title: Modulation of human mineralocorticoid receptor function by protein kinase A.
abstract: The mineralocorticoid receptor (MR) acts as a ligand-dependent transcription factor modulating specific gene expression in sodium-transporting epithelia. Physiological evidence suggest a cross-talk between the cAMP- and aldosterone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in HepG2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulates glucocorticoid response element (GRE)-containing promoters in a ligand-independent manner. This effect was strictly MR dependent since no activation of the reporter gene was observed in the absence of cotransfected hMR expression plasmid. Furthermore, a synergistic activation was achieved when cells were treated with both aldosterone and cAMP. This synergistic effect was also observed in the CV1 and the stable hMR-expressing M cells but was dependent on the promoter used. In particular, synergism was less pronounced in promoters containing several GREs. We show that (protein kinase-inhibiting peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and aldosterone induction, which indicates that a functional cAMP pathway is required for stimulation of transcription by aldosterone. Using MR-enriched baculovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA. Increased DNA binding of hMR is likely to reflect an increase in the number of active receptors, as measured by Scatchard analysis. Using a truncated MR, we show that the N-terminal domain is required for the effect. Finally, the N-terminal truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repressor.
authors.affiliation: INSERM Unité 490, Centre universitaire des Saints-Pères, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Massaad
authors.forename: N
authors.initials: N
authors.lastname: Houard
authors.forename: M
authors.initials: M
authors.lastname: Lombès
authors.forename: R
authors.initials: R
authors.lastname: Barouki
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D018161:Receptors, Mineralocorticoid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000187:drug effects; D014407:Tumor Cells, Cultured
chemical_list: D010455:Peptides; D018161:Receptors, Mineralocorticoid; D011994:Recombinant Proteins; C061479:protein kinase inhibitor peptide; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000450:Aldosterone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases