Corticotropin-releasing hormone or dexamethasone regulates rat proopiomelanocortin transcription through Tpit/Pitx-responsive element in its promoter

Abstract

Tpit/Pitx-responsive element (Tpit/PitxRE), which binds transcription factors Tpit and Pitx1, confers cell-type specific expression of proopiomelanocortin (POMC) gene in pituitary corticotrops where the gene expression is mainly regulated by corticotropin-releasing hormone (CRH) and glucocorticoids (Gcs). CRH stimulates POMC gene expression, which is mediated by the accumulation of intracellular cAMP and requires binding of Nur factors to Nur-responsive element (NurRE). Gcs antagonize NurRE-dependent POMC gene expression through direct interaction between glucocorticoid receptors and Nur factors. We examined whether Tpit/PitxRE and NurRE are involved in CRH/cAMP-induced activation and Gc-induced repression of POMC gene expression by reporter assay in AtT-20 corticotropic cells. Deletion and mutation of Tpit/PitxRE markedly reduced basal activity of the promoter, and those of NurRE decreased the levels of the CRH/cAMP-induced activation. Nifedipine, KN-62, and W-7, specific inhibitors of the L-type calcium channel, calmodulin-dependent protein kinase II, and calmodulin respectively, attenuated CRH/cAMP-induced activation of promoters with three copies of either Tpit/PitxRE or NurRE, indicating that both Tpit/PitxRE and NurRE mediate CRHinduced activation of POMC gene expression in a calciumdependent manner. Deletion and mutation of Tpit/PitxRE abolished dexamethasone (DEX)-induced repression of POMC gene expression, while those of NurRE did not, indicating that Tpit/PitxRE predominantly mediates Gc-induced repression of POMC transcription. However, DEX treatment attenuated activities of promoters with three copies of either Tpit/PitxRE or NurRE, suggesting that Gcs act at NurRE as well as Tpit/PitxRE to repress POMC gene expression. We conclude that Tpit/PitxRE is an important element by which CRH and Gcs regulate the POMC gene expression.

Role of NeuroD1 on the negative regulation of Pomc expression by glucocorticoid

“…As shown in Fig 5A and 5B, DEX did not decrease Tpit and Pitx1 mRNA expression in time-dependent manners. This is consistent to the previous report by Murakami et al [27] which demonstrated that DEX did not suppress mRNA expression of Tpit and Pitx1 . These data indicate that the DEX-mediated Pomc transcription suppression may not be mediated via the decrease of Tpit and Pitx1 mRNA expression.…”

“…Reverse transcription mixtures were subjected to qPCR with KAPA SYBR FAST Universal 2x qPCR Master Mix (KAPA Biosystems) for primers of Pomc , NeuroD1 , Pan1 ( E47 ), Rb , and GAPDH using a CFX connect Real Time PCR thermal cycler (Bio-Rad, Hercules, CA). The following primer sequences were used: mouse Pomc (forward, ; reverse, ), mouse NeuroD1 (forward, ; reverse, ), mouse Pan1 ( E47 ) (forward, ; reverse, ), mouse Rb (forward, ; reverse, ), mouse Tpit (forward, ; reverse, ), mouse Pitx1 (forward, ; reverse, ) [27], mouse Nur77 (forward, ; reverse, ) [31], mouse Nurr1 (forward, ; reverse, ) [32], mouse GAPDH (forward, ; reverse, ), rat Pomc (forward, ; reverse, ) [33] and rat GAPDH (forward, ; reverse, ) [34]. Reactions were incubated at 95°C for 1 min and then amplified using temperature parameters of 95°C for 15 sec; 60°C for 10 sec; 72°C for 20 sec.…”

“…In addition to NeuroD1 [7], Nur77, Nurr1, Tpit and Pitx are known to activate Pomc transcription [26,27]. Among them, Nur77 [26] and Tpit/PitxRE [27] have been demonstrated to be involved in the Gc-mediated Pomc repression. However, the function of NeuroD1 in the negative regulation of Pomc has not yet been demonstrated.…”

Functional characterization of the mouse melanocortin 3 receptor gene promoter

“…Transfected cells were washed in phosphate-buffered saline (PBS (−)), and lysed with the reporter lysis buffer provided in a DualLuciferase Reporter Assay System (Promega) according to the manufacturer's instructions and our previous study (Murakami et al, 2007). The lysates were assayed for luciferase activity using a T-20/20 Luminometer (Promega).…”

The β isoenzyme of Ca2+/calmodulin-dependent kinase type II as possible mediator of somatostatin functions in pituitary tumour cells

“…This compound competitively inhibits the binding of calmodulin to CaMKII by interacting with the calmodulin binding site and thus inactivates the enzyme (Hidaka and Yokokura 1996). KN-62 was applied at 10 μM, a concentration widely used in different systems, including rat anterior pituitary cells, to inhibit specifically CAMKII activity (Cui et al 1996;Roberson et al 2005;Murakami et al 2007). As shown in Fig.…”