This article has Open Peer Review reports available.
Angelica sinensis extract inhibits RANKL-mediated osteoclastogenesis by down-regulated the expression of NFATc1 in mouse bone marrow cells
- Lingbo Kong†1,
- Qinpeng Zhao†1,
- Xiaodong Wang1,
- Jinyu Zhu2,
- Dingjun Hao1Email author and
- Chongfei Yang2Email author
© Kong et al.; licensee BioMed Central Ltd. 2014
Received: 14 May 2014
Accepted: 10 December 2014
Published: 12 December 2014
Destructive erosion of bone or osteolysis is a major complication of inflammatory conditions such as rheumatoid arthritis (RA), periodontal disease, and periprosthetic osteolysis. Natural plant-derived products have received recent attention as potential therapeutic and preventative drugs in human disease.
The effect of Angelica sinensis (AS) extract on RANKL-induced osteoclast differentiation was examined in this study. The osteoclast precursor cell line bone marrow macrophages (BMMs) was cultured and stimulated with RANKL followed by treatment with AS at several doses. Gene expression profiles of c-Fos, c-Jun, NFATc1, TRAP, and OSCAR were sequentially evaluated.
AS extract inhibited RANKL-mediated osteoclast differentiation in BMMs in a dose-dependent manner without any evidence of cytotoxicity. AS extract strongly inhibited p38, ERK, JNK, p65 phosphorylation and I-κB degradation in RANKL-stimulated BMMs. AS extract also inhibited the mRNA expression of c-Fos, c-Jun, NFATc1, TRAP, and OSCAR in RANKL-treated BMMs. Moreover, RANKL-induced c-Fos, c-Jun and NFATc1 protein expression was suppressed by AS extract.
These results collectively suggested that AS extract demonstrated inhibitory effects on RANKL-mediated osteoclast differentiation in bone marrow macrophages in vitro, indicating that AS may therefore serve as a useful drug in the prevention of bone loss.
Osteoclasts are derived from hematopoietic stem cells, which are unique multinucleated cells responsible for bone resorption . Osteoblasts and stromal cells express receptor activators of the nuclear factor-kB (NF-kB) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) . M-CSF induces expression of RANKL receptor (RANK) as well as supports survival and proliferation in early stage precursors of osteoclast lineages in mouse bone marrow cells . RANKL is a member of the tumor necrosis factor (TNF) family and binds to the RANK receptor expressed in osteoclast precursor cells . The binding of RANKL and RANK on osteoclast progenitor cells triggers the activation of tumor necrosis factor receptor-associated factor 6 (TRAF6)  and subsequently the activation of NF-kB and mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and stressactivated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) [6, 7]. Although more detailed mechanism is still expected to be unveiled, the major signaling events triggered upon RANK ligation include recruitment of TRAF6, the activation of transcription factors NF-κB, c-Fos, AP-1, and nuclear factor of activated T cells (NFATc1) [2, 5, 8–11]. NFATc1 is a downstream transcription factor in the RANKL/RANK signal pathway and as a key molecule of osteoclastogenesis, NFATc1 induces a series of osteoclast-specific genes, including tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR) and cathepsin K −[12, 13]. c-Fos is also an essential transcription factor for osteoclastogenesis and positively regulates osteoclastogenesis via NFATc1 activation .
Many plant-derived natural products have been used in traditional medicine for the treatment of various diseases. Several compounds derived from natural products have been recently reported to possess inhibitory effects on osteoclast differentiation and function, leading to decreased bone loss in vivo . Angelica sinensis (AS) has been used to regulate menstruation, an inflammatory syndrome, in Asia for thousands of years. Recently, a component of AS extract, ligustilide has been reported to regulate several extracellular signaling pathways, including ERK1/2, p38 and SAPK/JNK . Additionally, our previous study demonstrated that AS extract could inhibit wear debris particles-induced bone resorption by attenuating proinflammatory cytokines . However, the effects of AS extract on RANKL and M-CSF functions vital to osteoclast differentiation are not clarified. In this study, we aimed to investigate the effects of AS extract on signaling pathways involved in osteoclast differentiation, activation, and survival in vitro.
Dry root slices of a popular Chinese herb, Angelica sinensis (Dang Gui), was obtained from Tong Ren Tang (Tong Ren Tang Group Co., Ltd.; Beijing, China) and extracted in water (85°C) for 4 h. The water-soluble fraction was cleared sequentially by centrifugation (3300 × g, 20 min, 4°C) and filtration through a 0.2 mm filter . From10 g dry Angelica sinensis root, about 0.8 g yellow, powdery substance was recovered after lyophilization.
