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.

Abstract

Corticotropin-relessing 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 homogenous 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 cAMPdependent 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 frtst 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 ,-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 ofliposomes 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.Adrenocorticotropin (ACTH) release is stimulated by a variety of hormones including corticotropin-releasing factor (CRF) (1, 2), vasopressin (3,4), and catecholamines (5). These hormones may evoke secretion by initiating multiple events within the corticotroph. CRF activates both adenylate cyclase and cAMP-dependent protein kinase in rat anterior pituitary membranes (6) and homogenates of a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogenous population of corticotrophs (7,8). These data suggest that cAMP is involved in the receptor-mediated release of ACTH. 8-Adrenergic agonists, which stimulate cAMP-dependent protein kinase activity and the secretion of ACTH from AtT-20 cells (7), also depolarize AtT-20 cells and increase the frequency of action potentials in these corticotrophs (9, 10). This electrical activity has been associated with transient calcium currents (9, 10), and (3-adrenergic agonists and forskolin, a direct activator at adenylate cyclase, increase cytosolic calcium levels in . In addition to cAMP and calcium, activators of protein kinase C evoke ACTH release (12), and recent studies (13) have suggested a role for phosphatidylinositol turnover in hormone secretion from corticotrophs.

Dimer-Specific Potentiation of NGFI-B (Nur77) Transcriptional Activity by the Protein Kinase A Pathway and AF-1-Dependent Coactivator Recruitment

“…Although the primary response to CRH leading to activation of the cAMP/PKA pathway and accumulation of intracellular Ca 2ϩ has been well characterized (38,39,66), a wide variety of POMC gene response elements and transcriptional effectors have been proposed to mediate the nuclear events triggered by these second messengers. These include cFos (4), CREB (36), and PCRH-REB-1 (32).…”

“…Upon binding its receptor on corticotrope cells, CRH induces a rapid increase in cyclic AMP (cAMP) levels (39) followed by activation of PKA (66). CRH induction of the cAMP pathway also results in increased intracellular Ca 2ϩ levels through an enhancement of the activities of L-and P-type Ca 2ϩ channels, which are known to be modulated by PKA (37,38).…”

TIF1β/KAP-1 Is a Coactivator of the Orphan Nuclear Receptor NGFI-B/Nur77

“…Total RNA was prepared with the RNAeasy columns (Qiagen) according to the manufacturer's instructions. Reverse transcription was performed with 1 g of this RNA using oligo(dT) [12][13][14][15][16][17][18] (Invitrogen) and SuperScript II RT (Invitrogen). The cDNA was then used for quantitative real time PCR (MX 3005; Stratagene, La Jolla, CA) with the SYBR Green kit (Qiagen, Valencia, CA).…”

“…Upon binding to its receptor CRHR-1 on corticotrophs (12), CRH induces a signaling cascade that ultimately leads to increased POMC gene transcription. CRH increases cAMP levels, followed by activation of the protein kinase A (PKA) and mitogen-activated protein kinase pathways (13)(14)(15). Nur factors regulate the POMC promoter via the Nur response element (NurRE) that binds homodimers or heterodimers of Nur factors containing at least NGFI-B (16,17).…”

The T-box Factor Tpit Recruits SRC/p160 Co-activators and Mediates Hormone Action

“…POMC gene expression is itself under the positive control of hypothalamic corticotropin-releasing hormone (CRH). Ex-tensive studies have characterized the early steps of corticotrophs response to CRH, which include activation of the cAMP/ PKA pathway, MAPK activation and accumulation of intracellular Ca 2ϩ (12)(13)(14)(15). We have demonstrated that nuclear receptors (NRs) of the NGFI-B subfamily (Nur factors) are important mediators of CRH action on POMC, and at least part of the CRH effects are mediated through their binding element in the promoter, the NurRE (16 -18).…”