Estrogen Sulfotransferase-mediated Reciprocal Regulation Between Inflammatory Stimuli And Estrogen Homeostasis

Estrogens, as one of the important sex hormones, play crucial roles in physiological events in mammals and exert their effects on various reproductive and non-reproductive tissues. Estrogen homeostasis is tightly regulated through balanced synthesis and metabolism. A critical metabolic pathway to deactivate estrogens is through the estrogen sulfotransferase (EST or SULT1E1)-mediated sulfation, because sulfonated estrogens fail to bind to and activate the estrogen receptors. Epidemiological evidence has suggested that estrogens play a role in the etiology of chronic inflammatory diseases. Elucidation of the regulation between inflammation and estrogen homeostasis may provide novel therapeutic strategies for the prevention and treatment of inflammation related disease.In this study, several experimental inflammatory animal models, transgenic animal models, and techniques of molecular biology and cell biology are employed to investigate EST as the mediator for the reciprocal regulation between inflammatory stimuli and estrogen homeostasis.1. Inflammatory stimuli induce the expression and activity of hepatic ESTThe results from real-time PCR and Northern blot both showed that treatment with lipopolysaccharide (LPS) in wild-type female mice markedly induced the mRNA expression of EST in the liver. Consistent with the mRNA induction of EST, increased estrogen sulfotransferase activity was observed in the liver cytosols prepared from LPS-treated mice. Moreover, treatment of 4-week old intact virgin female mice with LPS for 24 h resulted in a significantly reduced circulating estradiol level.The time and dose courses of the EST induction by LPS were further analyzed. EST gene expression was determined at 4,8,16 and 24 h after LPS injection. LPS administration caused an increase in the mRNA level of EST as early as 4 h after treatment. The induction of EST climaxed at 16 h. When treated with different doses of LPS (0.1,1,5,10 mg/kg) for 16 h, LPS induced EST mRNA expression in a dose-dependent manner.In the same LPS-treated mice, the hepatic expression of Cyp3all and Sultlal was significantly decreased, which was consistent with previous reports. LPS also had little effect on the hepatic expression of steroid sulfatase (Sts). These results suggested that the induction was EST-specific.To further demonstrate the effect of inflammation on the expression of EST, we challenged female mice with the cecal ligation and puncture (CLP)-induced sepsis model, as well as the methionine and choline deficient (MCD) diet-induced non-alcoholic steatohepatitis (NASH) model. These two inflammatory models both showed induction of hepatic EST.2. The mechanism of the induction of hepatic EST by inflammatory stimuliIt has been reported that EST is a transcriptional target of nuclear receptors liver Ⅹ receptor α (LXRα) and glucocorticoid receptor (GR). EST has also been shown to be induced by the agonists of constitutive androstane receptor (CAR). To determine whether the regulation of EST by LPS is nuclear receptor-dependent, we measured the expression of GR target gene Tat, LXRa target gene Srebp-1c, and CAR target gene Cyp2b10. The results showed that the expression of these genes were not induced but rather suppressed by LPS. Furthermore, the induction of EST by LPS was intact in LXRa and LXRβ double knockout mice and CAR knockout mice. These results together suggested that the induction of EST by LPS was independent of LXR, GR and CAR.LPS elicits its inflammatory actions through the Toll-like receptor 4 (TLR4). We used the C3H/HeJ mice that lack a functional TLR4 to determine whether TLR4 was necessary for the induction of EST by LPS. The results showed that the LPS-responsive induction of EST observed in the control TLR4 WT C3H/HeOuJ mice was completely abolished in the C3H/HeJ mice, suggesting the induction of EST by LPS was TLR4-dependent.NF-κB is an important inflammatory transcriptional factor downstream of TLR4. Electrophoretic mobility shift assay (EMSA), luciferase reporter gene assay and chromatin immunoprecipatiation (ChIP) assay indicated that EST gene promoter is a transcriptional target of NF-κB. NF-κB regulates EST expression through a NF-κB binding site in the promoter region of the EST gene. To functional validate the role of NF-κB in the induction of EST, we dosed WT female mice with LPS or subjected mice to CLP in the absence or presence of pyrrolidine dithiocarbamate (PDTC), a pharmacological inhibitor of NF-κB. Our results showed that pre-treatment of mice with PDTC attenuated the LPS-responsive and CLP-responsive induction of EST, suggesting that NF-κB was required for the optimal induction of EST by LPS and CLP.3. The role of EST in the reciprocal regulation between inflammatory stimuli and estrogen homeostasisThe primary function of EST is to sulfonate and deactivate estrogens. We showed that the hepatic expression of a panel of estrogen responsive genes was decreased in LPS-treated mice and CLP-induced mice. This effect was mediated by EST because it was abolished in EST-/-mice. Estrogens are known to promote uterine epithelial proliferation. To determine the functional consequence of EST induction by LPS, we pre-treated ovariectomized WT mice with LPS before challenging the mice with E2. The uterine responses we measured include uterine epithelial proliferation by BrdU labeling and immunostaining and estrogen-responsive gene expression by real-time PCR analysis. The results showed that E2 treatment caused a dramatic increase of BrdU labeling, but this effect was largely compromised in LPS-treated WT mice. The inhibitory effect of LPS on epithelial proliferation was abolished in EST-/-mice, indicating that the expression and regulation of EST was essential for the inhibition. Similar results were obtained when the uterine gene expression was evaluated.To further evaluate the effect of EST in inflammation, we went on to determine whether loss of EST would attenuate inflammation in the LPS and CLP models, presumably by having a compromised estrogen deprivation. The results showed that the LPS-responsive induction of TNFoc and IL-6 was attenuated in EST-/-mice compared to their WT counterparts. Similarly, the CLP-responsive induction of IL-1β and IL-6 was also attenuated in EST-/-mice. These results together suggested that loss of EST attenuated LPS-and CLP-induced responsive inflammatory response.In summary, we have revealed a novel EST-mediated reciprocal regulation between inflammatory stimuli and estrogen homeostasis. This mechanism may provide a novel therapeutic target for the prevention and treatment of inflammation related diseases.