CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression

Abstract

Hypothalamic growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) gene expression in anterior pituitary somatotrophs by binding to the GHRH receptor, a G-protein-coupled transmembrane receptor, and by mediating a cAMP-mediated protein kinase A (PKA) signal-transduction pathway. Two nonclassical cAMP-response element motifs (CGTCA) are located at nucleotides -187/-183 (distal cAMP-response element; dCRE) and -99/-95 (proximal cAMP-response element; pCRE) of the human GH promoter and are required for cAMP responsiveness, along with the pituitary-specific transcription factor Pit-1 (official nomenclature, POU1F1). Although a role for cAMP-response element binding protein (CREB) in GH stimulation by PKA has been suggested, it is unclear how the effect may be mediated. CREB binding protein (CBP) is a nuclear cofactor named for its ability to bind CREB. However, CBP also binds other nuclear proteins. We determined that CBP interacts with Pit-1 and is a cofactor for Pit-1-dependent activation of the human GH promoter. This pathway appears to be independent of CREB, with CPB being the likely target of phosphorylation by PKA.

Figure 1

Proximal end (–195/–1) of the hGH promoter. The 2 core CREs at –187/–183 and –99/–95 are underlined with a solid line, and the 2 Pit-1 binding sites at –122/–111 and –86/–75 are underlined with a dashed line. Nucleotides altered for mutational analysis are indicated below each site.

Figure 2

CBP and Pit-1 synergistically activate the proximal hGH promoter after stimulation of the hGHRH receptor by hGHRH, which requires both Pit-1 binding sites. CV-1 cells were transfected with hGHRH receptor and SV-40 expression vectors (pSG5) containing either Pit-1 or CBP cDNAs in the presence of (a) 195 bp of the hGH promoter; (b) mutation of the proximal Pit-1 binding site, GH1; (c) mutation of the distal Pit-1 binding site, GH2; or (d) mutation of both Pit-1 binding sites to nonbinding sites. Stimulation was with hGHRH(1-29)-NH₂ for 6 hours, or with BSA as a control. Data are expressed as mean fold activation ± SEM relative to EV Pit-1 plus EV CBP after stimulation with BSA. Significant activation was not seen without cotransfection of the GHRH receptor (data not shown).

Figure 3

CBP and Pit-1 synergistic activation of the hGH promoter after GHRH stimulation does not require the presence of CREs. CV-1 cells were transfected with hGHRH receptor and SV-40 expression vectors (pSG5) containing either Pit-1 or CBP cDNAs in the presence of (a) 195 bp of the hGH promoter (b) mutation of the pCRE, or (c) deletion of an additional 55 bp of the hGH promoter, with loss of the dCRE. Stimulation was with hGHRH(1-29)-NH₂ for 6 hours, or with BSA as a control. Data are expressed as mean fold activation ± SEM relative to EV Pit-1 plus EV CBP after stimulation with BSA. Significant activation was not seen without cotransfection of the GHRH receptor (data not shown).

Figure 4

CBP and Pit-1 synergistically activate the proximal hGH promoter by PKA. CV-1 cells were transfected with SV-40 expression vectors (pSG5) containing either Pit-1 or CBP cDNAs and 195 bp of the proximal hGH promoter in the presence of WT or mutant PKA catalytic subunit. W261C is a naturally occurring Pit-1 binding mutant, and AAA is a Pit-1 cDNA with loss of all phosphorylation sites (S115A/T119A/T220A). Data are expressed as mean fold activation ± SEM relative to EV Pit-1 plus EV CBP plus PKA mutant.

Figure 5

CBP and Pit-1 synergistic activation of the hGH promoter by PKA does not require the presence of CREs. CV-1 cells were transfected with SV-40 expression vectors (pSG5) containing either Pit-1 or CBP cDNAs in the presence of WT or mutant PKA catalytic subunit and in the presence of (a) 195 bp of the hGH promoter (b) mutation of the pCRE, or (c) deletion of an additional 55 bp of the hGH promoter, with loss of the dCRE. Data are expressed as mean fold activation ± SEM relative to EV Pit-1 plus EV CBP plus PKA mutant.

Figure 6

CREB independence of PKA activation of the hGH promoter. (a) CREB-deficient F9 cells were transfected with SV-40 expression vectors (pSG5) containing Pit-1 or CBP cDNAs and 195 bp of the proximal hGH promoter in the presence of WT or mutant PKA catalytic subunit. Data are expressed as fold activation relative to EV Pit-1 plus EV CBP plus PKA mutant. (b) CREB-deficient F9 cells and CREB-sufficient CV-1 cells were transfected with a common glycoprotein α-subunit reporter construct, which contains 2 well-defined CREs, and WT or mutant PKA catalytic subunit. Data are expressed as mean fold activation ± SEM relative to EV Pit-1 plus EV CBP plus PKA mutant.

Figure 7

Determination of CBP domains responsible for synergism with Pit-1 on activation of the hGH promoter. (a) Schematic representation of CBP and its deletion constructs. (b) CV-1 cells were transfected with SV-40 expression vectors (pSG5) containing WT Pit-1, WT or deletion construct CBP cDNAs, and 195 bp of the proximal hGH promoter in the presence of WT or mutant PKA catalytic subunit. Data are expressed as mean fold activation ± SEM relative to WT Pit-1 plus EV CBP plus PKA mutant. (c) CV-1 cells were transfected with SV-40 expression vectors (pSG5) containing WT Pit-1 and CBP cDNAs, CMV expression vector containing E1A, and 195 bp of the proximal hGH promoter in the presence of WT or mutant PKA catalytic subunit. Data are expressed as mean fold activation ± SEM relative to EV Pit-1 plus EV CBP plus PKA mutant.

Figure 8

Activation of a CRE reporter by hGHRH. CV-1 cells were transfected with hGHRH receptor, SV-40 expression vectors (pSG5) containing either Pit-1 or CBP cDNAs, and 4 copies of a CRE upstream of the TK promoter. Stimulation was with hGHRH(1-29)-NH₂ for 6 hours, or with BSA as a control. Data are expressed as mean fold activation ± SEM relative to EV Pit-1 plus EV CBP after stimulation with BSA.

Figure 9

Protein interactions between CBP and Pit-1. GST pull-down assay of radiolabeled fragments of the CBP protein and WT Pit-1. GST–Pit-1 fusion proteins were synthesized, purified, and exposed to ³⁵S-labeled CBP amino acids 1–450, CBP amino acids Δ8–1457, or unprogrammed reticulocyte lysate. After extensive washing, proteins trapped by the resin were resolved on SDS-PAGE and detected by autoradiography.

Figure 10

Activation of the proximal hGH promoter in Pit-1– and CBP-sufficient GH₃ cells after stimulation by hGHRH or PKA. GH₃ cells were transfected with 195 bp of the proximal hGH promoter or mutation of the proximal GH1 and/or distal GH2 Pit-1 binding sites with (a) cotransfection of hGHRH receptor and stimulation with hGHRH(1-29)-NH₂ for 6 hours, or with BSA as a control; or (b) cotransfection with WT or mutant PKA catalytic subunit. Data are expressed as relative light units (RLU) ± SEM.

Figure 11

Model of Pit-1–dependent stimulation of hGH gene expression by CBP.