J Periodontal Implant Sci.  2010 Feb;40(1):39-44. 10.5051/jpis.2010.40.1.39.

The synergistic regulatory effect of Runx2 and MEF transcription factors on osteoblast differentiation markers

• Lee JM
• Libermann
• Cho JY

• KMID: 2094714
• DOI: http://doi.org/10.5051/jpis.2010.40.1.39

Abstract

PURPOSE
Bone tissues for clinical application can be improved by studies on osteoblast differentiation. Runx2 is known to be an important transcription factor for osteoblast differentiation. However, bone morphogenetic protein (BMP)-2 treatment to stimulate Runx2 is not sufficient to acquire enough bone formation in osteoblasts. Therefore, it is necessary to find other regulatory factors which can improve the transcriptional activity of Runx2. The erythroblast transformation-specific (ETS) transcription factor family is reported to be involved in various aspects of cellular proliferation and differentiation.
METHODS
We have noticed that the promoters of osteoblast differentiation markers such as alkaline phosphatase (Alp), osteopontin (Opn), and osteocalcin (Oc) contain Ets binding sequences which are also close to Runx2 binding elements. Luciferase assays were performed to measure the promoter activities of these osteoblast differentiation markers after the transfection of Runx2, myeloid Elf-1-like factor (MEF), and Runxs+MEF. Reverse-transcription polymerase chain reaction was also done to check the mRNA levels of Opn after Runx2 and MEF transfection into rat osteoblast (ROS) cells.
RESULTS
We have found that MEF, an Ets transcription factor, increased the transcriptional activities of Alp, Opn, and Oc. The addition of Runx2 resulted in the 2- to 6-fold increase of the activities. This means that these two transcription factors have a synergistic effect on the osteoblast differentiation markers. Furthermore, early introduction of these two Runx2 and MEF factors significantly elevated the expression of the Opn mRNA levels in ROS cells. We also showed that Runx2 and MEF proteins physically interact with each other.
CONCLUSIONS
Runx2 interacts with MEF proteins and binds to the promoters of the osteoblast markers such as Opn nearby MEF to increase its transcriptional activity. Our results also imply that osteoblast differentiation and bone formation can be increased by activating MEF to elicit the synergistic effect of Runx2 and MEF.

Keyword

Cell differentiation; Core binding factor alpha 1 subunit; Osteoblasts

MeSH Terms

Alkaline Phosphatase
Animals
Antigens, Differentiation
Bone and Bones
Bone Morphogenetic Proteins
Cell Differentiation
Cell Proliferation
Core Binding Factor Alpha 1 Subunit
Erythroblasts
Humans
Luciferases
Osteoblasts
Osteocalcin
Osteogenesis
Osteopontin
Polymerase Chain Reaction
Proteins
Rats
RNA, Messenger
Transcription Factors
Transfection
Alkaline Phosphatase
Antigens, Differentiation
Bone Morphogenetic Proteins
Core Binding Factor Alpha 1 Subunit
Luciferases
Osteocalcin
Osteopontin
Proteins
RNA, Messenger
Transcription Factors

Figure

• 

  Figure 1 Synergistic effects of erythroblast transformation-specific (ETS) and Runx2 on the osteopontin (Opn) promoter. (A) Sequence analysis of the Opn promoter showed potential ETS and Runx2 binding elements. (B) Opn-luciferase (Opn-Luc) promoter construct (1,983 bp) was co transfected with ETS transcription factors as designated with or without Runx2 + CBFbeta into COS7 cells. Twenty-four hours after transfection the luciferase activity was measured as described in the Materials and Methods. In the Opn-Luc graph, the black bar is for only pCDNA3.1 vector and the gray bar is for Runx2 + CBFbeta as expression cDNA.

• 

  Figure 2 Synergistic effects of erythroblast transformation-specific (ETS) and Runx2 on the osteocalcin (Oc) promoter. (A) Sequence analysis of the Oc promoter showed potential ETS and Runx2 response elements. (B) Oc-luciferase (Oc-Luc) promoter construct (1,080 bp) was co-transfected with ETS transcription factors as designated with or without Runx2 + CBFbeta into COS7 cells. Twenty-four hours after transfection, the luciferase activity was measured. In Oc-Luc graph, the black bar is for pCDNA3.1 vector only and the grey bar is for Runx2 + CBFbeta as expression cDNA.

• 

  Figure 3 Synergistic effects of erythroblast transformation-specific (ETS) and Runx2 on the alkaline phosphatase (Alp) promoter. (A) Sequence analysis of the Alp promoter showed potential ETS and Runx2 binding elements. (B) Alp-luciferase (Alp-Luc) promoter construct (1.9 kb) was co-transfected with myeloid Elf-1-like factor (MEF) transcription factor with or without Runx2 + CBFbeta into COS7 cells. Twenty-four hours after transfection the luciferase activity was measured. In the Alp-Luc graph, the black bar is for pCDNA3.1 vector only and the grey bar is for Runx2 + CBFbeta as expression cDNA.

• 

  Figure 4 (A) Bone sialoprotein-luciferase (BSP-Luc) promoter construct was co-transfected with myeloid Elf-1-like factor (MEF) transcription factor with or without Runx2 + CBFbeta into COS7 cells. Twenty-four hours after transfection the luciferase activity was measured. In BSP-Luc graph, the black bar is for only pCDNA3.1 vector and grey bar is for Runx2 + CBFbeta as expression cDNAs. (B) ROS17/2.8 cells were transfected with Mock (pCDNA3.1), MEF, Runx2, or MEF together with Runx2. Runx2 was always introduced with CBFbeta cofactor on the mark of 'Runx2.' Twenty-four hours after transfection, the cells were harvested, RNA was collected and RT-PCR were performed with Opn primers as described in the Materials and Methods.

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