Abstract
Nrf2 (nuclear factor E2 p45-related factor 2) is believed to be a transcription factor essential for the regulation of many detoxifying and antioxidative genes in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of osteoblastic differentiation. nrf2 mRNA expression was significantly up-regulated in femur isolated from ovariectomized mice, whereas in situ hybridization analysis revealed that up-regulation of nrf2 mRNA was mainly found in osteoblasts attached on cancellous bone in femur of ovariectomized mice. Expression of Nrf2 protein was also seen in osteoblasts in neonatal mouse tibia and calvaria. In osteoblastic MC3T3-E1 cells stably transfected with nrf2 expression vector, significant inhibition was seen in the maturation-dependent increase in alkaline phosphatase activity as well as the mineralized matrix formation. Stable overexpression of nrf2 significantly impaired Runx2 (runt-related transcription factor 2)-dependent stimulation of osteocalcin promoter activity and recruitment of Runx2 on osteocalcin promoter without affecting the expression of runx2 mRNA. Coimmunoprecipitation and mammalian two-hybrid assay revealed a physical interaction between Runx2 and Nrf2, whereas cellular distribution of endogenous Runx2 was not apparently changed by nrf2 overexpression in MC3T3-E1 cells. Alternatively, Nrf2 bound to antioxidant-responsive element-like-2 sequence of osteocalcin promoter. The inhibition by nrf2 on runx2-dependent osteocalcin promoter activity was partially prevented by the introduction of reporter of deletion mutant for ARE-like-2 sequence of osteocalcin promoter. These data suggest that Nrf2 may negatively regulate cellular differentiation through inhibition of the Runx2-dependent transcriptional activity in osteoblasts.
Highlights
Icate balancing between bone formation and resorption by the two different cells in the bone marrow microenvironment
Bone loss was evident in cancellous bone (Fig. 1B), but not in cortical bone, by micro-CT analysis. mRNA was extracted from femur after removing bone marrows by flush out, followed by determination of nrf2 expression by semiquantitative RT-PCR. nrf2 mRNA expression was significantly up-regulated by 3-fold in femur isolated from ovariectomized mice when compared with that in shamoperated mice (Fig. 1C)
Several previous studies have demonstrated the functional expression of Nrf2 in bone as a transcription factor regulating basal and inducible expression of numerous detoxifying and antioxidant genes [31, 32], no direct evidence for a role in mechanisms underlying the cellular differentiation in osteoblasts is available in the literature to date
Summary
Icate balancing between bone formation and resorption by the two different cells in the bone marrow microenvironment. In mesenchymal stem cells isolated from bone of ovariectomized mice, marked reduction of mRNA expression is seen for a variety of osteoblastic differentiation marker genes, including type I collagen and ALP,2 in addition to runx2 (Runt-related transcription factor 2) [9].
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