Characterization of human mucin gene MUC4 promoter - Importance of growth factors and proinflammatory cytokines for its regulation in pancreatic cancer cells

The human mucin gene MUC4 encodes a large transmembrane mucin that is thoug ht to play important roles in tumor cell biology and that is overexpressed in human pancreatic carcinomas. In this report, we describe the structure a nd functional activity of the 5 ' -flanking region, including 1.0 kilobase of the promoter. The long 5 ' -untranslated region (2.7 kilobases) is chara cterized by a high content of GC in its 3 ' -end. The first TATA box was lo cated at -2672/-2668. Multiple transcription start sites and a high density of putative binding sites for Sp1 (GC and CACCC boxes), AP-1/-2/-4, cAMP-r esponsive element-binding protein, GATA, GR, and STAT transcription factors were found within the 5 ' -flanking region. Transcriptional activity of th e promoter was assessed using pGL3-luciferase deletion mutants in two MUC4- expressing (CAPAN-1 and CAPAN-2) and one nonexpressing (PANC-1) pancreatic cancer cell line. Two highly active fragments (-219/-1 and -2781/-2572) tha t drive MUC4 transcription in CAPAN-1 and CAPAN-2 cells were identified. Ge l retardation assays indicated that Sp1 and Sp3 bind to cognate cis-element s found in the 5 ' -flanking region and that Sp1 transactivates, whereas Sp 3 inhibits the GC-rich region (-464/-1) in CAPAN-2 cells. Activation of pro tein kinase C with phorbol ester and treatment of cells with epidermal grow th factor and transforming growth factor-alpha resulted in upregulation of the promoter. Tumor necrosis factor-alpha and interferon (IFN)-gamma inflam matory cytokines had no or mild effect on MUC4 transcriptional activity whe n used alone. However, a very strong synergistic effect (10-12-fold activat ion) between IFN-gamma and tumor necrosis factor-a or IFN-gamma and transfo rming growth factor-a was obtained in CAPAN-2 cells. Altogether these resul ts demonstrate that the 5 ' -flanking region of MUC4 contains epithelial ce ll-specific, positive, and negative regulatory cis-elements, that Sp1/Sp3 a re important regulators of MUC4 basal expression, and that its regulation i n pancreatic cancer cells involves complex interplay between several signal ing pathways.