An Otx-Related Homeodomain Protein Binds an LHβ Promoter Element Important for Activation During Gonadotrope Maturation

The hormone-secreting cell types of the anterior pituitary differentiate in a specific spatial and temporal manner. The α-subunit of the glycoprotein hormones appears at embryonic d 11.5 in the mouse, followed by steroidogenic factor-1, which distinguishes the gonadotrope progenitor cells, around embryonic d 14. Gonadotrope maturation is marked by the onset of LHβ-gene expression 2 d later. The αT3-1 and LβT2 immortalized mouse pituitary cell lines correspond to these later sequential stages of gonadotrope differentiation. In addition to the early markers of the gonadotrope lineage present in αT3-1 cells, LβT2 cells also express markers of a mature gonadotrope, including LHβ and FSHβ. Using transient transfections to compare expression among gonadotrope and nongonadotrope-derived cell types, we show that the rat 1.8-kb LHβ promoter directs reporter gene expression specifically to the mature gonadotrope LβT2 cell line. Promoter truncation and mutagenesis analyses indicate that the homeodomain (HD) element located at approximately −100 bp relative to the transcriptional start site is essential for this selectivity to LβT2 cells when compared with αT3-1 cells. In EMSAs, this HD site binds a protein present in LβT2 but not other gonadotrope-derived cells. Antibody supershift and competition experiments indicate that this LβT2 nuclear protein is a K50 HD protein related to the Otx family, though it is not a known pituitary homeobox transcription factor protein. These studies indicate a role for a novel Otx-related HD protein in gonadotrope maturation during development.