Estrogenic modulation of cytokine-­signaling pathways within the brain

Abstract

The gonadal steroid estradiol has widespread and diverse actions within the brain, although many of the cellular mechanisms responsible are unknown. This study extends a previous observation that estradiol can modulate cytokine signalling pathways within the hypothalamus. Adult female rats and mice were ovariectomized and then treated acutely (4 h) or chronically (1 week) with estradiol. Brains were removed, frozen and 300 μm coronal sections prepared. Specific regions were collected by micro-punch, and suppression of cytokine signaling (SOCS1 and SOCS3) mRNA levels measured by qPCR. Chronic estradiol treatment caused ~two fold increase (p< 0.05) in SOCS1 and SOCS3 mRNA levels in the medial preoptic area, arcuate nucleus, and cortex in the rat. In the mouse, both acute and chronic estradiol treatment significantly (p< 0.05) increased SOCS1 and SOCS3 mRNA levels (1.5-2-fold) in the medial preoptic area, but not in the cortex. SOCS mRNA levels in the arcuate nucleus were unable to be properly quantified due to fluctuating levels of β-actin mRNA (the reference gene).

Estradiol-induced changes in Socs expression would be expected to inhibit signal transducer and activator of transcription (STAT) responses; but there are some suggestions in the literature that estradiol increases STAT signaling. To investigate this possibility, parallel experiments were conducted in which estradiol treated animals were injected with the STAT3-activator leptin (1 mg/kg for 2h), and perfused with paraformaldehyde. Brain sections were than prepared and processed for immunohistochemistry. As expected, STAT3 positive cells were seen throughout the hypothalamus in response to leptin. However, acute or chronic estradiol treatments were unable to influence this response. In vitro cell culture experiments were used to further examine effects of estradiol on STAT signaling in response to two different cytokines, namely leptin and prolactin. Primary hypothalamic neuronal cultures were treated with estradiol in combination with leptin or prolactin (100 ng/ml) for 30 min.

Immunohistochemistry was carried out to characterize neurons in the cultures using the well-known neuronal marker βIII-tubulin. Some of these neurons expressed basal pSTAT3 levels, but failed to respond to leptin or estradiol treatment. In contrast, prolactin significantly increased the number of neurons expressing pSTAT5 in the cultures but estradiol treatment did not influence this response.

In conclusion, qPCR experiments showed treatment with estradiol can result in increased SOCS mRNA levels. The lack of effect of estradiol on leptin or prolactin-induced STAT activation supports the idea that these effects on Socs gene expression are direct rather than via STAT signalling pathways. Further experiments, such as testing for estradiol-induced Socs gene expression in the presence of a STAT inhibitor, are required to support this novel concept.