Phorbol ester up-regulates phospholipase D1 but not phospholipase D2 expression through a PKC/Ras/ERK/NFkappaB-dependent pathway and enhances matrix metalloproteinase-9 secretion in colon cancer cells.
Despite its importance in cell proliferation and tumorigenesis, very little is known about the molecular mechanism underlying the regulation of phospholipase D (PLD) expression. PLD isozymes are significantly co-overexpressed with cancer marker genes in colorectal carcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment, as a mitogenic signal in colon cancer cells, selectively increases PLD1 expression in transcription and post-transcription. Moreover, experiments using intraperitoneal injection of PMA into mice showed selective PLD1 induction in the intestine and lung tissues, which suggests its physiological relevance in vivo. Therefore, we have undertaken a detailed analysis of the effects of PMA on the promoter activity of PLD genes. Protein kinase C inhibitors, but not a protein kinase A inhibitor, were found to suppress the up-regulation of PLD1 but not PLD2. Dominant-negative mutants of Ras, Raf, and MEK suppressed the induction and activity of PLD1. Moreover, depletion of the supposedly involved proteins reduced the endogenous PLD1 protein level. An important role for NFkappaB as a downstream target of ERK in PMA-induced PLD1 induction was also demonstrated using the inhibitor, small interfering RNA, chromatin immunoprecipitation assay, and site-specific mutagenesis. Furthermore, inhibitors of these signaling proteins and depletion of PLD1 suppressed PMA-induced matrix metalloproteinase-9 secretion and PLD1 induction. In conclusion, we demonstrate for the first time that induction of PLD1 through a protein kinase C/Ras/ERK/NFkappaB-dependent pathway is involved in the secretion of matrix metalloproteinase-9 in colorectal cancer cells.
Kang
Dong Woo
DW
Department of Molecular Biology, College of Natural Science, and College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea.
Park
Mi Hee
MH
Lee
Young Jun
YJ
Kim
Hyung Sik
HS
Kwon
Taeg Kyu
TK
Park
Won-Sang
WS
Min
Do Sik
do S
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism*; D034741:RNA, Small Interfering; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*; D018631:ras Proteins / Q000378:metabolism*
D016328:NF-kappa B; D034741:RNA, Small Interfering; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D020780:Matrix Metalloproteinase 9; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate