Estradiol regulates corticotropin-releasing hormone gene (crh) expression in a rapid and phasic manner that parallels estrogen receptor-alpha and -beta recruitment to a 3',5'-cyclic adenosine 5'-monophosphate regulatory region of the proximal crh promoter - PubMed (original) (raw)

Estradiol regulates corticotropin-releasing hormone gene (crh) expression in a rapid and phasic manner that parallels estrogen receptor-alpha and -beta recruitment to a 3',5'-cyclic adenosine 5'-monophosphate regulatory region of the proximal crh promoter

Avin S Lalmansingh et al. Endocrinology. 2008 Jan.

Abstract

In the central nervous system, CRH regulates several affective states. Dysregulation of neuronal crh expression in the paraventricular nucleus of the hypothalamus correlates with some forms of depression, and amygdalar crh expression may modulate levels of anxiety. Because estrogens modulate these states, we sought to determine 17beta-estradiol (E2) effects on crh expression. CRH mRNA levels were measured in the AR-5 amygdaloid cell line by RT-PCR analysis. They increased by 1 min of E2 treatment, suggesting that crh behaves as an immediate-early gene. After peaking at 3 min, CRH mRNA returned to basal levels and then increased by 60 min. To dissect some of the molecular mechanisms underlying these events, we measured occupancy of the crh promoter by estrogen receptors (ERs) and coactivators, using chromatin immunoprecipitation. Because this promoter does not contain palindromic estrogen response elements, we targeted the region of a cAMP regulatory element (CRE), implicated in crh regulation. The temporal pattern of the mRNA response was mimicked by recruitment of ERalpha and -beta, phospho-CRE-binding protein, coactivators steroid receptor coactivator-1 and CRE-binding protein-binding protein (CBP), and an increase in histone 3 and 4 acetylation. Lastly, ERalpha and -beta loading were temporally dissociated, peaking at 1 and 3 min, respectively. The ER peaks were associated with coactivators and acetylation patterns. ERalpha associated with phospho-CRE-binding protein, CBP, steroid receptor coactivator-1, and increased acetylated histone 3. ERbeta associated with CBP and increased acetylated histone 4. The tight temporal correlation between E2-induced CRH mRNA levels and promoter occupancy by ERs strongly suggest that E2 regulates crh expression through an ERalpha- and/or ERbeta-CRE alternate pathway.

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Figures

Figure 1

In AR-5 cells, ERα- and -β IR are predominantly nuclear. A and B, Ac-H3 IR (1:100; A) and tubulin-α IR (1:1000; B); C and D, ERα IR (1:175; C) and ERβ IR (1:100; D) are primarily nuclear, with some cytoplasmic localization; E and F, preabsorption of ER antibodies with their antigenic peptide eliminates staining of ERα and ERβ, respectively; G and H, preabsorption with the converse subtype’s antigenic peptide has no effect on ERα (G) or ERβ (H) staining patterns; I, in the absence of primary antibody, no IR is apparent; J, phase contrast shows the presence of cells in the field shown in I. Scale bar in A = 20 μm and applies to all panels.

Figure 2

Detection of ERα and ERβ protein and mRNA expression in AR-5 cells. A–C, Western blot analysis of AR-5 lysates and RT-PCR of AR-5 mRNA revealed ERα IR and mRNA, respectively. A, Antibodies against ERα (MC-20, 1:1000; Santa Cruz) recognized a band with a molecular mass of approximately 62–63 kDa in AR-5 cells and uterus (positive control), but not in spleen. B, After stripping the blot, another antibody against ERα (C1355, 1:5000; Millipore) was used and recognized the same band in AR-5 cells and uterus but not in spleen. C, As an independent measure of ERα expression in AR-5 cells, we detected ERα mRNA levels by RT-PCR. Ethidium bromide staining of PCR products revealed a single ERα amplicon (792 nucleotides). D and E, Western blot analysis and RT-PCR also revealed ERβ IR and mRNA in AR-5 lysates. E, against ERβ (H150, 1:1000; Santa Cruz) recognized two bands with molecular masses of approximately 59 and 70 kDa in AR-5 cells and prostate (positive control) but not spleen. D, Antibodies Detection of ERβ mRNA by RT-PCR revealed the presence of four splice variants of predicted lengths: ERβ2 (788 nucleotides), ERβ1 (734 nucleotides), ERβ2δ3 (671 nucleotides), and ERβ1δ3 (617 nucleotides).

Figure 3

E2 treatment leads to both a rapid and a delayed increase in CRH mRNA levels that differ in the requirement for protein synthesis. AR-5 cells were cultured in media supplemented with stripped serum. A, Cells were treated with ethanolic vehicle (Veh) or E2 (10−7

) for 1, 3, 10, 30, and 60 min. B, cells were treated with E2 (10−7

) in the absence or presence of the protein synthesis inhibitor cycloheximide (10 mg/ml) for 1, 3, and 60 min. mRNA levels were measured using quantitative real-time RT-PCR. Error bars represent ±

sem

; n = 5. A: τ, Significantly different from 10 min by Bonferroni post hoc test; *, significantly different from 0 min by Bonferroni post hoc test; #, significantly different from 0 min by two-sample t tests with Bonferroni corrections as follows: 1 min (Z = 2.5491; P < 0.006), 3 min (Z = 2.6740; P < 0.004), and 60 min (Z = 2.6740; P < 0.004). B: #, Significant differences between E2 (60 min) and E2 plus cycloheximide (60 min) by two-sample t test (Z = 1.7457; P < 0.041; n = 3).

