ACTH radioimmunocytochemistry (RICH) on rat anterior pituitary cells

Summary Radioimmunocytochemistry (RICH) was applied to detect corticotrophs in adult rat pituitaries and 8-day-old anterior pituitary monolayers by incubating sections and cultures with ¹²⁵I-ACTH-anti ACTH immune complexes. After incubations autoradiography was made. In comparison, conventional immunostaining was carried out on adjacent sections and parallel cultures. It has been esteblished that RICH is suitable for detection of corticotrophs.     

A quantitative approach to trace the corticotrophs in culture after adrenalectomy

Summary Anterior pituitaries of adrenalectomized and sham operated adult rats were dispersed by trypsin and cultured for 4 and 8 days. Adrenalectomy caused a moderate increase in number of corticotrophs in both zero-time cell suspensions and cultures. There was a pronounced elevation of immunoreactive ACTH content in both cells and media and an enhanced secretory response to stimulation of cultures with stalk-median eminence extract containing cortiocotropin releasing (CRF) activity. Some cells identified as corticotrophs by a specific immunostaining incorporated tritiated thymidine into their nuclei suggesting their ability to enter the cell cycle. The relatively smaller increase in number of ACTH cells and the considerably higher ACTH producing capacity of the corticotrophs after adrenalectomy seem to be inconsistent with the quantal response model of hormone secretion recently introduced by Rodbard.     

RICH1 inhibits breast cancer stem cell traits through activating kinases cascade of Hippo signaling by competing with Merlin for binding to Amot-p80

Cancer stem cells (CSCs) are regarded as the root of tumor recurrence and distant metastasis, as well as the major cause of resistance to conventional cancer therapies. Elucidating the mechanism of regulating CSCs is of great significance for the development of CSCs-targeting therapy strategies. YAP/TAZ are identified as key regulators of CSCs-related traits on breast cancer cells; however, the upstream regulatory mechanism of Hippo kinases cascade involved in regulating YAP/TAZ remains elusive. In this study, we found that the low expression of RICH1 in breast cancer was associated with poor prognosis. Depletion of RICH1 promoted the stemness and disrupted the normal epithelial architecture of MCF10A cells. Besides, RICH1 inhibited the migration and invasion of breast cancer cells and sensitized these cells to chemotherapeutic drugs. Mechanistically, RICH1 activated the kinases cascade of Hippo signaling via displacing Amot-p80 from the complex with Merlin. Further studies revealed that the deletion of the BAR domain of RICH1 abolished the function of attenuating the binding of Amot-p80 and Merlin, illustrating that the competitive binding to Amot-p80 with Merlin was mediated by the BAR domain of RICH1. In conclusion, our work elucidated the role and molecular mechanism of RICH1 in stemness regulation of breast cancer, and might provide opportunities for CSCs-targeting therapy.Subject terms: Breast cancer, Cancer stem cells     

The ultrastructural characteristics of the corticotropic cells of the rat adenohypophysis

Ultrastructural characteristics of corticotropic cells have been studied on serial semithin and ultrathin sections of the hypophysis from intact rats by the immunofluorescence and electron microscopic methods. It is shown that the population of corticotropic cells is heterogeneous and consists of degranulated, moderate granulated, hypergranulated cells and transitional forms among them. Ultrastructurally corticotropic cells correspond to Siperstein-Nakayama's ACTH cells. Kurosumi-Kobayashi "corticotrophs" fail to react with the antiserum to ACTH, so they have not regard for its production.     

Human pituitary corticotrophs: their amphophilic stainability and immunohistochemical characterization

Immunohistochemical characterization of the human pituitary beta(R) cells was investigated through the findings of the immunoreactivities with anti-porcine ACTH, -rat TSH, -rat FSH sera. Immunostained corticotrophs are oval or round in shape and localized in the anteromedial wedge. It is shown on the adjacent sections that they correspond to the beta(R) cells with amphophilic stainability with PAS-iron hematoxylin. In this wedge, amphophilic cells are preponderant, but PAS-positive thyrotrophs and gonadotrophs are not numerous. Amphophilic stainability varies in degree from cell to cell: One cell contains numerous medium-size of secretory granules weakly stained with iron hematoxylin and strongly with PAS in the PAS-positive cytoplasm, and the other cell is filled with big secretory granules intensively stained with iron hematoxylin and weakly with PAS. The immunostained TSH, LH and FSH cells are different from the beta(R) corticotrophs, because anti-ACTH serum never reacts to the TSH, LH and FSH cells in the two adjacent sections. LH and FSH reactivities are observed in the single cells. It is concluded that human corticotrophs are amphophilic beta(R) cells filled with secretory granules, and that they have quite a different appearance from the rat chromophobic stellate corticotrophs with a row arrangement of secretory granules along the plasma membrane.     

