Abstract
cAMP is a key messenger of many hormones and neuropeptides, some of which modulate the composition of extracellular matrix. Treatment of human dermal fibroblasts with dibutyryl cyclic AMP and forskolin antagonized the inductive effects of transforming growth factor-beta (TGF-beta) on the expression of collagen, connective tissue growth factor, tissue inhibitor of matrix metalloproteinase-1, and plasminogen activator inhibitor type I, four prototypical TGF-beta-responsive genes. Increased intracellular cAMP prevented TGF-beta-induced Smad-specific gene transactivation, although TGF-beta-mediated Smad phosphorylation and nuclear translocation remained unaffected. However, increased cAMP levels abolished TGF-beta-induced interaction of Smad3 with its transcriptional co-activator cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300. Overexpression of the transcriptional co-activator CBP/p300 rescued Smad-specific gene transcription in the presence of cAMP suggesting that sequestration of limited amounts of CBP/p300 by the activated cAMP/CREB pathway is the molecular basis of this inhibitory effect. These findings were extended by two functional assays. Increased intracellular cAMP levels suppressed the inductive activity of TGF-beta to contract mechanically unloaded collagen lattices and resulted in an attenuation of fibroblast migration of mechanically induced cell layer wounds. Of note, cAMP and TGF-beta synergistically induced hyaluronan synthase 2 (HAS2) expression and hyaluronan secretion, presumably via putative CREB-binding sites adjacent to Smad-binding sites within the HAS2 promoter. Our findings identify the cAMP pathway as a potent but differential and promoter-specific regulator of TGF-beta-mediated effects involved in extracellular matrix homeostasis.
Highlights
Cellular signaling from the TGF- family of growth factors is initiated by binding of the ligands to transmembrane receptor serine/threonine kinases, TRI and TRII
The ability of forskolin to activate cAMP-dependent gene transactivation in HDF was verified in experiments using pCRE-luc, a reporter construct consisting of three cAMP-response element (CRE) cloned upstream of the SV40 promoter, driving the expression of the luciferase gene
Forskolin efficiently transactivated the CRE-dependent construct, 12–16-fold above unstimulated control values (Fig. 1B). These results demonstrate the functionality of the cAMP-protein kinase A (PKA)-cAMP-response element-binding protein (CREB) cascade in HDF and warrant further investigation into the interference of cAMPelevating agents with TGF--mediated signaling in our experimental system
Summary
Cell Culture and Biochemical Reagents—Human dermal fibroblasts (HDF) from neonatal foreskin were purchased from Cell Systems, St. HDF were seeded into 12-well tissue culture plates at a density of 250,000 cells per well. The repeated nature of this procedure resulted in a cell-free gap of ϳ0.8 mm between two adjoining areas of fibroblasts at 90 –95% confluence (see corresponding figures). At this point, cell culture medium was replaced two times with fresh medium to reduce the number of cells introduced into suspension reattaching to the cell-free zone during experimentation. Identification of CREB and Smad transcription factorbinding sites in the sets of sequences was performed by using the MatInspector module of the Genomatix data base, which employed the Matrix Family Library version 7.1 (June 2008) [25]. All analyses were conducted with high threshold values (core similarity 1.0, matrix similarity Ͼ 0.9)
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