Role of VEGF in intermittent hypoxia-exposed neuroblastoma cells-mediated osteoclastogenesis in RAW 264.7 cells.

<p>(<b>A</b>) Intermittent hypoxia-exposed (IH) neuroblastoma cells were transfected with NTC siRNA (siNTC), HIF-1α-siRNA (siHIF-1α), HIF-2α-siRNA (siHIF-2α) or HIF-1α siRNA+HIF-2α siRNA(1∶1 ratio) mix (siHIF-1α+siHIF-2α) and cultured under normoxia. After 36 h, the cells were washed and maintained in serum-free medium for 24 h.VEGF concentration in cell culture supernatants was determined by ELISA. Values are expressed as mean ± SD (n = 3). °°P<0.01 parental (N) versus IH; * P<0.05, **P<0.01 IH/siNTC versus IH/siHIF-1α or IH/siHIF-2α-siRNA or IH/siHIF-1α+siHIF-2α. (<b>B</b>) RAW 264.7 cells were exposed to growth medium (control), growth medium with RANKL, CM from parental SH-SY-5Y cells (N), IH cells or IH cells transfected with NTC siRNA (siNTC), HIF-1α-siRNA (siHIF-1α), HIF-2α-siRNA (siHIF-2α) or HIF-1α+HIF-2α siRNA (1∶1 ratio) mix (siHIF-1α+siHIF-2α) and were allowed to differentiate under normoxia. The medium was changed after three days. After six days, cells were washed twice with PBS, fixed with 3.7% formaldehyde and stained for TRAP. RANKL-treated RAW cells served as a positive control. TRAP-positive multinucleated cells containing three or more nuclei were counted under a light microscope. Values represent the number of TRAP positive cells counted in ten different fields for each experiment from three independent experiments and expressed as mean ± SD. °°P<0.01 control versus RANKL; **P<0.01 IH/siNTC versus IH/siHIF-1α or IH/siHIF-2α or IH/siHIF-1α+siHIF-2α. (<b>C</b>) IH cells were transfected with NTC siRNA (siNTC) or HIF-1α-siRNA (siHIF-1α) and CM were collected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105555#s2" target="_blank">Methods</a>. Additionally parental, IH-siNTC, and IH-siHIF-1α cells were treated with VEGF antibodies and CM were collected. RAW 264.7 cells were exposed to growth medium (control), growth medium with RANKL, CM from parental or IH cells treated with NTC siRNA, HIF-1α-siRNA and/or VEGF antibodies and grown for six days with medium change after three days under normoxia. TRAP activity was assessed in cell lysates by the para-nitrophenyl phosphate hydrolysis assay. Results were normalized with total protein content of cultures and expressed as ηmol/min/mg protein. CM from RANKL-treated cultures served as a positive control. Values are expressed as mean ± SD (n = 4). °°P<0.01 control versus RANKL; **P<0.01 IH versus IH/VEGF Ab or IH/siHIF-1α.</p

Intratibial tumor growth.

<p>(<b>A</b>) Parental (N) and intermittent hypoxia-exposed (IH) cells (5×10⁵ in 25 µl PBS) stably expressing luciferase were directly injected into the right tibia of five to six week-old SCID mice. Bioluminescent activity was imaged in IVIS-100 system at 35 day after the implantation and photon counts were collected to reflect live tumor volume. (<b>B</b>) Quantification of bioluminescence **<i>p</i><0.01, parental (N) versus IH cells. (<b>C</b>) Tibias were harvested, decalcified, sectioned, and stained for TRAP. A representative image is shown and the arrow indicates TRAP-positive osteoclasts. (<b>D</b>) Graphical representation of number of osteoclast-like cells, counted by microscopy after TRAP staining.**<i>p</i><0.01, parental (N) versus IH cells.</p

Effects of intermittent hypoxia exposure on the migration of neuroblastoma cells.

