Induction of mouse c-src in RAW264 cells is dependent on AP-1 and NF-κB and important for progression to multinucleated cell formation

Kumagai, Naoya; Ohno, Keita; Tameshige, Ryusuke; Hoshijima, Mitsuhiro; Yogo, Keiichiro; Ishida, Norihiro; Takeya, Tatsuo

doi:10.1016/j.bbrc.2004.10.094

Cited by 17 publications

(20 citation statements)

References 46 publications

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“…Analogously, in our study osteoclast precursors stayed firmly attached to osteoblasts when c-Src activity was inhibited by AZD0530. Osteoclast formation from RAW264 cells, in which c-Src was knocked out, was also completely inhibited, possibly due to hampered cell migration (31). In c-Src knockout mice, however, osteoclast formation was enhanced rather than inhibited (12).…”

Section: Discussionmentioning

confidence: 99%

The Src Inhibitor AZD0530 Reversibly Inhibits the Formation and Activity of Human Osteoclasts

Vries

Mullender

Duin

et al. 2009

Molecular Cancer Research

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Tumor cells in the bone microenvironment are able to initiate a vicious cycle of bone degradation by mobilizing osteoclasts, multinucleated cells specialized in bone degradation. c-Src is highly expressed both in tumors and in osteoclasts. Therefore, drugs like AZD0530, designed to inhibit Src activity, could selectively interfere with both tumor and osteoclast activity. Here we explored the effects of AZD0530 on human osteoclast differentiation and activity. The effect on osteoclasts formed in vivo was assessed in mouse fetal calvarial explants and in isolated rabbit osteoclasts, where it dose-dependently inhibited osteoclast activity. Its effect on formation and activity of human osteoclasts in vitro was determined in cocultures of human osteoblasts and peripheral blood mononuclear cells. AZD0530 was most effective in inhibiting osteoclast-like cell formation when present at the onset of osteoclastogenesis, suggesting that Src activity is important during the initial phase of osteoclast formation. Formation of active phosphorylated c-Src, which was highly present in osteoclast-like cells in cocultures and in peripheral blood mononuclear cell monocultures, was significantly reduced by AZD0530. Furthermore, it reversibly prevented osteoclast precursor migration from the osteoblast layer to the bone surface and subsequent formation of actin rings and resorption pits. These data suggest that Src is pivotal for the formation and activity of human osteoclasts, probably through its effect on the distribution of the actin microfilament system. The reversible effect of AZD0530 on osteoclast formation and activity makes it a promising candidate to temper osteoclastic bone degradation in bone diseases with enhanced osteoclast activity such as osteolytic metastatic bone disease. (Mol Cancer Res 2009;7(4):476-88)

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Section: Discussionmentioning

confidence: 99%

The Src Inhibitor AZD0530 Reversibly Inhibits the Formation and Activity of Human Osteoclasts

Vries

Mullender

Duin

et al. 2009

Molecular Cancer Research

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“…Luciferase reporter gene assays were conducted using the 1.9-kb murine c-Src wild-type (wt) and AP-1 point mutant luciferase reporter genes (Kumagai et al, 2004). Luciferase activity was determined upon normalization of transfection efficiency using a cotransfected ␤-galactosidase reporter gene (Katiyar et al, 2007).…”

Section: Cell Culture Virus Production and Reporter Gene Assaysmentioning

confidence: 99%

Disruption of c-Jun Reduces Cellular Migration and Invasion through Inhibition of c-Src and Hyperactivation of ROCK II Kinase

Jiao¹,

Katiyar²,

Liu³

et al. 2008

MBoC

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The spread of metastatic tumors to different organs is associated with poor prognosis. The metastatic process requires migration and cellular invasion. The protooncogene c-jun encodes the founding member of the activator protein-1 family and is required for cellular proliferation and DNA synthesis in response to oncogenic signals and plays an essential role in chemical carcinogenesis. The role of c-Jun in cellular invasion remains to be defined. Genetic deletion of c-Jun in transgenic mice is embryonic lethal; therefore, transgenic mice encoding a c-Jun gene flanked by LoxP sites (c-junf/f) were used. c-jun gene deletion reduced c-Src expression, hyperactivated ROCK II signaling, and reduced cellular polarity, migration, and invasiveness. c-Jun increased c-Src mRNA abundance and c-Src promoter activity involving an AP-1 site in the c-Src promoter. Transduction of c-jun−/− cells with either c-Jun or c-Src retroviral expression systems restored the defective cellular migration of c-jun−/− cells. As c-Src is a critical component of pathways regulating proliferation, survival, and metastasis, the induction of c-Src abundance, by c-Jun, provides a novel mechanism of cooperative signaling in cellular invasion.

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“…Osteoclast precursors were generated from mouse bone marrow cells by macrophage-colony stimulating factor (M-CSF) treatment as described previously. (20) Cells were fixed and stained for TRACP activity as described previously. (3) Isolation of mRNA, reverse transcription, and hybridization Pooled total RNA was isolated from RAW264 cells at 0, 2, 5, 12, 24, 36, 48, 60, and 72 h after treatment with RANKL or GST.…”

Section: Cell Culture and Osteoclastogenesis In Vitromentioning

confidence: 99%

CCR1 Acts Downstream of NFAT2 in Osteoclastogenesis and Enhances Cell Migration

Ishida

Hayashi

Hattori

et al. 2006

Journal of Bone and Mineral Research

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We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKLstimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKLdependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration. Introduction:We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells. Materials and Methods: RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay. Results and Conclusions:We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA-or CCR1 siRNA-treated cells were used. Moreover, treatment with a G␣ inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.

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Induction of mouse c-src in RAW264 cells is dependent on AP-1 and NF-κB and important for progression to multinucleated cell formation

Cited by 17 publications

References 46 publications

The Src Inhibitor AZD0530 Reversibly Inhibits the Formation and Activity of Human Osteoclasts

The Src Inhibitor AZD0530 Reversibly Inhibits the Formation and Activity of Human Osteoclasts

Disruption of c-Jun Reduces Cellular Migration and Invasion through Inhibition of c-Src and Hyperactivation of ROCK II Kinase

CCR1 Acts Downstream of NFAT2 in Osteoclastogenesis and Enhances Cell Migration

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