Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by
Vitamin D is an essential factor for ossification, including activation of calcium absorption in the intestine, inhibition of calcium release in the kidney, and promotion of osteogenesis in bone. Indeed, vitamin D deficiency is well known to induce bone-softening diseases, such as rickets in children and osteomalacia in adults [
Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation [
Previously, we produced a transgenic mouse line expressing luciferase under the control of a 10-kb human osteocalcin enhancer/promoter sequence. This mouse line was backcrossed with a hairless mouse line to enable us to monitor bone formation during growth, fracture repair, and aging using
All of the animal experiments described were approved by the Institutional Animal Care and Use Committee of Tottori University (Permission nos. 18-2-42 and 09-Y-64). All the mice received humane care in compliance with Tottori University’s guidelines for the care and use of laboratory animals in research.
MG-63 human osteosarcoma cells and HeLa cells were cultured in Eagle’s minimal essential medium and Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA), 100 U/mL penicillin (Meiji Seika Pharma Co. Ltd., Tokyo, Japan), and 0.1 mg/mL streptomycin (Meiji Seika Pharma Co. Ltd.) at 37°C under 5% CO2 in air. For luciferase reporter assays, 2
Hairless human osteocalcin enhancer/promoter-luciferase transgenic mice (OC-Luc Tg mice) [
Statistical analysis was performed using StatView (SAS Institute Inc., Cary, NC). The Student’s
We previously constructed a plasmid expressing luciferase under the control of a 10-kb human osteocalcin enhancer/promoter sequence (pOC-Luc) (Figure
Constructs for the generation of the transgenic mice. (a) Schematic map of the transgenes used in the study. pOC-Luc is composed of a 10-kb human osteocalcin enhancer/promoter sequence, with 60 bp of the 5′-untranslated sequence (white box), a luciferase gene (black box), an SV40 late polyadenylation signal (striped box), and an insulator sequence (gray box). (b) Enhanced expression of pOC-Luc by 1
The regulation of pOC-Luc was monitored by its luciferase activity following transfection into MG-63 cells. The MG-63 cell line is a well-characterized human osteoblast-like cell line that shows 1
We previously established a transgenic mouse line, OC-Luc Tg, harboring the human osteocalcin enhancer/promoter-luciferase gene derived from pOC-Luc [
To compare the effects of 1
Regulation of the human osteocalcin enhancer/promoter by vitamins D3 and D2
The bone-mobilizing activity of 1
In the transgenic mice harboring human osteocalcin enhancer/promoter luciferase reporter gene, strong osteogenic activity was observed by 1
The authors wish to thank Dr. Naohiro Hori and their laboratory members for technical assistance and valuable discussions. This study was supported in part by a City Area Program (basic stage), a Regional Innovation Cluster Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).