title: Up-regulation of retinoblastoma protein phosphorylation in gingiva after cyclosporine A treatment: an in vivo and in vitro study.
abstract: OBJECTIVE
  Cyclosporine A can induce gingival cell proliferation; however, the precise molecular regulation of the proliferation is uncertain. Therefore, this study was carried out to examine, in vivo and in vitro, the expression of genes and proteins associated with gingival cell proliferation after treatment with cyclosporine A.
  
  
  METHODS
  Forty Sprague Dawley rats with right maxillary posterior edentulous gingivae were assigned to a cyclosporine A group (30 mg/kg daily of cyclosporine A, administered orally) or a control group (administered mineral oil only). The animals were killed 4 wk after treatment. The edentulous gingivae were dissected out and analyzed for the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb1) mRNA and/or protein, and phosphorylated Rb1 (pRb1), by real-time RT-PCR or immunohistochemistry. In human gingival fibroblast (HGF) cultures, the expression of PCNA, CDK4, cyclin D1 and Rb1 proteins and Rb1 phosphorylation were determined by western blotting after cyclosporine A treatment (0-10(4) ng/mL).
  
  
  RESULTS
  Proliferating cell nuclear antigen and cyclin D1 mRNAs (Pcna and Ccnd1, respectively) were expressed more strongly in the gingivae of cyclosporine A-treated animals than in the gingivae of the controls. Immunohistochemical analyses showed that a greater number of gingival cells stained positive for cyclin D1, CDK4 and pRb1 in the cyclosporine A group than in the control group. Increased expression of cyclin D1, CDK4 and PCNA proteins was observed in HGFs after cyclosporine A treatment. The phosphorylation of Rb1 was enhanced in HGFs after treatment with cyclosporine A at concentrations of 10(2)-10(3) ng/mL.
  
  
  CONCLUSIONS
  The increases in cyclin D1, PCNA and CDK4, together with the enhanced phosphorylation of Rb1, suggest that cyclosporine A promotes cell-cycle progression through the G(1)/S transition in the gingiva.
authors.affiliation: Department of Periodontology, School of Dentistry, National Defense Medical Center and Tri-Service General Hospital, Taipei, Taiwan.
authors.forename: C-Y
authors.initials: CY
authors.lastname: Chiang
authors.forename: H-P
authors.initials: HP
authors.lastname: Tu
authors.forename: Y-T
authors.initials: YT
authors.lastname: Chen
authors.forename: Y-T
authors.initials: YT
authors.lastname: Chin
authors.forename: T-M
authors.initials: TM
authors.lastname: Lai
authors.forename: H-C
authors.initials: HC
authors.lastname: Chiu
authors.forename: S
authors.initials: S
authors.lastname: Nieh
authors.forename: E
authors.initials: E
authors.lastname: Fu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000032:analysis / Q000187:drug effects; D051358:Cyclin-Dependent Kinase 4 / Q000032:analysis / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology*; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D016160:Retinoblastoma Protein / Q000032:analysis / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: C530105:Ccnd1 protein, mouse; D007166:Immunosuppressive Agents; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D016572:Cyclosporine; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4