Repression of 5-aminolevulinate synthase gene by the potent tumor promoter, TPA, involves multiple signal transduction pathways.
The potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) induces activator protein-1 (AP-1) transcription factors, early response genes involved in a diverse set of transcriptional regulatory processes, and protein kinase C (PKC) activity. This work was designed to explore the signal transduction pathways involved in TPA regulation of 5-aminolevulinate synthase (ALAS) gene expression, the mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. We have previously reported that TPA causes repression of ALAS gene, but the signaling pathways mediating this effect remain elusive. The present study investigates the role of different cascades often implicated in the propagation of phorbol ester signaling. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in human hepatoma HepG2 cells. In these experimental conditions, we analyzed TPA action upon endogenous ALAS mRNA levels, as well as the promoter activity of a fusion reporter construct, harboring the TPA-responsive region of ALAS gene driving chloramphenicol acetyl transferase gene expression. We demonstrated that the participation of alpha isoform of PKC, phosphatidylinositol 3-kinase (PI3K), extracellular-signal regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) is crucial for the end point response. Remarkably, in this case, ERK activation is achieved in a Ras/Raf/MEK-independent manner. We also propose that p90RSK would be a convergent point between PI3K and ERK pathways. Furthermore, we elucidated the crosstalk among the components of the cascades taking part in TPA-mediated ALAS repression. Finally, by overexpression of a constitutively active p90RSK and the coactivator, cAMP-response element protein (CREB)-binding protein (CBP), we reinforced our previous model, that implies competition between AP-1 and CREB for CBP.
Guberman
Alejandra S
AS
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II Piso 4, Ciudad Universitaria, 1428 Buenos Aires, Argentina.
Scassa
María E
ME
Cánepa
Eduardo T
ET
D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis* / Q000235:genetics*; D015152:Blotting, Northern; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005822:Genetic Vectors; D006418:Heme / Q000737:chemistry; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010703:Phorbol Esters / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism; D051571:Protein Kinase C-alpha; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D010703:Phorbol Esters; D020033:Protein Isoforms; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D006418:Heme; D012313:RNA; D000624:5-Aminolevulinate Synthetase; D019869:Phosphatidylinositol 3-Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D013755:Tetradecanoylphorbol Acetate