Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.
BACKGROUND
Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear.


METHODS
We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin.


RESULTS
We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner.


CONCLUSIONS
Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.
George Whipple Lab for Cancer Research, Department of Pathology, The Wilmot Cancer center, University of Rochester Medical Center, Rochester, NY 14642, USA.
Shin-Jen
SJ
Lin
Hsin-Chiu
HC
Ho
Yi-Fen
YF
Lee
Ning-Chun
NC
Liu
Su
S
Liu
Gonghui
G
Li
Chih-Rong
CR
Shyr
Chawnshang
C
Chang
D000818:Animals; D000831:Animals, Newborn; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism / Q000473:pathology; D016723:Bone Remodeling; D001842:Bone and Bones / Q000378:metabolism / Q000473:pathology; D002454:Cell Differentiation; D002478:Cells, Cultured; D005260:Female; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000378:metabolism* / Q000473:pathology; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis; D010024:Osteoporosis / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D000943:Antigens, Differentiation; C490514:Nr2c2 protein, mouse; D012333:RNA, Messenger; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; D015675:Osteocalcin