Isolation and Characterization of MC3T3-E1 Preosteoblast Subclones with Distinct In Vitro and In Vivo Differentiation/Mineralization Potential

Wang, Dian; Christensen, Kurt D.; Chawla, Kanwal; Xiao, Guozhi; Krebsbach, Paul H.; Franceschi, Renny T.

doi:10.1359/jbmr.1999.14.6.893

Cited by 606 publications

(536 citation statements)

References 55 publications

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“…were similar to those of alkaline phosphatase (ALP), an osteoblast marker, 14 and bone sialoprotein (BSP), a common osteoblast differentiation marker gene, 14 especially at the late stages of differentiation (414 days; Figure 4a). The activation of the Wnt/β-catenin pathway during the differentiation of MC3T3E1 cells into osteoblasts was confirmed by the induction of fibroblast growth factor 18 (Fgf18), a direct and specific target of the Wnt/β-catenin pathway, 15 and by an increase in the level of β-catenin (Figures 4a and b).…”

Section: Cxxc5mentioning

confidence: 56%

CXXC5 is a negative-feedback regulator of the Wnt/β-catenin pathway involved in osteoblast differentiation

Yoon

Yun

et al. 2015

Cell Death Differ

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The positive roles of the Wnt/β-catenin pathway in osteoblast differentiation and bone mineral density (BMD) maintenance have been clearly demonstrated in both animal experiments and clinical investigations. CXXC finger protein 5 (CXXC5), a recently identified negative regulator of the Wnt/β-catenin pathway, showed altered cellular localization and function, which were dependent on the cell type in previous studies. However, the in vivo function of CXXC5 has not been clearly investigated yet. Here, we characterized CXXC5 as a negative regulator of osteoblast differentiation and bone formation. Deficiency of CXXC5 resulted in elevated BMD in mice without any severe gross developmental abnormalities. CXXC5 exerted a negative-feedback effect on the Wnt/β-catenin pathway via Wnt-dependent binding to Dishevelled (Dvl) during osteoblast differentiation. Suppression of the Dvl-CXXC5 interaction using a competitor peptide resulted in the activation of the Wnt/β-catenin pathway and osteoblast differentiation, and accelerated thickness growth of ex vivo-cultured calvariae. Overall, CXXC5 is a negative-feedback regulator induced by Wnt/β-catenin signaling that inhibits osteoblast differentiation and bone formation via interaction with Dvl. Cell Death and Differentiation (2015) 22, 912-920; doi:10.1038/cdd.2014.238; published online 30 January 2015Bone is an extremely dynamic tissue at the microscopic level. A dynamic process, called bone remodeling, takes place seamlessly in the bone to repair microdamage and to replace old bone with new bone. 1 The resorption of old bone and the formation of new bone must be in balance to maintain homeostasis and a constant mass of bone. Osteoblasts have been identified as an essential factor in regulation of the bone remodeling process, which produce bone matrix and differentiate into osteocytes for bone formation, as well as regulate differentiation and activation of osteoclasts for bone resorption. 2 The Wnt/β-catenin pathway is receiving increased attention as a main regulatory pathway for osteoblast differentiation. [2][3][4] Wnt-dependent nuclear accumulation of an effector protein of the pathway, β-catenin, is a major trigger of osteoblast differentiation and bone formation. 2 Many other intracellular and extracellular components of the Wnt/β-catenin pathway are known to regulate osteoblast differentiation. 4 Especially two negative regulators of this pathway, Dickkopf 1 and sclerostin, have been highlighted as osteoblast and osteocytespecific negative regulators of bone formation. 5 CXXC finger protein 5 (CXXC5) is a member of a small protein family in which the members contain CXXC-type zincfinger domain. 6 However, unlike other members of this family, CXXC5 lacks a KFGG motif, which is essential for non-methylated CpG recognition that regulates chromatin remodeling. 7 CXXC5 localizes to the cytosol or nucleus depending on particular cell type in different tissues. Localization of CXXC5 in the nucleus was observed in promyelocytic leukemia cells. 7 CXXC5 has a role as a nuclear t...

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Section: Cxxc5mentioning

confidence: 56%

CXXC5 is a negative-feedback regulator of the Wnt/β-catenin pathway involved in osteoblast differentiation

Yoon

Yun

et al. 2015

Cell Death Differ

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“…HA deposition is the hallmark of osteogenic differentiation in MC3T3 cell culture [67]. Silencing Runx2 and Osx over 7, 14, and 21 days produced a significant reduction in HA deposition (Fig.…”

Section: Discussionmentioning

confidence: 89%

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

Shrivats

Hsu

Averick

et al. 2015

Clinical Orthopaedics &Amp; Related Research

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Background Heterotopic ossification (HO) may occur after musculoskeletal trauma, traumatic brain injury, and total joint arthroplasty. As such, HO is a compelling clinical concern in both military and civilian medicine. A possible etiology of HO involves dysregulated signals in the bone morphogenetic protein osteogenic cascade. Contemporary treatment options for HO (ie, nonsteroidal antiinflammatory drugs and radiation therapy) have adverse effects associated with their use and are not biologically engineered to abrogate the molecular mechanisms that govern osteogenic differentiation. Questions/purposes We hypothesized that (1) nanogelmediated short interfering RNA (siRNA) delivery against Runt-related transcription factor 2 (Runx2) and osterix (Osx) genes will decrease messenger RNA expression; (2) inhibit activity of the osteogenic marker alkaline phosphatase (ALP); and (3) inhibit hydroxyapatite (HA) deposition in osteoblast cell cultures. Methods Nanogel nanostructured polymers delivered siRNA in 48-hour treatment cycles against master osteogenic regulators, Runx2 and Osx, in murine calvarial preosteoblasts (MC3T3-E1.4) stimulated for osteogenic differentiation by recombinant human bone morphogenetic protein (rhBMP-2). The efficacy of RNA interference (RNAi) therapeutics was determined by quantitation of messenger RNA knockdown (by quantitative reverse transcription-polymerase chain reaction), downstream protein knockdown (determined ALP enzymatic activity assay), and HA deposition (determined by OsteoImage TM assay). Results Gene expression assays demonstrated that nanogelbased RNAi treatments at 1:1 and 5:1 nanogel:short interfering RNA weight ratios reduced Runx2 expression by 48.59% ± 19.53% (p \ 0.001) and 43.22% ± 18.01% (both p \ 0.001). The same 1:1 and 5:1 treatments against both Runx2 and Osx reduced expression of Osx by 51.65% ± 10.85% and 47.65% ± 9.80% (both p \ 0.001). Moreover, repeated 48-hour RNAi treatment cycles against Runx2 and Osx rhBMP-2 administration reduced ALP activity after 4 and 7 days. ALP reductions after 4 days in culture by nanogel 5:1 and 10:1 RNAi treatments were 32.4% ± 12.0% and 33.6% ± 13.8% (both p \ 0.001). After 7 days in culture, nanogel 1:1 and 5:1 RNAi treatments produced 35.9% ± 14.0% and 47.7% ± 3.2% reductions in ALP activity. Osteoblast mineralization data after 21 days suggested that Two of the authors (ARS, EH) contributed equally.

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“…To this end, we used subclone 24 of MC3T3-E1 cells (MC-24), which were characterized originally by Franceschi as a nondifferentiating subclone of preosteoblasts. (25) While not undergoing spontaneous differentiation, like the more commonly used subclone 14 of MC3T3-E1 cells, (25) we show for the first time that this MC-24 subclone can be induced by BMP-2 to differentiate into osteoblasts that produce mineralized nodules.…”

Section: Discussionmentioning

confidence: 73%

“…(8,9) The experimental study of BMP-2 signaling and BMP responsiveness is challenging and is complicated by at least two issues: (1) cross-talk with numerous other pathways (15,16) and (2) lack of synchronization of cellular differentiation in spontaneously differentiating heterogeneous preosteoblast cultures. (25) Several studies have demonstrated that MAPKs are required for BMP-2-induction of the osteoblastic phenotype and mineral deposition in vitro. (18,21,30) In particular, the MAPK JNK is reported to be required for both late-stage spontaneous differentiation of osteoblasts and BMP-2-induced differentiation of preosteoblasts and pleuripotent cells in vitro.…”

Section: Discussionmentioning

confidence: 99%

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Activation of c-Jun NH2-terminal kinase 1 increases cellular responsiveness to BMP-2 and decreases binding of inhibitory Smad6 to the type 1 BMP receptor

Liu

Viggeswarapu

et al. 2010

Journal of Bone and Mineral Research

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Bone morphogenetic protein 2 (BMP-2) plays a critical role in the differentiation of precursor cells and has been approved for clinical application to induce new bone formation. To date, unexpectedly high doses of recombinant BMP-2 have been required to induce bone healing in humans. Thus, enhancing cellular responsiveness to BMP-2 potentially has critically important clinical implications. BMP responsiveness may be modulated in part by cross-talk with other signaling pathways, including mitogen-activated protein kinases (MAPKs). c-Jun NH 2 -terminal kinase (JNK) is a MAPK that has been reported to be required for late-stage differentiation of preosteoblasts and BMP-2-induced differentiation of preosteoblasts and pleuripotent cells. In this study we determined that MC3T3-E1-clone 24 cells (MC-24) can be induced by BMP-2 to differentiate into mineralizing osteoblast cultures. Using this inducible system, we employed both JNK loss-of-function and gain-of-function reagents to make three key observations: (1) JNK is required for phosphorylation of Smad1 by BMP-2 and subsequent activation of Smad1 signaling and osteoblast differentiation, (2) JNK1, but not JNK2, is required for BMP-2-induced formation of mineralized nodules, and (3) JNK1 activation decreases binding of inhibitory Smad6 to the type I BMP receptor (BMPR-I) and reciprocally increases binding of Smad1, both observations that would increase responsiveness to BMP-2. Understanding this and other pathways that lead to increased cellular responsiveness to BMPs could greatly aid more cost-effective and safe clinical delivery of these important molecules. ß

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Isolation and Characterization of MC3T3-E1 Preosteoblast Subclones with Distinct In Vitro and In Vivo Differentiation/Mineralization Potential

Cited by 606 publications

References 55 publications

CXXC5 is a negative-feedback regulator of the Wnt/β-catenin pathway involved in osteoblast differentiation

CXXC5 is a negative-feedback regulator of the Wnt/β-catenin pathway involved in osteoblast differentiation

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

Activation of c-Jun NH2-terminal kinase 1 increases cellular responsiveness to BMP-2 and decreases binding of inhibitory Smad6 to the type 1 BMP receptor

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