Reagents and antibodies
Human RANKL and M-CSF was obtained from Peprotech (London, UK). The XTT assay kit was obtained from Roche (Indianapolis, IN, USA). Antibodies for c-Fos, c-Jun and nuclear factor of activated T cells 1 (NFATc1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Western blot antibodies for phosphor-p65, p-65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-p38, p38, and I-κB were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); β-actin antibody was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).
All the animal work and approach have been approved by the IACUC of the Hong-Hui Hospital, Xi’an Jiaotong University College of Medicine and conducted strictly followed by “the institutional guidelines for the care and use of laboratory animals at the Jiaotong University College of Medicine”. Bone marrow cells were obtained by flushing the femurs and tibiae of 5-week-old ICR mice with α-minimum essential medium (α-MEM; Gibco BRL, Gaithersburg, MD, USA) and suspended in α-MEM supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, USA). Non-adherent cells were collected and cultured for 3 days in the presence of M-CSF (20 ng/ml). Floating cells were discarded and adherent cells on dish bottoms were classified as bone marrow derived macrophages (BMMs). BMMs were seeded at 3.5 × 104 cells/well in α-MEM/10% FBS, and were cultured in the presence of M-CSF (20 ng/ml) and RANKL (40 ng/ml) for 4 days in the presence or absence of AS extract. Osteoclasts were identified by staining for tartrate-resistant acid phosphatase (TRAP) activity, as described below. TRAP-positive multinucleated cells with greater than three nuclei were counted as osteoclasts. Cytotoxicity assay for AS extract treated BMMs were plated in 96-well plates at a density of 1 × 104 cells/well in triplicate. Cells were treated with M-CSF (20 ng/ml) and increasing concentrations of AS extract were added to the mix. Cells were incubated for 3 days. After 3 days, XTT reagent (50 μl) was added to each well. Wells were incubated for 4 h. The optical density at 450 nm was analyzed with an ELISA reader.
RAW 264.7 cells were seeded in 48-well plates at a density of 3 × 103cells/well in triplicate and cultured for 4 days in the presence of increasing concentrations of AS extract. After 4 days, the cells were fixed and stained with Hematoxylin Sigma-Aldrich, Inc. (St. Louis, MO, USA). Colonies with 50 or greater cells were counted.
Real time RT-PCR analysis for c-Fos, c-Jun, NFATc1, TRAP, osteoclast-associated receptor (OSCAR)
Total RNA was isolated with TRIzol reagent (Invitrogen Inc., USA) per the manufacturer’s instructions. RNA (1 μg) was reverse transcribed using oligo dT primers (10 μg) and dNTPs (10 mM). The mixture was incubated at 65°C for 5 min, and cDNA was produced by incubating at 42°C for 50 min with first strand buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 100 mM DTT, RNase inhibitor, and Superscript II reverse transcriptase (Invitrogen). The cDNA was amplified using the following primer sets: c-Fos, 5′-CTGGTGCAGCCCACTCTGGTC- 3′ (forward) and 5′-CTTTCAGCAGATTGGCAATCTC-3′ (reverse); c-Jun, 5′-ACT CGG ACC TTC TCA CGT CG- 3′ (forward) and 5′-TAG ACC GGA GGC TCA CTG TG −3′ (reverse); NFATc1, 5′-CTCGAAAGACAGCACTGGAGCAT-3′ (forward) and 5′-CGGCTGCCTTCCGTCTCATAG-3′ (reverse); TRAP, 5′-CTGGAGTGCACGATGCCAGCGACA-3′ (forward) and 5′-TCCGTGCTCGGCGATGGACCAGA-3′ (reverse); OSCAR, 5′-CTGCTGGTAACGGATCAGCTCCCCAGA-3′ (forward) and 5′-CCAAGGAGCCAGAACCTTCGAAACT-3′ (reverse); and GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA- 3′(reverse). PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen) in triplicates according to the manufacturer’s instructions. Relative levels of c-Fos, c-Jun, NFATc1, TRAP, and OSCAR were normalized to GAPDH.
Western blot analysis
BMMs or osteoclasts were lysed in a buffer containing 50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, and protease inhibitors. The lysates were centrifuged at 14,000 × g for 20 min and supernatants were collected. Protein concentrations of supernatants were determined. Cellular proteins (30 μg) were resolved by 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidene difluoride membranes (Milipore, Bedford, MA, USA). Non-specific interactions were blocked with 5% skim milk for 2 h and were then probed with the appropriate primary antibodies. Membranes were incubated with the appropriate secondary antibodies attached to horseradish peroxidase, and immunoreactivity was detected with enhanced chemiluminescence reagents. Densitometric values were quantified for each band with the Image Pro-plus program version 4.0.
