Cyclic adenosine 3′,5′-monophosphate-elevating agents inhibit transforming growth factor-β-induced SMAD3/4-dependent transcription via a protein kinase A-dependent mechanism

Abstract

Transforming growth factor-b (TGF-b) 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-b receptors to regulate the transcription of specific target genes. Crosstalks with other signaling pathways may contribute to the specificity of TGF-b effects. In this report, we have investigated the effects of cyclic adenosine 3 0 ,5 0 -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-b 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 E 2 (PGE 2 ), interfere with TGF-b-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 PKAdependent manner. These results suggest that suppression of TGF-b/SMAD signaling and resulting gene transactivation by cAMP-inducing agents occurs via PKAdependent, CREB-mediated, disruption of SMAD-CBP/ p300 complexes.

Lithium inhibits Smad3/4 transactivation via increased CREB activity induced by enhanced PKA and AKT signaling

“…This effect is most likely due to the squelching effects of CREB in p300/CREB complexes, resulting in the blockade of Smad-dependent transcription. This notion is supported by a previous report that, in TGF-β-stimulated human HaCaT keratinocytes, the inhibition of TGF-β-induced Smad3/4-dependent transcription of the PAI-1 gene by cAMP-elevating agents is mediated through PKA activation and disruption of the Smad-CBP/p300 complex (Schiller et al, 2003). Our results thus confirm CREB's inhibitory effects and further demonstrate that lithium's inhibitory effect on PAI-1 promoter transactivation is partially mediated through a PKA-dependent mechanism in an unstimulated state of cortical neurons.…”

“…It has been documented that CBP and p300 function as essential transcriptional coactivators for Smad-dependent gene expression and that competition for CBP/p300 could be a mechanism to mediate signal-induced transcriptional repression (Hottiger and Nabel, 1998;Schiller et al, 2003). CBP and p300 are also known to be coactivators of CREB (Chrivia et al, 1993).…”

Increased cAMP Levels Modulate Transforming Growth Factor-β/Smad-induced Expression of Extracellular Matrix Components and Other Key Fibroblast Effector Functions

“…As shown in Fig. 1A, exposure of HDF to exogenous forskolin resulted in rapid phosphorylation of CREB at serine 133, consistent with previous results obtained in HaCaT keratinocytes (20). Similarly, the membrane-permeable cAMP analog Bt 2 cAMP led to a comparable extent of CREB phosphorylation.…”

Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase

“…TGF-␤ signaling occurs via Smad proteins, and a requirement for Smad proteins, specifically Smad3, in TGF-␤ regulation of ␣-SMA gene expression has been suggested during myofibroblast transformation of rat lung fibroblasts (51). Recent data indicate that cAMP acts in a PKAdependent manner to inhibit TGF-␤͞Smad signaling and gene activation by disruption of transcriptional cofactor binding (52).…”