Hypoxia down-regulates Wnt/β-catenin signaling in human OS cells.

<p>A, MG-63 human OS cells were cultured under hypoxia over 72 hour, and increased HIF protein expression was observed by western blot. Protein levels of active β-catenin were decreased under hypoxic conditions relative to normoxic conditions (0 hour). Actin was the loading control. B, Axin2 mRNA levels were determined as a measure of Wnt/β-catenin signaling under hypoxic conditions (72 hour, 0.5% O₂) via quantitative reverse-transcription PCR (qrt-PCR) normalized to normoxia. Asterisks indicate statistical significance (*p<0.05).</p

HIFs are present and active in human OS cells.

<p>A, 143B and MG-63 human OS cells showed increases in HIF-2α protein expression by western blot when cultured for 72 hour under hypoxic conditions (0.5% O₂). The HIF-2α downstream target, Oct4, was also increased, indicating HIF signaling activity. β-tubulin was the loading control. B, MNNG/HOS human OS cells were cultured under hypoxia for up to 48 hour and compared to a normoxia (0 hour) control. Both HIF-1α and HIF-2α levels were increased as seen by western blot. β-tubulin was used as a loading control.</p

Further Wnt/β-catenin signaling inhibition sensitizes hypoxic OS cells to doxorubicin.

<p>MG-63 OS cells were treated with increasing concentrations of doxorubicin under hypoxic conditions in the presence of the tankyrase inhibitor XAV939, the porcupine inhibitor IWP-2, or DMSO alone. Half maximal inhibitory concentrations (IC₅₀) were calculated, and the percent IC₅₀ relative to DMSO alone was determined. Asterisks indicate statistical significance (*p<0.05).</p

Wnt/β-catenin signaling down-regulation is both dependent and independent of HIF expression.

<p>A, Levels of active β-catenin were determined by western blot at different hypoxia time points for each of the shRNA in MNNG/HOS cells. Overall levels of active β-catenin decreased regardless of HIF expression, although the magnitude of decrease was not equal across all lines. Active and total β-catenin protein levels were decreased at the 72 hour time point. Actin was the loading control. B, Decreased Wnt/β-catenin signaling activity under hypoxia was confirmed by measuring axin2 mRNA levels via qrt-PCR in MNNG/HOS cells. Hypoxia (72 hour, 0.5% O₂) resulted in decreased axin2 mRNA expression relative to normoxia. C, When analyzed relative to the shnon hypoxia mRNA, axin2 mRNA was increased in the shHIF-1α MNNG/HOS cell lines. Asterisks indicate statistical significance (*p<0.05, **p<0.01, ***p<0.001). shNON: non-targeting shRNA; HIF shRNAs used are indicated in parentheses.</p

Hypoxia results in chemoresistance of human OS cells to doxorubicin.

<p>A, Dose-response curves for the 143B and MNNG/HOS (mHOS) cell lines treated with increasing concentrations of doxorubicin under normoxic and hypoxic conditions (72 hour, 0.5% O₂). Luminescence (viability) was determined as a percent of untreated control (0 µM doxorubicin). B, Average half maximal inhibitory concentration (IC₅₀) values were obtained from the dose-response curves and compared between normoxic and hypoxic conditions for the cell lines MNNG/HOS (mHOS), 143B, and a patient-derived OS cell line, 206-2. Asterisks indicate statistical significance (**p<0.01, ***p<0.001).</p

<i>Murine</i> primers for genes analyzed.

<p><i>Murine</i> primers for genes analyzed.</p

Genetic and Functional Studies of the Intervertebral Disc: A Novel <i>Murine</i> Intervertebral Disc Model

<div><p>Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, <i>ex vivo</i> organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through <i>ex-vivo</i> genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as <i>ex vivo</i> genetic recombination (<i>mT/mG</i> reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced <i>ex vivo</i> genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of <i>TGFBR1^fl/fl</i> explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the <i>murine</i> intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.</p></div

IVD explant response to TGF-ß.

<p>(<b>a</b>) Real-time PCR analysis of murine IVD explant gene expression response to stimulation with exogenously administered TGF-ß3 (10 ng/mL for 24 hours) demonstrates significant increase in the gene expressions of <i>ACAN</i> and <i>TIMP1</i>. Values are normalized to <i>GAPDH</i>. Results are presented relative to IVDs cultured without TGF-ß3 where the expression value was set to 1. Values are normalized to <i>GAPDH</i>. (<b>b</b>) Murine IVDs from <i>TGFßR1^fl/fl</i> demonstrate significant upregulation in <i>ACAN</i> gene expression when cultured with cre-expressing adenovirus. Exogenous TGF-ß3 at 10 ng/mL for 24 hours was cultured with wild-type murine IVDs. Results are presented relative to IVDs cultured with control adenovirus where the expression value was set to 1. Values are normalized to <i>GAPDH</i>. * denotes statistical significance (<i>p</i><0.05). <i>ns</i> denotes not statistically significant (<i>p</i>>0.05).</p

Method of IVD organ harvest for ex vivo culture.

<p>(<b>a</b>) Anterior spine is exposed after removal of peritoneal sac and paraspinal musculature. (<b>b</b>) Excision of a lumbar IVD after the superior and inferior extents were freed with sharp dissection at 4× magnification.</p

IVD explant response to IL-1ß.

<p>(<b>a</b>) When treated with IL-1ß at 10 ng/mL for 12 hours, IVD explants demonstrate significant decreases in gene expression of <i>COL2</i> (<i>p = 7.4×10⁻¹¹</i>) and <i>ACAN</i> (<i>p = 3.0×10⁻⁵</i>); ADAMTS4 (<i>p</i> = 0.009) gene expression is increased. Values are normalized to <i>GAPDH</i>. Results are presented relative to IVDs cultured without IL-1ß where the expression value was set to 1 (<b>b</b>) Murine IVD explants demonstrate increased MAPK phosphorylation when treated with IL-1ß at 10 ng/mL for 24 hours. <b>*</b> denotes statistical significance (p<0.05).</p