Involvement of cAMP response element-binding protein in the regulation of cell proliferation and the prolactin promoter of lactotrophs in primary culture

Abstract

Hypothalamic hormones, including dopamine, regulate critical functions of pituitary cells via the cAMP-protein kinase A (PKA) pathway. The PKA-downstream transcription factor cAMP response element (CRE)-binding protein (CREB) is an integrating molecule that is also activated by many other protein kinase pathways. We investigated the involvement of CREB in the regulation of cell proliferation and the PRL promoter of rat lactotrophs in primary cell culture. Recombinant adenoviruses were used for efficient gene delivery into pituitary cells. Bromocriptine, a dopaminergic agonist known to decrease intracellular cAMP concentrations, caused inhibition of PRL promoter activity and lactotroph proliferation, which was accompanied by decreases in CRE-mediated transcription and CREB phosphorylation in lactotrophs. Expression of a dominant-negative form of CREB (MCREB), which was effective in suppressing CRE-mediated transcription induced by the adenylate cyclase activator forskolin, inhibited basal and forskolin-induced PRL promoter activity and PRL mRNA expression. MCREB expression lowered basal proliferative levels and blocked forskolin-induced proliferation of lactotrophs. Insulin-like growth factor I (IGF-I), a potent mitogen in lactotrophs, did not affect intracellular cAMP concentrations but transiently increased lactotroph CREB phosphorylation. MCREB expression also inhibited IGF-I-induced lactotroph proliferation. These results suggest that CREB is involved in the regulation of cell proliferation and the PRL promoter in normal lactotrophs and that dopamine inhibition of these lactotroph functions is at least in part due to inhibition of the cAMP-PKA-CREB pathway.

A novel Omega-class glutathione S-transferase gene in Apis cerana cerana: molecular characterisation of GSTO2 and its protective effects in oxidative stress

“…In addition, several important transcription factors required for regulating various environmental stresses, such as heat shock factors (HSF) and activating protein-1 (AP-1), were predicted (Ding et al 2005). The cAMPresponsive element binding protein (CREB) and the CCAAT/enhancer binding protein (C/EBP) were also found; and these elements regulate gene expression during cell differentiation, proliferation, and apoptosis (Ishida et al 2007;Carlezon et al 2005). We speculated that AccGSTO2 may be involved in organismal development and environmental stress responses.…”

Adenovirus vectors differentially modulate proliferation of pituitary lactotrophs in primary culture in a mitogen and infection time-dependent manner

“…The adenovirus vector Ad-bgal, which expresses bgal under the control of the human cytomegalovirus immediate early gene (CMV) promoter, was purchased from Clontech Laboratories. Ad-Tet.On, an adenovirus expressing CMV-driven rTetR-VP16, and Ad-TRE/bgal, an adenovirus expressing bgal in a tetracycline-regulated manner, have been described in detail previously (Ishida et al 2007a). An adenovirus expressing firefly luciferase driven by the CMV promoter (Ad-luc) was produced by inserting luciferase cDNA of pGL3-Basic into the NheI/XbaI sites in the pShuttle vector.…”

“…To produce an adenovirus carrying the rat prolactin (PRL) promoter but no cDNA for exogenous proteins (Ad-emp), a pGL3 vector containing a 3 . 2-kb fragment of the rat PRL promoter (Ishida et al 2007b) was digested with HindIII and XbaI to delete luciferase cDNA and then cloned into the NotI/BamHI sites in pTRE-Shuttle2-NruI.MCS.SmaI (Ishida et al 2007a). These modified pShuttle and pTREShuttle2 vectors were digested with I-CeuI and PI-SceI, and the genes of interest were inserted into a site created with the same restriction enzymes in Adeno-X viral DNA, a replicationdefective adenoviral genome.…”

“…After blocking nonspecific binding by using a blocking reagent supplied in the tyramide signal amplification (TSA) fluorescence system (Perkin-Elmer, Boston, MA, USA), the pituitary cells were immunostained for pCREB and PRL as described previously (Ishida et al 2007a). Fluorescence images of pCREB and PRL labeled with FITC-tyramide and AlexaFluor 594 respectively were captured with a digital CCD camera and stored in a personal computer.…”

“…Expression levels of mRNAs for estrogen-responsive genes were determined by qRT-PCR as described previously (Ishida et al 2007a). Total pituitary RNA was isolated using the RNeasy Mini Kit (Qiagen) and treated with RNase-free DNase I (Qiagen).…”