Stimulus-selective inhibition of rat osteocalcin promoter induction and protein-DNA interactions by the homeodomain repressor Msx2

Elizabeth P. Newberry, Jeanne M. Boudreaux, Dwight A. Towler

Research output: Contribution to journalArticle

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Abstract

Osteocalcin (OC) is a matrix calcium-binding protein expressed in osteoblasts and odontoblasts undergoing mineralization. OC expression is up- regulated in part by signals initiated by basic fibroblast growth factor (FGF2), cyclic AMP or forskolin (FSK), and calcitriol via defined elements and DNA-protein interactions in the OC promoter. We identified the OC gene as a target for transcriptional suppression by Msx2, a homeodomain transcription factor that controls ossification in the developing skull. In this study, we examine the effects of Msx2 expression on OC promotor activation (luciferase reporter) by FGF2/FSK and calcitriol in MC3T3-E1 osteoblasts. Expression of Msx2 decreases basal activity of the 1-kilobase (-1050 to +32) rat OC promoter by 80%; however, the promoter is still inducible 3-fold by calcitriol. By contrast, OC promoter induction by FGF2/FSK is completely abrogated by Msx2. Because intrinsic Msx2 DNA binding activity is not required for the Msx2 suppressor function, we assessed whether Msx2 represses OC activation by regulating DNA-protein interactions at the FGF2 response element (OCFRE) and compared these interactions with those occurring at the calcitriol response element (VDRE). Treatment of MC3T3-E1 cells with FGF2/FSK or calcitriol up-regulates specific DNA-protein interactions at the OCFRE or VDRE, respectively, as detected by gel shift assay. Preincubation of crude nuclear extracts with recombinant glutathione S-transferase (GST)-Msx2 dose- dependently inhibits OCFRE DNA binding activity, whereas GST has no effect. Msx2 itself does not bind the OCFRE. Residues 132-148 required for Msx2 core suppressor function in transfection assays are also required to inhibit OCFRE DNA binding activity. By contrast, GST-Msx2 has no effect on calcitriol- regulated DNA-protein interactions at the VDRE. Using gel shift as an assay, the OCFRE DNA-binding protein OCFREB was purified to about 50% homogeneity from MG63 osteosarcoma cells. Recombinant Msx2 inhibits purified OCFREB DNA binding activity, whereas the Msx2 variant lacking residues 132-148 is inactive. Thus, Msx2 abrogates up-regulation of the OC promoter by FGF2/FSK in part by inhibiting OCFREB binding to the OCFRE.

Original languageEnglish (US)
Pages (from-to)29607-29613
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number47
DOIs
StatePublished - Nov 21 1997

Fingerprint

Osteocalcin
Rats
Fibroblast Growth Factor 2
Calcitriol
Colforsin
DNA
Proteins
Glutathione Transferase
Assays
Osteoblasts
Response Elements
Up-Regulation
Gels
Chemical activation
Odontoblasts
Calcium-Binding Proteins
DNA-Binding Proteins
Osteosarcoma
Luciferases
Complex Mixtures

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Stimulus-selective inhibition of rat osteocalcin promoter induction and protein-DNA interactions by the homeodomain repressor Msx2. / Newberry, Elizabeth P.; Boudreaux, Jeanne M.; Towler, Dwight A.

In: Journal of Biological Chemistry, Vol. 272, No. 47, 21.11.1997, p. 29607-29613.

Research output: Contribution to journalArticle

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abstract = "Osteocalcin (OC) is a matrix calcium-binding protein expressed in osteoblasts and odontoblasts undergoing mineralization. OC expression is up- regulated in part by signals initiated by basic fibroblast growth factor (FGF2), cyclic AMP or forskolin (FSK), and calcitriol via defined elements and DNA-protein interactions in the OC promoter. We identified the OC gene as a target for transcriptional suppression by Msx2, a homeodomain transcription factor that controls ossification in the developing skull. In this study, we examine the effects of Msx2 expression on OC promotor activation (luciferase reporter) by FGF2/FSK and calcitriol in MC3T3-E1 osteoblasts. Expression of Msx2 decreases basal activity of the 1-kilobase (-1050 to +32) rat OC promoter by 80{\%}; however, the promoter is still inducible 3-fold by calcitriol. By contrast, OC promoter induction by FGF2/FSK is completely abrogated by Msx2. Because intrinsic Msx2 DNA binding activity is not required for the Msx2 suppressor function, we assessed whether Msx2 represses OC activation by regulating DNA-protein interactions at the FGF2 response element (OCFRE) and compared these interactions with those occurring at the calcitriol response element (VDRE). Treatment of MC3T3-E1 cells with FGF2/FSK or calcitriol up-regulates specific DNA-protein interactions at the OCFRE or VDRE, respectively, as detected by gel shift assay. Preincubation of crude nuclear extracts with recombinant glutathione S-transferase (GST)-Msx2 dose- dependently inhibits OCFRE DNA binding activity, whereas GST has no effect. Msx2 itself does not bind the OCFRE. Residues 132-148 required for Msx2 core suppressor function in transfection assays are also required to inhibit OCFRE DNA binding activity. By contrast, GST-Msx2 has no effect on calcitriol- regulated DNA-protein interactions at the VDRE. Using gel shift as an assay, the OCFRE DNA-binding protein OCFREB was purified to about 50{\%} homogeneity from MG63 osteosarcoma cells. Recombinant Msx2 inhibits purified OCFREB DNA binding activity, whereas the Msx2 variant lacking residues 132-148 is inactive. Thus, Msx2 abrogates up-regulation of the OC promoter by FGF2/FSK in part by inhibiting OCFREB binding to the OCFRE.",
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