h-89 and Adrenal-Cortex-Neoplasms

We investigated the effects of epidermal growth factor (EGF) and prostaglandins (PG) on the expression of aromatase (CYP19) in human adrenocortical carcinoma cell line NCI-H295R cells. EGF significantly increased aromatase activity and CYP19 gene transcript in NCI-H295R cells. Exon PII was selected from among several tissue-specific exon I regions. Promoter II that abuts on exon PII was activated by EGF. PGE(2) also significantly increased aromatase activity, CYP19 gene transcript, and promoter II activity. The results of experiments using protein kinase (PK) inhibitors suggest that the cAMP-PKA signaling pathway is involved in the up-regulation of aromatase expression by EGF. PGE(2) activated promoter II activity in 4 h, while 12 h was required for its activation by EGF. In addition, PGE(2) was secreted from NCI-H295R cells in response to EGF. Selective agonists for prostaglandin receptors EP(1) and EP(2) significantly increased aromatase activity, which was decreased by the corresponding antagonists. Finally, antagonists for EP(1) and EP(2) inhibited the up-regulation of aromatase expression following EGF. These results suggest that PGE(2) secondarily acts as an autocrine signal in the up-regulation of aromatase expression by EGF in NCI-H295R cells.

Topics: Adrenal Cortex Neoplasms; Alprostadil; Aromatase; Benzylamines; Cell Line, Tumor; Dinoprost; Dinoprostone; Epidermal Growth Factor; Exons; Flavonoids; Humans; Isoquinolines; Promoter Regions, Genetic; Prostaglandins; Prostaglandins A; Prostaglandins E; Protein Kinase Inhibitors; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Prostaglandin E, EP2 Subtype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stimulation, Chemical; Sulfonamides

ACTH signaling pathway includes the action of both protein kinases, mainly cAMP-dependent protein kinase (protein kinase A, PKA), and serine/threonine and tyrosine phosphatases. MAPK phosphatase-1 (MKP-1) is a dual activity protein phosphatase involved in the dephosphorylation of MAPK. To determine whether MKP-1 is a component of ACTH cascade, here we investigate the expression levels of MKP-1 gene in Y1 mouse adrenocortical tumor cells under ACTH stimulation. ACTH transiently increased MKP-1 mRNA and protein levels. MKP-1 mRNA increase occurred at 30 min, peaked at 1 h (6-fold), and returned to basal levels thereafter. The ACTH-mediated mRNA increase was blunted by actinomycin D and enhanced by cycloheximide. A cell permeable cAMP analog, 8-bromo-cAMP, also transiently induced MKP-1 mRNA (4-fold) and the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamid abolished this effect. In contrast, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamid only partially reduced the effect of ACTH, suggesting the participation of PKA-independent mechanisms in the hormone-induced MKP-1 expression. In addition, we show that the rise in intracellular Ca(2+) and protein kinase C activation had a potent synergic effect on ACTH- and 8-bromo-cAMP-mediated MKP-1 induction. In summary, our findings demonstrate that MKP-1 is another component of ACTH signaling cascade and indicate that this hormone may potentially down-regulate MAPKs.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Calcium Signaling; Cell Cycle Proteins; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Dose-Response Relationship, Drug; Dual Specificity Phosphatase 1; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Immediate-Early Proteins; Isoquinolines; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Synthesis Inhibitors; Protein Tyrosine Phosphatases; RNA, Messenger; Sulfonamides; Transcriptional Activation; Tumor Cells, Cultured

The mechanisms and factors leading to enhanced aldosterone secretion and ultimately to neoplastic transformation of the adrenal cortex are poorly defined. Angiotensin-II (Ang-II) and endothelin-1 (ET-1) have emerged as likely candidates among potential aldosterone secretagogues and adrenocortical growth-promoting factors. We therefore compared the effects of Ang-II and ET-1 on steroid hormone secretion of Conn's adenomas.. Ten Conn's adenomas that showed responsiveness to Ang-II blockade in vivo were recruited. Fragments of the tumors were collected immediately after surgical excision, and dispersed cells were obtained by collagenase digestion and mechanical disaggregation. Steroid hormones secreted by dispersed Conn's adenoma cells were assayed by quantitative high-performance liquid chromatography or radioimmunoassay.. Both Ang-II and ET-1 (10(-9) mol/L) similarly enhanced the overall steroid hormone production. ET-1 raised the release of pregnenolone (as evaluated by blocking its further metabolism by cyanoketone), corticosterone, 18-hydroxycorticosterone, and aldosterone, without affecting that of 11-deoxycortisol, cortisol, and 11-deoxycorticosterone. The hormonal responses to ET-1 were partially reversed by 10(-7) mol/L of either the ETA-receptor antagonist BQ-123 or the ETB-receptor antagonist BQ-788 and were abolished when both antagonists were used together. The aldosterone response to the selective activation of ETA and ETB receptors was studied in three Conn's adenomas by exposing dispersed cells to ET-1 (10(-9) mol/L) plus BQ-788 (10(-7) mol/L) and to the ETB-receptor agonist BQ-3020 (10(-8) mol/L). Both treatments raised aldosterone output by about 2-fold. ETA receptor-mediated aldosterone response was abolished by the protein kinase (PK) C inhibitor calphostin C (10(-5) mol/L). ETB receptor-mediated secretory response was lowered by either calphostin C and the cyclooxygenase (COX) inhibitor indomethacin (10(-5) or 10(-4) mol/L) and was completely suppressed when these two were combined. The PKA inhibitor H-89 and the lipoxygenase inhibitor phenidone were ineffective.. Collectively, our findings indicate that Ang-II and ET-1 equipotently stimulate both early and late steps of aldosterone synthesis in Conn's adenoma cells. The secretagogue effect of ET-1 occurs via the activation of ETA and ETB receptors, which are coupled with the PKC-dependent and the PKC- and COX-dependent signaling pathways, respectively.

Topics: Adrenal Cortex Hormones; Adrenal Cortex Neoplasms; Adrenocortical Adenoma; Adult; Aged; Aldosterone; Angiotensin II; Endothelin-1; Endothelins; Female; Humans; Indomethacin; Isoquinolines; Male; Middle Aged; Naphthalenes; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Piperidines; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Pyrazoles; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Signal Transduction; Sulfonamides; Tumor Cells, Cultured