20 corticotroph tumor cells, it is readily detected in primary rat anterior pituitary cells. PAM isoform expression, which is tissue-specific and developmentally regulated, affects the efficiency with which sf-CD is produced. sf-CD levels are also modulated by the phosphorylation status of the cytosolic domain and by the ability of the cytosolic domain to interact with cytosolic proteins. sf-CD is produced by primary rat anterior pituitary cells in response to secretogogue, suggesting that sf-CD acts as a signaling molecule relaying information about secretion from the secretory granule to the nucleus.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Cell Nucleus/metabolism , Mixed Function Oxygenases/metabolism , Multienzyme Complexes/metabolism , Neuropeptides/metabolism , Pituitary Gland, Anterior/enzymology , Secretory Vesicles/enzymology , Amyloid Precursor Protein Secretases/genetics , Animals , Cell Line, Tumor , Cell Nucleus/genetics , Female , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mixed Function Oxygenases/genetics , Multienzyme Complexes/genetics , Neuropeptides/genetics , Protein Structure, Tertiary , Rats , Secretory Vesicles/genetics , Signal Transduction/physiologyABSTRACT
Retinoic acid (RA) plays a critical role in embryonic development, growth, and reproduction. RA is synthesized from retinoids via oxidation processes, and the oxidation of retinal to RA is catalyzed by the retinaldehyde dehydrogenases (RALDHs). We previously reported that RALDH1 mRNA was expressed in the anterior pituitary glands of adult rats and suppressed by administration of 17beta-estradiol in vivo. However, little is known about the mechanism regulating pituitary RALDH1 expression. In order to characterize the mechanism of estrogen-induced RALDH1 reduction, we examined the effect of 17beta-estradiol on the regulation of pituitary RALDH1 gene expression and protein production both in vivo and in vitro. Using quantitative real-time PCR and immunoblot analysis, we found that levels of RALDH1 gene expression and protein production markedly decreased after 1-week treatment with 17beta-estradiol in male rats. In immunohistochemical analysis, RALDH1-immunoreaction was observed in prolactin cells and folliculo-stellate cells. In 17beta-estradiol-treated rats, RALDH1-immunoreactivity was lower in prolactin cells, but not in folliculo-stellate cells. Treatment of isolated anterior pituitary cells with 17beta-estradiol (10(-14) - 10(-8) M) decreased expression of RALDH1 mRNA in a dose-dependent manner. Estradiol-induced suppression of RALDH1 expression was completely blocked by the estrogen receptor (ER) antagonist ICI 182, 780. The ERalpha-selective agonist propylpyrazole triol (10(-8) M) mimicked the effect of 17beta-estradiol on RALDH1 expression, but the ERbeta-selective agonist diarylpropionitrile (10(-8) M) did not. These results strongly suggest that RALDH1 mRNA expression is suppressed by 17beta-estradiol through ERalpha, and that estrogen regulates the expression of RALDH1 and production of RA in the anterior pituitary gland.
Subject(s)
Estrogen Receptor alpha/physiology , Gene Expression Regulation, Enzymologic , Pituitary Gland, Anterior/metabolism , Retinal Dehydrogenase/genetics , Aldehyde Dehydrogenase 1 Family , Animals , Cells, Cultured , Estradiol/agonists , Estradiol/pharmacology , Estrogen Receptor alpha/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hormone Antagonists/pharmacology , Male , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/enzymology , Prolactin/metabolism , RNA, Messenger/metabolism , Rats , Rats, Transgenic , Rats, Wistar , Retinal Dehydrogenase/metabolism , S100 Calcium Binding Protein beta Subunit , S100 Proteins/genetics , S100 Proteins/metabolismABSTRACT
17beta-estradiol (E2) exerts inhibitory actions on the nitric oxide pathway in rat adult pituitary glands. Previously, we reported that in vivo E2 acute treatment had opposite effects on soluble guanylyl cyclase (sGC) subunits, increasing alpha1- and decreasing beta1-subunit protein and mRNA expression and decreasing sGC activity in immature rats. Here we studied the E2 effect on sGC protein and mRNA expression in anterior pituitary gland from adult female rats to address whether the maturation of the hypothalamus-pituitary axis influences its effects and to corroborate whether these effects occur in physiological conditions such as during estrous cycle. E2 administration causes the same effect on sGC as seen in immature rats, and these effects are estrogen receptor dependent. These results suggest that E2 is the main effector of these changes. Since the sGC alpha-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC alpha-subunit are expressed. Here we show for the first time that sGCalpha2 and sGCalpha2 inhibitory (alpha2i) isoforms are expressed in this gland, but only sGCalpha2i mRNA increased after E2 acute treatment. Finally, to test whether E2 effects take place under a physiological condition, sGC subunit expression was monitored over estrous cycle. sGCalpha1, -beta1, and -alpha2i fluctuate along estrous cycle, and these changes are directly related with E2 level fluctuations rather than to NO level variations. These findings show that E2 physiologically regulates sGC expression and highlight a novel mechanism by which E2 downregulates sGC activity in rat anterior pituitary gland.
