Consensus SXR/PXR responsive element motifs in the first introns of SXR and PXR genes.

<p>(A) Consensus SXR/PXR responsive element motifs, variant direct repeat 5, were identified in the first intron of both murine PXR gene (chromosome 11) and human SXR gene (chromosome 17). The bold letters indicate consensus SXR/PXR binding motif. (B) Generation of reporter plasmid containing three copies of PXR responsive element (PXRE) and PXRE with mutation (PXREmut). Underlined letters indicate mutated nucleotides. (C) Cos7 cells were transfected with PXR or SXR expression vector and reporter plasmid containing murine PXR responsive element or mutated PXR responsive element, and β-galactosidase expression vector (β-gal). The cells were then treated with indicated concentrations of PXR agonist pregnane-16α-carbonitrile (PCN) or SXR agonist rifampicin (RIF) or vehicles: DMSO for PCN and ethanol (Et) for RIF. Data are shown as relative light units (R.L.U.) normalized by β-galactosidase activity. ** P < 0.01, ***P < 0.001 in Dunnett’s test with vehicle treated group as a control.</p

SXR-dependent induction of Fam20a by SXR ligands.

<p>(A) ATDC5 cells were infected with adeno-SXR or adeno-DsRed and cultured in phenol red-free DMEM with charcoal/dextran-treated FCS (5%) containing rifampicin (RIF) (10 μM), vitamin K2 (VK) (10 μM), or ethanol (Et). Total RNA was extracted and gene expression was analyzed by microarray followed by hierarchical cluster analysis. A heat-map visualization of clusters including SXR-dependent ligand-induced genes is shown. Red grids indicate high expression and green grids indicate low expression. (B) SXR-dependent induction of Fam20a was validated by quantitative real-time PCR. Expression of GAPDH was used as an internal control. ***P < 0.001 in Dunnett’s test with adeno-SXR-infected ethanol-treated cells as a control. (C) Expression of Fam20a was decreased in primary articular chondrocytes derived from PXR knockout mice. Primary chondrocytes were purified from the femoral and knee joints of newborn PXR knockout mice and wild type mice. ***P < 0.001.</p

Age-dependent wearing of articular cartilage of the knee joint.

<p>(A) Width of lateral articular cartilage of the tibia in 4-month-old wild type (WT; n = 8) and PXR knockout (KO; n = 8) mice, 8 month-old wild type (WT; n = 6) and PXR knockout (KO; n = 6) mice, and 13 month-old wild type (WT; n = 5) and PXR knockout (KO; n = 4) mice is shown. (B) Gap between femoral and tibial articular cartilage of 8-month-old wild-type (WT; n = 6) and PXR knockout (KO; n = 6) mice and 13 month-old wild-type (WT; n = 5) and PXR knockout (KO; n = 4) mice are shown. **P < 0.01.</p

Wearing of articular cartilage of the knee joint in PXR knockout mouse.

<p>Representative microscopic image of articular cartilage of 8-month-old and 13-month-old wild-type and PXR knockout mice are shown. Arrowheads indicate lateral articular cartilage of the tibia.</p

Pregnane X Receptor Knockout Mice Display Aging-Dependent Wearing of Articular Cartilage

<div><p>Steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), are nuclear receptors that are expressed at high levels in the liver and the intestine where they function as xenobiotic sensors that induce expression of genes involved in detoxification and drug excretion. Recent evidence showed that SXR and PXR are also expressed in bone tissue where they mediate bone metabolism. Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints. Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice. We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2. Lastly, we demonstrated the biological significance of Fam20a expression in chondrocytes by evaluating osteoarthritis-related gene expression of primary articular chondrocytes. Consistent with epidemiological findings, our results indicate that SXR/PXR protects against aging-dependent wearing of articular cartilage and that ligands for SXR/PXR have potential role in preventing osteoarthritis caused by aging.</p></div

Pharmacodynamic Effect of Emixustat on Rod Function.

<p>The recovery of rod photoreceptor function following a photobleach was measured by ERG. Mice were treated with varied doses of emixustat (n = 4/dose group), or vehicle (n = 4/group), and ERG responses were recorded every 2 minutes for 50 minutes using a light stimulus 0.01 cd*s/m². Panel A shows representative ERG waveforms in vehicle- and emixustat-treated mice. Arrows in panel A indicate the b-wave amplitude peaks which are plotted as a function of recovery time after photobleach in panel B. B-wave response amplitudes (μV), at each corresponding time point of recovery, are shown as mean values ± SEMfor each of the treatment groups (panel B). Emixustat treatment caused a dose-dependent suppression of b-wave response amplitudes. The dose required for half-maximal suppression of ERG b-wave recovery was determined to be 0.21 mg/kg.</p

Protection from Light Damage.

