Regulation of Sox9 activity by crosstalk with nuclear factor-kappaB and retinoic acid receptors.
BACKGROUND
Sox9 and p300 cooperate to induce expression of cartilage-specific matrix proteins, including type II collagen, aggrecan and link protein. Tumour necrosis factor (TNF)-alpha, found in arthritic joints, activates nuclear factor-kappaB (NF-kappaB), whereas retinoic acid receptors (RARs) are activated by retinoid agonists, including all-trans retinoic acid (atRA). Like Sox9, the activity of NF-kappaB and RARs depends upon their association with p300. Separately, both TNF-alpha and atRA suppress cartilage matrix gene expression. We investigated how TNF-alpha and atRA alter the expression of cartilage matrix genes.


METHODS
Primary cultures of rat chondrocytes were treated with TNF-alpha and/or atRA for 24 hours. Levels of transcripts encoding cartilage matrix proteins were determined by Northern blot analyses and quantitative real-time PCR. Nuclear protein levels, DNA binding and functional activity of transcription factors were assessed by immunoblotting, electrophoretic mobility shift assays and reporter assays, respectively.


RESULTS
Together, TNF-alpha and atRA diminished transcript levels of cartilage matrix proteins and Sox9 activity more than each factor alone. However, neither agent altered nuclear levels of Sox9, and TNF-alpha did not affect protein binding to the Col2a1 48-base-pair minimal enhancer sequence. The effect of TNF-alpha, but not that of atRA, on Sox9 activity was dependent on NF-kappaB activation. Furthermore, atRA reduced NF-kappaB activity and DNA binding. To address the role of p300, we over-expressed constitutively active mitogen-activated protein kinase kinase kinase (caMEKK)1 to increase p300 acetylase activity. caMEKK1 enhanced basal NF-kappaB activity and atRA-induced RAR activity. Over-expression of caMEKK1 also enhanced basal Sox9 activity and suppressed the inhibitory effects of TNF-alpha and atRA on Sox9 function. In addition, over-expression of p300 restored Sox9 activity suppressed by TNF-alpha and atRA to normal levels.


CONCLUSIONS
NF-kappaB and RARs converge to reduce Sox9 activity and cartilage matrix gene expression, probably by limiting the availability of p300. This process may be critical for the loss of cartilage matrix synthesis in inflammatory joint diseases. Therefore, agents that increase p300 levels or activity in chondrocytes may be useful therapeutically.
Rockel
Jason S
JS
Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, Department of Anatomy and Cell Biology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada. jeff.dixon@schulich.uwo.ca
Kudirka
Julie C
JC
Guzi
Andrew J
AJ
Bernier
Suzanne M
SM
D000818:Animals; D002356:Cartilage / Q000378:metabolism; D002478:Cells, Cultured; D024043:Collagen Type II / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D050881:E1A-Associated p300 Protein / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006609:High Mobility Group Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048728:MAP Kinase Kinase Kinase 1 / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D020239:Receptor Cross-Talk / Q000502:physiology*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C511513:COL2A1 protein, rat; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D006609:High Mobility Group Proteins; D046912:Multiprotein Complexes; D016328:NF-kappa B; D018168:Receptors, Retinoic Acid; D055758:SOX9 Transcription Factor; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D014212:Tretinoin; D004247:DNA; D050881:E1A-Associated p300 Protein; C494362:Ep300 protein, rat; D048728:MAP Kinase Kinase Kinase 1