Examination of nuclear receptor expression in osteoblasts reveals rorβ as an important regulator of osteogenesis

Abstract

A complex network of transcription factors contributes to the establishment and maintenance of the osteoblastic phenotype. Although relatively few transcription factors, such as Runx2 and osterix, are essential to the process of osteoblastic differentiation, others serve the purpose of fine-tuning in response to various environmental and hormonal cues. The nuclear receptor (NR) superfamily of transcription factors are involved in numerous aspects of bone biology. In this study, we characterized the expression pattern of the entire NR superfamily in differentiating primary murine calvarial cells in order to identify novel NR regulatory patterns. Dynamic patterns of NR expression were observed throughout the differentiation process. Interestingly, retinoic acid receptor-related orphan receptor β(Rorβ) expression was markedly suppressed at later stages of differentiation. To gain further insight into the function of NRs in bone biology, the NR superfamily was also profiled in mouse bone marrow precursor cells isolated from either young (6 month) or aging, osteoporotic (18–22 month) mice. Of interest, Rorβ was potently overexpressed in the aged cohort. Collectively, these data provided evidence that Rorβ expression is inversely correlated with osteogenic potential, suggesting Rorβ may be an important and unexplored regulator of osteogenesis. To validate this hypothesis, a cell model stably expressing Rorβ in mouse osteoblastic MC3T3-E1 cells was produced (MC3T3-Rorβ). These cells displayed markedly suppressed bone nodule formation as well as reduced osteocalcin and osterix gene expression. Since these genes are Runx2 targets, we reasoned that Rorβ may interfere with Runx2 activity. Consistent with this, transient transfection analysis demonstrated that Rorβ inhibited Runx2-dependent activation of a Runx2-reporter construct. In summary, our data provide a comprehensive profile of NR expression during osteoblast differentiation and identify Rorβ as a novel regulator of osteogenesis and potentially of age-related bone loss through antagonism of Runx2 activity.

Vitamin A Is a Negative Regulator of Osteoblast Mineralization

“…The negative effect of endogenous RA on osteoblast mineralization was further demonstrated by the finding that blocking RAR-signaling with a pan-RAR antagonist increased mineralization. This together with the recent observations that all three RAR receptors are downregulated during osteoblast differentiation support the view that RAR signaling is a negative regulator of osteoblast mineralization [28]. Previous studies on the effects of exogenous RA on mineralizing cultures of osteoblasts are consistent with our results [6], [29]–[32] except one study which used a very high, supraphysiological concentration of RA (10 µM) [33].…”

The role of vitamin A and retinoic acid receptor signaling in post-natal maintenance of bone

“…RORβ expression is negatively correlated with osteoblast differentiation, inhibits Runx2 transcriptional activity and enhances expression of genes normally downregulated in osteogenesis (e.g. Dcn, Mgp) (37).…”

Identification of Rorβ targets in cultured osteoblasts and in human bone

“…Previous examination of the role of Rorβ in MC3T3-E1 mouse osteoblastic cells demonstrated a suppression of the osteogenic phenotype in bone mineralization assays [5]; however, the molecular and cellular mechanisms of how Rorβ exerts its anti-osteogenic role are unknown. Therefore, we utilized Illumina microarray technology to identify global gene expression patterns between the MC3T3- GFP (control) and MC3T3– Rorβ - GFP (experimental) cell models [5].…”

“…The MC3T3- GFP and MC3T3– Rorβ -green fluorescent protein ( GFP ) cell lines were cultured in αMEM growth medium (Invitrogen, Carlsbad, CA) supplemented with 1X antibiotic/antimycotic (Invitrogen), 10% (v/v) fetal bovine serum (Hyclone, Logan, UT) and 400 ug/mL G418 antibiotic (Invitrogen) [5]. These cells stably express either GFP (control cells) or Rorβ-GFP (experimental cells), respectively.…”

“…Constitutive Rorβ expression inhibits mineralization of mouse calvarial osteoblasts. Interestingly, Rorβ expression is highly upregulated in osteoblastic precursors cells isolated from the bone marrow of aged (18-22 month-old), osteoporotic mice [5]. Collectively, these data suggest that Rorβ may function to inhibit Runx2-dependent processes not only during differentiation but also in an aging context.…”