title: Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells.
abstract: OBJECTIVE
  Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine-induced cytotoxicity and ECM degradation.
  
  
  METHODS
  Cytotoxicity and cell death were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT-PCR and western blot analysis.
  
  
  RESULTS
  Treatment with nicotine reduced cell viability and increased the proportion of annexin V-negative, propidium iodide-positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α and glucose-regulated protein-78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP-1, MMP-2, MMP-8 and MMP-9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine-induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor-κB.
  
  
  CONCLUSIONS
  These results indicate that nicotine-induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.
authors.affiliation: Department of Maxillofacial Tissue Regeneration, School of Dentistry and Institute of Oral Biology, Kyung Hee University, Seoul, Korea.
authors.forename: S-I
authors.initials: SI
authors.lastname: Lee
authors.forename: K-L
authors.initials: KL
authors.lastname: Kang
authors.forename: S-I
authors.initials: SI
authors.lastname: Shin
authors.forename: Y
authors.initials: Y
authors.lastname: Herr
authors.forename: Y-M
authors.initials: YM
authors.lastname: Lee
authors.forename: E-C
authors.initials: EC
authors.lastname: Kim
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D022762:CCAAT-Enhancer-Binding Proteins / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002934:Cinnamates / Q000494:pharmacology; D024042:Collagen Type I / Q000187:drug effects; D004549:Elastin / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D015852:Eukaryotic Initiation Factor-2 / Q000037:antagonists & inhibitors / Q000187:drug effects; D005109:Extracellular Matrix / Q000187:drug effects* / Q000201:enzymology; D005353:Fibronectins / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020781:Matrix Metalloproteinase 1 / Q000187:drug effects; D020778:Matrix Metalloproteinase 2 / Q000187:drug effects; D020784:Matrix Metalloproteinase 8 / Q000187:drug effects; D020780:Matrix Metalloproteinase 9 / Q000187:drug effects; D020782:Matrix Metalloproteinases / Q000187:drug effects; D016328:NF-kappa B / Q000187:drug effects; D009538:Nicotine / Q000037:antagonists & inhibitors / Q000633:toxicity*; D010513:Periodontal Ligament / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D011494:Protein Kinases / Q000032:analysis; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D051742:Transcription Factor CHOP / Q000187:drug effects / Q000235:genetics
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D002934:Cinnamates; D024042:Collagen Type I; C497249:DDIT3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D015852:Eukaryotic Initiation Factor-2; D005353:Fibronectins; D006360:Heat-Shock Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; C496827:salubrinal; D051742:Transcription Factor CHOP; D009538:Nicotine; D004549:Elastin; D011494:Protein Kinases; D051057:Proto-Oncogene Proteins c-akt; D020782:Matrix Metalloproteinases; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C488762:MMP8 protein, human; D020784:Matrix Metalloproteinase 8; D020780:Matrix Metalloproteinase 9; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1; D013890:Thiourea