Fibulin-5 (FBLN5), an extracellular matrix glycoprotein required for normal elastogenesis, is coordinately expressed with elastin during lung injury and repair. We found that treatment with transforming growth factor- (TGF-) induced a rapid but transient increase in FBLN5 heterogeneous nuclear RNA (hnRNA) followed by a sustained increased in the steady-state level of FBLN5 mRNA. The transcription start site of the human FBLN5 gene was localized at 221 nucleotides upstream of the translation start site using primer extension, Northern blots and functional analysis of transcriptional activity in reporter plasmids containing 5'-flanking regions. TGF- markedly increased FBLN5 promoter activity in transient transfection assays. Two putative Smad-binding sites were identified within the proximal promoter and are required for this TGF- induction. Electrophoretic gel mobility shift assay revealed that TGF- strongly increased binding of Smad2 and Smad3 nuclear complexes to the proximal FBLN5 promoter and induced a Smad2/3 dependent binding of slow migrating nuclear protein complex. FBLN5 mRNA induction by TGF- was blocked by pretreatment with TGF- receptor inhibitor SB-431542, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 and actinomycin D. Basal and TGF--induced FBLN5 hnRNA and mRNA were strongly and proportionally decreased by LY294002, as was TGF--induced phosphorylation of Akt, but not Smad3 as measured by Western analysis. In addition, LY294002 markedly and proportionally decreased FBLN5 promoter activity in transient transfection analyses with TGF--treated or untreated lung fibroblasts. These studies demonstrate that induction of FBLN5 gene expression in lung fibroblasts is mediated via canonical TGF-/Smads signaling and requires the PI3-kinase/Akt pathway.