title: CREB Cooperates with BMP-stimulated Smad signaling to enhance transcription of the Smad6 promoter.
abstract: Growth plate chondrocytes integrate a multitude of growth factor signals during maturation. PTHrP inhibits maturation through stimulation of PKA/CREB signaling while the bone morphogenetic proteins (BMPs) stimulate maturation through Smad mediated signaling. In this manuscript, we show that interactions between CREB and the BMP associated Smads are promoter specific, and demonstrate for the first time the requirement of CREB signaling for Smad mediated activation of a BMP responsive region of the Smad6 promoter. The 28 base pairs (bp) BMP responsive element of the Smad6 promoter contains an 11 bp Smad binding region and an adjacent 17 bp region in which we characterize a putative CRE site. PKA/CREB gain of function enhanced BMP stimulation of this reporter, while loss of CREB function diminished transcriptional activity. In contrast, ATF-2 and AP-1 transcription factors had minimal effects. Electrophoretic mobility shift assay (EMSA) confirmed CREB binding to the Smad6 promoter element. Mutations eliminating binding resulted in loss of transcriptional activity, while mutations that maintained CREB binding had continued reporter activation by CREB and BMP-2. The Smad6 gene was similarly regulated by CREB. Dominant negative CREB reduced BMP-2 stimulated Smad6 gene transcription by 50%, but markedly increased BMP-2 mediated stimulation of colX and Ihh expression. In contrast, PTHrP which activates CREB signaling, blocked the stimulatory effect of BMP-2 on colX and Ihh, but minimally inhibited the stimulatory effect of BMP on Smad6. These findings are the first to demonstrate a cooperative association between CREB and BMP regulated Smads in cells from vertebrates and demonstrate that promoter-specific rather than generalized interactions between PKA/CREB and BMP signaling regulate gene expression in chondrocytes.
authors.affiliation: Center for Musculoskeletal Research, University of Rochester, School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Andreia M
authors.initials: AM
authors.lastname: Ionescu
authors.forename: Hicham
authors.initials: H
authors.lastname: Drissi
authors.forename: Edward M
authors.initials: EM
authors.lastname: Schwarz
authors.forename: Mitsuyasu
authors.initials: M
authors.lastname: Kato
authors.forename: J Edward
authors.initials: JE
authors.lastname: Puzas
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: McCance
authors.forename: Randy N
authors.initials: RN
authors.lastname: Rosier
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Zuscik
authors.forename: Regis J
authors.initials: RJ
authors.lastname: O'Keefe
mesh_terms: D000818:Animals; D001483:Base Sequence; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D024121:Collagen Type X / Q000187:drug effects / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D053823:Hedgehog Proteins; D008969:Molecular Sequence Data; D044162:Parathyroid Hormone-Related Protein / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051905:Smad6 Protein; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*; D016212:Transforming Growth Factor beta
chemical_list: C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024121:Collagen Type X; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D053823:Hedgehog Proteins; D044162:Parathyroid Hormone-Related Protein; D051905:Smad6 Protein; C497804:Smad6 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases