Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2αA) is inhibited by tumor necrosis factor-α

Linda Gilbert, Xiaofei He, Paul Farmer, Janet Rubin, Hicham Drissi, Andre J van Wijnen, Jane B. Lian, Gary S. Stein, Mark S. Nanes

Research output: Contribution to journalArticle

309 Citations (Scopus)

Abstract

The transcription factor RUNX2 (Cbfa1/AML3/Pebp2αA) is a critical regulator of osteoblast differentiation. We investigated the effect of the inflammatory cytokine tumor necrosis factor α (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 IC50 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.

Original languageEnglish (US)
Pages (from-to)2695-2701
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number4
DOIs
StatePublished - Jan 25 2002
Externally publishedYes

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Osteoblasts
Tumor Necrosis Factor-alpha
Messenger RNA
Transcription
Protein Isoforms
Nuclear Proteins
Nucleotides
Electrophoretic mobility
Osteocalcin
Electrophoretic Mobility Shift Assay
Cycloheximide
Luciferases
Osteogenesis
Skull

ASJC Scopus subject areas

  • Biochemistry

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Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2αA) is inhibited by tumor necrosis factor-α. / Gilbert, Linda; He, Xiaofei; Farmer, Paul; Rubin, Janet; Drissi, Hicham; van Wijnen, Andre J; Lian, Jane B.; Stein, Gary S.; Nanes, Mark S.

In: Journal of Biological Chemistry, Vol. 277, No. 4, 25.01.2002, p. 2695-2701.

Research output: Contribution to journalArticle

Gilbert, L, He, X, Farmer, P, Rubin, J, Drissi, H, van Wijnen, AJ, Lian, JB, Stein, GS & Nanes, MS 2002, 'Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2αA) is inhibited by tumor necrosis factor-α', Journal of Biological Chemistry, vol. 277, no. 4, pp. 2695-2701. https://doi.org/10.1074/jbc.M106339200
Gilbert, Linda ; He, Xiaofei ; Farmer, Paul ; Rubin, Janet ; Drissi, Hicham ; van Wijnen, Andre J ; Lian, Jane B. ; Stein, Gary S. ; Nanes, Mark S. / Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2αA) is inhibited by tumor necrosis factor-α. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 4. pp. 2695-2701.
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abstract = "The transcription factor RUNX2 (Cbfa1/AML3/Pebp2αA) is a critical regulator of osteoblast differentiation. We investigated the effect of the inflammatory cytokine tumor necrosis factor α (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 IC50 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.",
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AU - Farmer, Paul

AU - Rubin, Janet

AU - Drissi, Hicham

AU - van Wijnen, Andre J

AU - Lian, Jane B.

AU - Stein, Gary S.

AU - Nanes, Mark S.

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