HOXA5 Expression Is Elevated in Breast Cancer and Is Transcriptionally Regulated by Estradiol.

HOXA5 is a homeobox-containing gene associated with the development of the lung, gastrointestinal tract, and vertebrae. Here, we investigate potential roles and the gene regulatory mechanism in HOXA5 in breast cancer cells. Our studies demonstrate that HOXA5 expression is elevated in breast cancer tissues and in estrogen receptor (ER)-positive breast cancer cells. HOXA5 expression is critical for breast cancer cell viability. Biochemical studies show that estradiol (E2) regulates HOXA5 gene expression in cultured breast cancer cells in vitro. HOXA5 expression is also upregulated in vivo in the mammary tissues of ovariectomized female rats. E2-induced HOXA5 expression is coordinated by ERs. Knockdown of either ERα or ERß downregulated E2-induced HOXA5 expression. Additionally, ER co-regulators, including CBP/p300 (histone acetylases) and MLL-histone methylases (MLL2, MLL3), histone acetylation-, and H3K4 trimethylation levels are enriched at the HOXA5 promoter in present E2. In summary, our studies demonstrate that HOXA5 is overexpressed in breast cancer and is transcriptionally regulated via estradiol in breast cancer cells.

Sex differences in the expression of morphine withdrawal symptoms and associated activity in the tail of the ventral tegmental area.

Recent studies, in male rodents, have begun to elucidate a role for the GABAergic neurons in the tail of the ventral tegmental area (tVTA) in morphine withdrawal. To date, the mechanisms underlying morphine withdrawal have been studied almost exclusively in male animals. As a result, there is a considerable gap in our current understanding of the processes underlying sex differences in morphine withdrawal behaviors and its effects on cellular activity in the tVTA in females. The purpose of the present study was to investigate the influence of sex on the expression and duration of spontaneous somatic morphine withdrawal syndrome, and to characterize the relationship between spontaneous somatic withdrawal symptoms and cellular activation (measured as phosphorylated CREB; pCREB), in the GABAergic tVTA in male and female rats. Morphine-dependent adult male and female Long Evans rats underwent 72 h of spontaneous withdrawal, and somatic withdrawal symptoms were assessed every 12 h. Male morphine-dependent rats expressed more severe symptoms during the early phases of withdrawal compared to females. Although, females demonstrated lower overall symptom severity, their symptoms persisted for a longer period of time, thus demonstrating higher withdrawal-symptom severity than males during late withdrawal. pCREB activity in the tVTA was elevated in morphine-withdrawn rats and was positively correlated with the severity of withdrawal symptoms. These results demonstrate sex differences in the timing of the expression of somatic withdrawal. Our data add to the growing body of evidence demonstrating a role for the tVTA in morphine withdrawal and begin to establish a sex-dependent behavioral and molecular profile within this brain region.

Exposure to morphine-associated cues increases mu opioid receptor mRNA expression in the nucleus accumbens of Wistar Kyoto rats.

The Wistar-Kyoto (WKY) rat has been proposed as a model of anxiety vulnerability as it exhibits pronounced behavioral inhibition, passive avoidance, exaggerated startle response, enhanced HPA-axis activation, and active avoidance that is resistant to extinction. Accumulating evidence suggests that WKY rats respond differently to rewarding stimuli when compared to outbred strains of rat. Conditioned responding to drug-associated cues is linked with alterations in the activation of mu opioid receptors (MOR) and kappa opioid receptors (KOR) in the nucleus accumbens (NAc). Furthermore, alterations in KOR expression/activation in the NAc of WKY rats are implicated in the regulation of some of the components that make up the unique behavioral phenotype of this strain. The purpose of this study was to extend upon previous work from our laboratory by investigating conditioned morphine reward in adult male WKY and SD rats, and to examine levels of KOR mRNA and MOR mRNA in the NAc at baseline and after acquisition of morphine CPP. Our results demonstrate that SD rats displayed morphine-induced CPP to each of the six doses of morphine tested (0.5, 1.25, 2.5, 5, 7.5, or 10mg/kg). Interestingly, WKY rats demonstrated CPP for only the 1.25, 2.5, and 5mg/kg doses, yet no preference at the lowest (0.5mg/kg) or highest (7.5 and 10mg/kg) doses. qPCR analysis of MOR and KOR in the NAc revealed no strain differences in basal levels of MOR, but higher levels of KOR in WKY rats compared to those of SD rats. Interestingly, after completion of the CPP task, WKY rats had overall higher levels of NAc MOR mRNA compared to SD rats; the initial basal differences in NAc KOR levels persisted without change due to CPP in either strain. These results demonstrate that the WKY rat exhibits a unique pattern of behavioral responding to morphine and implicates differences in NAc KOR signaling as a potential source of aversion to higher doses of morphine. Additionally, the CPP-induced upregulation of NAc MOR mRNA in WKY rats warrants further investigation in terms of its potential role as a factor constituting a unique vulnerability to subsequent drug exposure.

