Estradiol increases signal transducer and activator of transcription (STAT3) phosphorylation and DNA binding in brain nuclear extract after MCAO occlusion

Introduction

Estradiol is neuroprotective in animal models of cerebral ischemia ¹ . Although the precise mechanisms are unknown, estrogen's beneficial properties are, in part, linked to rapid activation of signal transduction pathways that lead to the transcription of neuroprotective genes ² . We tested the hypothesis that estradiol's neuroprotective effects are partially mediated via signal transducer and activator of transcription 3 (STAT3). STAT3 resides in the cytoplasm in a latent form, but is rapidly activated by tyrosine-705 phosphorylation in response to extracellular stimuli ³ , including estrogen ⁴ . Upon activation, STAT3 translocates to the nucleus where it binds to and upregulates neuroprotective genes, such as bcl-2 ⁵ .

Methods

Seven days prior to MCAO occlusion, ovariectomized (O) female Wistar rats were implanted subcutaneously with 25 mg 17b-estradiol (E). Rats were subjected to 2-hour MCAO and sacrificed after 3 hours of reperfusion. Laser-Doppler perfusion, body and head temperatures and arterial blood pressure were monitored and kept within normal range. Blood was taken from the heart at the time of euthanasia for measurement of plasma estradiol. The neocortex from the ipsilateral and contralateral hemispheres (between coronal levels 2 and −3 mm from Bregma) was dissected and homogenized to prepare nuclear and cytosolic protein extracts. Equal protein amounts from each fraction were probed with anti-phosphotyrosine(705)-STAT3 (p-STAT3) and anti-total STAT3 (T-STAT3) antibodies. Optical density from phospho-STAT3 was normalized to total STAT3 to calculate the degree of STAT3 phosphorylation. STAT3 promoter binding was assessed by electrophoretic mobility shift assay (EMSA) in nuclear extracts.

Results

Ischemia strongly induced phospho-STAT3 (5.85 ± 1.96 fold, mean ± sem) in the ipsilateral neocortex at 3 hours of reperfusion. Estradiol increased STAT3 phosphorylation in the ischemic side by 2.6 ± 0.92 fold, mean ± sem. STAT3 was observed in the nuclear, but not in the cytosolic fractions after MCAO, suggesting that estradiol enhances cytosolic to nuclear translocation of STAT3 after ischemia. EMSA revealed binding to a consensus STAT3 binding element in brain nuclear extracts from estradiol-treated animals.

Conclusion

Estradiol enhances STAT3 phosphorylation, translocation and DNA binding in brain nuclear extracts after MCAO. Increased STAT3 phosphorylation by estradiol may contribute to its neuroprotective effect by upregulating neuroprotective genes (See Figure 1).

Figure 1