The human α11 integrin promoter drives fibroblast-restricted expression in vivo and is regulated by TGF-β1 in a Smad- and Sp1-dependent manner

Integrin alpha 11 beta 1 is expressed by ectomesenchymally- and mesodermally-derived fibroblasts and is the major collagen receptor on embryonic fibroblasts. We have previously characterized a 3 kb human alpha 11 promoter region in vitro. In the current study we generated promoter-LacZ reporter transgenic mice to examine the ability of the 3 alpha 11 promoter to drive tissue-specific expression also in vivo. Our data show that the 3 kb alpha 11 promoter contains most of the regulatory elements that direct ectomesenchymal and mesodermal fibroblast-specific expression. Not much is known about integrin alpha 11 regulation by TGF-beta family members and the potential role of alpha 11 in TGF-beta 1 driven processes such as fibrosis and wound contraction. In the current study we show that TGF-beta 1 induces alpha 11 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 alpha 11 promoter-luciferase reporter constructs demonstrated that TGF-beta 1 responsive elements are located within the 3 kb alpha 11 promoter. Serial deletions located TGF-beta 1 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-beta 1-dependent alpha 11 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 Spl -binding site SBS1 (nt -140/-134) takes part in the responsiveness to TGF-beta 1 in a Smad2-dependent manner. In summary, our data confirm that 3 kb of the alpha 11 promoter is efficient in driving tissue-specific expression in vivo. We also demonstrate that this promoter confers TGF-beta 1 responsiveness which appears to rely on both a Smad-binding element at nt -182/-176 and a Spl -binding site at nt -140/-134. Our data furthermore indicate that additional elements needed for TGF-beta 1 responsiveness are located upstream in the -2962/-330 promoter region. (C) 2009 Elsevier B.V. All rights reserved.