8 research outputs found
Molecular targets of ZAS3 in RANK signaling.
<p>Shown is a schematic diagram depicting the multiple targets of ZAS3 involved in the regulation of RANK signaling, important for osteoclastogenic differentiation and function. Binding of RANKL to RANK activates cytoplasmic ZAS3 that (i) induces expression of TRAF6 and association of ZAS3 and TRAF6 leading to the recruitment of TGF-β-activated kinase 1 (TAK1) binding protein 2/3 (TAB2/3) to polyubiquitinated TRAF6, which in turn activates TAB1/TAK1, inhibitor of NF-kB alpha (IkBα) kinase (IKK) complexes, and nuclear translocation of NF-kB p50/p65; (ii) Simultaneously, ZAS3 activates mitogen-activated protein kinases to activate the transcription factor AP-1 via phosphorylation of p38 and assembly of c-Jun/Fos; (iii) ZAS3 associates with c-Jun to activate AP1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017161#pone.0017161-Oukka2" target="_blank">[13]</a>; (iv) ZAS3 itself can act as a transcription factor, i.e., it translocates into the nucleus and binds to gene regulatory elements to activate transcription of osteoclast-associated genes, such as TRAP and CTSK, and probably NFATc1; and (iv) through the activation of NF-kB, ZAS3 also activates NFATc1 that is shown to integrate sRANKL signaling in the terminal differentiation of osteoclasts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017161#pone.0017161-Asagiri1" target="_blank">[30]</a>. Additionally, ZAS3 mobilizes intracellular calcium, probably through one of its target genes, the calcium binding protein <i>S100A4/mts1 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017161#pone.0017161-Hjelmsoe1" target="_blank">[4]</a>, to activate calcineurin causing the dephosphorylation and nuclear translocation of NFATc1. ZAS3 individually, or in a transcriptional complex in conjunction with NTAFc1 and NF-kB through association with AP-1 drives the transcription program for osteoclast differentiation. Important signaling molecules, transcription factors, or enzymes involved in RANK signaling whose expression levels or protein-protein interactions shown here to be regulated by ZAS3 are indicated with asterisks.</p
ZAS3 is essential for RANKL-mediated osteoclastogenesis of bone marrow precursors.
<p>(A) Western blot analysis of total protein lysates prepared from bone marrow macrophages (BMM) cultured without (−) or with (+) sRANKL (50 ng/ml) for 3 days. Bone marrow aspirates isolated from WT mice were cultured in complete medium with M-CSF (50 ng/ml) for 3 days, and non-adherent cells were harvested and further cultured with M-CSF (50 ng/ml) and sRANKL (50 ng/ml) for 3 days. (B) Immunohistochemical analysis of ZAS3 (red) in bone marrow macrophages (BMM) cultured with M-CSF and sRANKL (50 ng/ml each) at an early phase of osteoclastogenesis. White arrow indicates a mononucleated cell in which ZAS3 shows prominently nuclear localization. Yellow arrow highlights the membrane proximity of ZAS3 in a cell that contained two nuclei and therefore, had undergone the first cell fusion. Nuclei were stained with DAPI (blue). (C) TRAP staining of BMM cultured in the presence of M-CSF and sRANKL (50 ng/ml each). More TRAP+ (cells stained red) and OLC were observed from <i>ZAS3+/+</i> than <i>ZAS3−/−</i> BMM at day 4 (upper panels) and day 6 (lower panel). At day 6, most <i>ZAS3+/+</i> BMM had differentiated into large multinucleated TRAP positive cells while <i>ZAS3−/−</i> BMM was inefficient to do so and failed to form large spreading TRAP+ OLC. Scale bar, 100 µm. Data were representatives of 1 out of 3 independent experiments. (D) Bar charts showing the number of TRAP+ multinucleated OLC (more than 3 nuclei) formed in response to sRANKL and M-CSF stimulation (initially plated 5000 cells per well of an 8 well slide). Data were expressed as the mean ± SD from 3 independent experiments. (E) Western blot analysis of protein lysates of BMM cultured with M-CSF and sRANKL for 6 days with indicated antibodies. +/+ <i>ZAS3</i> WT mice, −/− <i>ZAS3</i> KO mice, and +/− heterozygous ZAS3 mice.</p
The bones of adult <i>ZAS3</i> knockout mice have increased bone strength, thickness, and mineralization.
