Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2alpha A) is inhibited by tumor necrosis factor-alpha.
The transcription factor RUNX2 (Cbfa1/AML3/Pebp2alphaA) is a critical regulator of osteoblast differentiation. We investigated the effect of the inflammatory cytokine tumor necrosis factor alpha (TNF) on the expression of RUNX2 because TNF is known to inhibit differentiation of osteoblasts from pluripotent progenitor cells. TNF treatment of fetal calvaria precursor cells or MC3T3-E1 clonal pre-osteoblastic cells caused a dose-dependent suppression of RUNX2 steady state mRNA as measured by reverse transcription-PCR. The IC(50) for TNF inhibition was 0.6 ng/ml. TNF suppression of RUNX2 mRNA was confirmed using Northern analysis. The effect of TNF was studied using isoform-specific primers that flanked unique regions of two major RUNX2 isoforms. TNF suppressed expression of the mRNA coding for the shorter MRIPV isoform by >90% while inhibiting expression of the mRNA for the longer MASNS isoform by 50%. RUNX2 nuclear content was evaluated by electrophoretic mobility shift assay using a rat osteocalcin promoter binding sequence as probe and by Western analysis. TNF reduced nuclear RUNX2 protein. Inhibition of new protein synthesis with cycloheximide failed to prevent TNF inhibition of RUNX2 mRNA, suggesting that a newly translated protein did not mediate the TNF effect. RUNX2 mRNA half-life was 1.8 h and reduced to 0.9 h by TNF. The effect of TNF on RUNX2 gene transcription was evaluated using a 0.6-kb RUNX2 promoter-luciferase reporter in MC3T3-E1 cells. TNF caused a dose-dependent inhibition of transcription to 50% of control values. The inhibitory effect of TNF was preserved with deletions to nucleotide -108 upstream of the translational start site; however, localization downstream of nucleotide -108 was obscured by loss of basal activity. Our results indicate that TNF regulates RUNX2 expression at multiple levels including destabilization of mRNA and suppression of transcription. The disproportionate inhibition of RUNX2 nuclear protein suggests that additional post-transcriptional mechanisms may be occurring. Suppression of RUNX2 by TNF may decrease osteoblast differentiation and inhibit bone formation in TNF excess states.
Gilbert
Linda
L
Division of Endocrinology and Metabolism, Emory University School of Medicine and Atlanta Veterans Affairs Medical Center, Atlanta, Georgia 30033, USA.
He
Xiaofei
X
Farmer
Paul
P
Rubin
Janet
J
Drissi
Hicham
H
van Wijnen
Andre J
AJ
Lian
Jane B
JB
Stein
Gary S
GS
Nanes
Mark S
MS
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D020128:Inhibitory Concentration 50; D051379:Mice; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D050659:Core Binding Factor Alpha 1 Subunit; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D015675:Osteocalcin; D003513:Cycloheximide