Reconstitution of the Protein Kinase A Response of the Rat Prolactin Promoter: Differential Effects of Distinct Pit-1 Isoforms and Functional Interaction with Oct-1

Abstract

PRL gene transcription is primarily regulated by dopamine, which lowers cAMP levels and inhibits protein kinase A (PKA) activity. Current data indicate that the cAMP/PKA response maps to the most proximal Pit-1/Pit-1beta binding site footprint I (FP I) on the rat PRL (rPRL) promoter. Pit-1, a POU-homeo domain transcription factor, is specifically expressed in the anterior pituitary and is required both for the normal development of anterior pituitary cell types, somatotrophs, lactotrophs, and thyrotrophs, and for the expression of their hormones: GH, PRL, and TSHbeta. Pit-1 has been shown to functionally interact, via FP I, with several transcription factors, including Oct-1, a ubiquitous homeobox protein, and thyrotroph embryonic factor, which is found in lactotrophs, to activate basal rPRL promoter activity. Pit-1beta/GHF-2, a distinct splice isoform of Pit-1, acts to inhibit Ras-activated transcription from the rPRL promoter, which is mediated by a functional interaction between Pit-1 and Ets-1 at the most distal Pit-1 binding site (FP IV). In this manuscript we show 1) that the Pit-1beta isoform not only fails to block PKA activation, but is, in fact, a superior mediator of the PKA response; 2) that the PKA response requires intact POU-specific and POU-homeo domains of Pit-1; and 3) that Oct-1, but not thyrotroph embryonic factor, functions as a Pit-1-interacting factor to mediate an optimal PKA response.

Repression of the prolactin promoter: a functional consequence of the heterodimerization between Pit-1 and Pit-1 β

“…Our previous studies showed that Pit-1 functioned as a repressor of the PRL promoter only in pituitary cells (Diamond & Gutierrez-Hartmann 1996, Diamond et al 1999, Ferry et al 2005. We speculated that this pituitary-specific repression might be the result of the heterodimerization between Pit-1 and Pit-1 .…”

“…Plasmid pA3 PRL luc, firefly luciferase driven by the proximal (-425) rat (r) PRL promoter, was described previously (Conrad & Gutierrez-Hartmann 1992, Diamond & Gutierrez-Hartmann 1996, Diamond et al 1999. Plasmids pRSV HA Pit-1, pRSV HA Pit-1 , pRSV PKA and pRSV -globin, respectively driven by the RSV promoter, were described previously (Diamond & Gutierrez-Hartmann 1996, Diamond et al 1999.…”

“…Plasmids pRSV HA Pit-1, pRSV HA Pit-1 , pRSV PKA and pRSV -globin, respectively driven by the RSV promoter, were described previously (Diamond & Gutierrez-Hartmann 1996, Diamond et al 1999. Plasmid RSV CBP was the generous gift of Dr Chee-Gun Lee.…”

“…A 1:3 Pit-1:Pit-1 plasmid DNA ratio that results in equal levels of protein expression was used (Diamond & Gutierrez-Hartmann 1996, Diamond et al 1999. We controlled the nonspecific effect of the RSV promoters upon transcription factor availability by including amounts of pRSV -globin plasmid DNA in all assays to render the total pRSV DNA concentration constant.…”

The Regulation of Pituitary Prolactin Secretion: Hypothalamic, Intrapituitary and Intracellular Factors and Signaling Mechanisms

“…Involvement of different membrane enzymes in the context of varied levels of activation of G-proteins as well as Ca 2+ -and protein kinase C-dependent processes are equally important to generate the characteristic cAMP signal and cut-off at certain level by the cAMP-degrading phosphodiesterases. (Antony, 2000;Diamond 1999;Gonzalez-Iglesias, 2006) The level of Ca 2+ concentration in cytosol and the cyclic nucleotide signaling pathways are connected in regulation of physiological processes through messenger generation and also influencing additional other signaling pathways and vice versa, there is a distinct molecular machinery responsible to modulate the calcium concentration ([Ca 2+ ]i) (Bruce 2003).…”