Molecular characterization of mammalian pituitary transcription factors

Determination and differentiation of the hormone-secreting cell types of the anterior pituitary gland is regulated by several homeodomain transcription factors. Defects in the LHX3, LHX4, HESX1, PTX1, PTX2, PROP-1, and PIT-1 homeodomain proteins lead to abnormal pituitary development and pituitary disease in mammals. LHX3 is a LIM homeodomain transcription factor that is essential for development of the anterior pituitary lobe. The studies presented here were undertaken to better understand the role(s) that LHX3 plays in anterior pituitary cell type differentiation and specification. The LHX3 gene encodes two isoforms, LHX3a and LHX3b, which differ in their amino termini but share common cysteine-rich zinc finger-like LIM domains, a characteristic DNA-binding homeodomain, and a conserved carboxyl terminus. Lhx3a and Lhx3b are differentially expressed in anterior pituitary cells; Lhx3b is highly expressed in pituitary cell lines representing the gonadotrope, lactotrope, somatotrope, and thyrotrope lineages, while Lhx3a expression is restricted to the alpha glycoprotein subunit gene-expressing thyrotrope and gonadotrope lineages. LHX3a and LHX3b differ in their ability to bind specific DNA elements and trans-activate pituitary hormone genes; the b-specific domain regulates gene trans-activation by inhibiting LHX3 binding to DNA. Further, transfer of the b-specific domain to other transcription factors demonstrates that this domain specifically inhibits the function of homeodomain-containing proteins by inhibiting binding to regulatory elements. Analysis of LHX3 in human pituitary disease demonstrates that mutations in LHX3 do not prevent DNA binding or interaction with partner proteins but do impair the gene trans-activation function of LHX3. Further, the studies in this report identify a novel, functional LHX3 protein, M2-LHX3, and demonstrate this molecule is generated by an internal translation initiation codon. The alternate LHX3a- and b-specific coding sequences regulate differential usage of this internal start codon. Identification of M2-LHX3 enabled characterization of novel activation and repression domains in the LHX3 carboxyl terminus. In addition to characterizing the LHX3 proteins, the studies presented here describe cloning of the porcine Prop-1 transcription factor gene and analysis of its protein products. Together, these studies characterize the molecular actions of transcription factors required for development and function of the anterior pituitary gland