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.     

Atrial natriuretic factor does not affect basal, forskolin- and CRF-stimulated adenylate cyclase activity, cAMP formation or ACTH secretion, but does stimulate cGMP synthesis in anterior pituitary

The report that ANF inhibits basal and CRF-stimulated adenylate cyclase activity in anterior pituitary homogenates suggested that the atrial peptide could inhibit ACTH secretion. This possibility was investigated in the ACTH-secreting AtT-20 mouse pituitary tumor cell line as well as homogenates or primary cell cultures from rat anterior hypophysis. ANF (up to 5 X 10(-7) M) was found to be completely ineffective in stimulating basal, CRF- and/or forskolin-stimulated adenylate cyclase activity, cAMP accumulation and ACTH secretion. Similarly, ANF had no effect on spontaneous or GRF-induced GH release from cells in primary culture. ANF receptors, however, are present in AtT-20 cells and anterior pituitary cells as evidenced by the ability of the peptide to stimulate intracellular cGMP accumulation. The data, therefore, suggests that ANF does not have a negative modulatory action on the secretory function of anterior pituitary. The role of cGMP in any other action(s) of ANF remains unknown.     

Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes.

Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF-stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression.     

Stimulatory effect of leukemia inhibitory factor on ACTH secretion of dispersed rat pituitary cells

Cytokines are recognized to play an important role in modulating the immune and neuroendocrine system. We recently reported leukemia inhibitory factor (LIF) increased ACTH secretion and pro-opiomelanocortin mRNA level in the murine corticotroph tumor cell line (AtT-20). In this study, the expression of LIF in normal rat pituitary could be demonstrated by ribonuclease protection assay. LIF (1 nM) caused a slight, but significant increase in ACTH secretion (43.7% increase versus control, P<0.01), while showing statistically no significant change of growth hormone and prolactin level in dispersed rat pituitary cells. CRH (10 nM) also induced ACTH secretion 2.5-fold (P<0.01), and co-treatment of LIF and CRH exhibited 2.8-fold increase of ACTH secretion but no statistical difference from CRH treated group. These findings suggest that LIF also has same enhancing effect of ACTH secretion in primary pituitary cultured cells of rat as in AtT-20 cell and LIF acts as a paracrine or autocrine factor to modulate neuroendocrine function in the pituitary.     

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.     

Prolonged somatostatin pretreatment desensitizes somatostatin's inhibition of receptor-mediated release of adrenocorticotropin hormone and sensitizes adenylate cyclase

Addition of somatostatin-14 (SRIF) inhibits corticotropin releasing factor (CRF) and forskolin-stimulated cyclic AMP formation and ACTH release from tumor cells of the mouse anterior pituitary (AtT-20/D16-16). After long-term pretreatment of these cells with SRIF, the ability of SRIF to inhibit CRF and forskolin-stimulated cyclic AMP accumulation or ACTH secretion is markedly reduced. SRIF pretreatment also increases the formation of cyclic AMP in response to forskolin. This increase is delayed in onset, slow to recover, and blocked by the protein synthesis inhibitor, cycloheximide. SRIF pretreatment did not affect basal cyclic AMP and cyclic GMP levels or phosphodiesterase activity. It is proposed that prolonged treatment of AtT-20 cells with SRIF desensitizes SRIF receptors and induces a compensatory sensitization of adenylate cyclase through a process requiring protein synthesis.     

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid inhibits adrenocorticotropin secretion from anterior pituitary cells

Mouse clonal ACTH-secreting corticotrophs (AtT-20 cells) possess a membrane Ca2+-activated Cl- conductance which is partially blocked by the disulfonic stilbene derivative 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). In the current study the effect of SITS on the ACTH secretory process was evaluated. SITS markedly blocked basal and forskolin-stimulated ACTH secretion from AtT-20 cells (IC50 = 2.7 x 10(-4) M). Both CRF-induced ACTH secretion and forskolin-stimulated GH secretion from acutely dispersed rat anterior pituitary cells were inhibited by SITS (IC50 = 2.4 and 1.3 x 10(-4) M, respectively). SITS did not alter unstimulated or forskolin-elicited cAMP synthesis in AtT-20 cells, and in fact, could inhibit ACTH secretion in response to cAMP-independent agonists such as the calcium channel activator BAY-K-8644 or the protein kinase-C activator 12-tetradecanoyl-phorbol-13-acetate (IC50 = 2.6 and 2.4 x 10(-4) M, respectively). SITS did not alter the secretion of amylase from isolated exocrine pancreatic acinar cells. Its action was also fully reversible; after its removal from the incubation medium, cells secreted ACTH without a change in response to forskolin activation. Increasing extracellular Ca2+ or the addition of up to 10(-3) M tetraethylammonium or 4-aminopyridine did not reverse the inhibitory pattern of SITS action, suggesting that its inhibitory effect is most likely not due to hyperpolarization of AtT-20 cell membranes. The inability of amiloride to inhibit ACTH secretion further suggests that inhibition of ACTH secretion provoked by SITS is not due to a blockade of Cl-/HCO3- exchange. On the other hand, SITS was able to block 44% of basal 36Cl uptake by AtT-20 cells. Exchange of incubation medium chloride for gluconate or a reduction in the osmotic strength of the medium reduced both basal and secretagogue-stimulated ACTH secretion. The data suggest that SITS may modulate chloride-dependent, osmotically driven secretion from AtT-20 cells.     

