title: Retinoblastoma suppression of matrix metalloproteinase 1, but not interleukin-6, through a p38-dependent pathway in rheumatoid arthritis synovial fibroblasts.
abstract: OBJECTIVE
  Rheumatoid arthritis (RA) is characterized by increased synovial lining cellularity, inflammation, and destruction of cartilage and bone. During the pathogenesis of RA, synovial fibroblasts reenter the cell cycle and multiply in number. RA synovial fibroblasts express high levels of the MAP kinase p38, which may contribute to the production of interleukin-6 (IL-6) and matrix metalloproteinases (MMPs). IL-6 and MMP-1 promote inflammation and joint destruction, respectively. Taken together, these findings indicate that in RA the enhanced cell cycle activity and production of IL-6 and MMP-1 may be linked. Therefore, we sought to determine if the tumor suppressor gene product retinoblastoma (Rb), a negative regulator of cell cycle activity, inhibits IL-6, MMP-1, and p38 in RA synovial fibroblasts.
  
  
  METHODS
  RA and non-RA synovial fibroblasts were examined by enzyme-linked immunosorbent assay (ELISA) for the relative expression of inactive hyperphosphorylated Rb (inactive Rb/total Rb). Ectopic Rb expression was mediated by infection with a replication-defective adenovirus that expresses Rb (Ad-Rb). A control replication-defective adenovirus that expresses beta-galactosidase (Ad-beta-gal) was used. Cell cycle activity was determined by flow cytometry. IL-6 and MMP-1 expression was examined by real-time polymerase chain reaction and ELISA. Expression and activation of p38 were determined by kinase assays and ELISA. The activity of p38 was enhanced by infecting RA synovial fibroblasts with a replication-defective adenovirus that expresses a constitutively active form of MAPK kinase 3 (Ad-CA-MKK3), an upstream activator of p38.
  
  
  RESULTS
  Quiescent RA, compared with non-RA synovial fibroblasts, displayed a 200% (P < 0.02) increase in the inactive Rb isoform. Proliferating RA synovial fibroblasts exhibited a 60% (P < 0.12) increase in the inactive Rb isoform compared with non-RA synovial fibroblasts. Increased levels of the active Rb isoform inhibited cell cycle progression and suppressed IL-6 and MMP-1 secretion in RA synovial fibroblasts, although the steady-state levels of IL-6 and MMP-1 messenger RNA remained unchanged. However, Rb overexpression had no effect on spontaneous or IL-1beta-induced production of IL-6 or MMP-1 in non-RA synovial fibroblasts. Ectopic Rb expression reduced the activity of p38. Ad-CA-MKK3 infection in RA synovial fibroblasts increased p38 phosphorylation, and MMP-1 but not IL-6 secretion. In contrast, Rb overexpression inhibited Ad-CA-MKK3-mediated phosphorylation of p38 and subsequent increase in MMP-1.
  
  
  CONCLUSIONS
  Rb-mediated suppression of IL-6 and MMP-1 occurs at a posttranscriptional level. However, Ad-Rb reduction of MMP-1 but not IL-6 requires inhibition of the p38 pathway. These results suggest that Rb negatively regulates p38 activation, leading to decreased MMP-1 secretion in RA synovial fibroblasts.
authors.affiliation: Saint Louis University, School of Medicine, St. Louis, Missouri 63104, USA.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Bradley
authors.forename: John C
authors.initials: JC
authors.lastname: Scatizzi
authors.forename: Stefano
authors.initials: S
authors.lastname: Fiore
authors.forename: Eli
authors.initials: E
authors.lastname: Shamiyeh
authors.forename: Alisa E
authors.initials: AE
authors.lastname: Koch
authors.forename: Gary S
authors.initials: GS
authors.lastname: Firestein
authors.forename: Laura L
authors.initials: LL
authors.lastname: Gorges
authors.forename: Kevin
authors.initials: K
authors.lastname: Kuntsman
authors.forename: Richard M
authors.initials: RM
authors.lastname: Pope
authors.forename: Terry L
authors.initials: TL
authors.lastname: Moore
authors.forename: Jiahuai
authors.initials: J
authors.lastname: Han
authors.forename: Harris
authors.initials: H
authors.lastname: Perlman
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000503:physiopathology; D002453:Cell Cycle / Q000502:physiology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D013583:Synovial Membrane / Q000166:cytology; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D015850:Interleukin-6; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020781:Matrix Metalloproteinase 1