All data are expressed as means ± standard deviation (SD). Statistical analysis was done using SPSS software package ver. 11.0 (SPSS, Chicago, IL); one-way ANOVA was used for comparison among the different groups. Post hoc testing of differences between groups was performed by using Duncan’s test when the ANOVA was significant. All results were considered to be significant at the 5% critical level (P < 0.05).
Inhibition of osteoclast differentiation by AS extract
The cytotoxic effect of AS extract
AS extract generated a highly negative effect on osteoclastogenesis. We measured the effects of AS extract on bone marrow cells with the XTT assay to exclude the possibility that the inhibition was due to cytotoxicity. AS extract demonstrated no cytotoxic effects at the same doses which effectively inhibited osteoclast formation (Figure 1C). Also, AS extract did not affect in RAW 264.7 cells colony formation (Figure 1D), suggesting that osteoclastogenesis suppression by AS extract was not due to toxic effects on BMMs.
AS extract inhibits a variety of signals transduced by RANKL
RANKL induced c-Fos, c-Jun, NFATc1, TRAP, and OSCAR mRNA expression is reduced by AS extract
AS extract also inhibits c-Fos, c-Jun and NFATc1 protein expression
Western blots were examined to verify the effects of AS extract on c-Fos, c-Jun and NFATc1 protein expression. c-Fos, c-Jun and NFATc1 protein levels were increased in response to RANKL, but c-Fos, c-Jun and NFATc1 expression was significantly inhibited by AS extract (Figure 3B). These results demonstrated that the inhibitory effects of AS extract involved the inhibition of major transcription factors such as c-Fos, c-Jun and NFATc1.
Bone resorption by osteoclasts is frequently caused by excessive RANKL signaling which has been a valuable target for the treatment of pathological bone loss. RANKL activates a series of major intracellular signal transducing pathways including NFATc1, NF-κB, phosphoinositide 3 kinase (PI3K)-Akt, JNK, ERK, and p38 MAPK. AS is one of the most popular Chinese herbs in both clinical field and research field. Su et al.  reported that a component of AS extract: ligustilide suppresses the LPS-induced production of NO, PGE2 and TNF-α by inhibiting both IKK/NF-κB and MAPK/AP-1 signaling. Recently we  demonstrated that AS extract could inhibit bone resorption by attenuating proinflammatory cytokines. However, the effect of AS extract on RANKL-induced osteoclast formation, especially RANKL-mediated intracellular signal pathway still is an interesting question that remains to be investigated.
The MAPK group of enzymes selectively phosphorylates serine and threonine residues in response to extracellular stimuli and transmits the stimuli from the cell surface to the nucleus . MAPK is primarily composed of JNK, ERK, and p38 in mammalian cells. RANKL and RANK receptor binding expressed in osteoclast precursors provides a link between distinct signaling molecules such as JNK, ERK, and p38 MAPK . p38 MAPK signaling is particularly crucial in the early stages of osteoclast differentiation as it promotes the activity of microphthalmia-associated transcription factor (MITF) and TRAP expression. Inhibition of p38 MAPK with SB203580 has a negative effect on osteoclast formation . AS extract suppressed RANKL-induced the phosphorylation of p38, ERK and JNK activity in the present study (Figure 2).
The activation of signaling molecules induces transcription factors such as NF-κB, NFATc1, and AP-1 that are essential for osteoclast differentiation . NF-κB is an important signal mediator for inflammatory and immune reactions and is a major transcription factor for RANKL-activated osteoclastogenesis . I-κB is attached to NF-κB preventing it from migrating into the nucleus, and phosphorylation with I-κB kinase (IKK) separates the two proteins. Subsequent ubiquitination and proteosome degradation of I-κB allows the transfer of NF-κB into the nucleus and transcription of the target gene . Although our previous study showed that one factor of AS extract: ligustilide decreases NF-κB luciferase activity in a reporter assay , we also confirmed that AS extract inhibits NF-κB/I-κB protein expression in this experiments. These results are consistent with RANKL activation of NF-κB in osteoclastic precursor cells through IKK activation and susequent I-κB phosphorylation and degradation (Figure2).
