title: Inhibition of proteoglycan and type II collagen synthesis of disc nucleus cells by nicotine.
abstract: OBJECTIVE
  Systemic nicotine has been hypothesized to cause degeneration of the intervertebral disc which in turn decreases vascular supply to the disc through a cholinergic receptor-mediated process. Another possible mechanism may be through direct regulatory effects on disc cells. In this study, the authors tested the hypothesis that nicotine adversely affects nucleus pulposus cells by directly inhibiting proteoglycan synthesis and gene expression of type II collagen (Phase I study). They also assessed the hypothesis that nicotine inhibits the bone morphogenetic protein (BMP)-2-induced upregulation of extracellular matrix (Phase II study).
  
  
  METHODS
  Cells were isolated from nucleus pulposus obtained in rat lumbar discs and cultured on a monolayer. Media were treated with nicotine and/or recombinant human (rh)BMP-2 for 7 days. Sulfated glycosaminoglycan (SO4-GAG) in media was quantified using 1,9-dimethylmethylene blue (DMMB) assay. Gene assay of types I and II collagen, Sox9, and glyceraldehyde-3-phosphate dehydrogenase were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) and real time PCR. In the Phase I study, nicotine-treated (100 microg/ml) and non-treated cells were compared. The s-GAG production and messenger RNA (mRNA) of type II collagen and Sox9 decreased significantly in the nicotine-treated group. In the Phase II study, five groups were compared: 1) non-treatment; 2) rhBMP-2 only (100 ng/ml); and 3-5) with rhBMP-2 (100 ng/ml) and increasing doses of nicotine (1 [third group], 10, [fourth group], 100 [fifth group] microg/ml). The SO4-GAG production and mRNA of type II collagen and Sox9 decreased significantly in the groups treated with rhBMP-2 combined with 10 and 100 microg/ml of nicotine compared with the group treated with rhBMP-2.
  
  
  CONCLUSIONS
  The results of this study raise the possibility that nicotine may contribute to the process of disc degeneration by a direct effect on the nucleus pulposus cells, possibly by antagonizing the effect of BMP-2.
authors.affiliation: Department of Neurosurgery, Chonbuk National University Medical School, Institute for Medical Science, Chonju, Korea.
authors.forename: Keun Su
authors.initials: KS
authors.lastname: Kim
authors.forename: S Tim
authors.initials: ST
authors.lastname: Yoon
authors.forename: Jin Soo
authors.initials: JS
authors.lastname: Park
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Moon Soo
authors.initials: MS
authors.lastname: Park
authors.forename: William C
authors.initials: WC
authors.lastname: Hutton
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D024043:Collagen Type II / Q000096:biosynthesis*; D003470:Culture Media; D005731:Ganglionic Stimulants / Q000494:pharmacology* / Q000633:toxicity; D005786:Gene Expression Regulation / Q000187:drug effects*; D007403:Intervertebral Disc / Q000166:cytology* / Q000502:physiology*; D008159:Lumbar Vertebrae; D008297:Male; D009538:Nicotine / Q000494:pharmacology* / Q000633:toxicity; D011509:Proteoglycans / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D024043:Collagen Type II; D003470:Culture Media; D005731:Ganglionic Stimulants; D011509:Proteoglycans; D012333:RNA, Messenger; D009538:Nicotine