In this report, we show that the transcriptional coactivator CBP is critical to Egr-1-mediated activation of Lhb expression in pituitary gonadotrophs and plays a critical role in reproductive function in female mice. In vitro
studies were conducted in LβT2 gonadotrophs, as these cells have been used extensively to determine mechanisms of Lhb gene regulation (12
). The proximal Lhb promoter contains an enhancer region with two Egr-1 binding sites and two SF-1 binding sites separated by a Pitx1 binding site (19
). In LβT2 cells, one transcriptional coactivator, small nuclear RING finger protein (SNURF), has been shown to interact with Sp1 (found in the distal enhancer region) and SF-1 but not Egr-1 (8
CBP functions as a coactivator for many transcription factors and can be phosphorylated by a number of different signaling pathways (21
). While CBP and the related protein p300 have overlapping functions and high sequence homology, p300 lacks the consensus PKC phosphorylation site, conferring unique functions to the two proteins. Mice heterozygous for either a CBP or p300 knockout are viable, but homozygotes are embryonic lethal, as are CBP p300 compound heterozygotes (16
). There is considerable evidence that CBP and p300 are present in cells at limiting concentrations, providing a potential mechanism for tight regulation of Lhb expression (22
). In gonadotrophs, GNRH signaling via PKA phosphorylation results in CREB phosphorylation. This event may recruit CBP to the CRE on the Cga gene promoter in rodents but not in humans, as the CRE is not present in the proximal human Cga promoter (37
). According to our model, GNRH acting via PKC signaling pathways activates CBP via phosphorylation, allowing CBP to interact with Egr-1 on the Lhb promoter, thus permitting expression of Lhb ().
Model of proposed CBP interactions on the cAMP response element (CRE) and proximal Lhb promoter.
While our studies have not determined the exact mechanism by which CBP phosphorylation promotes interaction with Egr-1, a previous study using CBP protein fragments fused to GAL4 in a two-hybrid system showed that the N terminus of Egr-1 was able to physically interact with both the N and C termini of CBP, including the region containing Ser436 (38
). Nuclear magnetic resonance (NMR) structure studies suggest a potential mechanism by which CBP phosphorylation allows the protein to interact with transcription factors. The TAZ1 domains of CBP and p300, which share 92% sequence identity, contain a serine at position 436 in CBP, versus a glycine at position 422 in p300. The fourth α-helix of the TAZ1 domain was found to be longer in a CBP-transcription factor complex, owing to the presence of the helix-destabilizing glycine residue at the end of the p300 TAZ1 domain (9
). Thus, CBP phosphorylation in this domain may alter the protein conformation in a way that would allow it to interact with transcription factors such as Egr-1.
Using complementary methods of CBP overexpression and knockdown via shRNA, we demonstrated that CBP is required for GNRH-mediated Lhb expression in the LβT2 cell line. This is consistent with the results of other studies that have identified CBP as an important regulator of pituitary glycoprotein hormone subunit genes. In GH3
cells, CBP was shown to mediate thyroid-releasing hormone stimulation of both the alpha glycoprotein (Cga) and thyroid-stimulating hormone beta (Tshb) promoters, with distinct CBP domains required for activation of each gene (20
). CBP was also shown to mediate T3-dependent repression of human Cga expression in a heterologous cell line (54
). In each case, CBP was shown to interact with promoter-specific transcription factors (P-Lim, Pit-1, and p53) to regulate expression of both alpha and beta glycoprotein subunits via distinct domains.
While we showed that CBP is required for GNRH-mediated Lhb expression, treating LβT2 cells with GNRH did not change levels of CBP mRNA or protein but did result in rapid phosphorylation at Ser436. In order to test the functional significance of the serine phosphorylation site, we studied GNRH responsiveness in LβT2 cells expressing either WT or S436A mutant CBP. Initial experiments did not indicate a difference in GNRH responsiveness between WT protein and phosphorylation mutant protein. This is possibly because in cells transfected with mutant CBP, enough endogenous CBP was present to permit normal functioning of the cell. This is also consistent with results of prior studies that suggest that CBP can signal efficiently in limited concentrations (7
). By transducing cells with viral shRNA directed against the 5′UTR of CBP prior to overexpression, we were able to eliminate endogenous CBP while preserving WT and S436A transcripts produced by the pcDNA vector. Using this approach, we were able to demonstrate that while basal levels were unaffected by the loss of WT CBP, cells expressing the S436A mutant CBP are unable to increase Lhb transcription in response to GNRH.
