Effects of DLX2 overexpression on the osteogenic differentiation of MC3T3‑E1 cells

Sun,Hao; Liu,Zhixu; Li,Biao; Dai,Jiewen; Wang,Xudong

doi:10.3892/etm.2015.2378

Effects of DLX2 overexpression on the osteogenic differentiation of MC3T3‑E1 cells

Authors:
- Hao Sun
- Zhixu Liu
- Biao Li
- Jiewen Dai
- Xudong Wang
View Affiliations

Affiliations: Department of Oral and Cranio-maxillofacial Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, P.R. China
Published online on: March 20, 2015 https://doi.org/10.3892/etm.2015.2378
Pages: 2173-2179

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Distal‑less genes (DLX) play important roles in regulating organism development. DLX2 is crucial for the differentiation and development of the primordium, which determines the subsequent development and phenotype of the maxillofacial skeletal patterns, and is the primary candidate gene that regulates the development of the first branchial arch. The aim of the present study was to investigate the effects of DLX2 overexpression on the osteogenic differentiation of MC3T3‑E1 cells in vitro. A DLX2‑expression retrovirus vector was constructed by subcloning with a murine stem cell virus (MSCV) and verified by sequencing. MC3T3‑E1 cells were transfected with pMSCV‑DLX2 and stable clones were selected with puromycin. The mRNA and protein expression levels of DLX2 were determined using quantitative polymerase chain reaction (PCR) and western blot analysis, respectively. In addition, the expression levels of the osteogenic biomarkers, alkaline phosphatase (ALP), osteocalcin (OCN), runt‑related transcription factor (RUNX)2 and Msh homeobox (MSX)2, were assessed by quantitative PCR. ALP detection and Alizarin red staining were conducted to evaluate the effect of DLX2 overexpression on osteogenic differentiation. The data were analyzed by analysis of variance using the Student‑Newman‑Keuls method. Successful pMSCV‑DLX2 construction, as verified by direct sequencing, enabled DLX2 overexpression in vitro. Enhanced ALP activity and Alizarin red staining were observed in the MC3T3‑E1‑DLX2 cells when compared with the control group. During osteogenic induction, DLX2 overexpression was demonstrated to upregulate ALP and MSX2 expression at the early stage and OCN expression at the late stage, while no statistically significant difference was observed in RUNX2 expression when compared with the control group. Therefore, DLX2 overexpression in vitro induced the osteogenic differentiation of MC3T3‑E1 cells via upregulating bone formation‑associated genes, such as ALP and MSX2.

