Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis
© Lin et al.; licensee BioMed Central Ltd. 2012
Received: 18 February 2012
Accepted: 29 May 2012
Published: 7 June 2012
Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear.
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.
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.
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.
The TR4 nuclear receptor belongs to the nuclear receptor superfamily, which is comprised of transcription factors that are related by sequence and structure . As transcription factors, nuclear receptors control the expression of target genes and thereby direct developmental, physiological, and behavioral responses from the cellular level to that of the whole organism. TR4 is closely related to the TR2, retinoid X receptor, COUP-TF, and HNF4 in sequence and structure , and all of them bind to AGGTCA DNA sequence motifs in direct repeat (DR) orientation, with variable spacing, in the promoters of their target genes [1, 3–6]. Via knocking out TR4 (TR4−/−), a complex set of phenotypic abnormalities were found to exist in the TR4−/− mouse, including significant growth retardation , defects in female reproductive function  and maternal behavior , impaired cerebella function [9, 10], reduced sperm production , and reduced myelination , as well as abnormalities in glucose , lipid metabolism , and foam cell formation .
In this study, we reported that TR4 is involved in regulation of osteoblast activity via regulating osteocalcin expression. TR4 might represent a novel transcriptional factor involved in the bone remodeling network and provide a potential linkage to the pathogenesis of osteoporosis in human disease.
All animal procedures were approved by the Animal Care and Use Committee of the University of Rochester. TR4−/− mice used in this study were generated from heterozygous breeding pairs provided by Lexicon Genetics and genotyped as previously described .
Bone marrow cells were isolated from 10 weeks old TR4 wild type (TR4+/+) and TR4−/− mice. Cells were cultured in 2 mL of a modified essential medium (α-MEM) containing 10% fetal bovine serum (FBS) at 5x106 cells/well in 6-well plates. After 7 days in culture, the media was replaced with media containing 10 mM β-glycerophosphate and 50 mg/mL ascorbic acid. This media was changed every 2 days thereafter. On day 21 after plating, cells were fixed for alkaline phosphatase (ALP) and alizarin red staining or harvested for mRNA isolation using the RNeasy Mini Kit (Qiagen).
Mouse calvaria cells were obtained from neonatal mice 2–4 days after birth using sequential collagenease digestion. Briefly, calvaria were digested with trypsin/EDTA, then 1 mg/mL collagenase. Cells were collected by centrifugation, and cultured in proliferation media (DMEM with 20% FBS). Calvaria cells were cultured until 80% confluence and proliferation media was replaced by differentiation media (α-MEM containing 10% FCS, 2 mmol/L glutamine, 50 mg/mL ascorbic acid, and 10 mmol/L b-glycerol phosphate).
Total RNA was isolated from TR4−/− and TR4+/+ mice calvaria cultures using TRIzol Reagent (Invitrogen). The relative abundance of target mRNA was quantified relative to the control β-actin gene expression from the same reaction. Real-time PCR quantification amplifications of reverse-transcribed first strand DNA samples were performed using the iCycler iQ PCR cycler (Bio-Rad). Relative quantification of PCR products were based on value differences between the target and β-actin control using the 2ΔΔCT method.
The electrophoretic mobility shift assay (EMSA) & chromatin immunoprecipitation (ChIP) assay
TR4 protein for EMSA was transcribed and translated in TNT reticulocyte lysates (Promega). For the antibody super shift assay, the mixtures were incubated for 15 min in the presence or absence of a mouse anti-TR4 monoclonal antibody. The protein/DNA complexes were analyzed on a 5% native polyacrylamide gel. The results were visualized by autoradiography (Storm PhosphorImaging System, Amersham Pharmacia).
ChIP assays were performed in MC3T3 cells. Immunoprecipitations were performed at 4°C overnight, with 2 μg monoclonal antibody.
Bone tissue preparation
The femurs were collected and excess muscle and soft tissues were excised. The bone specimens were fixed in 10% neutral buffered formalin. The specimens were decalcified for 21 days in 14% EDTA (pH 7.2), embedded in paraffin, and sectioned at thickness of 3 μm. The sections were stained as described previously .