Our studies also demonstrate that CBP likely promotes Lhb expression via interactions with Egr-1, the most critical transcription factor for Lhb. We identified Egr-1 as a target of CBP coactivation for several reasons. First, CBP has been shown to directly interact with Egr-1. CBP overexpression increases Egr-1-mediated activation of a 5-lipooxygenase reporter construct in COS cells; conversely, Egr-1 can also activate CBP, resulting in acetylation and stabilization of Egr-1 (38
). Additionally, both CBP and Egr-1 are activated by PKC. PKC activation has been shown to increase Egr-1 expression levels in gonadotrophs following GNRH administration (5
), while atypical PKC has been shown to phosphorylate CBP in hepatocytes following administration of insulin (21
). Consistent with studies showing CBP–Egr-1 interactions, we found that treatment of LβT2 cells with GNRH increases binding of CBP to the proximal Lhb promoter in the region containing Egr-1 binding sites. This promoter region also contains binding sites for other transcription factors, including a response element for SF-1, which is known to interact with CBP. However, we showed that CBP was not able to bind the proximal Lhb promoter after Egr-1 knockdown. Thus, while it is possible that CBP interacts with other transcription factors on the Lhb promoter, CBP is not able to bind the promoter without Egr-1. This underscores the critical role for Egr-1–CBP interaction on the proximal Lhb promoter in response to GNRH.
We also found that phosphorylation of CBP at Ser436 was critical for Egr-1 activation, as we showed in a modified two-hybrid system that treatment of cells with GNRH resulted in activation of Egr-1 in cells expressing WT CBP, but not in cells expressing S436A mutant CBP. This experiment showed that CBP activated Egr-1 via interactions on the promoter, rather than by increasing Egr-1 mRNA levels, as the quantity of Egr-1 in this system remains fixed.
In the present study, we extended our findings from LβT2 cells and demonstrated the requirement of CBP for gonadotroph function in vivo
. While the S436A mutation impairs fertility only in female mice, we detected a lack of GNRH responsiveness in both males and females. Breeding pairs containing an S436A mouse had both fewer litters over time and fewer pups per litter. In contrast, breeding pairs containing an S436A male exhibited normal fertility. This was not entirely surprising, as Lee et al. previously demonstrated female infertility but not male infertility in Egr-1 knockout mice (29
). The reduction in litter size seen with S436A mice paired with normal fertility in male mutant mice suggests that there is a threshold of Lhb expression required for normal gonadal function.
We then tested peak LH responses in males and females and measured estrous cycling in females to determine if there was adequate LH secretion to produce a proestrus surge. While S436A mice did exhibit estrous cyclicity, they spent more time in diestrus and had fewer proestrus surges than WT mice. This is consistent with our findings that S436A female mice can produce offspring, but with greater intervals between litters. Even so, the comparison of times between litters underestimates the extent to which fertility is impaired in S436A female mice, as several mice in the study never produced a second litter. Serum LH measurements showed that S436A mice were able to increase LH levels in proestrus but to a much lower magnitude than WT mice. Lower LH levels in proestrus may result in the release of fewer eggs per cycle, which is also consistent with our finding of reduced litter size. Ovarian histology corroborates these data, as ovaries from S436A mice have fewer corpora lutea and more atretic follicles than WT mice, indicating follicle formation without ovulation. Biochemically, our findings in male S436A mice were analogous to findings in female mice in that male S436A mice had a subnormal response to GNRH. As fertility and testicular histology were normal, this suggests that male mice have a lower threshold for LH levels needed for normal reproductive function. Our biochemical evaluation of S436A mutant mice also showed that the mutation did not alter baseline FSH levels or the FSH response to GNRH, thus emphasizing the specificity of the effects of the mutation to Lhb. Taken together, findings for male and female S436A mice indicate a defect specific to Lhb, specifically at the level of the pituitary gland, as S436A mice were unable to respond normally to GNRH.
Our findings are the first to identify the critical role of CBP in gonadotroph function. As a transcriptional coactivator, CBP can be activated by a number of different signaling pathways, including MAPK and PKC. As such, CBP may act as an integrator of signals in the gonadotroph, providing an additional level of responsiveness in the pituitary gland. Our group has previously shown that CBP can be phosphorylated in response to insulin at Ser436 via atypical PKC signaling (21
). Phosphorylation of CBP via insulin results in dissociation of CBP from the CREB-CBP-TORC2 complex, and in gonadotrophs, it results in recruitment to the Lhb promoter and activation of Lhb gene expression. We and others have shown that insulin can increase GNRH-mediated Lhb expression and secretion with some evidence of a costimulatory effect of insulin and GNRH in LβT2 cells via activation of AKT and extracellular signal-regulated kinase (ERK) (1
). This suggests that energy-homeostatic information can be integrated at the level of the pituitary gland, possibly through CBP, to modulate reproductive function. In vivo
studies demonstrate the importance of nutritional signals in the reproductive axis. Short-term food restriction suppresses pulsatile LH secretion in adult male rhesus monkeys, while in mice, loss of insulin signaling in the brain impairs fertility (3
). Excess insulin signaling may directly impact gonadotroph function as well. In a previous study, we showed that obese, hyperinsulinemic mice have elevated LH levels and female infertility that can be corrected with selective loss of insulin signaling in the gonadotroph (2
). It remains to be determined, however, if insulin is able to activate Lhb expression in the gonadotroph via CBP phosphorylation. If so, this would reveal a potential mechanism for abnormal LH levels and reproductive dysfunction in polycystic ovary syndrome. As we have previously shown that metformin is able to bypass insulin resistance and directly phosphorylate CBP in the livers of obese mice, future studies will be required to determine if insulin and metformin act directly on gonadotrophs to alter reproductive function in obesity-related infertility.