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...
Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial–temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell–cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell–cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell–cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo.
Highlights d Enlarged thymus in K5D1 mice produces immunocompetent and self-tolerant T cells d Enlarged thymus in K5D1 mice enables proteomic analysis of cTECs and mTECs d Trans-omics profiles identify signature molecules that characterize cTECs and mTECs d b5t deficiency specifically affects proteasomal subunit composition in cTECs
N-methyl-D-aspartate (NMDA) receptors for the central neurotransmitter l-glutamate (Glu) have been shown to be present in both osteoblasts and osteoclasts. Sustained exposure to the NMDA channel antagonist dizocilpine (MK-801) significantly prevented increases in both alkaline phosphatase activity and Ca2+ accumulation in a concentration-dependent manner in osteoblasts cultured for 7-28 days in vitro (DIV), without significantly affecting cell survivability. Osteocalcin expression was markedly reduced in the presence of MK-801 in osteoblasts cultured for 28 DIV. Both an NMDA domain antagonist and a glycine domain antagonist similarly prevented Ca2+ accumulation in osteoblasts exposed for 28 consecutive DIV. MK-801 was effective in significantly inhibiting Ca2+ accumulation determined at 28 DIV in osteoblasts exposed before 7 DIV but was ineffective in cells exposed after 11-21 DIV. Sustained exposure to MK-801 significantly inhibited DNA binding activity and expression of core binding factor alpha-1 (CBFA1) in osteoblasts exposed after 7 DIV up to 28 DIV, but not in those exposed before 7 DIV. These results suggest that heteromeric NMDA receptor channels may be functionally expressed to regulate mechanisms underlying cellular differentiation rather than proliferation and/or maturation through modulation of expression of CBFA1 in cultured rat calvarial osteoblasts.
Loss- and gain-of function approaches modulating canonical Wnt/β-catenin activity have established a role for the Wnt/β-catenin pathway during tooth development. Here we show that Wnt/β-catenin signaling is required in the dental mesenchyme for normal incisor development, as locally restricted genetic inactivation of β-catenin results in a splitting of the incisor placode, giving rise to two incisors. Molecularly this is first associated with down-regulation of Bmp4 and subsequent splitting of the Shh domain at a subsequent stage. The latter phenotype can be mimicked by ectopic application of the BMP antagonist Noggin. Conditional genetic inactivation of Bmp4 in the mesenchyme reveals that mesenchymal BMP4 activity is required for maintenance of Shh expression in the dental ectoderm. Taken together our results indicate that β-catenin together with Lef1 and Tcf1 are required to activate Bmp4 expression in order to maintain Shh expression in the dental ectoderm. This provides a mechanism whereby the number of incisors arising from one placode can be varied through local alterations of a mesenchymal signaling circuit involving β-catenin, Lef1, Tcf1 and Bmp4.
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