<p>(<b>A</b>) Intermittent hypoxia-exposed (IH) cells were transfected with either NTC siRNA (siNTC), HIF-1α-siRNA (siHIF-1α), HIF-2α siRNA (siHIF-2α) or HIF-1α+HIF-2α siRNAs (1∶1 ratio) mix (siHIF-1α+siHIF-2α). Parental (N) and transfected cells were cultured at normoxia for 36 h, a scrape wound was made across the culture dish with a plastic pipette tip. The cells were washed twice with PBS and the medium was replaced. Cells were cultured to allow wound closure for 12 h, at which time pictures were taken with the Olympus microscope. Wound width was measured using a calibrated eyepiece graticule. A representative image is shown. (<b>B</b>) Graphical representation of monolayer wound-induced migration data. Values are expressed as mean ± SD (n = 4). °°P<0.01 Parental (N) versus IH; * P<0.05, **P<0.01 IH-siNTC versus IH-siHIF-1α or IH-siHIF-2α or IH-siHIF-1α+siHIF-2α.</p

Schematic representation of the role of intermittent hypoxia in enhancement of neuroblastoma-mediated osteoclastogenesis.

<p>Schematic representation of the role of intermittent hypoxia in enhancement of neuroblastoma-mediated osteoclastogenesis.</p

Effects of intermittent hypoxia exposure to neuroblastoma cells on <i>in vitro</i> osteoclastogenesis.

<p>RAW 264.7cells were supplemented with CM from the SH-SY5Y neuroblastoma cells and characterized for the osteoclastogenic response. (<b>A</b>) RAW 264.7cells were exposed to growth medium alone (control), growth medium with RANKL (100 ηg/ml) or CM from parental SH-SY5Y cells (N), intermittent hypoxia exposed (IH) cells, or stable transfectants (HIF-1α, shLuc and shHIF-1α) and were allowed to differentiate for six days under normoxic condition. The medium was changed after three days. CM from knockdown cells (shLuc and shHIF-1α) that were grown under hypoxia (1% O₂, 24 h) were also evaluated. Cells were washed twice with PBS, fixed with 3.7% formaldehyde and stained for TRAP with Acid Phosphatase, Leukocyte (TRAP) kit according manufacturer's instructions. A representative image is shown. Arrow indicates cells multinucleated and TRAP-positive. (<b>B</b>) Multinucleated and TRAP-positive cells in each well were counted. Graphical representation of the number of osteoclast-like cells is shown. Values represent number of TRAP positive cells counted in ten different fields for each experiment from three independent experiments and expressed as mean ± SD. °°P<0.01 control versus RANKL; * P<0.05, **P<0.01 Parental (N) versus IH or HIF 1α; # P<0.05 shLuc/normoxia versus shLuc/hypoxia or shHIF 1α/hypoxia. (<b>C</b>) RAW 264.7cells were exposed to growth medium (control), growth medium with RANKL (100 ηg/ml) or CM from parental SH-SY5Y cells (N), IH cells, stable transfectants (HIF-1α, shLuc and shHIF-1α), or knockdown cells (shLuc and shHIF-1α) that were exposed to hypoxia (1% O₂, 24 h) and were grown under normoxic condition for six days. The medium was changed after three days. Cells were assessed by real RT-PCR for the expression of TRAP, CaSR and Cath K mRNAs. Values are from three independent experiments in triplicates and expressed as mean ± SD. °°P<0.01 control versus RANKL; * P<0.05, **P<0.01 Parental (N) versus IH or HIF 1α; # P<0.05, ##P<0.01 shLuc/normoxia versus shLuc/hypoxia or shHIF 1α/hypoxia.</p

Effects of intermittent hypoxia exposure to neuroblastoma cells on CaSR expression in RAW 264.7 cells.

