A novel tumor necrosis factor alpha-responsive CCAAT/enhancer binding protein site regulates expression of the cartilage-derived retinoic acid-sensitive protein gene in cartilage.
OBJECTIVE
Inflammatory processes in rheumatoid arthritis are primarily regulated by the cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta). Previous studies in our laboratory have shown that IL-1beta represses expression of the cartilage characteristic genes, cartilage-derived retinoic acid-sensitive protein (cd-rap) and type II collagen (COL2A1); this mechanism of repression involves activation of a CCAAT/enhancer binding protein (c/EBP) site within promoter regions. The aim of this study was to investigate novel TNFalpha-mediated mechanisms that regulate the expression of cd-rap.


METHODS
Rat chondrosarcoma cells were transiently transfected with complementary DNA constructs encoding cd-rap, in the presence of TNFalpha. The expression of c/EBPbeta, SOX9, and p300 in rat chondrosarcoma cells and primary human articular chondrocytes after treatment with TNFalpha was examined by reverse transcription-polymerase chain reaction and Western blotting. The effect of TNFalpha on endogenous binding of c/EBPbeta or SOX9 to the cd-rap promoter was examined by chromatin immunoprecipitation assays.


RESULTS
We identified a new c/EBP binding site in the cd-rap promoter (from position -1059 bp to position -1046 bp). Binding of c/EBP to this site was regulated by TNFalpha but not IL-1beta, resulting in down-regulation of cd-rap expression. This effect was reversed by mutational inactivation of the c/EBP motif. In addition, the activation potential of SOX9 and CREB binding protein/p300 on the cd-rap promoter was enhanced after mutation of the new c/EBP binding site, indicating that blockage of this site would increase transcription.


CONCLUSIONS
TNFalpha regulates the expression and/or DNA-binding potential of key positive-acting and negative-acting transcription factors that control the expression of the cartilage matrix gene, cd-rap.
Imamura
Toshihiro
T
Washington University, School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110, USA.
Imamura
Chisako
C
McAlinden
Audrey
A
Davies
Sherri R
SR
Iwamoto
Yukihide
Y
Sandell
Linda J
LJ
D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000502:physiology*; D002358:Cartilage, Articular / Q000166:cytology; D002478:Cells, Cultured; D002813:Chondrosarcoma; D005786:Gene Expression Regulation; D006801:Humans; D011506:Proteins / Q000235:genetics*; D051381:Rats
D022763:CCAAT-Enhancer-Binding Protein-alpha; C099322:Mia protein, rat; D011506:Proteins