Studying the regulatory mechanism of the glycoprotein hormone alpha subunit (alpha GSU) gene in thyrotropes is essential for understanding the synthesis of functional thyroid-stimulating hormone (TSH). Here, we investigated the influence of a homeodomain transcription factor Msx1 (Msh homeobox 1) on alpha GSU expression in thyrotropes. The transient expression of Msx1 inhibited the activity of an alpha GSU reporter gene, as well as its endogenous mRNA level in thyrotrope-derived alpha TSH cells. Luciferase reporter assays with serial deletion constructs and a close examination of the sequences revealed that the putative Msx1 binding site (PMS) in the alpha GSU promoter is not responsible for Msx1-mediated transcriptional repression. We also identified the TATA-box binding protein (TBP) as an interacting protein in thyrotropes. Interaction of TBP with Msx1 attenuates the inhibitory effect of Msx1 on alpha GSU gene expression in a DNA binding-independent manner. Furthermore, transient transfection studies with mutant Msx1 revealed that the interaction of TBP and Msx1 is critical for Msx1-mediated transcriptional repression of the alpha GSU. These results suggest that Msx1 functions as a transcriptional repressor of alpha GSU and that its interaction with TBP is an integral part of the mechanism by which Msxl regulates the inhibition of alpha GSU gene expression. (C) 2015 Elsevier Inc. All rights reserved.