Proliferation of pituitary corticotrophs following adrenalectomy as revealed by immunohistochemistry combined with bromodeoxyuridine-labeling

Proliferation of corticotrophs following adrenalectomy (ADX) was studied by a combination of bromodeoxyuridine (BrdU)-labeling and immunohistochemistry. Rats were adrenalectomized, allowed to survive for 1, 3, 7, and 14 days and given 100 mg/kg body wt BrdU 3 h before sacrifice. BrdU and adrenocorticotropic hormone (ACTH) were detected in the same sections of the anterior pituitary using double-labeling immunohistochemistry. BrdU-labeled cells in the pituitary showed a tendency to increase until 1 week after ADX and slightly decreased at 2 weeks. Corticotrophs were increased to about 1.5 times of the control level 1–2 weeks after ADX. The number of cells double-labeled with both BrdU and ACTH increased markedly after ADX, suggesting active mitosis of existing corticotrophs. On the other hand, the ratios of these double-stained cells to all BrdU-labeled cells and to all corticotrophs were 5–7% and 0.9–1.3%, respectively, even after ADX, suggesting that the majority of corticotrophs which were increased after ADX were recruited from some other type of immature cells. The extent to which the two mechanisms are involved in hyperplasia of corticotrophs after ADX remains to be elucidated.     

Effects of corticotrophin-releasing hormone on corticotrophs in anterior pituitary gland allografts in hypophysectomized,orchidectomized hamsters

Summary We investigated the effects of corticotrophin-releasing hormone (CRH) on the percentage of anterior pituitary gland (APG) cells which are corticotrophs as well as the size and shape of corticotrophs. Pituitary glands were removed from 7-week-old male hamsters and placed beneath the renal capsules of hamsters that had been hypophysectomized and orchidectomized 3 weeks previously. Beginning 6 days after each host had received a single allograft, each was injected subcutaneously twice daily with 4 g CRH or vehicle for 16 days. Six hosts in each group were decapitated 16 h after the last injection. Sections of anterior pituitary tissue were stained for ACTH and with hematoxylin. The percentage of corticotrophs among APG cells was greater in allografts exposed to exogenous CRH (20%) than in allografts exposed to vehicle (15%). Exposure to exogenous CRH increased the cross-sectional area of corticotroph cells in allografts to values greater than those measured for corticotrophs in allografts exposed to vehicle, without altering the shape of cells. Results of subsequent studies suggested that hamsters with allografts injected with vehicle do not release ACTH and that exogenous CRH causes an abrupt release of ACTH from allografts. These results indicate that CRH releases ACTH from ectopic corticotrophs and that administration of CRH can increase corticotroph size and the percentage of APG cells that are corticotrophs.     

Three-dimensional and quantitative observation of the hypertrophy of rat corticotrophs following adrenalectomy

Summary The three-dimensional form of corticotrophs in the anterior pitiutary gland of rats was studied by reconstruction from serial semi-thin sections both in control rats and rats one week after adrenalectomy. The corticotrophs have large depressions and cup-shaped cavities on their surface, and these features became more conspicuous after adrenalectomy. The hypertrophy of corticotrophs in adrenalectomized rats was quantified by measuring the area and perimeter of all serially sectioned profiles. The volume of the whole cell increased from 1129±114 m³ to 2902±201 m³ (P<0.01) after adrenalectomy, while the surface area of the cells increased from 690±45 m² to 1431±116 m² (P<0.01). The volume of the nucleus increased from 87±11 m³ to 172±14 m³ (P<0.05). Though the complexity of the form of corticotrophs seems to be increased after adrenalectomy, the ratios (adrenalectomized/control) of cell volume and surface area were 2.57 and 2.07, respectively; this indicates that the increase of the cell volume was greater than that of the surface area.     