Endocrine disrupting chemical, bisphenol-A, induces breast cancer associated gene HOXB9 expression in vitro and in vivo.

HOXB9 is a homeobox-containing gene that plays a key role in mammary gland development and is associated with breast and other types of cancer. Here, we demonstrate that HOXB9 expression is transcriptionally regulated by estradiol (E2), in vitro and in vivo. We also demonstrate that the endocrine disrupting chemical bisphenol-A (BPA) induces HOXB9 expression in cultured human breast cancer cells (MCF7) as well as in vivo in the mammary glands of ovariectomized (OVX) rats. Luciferase assay showed that estrogen-response-elements (EREs) in the HOXB9 promoter are required for BPA-induced expression. Estrogen-receptors (ERs) and ER-co-regulators such as MLL-histone methylase (MLL3), histone acetylases, CBP/P300, bind to the HOXB9 promoter EREs in the presence of BPA, modify chromatin (histone methylation and acetylation) and lead to gene activation. In summary, our results demonstrate that BPA exposure, like estradiol, increases HOXB9 expression in breast cells both in vitro and in vivo through a mechanism that involves increased recruitment of transcription and chromatin modification factors.

Local field potentials in the ventral tegmental area during cocaine-induced locomotor activation: Measurements in freely moving rats.

The ventral tegmental area (VTA) has been established as a critical nucleus for processing behavioral changes that occur during psychostimulant use. Although it is known that cocaine induced locomotor activity is initiated in the VTA, not much is known about the electrical activity in real time. The use of our custom-designed wireless module for recording local field potential (LFP) activity provides an opportunity to confirm and identify changes in neuronal activity within the VTA of freely moving rats. The purpose of this study was to investigate the changes in VTA LFP activity in real time that underlie cocaine induced changes in locomotor behavior. Recording electrodes were implanted in the VTA of rats. Locomotor behavior and LFP activity were simultaneously recorded at baseline, and after saline and cocaine injections. Results indicate that cocaine treatment caused increases in both locomotor behavior and LFP activity in the VTA. Specifically, LFP activity was highest during the first 30 min following the cocaine injection and was most robust in Delta and Theta frequency bands; indicating the role of low frequency VTA activity in the initiation of acute stimulant-induced locomotor behavior. Our results suggest that LFP recording in freely moving animals can be used in the future to provide valuable information pertaining to drug induced changes in neural activity.

Bisphenol-A induces expression of HOXC6, an estrogen-regulated homeobox-containing gene associated with breast cancer.

HOXC6 is a homeobox-containing gene associated with mammary gland development and is overexpressed in variety of cancers including breast and prostate cancers. Here, we have examined the expression of HOXC6 in breast cancer tissue, investigated its transcriptional regulation via estradiol (E2) and bisphenol-A (BPA, an estrogenic endocrine disruptor) in vitro and in vivo. We observed that HOXC6 is differentially over-expressed in breast cancer tissue. E2 induces HOXC6 expression in cultured breast cancer cells and in mammary glands of Sprague Dawley rats. HOXC6 expression is also induced upon exposure to BPA both in vitro and in vivo. Estrogen-receptor-alpha (ERα) and ER-coregulators such as MLL-histone methylases are bound to the HOXC6 promoter upon exposure to E2 or BPA and that resulted in increased histone H3K4-trimethylation, histone acetylation, and recruitment of RNA polymerase II at the HOXC6 promoter. HOXC6 overexpression induces expression of tumor growth factors and facilitates growth 3D-colony formation, indicating its potential roles in tumor growth. Our studies demonstrate that HOXC6, which is a critical player in mammary gland development, is upregulated in multiple cases of breast cancer, and is transcriptionally regulated by E2 and BPA, in vitro and in vivo.