<p>The ability of emixustat to provide protection from light damage was assessed as described in <i>Methods</i>. Mice received a single dose of emixustat, or vehicle, prior to light exposure (8,000 lux white light, 1 hour). Histological analyses and determination of ONL thickness was performed following a 2-week recovery period. Panel A shows tissue sections prepared from untreated, dark-adapted mice, untreated light-exposed mice, and mice pre-treated with either 0.3 or 1.0 mg/kg emixustat (ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer). Quantitative analysis of ONL layer thickness (panel B) was used to calculate the ED₅₀ for preservation of the ONL in emixustat-treated mice (ED₅₀ = 0.20 mg/kg). There was a statistically significant preservation of ONL thickness in mice treated with 1.0 or 3.0 mg/kg emixustat compared to the vehicle-treated, light control group (*, p<0.01).</p

Processing of Vitamin A in The Visual Cycle.

<p>Enzymatic processing within the visual cycle begins with delivery of vitamin A (all-<i>trans</i>-retinol) from the blood circulation. Upon entry into the RPE, all-<i>trans</i>-retinol is converted to a retinyl ester through the activity of lecithin retinol acyl transferase (1). The resulting all-<i>trans</i>-retinyl ester pool represents a storage form of vitamin A upon which RPE65 acts to generate 11-<i>cis</i>-retinol (2); 11-<i>cis</i>-retinol is then oxidized by an 11-<i>cis</i>-specific retinol dehydrogenase to form the visual chromophore, 11-<i>cis</i>-retinal (3). The visual chromophore is delivered to rod and cone outer segments (4) where it combines with opsins to form visual pigments (e.g., rhodopsin). Light activation of rhodopsin initiates visual transduction processes and liberates all-<i>trans</i>-retinal as a photoproduct. Reduction of all-<i>trans</i>-retinal, via all-<i>trans</i>-retinal dehydrogenase, produces all-<i>trans</i>-retinol (5), which is transferred back to the RPE for recycling. The continued activity of RPE65 in the light state ensures sustained levels of rhodopsin, closure of ion channels through transducin activation, and reduced oxygen demand.</p

Reduced Retinal Neovascularization.

<p>The effect of emixustat on retinal neovascularization was studied in the mouse OIR model. Seven day-old mouse pups were subjected to hyperoxia (75% oxygen) for 5 days. On P12, the mice were returned to room air and daily treatments with ruboxistaurin (10 mg/kg), emixustat (0.03–3.0 mg/kg), or appropriate vehicles were administered as described in <i>Methods</i>. Retinal flat mounts were prepared and areas of NV were quantified; these data were compared to data from control mice that were maintained in a normoxic environment (21% oxygen). Mice that were moved from a hyperoxic to normoxic environment, without treatment, showed a significant extent of retinal NV (~30% of the retinal area). Treatment with the ruboxistaurin (positive control) reduced the area of NV to ~20% of the total retinal area. In mice treated with emixustat, a dose-dependent reduction in retinal NV was observed. The reduction in NV at the highest emixustat dose (3.0 mg/kg/day) approached a level that was comparable to that obtained with ruboxistaurin (Fig 6A; *, t-test, p<0.05). The ED₅₀ for reduction of retinal NV in emixustat-treated mice was 0.46 mg/kg/day. Representative retinal flat mounts from an untreated, normoxic control, an untreated OIR control, and in a 3 mg/kg emixustat-treated mouse are shown in panels B, C, and D, respectively. Areas of NV, outlined in red tracings, were identified and quantified using Adobe Photoshop software.</p

Reduced Lipofuscin Autofluorescence and A2E Accumulation.

<p>The effect of emixustat on lipofuscin autofluorescence and A2E levels was examined in an animal model of autosomal recessive Stargardt disease (<i>Abca4</i>^-/- mice). Histological analysis of lipofuscin autofluorescence in untreated, strain- and age-matched wild-type mice, and <i>Abca4</i>^-/- mice treated with either vehicle or emixustat (0.3 or 3.0 mg/kg) is shown in panels A—D, respectively. All mice were 5 months of age. Lipofuscin fluorophores were extracted from RPE eyecups and analyzed by HPLC as described in <i>Methods</i>. Representative chromatograms from eyecup extracts of mice treated with emixustat (3 mg/kg for 3 months) and vehicle (red and black tracings, respectively) are shown in panel E. UV-vis spectra associated with the indicated peaks (numbered 1–5) are shown in the panel inset. Peak 1 in the chromatogram was determined to be A2E based upon spectral identity and co-elution with an authentic A2E standard. Quantitative analysis of A2E levels (based on area units of peak 1) in <i>Abca4</i>^-/- mice is shown in panel F (peaks 2–4 were not quantified). Numbers of mice analyzed for A2E quantitation are as follows: Day 0: n = 17; Vehicle: n = 6; 0.03 mg/kg: n = 6; 0.1 mg/kg: n = 7; 0.3 mg/kg, n = 8 1.0 mg/kg, n = 8; 3 mg/kg, n = 8. A2E levels increased from ~5 to ~20 pmoles/eye over a 3-month period in vehicle-treated <i>Abca4</i>^-/- mice. <i>Abca4</i>^-/- mice treated with emixustat showed a dose-dependent reduction of A2E which was statistically significant at doses ≥ 0.30 mg/kg/day, relative to vehicle treated <i>Abca4</i>^-/- mice (*, p<0.05). The ED₅₀ for the effect of emixustat on reducing accumulation of A2E was 0.47 mg/kg/day.</p