Lectin cytochemistry of cell types in human and canine pituitary

Summary Labeled lectins specific for different sugars were employed to identify different cell types in pituitaries from six human autopsies and seven dogs. To determine the lectins bound by each cell type, fixed-paraffin embedded sections serial to those stained with lectins were immunostained for specific hormones and the serial pairs were examined in a comparison microscope. In human pituitaries corticotrophs stained selectively with lectins having affinity for -l-fucose and the core region of complex type N-glycosyl-proteins. Some corticotrophs also stained for the presence of terminal -galactose. Thyrotrophs stained selectively with a periodate oxidation-borohydride reduction-concanavalin A sequence. Some mammotrophs evidenced content of glycoconjugate with terminal -galactose. Dendritic cells stained selectively for abundant glycogen with the periodate-reduction-concanavalin A sequence and a lectin from Griffonia simplicifolia. Adenohypophyseal cells of dog pituitary differed in showing absence of terminal -galactose in corticotrophs, presence of terminal -galactose in thyrotrophs, presence of glycoconjugate with N-glycosidically bound oligosaccharide in thyrotrophs and gonadotrophs and presence of terminal -galactose with a different lectin affinity in mammotrophs. The main contributions of lectin histochemistry applied to the pituitary include providing an additional histologic method for identification of some cell types, and localizing glycosylated prohormone or other biochemically unrecognized non-hormone glycoconjugates whose function in pituitary cells remains to be explained.This research was supported by NIH Grants AM-10956 and HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant #79     

Corticotroph cells in primary cultures of rat adenohypophysis: A light and electron microscopic immunocytochemical study

Summary Monolayer cultures of trypsin-dispersed cells of the rat adenohypophysis were grown for 5 to 54 days. ACTH was localized by immunocytochemistry using an antiserum to synthetic ACTH^1–28 prepared in rabbit and sheep anti-goat immunoglobulin coupled with peroxidase. ACTH content of the culture medium was measured by radioimmunoassay.Corticotrophs were found in the cultures and ACTH in the medium at each cultivation time. The corticotrophs retained their essential morphological characteristics. Immunological staining was found in the secretory granules, some tubular or saccular structures, parts of the rough endoplasmic reticulum, and the cytoplasmic matrix. Immature secretory granules in the Golgi apparatus as well as some Golgi elements showed different degrees of immunoreactivity. In agreement with the high ACTH content of the culture medium the number, size and shape of the secretory granules, the active Golgi apparatus, the high amount of extragranular ACTH as well as pictures suggesting granule extrusion claim for a high ACTH synthesis and transport (and low ACTH storage) in the cultured corticotrophs.     

Presence of angiotensin II in the adult male rat anterior pituitary gland: immunocytochemical study after cryoultramicrotomy

Angiotensin II (AII)-like immunoreactivity and binding sites have recently been demonstrated at the pituitary level. This peptide also exerts a stimulatory effect on anterior pituitary hormone release. Immunocytochemistry on ultrathin sections obtained by cryoultramicrotomy was used with the aim of localizing endogenous AII-like material at the cellular and subcellular levels of the anterior pituitary gland. AII-like immunostaining was observed only in gonadotrophs, lactotrophs, and corticotrophs. In gonadotrophs, AII-like immunoreactivity was restricted only to secretion granules. In the two other immunoreactive cells, lactotrophs and corticotrophs, immunostaining was observed in the cytoplasm and in the nucleus. In the cytoplasm, AII-like material was visualized in the cytoplasmic matrix and in the secretory granules. In the nucleus, immunostaining was distributed in the euchromatin in the vicinity of the heterochromatin. AII-like immunoreactivity was also seen at the plasma membrane, but only scarcely. No reaction product was found when anti-AII serum preincubated with AII was used. These immunocytochemical results (1) provide evidence that gonadotrophs are only a site of synthesis and/or storage of AII-like material, (2) indicate that lactotrophs and corticotrophs are cells for AII and (3) provide cytological evidence for a direct participation of AII in the regulation of the lactotropic and corticotropic function.     

Solution structure of the catalytic domain of RICH protein from goldfish

Regeneration-induced CNPase homolog (RICH) is an axonal growth-associated protein, which is induced in teleost fish upon optical nerve injury. RICH consists of a highly acidic N-terminal domain, a catalytic domain with 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity and a C-terminal isoprenylation site. In vitro RICH and mammalian brain CNPase specifically catalyze the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains unknown. Here, we report the NMR structure of the catalytic domain of goldfish RICH and describe its binding to CNPase inhibitors. The structure consists of a twisted nine-stranded antiparallel beta-sheet surrounded by alpha-helices on both sides. Despite significant local differences mostly arising from a seven-residue insert in the RICH sequence, the active site region is highly similar to that of human CNPase. Likewise, refinement of the catalytic domain of rat CNPase using residual dipolar couplings gave improved agreement with the published crystal structure. NMR titrations of RICH with inhibitors point to a similar catalytic mechanism for RICH and CNPase. The results suggest a functional importance for the evolutionarily conserved phosphodiesterase activity and hint of a link with pre-tRNA splicing.     