Non-scanning fiber-optic near-infrared beam led to two-photon optogenetic stimulation in-vivo.

Stimulation of specific neurons expressing opsins in a targeted region to manipulate brain function has proved to be a powerful tool in neuroscience. However, the use of visible light for optogenetic stimulation is invasive due to low penetration depth and tissue damage owing to larger absorption and scattering. Here, we report, for the first time, in-depth non-scanning fiber-optic two-photon optogenetic stimulation (FO-TPOS) of neurons in-vivo in transgenic mouse models. In order to optimize the deep-brain stimulation strategy, we characterized two-photon activation efficacy at different near-infrared laser parameters. The significantly-enhanced in-depth stimulation efficiency of FO-TPOS as compared to conventional single-photon beam was demonstrated both by experiments and Monte Carlo simulation. The non-scanning FO-TPOS technology will lead to better understanding of the in-vivo neural circuitry because this technology permits more precise and less invasive anatomical delivery of stimulation.

Histone methyltransferase EZH2 is transcriptionally induced by estradiol as well as estrogenic endocrine disruptors bisphenol-A and diethylstilbestrol.

Enhancer of Zeste homolog 2 (EZH2), a methyltransferase specific to histone 3 lysine 27, is a critical player in gene silencing and is overexpressed in breast cancer. Our studies demonstrate that EZH2 is transcriptionally induced by estradiol in cultured breast cancer cells and in the mammary glands of ovariectomized rats. EZH2 promoter contains multiple functional estrogen-response elements. Estrogen receptors (ERs) and ER coregulators such as mixed lineage leukemia (MLL) histone methylases (MLL2 and MLL3) and histone acetyltransferase CBP/P300 bind to the EZH2 promoter in the presence of estradiol and regulate estradiol-induced EZH2 expression. EZH2 expression is also increased upon exposure to estrogenic endocrine disrupting chemicals (EDCs) such as bisphenol-A (BPA) and diethylstilbestrol (DES). Similar to estradiol, BPA and DES-induced EZH2 expression is coordinated by ERs, MLLs and CBP/P300. In summary, we demonstrate that EZH2 is transcriptionally regulated by estradiol in vitro and in vivo, and its expression is potentially dysregulated upon exposure to estrogenic EDCs.

Bisphenol-A and diethylstilbestrol exposure induces the expression of breast cancer associated long noncoding RNA HOTAIR in vitro and in vivo.

Antisense transcript, long non-coding RNA HOTAIR is a key player in gene silencing and breast cancer and is transcriptionally regulated by estradiol. Here, we have investigated if HOTAIR expression is misregulated by bisphenol-A (BPA) and diethylstilbestrol (DES). Our findings demonstrate BPA and DES induce HOTAIR expression in cultured human breast cancer cells (MCF7) as well as in vivo in the mammary glands of rat. Luciferase assay showed that HOTAIR promoter estrogen-response-elements (EREs) are induced by BPA and DES. Estrogen-receptors (ERs) and ER-coregulators such as MLL-histone methylases (MLL1 and MLL3) bind to the HOTAIR promoter EREs in the presence of BPA and DES, modify chromatin (histone methylation and acetylation) and lead to gene activation. Knockdown of ERs down-regulated the BPA and DES-induced expression of HOTAIR. In summary, our results demonstrate that BPA and DES exposure alters the epigenetic programming of the HOTAIR promoters leading to its endocrine disruption in vitro and in vivo.

Acute estradiol treatment affects the expression of cocaine-induced conditioned place preference in ovariectomized female rats.