Somatostatin inhibits multireceptor stimulation of cyclic AMP formation and corticotropin secretion in mouse pituitary tumor cells.

The AtT-20/D16-16 mouse pituitary tumor cell secretes corticotropin (ACTH) in response to corticotropin-releasing factor (CRF), (-)-isoproterenol, and vasoactive intestinal peptide (VIP). These responses are associated with a rapid increase in cyclic AMP formation. Somatostatin (SRIF) markedly decreases the stimulatory effect of CRF, (-)-isoproterenol, and VIP on both cyclic AMP formation and immunoreactive ACTH secretion. Forskolin and cholera toxin, adenylate cyclase activators, also stimulate cyclic AMP formation and ACTH secretion in AtT-20 cells and these responses are all inhibited by SRIF. The ACTH secretory responses to melittin and to the calcium ionophore A23187, neither of which increases cyclic AMP in AtT-20 cells, were not inhibited by SRIF. SRIF did not affect the binding of a tritiated beta-adrenergic receptor antagonist to AtT-20 membranes nor did it decrease basal cyclic AMP formation even in the presence of excess phosphodiesterase inhibitor, indicating that the reduction of cyclic AMP levels by SRIF did not involve either an interference with beta-adrenergic agonist binding to receptors or stimulation of cyclic AMP degradation. These results indicate that the inhibition of CRF-, (-)-isoproterenol-, and VIP-stimulated ACTH secretion by SRIF may be regulated by its inhibitory action on adenylate cyclase.     

Leukemia inhibitory factor (LIF) modulates pro-opiomelanocortin (POMC) gene regulation in stably transfected AtT-20 cells overexpressing LIF

Leukemia inhibitory factor (LIF) levels are elevated in sepsis and correlate with shock and poor prognosis. We have previously shown that lipopolysaccharide (LPS) administration induces hypothalamic and pituitary LIF expression in vivo, which is associated with the acute rise in circulating adrenocorticotrophic hormone (ACTH) levels. As AtT-20 cells respond to LIF, we established murine LIF (mLIF) stably transfected AtT-20 cell lines to study LIF regulation of pro-opiomelanocortin (POMC) expression and ACTH secretion. Our results show that mLIF transfectants accumulated mLIF (up to 15.6±3.2 ng/mL after 24h) as well as increased ACTH secretion (up to 2.4-fold above control cells) in conditioned medium. The magnitude of ACTH induction correlated with mLIF concentrations in different transfectants (r=0.75–0.88,p<0.05). Moreover, mLIF transfectants showed a higher sensitivity to CRH stimulation with an increased ACTH production within 8 h (p<0.05), whereas control cells were responsive to CRH at 24h. Additionally, mLIF transfectants exhibited a maximum threefold ACTH induction, compared to 1.7-fold in control cells. Furthermore, mLIF transfectants have a blunted dexamethasone-mediated inhibition of ACTH (35% inhibition in control cells vs no inhibition in mLIF-transfected cells at 24h). These findings support and extend the previous observations of LIF acting at the pituitary level, and indicate that mLIF stably-transfected AtT-20 cells are a useful model for studying mLIF-mediated gene regulation in pituicytes.     

Corticotropin-release inhibitory factor Evidence for dual stimulatory and inhibitory hypothalamic regulation over adrenocorticotropin secretion and biosynthesis.

The hypothalamus is currently thought to stimulate the synthesis and secretion of ACTH uniquely by secreting neuropeptides into the hypophysial-portal circulation, of which the most important are CRH and arginine vasopressin. However, analysis of the effects of pituitary isolation on the pituitary-adrenal axis in a variety of species suggests that the hypothalamus exerts both stimulatory and inhibitory regulation over ACTH secretion and POMC biosynthesis, and that the inhibitory control is dominant. Because none of the currently known inhibitory factors in the hypophysial-portal circulation consistently decreases basal ACTH secretion and POMC mRNA levels in normal anterior pituitary cells, it is suggested that this inhibition is mediated by a currently unidentified hypothalamic substance, presumably a neuropeptide, which we have termed corticotropin-release inhibitory factor (CRIF). The possible roles in clinical medicine of agonists and antagonists of this putative CRIF are discussed.     

Dynorphin stimulates corticotropin release from mouse anterior pituitary AtT-20 cells through nonopioid mechanisms

Dynorphin (Dyn) peptides were previously shown to increase plasma corticotropin (ACTH) in the ovine fetus, but the site of its action remains unclear. In the present study, Dyn A(1-17) was found to stimulate ACTH release from mouse anterior pituitary tumor AtT-20 cells in a dose-dependent manner. Naloxone did not block the effect of Dyn A(1-17) and the selective kappa-opioid receptor agonist U50488H did not stimulate ACTH release. Dyn A(2-17), a degradative peptide fragment that does not bind to opioid receptors, also stimulated ACTH release from AtT-20 cells. Although the nonopioid effects of Dyn have previously been attributed to N-methyl-D-aspartate (NMDA) receptors, the ACTH-releasing effects of Dyn A(1-17) in AtT-20 cells were not affected by co-administration of NMDA receptor antagonist LY235959. The ACTH response to Dyn A(1-17) could not be blocked by alpha-helical CRH (CRH antagonist) and was additive with a maximal stimulatory dose of CRH, suggesting different mechanisms of action. These results show that the release of ACTH by Dyn A(1-17) in AtT-20 cells is not mediated by kappa-opioid receptors or by the NMDA receptor.     

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.