Subject(s)
Estrous Cycle/genetics , Gene Expression Regulation, Enzymologic , Guanylate Cyclase/genetics , Pituitary Gland, Anterior/enzymology , Receptors, Cytoplasmic and Nuclear/genetics , Animals , Cells, Cultured , Cyclic GMP/metabolism , Enzyme Activation/drug effects , Estradiol/analogs & derivatives , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Estrous Cycle/metabolism , Estrous Cycle/physiology , Female , Fulvestrant , Gene Expression Regulation, Enzymologic/drug effects , Guanylate Cyclase/metabolism , Nitric Oxide/pharmacology , Nitric Oxide/physiology , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/physiology , Soluble Guanylyl CyclaseABSTRACT
Ecto-peptidases modulate the action of peptides in the extracellular space. The relationship between peptide receptor and ecto-peptidase localization, and the physiological role of peptidases is poorly understood. Current evidence suggests that pyroglutamyl peptidase II (PPII) inactivates neuronally released thyrotropin-releasing hormone (TRH). The impact of PPII localization in the anterior pituitary on the endocrine activities of TRH is unknown. We have studied whether PPII influences TRH signaling in anterior pituitary cells in primary culture. In situ hybridization (ISH) experiments showed that PPII mRNA was expressed only in 5-6% of cells. ISH for PPII mRNA combined with immunocytochemistry for prolactin, beta-thyrotropin, or growth hormone, showed that 66% of PPII mRNA expressing cells are lactotrophs, 34% somatotrophs while none are thyrotrophs. PPII activity was reduced using a specific phosphorothioate antisense oligodeoxynucleotide or inhibitors. Compared with mock or scrambled oligodeoxynucleotide-treated controls, knock-down of PPII expression by antisense targeting increased TRH-induced release of prolactin, but not of thyrotropin. Similar data were obtained with either a transition-state or a tight binding inhibitor. These results demonstrate that PPII expression in lactotrophs coincides with its ability to control prolactin release. It may play a specialized role in TRH signaling in the anterior pituitary. Anterior pituitary ecto-peptidases may fulfill unique functions associated with their restricted cell-specific expression.
Subject(s)
Aminopeptidases/physiology , Pituitary Gland, Anterior/enzymology , Prolactin/metabolism , Pyrrolidonecarboxylic Acid/analogs & derivatives , Thyrotropin-Releasing Hormone/physiology , Animals , Cells, Cultured , Female , In Situ Hybridization , RNA, Messenger/metabolism , Rats , Rats, WistarABSTRACT
The vertebrate pituitary gland is a key endocrine control organ that contains six distinct hormone secreting cell types. In this study, we analyzed the role of direct cell-to-cell Delta-Notch signaling in zebrafish anterior pituitary cell type specification. We demonstrate that initial formation of the anterior pituitary placode is independent of Notch signaling. Later however, loss of Notch signaling in mind bomb (mib) mutant embryos or by DAPT treatment leads to increased numbers of lactotropes and loss of corticotropes in the anterior pars distalis (APD), increased number of thyrotropes and loss of somatotrope cell types in the posterior pars distalis (PPD), and fewer melanotropes in the posterior region of the adenohypophysis, the pars intermedia (PI). Conversely, Notch gain of function leads to the opposite result, loss of lactotrope and thyrotrope cell specification, and an increased number of corticotropes, melanotropes, and gonadotropes in the pituitary. Our results suggest that Notch acts on placodal cells, presumably as a permissive signal, to regulate progenitor cell specification to hormone secreting cell types. We propose that Notch mediated lateral inhibition regulates the relative numbers of specified hormone cell types in the three pituitary subdomains.
