Defective glucocorticoid regulation of proopiomelanocortin gene expression and peptide secretion in a small cell lung cancer cell line

A human small cell lung cancer cell line (COR L103) that actively expresses the proopiomelanocortin (POMC) gene has been used as a model of extrapituitary ACTH-secreting tumors to investigate the phenomenon of resistence of ACTH production to glucocorticoids. After both short term (24 h) and long term (10 days) exposure to hydrocortisone at concentrations of 500 and 1000 nM, the accumulation of intracellular POMC mRNA, ACTH, and ACTH precursor peptides in the culture medium was not suppressed. These finding contrast with those in the pituitary corticotroph cell line AtT20, in which POMC mRNA, ACTH, and ACTH precursors were suppressed under the same conditions. Two other genes that are regulated by glucocorticoids in other cell types, the tyrosine amino transferase gene and the glucocorticoid receptor gene, were expressed in COR L103 cells. However, neither gene appeared to be regulated by hydrocortisone in this small cell lung cancer cell line. Further studies demonstrated that glucocorticoid receptor binding could be detected in the nucleus and cytoplasm, with a Kd of 5 X 10(-9) M. It is concluded that nonsuppression of POMC by glucocorticoids is probably part of a more global defect of glucocorticoid signaling in these cells, but that this defect lies distal to steroid binding in the nucleus.     

Mimecan in pituitary corticotroph cells may regulate ACTH secretion and the HPAA

Mimecan is a protein of unknown function that is expressed in the pituitary tissues of mouse and human. In this study, we observed the function of mimecan on the proopiomelanocortin (POMC) gene in the pituitary and the hypothalamo-pituitary-adrenal axis (HPAA). Incubating pituitary corticotroph AtT-20 cells with recombinant mimecan protein stimulated adrenocorticotrophic hormone (ACTH) secretion without significantly up-regulating POMC gene expression. In addition, pituitary corticotroph AtT-20 cell corticotropin-releasing hormone receptor 1 (CRHR1) gene expression was induced by mimecan. Interestingly, long-term mimecan overexpression in corticotroph cells increased CRHR1 mRNA levels while slightly decreasing POMC mRNA expression and ACTH secretion. Using mimecan knockout mice, we found that, although the serum ACTH concentration was not significantly different between wild type and mimecan knockout mice under basal conditions, the serum ACTH level was relatively lower in mimecan knockout mice after treatment with corticotropin-releasing hormone (CRH). Meanwhile, we observed that POMC and CRHR1 gene expression decreased in primary cultured knockout mouse pituitary cells compared with wild type cells. Taken together, these data suggest that mimecan expressed in pituitary corticotroph cells mainly regulates ACTH secretion in the pituitary and coordinates the HPAA.     

Interleukin-6 is inhibited by glucocorticoids and stimulates ACTH secretion and POMC expression in human corticotroph pituitary adenomas.

Interleukins and their receptors are expressed intrinsically in the anterior pituitary and regulate hormone production and cell proliferation. It has previously been shown that interleukin-6 (IL-6) regulates hormone secretion in normal pituitary cells and cell lines. Here we examined the effects of IL-6 on propiomelanocortin (POMC) expression and ACTH production in corticotroph adenoma cells in vitro. We found that IL-6 stimulates both ACTH secretion and POMC gene expression in corticotroph adenoma cell cultures. This first demonstration of the stimulatory action of IL-6 on human corticotroph adenoma cell function provides further evidence for a direct action of IL-6 on corticotroph pituitary cells. We have confirmed previous reports of IL-6 production by corticotroph adenoma cells and in addition, demonstrated for the first time that the synthetic glucocorticoid dexamethasone is a potent suppressor of intratumoral IL-6 production. This intratumoral produced IL-6 may be in part responsible, in an autocrine manner, for the stimulation of ACTH synthesis and secretion. Our results suggest that IL-6 might play a role in the pathogenesis of Cushing's disease. However, elevated glucocorticoid levels in patients with Cushing's disease may prevent excessive action of IL-6 on ACTH production and tumor progression of corticotroph adenomas in vivo.     

Post-translational processing of pro-opiomelanocortin in the Brattleboro (di/di) rat pituitary

In homozygous (di/di) Brattleboro rats, pro-opiomelanocortin (POMC) synthesis and processing, and the release of its products, is by definition not under the control of hypothalamic arginine vasopressin (AVP) in contrast with normal rats or heterozygote (di/+) littermates. To explore the role of AVP on these parameters, we have compared homozygotes and heterozygotes in terms of POMC mRNA levels, pituitary content of immunoreactive (ir)-ACTH, ir-beta-endorphin (beta-EP), alpha-melanocyte-stimulating hormone (alpha-MSH) and ir-N-acetyl-EP (NacEP), plasma levels of ir-ACTH and ir-beta-EP, and RP-HPLC profiles of the various forms of ir-alpha-MSH and ir-NacEP. Homozygous rats had immeasurably low levels of hypothalamic ir-AVP, in contrast with their heterozygote littermates; hypothalamic ir-corticotropin releasing factor did not differ between strains. No difference between-strains was seen in levels of POMC mRNA; elevated levels of all pituitary peptides, except ir-ACTH, were found in di/di rats; plasma levels of both ir-ACTH and ir-beta-EP were lower in di/di rats; pituitary ir-alpha-MSH RP-HPLC profiles were similar in both strains, but those for ir-NacEP showed a striking increase in Nac alpha-EP in di/di rats. We interpret these data as evidence for decreased degradation/retarded release of POMC products from the di/di anterior pituitary, for increased processing of POMC products to shorter forms in both anterior pituitary and neuro-intermediate lobe, and thus for a role of AVP in the processing of POMC, as well as POMC synthesis and ACTH/beta-EP release.     