Mice lacking TR4 develop skeletal abnormalities
Reduced osteoblast differentiation in TR4−/− mice
Reduction of osteoblastic differentiation and associated genes in calvaria from TR4−/− mice
Quantitative real-time PCR analysis was then performed to determine the expression of genes associated with osteoblast activity. As shown in Figure 3B, all the expression levels of osteoblast marker genes, including PTH, PTHR, osteocalcin, and type I collagen alpha 1 were found to be dramatically reduced in osteoblasts from TR4−/− mice as compared to those from TR4+/+ mice. This reduction of osteoblastic gene expression also reflected undetectable serum concentrations of osteocalcin, a bio-marker for osteoblast activity, found in TR4−/− mice (Figure 3C), suggesting TR4 may be able to regulate bone differentiation through those differentiation markers.
TR4 modulates osteocalcin gene at transcriptional level
Furthermore, we found deletion of the N- and C-terminals of TR4 (TR4▽N and TR4▽C, respectively) resulted in loss of the TR4 ability to transactivate OC-Luc (Figure 4B), suggesting the full-length of TR4 is required for this regulation. This conclusion was further confirmed showing TR4-mediated osteocalcin promoter transcriptional activation was reduced in the TR4-siRNA knockdown cells in a dose dependent manner (Figure 4C). Importantly, as the third G within the DR motif is important for TR4 binding, we therefore mutated this position from GGGTGAatgAGGACA to GGATGAatgAGTACA and as expected, we found mutated TR4RE in OC-Luc (OCm-Luc) was no longer able to transactivate luciferase activity (Figure 4D).
Together, results from Figure 4A-D suggested that TR4 modulated bone formation via regulating osteocalcin gene expression at the transcriptional level.
TR4 binds to the regulatory sequence of osteocalcin gene in vitro and in vivo
To further test the TR4-TR4RE interaction in vitro, EMSA was applied. As shown in Figure 5B, TR4 can form a complex with TR4RE, and this TR4-TR4RE can be super shifted by adding TR4 antibody (lane 2) . The specific TR4-DNA-protein complex was further competed out by 100 X unlabeled TR4RE (lane 3), but not mutant TR4RE (TR4REm) (lane 4 and 5), suggesting TR4 can physically bind to osteocalcin promoter region to transcribe its expression.
We found that TR4 directly influences osteoblast activity with reduced osteoblast differentiation found in bone marrow progenitor cells isolated from TR4−/− mice as compared to TR4+/+ mice. This reduction of bone marrow progenitor cells differentiation could then contribute to osteoporosis developed in their later life. Our data are in agreement with the critical role of bone marrow progenitor cells in the senile osteoporosis where depletion and/or inhibition of bone marrow progenitor cells differentiation and activation could cause osteoporosis. TR4 represents a novel factor that controls differentiation of bone marrow progenitor cells allowing for osteoblast formation. And the depletion of TR4 in mice results in premature aging accompanied with osteoporosis in both genders. TR4−/− mice display a significant adipocyte accumulation in their trabecular bones, indicating that blocking TR4 function can also alter the balance between adipogenesis and osteogenesis, and that bone formation is inhibited. These findings make TR4 a candidate gene to monitor bone activity.
TR4 transcript levels did not alter when undifferentiated embryonic stem cells were induced into adipocytes and osteoblasts , suggesting that the environmental/exogenous factors that control TR4 activity might be critical for controlling the bone marrow progenitor cells activity to affect osteoblast differentiation. Therefore, identifying its upstream modulator(s) offers novel therapeutic and/or preventive approaches to target senile osteoporosis. However, in contrast to TR4, it is reported that attenuation of PPAR gamma function could promote osteogenesis, such that bone formation can be enhanced . Since both TR4 and PPAR gamma are members of the nuclear receptor superfamily and share the similar regulatory DNA response elements and some upstream modulator(s), it will be of interest to dissect the activity/specificity of both receptors in the process of early bone marrow progenitor cells differentiation that might have a great impact on the osteoblast activity.
Together, these results demonstrate TR4 may function as a novel transcriptional factor to play its pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.
This work was supported by George Whipple Professorship endowment, NIH Grants CA127548 and CA156780, and Taiwan Department of Health Clinical Trial and Research Center of Excellence Grant DOH99-TD-B-111-004 (china Medical University, Taichung, Taiwan).
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