title: The human alpha11 integrin promoter drives fibroblast-restricted expression in vivo and is regulated by TGF-beta1 in a Smad- and Sp1-dependent manner.
abstract: Integrin alpha11beta1 is expressed by ectomesenchymally- and mesodermally-derived fibroblasts and is the major collagen receptor on embryonic fibroblasts. We have previously characterized a 3kb human alpha11 promoter region in vitro. In the current study we generated promoter-LacZ reporter transgenic mice to examine the ability of the 3kb alpha11 promoter to drive tissue-specific expression also in vivo. Our data show that the 3 kb alpha11 promoter contains most of the regulatory elements that direct ectomesenchymal and mesodermal fibroblast-specific expression. Not much is known about integrin alpha11 regulation by TGF-beta family members and the potential role of alpha11 in TGF-beta1 driven processes such as fibrosis and wound contraction. In the current study we show that TGF-beta1 induces alpha11 transcription in the fibrosarcoma cell line HT1080 as well as in primary fibroblasts. Co-transfection of an expression plasmid encoding constitutively active ALK5 together with alpha11 promoter-luciferase reporter constructs demonstrated that TGF-beta1 responsive elements are located within the 3kb alpha11 promoter. Serial deletions located TGF-beta1 responsiveness to the proximal promoter (nt -176/+25) as well as to the region extending to nt -330. Transfection and expression of the inhibitory Smad7 in the cells attenuated the TGF-beta1-dependent alpha11 induction both at the RNA and the protein level. Mutation and deletion analyses identified a Smad-binding element, SBE2 (nt -182/-176), as an important Smad3-binding site in this part of the promoter. Further analyses suggested that the Sp1-binding site SBS1 (nt -140/-134) takes part in the responsiveness to TGF-beta1 in a Smad2-dependent manner. In summary, our data confirm that 3kb of the alpha11 promoter is efficient in driving tissue-specific expression in vivo. We also demonstrate that this promoter confers TGF-beta1 responsiveness which appears to rely on both a Smad-binding element at nt -182/-176 and a Sp1-binding site at nt -140/-134. Our data furthermore indicate that additional elements needed for TGF-beta1 responsiveness are located upstream in the -2962/-330 promoter region.
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway.
authors.forename: Ning
authors.initials: N
authors.lastname: Lu
authors.forename: Sergio
authors.initials: S
authors.lastname: Carracedo
authors.forename: Jonna
authors.initials: J
authors.lastname: Ranta
authors.forename: Rainer
authors.initials: R
authors.lastname: Heuchel
authors.forename: Raija
authors.initials: R
authors.lastname: Soininen
authors.forename: Donald
authors.initials: D
authors.lastname: Gullberg
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005347:Fibroblasts; D006801:Humans; D039001:Integrin alpha Chains / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051785:Smad Proteins / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000502:physiology*
chemical_list: C121535:ITGA11 protein, human; D039001:Integrin alpha Chains; D051785:Smad Proteins; D016329:Sp1 Transcription Factor; D053773:Transforming Growth Factor beta1; D012313:RNA