Analysis of homogeneous populations of anterior pituitary folliculostellate cells by laser capture microdissection and reverse transcription-polymerase chain reaction

Pituitary folliculostellate (FS) cells are usually located between the secretory cells in the anterior pituitary, and they produce many peptides that exert a paracrine effect on hormone-producing pituitary cells. Previous approaches have been unsuccessful in obtaining homogeneous populations of FS cells. We used a combination of immunostaining with S100 protein followed by laser capture microdissection (Immuno-LCM) to obtain purified populations of rat FS cells. These cells were analyzed along with a mouse FS cell line (TtT/GF) by RT-PCR for gene expression. RT-PCR analyses showed that both FS cell populations expressed the mRNAs for glial fibrillary acidic protein, S100 protein, transforming growth factor-beta1 (TGFbeta1), TGFbeta receptor, interleukin-6, leptin, leptin receptor, pituitary adenylate cyclase-activating polypeptide (PACAP), and PACAP receptors. Both FS cell populations were negative for PRL, GH, and POMC, supporting the homogeneity of the rat FS cell population. TGFbeta1, but not PACAP-38, treatment stimulated cell proliferation in both FS cell populations. TGFbeta1 increased leptin, but not interleukin-6, mRNA expression in rat FS cells. However, TGFbeta1 inhibited leptin RNA expression in the TtT/GF cell line, as shown by RT-PCR and Northern blot analysis. These results indicate that 1) homogeneous populations of FS cells can be prepared by Immuno-LCM; 2) TGFbeta1 stimulates the proliferation of normal rat FS cells and the TtT/GF cell line; and 3) the effects of TGFbeta1 to stimulate leptin mRNA expression in rat FS cells but inhibit leptin mRNA expression in TtT/GF cells probably reflect alterations in signal transduction in the TtT/GF cell line.     

Comparison of ACTH and ACTH precursor peptides secreted by human pituitary and lung tumour cells in vitro

The molecular forms of ACTH secreted by established human small cell lung cancer (SCLC) cells and primary cultures derived from a bronchial carcinoid tumour, a pituitary adenoma and hyperplastic pituitary tissue have been characterized by Sephadex G-75 chromatography and quantified with two novel immunoradiometric assays for ACTH and ACTH precursor peptides. Pro-opiomelanocortin (POMC; Mr 31,000) and pro-ACTH (Mr 22,000) were secreted by all cell types. No smaller peptides were identified in the culture media from SCLC and bronchial carcinoid cells, implying a deficiency in the enzymes and/or intracellular organelles required for extensive POMC processing. A more heterogeneous profile of ACTH-containing peptides was produced by cells of pituitary origin, indicating more extensive proteolytic processing of POMC. However, the major peptide secreted by cells from a large aggressive pituitary adenoma was unprocessed POMC (Mr 31,000). These results suggest that both lung and pituitary cells in vitro retain their in-vivo pattern of POMC processing and provide valuable models in which to study the regulation of ACTH synthesis and secretion.     

Effects of protein kinase-C-related adrenocorticotropin secretagogues and interleukin-1 on proopiomelanocortin gene expression in rat anterior pituitary cells

To examine the effects of the cAMP-independent protein kinase-C system and interleukin-1 (IL-1) on secretion of ACTH and POMC gene expression in cultured rat anterior pituitary (AP) cells, AP cells were incubated with CRF, 8-bromo-cAMP, arginine vasopressin, angiotensin II, norepinephrine, and phorbol 12-myristate 13-acetate. After 15 h of incubation, CRF and 8-bromo-cAMP increased both ACTH release and the POMC mRNA level. Arginine vasopressin, angiotensin II, norepinephrine, or phorbol 12-myristate 13-acetate stimulated ACTH release but failed to increase basal or CRF-stimulated POMC mRNA levels. Human recombinant IL-1 alpha and -beta increased neither ACTH release nor POMC mRNA levels after 3 h of incubation. After 15 h of incubation, 100 pM to 10 nM IL-1 alpha and -beta increased ACTH release. However, POMC mRNA levels were significantly elevated only by 10 pM IL-1 beta. These results suggest that the CRF-cAMP system plays a major role in both ACTH release and expression of the POMC gene in AP cells, but the cAMP-independent protein kinase-C system contributes only to ACTH release; that acute stimulation of ACTH release from AP with IL-1 administration is not due to direct action of IL-1 at the pituitary level; that chronic exposure of AP cells to IL-1 alpha or -beta can stimulate ACTH release; and that the direct effects of IL-1 alpha and -beta on POMC gene expression, if any, seem to be minimal.