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

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

Marrow Adipose Tissue: Skeletal Location, Sexual Dimorphism, and Response to Sex Steroid Deficiency

Marrow adipose tissue (MAT) is unique with respect to origin, metabolism, and function. MAT is characterized with high heterogeneity which correlates with skeletal location and bone metabolism. This fat depot is also highly sensitive to various hormonal, environmental, and pharmacologic cues to which it responds with changes in volume and/or metabolic phenotype. We have demonstrated previously that MAT has characteristics of both white (WAT) and brown (BAT)-like or beige adipose tissue, and that beige phenotype is attenuated with aging and in diabetes. Here, we extended our analysis by comparing MAT phenotype in different locations within a tibia bone of mature C57BL/6 mice and with respect to the presence of sex steroids in males and females. We report that MAT juxtaposed to trabecular bone of proximal tibia (pMAT) is characterized by elevated expression of beige fat markers including Ucp1, HoxC9, Prdm16, Tbx1, and Dio2, when compared with MAT located in distal tibia (dMAT). There is also a difference in tissue organization with adipocytes in proximal tibia being dispersed between trabeculae, while adipocytes in distal tibia being densely packed. Higher trabecular bone mass (BV/TV) in males correlates with lower pMAT volume and higher expression of beige markers in the same location, when compared with females. However, there is no sexual divergence in the volume and transcriptional profile of dMAT. A removal of ovaries in females resulted in decreased cortical bone mass and increased volume of both pMAT and dMAT, as well as volume of gonadal WAT (gWAT). Increase in pMAT volume was associated with marked increase in Fabp4 and Adiponectin expression and relative decrease in beige fat gene markers. A removal of testes in males resulted in cortical and trabecular bone loss and the tendency to increased volume of both pMAT and dMAT, despite a loss of gWAT. Orchiectomy did not affect the expression of white and beige adipocyte gene markers. In conclusion, expression profile of beige adipocyte gene markers correlates with skeletal location of active bone remodeling and higher BV/TV, however bone loss resulted from sex steroid deficiency is not proportional to MAT expansion at the same skeletal location

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

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

Partial Agonist, Telmisartan, Maintains PPARγ Serine 112 Phosphorylation, and Does Not Affect Osteoblast Differentiation and Bone Mass

<div><p>Peroxisome proliferator activated receptor gamma (PPARγ) controls both glucose metabolism and an allocation of marrow mesenchymal stem cells (MSCs) toward osteoblast and adipocyte lineages. Its activity is determined by interaction with a ligand which directs posttranscriptional modifications of PPARγ protein including dephosphorylation of Ser112 and Ser273, which results in acquiring of pro-adipocytic and insulin-sensitizing activities, respectively. PPARγ full agonist TZD rosiglitazone (ROSI) decreases phosphorylation of both Ser112 and Ser273 and its prolonged use causes bone loss in part due to diversion of MSCs differentiation from osteoblastic toward adipocytic lineage. Telmisartan (TEL), an anti-hypertensive drug from the class of angiotensin receptor blockers, also acts as a partial PPARγ agonist with insulin-sensitizing and a weak pro-adipocytic activity. TEL decreased ^S273pPPARγ and did not affect ^S112pPPARγ levels in a model of marrow MSC differentiation, U-33/γ2 cells. In contrast to ROSI, TEL did not affect osteoblast phenotype and actively blocked ROSI-induced anti-osteoblastic activity and dephosphorylation of ^S112pPPARγ. The effect of TEL on bone was tested side-by-side with ROSI. In contrast to ROSI, TEL administration did not affect bone mass and bone biomechanical properties measured by micro-indentation method and did not induce fat accumulation in bone, and it partially protected from ROSI-induced bone loss. In addition, TEL induced “browning” of epididymal white adipose tissue marked by increased expression of UCP1, FoxC2, Wnt10b and IGFBP2 and increased overall energy expenditure. These studies point to the complexity of mechanisms by which PPARγ acquires anti-osteoblastic and pro-adipocytic activities and suggest an importance of Ser112 phosphorylation status as being a part of the mechanism regulating this process. These studies showed that TEL acts as a full PPARγ agonist for insulin-sensitizing activity and as a partial agonist/partial antagonist for pro-adipocytic and anti-osteoblastic activities. They also suggest a relationship between PPARγ fat “browning” activity and a lack of anti-osteoblastic activity.</p></div