Cyclic adenosine 3',5'-monophosphate-elevating agents inhibit transforming growth factor-beta-induced SMAD3/4-dependent transcription via a protein kinase A-dependent mechanism.
Transforming growth factor-beta (TGF-beta) plays complex roles in carcinogenesis, as it may exert both tumor suppressor and pro-oncogenic activities depending on the stage of the tumor. SMAD proteins transduce signals from the TGF-beta receptors to regulate the transcription of specific target genes. Crosstalks with other signaling pathways may contribute to the specificity of TGF-beta effects. In this report, we have investigated the effects of cyclic adenosine 3',5'-monophosphate (cAMP), a key second messenger in the cellular response to various hormones, on SMAD-dependent signaling in human HaCaT keratinocytes. Using either an artificial SMAD3/4-dependent reporter construct or the natural TGF-beta target, plasminogen activator inhibitor-1, we show that membrane-permeable dibutyryl cAMP, and other intracellular cAMP-elevating agents such as the phosphodiesterase inhibitor isobutyl-methylxanthine, the adenylate cyclase activator forskolin, or exogenous prostaglandin E2 (PGE2), interfere with TGF-beta-induced SMAD-specific gene transactivation. Inhibition of protein kinase A (PKA), the main downstream effector of cAMP, with H-89, suppressed cAMP-dependent repression of SMAD-driven gene expression. Inversely, coexpression of either an active PKA catalytic subunit or that of the cAMP response element (CRE)-binding protein (CREB) blocked SMAD-driven gene transactivation. cAMP-elevating agents did not inhibit nuclear translocation and DNA binding of SMAD3/4 complexes, but abolished the interactions of SMAD3 with the transcription coactivators CREB-binding protein (CBP) and p300 in a PKA-dependent manner. These results suggest that suppression of TGF-beta/SMAD signaling and resulting gene transactivation by cAMP-inducing agents occurs via PKA-dependent, CREB-mediated, disruption of SMAD-CBP/p300 complexes.
Schiller
Meinhard
M
INSERM U532, Institut de Recherche sur la Peau, Université Paris VII, Hôpital Saint-Louis, F-75010 Paris, France.
Verrecchia
Frank
F
Mauviel
Alain
A
D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D004247:DNA; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases