Fibulin-5 gene expression in human lung fibroblasts is regulated by TGF-beta and phosphatidylinositol 3-kinase activity.
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-beta (TGF-beta) 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 by using primer extension, Northern blots, and functional analysis of transcriptional activity in reporter plasmids containing 5'-flanking regions. TGF-beta 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-beta induction. Electrophoretic gel mobility shift assay revealed that TGF-beta 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-beta was blocked by pretreatment with TGF-beta receptor inhibitor SB-431542, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY-294002, and actinomycin D. Basal and TGF-beta-induced FBLN5 hnRNA and mRNA were strongly and proportionally decreased by LY-294002, as was TGF-beta-induced phosphorylation of Akt, but not Smad3, as measured by Western blot analysis. In addition, LY-294002 markedly and proportionally decreased FBLN5 promoter activity in transient transfection analyses with TGF-beta-treated or untreated lung fibroblasts. These studies demonstrate that induction of FBLN5 gene expression in lung fibroblasts is mediated via canonical TGF-beta/Smad signaling and requires the PI3-kinase/Akt pathway.
Kuang
Ping-Ping
PP
The Pulmonary Center and Department of Biochemistry, Boston University School of Medicine, Boston Veteran Administration Medical Center, Boston, Massachusetts 02118, USA. pkuang@lung.bumc.bu.edu
Joyce-Brady
Martin
M
Zhang
Xiao-Hui
XH
Jean
Jyh-Chang
JC
Goldstein
Ronald H
RH
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008168:Lung / Q000166:cytology*; D051379:Mice; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051785:Smad Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; C492373:FBLN5 protein, human; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D012313:RNA; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt