Fetal alcohol exposure alters proopiomelanocortin gene expression and hypothalamic-pituitary-adrenal axis function via increasing MeCP2 expression in the hypothalamus.

Proopiomelanocortin (POMC) is a precursor gene of the neuropeptide β-endorphin in the hypothalamus and is known to regulate various physiological functions including stress response. Several recent reports showed that fetal alcohol exposure programs the hypothalamus to produce lower levels of POMC gene transcripts and to elevate the hypothalamic-pituitary-adrenal (HPA) axis response to stressful stimuli. We investigated the role of methyl CpG binding protein (MeCP2) in the effects of prenatal ethanol on POMC gene expression and hypothalamic-pituitary-adrenal (HPA) axis function. Pregnant Sprague Dawley rats were fed between GD 7 and 21 with a liquid diet containing 6.7% alcohol, pair-fed with isocaloric liquid diet, or fed ad libitum with rat chow, and their male offsprings were used at 60 days after birth in this study. Fetal alcohol exposure reduced the level of POMC mRNA, but increased the level of DNA methylation of this gene in the arcuate nucleus (ARC) of the hypothalamus where the POMC neuronal cell bodies are located. Fetal alcohol exposed rats showed a significant increase in MeCP2 protein levels in POMC cells, MeCP2 gene transcript levels as well as increased MeCP2 protein binding on the POMC promoter in the arcuate nucleus. Lentiviral delivery of MeCP2 shRNA into the third ventricle efficiently reduced MeCP2 expression and prevented the effect of prenatal ethanol on POMC gene expression in the arcuate nucleus. MeCP2-shRNA treatment also normalized the prenatal ethanol-induced increase in corticotropin releasing hormone (CRH) gene expression in the hypothalamus and elevated plasma adrenocorticotrophic hormone (ACTH) and corticosterone hormone responses to lipopolysaccharide (LPS) challenge. These results suggest that fetal alcohol programming of POMC gene may involve recruitment of MeCP2 on to the methylated promoter of the POMC gene to suppress POMC transcript levels and contribute to HPA axis dysregulation

Primer sequences.

<p>Forward primer (FP), Reverse primer (RP), Methyl forward primer (MFP), Methyl reverse primer (MRP), Un methyl forward primer (UFP), Un methyl reverse primer (URP), Promoter forward primer (PFP), Promoter reverse primer (PRP).</p><p>Primer sequences.</p

Effects of lentiviral knockdown of MeCP2 on POMC gene expression levels in fetal alcohol exposed rat offspring.

<p><b>A</b>. A representative western blot gel showing MeCP2 protein levels (top) and actin levels (bottom) in MBH of scr sh (scr) or MeCP2 sh (MeCP) RNA-treated AD, PF and AF rat offspring. Histograms showing MeCP2 and actin ratio values in MBH of AD, PF and AF rats.treated with scr sh or MeCP2 sh RNA. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, MeCP2 sh vs. scr-sh; ^a, P<0.05, AF vs. AD or PF. <b>B</b>. POMC mRNA levels in MBH of scr-sh and MeCP2 sh RNA of AD, PF and AF rat offspring. POMC mRNA amounts were normalized with GAPDH and expressed as relative mRNA level. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, AF- vs. AD- or PF-scr-sh-treated groups; ^a, P<0.05, MeCP2 sh vs. scr-sh (AF group).</p

Changes in MeCP2 gene and protein levels in MBH of fetal alcohol exposed rat offspring.

<p><b>A</b>. MeCP2 mRNA levels in MBH of AD, PF and AF rat offspring. MeCP2 mRNA levels were measured by quantitative RT-PCR and the amounts were normalized with GAPDH values and expressed as relative mRNA levels. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, <i>P</i><0.05, AF vs. AD or PF. <b>B</b>. A representative western blot gel demonstrating changes in MeCP2 protein levels (top) and actin levels (loading control, bottom) in MBH of AD, PF and AF rat offspring along with quantification measurements were represented as relative protein level (MeCP2/actin). Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; **, <i>P</i><0.01, AF vs. AD or PF. <b>C</b>. MeCP2 protein levels in β-endorphin neurons in the ARC of AD, PF and AF rat offspring were measured by double immunofluorescence methods (MeCP2 proteins are shown in green and β-endorphin shown in red). Representative photographs show double-labeled cells in each group. Histograms show the mean ± SEM (n = 6) values of percent β-EP cells expressing MeCP2 and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; ***, <i>P</i><0.001, AF vs. AD or PF.</p

Changes in MeCP2 binding onto POMC promoter in ARC and PVN of fetal alcohol exposed rat offspring.