Women and female rodents are more responsive to the subjective effects of psychostimulant drugs of abuse compared to males. A growing body of literature supports a role for estradiol as a mechanism underlying these sex differences. However, little is known about the influence of acute elevations in levels of estradiol on drug conditioned behaviors. The aim of the present study was to evaluate the influence of an acute increase in systemic estradiol levels on the expression of cocaine conditioned place preference (CPP). Using a six day conditioning procedure, ovariectomized (OVX) female rats were conditioned with one of four doses of cocaine (2.5, 5, 10, or 15mg/kg) to associate one of two large chambers of a CPP apparatus with cocaine or saline. Thirty minutes prior to the start of the CPP preference test, rats were pretreated with either 5µg estradiol benzoate (EB) or peanut oil (PO). PO-treated rats expressed a significant preference for only the mid-range conditioning doses of cocaine (5 and 10mg/kg). However, acute EB treatment resulted in a rightward shift in the cocaine dose-response curve; rats demonstrated a significant preference at only the moderate and high conditioning doses of cocaine (10 and 15mg/kg). These findings demonstrate that acute elevations in estradiol may dampen the expression of conditioned responses to cocaine's secondary rewards at lower conditioning doses of the drug and facilitate CPP at higher doses while estradiol deficiency decreases the threshold dose of cocaine necessary to induce CPP.

Sex differences in the neurobiology of drug addiction.

Epidemiological data demonstrate that while women report lower rates of drug use than men, the number of current drug users and abusers who are women continues to increase. In addition women progress through the phases of addiction differently than men; women transition from casual drug use to addiction faster, are more reactive to stimuli that trigger relapse, and have higher rates of relapse then men. Sex differences in physiological and psychological responses to drugs of abuse are well documented and it is well established that estrogen effects on dopamine (DA) systems are largely responsible for these sex differences. However, the downstream mechanisms that result from interactions between estrogen and the effects of drugs of abuse on the DA system are just beginning to be explored. Here we review the basic neurocircuitry which underlies reward and addiction; highlighting the neuroadaptive changes that occur in the mesolimbic dopamine reward and anti-reward/stress pathways. We propose that sex differences in addiction are due to sex differences in the neural systems which mediate positive and negative reinforcement and that these differences are modulated by ovarian hormones. This forms a neurobehavioral basis for the search for the molecular and cellular underpinnings that uniquely guide motivational behaviors and make women more vulnerable to developing and sustaining addiction than men.

MLL histone methylases regulate expression of HDLR-SR-B1 in presence of estrogen and control plasma cholesterol in vivo.

High-density lipoprotein receptors scavenger receptor class B type I [HDLR-SR-B1 (SR-B1)] is a key player in reverse cholesterol transport and maintaining blood cholesterol. We demonstrated that human SR-B1 is transcriptionally activated by 17ß-estradiol (E2) in HEPG2 and JAR cells. SR-B1 promoter contains multiple estrogen response elements (ERE half-sites) along with some Sp1 binding sites. Knockdown of estrogen receptor (ER)α and ERß down-regulated E2-induced SR-B1 expression. ERs were bound to SR-B1 promoter EREs in an E2-dependent manner. Along with ERs, mixed-lineage leukemia (MLL) histone methylases, especially MLL1 and MLL2, play key roles in E2-mediated SR-B1 activation. MLL1 and MLL2 bind to SR-B1 promoter in an E2-dependent manner and control the assembly of transcription pre-initiation complex and RNA polymerase II (RNAPII) recruitment. ERs and MLLs play critical roles in determining the cholesterol uptake by steroidogenic tissues/cells, and their knockdown suppressed the E2-induced cholesterol uptake efficiencies of the cells. Intriguingly, MLL2 knockdown in mice resulted in a 33% increase in plasma cholesterol level and also reduced SR-B1 expression in mice liver, demonstrating its crucial functions in controlling plasma cholesterol in vivo.

Assessing learned associations between conditioned cocaine reward and environmental stimuli in the Wistar Kyoto rat.

