Progress 10/01/12 to 09/30/17
Outputs
Target Audience:Members of the scientific community with a particular interest in endocrinology Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Journal publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The proposed research relates to goal #3 of the USDA national goal areas, a healthy, well-nourished population. A high level of plasma prolactin, hyperprolactinemia, is known to be one of the major reasons for reproductive dysfunction such as amenorrhea, galactorrhea and infertility in women. Many of the patients with hyperprolactinemia also show prolactin-secreting pituitary adenomas (prolactinomas). The idea that a woman's lifestyle choices and exposure to environmental toxins during pregnancy may affect her offspring's risk of various cancers is a newly emerging concept. Statistics from the Centers for Disease Control indicate that a significant number of women drink or binge-drink while pregnant. Therefore, the reports that rats exposed to alcohol during fetal development have increased susceptibility to tumorigenesis in adulthood suggest that offspring from this group of women may be at increased risk for prolactinomas. Recent evidence indicated that alcohol exposure during the fetal period increases the susceptibility to tumor development in the pituitary tissue, but the cause is unknown. Hence our aims have been to determine whether prenatal ethanol exposure alters D2R expression and increases lactotropes' susceptibility to the mitogenic action of estradiol; ii) To determine whether prenatal ethanol exposure causes sustained alteration in CpG methylation of the promoter sequences of D2R gene to reduce this receptor expression in lactotropes; iii) To test whether hypermethylation of the D2R gene is involved in the increased susceptibility of lactotropes to the mitogenic action of estradiol in fetal alcohol exposed animals. The original Specific Aims have not been changed. Our previous research suggested the possibility that ethanol may interact with D2 receptor to increase PRL production and lactotropic cell proliferation in the pituitary. In order to further evaluate the cause of D2 receptor deregulation after ethanol, we initiated studies to investigate the role of epigenetic programming in ethanol action. For this purpose, we shifted our adult alcohol feeding animal model to a prenatal ethanol feeding animal model, since this animal model provides opportunity to study a stable epigenetic mark that could be easily demonstrated experimentally. Prenatal ethanol exposure produces epigenetic marks in DNA during embryonic development changing DNA expression patterns that often exist during adulthood in animals. Methylation of DNA promoters can suppress gene expression and can be related to cancer susceptibility. The primary regulator of prolactin cells in the mammalian pituitary is the action of dopamine at the D2 receptor (D2R). Deregulation in this pathway can induce susceptibility to prolactinoma development. The aim of this work was to study the effect of fetal alcohol administration on the methylation of D2R promoters in the anterior pituitary glands of male and female Fischer 344 (F344) rats in order to determine their susceptibility to tumorigenesis. Pregnant rats were fed between GD 7 and 21 with liquid diet containing alcohol (alcohol-fed), pair-fed with isocaloric liquid diet (pair-fed), or fed ad libitum with rat chow (ad lib-fed). The rat pups were weaned and group- housed by sex until sacrifice. At 90 days of age, these rats were sacrificed and pituitary tissues were obtained for subsequent analyses. The methylation status of the D2R promoter was evaluated by real-time PCR using the probes specific to the methylated and unmethylated states of the region adjacent to the D2R transcriptional start and compared to the total D2R gene expression level measured by real-time PCR. The proximal D2R promoter in the pituitary of alcohol-fed rat was significantly hypermethylated in comparison to the control rat pituitary, and the D2R gene expression level was decreased in both male and female offspring. In addition, mRNA levels of prolactin were increased in the pituitary of alcohol-fed rats as compared to the pituitary of control groups. We also found 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. We have also determined how alcohol increases splicing of dopamine D2 receptors to reduce the amount of D2R short form (D2S), which activate Gi to reduce prolactin production and prolactin cell proliferation and prolactinomas development. We tested whether microRNAs (miRs) are involved in fetal alcohol induced alteration in D2R splicing and tumorigenesis. FAE increased the expression levels of miR-9 and miR-326 and did not produce any significant changes in miR-153 or miR-200a levels in the pituitary. FAE effects on miR-9 and miR-326 were associated with reduced levels of D2R and D2S, increased levels of PRL in the pituitary, and in plasma. These effects of FAE on D2R, D2S and PRL were enhanced following estradiol treatment. In PRL producing MMQ cells, ethanol increased miR-9 but not miR-326, reduced levels of D2R and D2S and increased levels of PRL. Treatment of MMQ cells with an anti-miR-9 oligo reduced ethanol effects on miR-9, D2R, D2S and PRL. MiR-9 mimic oligos reduced the luciferase activity of reporter vector containing D2R 3'UTR, but failed to reduce the mutant luciferase activity. These data suggest that FAE programs the pituitary to produce increased amounts of miR-9 expression that represses the D2R gene and its spliced variant D2S by targeting its 3' UTR leading to an increase in PRL production and secretion. In a separate study, we evaluated the effect of opioid drugs to control the pituitary tumor growth. We have recently identified a tumor suppressive effect of the combination treatment of a mu-opioid receptor antagonist naltrexone and a delta-opioid receptor agonist D-Ala2-,N-Me-Phe4,Gly-ol Enkephalin (DPDPE) in a rat model of breast cancer. Hence, we tested whether Naltrexone and DPDPE combination therapy is useful to control pituitary tumor growth in fetal alcohol exposed rats. Pregnant Fischer 344 rats were fed between gestational days 7 and 21 with a liquid diet containing alcohol (AF), pair-fed with isocaloric liquid diet (PF), or fed ad libitum with rat chow (AD). At 60 days of age, female offspring rats were ovariectomized and implanted estradiol-17ß capsule (E2). 6 weeks after E2 treatment, animals were injected with naltrexone and DPDPE or vehicle alone for a period of 2 weeks. The time-dependent growth of the pituitary tumor was visualized using magnetic resonance imaging (MRI). MRI images were obtained by using M2 MRI Scanning System, 60mm Radio Frequency Coil, T2 Weighted GRE-SP (Gradient Echo Spoiled) sequence, NRG-console software. At the end of the treatment, pituitary weights and immune functions were determined. Both imaging data and weight data indicated that estrogen-induced increase in the weight and volume of the pituitary gland were increased in animals exposed to fetal alcohol. Naltrexone and DPDPE treatment reduced the weight and volume of the pituitary gland in both fetal alcohol exposed and control-fed animals. The treatment of opioid drugs also increased the cytotoxic activity in the blood and in the spleen. These data provide a novel possibility in treating pituitary tumors and possibly other cancers using a combination therapy of naltrxone and DPDPE in fetal alcohol exposed or other patients.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Gangisetty O, Wynne O, Jabbar S, Nasello C, Sarkar DK. Fetal Alcohol Exposure Reduces Dopamine Receptor D2 and Increases Pituitary Weight and Prolactin Production via Epigenetic Mechanisms. PLoS One. 2015 Oct 28;10(10):e0140699. doi: 10.1371/journal.pone.0140699. PubMed PMID: 26509893; PubMed Central PMCID: PMC4624904.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Maglakelidze G, Wynne O, Sarkar DK. A combined opiate agonist and antagonist treatment reduces prolactin secreting pituitary tumor growth. J Cell Commun Signal. 2017 Jan 31. doi: 10.1007/s12079-017-0374-x. PubMed PMID: 28144772.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Gangisetty O, Jabbar S, Wynne O, Sarkar DK. MicroRNA-9 regulates fetal alcohol induced changes in D2 receptor to promote prolactin production. J Endocrinol. 2017 Jul 14. pii: JOE-17-0135. doi: 10.1530/JOE-17-0135. PubMed PMID: 28710248.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Members of the scientific community with a particular interest in endocrinology Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Continue evaluating how fetal alcohol progam the pituitary gland to produce excess levels of prolactin hormones and increase the sensitivity to develop tumors in the gland.
Impacts
What was accomplished under these goals?
