Transforming growth factor-β up-regulates the β₅ integrin subunit expression via Sp1 and Smad signaling

Integrin-mediated cell-matrix interactions play important roles in regulating cell function. Since transforming growth factor-β (TGF-β) modulates many osteoblast activities, we hypothesized that the growth factor acts in part by modulating integrin expression. TGF-β increased cell adhesion to vitronectin and up-regulated the surface level of α(v)β₅ via increasing β₅ protein synthesis by a transcriptional mechanism. Promoter activity analysis demonstrated that a TGF-β-responsive element resides between nucleotides -63 and -44. Electrophoretic mobility shift assay and immunoprecipitation/Western studies indicated that the nuclear complex formed using the -66/-42 oligonucleotide contained both Sp1/Sp3 and Smad proteins. Since nuclear Sp1/Sp3 levels were not altered, whereas Smad levels were increased by TGF-β, we investigated the roles of Smad proteins in the up-regulation of β₅ gene activation. Co-transfection of cells with β₅ promoter reporter construct and expression vectors for Smad3, Smad4, and Sp1 increased the stimulatory effect of TGF-β. Furthermore, expression of dominant negative Smad3 or Smad4 in cells decreased or abolished the stimulation of β5 promoter activity by TGF-β. Smad4 mutant also inhibited the up-regulation of surface β₅ level by TGF-β. Thus, TGF-β increases expression of the integrin β₅ gene by mechanisms involving Sp1/Sp3 and Smad transcription factors.