NFATc1 is a master regulator of osteoclastogenesis which autoamplifies and conducts the expression of osteoclast specific genes such as activator protein-1 (AP-1), TRAP, calcitonin receptor, OSCAR, and cathepsin K [10, 23–25]. The RANKL-induced NFATc1 expression is mediated by the activation of AP-1 consisting of c-Fos and c-Jun [26, 27]. c-Fos was reported to be critical for transcriptional activation of NFATc1 in RANKL-induced osteoclastogenesis . Putative c-Fos binding sites were mapped in the promoter region of NFATc1, and the NFATc1 expression was abolished in the osteoclast precursors lacking c-Fos .On the other hand, Ikeda et al.  reported that RANKL could not induce expression of NFATc1 in dominant-negative c-Jun transgenic mouse. It is possible that the induction of NFATc1 may be cooperatively up-regulated by c-Fos and c-Jun, which make up each other for the induction . The present study demonstrates the reduced both c-Fos and c-Jun expression in AS extract treated BMM cells (Figure 3). Therefore, the reduced c-Fos and c-Jun expression is suggested to cause the impaired activation of NFATc1, followed by the inhibition of RANKL-induced osteoclast formation. Additionally mRNA levels of major osteoclast marker TRAP and OSCAR was also inhibited by AS extract (Figure 3). These data suggest that the AP-1 transcription factor is the targets of AS extract-induced inhibition of osteoclastogenesis.
In summary, the present study demonstrated that AS inhibited osteoclastogenesis from macrophages and bone resorption in vitro. AS also reduced the RANKL-induced expression of osteoclastic marker genes. In addition, AS attenuated RANKL-induced ERK, p38, JNK, NF-κB, AP-1 and NFATc1 activation. Although additional experiments are needed to confirm the efficacy of AS in treating disease conditions in vivo, our results indicate that AS has potential as a therapy for disorders associated with bone loss.
The authors wish to thank Guo Hua (Honghui hospital) for overseeing the conduct of the study, Rui Ma, the Fourth Military Medical University (Xi’an, China) for statistical analysis assistance and Shu Zhu for overseeing data management. We thank Lifeng Yu for regulatory and quality activities and for his technical support. We thank Xiaorui Cao, PhD, for his assistance with manuscript preparation and review.
- Han Bok K, Byeong Ki L: Inhibition of osteoclast differentiation and bone resorption by rotenone, through down-regulation of RANKL-induced c-Fos and NFATc1 expression. Bone. 2010, 46: 724-731. 10.1016/j.bone.2009.10.042.View ArticleGoogle Scholar
- Boyle WJ, Simonet WS, Lancey DL: Osteoclast differentiation and activation. Nature. 2003, 423: 337-342. 10.1038/nature01658.View ArticlePubMedGoogle Scholar
- Arai F, Miyamoto T, Ohneda O, Inada T, Sudo T, Brasel K: Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors. J Exp Med. 1999, 190: 1741-1754. 10.1084/jem.190.12.1741.View ArticlePubMedPubMed CentralGoogle Scholar
- Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ: Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev. 1999, 20: 345-357. 10.1210/edrv.20.3.0367.View ArticlePubMedGoogle Scholar
- Kobayashi N, Kadono Y, Naito A, Matsumoto K, Yamamoto T, Tanaka S: Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis. EMBO J. 2001, 20: 1271-1280. 10.1093/emboj/20.6.1271.View ArticlePubMedPubMed CentralGoogle Scholar
- Hotokezaka H, Sakai E, Kanaoka K, Saito K, Matsuo K, Kitaura H: U0126 and PD98059, specific inhibitors of MEK, accelerate differentiation of RAW264.7 cells into osteoclast-like cells. J Biol Chem. 2002, 277: 47366-47372. 10.1074/jbc.M208284200.View ArticlePubMedGoogle Scholar
- Matsuo K, Galson DL, Zhao C, Peng L, Laplace C, Wang KZ: Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos. J Biol Chem. 2004, 279: 26475-26480. 10.1074/jbc.M313973200.View ArticlePubMedGoogle Scholar
- David JP, Sabapathy K, Hoffmann O, Idarraga MH, Wagner EF: JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and –independent mechanisms. J Cell Sci. 2002, 115: 4317-4325. 10.1242/jcs.00082.View ArticlePubMedGoogle Scholar
- Lee SE, Woo KM, Kim SY, Kim HM, Kwack K, Lee ZH: The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation. Bone. 2002, 30: 71-77.View ArticlePubMedGoogle Scholar
- Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H: Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell. 2002, 3: 889-901. 10.1016/S1534-5807(02)00369-6.View ArticlePubMedGoogle Scholar
- Matsuo K, Owens JM, Tonko M, Elliott C, Chambers TJ, Wagner EF: Fos is a transcriptional target of c-Fos during osteoclast differentiation. Nat Genet. 2000, 24: 184-187. 10.1038/72855.View ArticlePubMedGoogle Scholar
- Nakashima T, Takayanagi H: Osteoimmunology: cross talk between the immune and bone systems. J Clin Immunol. 2009, 29: 555-567. 10.1007/s10875-009-9316-6.View ArticlePubMedGoogle Scholar
- Zhao Q, Wang X, Liu Y, He A, Jia R: NFATc1: function in osteoclasts. Int J Biochem Cell Biol. 2010, 42: 576-579. 10.1016/j.biocel.2009.12.018.View ArticlePubMedGoogle Scholar
- Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, Fleisch HA: c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science. 1994, 266: 443-448. 10.1126/science.7939685.View ArticlePubMedGoogle Scholar
- Su YW, Chiou WF, Chao SH: Ligustilide prevents LPS-induced iNOS expression in RAW 264.7 macrophages by preventing ROS production and down-regulating the MAPK, NF-κB and AP-1signaling pathways. Int Immuno Pharmacol. 2011, 11: 1166-1172. 10.1016/j.intimp.2011.03.014.View ArticleGoogle Scholar
- Yang C, Niu S, Lifeng Y: The aqueous extract of Angelica sinensis, a popular Chinese herb, inhibits wear debris-Induced Inflammatory osteolysis in mice. J Surg Res. 2012, 176: 476-483. 10.1016/j.jss.2011.08.011.View ArticlePubMedGoogle Scholar
- Wang H, Li W, Li J: The aqueous extract of a popular herbal nutrient supplement, Angelica sinensis, protects mice against lethal endotoxemia and sepsis. J Nutr. 2006, 136: 360-PubMedPubMed CentralGoogle Scholar
- Kim K, Kim JH, Lee J, Jin HM, Lee SH, Fisher DE: Nuclear factor of activated T cells c1 induces osteoclast-associated receptor gene expression during tumor necrosis factor-related activation-induced cytokine mediated osteoclastogenesis. J Biol Chem. 2005, 280: 35209-35216. 10.1074/jbc.M505815200.View ArticlePubMedGoogle Scholar
- Matsumoto M, Sudo T, Saito T, Osada H, Tsujimoto M: Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). J Biol Chem. 2000, 275: 31155-31161. 10.1074/jbc.M001229200.View ArticlePubMedGoogle Scholar
- Takayanagi H: Osteoimmunology. Nat Rev Immunol. 2007, 7: 292-304. 10.1038/nri2062.View ArticlePubMedGoogle Scholar
- Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, Bravo R: Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2. Nat Med. 1997, 3: 1285-1289. 10.1038/nm1197-1285.View ArticlePubMedGoogle Scholar
- Hayden MS, Ghosh S: Signaling to NF-κB. Genes Dev. 2004, 18: 2124-2195.View ArticleGoogle Scholar
- Crotti TN, Flannery M, Walsh NC, Fleming JD, Goldring SR, McHugh KP: NFATc1 regulation of the human beta 3 integrin promoter in osteoclast differentiation. Gene. 2006, 372: 92-102.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim Y, Sato K, Asagiri M, Morita I, Soma K, Takayanagi H: Contribution of nuclear factor of activated T cells c1 to the transcriptional control of immunoreceptor osteoclast-associated receptor but not triggering receptor expressed by myeloid cells-2 during osteoclastogenesis. J Biol Chem. 2005, 280: 32905-32913. 10.1074/jbc.M505820200.View ArticlePubMedGoogle Scholar
- Matsumoto M, Koqawa M, Wada S, Takayangi H, Tsujimoto M, Katayama S: Essential role of p38 mitogen-activated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1. J Biol Chem. 2004, 279: 45969-45979. 10.1074/jbc.M408795200.View ArticlePubMedGoogle Scholar
- Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV: Criticalroles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Invest. 2004, 114: 475-484. 10.1172/JCI200419657.View ArticlePubMedPubMed CentralGoogle Scholar
- Wagner EF, Eferl R: Fos/AP-1 proteins in bone and the immune system. Immunol Rev. 2005, 208: 126-140. 10.1111/j.0105-2896.2005.00332.x.View ArticlePubMedGoogle Scholar
- Mohamed S: Interleukin-10 inhibits RANKL-mediated expression of NFATc1 in part via suppression of c-Fos and c-Jun in RAW264.7 cells and mouse bone marrow cells. Bone. 2007, 41: 592-602. 10.1016/j.bone.2007.05.016.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/481/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.