title: TGFβ regulates Galectin-3 expression through canonical Smad3 signaling pathway in nucleus pulposus cells: implications in intervertebral disc degeneration.
abstract: Galectin-3 is highly expressed in notochordal nucleus pulposus (NP) and thought to play important physiological roles; however, regulation of its expression remains largely unexplored. The aim of the study was to investigate if TGFβ regulates Galectin-3 expression in NP cells. TGFβ treatment resulted in decreased Galectin-3 expression. Bioinformatic analysis using JASPAR and MatInspector databases cross-referenced with published ChIP-Seq data showed nine locations of highly probable Smad3 binding in the LGALS3 proximal promoter. In NP cells, TGFβ treatment resulted in decreased activity of reporters harboring several 5' deletions of the proximal Galectin-3 promoter. While transfection of NP cells with constitutively active (CA)-ALK5 resulted in decreased promoter activity, DN-ALK5 blocked the suppressive effect of TGFβ on the promoter. The suppressive effect of Smad3 on the Galectin-3 promoter was confirmed using gain- and loss-of-function studies. Transfection with DN-Smad3 or Smad7 blocked TGFβ mediated suppression of promoter activity. We also measured Galectin-3 promoter activity in Smad3 null and wild type cells. Noteworthy, promoter activity was suppressed by TGFβ only in wild type cells. Likewise, stable silencing of Smad3 in NP cells using sh-Smad3 significantly blocked TGFβ-dependent decrease in Galectin-3 expression. Treatment of human NP cells isolated from tissues with different grades of degeneration showed that Galectin-3 expression was responsive to TGF-β-mediated suppression. Importantly, Galectin-3 synergized effects of TNF-α on inflammatory gene expression by NP cells. Together these studies suggest that TGFβ, through Smad3 controls Galectin-3 expression in NP cells and may have implications in the intervertebral disc degeneration.
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, U.S.A.; Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, P.R. China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, P.R. China.
authors.forename: Wen
authors.initials: W
authors.lastname: Yuan
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, U.S.A.; Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, P.R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, U.S.A.; Department of Orthopaedics, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, P.R. China.
authors.forename: Hua
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, U.S.A.
authors.forename: Maxwell G
authors.initials: MG
authors.lastname: Hunt
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, U.S.A.; Department of Orthopaedic Surgery, Shaghai First People's Hospital, Shanghai, P.R. China.
authors.forename: Chao
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, U.S.A.
authors.forename: Irving M
authors.initials: IM
authors.lastname: Shapiro
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, U.S.A.. Electronic address: makarand.risbud@jefferson.edu.
authors.forename: Makarand V
authors.initials: MV
authors.lastname: Risbud
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D037502:Galectin 3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D055959:Intervertebral Disc Degeneration / Q000235:genetics; D000070614:Nucleus Pulposus / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D037502:Galectin 3; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha
keywords: Disc degeneration; Galectin-3; Intervertebral disc; Nucleus pulposus; Smad3; TGFβ