Clinical studies demonstrate that anxiety disorders increase the risk of substance use disorder. However, few studies have directly assessed anxiety as a vulnerability factor in processing of rewarding stimuli. The Wistar­Kyoto (WKY) rat has been proposed as a model of anxiety vulnerability because it exhibits extreme behavioral inhibition in novel and social environments; yet, it displays paradoxical rapid active avoidance learning that is resistant to extinction. The present study was designed to characterize the acquisition and persistence of cocaine conditioned place preference (CPP) in WKY rats. In the first of a series of three experiments, adult male WKY and Sprague Dawley (SD) rats were given six pairings of cocaine (3, 5, 10, 15 mg/kg) or saline on alternating days. SD rats developed cocaine-induced CPP to each of the four doses of cocaine tested. In contrast, WKY rats demonstrated CPP when conditioned with 3, 5, and 10 mg/kg, but displayed no preference to the 15 mg/kg dose. Next, separate groups of rats were subject to an extended CPP paradigm, which included acquisition, extinction and reinstatement phases. Rats were conditioned with cocaine and saline on alternating days using either a 6/6 (as above) or 4/4 conditioning regimen. Both SD and WKY rats acquired a lasting CPP with the 6/6 conditioning regimen. Results from the 4/4 conditioning regimen show that SD, but not WKY, rats acquired CPP. Preference scores for SD rats during the cocaine primed reinstatement test were significantly different from pretest scores indicating reinstatement of CPP in this group. Paradoxically, WKY rats demonstrated a latent sensitization to the conditioned rewarding effects of cocaine during the drug-primed reinstatement test. Taken together, WKY rats appear to be more sensitive to high doses of cocaine and need more experience with the drug to acquire a preference than SD rats.

Evaluating underlying neuronal activity associated with escape/avoidance behavior in response to noxious stimulation in adult rats.

The place escape/avoidance paradigm (PEAP) is a behavioral test designed to quantify the level of unpleasantness evoked by painful stimuli by assessing the willingness of a subject to escape/avoid a preferred area when it is associated with noxious stimulation. Previous studies have demonstrated that escape/avoidance behavior is dependent on activity in the anterior cingulate cortex (ACC), a region of the limbic system involved in processing the emotional component of pain in humans and animals. Analysis of c-Fos expression in the ACC confirmed that the escape/avoidance response to noxious stimuli corresponds to changes in neural activation in this region. Behavioral tests such as the PEAP may be more sensitive to changes in supraspinal pain processing and could contribute to the development of novel analgesics in the future.

Influence of sex on reinstatement of cocaine-conditioned place preference.

To explore sex differences in reinstatement of conditioned place preference, we subjected intact male and female Long Evans rats to an extended conditioned place preference (CPP) paradigm, which included observations of acquisition, extinction, and reinstatement of a preference to cocaine-paired stimuli. In a series of experiments, separate groups of animals were given six 30-min pairings of one chamber with cocaine (3, 5, 10, 15, 20, 25mg/kg) and six of the other with saline on alternate days. Overall, there were no sex differences in acquisition of cocaine CPP at any of the six doses tested (p>0.05). All animals established cocaine CPP at each of the six doses tested during the acquisition test, with the exception of the group of females conditioned with 5mg/kg. Preferences for the cocaine-paired chamber were successfully extinguished for both males and females after an extinction-training period. CPP reinstatement was achieved by the groups of males and females given training and priming doses of 10, 15, 20, and 25mg/kg (p<0.05). Overall, our reinstatement data demonstrate that reinstatement of cocaine CPP is greater for female versus male animals. Females showed a greater magnitude of reinstatement of cocaine CPP when trained and primed with 15 and 25mg/kg as compared to males (p<0.05). Further, at the three highest doses tested (15, 20, and 25mg/kg), females showed a greater magnitude of CPP in the reinstatement phase of CPP compared to that of the initial acquisition phase (p<0.05). The reinstatement data for the males show that the 20mg/kg dose resulted in the highest levels of reinstatement preference for male rats. These results indicate that sex differences in reinstatement to conditioned behavior maybe due, in part, to females forming a stronger association for the salience of the drug and the environment in which it was administered.