The role of extracellular signal-regulated kinase (ERK) in mediating the ability of thyrotropin-releasing hormone (TRH) to stimulate the prolactin gene has been well elucidated. ERK is inactivated by a dual specificity phosphatase, mitogen-activated protein kinase phosphatase (MKP). In this study, we examined the induction of MKP-1 protein by thyrotropin-releasing hormone (TRH) in pituitary GH3 cells and investigated the possible role for MKP-1 in TRH-induced prolactin gene expression. MKP-1 protein was induced significantly from 60 min after TRH stimulation, and remained elevated at 4 h. The effect of TRH on MKP-1 expression was completely prevented in the presence of the MEK inhibitor, U0126. In the experiments using triptolide, a potent blocker for MKP-1, MKP-1 induction by TRH was completely inhibited in a dose-dependent manner. TRH-induced ERK activation was significantly enhanced in this condition. Prolactin promoter activity, activated by TRH, was reduced to the control level in the presence of triptolide in a dose-dependent manner, was reduced to the control level in the presence of triptolide in a dose-dependent manner. In GH3 cells, which were transfected with MKP-1 specific siRNA, both the basal and TRH-stimulated activities of the prolactin promoter were significantly reduced compared to the cells transfected with negative control siRNA. Our present results support a critical role of MKP-1 in TRH-induced, ERK-dependent, prolactin gene expression. (C) 2009 Elsevier Inc. All rights reserved.