Stage-specific expression of Dlx-5 during osteoblast differentiation: Involvement in regulation of osteocalcin gene expression

H. M. Ryoo, H. M. Hoffmann, T. Beumer, B. Frenkel, D. A. Towler, G. S. Stein, J. L. Stein, A. J. Van Wijnen, J. B. Lian

Research output: Contribution to journalArticle

228 Citations (Scopus)

Abstract

Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx- 1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx- 2, which is found predominantly in proliferating osteoblasts. Several approaches were pursued to determine functional consequences of Dlx-5 expression on osteocalcin transcription. Constitutive expression of Dlx-5 in ROS 17/2.8 cells decreased osteocalcin promoter activity in transient assays, and conditional expression of Dlx-5 in stable cell lines reduced endogenous mRNA levels. Consistent with this finding, antisense inhibition of Dlx-5 increased osteocalcin gene transcription. Osteocalcin promoter deletion analysis and binding of the in vitro translation product of Dlx-5 demonstrated that repressor activity was targeted to a single homeodomain- binding site, located in OC-Box I (-99 to -76). These findings demonstrate that Dlx-5 represses osteocalcin gene transcription. However, the coupling of increased Dlx-5 expression with progression of osteoblast differentiation suggests an important role in promoting expression of the mature bone cell phenotype.

Original languageEnglish (US)
Pages (from-to)1681-1694
Number of pages14
JournalMolecular Endocrinology
Volume11
Issue number11
DOIs
StatePublished - Oct 22 1997

Fingerprint

Osteocalcin
Gene Expression Regulation
Osteoblasts
Bone and Bones
Homeobox Genes
Genes
Phenotype
Messenger RNA
Northern Blotting
Cartilage
Tooth
Complementary DNA
Binding Sites
Cell Line
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

Cite this

Stage-specific expression of Dlx-5 during osteoblast differentiation : Involvement in regulation of osteocalcin gene expression. / Ryoo, H. M.; Hoffmann, H. M.; Beumer, T.; Frenkel, B.; Towler, D. A.; Stein, G. S.; Stein, J. L.; Van Wijnen, A. J.; Lian, J. B.

In: Molecular Endocrinology, Vol. 11, No. 11, 22.10.1997, p. 1681-1694.

Research output: Contribution to journalArticle

Ryoo, HM, Hoffmann, HM, Beumer, T, Frenkel, B, Towler, DA, Stein, GS, Stein, JL, Van Wijnen, AJ & Lian, JB 1997, 'Stage-specific expression of Dlx-5 during osteoblast differentiation: Involvement in regulation of osteocalcin gene expression', Molecular Endocrinology, vol. 11, no. 11, pp. 1681-1694. https://doi.org/10.1210/me.11.11.1681
Ryoo, H. M. ; Hoffmann, H. M. ; Beumer, T. ; Frenkel, B. ; Towler, D. A. ; Stein, G. S. ; Stein, J. L. ; Van Wijnen, A. J. ; Lian, J. B. / Stage-specific expression of Dlx-5 during osteoblast differentiation : Involvement in regulation of osteocalcin gene expression. In: Molecular Endocrinology. 1997 ; Vol. 11, No. 11. pp. 1681-1694.
@article{b6de9771f0a54b9192445d44c571c795,
title = "Stage-specific expression of Dlx-5 during osteoblast differentiation: Involvement in regulation of osteocalcin gene expression",
abstract = "Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx- 1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx- 2, which is found predominantly in proliferating osteoblasts. Several approaches were pursued to determine functional consequences of Dlx-5 expression on osteocalcin transcription. Constitutive expression of Dlx-5 in ROS 17/2.8 cells decreased osteocalcin promoter activity in transient assays, and conditional expression of Dlx-5 in stable cell lines reduced endogenous mRNA levels. Consistent with this finding, antisense inhibition of Dlx-5 increased osteocalcin gene transcription. Osteocalcin promoter deletion analysis and binding of the in vitro translation product of Dlx-5 demonstrated that repressor activity was targeted to a single homeodomain- binding site, located in OC-Box I (-99 to -76). These findings demonstrate that Dlx-5 represses osteocalcin gene transcription. However, the coupling of increased Dlx-5 expression with progression of osteoblast differentiation suggests an important role in promoting expression of the mature bone cell phenotype.",
author = "Ryoo, {H. M.} and Hoffmann, {H. M.} and T. Beumer and B. Frenkel and Towler, {D. A.} and Stein, {G. S.} and Stein, {J. L.} and {Van Wijnen}, {A. J.} and Lian, {J. B.}",
year = "1997",
month = "10",
day = "22",
doi = "10.1210/me.11.11.1681",
language = "English (US)",
volume = "11",
pages = "1681--1694",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
number = "11",

}

TY - JOUR

T1 - Stage-specific expression of Dlx-5 during osteoblast differentiation

T2 - Involvement in regulation of osteocalcin gene expression

AU - Ryoo, H. M.

AU - Hoffmann, H. M.

AU - Beumer, T.

AU - Frenkel, B.

AU - Towler, D. A.

AU - Stein, G. S.

AU - Stein, J. L.

AU - Van Wijnen, A. J.

AU - Lian, J. B.

PY - 1997/10/22

Y1 - 1997/10/22

N2 - Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx- 1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx- 2, which is found predominantly in proliferating osteoblasts. Several approaches were pursued to determine functional consequences of Dlx-5 expression on osteocalcin transcription. Constitutive expression of Dlx-5 in ROS 17/2.8 cells decreased osteocalcin promoter activity in transient assays, and conditional expression of Dlx-5 in stable cell lines reduced endogenous mRNA levels. Consistent with this finding, antisense inhibition of Dlx-5 increased osteocalcin gene transcription. Osteocalcin promoter deletion analysis and binding of the in vitro translation product of Dlx-5 demonstrated that repressor activity was targeted to a single homeodomain- binding site, located in OC-Box I (-99 to -76). These findings demonstrate that Dlx-5 represses osteocalcin gene transcription. However, the coupling of increased Dlx-5 expression with progression of osteoblast differentiation suggests an important role in promoting expression of the mature bone cell phenotype.

AB - Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx- 1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx- 2, which is found predominantly in proliferating osteoblasts. Several approaches were pursued to determine functional consequences of Dlx-5 expression on osteocalcin transcription. Constitutive expression of Dlx-5 in ROS 17/2.8 cells decreased osteocalcin promoter activity in transient assays, and conditional expression of Dlx-5 in stable cell lines reduced endogenous mRNA levels. Consistent with this finding, antisense inhibition of Dlx-5 increased osteocalcin gene transcription. Osteocalcin promoter deletion analysis and binding of the in vitro translation product of Dlx-5 demonstrated that repressor activity was targeted to a single homeodomain- binding site, located in OC-Box I (-99 to -76). These findings demonstrate that Dlx-5 represses osteocalcin gene transcription. However, the coupling of increased Dlx-5 expression with progression of osteoblast differentiation suggests an important role in promoting expression of the mature bone cell phenotype.

UR - http://www.scopus.com/inward/record.url?scp=0030801264&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030801264&partnerID=8YFLogxK

U2 - 10.1210/me.11.11.1681

DO - 10.1210/me.11.11.1681

M3 - Article

C2 - 9328350

AN - SCOPUS:0030801264

VL - 11

SP - 1681

EP - 1694

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0888-8809

IS - 11

ER -