PKC-dependent stimulation of the human MCT1 promoter involves transcription factor AP2.
Monocarboxylate transporter (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 microM, 24 h), upregulates butyrate transport and MCT1 protein expression in human intestinal Caco-2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the present study, we showed that PMA (0.1 microM, 24 h) increased the MCT1 promoter activity (-871/+91) by approximately fourfold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1 h and persisted until 24 h, suggesting that the effects of PMA are attributable to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC-zeta isoform. 5'-deletion studies demonstrated that the MCT1 core promoter region (-229/+91) is the PMA-responsive region. Site-directed mutagenesis studies showed the predominant involvement of potential activator protein 2 (AP2) binding site in the activation of MCT1 promoter activity by PMA. In addition, overexpression of AP2 in Caco-2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine.
Saksena
Seema
S
Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. saksena@uic.edu
Dwivedi
Alka
A
Gill
Ravinder K
RK
Singla
Amika
A
Alrefai
Waddah A
WA
Malakooti
Jaleh
J
Ramaswamy
Krishnamurthy
K
Dudeja
Pradeep K
PK
D001483:Base Sequence; D018938:Caco-2 Cells; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology*; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D027501:Monocarboxylic Acid Transporters / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D027981:Symporters / Q000235:genetics* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D050656:Transcription Factor AP-2 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D015854:Up-Regulation; D000099221:Protein Kinase C zeta; D000099233:Monocarboxylate Transport Protein 1
D017868:Cyclic AMP-Dependent Protein Kinases; D020536:Enzyme Activators; D007211:Indoles; D008301:Maleimides; D027501:Monocarboxylic Acid Transporters; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D027981:Symporters; D013755:Tetradecanoylphorbol Acetate; D050656:Transcription Factor AP-2; D000099221:Protein Kinase C zeta; D000099233:Monocarboxylate Transport Protein 1; C088060:bisindolylmaleimide