Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes.
We previously isolated a rheumatoid arthritis-related antigen (RA-A47) protein that had reactivity with RA sera from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8. Sequencing analysis of ra-a47 cDNA revealed RA-A47 as a product of the colligin-2 gene, which is also known as the human heat shock protein (HSP) 47 gene. Expression of hsp47 has been shown to be cooperatively altered with that of collagen genes upon stimulation. In this study, it was confirmed that the mRNA expression of ra-a47 and COL2A1, a type II collagen gene, was upregulated on stimulation with transforming growth factor (TGF) beta in chondrocytes. However, in contrast, inflammatory cytokines such as tumor necrosis factor (TNF) alpha, interferon (IFN) beta, and interleukin (IL)-6 downregulated the expression of ra-a47 mRNA, whereas the expression of COL2A1 mRNA was not repressed, or even upregulated, in HCS-2/8 cells. Of note, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP)-9 mRNAs were strongly stimulated by TNFalpha. We also found that cell-surface type II collagen disappeared upon such a stimulation, suggesting that decrement of RA-A47 may inhibit the secretion of type II collagen and lead to its accumulation inside the cells. RA-A47 was detected in the cultured medium of TNFalpha-treated HCS-2/8 cells and of IL-1-treated rabbit chondrocytes by Western blot analysis. Under the same conditions, RA-A47 was detected on the cell surface by immunofluorescence staining. These findings demonstrate that the RA-A47 chaperone protein is specifically downregulated, causing the intracellular accumulation of unsecretable type II collagen, while the extracellular matrix (ECM) is degraded by MMPs and iNOS through the stimulation of chondrocytes by TNFalpha. The altered localization of RA-A47 to the surface or outside of cells may represent the mechanism for the recognition of RA-A47 as an autoantigen during rheumatoid arthritis.
Hattori
T
T
Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Okayama, Japan.
Kubota
S
S
Yutani
Y
Y
Fujisawa
T
T
Nakanishi
T
T
Takahashi
K
K
Takigawa
M
M
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000502:physiology*; D003094:Collagen / Q000235:genetics; D016207:Cytokines / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D006023:Glycoproteins; D050958:HSP47 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000032:analysis / Q000235:genetics*; D006801:Humans; D016899:Interferon-beta / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D007700:Kinetics; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002352:Carrier Proteins; D016207:Cytokines; D006023:Glycoproteins; D050958:HSP47 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007375:Interleukin-1; D015850:Interleukin-6; D012333:RNA, Messenger; C494660:SERPINH1 protein, human; D014409:Tumor Necrosis Factor-alpha; C046289:colligin; D016899:Interferon-beta; D003094:Collagen