Dissimilar responsiveness of cultured corticotrophs and melanotrophs to tripeptide aldehydes

Cultured cells from adult rat anterior pituitaries or intermediate lobes were treated with the proteinase inhibitor tripeptide aldehydes BOC-DPhe-Pro-Arg-H (Boc-fPRH) and DPhe-Pro-Arg-H (fPRH), ovine corticotropin-releasing factor (oCRF), and bromocriptine. One millimolar fPRH stimulated basal, and slightly enhanced oCRF-induced ACTH release by melanotrophs in short-term experiments. The basal release of alpha-MSH was also stimulated by the drug. In long-term experiments, fPRH elevated markedly both the release and the intracellular level of ACTH; BOC-fPRH caused an increased alpha-MSH release. Tritiated fPRH had no preference for POMC-producing cells and BOC-fPRH or fPRH were harmless to the cell morphology. In anterior pituitary cell cultures, fPRH diminished slightly basal and oCRF-induced ACTH release. Bromocriptine was ineffective on corticotrophs, however, in melanotrophs it inhibited ACTH release markedly with or without fPRH in the medium. The dissimilar responsiveness of the corticotrophs and melanotrophs to the peptide aldehydes may be interpreted in terms of their differing membrane receptors or intracellular mechanism of stimulus-secretion coupling.     

Lectin cytochemistry of cell types in human and canine pituitary

Labeled lectins specific for different sugars were employed to identify different cell types in pituitaries from six human autopsies and seven dogs. To determine the lectins bound by each cell type, fixed-paraffin embedded sections serial to those stained with lectins were immunostained for specific hormones and the serial pairs were examined in a comparison microscope. In human pituitaries corticotrophs stained selectively with lectins having affinity for alpha-L-fucose and the core region of complex type N-glycosyl-proteins. Some corticotrophs also stained for the presence of terminal beta-galactose. Thyrotrophs stained selectively with a periodate oxidation-borohydride reduction-concanavalin A sequence. Some mammotrophs evidenced content of glycoconjugate with terminal beta-galactose. Dendritic cells stained selectively for abundant glycogen with the periodate-reduction-concanavalin A sequence and a lectin from Griffonia simplicifolia. Adenohypophyseal cells of dog pituitary differed in showing absence of terminal beta-galactose in corticotrophs, presence of terminal beta-galactose in thyrotrophs, presence of glycoconjugate with N-glycosidically bound oligosaccharide in thyrotrophs and gonadotrophs and presence of terminal beta-galactose with a different lectin affinity in mammotrophs. The main contributions of lectin histochemistry applied to the pituitary include providing an additional histologic method for identification of some cell types, and localizing glycosylated prohormone or other biochemically unrecognized non-hormone glycoconjugates whose function in pituitary cells remains to be explained.     

Expression of IGFBP7 mRNA in Corticotrophs in the Anterior Pituitary of Adrenalectomized Rats

The number of corticotrophs increases in the anterior pituitary (AP) gland in adrenalectomized (AdX) rats. In this study, aimed at identifying the growth factor implicated in this proliferation, we analyzed proteins secreted from a cDNA library of the AP of AdX rats, using the signal sequence trap method. A PCR analysis of several cDNAs that coded for insulin-like growth factor binding protein (IGFBP) 5, IGFBP7, and vacuolar H⁺-ATPase accessory subunit Ac45 revealed an increased and decreased expression level of IGFBP7 mRNA in the AP of AdX rats and AdX rats injected with dexamethasone, respectively. IGFBP7 mRNA was predominately expressed in the corticotrophs of the APs of both sham-operated and AdX rats. The AP of AdX rats contained an increased number of IGFBP7 mRNA–expressing cells and corticotrophs compared with that of sham-operated rats, but the ratio of IGFBP7 mRNA–positive corticotrophs per total number of corticotrophs did not significantly change in either group. Histochemical analysis of labeled proliferating cell nuclear antigen (PCNA) and sex-determining region Y box-2 (SOX2) revealed the presence of several PCNA-positive signals and the absence of SOX2 cells among the corticotrophs, suggesting that IGFBP7 mRNA–expressing corticotrophs are derived from in situ corticotrophs and that they increase in number as corticotrophs increase. The possible roles of IGFBP7 in the corticotrophs are also discussed. (J Histochem Cytochem 58:969–978, 2010)     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.     

