Cutaneous Effects of In Utero and Lactational Exposure of C57BL/6J Mice to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

To determine the cutaneous effects of in utero and lactational exposure to the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), pregnant C57BL/6J mice were exposed by gavage to a vehicle or 5 µg TCDD/kg body weight at embryonic day 12 and epidermal barrier formation and function were studied in their offspring from postnatal day 1 (P1) through adulthood. TCDD-exposed pups were born with acanthosis. This effect was AHR-dependent and subsided by P6 with no evidence of subsequent inflammatory dermatitis. The challenge of adult mice with MC903 showed similar inflammatory responses in control and treated animals, indicating no long-term immunosuppression to this chemical. Chloracne-like sebaceous gland hypoplasia and cyst formation were observed in TCDD-exposed P21 mice, with concomitant microbiome dysbiosis. These effects were reversed by P35. CYP1A1 and CYP1B1 expression in the skin was increased in the exposed mice until P21, then declined. Both CYP proteins co-localized with LRIG1-expressing progenitor cells at the infundibulum. CYP1B1 protein also co-localized with a second stem cell niche in the isthmus. These results indicate that this exposure to TCDD causes a chloracne-like effect without inflammation. Transient activation of the AhR, due to the shorter half-life of TCDD in mice, likely contributes to the reversibility of these effects.

AHR Regulates Metabolic Reprogramming to Promote SIRT1-Dependent Keratinocyte Differentiation.

Activation of the transcription factor, AHR, in normal human epidermal keratinocytes increased AHR binding in the gene regions of the glucose transporter, SLC2A1, and the glycolytic enzyme, ENO1. This increased chromatin binding corresponded with AHR-dependent decreases in levels of SLC2A1 and ENO1 mRNA, protein, and activities. Studies of the ENO1 promoter showed activation of the AHR decreases the transcription of ENO1. Glycolysis was lowered by activation of the AHR as measured by decreases in glucose uptake and the production of pyruvate and lactate. Levels of ATP were also decreased. Downregulation of glucose metabolism, either by activation of the AHR, inhibition of glycolysis, inhibition of glucose transport, or inhibition of enolase, increased SIRT1 protein levels in normal human epidermal keratinocytes and the immortalized keratinocyte cell line, N/TERT-1. This increase in SIRT1 was abrogated by the addition of exogenous pyruvate. Moreover, keratinocyte differentiation in response to downregulation of glycolysis, either by activation of the AHR, inhibition of glucose transport, or inhibition of enolase, was dependent on SIRT1. These results indicate that regulation of glycolysis controls keratinocyte differentiation, and that activation of the AHR, by lowering the expression of SLC2A1 and ENO1, can determine this fate.

NRF2 regulates core and stabilizing circadian clock loops, coupling redox and timekeeping in Mus musculus.

Diurnal oscillation of intracellular redox potential is known to couple metabolism with the circadian clock, yet the responsible mechanisms are not well understood. We show here that chemical activation of NRF2 modifies circadian gene expression and rhythmicity, with phenotypes similar to genetic NRF2 activation. Loss of Nrf2 function in mouse fibroblasts, hepatocytes and liver also altered circadian rhythms, suggesting that NRF2 stoichiometry and/or timing of expression are important to timekeeping in some cells. Consistent with this concept, activation of NRF2 at a circadian time corresponding to the peak generation of endogenous oxidative signals resulted in NRF2-dependent reinforcement of circadian amplitude. In hepatocytes, activated NRF2 bound specific enhancer regions of the core clock repressor gene Cry2, increased Cry2 expression and repressed CLOCK/BMAL1-regulated E-box transcription. Together these data indicate that NRF2 and clock comprise an interlocking loop that integrates cellular redox signals into tissue-specific circadian timekeeping.

Long-term alteration of anxiolytic effects of ovarian hormones in female mice by a peripubertal immune challenge.

Recent reports indicate that exposure to some stressors, such as shipping or immune challenge with the bacterial endotoxin, lipopolysaccharide (LPS), during the peripubertal period reduces sexual receptivity in response to ovarian hormones in adulthood. We hypothesized that a peripubertal immune challenge would also disrupt the response of a non-reproductive behavior, anxiety-like behavior, to ovarian hormones in adulthood. Female C57Bl/6 mice were injected with LPS during the peripubertal period and tested for anxiety-like behavior in adulthood, following ovariectomy and ovarian hormone treatment. Treatment with estradiol followed by progesterone reduced anxiety-like behavior in control, but not LPS-treated females. We next determined if the disruptive effect of LPS on adult behavior were limited to the peripubertal period by treating mice with LPS either during this period or in adulthood. LPS treatment during the peripubertal period disrupted the anxiolytic effect of ovarian hormones, whereas treatment in adulthood did not. We further tested if this model of peripubertal immune challenge was applicable to an outbred strain of mice (CD-1). Similar to C57Bl/6 mice, LPS treatment during the peripubertal period, but not later, disrupted the anxiolytic effect of estradiol and progesterone. These data suggest that a peripubertal immune challenge disrupts the regulation of anxiety-like behavior by ovarian hormones in a manner that persists at least for weeks after the termination of the immune challenge.

