title: Cartilage intermediate layer protein (CILP) regulation in intervertebral discs. The effect of age, degeneration, and bone morphogenetic protein-2.
abstract: METHODS
  An in vitro study using rabbit intervertebral disc tissue and disc cells.
  
  
  OBJECTIVE
  To evaluate the effects of disc degeneration, age, and bone morphogenetic proteins-2 (BMP-2) on cartilage intermediate layer protein (CILP) expression and elucidate the molecular mechanism by which BMP-2 regulates CILP expression.
  
  
  BACKGROUND
  CILP is implicated in several diseases that affect cartilage. The CILP polymorphism acts as a modulator of lumbar disc disease susceptibility. However, regulation of the CILP gene in disc tissue remains poorly understood.
  
  
  METHODS
  Intact discs from young rabbits were punctured to induce disc degeneration. These young rabbits and other older rabbits were used to measure the expression of CILP, proteoglycan, and collagen II using Western blot and real-time PCR. Primary disc cells from the rabbits were treated with rhBMP-2, or siRNAs, and the gene expression was analyzed by Western blot and real-time PCR. The activity of the CILP promoter was measured by using the Dual Luciferase Reporter Assay System.
  
  
  RESULTS
  Our study demonstrates that the intervertebral disc expresses significant levels of CILP and that the expression of CILP increases substantially with increasing age and disc degeneration. In contrast, the expression of proteoglycan and collagen II decrease with increasing age and disc degeneration. BMP-2 induces the expression of CILP protein and stimulates the activity of the CILP promoter in rabbit primary disc cells. The induction of CILP by BMP-2 can be augmented with age. Knockdown of Smad1 by siRNA abolishes the stimulatory effects of BMP-2 on CILP expression in the primary disc cells.
  
  
  CONCLUSIONS
  Our data demonstrate that disc degeneration, age, and BMP-2 are regulators of the CILP gene. BMP-2 induces CILP expression by activating the Smad1 signal pathway.
authors.affiliation: Emory Spine Center, Emory University School of Medicine and VA Medical Center, Atlanta, GA, USA. zwang2@emory.edu
authors.forename: Zili
authors.initials: Z
authors.lastname: Wang
authors.forename: Jin Hwan
authors.initials: JH
authors.lastname: Kim
authors.forename: Kosaku
authors.initials: K
authors.lastname: Higashino
authors.forename: Sung Soo
authors.initials: SS
authors.lastname: Kim
authors.forename: Susheng
authors.initials: S
authors.lastname: Wang
authors.forename: Shoji
authors.initials: S
authors.lastname: Seki
authors.forename: William C
authors.initials: WC
authors.lastname: Hutton
authors.forename: S Tim
authors.initials: ST
authors.lastname: Yoon
mesh_terms: D000367:Age Factors; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000502:physiology*; D002478:Cells, Cultured; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D020868:Gene Silencing; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D007403:Intervertebral Disc / Q000187:drug effects / Q000378:metabolism*; D055959:Intervertebral Disc Degeneration / Q000235:genetics*; D064235:Matrilin Proteins; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011817:Rabbits; D051898:Smad1 Protein / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D055396:Bone Morphogenetic Protein 2; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D064235:Matrilin Proteins; D011509:Proteoglycans; D034741:RNA, Small Interfering; D051898:Smad1 Protein