This research relates to goal #3 of the USDA national goal areas, a healthy, well-nourished population. A high level of plasma prolactin, hyperprolactinemia, is known to be one of the major reasons for reproductive dysfunction such as amenorrhea, galactorrhea and infertility in women. Many of the patients with hyperprolactinemia also show prolactin-secreting pituitary adenomas (prolactinomas). The idea that a woman's lifestyle choices and exposure to environmental toxins during pregnancy may affect her offspring's risk of various cancers is a newly emerging concept. Statistics from the Centers for Disease Control indicate that a significant number of women drink or binge-drink while pregnant. Therefore, the reports that rats exposed to alcohol during fetal development have increased susceptibility to tumorigenesis in adulthood suggest that offspring from this group of women may be at increased risk for prolactinomas. Recent evidence indicated that alcohol exposure during the fetal period increases the susceptibility to tumor development in the pituitary tissue, but the cause is unknown. In this study we determined whether FAE induced abnormalities in the pituitary involve microRNAs (miRs). We employed an animal model in which Fisher 344 female rats were fed with a liquid diet containing 6.7% alcohol or control diets between gestational day 7 and 21. FAE increased expression levels of miR-9 and miR-326 in the pituitary during the adult period. FAE effects on miR-9 and miR-326 were associated with reduced mRNA levels of dopamine D2 receptor (D2R) and D2R short spliced variant (D2S), increased mRNA levels of PRL in the pituitary, and protein levels of PRL in plasma. These effects of FAE on miRs, D2R, D2S and PRL were enhanced following estradiol treatment. In PRL producing MMQ cells, ethanol increased miR-9 but not miR-326, reduced levels of D2R and D2S and increased levels of PRL. Treatment of MMQ cells with an anti-miR-9 reduced ethanol effects on miR-9, D2R, D2S and PRL In MMQ cells, miR-9 mimic oligo effectively reduced the luciferase activity of reporter vector containing D2R 3'UTR but failed to reduce the mutant luciferase activity. In vivo, anti-miR-9 also blocked FAE effects on D2R, D2S and PRL. These data suggest that FAE programs the pituitary to produce increased amount of miR-9 expression that represses D2R gene and its spliced variant D2S by targeting its 3'UTR leading to an increase in PRL production and secretion.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Scientific Community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training a postdoctoral fellow and a graduate student How have the results been disseminated to communities of interest?Research report was presented in the National Meetings of Research Society of Alcoholism and American Associtation of Cancer Research. What do you plan to do during the next reporting period to accomplish the goals?Continue working to identify how alcohol epigenetically program the pituiary to promote carcinogenesis.
Impacts
What was accomplished under these goals?
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 during gestational day 7 to day 21. Control rats were pair-fed with isocaloric liquid diet or fed ad libitum 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 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. Altered fetal programming due to a suboptimal in utero environment increases susceptibility to many diseases later in life. While many reports have linked alcohol consumption to increased risk of pituitary tumors in women and rodents, alcohol exposure in utero may also affect susceptibility to pituitary tumorigenesis during adulthood. Alcohol ingestion inhibits folic acid absorption and decreases the availability of the methyl donor, which is critical for methylation processes and significantly affects D2R expression and function. Abnormality in D2R function is connected with the development of hyperprolactinemia and prolactinomas. Our study report 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. We believe that identification of the causes of pituitary pathology will pave the way for the development of novel therapeutic tools in the treatment and prevention of hyperprolactinemia and prolactinomas which will also help the pharmaceutical and/or nutraceutical industry in New Jersey.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Gangisetty O, Wynne O, Jabbar S, Nasello C, Sarkar DK. Fetal Alcohol Exposure Reduces Dopamine Receptor D2 and Increases Pituitary Weight and Prolactin Production via Epigenetic Mechanisms. PLoS One. 2015 Oct 28;10(10):e0140699.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One graduate student and one postdoctoral fellow. How have the results been disseminated to communities of interest? We will be presenting these research findings in an upcoming national meeting in June. What do you plan to do during the next reporting period to accomplish the goals? We want to continue doing in-depth molecular studies to tease apart how prental ethanol-induced epigenetic modification in D2R, creating a cellular substrate vulnerable to reproductive dysfunction and pituitary tumors in adulthood. We are also beginning to characterize the tumor phenotypes caused by the dopamine receptor gene modification.
Impacts
What was accomplished under these goals?
In this study, we determined whether fetal ethanol exposures increase the susceptibility to estrogen-induced prolactinomas. Furthermore, we evaluated the role of dopamine receptor D2 (D2R) which inhibits PRL production and lactotroph cell growth. Pregnant Fischer 344 rats were fed between gestational days 7 and 21 with a liquid diet containing alcohol (AF), pair-fed with isocaloric liquid diet (PF), or fed ad libitum with rat chow (AD). At 60 days of age, female offspring rats were ovariectomized and implanted estradiol-17ß capsule. Rats were sacrificed after 60 days, pituitary tissues were collected and used for cell proliferation, gene expression and promoter methylation measurements. The pituitary weight and volume were increased in AF rats compared to PF and AD rats. The pituitaries of AF rats showed increased PRL and reduced D2R mRNA levels compared to control rats. We hypothesized the role of epigenetic mechanism in regulating D2R expression in FAE rats. We evaluated the promoter methylation of D2R using methylation specific PCR (MSP) and pyrosequencing methods. The proximal D2R promoter is hypermethylated in the pituitary of AF rats compared to control rats. We also assessed the expression of DNA methyl transferases and methyl CpG binding protein (MeCP2) and found that these proteins were increased in AF rats compared to control rats. We further treated rats with epigenetic modulators such as 5-Aza deoxy cytidine (5-AZADC) a DNA methylation inhibitor or Trichostatin-A (TSA) a HDAC inhibitor at postnatal day 2, 4 and 6 prior to inducing prolactinoma. We found that pituitary tumor weights were reduced in AF rats treated with 5AZADC or TSA confirming the role of epigenetic modulation in altering growth and development of pituitary gland. Since, post-transcriptional regulation of DRD2 expression can be regulated by microRNAs, we tested whether fetal alcohol exposures programming of D2 receptors involves microRNAs. We found that the expression of D2R targeted microRNAs Mir-9, Mir-153, Mir-200a and Mir-326 were determined using Taqman real time PCR assay. The expression level of Mir-9, Mir-326 but not Mir-153 or Mir-200a was increased in pituitaries of AF rats compared to AD and PF controls. We also assessed the effect of Mir-9 overexpression on D2R mRNA levels by treating MMQ cells, lactotroph derived pituitary tumor cell line, with or without alcohol. We found that antiMir-9 inhibitor effectively reduced Mir-9 expression and increased D2R expression. The D2R regulated PRL expression was also reduced with antiMir-9 inhibitor in MMQ cells treated with alcohol. These data suggest that fetal alcohol exposure epigenetically programs the pituitary to increase PRL expression possibly by enhancing mir-9 expression causing the repression of D2R gene and its cell growth inhibitory function in the pituitary gland. Our study reports thenovel possibility that fetal alcohol exposure programs the pituitary to increase the susceptibility to tumor development, possibly by enhancing the promoter hypermethylaion of D2R gene and enhancing mRNA expression of mir-9 causing repression of D2R gene and it's cell growth inhibitory function in the pituitary gland. In-depth molecular studies are required to tease apart how prenatal ethanol-induced epigenetic modifications in D2R, creating a cellular substrate vulnerable to reproductive dysfunction and pituitary tumors in adulthood. We believe that identification of the causes of pituitary pathology will pave the way for the development of novel therapeutic tools in the treatment and prevention of hyperprolactinemia and prolactinomas, which will also help the pharmaceutical and/or nutraceutical industry in New Jersey.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One graduate student and three postdoctoral fellow, How have the results been disseminated to communities of interest? We have presented our findings in a scientific conference. What do you plan to do during the next reporting period to accomplish the goals? We want to continue doing in-depth molecular studies to tease apart how prenatal ethanol-induced epigenetic modifications in D2R, creating a cellular substrate vulnerable to reproductive dysfunction and pituitary tumors in adulthood.
Impacts
What was accomplished under these goals?
We have recently initiated study and found that prenatally alcohol-exposed rats showed a significant increase in the weight and the number of mitotic lactotropes in the pituitary, as well as an elevated mRNA levels of prolactin and D2R spliced variant D2L, but had decreased D2R. In order to determine whether methylation of D2R gene is responsible for the reduction of this gene, we measure the methylation status of the D2R promoter. The proximal D2R promoter in the pituitary of the prenatally alcohol-exposed rat was significantly hypermethylated in comparison to the control rat pituitary. We also found that the expression of a D2R targeted microRNA Mir-9, but not Mir-200a or Mir-153, was increased in the pituitary of fetal alcohol exposed rats treated with estrogen. These data suggest that fetal alcohol exposure programs the pituitary to increase the susceptibility to tumor development possibly by enhancing the promoter hypermethylaion of D2R gene and enhancing mRNA expression of mir-9, causing repression of D2R gene and it’s cell growth inhibitory function in the pituitary gland.
Publications
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