Abstract
The regulation of thyrotropin (TSH) by thyroid hormone (T3) negatively and by TSH-releasing hormone (TRH) positively is crucial to the thyroid hormone homeostasis. Recently we studied the molecular mechanism of T3/TRinduced TSH beta gene suppression using CV1 cells expressed with Pit1 and GATA2: the activator ofTSH beta gene is GATA2 and Pit1 protects GATA2 from inhibition by T3/TR and other suppressors. T3-boundTR causes histone deacetylation by recruiting HDAC3 or dissociating some HAT activities. TRAP220 is critical to activating function of GATA2 and T3-bound TR may squelch TRAP220 form the mediator complex, resulting in TSH beta gene suppression. We have now investigated the mechanism how TRH stimulates TSH beta gene. As we previously reported, the wild-type TSH beta-CAT reporter gene requires GATA2 and Pit1 together for activation, but GATA2 alone can activate the TSH beta-D4-CAT reporter gene which possesses only the Pit1-binding site and the GATA responsive elements in the TSH beta gene promoter. TRH together with TRH receptor stimulated TSH beta-D4-CAT dose-dependently only in the presence of GATA2. Pholbol ester TPA, but not Forskolin, also stimulated the reporter gene and the inhibitors against protein kinase C (PKC), but not mAPK nor PKA, blocked the TRH effects. The transactivation by 100nmTRH was inhibited by T3 dose-dependently and blocked completely by 10nm T3. We previously demonstrated that the zinc finger region of GATA2 is important to T3/TR-mediated TSH beta gene suppression. This region is also critical to the TRH-induced activation. Interestingly, gel-shift study revealed that TRH and TPA significantly enhanced binding of GATA2 to the TSH beta gene promoter. Our study clarified (1) TRH significantly stimulates TSH beta gene which is activated by GATA2, (2) the TRH signaling pathway is via PKC, and (3) TRH enhances GATA2 binding to the promoter, potentiating its activating effects.