Esophagin is a member of the small proline-rich protein family of cell envelope precursor proteins expressed during squamous differentiation. Esophagin expression is confined to the suprabasal layers of the epithelium and is shown to be highly restricted to internal stratified squamous epithelia, including the esophagus, oral mucosa cervix, and vagina. Loss of expression of esophagin is observed in squamous carcinomas and adenocarcinomas of the esophagus, and decreased expression is present in areas of dysplasia, suggesting that dysregulation of esophagin expression is an early marker for cell transformation. Elucidation of the promoter elements that mediate this pattern of expression is an important step in enhancing our understanding of the intracellular pathways that control the differentiation of esophageal epithelium. The sequencing and analysis of the esophagin gene, including 2.7kb of the promoter region, has been completed. A minimal promoter region sufficient for in vitro expression in primary esophageal keratinocytes has been defined by using a panel of deletion constructs and a luciferase reporter system. In addition, several positive and negative regulatory regions, including calcium-, PMA-, and UV-irradiation-responsive regions, have been identified. Evaluation of the promoter sequence using MatInspector 2.1 and the TRANSFAC database has revealed putative regulatory elements for a number of transcription factors believed to be involved in squamous differentiation, including AP-1, POU factors, Sp1, NF-kappaB, CREB, C/EBP-beta, and ets factors, as well as for EBV and HPV viral proteins. A putative role for C/EBP-beta and Oct-1 binding sites in the minimal promoter region -116 is suggested by site-directed mutagenesis and in vitro analysis of luciferase reporter constructs. In addition, calcium-induced differentiation in primary esophageal epithelial cells is shown to be accompanied by an increase in Sp1 and Oct-1 expression. No change in the expression of ets factors or AP-1 proteins was observed during calcium induction. The temporal pattern of esophagin expression during induction by calcium has been established in primary cultures of esophageal keratinocytes and by UV-irradiation and PMA in the KYSE 450 esophageal squamous cell carcinoma cell line. These studies provide a model for evaluating the regulatory factors that are involved in normal squamous differentiation in the esophagus.