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.In bone tissues, both formation and maintenance are sophisticatedly regulated by bone-forming osteoblasts and bone-resorbing osteoclasts (1-3). The osteoblast lineage is derived from primitive multipotent mesenchymal stem cells with potentiality to differentiate into bone marrow stromal cells, chondrocytes, muscles, and adipocytes (4), whereas osteoclasts are multinucleated cells derived from the fusion of mononuclear hematopoietic precursors (3). The development and differentiation of these two distinct cells are under tight regulation by a number of endogenous substances. These include growth factors, cytokines, and hormones, which are individually secreted through endocrine, paracrine/autocrine, and neurocrine systems essential for the delicate balancing between bone formation and resorption by the two different cells in the bone marrow microenvironment. An imbalance between these two cells leads to pathogenesis and etiology of certain bone metabolic diseases, including osteoporosis and osteopetrosis (5, 6). Ovariectomy has been employed to experimentally establish model animals with bone loss seen in postmenopausal osteoporosis caused by estrogen deficiency (7). In ovariectomized animals, osteoblast indices, such as osteoblast numbe...
Background: NAT8L (N-acetyltransferase 8-like) synthesizes N-acetylaspartate and is required for myelination in the brain. Its function in other tissues was undefined. Results: Nat8l is highly expressed in adipose tissues and impacts adipogenic marker gene expression, lipid turnover, and energy metabolism in brown adipocytes. Conclusion: Nat8l expression influences cellular bioenergetics in adipocytes. Significance: These findings establish a novel pathway in brown adipocyte metabolism.
A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens (NAc) of mice with methamphetamine (METH) treatment. Previously we reported that suppression of Shati/Nat8l enhanced METH-induced behavioral alterations via dopaminergic neuronal regulation. However, the physiological mechanisms of Shati/Nat8l on the dopaminergic system in the brain are unclear. In this study, we injected adeno-associated virus (AAV) vector containing Shati/Nat8l into the NAc or dorsal striatum (dS) of mice, to increase Shati/Nat8l expression. Overexpression of Shati/Nat8l in the NAc, but not in the dS, attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference in mice. Moreover, the Shati/Nat8l overexpression in the NAc attenuated the elevation of extracellular dopamine levels induced by METH in in vivo microdialysis experiments. These behavioral and neurochemical alterations due to Shati/Nat8l overexpression in the NAc were inhibited by treatment with selective group II metabotropic glutamate receptor type 2 and 3 (mGluR2/3) antagonist LY341495. In the AAV vector-injected NAc, the tissue contents of both N-acetylaspartate and N-acetylaspartylglutamate (NAAG), endogenous mGluR3 agonist, were elevated. The injection of peptidase inhibitor of NAAG or the perfusion of NAAG itself reduced the basal levels of extracellular dopamine in the NAc of naive mice. These results indicate that Shati/Nat8l in the NAc, but not in the dS, plays an important suppressive role in the behavioral responses to METH by controlling the dopaminergic system via activation of group II mGluRs.
Previous studies have demonstrated functional expression of different glutamate receptor subtypes (GluRs) in both osteoblasts and osteoclasts. In the present study, we investigated the possible functional expression by osteoclasts of different glutamatergic signaling machineries including GluRs. In disagreement with the aforementioned prevailing view, no mRNA expression was found for all GluRs examined in primary cultured mouse osteoclasts differentiated from bone marrow precursors. Constitutive expression of mRNA was seen with glutamate transporters, such as excitatory amino acid transporters and cystine/glutamate antiporter, in primary osteoclasts. Glutamate significantly inhibited osteoclastogenesis at a concentration over 500 mol/L in both primary osteoclasts and preosteoclastic RAW264.7 cells without affecting the cell viability in a manner sensitive to the antiporter inhibitor. In RAW264.7 cells stably overexpressing the cystine/glutamate antiporter, the inhibition by glutamate was more conspicuous than in cells transfected with empty vector alone. The systemic administration of glutamate significantly prevented the decreased bone mineral density in both femur and tibia in addition to increased osteoclastic indices in ovariectomized mice in vivo. These results suggest that glutamate may play a pivotal role in mechanisms associated with osteoclastogenesis through the cystine/glutamate antiporter functionally expressed by osteoclasts devoid of any GluRs cloned to date. (Am J
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