Figure 4

ERα and ERβ selectively occupy the crh CRE region in response to E2. A, Distribution of putative ER regulatory elements within the rat crh promoter; B, E2 increases ERα and ERβ occupancy in the region of the crh CRE and c-fos ERE. PCR amplicons correspond to the promoter regions, as indicated. AR-5 cells were treated for 60 min. V, Vehicle.

Figure 5

E2 treatment leads to rapid and dynamic recruitment of ERα, ERβ, and SRC-1 to the crh promoter in the region of the proximal CRE. The same conditions and time points were used as in the CRH mRNA experiment shown in Fig. 3. Here, ChIPs were performed with ERα, ERβ, and SRC-1 antibodies. The region of the crh promoter containing the CRE was amplified by quantitative PCR. Cells were treated for the times indicated. Data represent the fold of E2-treated cells compared with vehicle. Error bars represent ±

sem

; n = 6. *, P < 0.05, Bonferroni post hoc test for significant time effects; #, significant difference from the 0-min time point by two-sample t test with Bonferroni corrections as follows: ERα, 1 min (Z = 2.8723; P < 0.002), 3 min (Z = 2.9406; P < 0.003), 30 min (Z = 2.5491; P < 0.006), and 60 min (Z = 2.5491; P < 0.006); ERβ 1 min (Z = 2.8627; P < 0.003), 3 min (Z = 2.9406; P < 0.003), 10 min (Z =2.5491; P < 0.006), 30 min (Z = 2.5491; P < 0.006), and 60 min (Z = 2.5491; P < 0.006); SRC-1 1 min (Z = 2.6740; P < 0.004) 3 min (Z = 2.5491; P < 0.006), and 60 min (Z = 2.3910; P < 0.009).

Figure 6

E2 elicits rapid and dynamic loading of pCREB and CBP at the crh promoter in the region of the CRE. The AR-5 whole cell lysates used in Fig. 3 were probed with total CREB, phospho-CREB, and CBP antibodies, in parallel. The same region of the CRE was amplified as in Fig. 4. Data represent the fold of E2-treated cells compared with vehicle. Error bars represent ±

sem

, n = 4. (#) Significant difference from the 0 min time point by two-sample t test with Bonferroni corrections as follows: (pCREB) 1 min (Z = 2.5491; P < 0.006), 30 min (Z = 2.3910; P < 0.009), and 60 min (Z = 2.5491; P < 0.006); (Total CREB) 60 min (Z = 2.5491; P < 0.006); (CBP) 3 min (Z = 2.7765, P < 0.004), and 60 min (Z = 2.6740; P < 0.004). Note that the scale is different from the scale used in Figs. 3 and 5 (maximum of 20 vs. 30). Veh, Vehicle.

Figure 7

E2 increases acetylation of H3 and H4 at the crh promoter in the region of the CRE. Aliquots of the AR-5 whole-cell lysates used for Figs. 4 and 5 were probed with Ac-H3 and Ac-H4 antibodies in parallel. Data represent the fold of E2-treated compared with vehicle (Veh). Error bars represent ±

sem

; n = 4. #, Significant difference from the 0-min time point by two-sample t test with Bonferroni corrections as follows: Ac-H3 1 min (Z = 2.5491; P < 0.006), 30 min (Z = 2.5491; P < 0.006), and 60 min (Z = 2.5491; P < 0.006); Ac-H4 60 min (Z = 2.3910; P < 0.009).

Figure 8

Compilation of time courses indicates a sequential and ordered assembly of ERα and ERβ complexes and H3 and H4 acetylation states. A, Data shown in Figs. 3 and 5, superimposed; B, data shown in Figs. 5–7, superimposed. pCREB and SRC-1 occupancy peak at 1 min, as does Ac-H3. CBP peaks at 3 min, as does Ac-H4. Total CREB is omitted because the increase was not significant at any of the times shown.

Figure 9

Putative ER complexes at the crh CRE. ERα interacts with the SRC-1 coactivator present in a pCREB transcriptional complex. An ERα-SRC-1 complex could bind directly to pCREB or via an intermediary factor, as shown. The ERα complex would promote acetylation of H3 in the region of the CRE. In distinction, ERβ interacts with CBP either via direct or indirect interactions. CBP, in turn, would associate with a CREB family member bound to the CRE. In this case, CBP would promote acetylation of H4.

References

1. Rivier C, Rivier J, Vale W 1982 Inhibition of adrenocorticotropic hormone secretion in rat by immunoneutralization of corticotropin-releasing factor. Science 218:377–379 - PubMed
1. Sawchenko PE 1987 Evidence for a local site of action for glucocorticoids in inhibiting CRF and vasopressin expression in the paraventricular nucleus. Brain Res 403:213–223 - PubMed
1. Sawchenko PE 1987 Adrenalectomy-induced enhancement of CRF and vasopressin immunoreactivity in parvocellular neurosecretory neurons: anatomic, peptide, and steroid specificity. J Neurosci 7:1093–1106 - PMC - PubMed
1. Jingami H, Matsukura S, Numa S, Imura H 1985 Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/β-lipotropin precursor mRNA in the pituitary in rats. Endocrinology 117:1314–1320 - PubMed
1. Kovacs KJ, Mezey E 1987 Dexamethasone inhibits corticotropin-releasing factor gene expression in the rat paraventricular nucleus. Neuroendocrinology 46:365–368 - PubMed

Estradiol regulates corticotropin-releasing hormone gene (crh) expression in a rapid and phasic manner that parallels estrogen receptor-alpha and -beta recruitment to a 3',5'-cyclic adenosine 5'-monophosphate regulatory region of the proximal crh promoter - PubMed (original) (raw)