<p>(A) Biomechanical properties of femurs were evaluated by three-point bending test. Load to fracture (Fx) was significantly increased in both male and female <i>ZAS3−/−</i> mice compared to +/+ and +/− control mice (*, <i>p</i><0.05). (B) & (C) Micro-CT reconstruction images of femora of 4-month-old mice. Bar represents 1 mm. (D) Sagittal section of distal femora from 5-month-old mice stained with von Kossa's method plus MacNeal tetrachrome counterstain. Mineralized bone was stained black and collagen type 1 was stained light blue. Bar represents 1 mm. +/+ <i>ZAS3</i> WT mice, −/− <i>ZAS3</i> KO mice, and +/− heterozygous ZAS3 mice.</p
Silencing of ZAS3 inhibits RANKL-mediated osteoclastogenesis in RAW 264.7 cells.
<p>RAW264.7 cells were transfected with (1 µM, 5 µM, or 10 µM) scramble siRNA or a pool of four <i>ZAS3</i> siRNA and incubated with complete medium supplemented with sRANKL (50 ng/ml). RNA and total protein lysates were prepared 4 days later and analyzed by (A) RT-PCR and (B) Western blot analysis using gene-specific primer sets and antibodies, respectively. (C) The above transfected cells were incubated with sRANKL (50 ng/ml) for 6 days and the numbers of OLC (TRAP positive cells with three or more nuclei) were counted. The percentage of OLC formed by scramble siRNA transfected cells was tentatively assigned as 100. Cells transfected with <i>ZAS3</i> siRNA formed relatively much less (10% versus scramble) OLC. Data are expressed as mean ± SD from three independent experiments. (D) RAW 264.7 cells were mock transfected, transfected with scramble siRNA, or with <i>ZAS3</i> siRNA, and incubated with sRANKL (50 ng/ml) for 4 days. Subsequently, EMSA was performed with nuclear extracts and <sup>32</sup>P AP-1 consensus sequences. The amount of siRNA (1, 5, and 10 in micromoles) used in transfection is shown on the top of the lanes.</p
ZAS3 promotes RANKL-mediated osteoclastogenesis and expression of osteoclast-associated genes.
<p>RAW264.7 cells (from 500 to 2500 cells) were stably transfected with the empty vector (EV) or the ZAS3 expression construct. (A) Indicated cells were plated onto wells of a 24-well plate and cultured in complete medium supplemented with sRANKL (50 ng/ml) for 6 days, and stained for TRAP. TRAP+ cells with 3 or more nucleic were considered as OLC and counted. Bar charts show the numbers of OLC. (B) Indicated cells were incubated with sRANKL (50 ng/ml) for 0, 2, 4, or 8 days. The induction of ZAS3, TRAP and CTSK (osteogenic markers), and HSP90 (loading control) was examined in total cell lysates by Western blot analysis. (C) Indicated cells were incubated with sRANKL (50 ng/ml) for 6 days and the induction of TRAF-6, RelB, c-Fos, and c-Jun was analyzed by Western blot analysis. (D) EMSA was performed with <sup>32</sup>P-GTTCT<u>GGGGAAGTCC</u>AGTGCTCACATGACC DNA probe corresponding to the mouse TRAP gene enhancer (ZAS3 binding site is shown in underline) and nuclear extracts prepared from indicated cells. The major DNA-protein complexes are designed C1 to C4. Complex C4 (indicated with a black arrow) was observed only in the ZAS3 transfected samples (lane 3). The formation of C2 (indicated with a grey arrow) and C4 was abolished by the addition of ZAS3 antibodies to the binding reactions (lanes 4 and 5).</p
Dynamic femoral bone parameters of adult ZAS3 WT and KO mice.
<p>BV/TV, bone volume/total volume; Tr.N, trabecular number; Tr.Sp, trabecular separation; Tr.Th, trabecular thickness; MAR, mineral apposition rate; MS/BS, mineralization to bone surface; BFR/BS, bone formation rate; CS, cross section; and OC/BS, number of osteoclast/bone surface.</p
Expression and subcellular localization of ZAS3 associate with osteoclastogenesis of RAW 264.7 preosteoclasts.
<p>(A) Morphological changes and increase in TRAP expression of RAW264.7 cells upon sRANKL-mediated osteoclastogenesis. RAW 264.7 cells were cultured for 0, 2, 4 or 6 days and stained for TRAP. Scale bar, 50 µm and arrows show representative TRAP+ cells. (B) Western blot analysis to show the presence of ZAS3 in RAW 264.7 cells at days 0, 2, 4 and 6 incubated with sRANKL (50 ng/ml). The protein filter was also incubated with histone H1 antibodies as a loading control. (C) Immunohistochemical fluorescence microscopy showing the expression of ZAS3 in RAW 264.7 cells at various stages of osteoclast differentiation. Number shown was the number of nucleus or nuclei presence in the cell indicated. During osteoclastogenesis, multinucleated giant cells are progressively formed by cell fusion. Therefore, increasing number of nuclei represented cells were at more advanced stage of osteoclastogenesis.</p
Biomechanical property and dimensions of femur bones.
<p>Biomechanical property and dimensions of femur bones.</p