Dissimilar responsiveness of cultured corticotrophs and melanotrophs to tripeptide aldehydes

Summary Cultured cells from adult rat anterior pituitaries or intermediate lobes were treated with the proteinase inhibitor tripeptide aldehydes BOC-DPhe-Pro-Arg-H (Boc-tPRH) and DPhe-Pro-Arg-H (fPRH), ovine corticotropin-releasing factor (oCRF), and bromocriptine. One millimolar fPRH stimulated basal, and slightly enhanced oCRF-induced ACTH release by melanotrophs in short-term experiments. The basal release of alpha-MSH was also stimulated by the drug. In long-term experiments, fPRH elevated markedly both the release and the intracellular level of ACTH; BOC-fPRH caused an increased alpha-MSH release. Tritiated fPRH had no preference for POMC-producing cells and BOC-fPRH or fPRH were harmless to the cell morphology. In anterior pituitary cell cultures, fPRH diminished slightly basal and oCRF-induced ACTH release. Bromocriptine was ineffective on corticotrophs, however, in melanotrophs it inhibited ACTH release markedly with or without fPRH in the medium. The dissimilar responsiveness of the corticotrophs and melanotrophs to the peptide aldehydes may be interpreted in terms of their differing membrane receptors or intracellular mechanism of stimulus-secretion coupling.In honour of Prof. P. van Duijn     

Activation of protein kinase C differentially regulates corticotropin-releasing factor-stimulated peptide secretion and cyclic AMP formation of intermediate and anterior pituitary cells in culture

The present study was aimed at investigating the effect of protein kinase C (PKC) activation on CRF receptor function of proopiomelanocortin (POMC) cells in culture. Incubation of tissues with the phorbol ester PMA selectively potentiated corticotropin-releasing factor (CRF)-stimulated ACTH secretion and cyclic AMP formation of anterior pituitary (AP) cells, while, in sharp contrast, it failed to similarly affect intermediate pituitary (IP) cells and AtT-20 corticotrophs exposed to CRF. Unexpectedly, however, long-term treatment of cultures with PMA, which depletes cell stores of PKC, resulted in a similar dramatic attenuation of stimulated peptide release from both corticotrophs and melanotrophs, while being without significant effect on cyclic AMP production. Exposure of cells to PMA did not change either basal or CRF-enhanced levels of POMC mRNA. We conclude that activation of PKC fails to synergize with CRF-mediated signalling in IP and AtT-20 cells, although optimal CRF receptor expression requires the presence of a functional kinase C pathway, thus suggesting cross-talks between both messenger systems.     

Cytochemical identification of human and murine pituitary corticotrophs and somatotrophs as APUD cells

Summary Cytochemical techniques, performed sequentially on single sections, have confirmed the identity of human and murine pituitary corticotrophs as APUD cells. These same methods indicate that in both species the acidophil somatotroph, identified as such by immunofluorescence using anti-human GH, possesses APUD qualities not only with respect to amine-handling, but also in terms of enzyme content.Inclusion of the somatotroph in the APUD series of cells which produce polypeptide hormones has far-reaching implications.     

Fine structural criteria for identifying rat corticotrophs

Summary The fine structural characteristics of normal rat corticotrophs stained with anti-porcine ACTH^1–39 serum were studied. At the ultrastructure level immunoreactive corticotrophs appear to comprise four distinct cell types: (1) large stellate cells (Siperstein cells) containing granules (170–250 nm in diameter) arranged in a peripheral row and usually embracing an acidophil; (2) elongate spindle-shaped cells (Moriarty cells) in which the secretory granules (170–250 nm in diameter) are distributed in a row or in small clusters in the peripheral cytoplasm; (3) oval or polygonal cells filled only with small secretory granules (130–170 nm in diameter), resembling the acidophil of small granules type (Yoshimura et al. 1974); and (4) polygonal or stellate cells filled with secretory granules of varying diameters (180–300 nm in diameter) and occasionally embracing an acidophil. The first type is the most common, but the others are infrequent. It is concluded that the criteria of Siperstein and Miller (1970) do not necessarily include all categories of rat corticotrophs.