Response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids depends on the integrity of the cAMP pathway.
The phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated at the transcriptional level by a variety of effectors in a tissue-specific fashion. In order to study the parameters involved in the tissue-specific hormonal regulation of the PEPCK gene, we have used a transient expression test in well-differentiated rat hepatoma cells as well as in dedifferentiated variants. In this test, the PEPCK promoter is induced by glucocorticoids in well-differentiated FGC4 cells, but not in H5 dedifferentiated variants, in spite of the presence in H5 cells of the glucocorticoid receptor. Study of the PEPCK promoter using electrophoretic mobility shift assays reveals binding sites for the liver-enriched transcription factors HNF1, vHNF1, HNF3, HNF4, and CAAT/enhancer binding protein members. Overexpression of the liver-enriched transcription factors absent in the dedifferentiated variants, such as HNF1 and HNF4, is not sufficient to restore glucocorticoid response of the PEPCK promoter in the variants. Moreover, systematic analysis of the PEPCK promoter reveals that the presence of a region covering a cAMP-responsive element (CRE1 at -80) and a CAAT box is necessary for full response of the PEPCK promoter to glucocorticoids in well-differentiated rat hepatoma cells. In a cotransfection test, overexpression of the regulatory subunit of protein kinase A (PKA), causing sequestering of PKA, abolishes the glucocorticoid response of the promoter in well-differentiated cells. On the other hand, in dedifferentiated variants, overexpression of the catalytic subunit of PKA restores the response to glucocorticoids. The action of PKA on the glucocorticoid response requires the presence of the CRE1 element and is promoter specific because it does not concern nonhepatic promoters such as the long terminal repeats of the mouse mammary tumor virus. These results suggest that the full response of the PEPCK promoter to glucocorticoids requires activation of another signal transduction pathway, the cAMP-mediated pathway.
Angrand
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Unité de Génétique de la Différenciation, URA 1149 du Centre National de la Recherche Scientifique, Département de Biologie Moléculaire, Institut Pasteur, Paris, France.
Coffinier
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Weiss
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D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D014644:Genetic Variation; D005943:Gluconeogenesis / Q000187:drug effects; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D014157:Transcription Factors; D003907:Dexamethasone; D000242:Cyclic AMP; D010729:Phosphoenolpyruvate Carboxykinase (GTP)