Subject(s)
Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/physiology , Receptors, Notch/genetics , Zebrafish Proteins/genetics , Animals , Cell Differentiation/physiology , Embryo, Nonmammalian/physiology , Embryonic Development , Gene Expression Regulation, Developmental , Immunohistochemistry , In Situ Hybridization , Pituitary Gland, Anterior/enzymology , RNA, Messenger/genetics , Signal Transduction , Zebrafish/geneticsABSTRACT
Chronic exposure of rats to ethanol results in significant changes in pituitary hormone secretion. However, identification of the site(s) and mechanism of action of ethanol to induce these effects remains elusive. Free radical damage at the adenohypophyseal level may play a role in the decline in serum gonadotropin levels in ethanol-fed rats. Since 24-h changes in redox state occurred, we analyzed the 24-h changes in pituitary gene expression of the prooxidant enzymes nitric oxide synthase (NOS) 1 and 2, and of heme oxygenase-1 (HO-1) enzyme, and in plasma NO(2)(-) and NO(3)(-) (NO(x)) levels, in ethanol and control rats. Male rats, 35-day-old, received a liquid diet for 4 weeks. The ethanol-fed group received a similar diet to controls except for that maltose was isocalorically replaced by ethanol. Animals were killed at six time intervals during a 24-h cycle. Anterior pituitary mRNA levels encoding NOS1, NOS2 and HO-1 were measured by real-time PCR analysis. Plasma NO(x) concentration was determined by the Griess reaction. Ethanol feeding of prepubertal rats changed significantly the 24-h pattern of expression of NOS1, NOS2 and HO-1 in the adenohypophysis and augmented NOS2 and HO-1 mRNA levels. Peak values for the three enzymes in ethanol-fed rats occurred at the beginning of the scotophase (i.e., at 21:00 h). Ethanol feeding augmented mean values plasma NO(x) levels with a maximum at 13:00 h while in controls a biphasic pattern was observed, with peaks at 09:00 h and 17:00-21:00 h. One of the mechanisms by which ethanol augments oxidative damage in the adenohypophysis may include overproduction of nitric oxide and carbon monoxide.
Subject(s)
Circadian Rhythm/drug effects , Ethanol/adverse effects , Gene Expression Regulation, Enzymologic/drug effects , Heme Oxygenase-1/genetics , Nitric Oxide Synthase/genetics , Pituitary Gland, Anterior/drug effects , Alcohol-Induced Disorders, Nervous System/enzymology , Alcohol-Induced Disorders, Nervous System/genetics , Alcohol-Induced Disorders, Nervous System/physiopathology , Animals , Carbon Monoxide/metabolism , Central Nervous System Depressants/adverse effects , Circadian Rhythm/genetics , Free Radicals/metabolism , Gene Expression Regulation, Enzymologic/genetics , Gonadotropins, Pituitary/metabolism , Male , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type II/genetics , Nitrites/blood , Oxidation-Reduction , Oxidative Stress/drug effects , Oxidative Stress/genetics , Pituitary Diseases/chemically induced , Pituitary Diseases/enzymology , Pituitary Diseases/genetics , Pituitary Gland, Anterior/enzymology , Pituitary Gland, Anterior/physiopathology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Wistar , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
Cessation of lactation causes a massive loss of surplus lactotrophs in the rat pituitary gland. The factors and mechanisms involved in this phenomenon have not yet been elucidated. Besides its inhibitory control on prolactin secretion and lactotroph proliferation, evidence suggests that dopamine (DA) may be a proapoptotic factor for lactotrophs. We therefore tested the proapoptotic effect of DA on pituitary glands from virgin, lactating, and postlactating rats. By measuring mitochondrial membrane potential loss, caspase-3 activation, and nuclear fragmentation, we show that DA induces apoptosis specifically in lactotrophs from postlactating rats. We then determined that this effect was partly mediated by the DA transporter (DAT) rather than the D(2) receptor, as corroborated by the detection of DAT expression exclusively in lactotrophs from postlactating rats. We also observed tyrosine hydroxylase (TH) expression in postlactating lactotrophs that was accompanied by an increase in DA content in the anterior pituitary gland of postlactating compared with virgin rats. Finally, we observed that cells expressing TH coexpressed DAT and cleaved caspase-3. These findings show that DA may play a role in lactotroph regression during the postlactation period by inducing apoptosis. The fact that this process requires DAT and TH expression by lactotrophs themselves suggests that it may be "autocrine" in nature.
Subject(s)
Apoptosis/drug effects , Dopamine Plasma Membrane Transport Proteins/genetics , Dopamine/pharmacology , Lactation/drug effects , Lactotrophs/metabolism , Tyrosine 3-Monooxygenase/genetics , Animals , Caspase 3/metabolism , Cells, Cultured , Dopamine Plasma Membrane Transport Proteins/metabolism , Dopamine Plasma Membrane Transport Proteins/physiology , Female , Gene Expression Regulation/drug effects , Lactation/genetics , Lactation/metabolism , Models, Biological , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/enzymology , Pituitary Gland, Anterior/metabolism , Rats , Rats, Sprague-Dawley , Tyrosine 3-Monooxygenase/metabolismABSTRACT
A recent report provides new evidence for the presence of glucokinase (GK) in the anterior pituitary. In the present study, immunohistochemistry was used to identify the cells containing GK in the pituitary of rats and monkeys. In rats, GK was detected as a generalized cytoplasmic staining in a discrete population of cells in the anterior pituitary. In colocalization experiments, the majority of cells expressing follicle-stimulating hormone (FSH) or luteinizing hormone (LH) also contained GK. In addition to the gonadotropes, GK was observed in a subpopulation of corticotropes and thyrotropes. GK was not detected in cells expressing growth hormone or prolactin. In monkeys, GK was also observed in a discrete population of cells. Intracellular distribution differed from the rat in that GK in most cells was concentrated in a perinuclear location that appeared to be associated with the Golgi apparatus. However, similar to rats, colocalization experiments showed that the majority of cells expressing FSH or LH also contained GK. In addition to the gonadotropes, GK was observed in a subpopulation of corticotropes and thyrotropes. In the monkey, only a few cells had generalized cytoplasmic staining for GK. These experiments provide further evidence for the presence of GK in the anterior pituitary. Although some corticotropes and thyrotropes contained GK, the predominant cell type expressing GK was gonadotropes. In view of the generally accepted role of GK as a glucose sensor in a variety of cells including the insulin-producing pancreatic beta-cells as the prototypical example, it is hypothesized that hormone synthesis and/or release in pituitary cells containing GK may be directly influenced by blood glucose.
Subject(s)
Glucokinase/metabolism , Immunohistochemistry/methods , Pituitary Gland, Anterior/enzymology , Animals , Female , Follicle Stimulating Hormone/metabolism , Gonadotropins, Pituitary/metabolism , Luteinizing Hormone/metabolism , Macaca fascicularis , Male , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/metabolism , Rats , Rats, Sprague-Dawley , Thyrotropin/metabolismABSTRACT
We previously showed that long-term exposure of anterior pituitary cells to nitric oxide (NO) induces apoptosis. The intracellular signals underlying this effect remained unclear. In this study, we searched for possible mechanisms involved in the early stages of the NO apoptotic cascade. Caspase 3 was activated by NO with no apparent disruption of mitochondrial membrane potential. NO caused a rapid increase of reactive oxygen species (ROS), and this increase seems to be dependent of mitochondrial electron transport chain. The antioxidant N-acetyl-cysteine avoided ROS increase, prevented the NO-induced caspase 3 activation, and reduced the NO apoptotic effect. Catalase was inactivated by NO, while glutathione peroxidase (GPx) activity and reduced glutathione (GSH) were not modified at first, but increased at later times of NO exposure. The increase of GSH level is important for the scavenging of the NO-induced ROS overproduction. Our results indicate that ROS have an essential role as a trigger of the NO apoptotic cascade in anterior pituitary cells. The permanent inhibition of catalase may strengthen the oxidative damage induced by NO. GPx activity and GSH level augment in response to the oxidative damage, though this increase seems not to be enough to rescue the cells from the NO effect.
Subject(s)
Apoptosis/physiology , Nitric Oxide/physiology , Pituitary Gland, Anterior/metabolism , Reactive Oxygen Species/metabolism , Animals , Antioxidants/pharmacology , Caspase 3/metabolism , Enzyme Activation , Female , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Membrane Potentials , Mitochondria/physiology , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/enzymology , Rats , Rats, WistarABSTRACT
Retinoic acid (RA) plays an important role in cell growth and tissue development and is also a regulating factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine hormone is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases catalyzing the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. In this study, we examined the expression of RALDH1, RALDH2, and RALDH3 mRNA in the rat embryonic pituitary gland. By in situ hybridization with digoxigenin-labeled cRNA probes, we detected mRNA expression for RALDH2 and RALDH3, but not RALDH1. The expression of RALDH2 and RALDH3 was located in Rathke's pouch at embryonic day 12.5 (E12.5) and subsequently in the developing anterior pituitary gland. We also used quantitative real-time polymerase chain reaction to analyze RALDH2 and RALDH3 mRNA expression levels during the development of the pituitary gland. We found that pituitary RALDH2 and RALDH3 mRNA levels were high at E17.5 and decreased markedly after birth. Our study is the first to show that RALDH2 and RALDH3, but not RALDH1, are expressed in the embryonic anterior pituitary gland of the rat.
Subject(s)
Aldehyde Oxidoreductases/genetics , Aldehyde Oxidoreductases/metabolism , Pituitary Gland, Anterior/embryology , Pituitary Gland, Anterior/enzymology , Retinal Dehydrogenase/genetics , Aldehyde Dehydrogenase 1 Family , Animals , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Pituitary Gland, Anterior/cytology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Retinal Dehydrogenase/metabolismABSTRACT
Angiotensin 1-7 (Ang 1-7) is a peptide originated from Ang II. It is known that in vessels Ang 1-7 shows opposite effects to Ang II. Ang 1-7 can modify processes of proliferation. However, Ang 1-7 action in pituitary gland cells was never studied. Moreover, the specific binding sites for Ang 1-7 are still unknown. The aim of this study was to examine the effects of Ang 1-7 on tyrosine kinases (PTKs) activity in the anterior pituitary. The reaction of phosphorylation was carrying out in presence of different concentration of Ang 1-7 and losartan (antagonist of AT1 receptor) and PD123319 (antagonist of AT2). Our results show that Ang 1-7 inhibited activity of PTK to 60% of basic activity. Losartan did not change the Ang 1-7-induced changes in PTKs activity. The presence of PD123319 together with Ang 1-7 caused stronger inhibition PTKs activity than Ang 1-7 alone. These observations suggest that Ang 1-7 binds to the novel, unknown, specific for this peptide receptor.
Subject(s)
Angiotensins/pharmacology , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/enzymology , Protein-Tyrosine Kinases/metabolism , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II Type 2 Receptor Blockers , Animals , Male , Rats , Rats, Wistar , Receptor, Angiotensin, Type 1/metabolism , Receptor, Angiotensin, Type 2/metabolismABSTRACT
Previous studies showed that 17 beta-estradiol (17 beta-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17 beta-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17 beta-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17 beta-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17 beta-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing alpha1 and decreasing beta1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6-9 h after 17 beta-E2 administration. 17beta-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17 beta-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17 beta-E2, because they were not observed with the administration of other steroids such as progesterone and 17 alpha-estradiol. This inhibitory action of 17beta-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17 beta-E2 action. 17 beta-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17 beta-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC beta 1 subunit and sGC activity in a specific, ER-dependent manner.
Subject(s)
Estradiol/pharmacology , Guanylate Cyclase/genetics , Nitric Oxide/pharmacology , Pituitary Gland, Anterior/enzymology , Animals , Cyclic GMP/biosynthesis , Down-Regulation/drug effects , Estradiol/analogs & derivatives , Female , Fulvestrant , Gene Expression/drug effects , Guanylate Cyclase/analysis , Guanylate Cyclase/metabolism , Isoenzymes/analysis , Isoenzymes/genetics , Isoenzymes/metabolism , RNA, Messenger/analysis , Rats , Rats, Wistar , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/physiology , SolubilityABSTRACT
Enzymatic activity of glucokinase was demonstrated, quantitated, and characterized kinetically in rat and mouse pituitary extracts using a highly specific and sensitive spectrometric assay. A previously proposed hypothesis that the glucokinase gene might be expressed in the pituitary corticotrophic cells was therefore reexamined using mRNA in situ hybridization and immunohistochemical techniques. No evidence was found that corticotrophs are glucokinase positive, and the identity of glucokinase-expressing cells remains to be determined. The findings do, however, suggest a novel hypothesis that a critical subgroup of anterior pituitary cells might function as glucose sensor cells and that direct fuel regulation of such cells may modify the classical indirect neuroendocrine pathways that are known to control hormone secretion from anterior pituitary cells.