Phorbol 12-myristate 13-acetate up-regulates the transcription of MUC2 intestinal mucin via Ras, ERK, and NF-kappa B.
MUC2 is a secretory mucin normally expressed by goblet cells of the intestinal epithelium. It is overexpressed in mucinous type colorectal cancers but down-regulated in colorectal adenocarcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment of colon cancer cell lines increases MUC2 expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5'-flanking region of the MUC2 gene using stably and transiently transfected promoter reporter vectors. Protein kinase C inhibitors (bisindolylmaleimide, calphostin C) and inhibitors of mitogen-activated protein/extracellular signal regulated kinase kinase (MEK) (PD98059 and U0126) suppressed up-regulation of MUC2. Src tyrosine kinase inhibitor PP2, a protein kinase A inhibitor (KT5720), and a p38 inhibitor (SB 203580) did not affect transcription. Western blotting and reverse transcription-PCR analysis confirmed these results. In addition, co-transfections with mutants of Ras, Raf, and MEK showed that the induction of MUC2 promoter activity by PMA required these three signaling proteins. Our results demonstrate that PMA activates protein kinase C, stimulating MAP kinase through a Ras- and Raf-dependent mechanism. An important role for nuclear factor kappaB (NF-kappaB) was also demonstrated using the inhibitor caffeic acid phenethyl ester and electrophoretic mobility shift assays. Such identification of pathways involved in MUC2 up-regulation by PMA in the HM3 colon cancer cell line may serve as a model for the effects of cytokines and growth factors, which regulate MUC2 expression during the progression of colorectal cancer.
Lee
Hae-Wan
HW
Gastrointestinal Research Laboratory, Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Ahn
Dae-Ho
DH
Crawley
Suzanne C
SC
Li
Jian-Dong
JD
Gum
James R
JR
Basbaum
Carol B
CB
Fan
Nancy Q
NQ
Szymkowski
David E
DE
Han
Sang-Young
SY
Lee
Bong H
BH
Sleisenger
Marvin H
MH
Kim
Young S
YS
D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005799:Genes, Dominant; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D055262:Mucin-2; D009077:Mucins / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D018631:ras Proteins / Q000378:metabolism*
D004791:Enzyme Inhibitors; C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins; D016328:NF-kappa B; D012313:RNA; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate