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 α11β1 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 α11 promoter region in vitro. In the current study we generated promoter-LacZ reporter transgenic mice to examine the ability of the 3 kb α11 promoter to drive tissue-specific expression also in vivo. Our data show that the 3 kb α11 promoter contains most of the regulatory elements that direct ectomesenchymal and mesodermal fibroblast-specific expression.Not much is known about integrin α11 regulation by TGF-β family members and the potential role of α11 in TGF-β1 driven processes such as fibrosis and wound contraction. In the current study we show that TGF-β1 induces α11 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 α11 promoter-luciferase reporter constructs demonstrated that TGF-β1 responsive elements are located within the 3 kb α11 promoter. Serial deletions located TGF-β1 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-β1-dependent α11 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-β1 in a Smad2-dependent manner.In summary, our data confirm that 3 kb of the α11 promoter is efficient in driving tissue-specific expression in vivo. We also demonstrate that this promoter confers TGF-β1 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-β1 responsiveness are located upstream in the −2962/−330 promoter region.