<p>(<b>A</b>) RAW 264.7 cells were exposed to growth medium (control), growth medium with RANKL or CM from parental SH-SY5Y cells (N), or intermittent hypoxia-exposed (IH) cells and cultured for six days under normoxia. The medium was changed after three days. Cells were fixed and labeled with CaSR antibodies and Alexa-594 antimouse-conjugated antibodies. Nuclei were stained with DAPI. RANKL-treated RAW cells served as a positive control for CaSR expression during osteoclastogenesis. A representative image is shown. Bar: 100 µm. (<b>B</b>) Cell lysates were analyzed for the levels of CaSR protein by Western blotting. The band intensity was measured and the protein level was normalized to the corresponding β-actin level. The results are expressed as relative quantity to the control (first lane of the blot).</p

Intermittent Hypoxia Effect on Osteoclastogenesis Stimulated by Neuroblastoma Cells

<div><p>Background</p><p>Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions.</p><p>Methods</p><p>We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and <i>in vitro</i> differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and <i>in vivo</i> bioluminescence imaging was used in the analysis of tumor growth in bone.</p><p>Results</p><p>Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells.</p><p>Conclusions</p><p>Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.</p></div

Effects of intermittent hypoxia exposure to neuroblastoma cells on modulation of osteoclastogenic signaling pathways in RAW 264.7cells.

<p>(<b>A</b>) RAW 264.7 cells were exposed to growth medium (control), growth medium with RANKL (100 ηg/ml), CM from parental SH-SY5Y cells, or intermittent hypoxia-exposed (IH) cells. The cells were then allowed to grow for six days under normoxia with medium change after three days. The JNK, ERK and p38 activation states were determined by immunoblot analysis using antibodies specifically directed against the phosphorylated forms of the enzymes, compared to data obtained with antibodies directed against the unphosphorylated states of the kinases. (<b>B</b>) RAW 264.7 cells were exposed to CM from parental SH-SY5Y cells (N), IH cells or IH cells treated with 0.1 or 1 µM U0126 (MEK signaling pathway inhibitor), 2.5 or 5 µM SP600125 (JNK signaling pathway inhibitor) and 0.1 or 1 µM SB203580 (p38 signaling pathway) and cultured under normoxia. The medium was changed after three days. After six days, cultures were analyzed for TRAP-positive multinucleated cells containing three or more nuclei under a light microscope. A representative image is shown. (<b>C</b>) Graphical representation of the number of TRAP-positive multinucleated cells. Values represent the number of TRAP positive cells counted in ten different fields for each experiment from three independent experiments and expressed as mean ± SD. * P<0.05, **P<0.01 IH versus IH treated with SP600125 (2.5 or 5 µM) or SB203580 (0.1 or 1 µM).</p

Effect of HIF-1α siRNA on differentiation of human neuroblastoma cells.

<p>(<b>A</b>) Normoxic (N) and intermittent hypoxia (IH) conditioned neuroblastoma cells were treated with non-targeted control (NTC) or HIF-1α siRNA smart pool for 36 h and phase-contrast images were taken under bright field using an Olympus CKX41 inverted microscope (bar, 50 µm). (<b>B</b>) Graph illustrates quantification of neurite lengths of cells treated with NTC or HIF-1α siRNA. **p<0.01, intermittent hypoxia conditioned cells treated with HIF-1α siRNA versus NTC or untreated. (<b>C</b>) Western blotting. Cells were treated with NTC or HIF-1α siRNA smart pool. After 36 h, cells were lysed and cell extracts were subjected to western blotting analysis for HIF-1α, NF-M and Neu N.</p

Effects of intermittent hypoxia on neural crest /SNS markers.

<p>Upregulation of markers for neural crest genes. (<b>A</b>) Western Blotting. Cell lysates of normoxic (N) and intermittent hypoxia (IH) conditioned neuroblastoma cells were analyzed by western blotting for the levels of c-Kit and TH. Real-time PCR. PCR analysis was performed in normoxic (N) and intermittent hypoxia (IH) conditioned neuroblastoma cells using primers specific to Notch-1 (<b>B</b>), ID2 (<b>C</b>) and HES-1(<b>D</b>) gene transcripts. **P<0.01, intermittent hypoxia versus normoxia. Downregulation of SNS markers. Real-time PCR. PCR analysis was performed in normoxic (N) and intermittent hypoxia (IH) conditioned neuroblastoma cells using primers specific to NPY (<b>E</b>), HASH-1(<b>F</b>) and dHAND (<b>G</b>). **P<0.01, intermittent hypoxia versus normoxia.</p