View Figures

View References

1	Depew MJ, Simpson CA, Morasso M and Rubenstein JL: Reassessing the Dlx code: the genetic regulation of branchial arch skeletal pattern and development. J Anat. 207:501–561. 2005. View Article : Google Scholar : PubMed/NCBI
2	Jin Y: Mouse Development Biology and Embryo Research Method. Volume 21st. People's Medical Publishing House; Beijing: pp. 70–73. 2005
3	Ferguson CA, Tucker AS and Sharpe PT: Temporospatial cell interactions regulating mandibular and maxillary arch patterning. Development. 127:403–412. 2000.PubMed/NCBI
4	Depew MJ, Lufkin T and Rubenstein JL: Specification of jaw subdivisions by Dlx genes. Science. 298:381–385. 2002. View Article : Google Scholar : PubMed/NCBI
5	Qiu M, Bulfone A, Ghattas I, et al: Role of the Dlx homeobox genes in proximodistal patterning of the branchial arches: mutations of Dlx-1, Dlx-2 and Dlx-1 and −2 alter morphogenesis of proximal skeletal and soft tissue structures derived from the first and second arches. Dev Biol. 185:165–184. 1997. View Article : Google Scholar : PubMed/NCBI
6	Qiu M, Bulfone A, Martinez S, et al: Null mutation of Dlx-2 results in abnormal morphogenesis of proximal first and second branchial arch derivatives and abnormal differentiation in the forebrain. Genes Dev. 9:2523–2538. 1995. View Article : Google Scholar : PubMed/NCBI
7	Merlo GR, Zerega B, Paleari L, et al: Multiple functions of Dlx genes. Int J Dev Biol. 44:619–626. 2000.PubMed/NCBI
8	Panganiban G and Rubenstein JL: Developmental functions of the Distal-less/Dlx homeobox genes. Development. 129:4371–4386. 2002.PubMed/NCBI
9	Li H, Marijanovic I, Kronenberg MS, et al: Expression and function of Dlx genes in the osteoblast lineage. Dev Biol. 316:458–470. 2008. View Article : Google Scholar : PubMed/NCBI
10	Harris SE, Guo D, Harris MA, et al: Transcriptional regulation of BMP-2 activated genes in osteoblasts using gene expression microarray analysis: role of Dlx2 and Dlx5 transcription factors. Front Biosci. 8:s1249–s1265. 2003. View Article : Google Scholar : PubMed/NCBI
11	Lee MH, Kwon TG, Park HS, et al: BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun. 309:689–694. 2003. View Article : Google Scholar : PubMed/NCBI
12	Muraglia A, Perera M, Verardo S, et al: DLX5 overexpression impairs osteogenic differentiation of human bone marrow stromal cells. Eur J Cell Biol. 87:751–761. 2008. View Article : Google Scholar : PubMed/NCBI
13	Karsenty G and Wagner EF: Reaching a genetic and molecular understanding of skeletal development. Dev Cell. 2:389–406. 2002. View Article : Google Scholar : PubMed/NCBI
14	Beck GR Jr, Zerler B and Moran E: Phosphate is a specific signal for induction of osteopontin gene expression. Proc Natl Acad Sci USA. 97:8352–8357. 2000. View Article : Google Scholar : PubMed/NCBI
15	Hu Z, Peel SA, Ho SK, et al: Role of bovine bone morphogenetic proteins in bone matrix protein and osteoblast-related gene expression during rat bone marrow stromal cell differentiation. J Craniofac Surg. 16:1006–1014. 2005. View Article : Google Scholar : PubMed/NCBI
16	Vaes BL, Dechering KJ, Feijen A, et al: Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development. J Bone Miner Res. 17:2106–2118. 2002. View Article : Google Scholar : PubMed/NCBI
17	Glowacki J, Rey C, Glimcher MJ, et al: A role for osteocalcin in osteoclast differentiation. J Cell Biochem. 45:292–302. 1991. View Article : Google Scholar : PubMed/NCBI
18	Hu JZ, Jiang XQ, Zhang ZY, et al: Effects of Nell-1 gene on bone formation-related gene expression during osteoblastic differentiation of rat bone marrow stromal cells by real-time PCR. Zhongguo Kou Qiang He Mian Wai Ke Za Zhi. 6:48–53. 2008.[(In Chinese)].
19	Hu JZ, Jiang XQ, Zhang ZY and Zhang XL: Effect of Nell-1 gene on osteogenic differentiation of rat bMSCs: An in vitro study. Zhongguo Kou Qiang He Mian Wai Ke Za Zhi. 7:38–43. 2009.[(In Chinese)].
20	Li P, Yu SH, Chen D, et al: Overexpression of Runx2 induces osteogenic differentiation in C2C12 cells. Zhongguo Sheng Wu Hua Xue Yu Fen Zi Sheng Wu Xue Bao. 26:236–242. 2010.[(In Chinese)].
21	Cheng SL, Shao JS, Charlton-Kachigian N, et al: MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 278:45969–45977. 2003. View Article : Google Scholar : PubMed/NCBI
22	Bendall AJ and Abate-Shen C: Roles for Msx and Dlx homeoproteins in vertebrate development. Gene. 247:17–31. 2000. View Article : Google Scholar : PubMed/NCBI
23	Ryoo HM, Lee MH and Kim YJ: Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells. Gene. 366:51–57. 2006. View Article : Google Scholar : PubMed/NCBI