Figure 4.

In the mouse hypothalamus, hepatitis virus B X-associated protein 2 small inhibitory RNA affects only TRβ1-mediated activation of thyrotropin-releasing hormone transcription. (A) Dose-dependent effect of XAP2 siRNA on basal TRH transcription. TRH-Luc transcription was followed in hypothyroid 2-day-old mice, 20 h after hypothalamic injection of 1 μg TRH-Luc, 100 ng TRβ1 expression vector and 0, 20 or 200 nM of siXAP2-1 or 200 nM of scrambled siRNA (siCT) complexed with JetSI. Values are means±s.e.m., n⩾30; ^***P<0.001. (B) Two different XAP2 siRNAs affect the activation but not the T₃-dependent repression of TRH transcription. TRH-Luc transcription was measured in hypothyroid 2-day-old mice, 20 h after hypothalamic injection of 1 μg reporter construct, 100 ng of TRβ1 expression vector and 200 nM of siXAP2-1, siXAP2-2 or scrambled siRNA (siCT) complexed with JetSI. After transfection, mice were treated with T₃ (2.5 μg/g of body weight, filled bars) or saline (open bars). Values are means±s.e.m., n⩾6; ns: not significant; ^*P<0.05; ^**P<0.01. (C) XAP2 siRNA affects activation of TRH transcription mediated by TRβ1 but not by TRβ2. TRH-Luc transcription was measured in hypothyroid 2-day-old mice, 20 h after hypothalamic injection of 1 μg reporter construct, 100 ng of either TRβ1 or TRβ2 expression vectors and 200 nM of siXAP2-1 or scrambled siRNA (siCT) complexed with JetSI. After transfection, mice were treated with T₃ (2.5 μg/g of body weight, filled bars) or saline (open bars). Values are means±s.e.m., n⩾14; ns: not significant; ^**P<0.01; ^***P<0.001. Each experiment was repeated at least three times. siRNA, small inhibitory RNA; T₃, triiodothyronine; TRβ1, TRβ2, thyroid hormone receptor subtypes; TRH, thyrotropin-releasing hormone; XAP2, hepatitis virus B X-associated protein 2.