Sex differences in epigenetic regulation of the estrogen receptor-alpha promoter within the developing preoptic area.

Sex differences in the brain are largely organized by a testicular hormone surge that occurs in males shortly after birth. Although this hormone surge is transient, sex differences in brain and behavior are lasting. Here we describe a sex difference in DNA methylation of the estrogen receptor-alpha (ERalpha) promoter region within the developing rat preoptic area, with males exhibiting more DNA methylation within the ERalpha promoter than females. More importantly, we report that simulating maternal grooming, a form of maternal interaction that is sexually dimorphic with males experiencing more than females during the neonatal period, effectively masculinizes female ERalpha promoter methylation and gene expression. This suggests natural variations in maternal care that are directed differentially at males vs. females can influence sex differences in the brain by creating sexually dimorphic DNA methylation patterns. We also find that the early estradiol exposure may contribute to sex differences in DNA methylation patterns. This suggests that early social interaction and estradiol exposure may converge at the genome to organize lasting sex differences in the brain via epigenetic differentiation.

Dopaminergic activation of estrogen receptors induces fos expression within restricted regions of the neonatal female rat brain.

Steroid receptor activation in the developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. Recent data suggests that dopamine can regulate expression of progestin receptors within restricted regions of the developing rat brain by activating estrogen receptors in a ligand-independent manner. It is unclear whether changes in neuronal activity induced by dopaminergic activation of estrogen receptors are also region specific. To investigate this question, we examined where the dopamine D1-like receptor agonist, SKF 38393, altered Fos expression via estrogen receptor activation. We report that dopamine D1-like receptor agonist treatment increased Fos protein expression within many regions of the developing female rat brain. More importantly, prior treatment with an estrogen receptor antagonist partially reduced D1-like receptor agonist-induced Fos expression only within the bed nucleus of the stria terminalis and the central amygdala. These data suggest that dopaminergic activation of estrogen receptors alters neuronal activity within restricted regions of the developing rat brain. This implies that ligand-independent activation of estrogen receptors by dopamine might organize a unique set of behaviors during brain development in contrast to the more wide spread ligand activation of estrogen receptors by estrogen.

Evidence that female endocrine state influences catecholamine responses to male courtship song in European starlings.

Little is known about the neural control of female responses to male courtship. Female European starlings in breeding condition with high concentrations of estrogen select mates based on variation in song and approach nest boxes broadcasting male song. In contrast, outside of the breeding season (when estrogen is low) females do not display the same response to male song. The catecholamines dopamine and norepinephrine regulate behaviors important for mate choice such as arousal, attention, sexual motivation, and goal-directed approach responses, suggesting a role for catecholamines in female responses to male song. In the present study, treating females with a dopamine agonist inhibited, whereas an antagonist stimulated female interest in nest boxes broadcasting male song. In a second study immunocytochemistry was used to examine the distribution of the phosphorylated (i.e., active) form of tyrosine hydroxylase (pTH), the rate-limiting enzyme for catecholamine synthesis. Exposure to male song in breeding condition females reduced pTH density in brain regions involved in social behavior (lateral septum, ventromedial nucleus of the hypothalamus) and a region involved in visual processing (nucleus of Edinger-Westphal) but not song control regions. Opposite patterns of pTH labeling densities were observed in the same regions in response to song in non-breeding condition females. pTH in the ventral tegmental area was also affected by song and female endocrine condition. Overall, the present data support an inhibitory role for dopamine in female responses to courtship and suggest that endocrine state and catecholamines interact to regulate this behavior.

Dopaminergic activation of estrogen receptors in neonatal brain alters progestin receptor expression and juvenile social play behavior.

Steroid receptor activation in developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. We report that estrogen receptors can be activated in a ligand-independent manner within developing brain by membrane dopamine receptors. Neonatal treatment with either estradiol or a dopamine D1 receptor agonist can increase the expression of an estrogen receptor-regulated gene (i.e. progestin receptors) and later juvenile social play. More importantly, increases in social play behavior induced by neonatal treatment with estradiol or a dopamine D1 receptor agonist can be prevented by prior treatment with an estrogen receptor antagonist. This suggests that changes in dopamine transmission in developing brain can activate estrogen receptors in a ligand-independent manner to influence gene expression and have lasting consequences on social behavior.