The effect of β-catenin silencing on the expression of adipocyte, osteoblast, and Wnt-signaling gene markers.

<p>U-33/γ2 cells were transiently transfected with either 200 ng of β-cat siRNA, which consisted of four β-catenin-specific 20–25 nt oligonucleotides, or 200 ng of scrambled siRNA (sc siRNA) for negative control. Seventy two hours after transfection proteins and RNA were extracted. A. Western blot analysis of β-catenin protein levels. Each lane was loaded with 50 µg of total protein lysate. B–D. Analysis of gene expression of adipocyte-specific (B), osteoblast-specific (C), and Wnt signaling (D) gene markers. All values are expressed as fold change compared to control transfected with sc siRNA and represented by value 1. * p<0.05.</p

Stabilization of β-catenin protein using LiCl does not affect PPARγ2 anti-osteoblastic activity.

<p>U-33/γ2 cells were treated with either vehicle, 1 µM Rosi, 10 mM LiCl, or in combination for 72 h. A. Alkaline phosphatase activity. B–D. Relative expression of osteoblast-specific gene markers and Wnt10b. Fold change in transcript levels was calculated as compared to vehicle treated cells. Statistical differences are shown between Rosi-treated samples and samples receiving combined treatment (NS – non-significant). V – vehicle; R – Rosi; L – LiCl; LR – LiCl+Rosi.</p

β-Catenin Directly Sequesters Adipocytic and Insulin Sensitizing Activities but Not Osteoblastic Activity of PPARγ2 in Marrow Mesenchymal Stem Cells

<div><p>Lineage allocation of the marrow mesenchymal stem cells (MSCs) to osteoblasts and adipocytes is dependent on both Wnt signaling and PPARγ2 activity. Activation of PPARγ2, an essential regulator of energy metabolism and insulin sensitivity, stimulates adipocyte and suppresses osteoblast differentiation and bone formation, and correlates with decreased bone mass and increased fracture rate. In contrast, activation of Wnt signaling promotes osteoblast differentiation, augments bone accrual and reduces total body fat. This study examined the cross-talk between PPARγ2 and β-catenin, a key mediator of canonical Wnt signaling, on MSC lineage determination. Rosiglitazone-activated PPARγ2 induced rapid proteolytic degradation of β-catenin, which was prevented by either inhibiting glycogen synthase kinase 3 beta (GSK3β) activity, or blocking pro-adipocytic activity of PPARγ2 using selective antagonist GW9662 or mutation within PPARγ2 protein. Stabilization of β-catenin suppressed PPARγ2 pro-adipocytic but not anti-osteoblastic activity. Moreover, β-catenin stabilization decreased PPARγ2-mediated insulin signaling as measured by insulin receptor and FoxO1 gene expression, and protein levels of phosphorylated Akt (pAkt). Cellular knockdown of β-catenin with siRNA increased expression of adipocyte but did not affect osteoblast gene markers. Interestingly, the expression of Wnt10b was suppressed by anti-osteoblastic, but not by pro-adipocytic activity of PPARγ2. Moreover, β-catenin stabilization in the presence of activated PPARγ2 did not restore Wnt10b expression indicating a dominant role of PPARγ2 in negative regulation of pro-osteoblastic activity of Wnt signaling. In conclusion, β-catenin and PPARγ2 are in cross-talk which results in sequestration of pro-adipocytic and insulin sensitizing activity. The anti-osteoblastic activity of PPARγ2 is independent of this interaction.</p> </div

PPARγ2 mutation, abrogating the pro-adipocytic but not the anti-osteoblastic activity, protects β-catenin protein from degradation.

<p>A. Western blot analysis of protein levels of non-mutated (WT) and mutated (D409A) forms of PPARγ2 analyzed 72 h after transfection of HEK293 cells. β-actin was used as a loading control. Each lane was loaded with 50 µg of total protein lysate. EV – empty vector control. B. Western blot analysis of β-catenin protein levels after treatment with 1 µM Rosi for 72 h. Hek293 cells were transfected with β-catenin expression construct and either empty expression vectors (pSPORT6 and pEF-BOS), or non-mutated (WT), or mutated (D409A) PPARγ2 expression constructs. Each lane was loaded with 50 µg of total protein lysate. C. Effect of D409A mutation on transcriptional activity of PPARγ2. Hek293 cells were transiently transfected with above constructs and co-transfected with p2AOx luciferase reporter gene construct under the control PPARγ response elements. Cells were treated with either vehicle or 1 µM Rosi for 48 h and lysates were analyzed for luciferase activity. Promoter activity of firefly luciferase was normalized to renilla luciferase which was used as a transfection control. D – G. Effect of D409A mutation on expression of adipocyte-specific (D) and osteoblast-specific (E – G) gene markers, and Wnt10b (H). U-33/c cells were transiently transfected with either empty vector (pEF-BOS), or non-mutated (WT), or mutated (D409A) PPARγ2 expression constructs and treated with either vehicle or 1 µM Rosi for 72 h. Relative transcript levels were calculated as fold change as compared to vehicle treated cells in each transfection. V – vehicle; R- Rosi; * p<0.05 V <i>vs.</i> R.</p

GSK3β antagonist LiCl protects β-catenin protein from PPARγ2-mediated degradation and preserves β-catenin transcriptional activity.

<p>U-33/γ2 cells were treated with either vehicle, 1 µM Rosi, 10 mM LiCl, or in combination for 72 h. A. Western blot analysis of total levels of β-catenin protein. β-actin was used as a loading control. Each lane was loaded with 50 µg of total protein lysate. B. Relative expression of β-catenin mRNA as compared to vehicle treated cells. C. Immunocytochemistry of β-catenin protein. Green: β-catenin; purple: DAPI staining of nuclei. D. Percentage of β-catenin positive cells (T) and cells positive for β-catenin in the nucleus (N). E. Transcriptional activity of β-catenin measured in U-33/γ2 cells treated as above for 48 h using TOP-Flash construct in luciferase gene reporter assay. Promoter activity of firefly luciferase was normalized to renilla luciferase which was used as a transfection control (* p<0.05). V – vehicle; R – Rosi; L – LiCl; L+R or LR – LiCl+Rosi.</p

Rosi-mediated activation of PPARγ2 degrades the pool of active, protein-unbound β-catenin.

<p>A. Western blot analysis of protein-unbound (Active) and protein-bound (Inactive) fractions of β-catenin isolated from U-33/γ2 cells treated with either vehicle (DMSO) or 1 µM Rosi for 1 h. Protein loading per lane: 3 µg of protein-bound and 50 µg of protein-unbound fraction. B. Relative expression of β-catenin mRNA analyzed after 1 h treatment of U-33/γ2 cells with either vehicle or 1 µM Rosi. C. Western blot analysis of total β-catenin protein levels isolated from U-33/c cells and U-33/γ2 cells treated with either vehicle or 1 µM Rosi for 72 h. Each lane was loaded with 50 µg of total protein lysate. D. Relative expression of β-catenin mRNA analyzed in U-33/c and U-33/γ2 cells after 72 h treatment with either vehicle or 1 µM Rosi. Gene expression is presented as fold difference as compared to levels of β-catenin transcript in vehicle treated U-33/c cells (* p<0.05). E. Immunofluorescent visualization of β-catenin and PPARγ2 proteins in untreated U-33/γ2. V – vehicle; R – Rosi; A – active; I – inactive.</p

Selective antagonist GW9662 of PPARγ2 pro-adipocytic activity increases β-catenin protein stability.

<p>U-33/γ2 cells were treated with either vehicle, 1 µM Rosi, 10 µM GW9662, or in combination for 72 h. A. Adipocyte differentiation was assessed by measuring the number of Oil Red O positive cells. B – C. Relative expression of adipocyte-specific gene markers. D. Osteoblast differentiation was assessed by measuring alkaline phosphatase activity. E – G. Relative expression of osteoblast-specific gene markers and Wnt10b. Fold change in transcript levels was calculated as compared to vehicle treated cells. H. Immunocytochemistry of β-catenin protein. Green: β-catenin; purple: DAPI staining of nuclei. I. Percentage of β-catenin positive cells (T) and cells positive for β-catenin in the nucleus (N). J. Western blot analysis of total β-catenin protein levels. Each lane was loaded with 50 µg of total protein lysate. K. Transcriptional activity of β-catenin measured with luciferase gene reporter assay using TOP-Flash construct. Promoter activity of firefly luciferase was normalized to renilla luciferase which was used as a transfection control. Statistically significant differences are shown between Rosi-treated samples and samples receiving combined treatment (* p<0.05; NS – non-significant). V – vehicle; R – Rosi; G – GW9662; GR – GW9662+ Rosi.</p

High-Performanced Cathode with a Two-Layered R–P Structure for Intermediate Temperature Solid Oxide Fuel Cells

Driven by the mounting concerns on global warming and energy crisis, intermediate temperature solid-oxide fuel cells (IT-SOFCs) have attracted special attention for their high fuel efficiency, low toxic gas emission, and great fuel flexibility. A key obstacle to the practical operation of IT-SOFCs is their sluggish oxygen reduction reaction (ORR) kinetics. In this work, we applied a new two-layered Ruddlesden–Popper (R-P) oxide, Sr₃Fe₂O_7‑δ (SFO), as the material for oxygen ion conducting IT-SOFCs. Density functional theory calculation suggested that SFO has extremely low oxygen ion formation energy and considerable energy barrier for O^2– diffusion. Unfortunately, the stable SrO surface of SFO was demonstrated to be inert to O₂ adsorption and dissociation reaction, and thus restricts its catalytic activity toward ORR. Based on this observation, Co partially substituted SFO (SFCO) was then synthesized and applied to improve its surface vacancy concentration to accelerate the oxygen adsorptive reduction reaction rate. Electrochemical performance results suggested that the cell using the SFCO single phase cathode has a peak power density of 685 mW cm^–2 at 650 °C, about 15% higher than those when using LSCF cathode. Operating at 200 mA cm^–2, the new cell using SFCO is quite stable within the 100-h’ test

Supplement Number 1

The X-ray diffraction (XRD) pattern of the NiO sample, the infrared thermography results under different excitation powers, the temperature distribution in the simulated NiO sample at different time and the (001)-oriented NiO sample experimental results

Supplement Number 2

The temperature variation in the simulated NiO sample from 0 s to 500