<p><b>A</b>. Schematic representation of POMC promoter with CpG sites and primers flanking MeCP2 binding site. <b>B</b>. The levels of MeCP2 binding on POMC promoter in ARC and PVN of AD, PF and AF rat offspring were measured by ChIP assay. MeCP2 bound DNA pulled by its specific antibody was amplified by real time PCR using primers specific for the POMC promoter. MeCP2 enriched DNA was normalized with GAPDH and expressed as fold enrichment (MeCP2/GAPDH). AD and AF are ARC samples of <i>ad libitum</i>-fed and alcohol-fed rats. ADP and AFP are PVN samples of <i>ad libitum</i>-fed and alcohol-fed rats. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, <i>P</i><0.05, AF vs. AD; ^a, P<0.001, ADP and AFP versus AD or AF.</p

Fetal Alcohol Exposure Reduces Dopamine Receptor D2 and Increases Pituitary Weight and Prolactin Production via Epigenetic Mechanisms

<div><p>Recent evidence indicated that alcohol exposure during the fetal period increases the susceptibility to tumor development in mammary and prostate tissues. Whether fetal alcohol exposure increases the susceptibility to prolactin-producing tumor (prolactinoma) development in the pituitary was studied by employing the animal model of estradiol-induced prolactinomas in Fischer 344 female rats. We employed an animal model of fetal alcohol exposure that simulates binge alcohol drinking during the first two trimesters of human pregnancy and involves feeding pregnant rats with a liquid diet containing 6.7% alcohol during gestational day 7 to day 21. Control rats were pair-fed with isocaloric liquid diet or fed <i>ad libitum</i> with rat chow diet. Adult alcohol exposed and control female offspring rats were used in this study on the day of estrus or after estrogen treatment. Results show that fetal alcohol-exposed rats had increased levels of pituitary weight, pituitary prolactin (PRL) protein and mRNA, and plasma PRL. However, these rats show decreased pituitary levels of dopamine D2 receptor (D2R) mRNA and protein and increased pituitary levels of D2R promoter methylation. Also, they show elevated pituitary mRNA levels of DNA methylating genes (DNMT1, DNMT3b, MeCP2) and histone modifying genes (HDAC2, HDAC4, G9a). When fetal alcohol exposed rats were treated neonatally with a DNA methylation inhibitor 5-Aza deoxycytidine and/or a HDAC inhibitor trichostatin-A their pituitary D2R mRNA, pituitary weights and plasma PRL levels were normalized. These data suggest that fetal alcohol exposure programs the pituitary to increase the susceptibility to the development of prolactinomas possibly by enhancing the methylation of the D2R gene promoter and repressing the synthesis and control of D2R on PRL-producing cells.</p></div

Effects of epigenetic modulatory drugs on fetal alcohol induced changes in pituitary D2R mRNA levels, protein levels, pituitary weight and plasma PRL levels.

<p>AD, PF and AF rat offspring were treated with DNA methyl transferase inhibitor 5-Aza deoxycytidine (5-AZAdC), HDACC inhibitor Trichostatin-A (TSA) alone or together during postnatal day 2–6 and after 60 days, these rats were ovariectomized and estrogen treated for 60 days. Mean ± SEM values of pituitary D2R mRNA levels (A), pituitary weight (B) and plasma PRL levels (C) are shown in the histograms. Data are mean ± SEM of n = 6 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, between AF and controls (AD or PF). @, P<0.05 between the treatment and control in AF group.</p

Effect of fetal alcohol exposure on the level of genes regulating DNA methylation and histone modification in the pituitary.

<p>Pituitary mRNA levels of DNMT1 (A), DNMT3a (B), DNMT3b (C), MeCP2 (D), HDAC2 (E), HDAC4 (F), G91 (G) and Set7 (H) of AD, PF. AF rats after ovariectomy and estrogen treatment for 60 days. Data are mean ± SEM of n = 6 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, **, P<0.01, and ***, P<0.001 between AF and controls (AD or PF).</p

Schematic diagram illustrating fetal alcohol exposure induced epigenetic changes regulating D2R expression and its control of PRL synthesis and lactotropic cell growth in the pituitary.

<p>In control rats, D2R mRNA expression is regulated by the epigenetic mechanism involving DNA demethylation and histone deacetylation of the D2R promoter. D2R participates in mediating the inhibitory action of dopamine on PRL synthesis and cell proliferation in lactotropic cells of the pituitary gland (A). In fetal alcohol exposed (FAE) rats, increased D2R promoter methylation and histone deacetylation result in reduced D2R expression, The lower number of D2Rs prevents dopamine to act on lactotropes causing more PRL production and increased cell proliferation (B). DNA wrapped with histone octomer is represented as black thread with cylindrical structures. Methylated CpG is represented as pentagon structure in the DNA. Acetyl groups of histones are represented as triangles and methyl groups as pentagons on N terminal tails of histone.</p

Effect of fetal alcohol exposure on the sensitivity of pituitary lactotropes to the estrogen tumor-promoting action.

<p>Alcohol-fed (AF) or control-fed (PF, AD) rats offspring were ovariectomized and implanted with a β-estradiol implants at 60 days of age and after 60 (E2-60 d) or 90 days (E2-90 d) were used for this study. Mean ± SEM values of pituitary weight at 60 d (A) and 90 d (B), pituitary PRL mRNA level at 60 d (C) and 90 d (D), pituitary PRL protein level at 60 d (E) and 90 d (F), and plasma PRL levels at 60 d (G) and 90 d (H) are shown in the histograms. Data are mean ± SEM of n = 4–8 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05 and ***, P<0.001 between AF and controls (AD or PF).</p