Increased heme-oxygenase 1 expression in mesenchymal stem cell-derived adipocytes decreases differentiation and lipid accumulation via upregulation of the canonical Wnt signaling cascade

Abstract

Introduction: Heme oxygenase (HO), a major cytoprotective enzyme, attenuates oxidative stress and obesity. The canonical Wnt signaling cascade plays a pivotal role in the regulation of adipogenesis. The present study examined the interplay between HO-1and the Wnt canonical pathway in the modulation of adipogenesis in mesenchymal stem cell (MSC)-derived adipocytes.

Methods: To verify the role of HO-1 in generating small healthy adipocytes, cobalt protoporphyrin (CoPP), inducer of HO-1, was used during adipocyte differentiation. Lipid accumulation was measured by Oil red O staining and lipid droplet size was measured by BODIPY staining.

Results: During adipogenesis in vitro, differentiating pre-adipocytes display transient increases in the expression of genes involved in canonical Wnt signaling cascade. Increased levels of HO-1 expression and HO activity resulted in elevated levels of β-catenin, pGSK3β, Wnt10b, Pref-1, and shh along with increased levels of adiponectin (P < 0.05). In addition, induction of HO-1 resulted in a reduction in C/EBPα, PPARγ, Peg-1/Mest, aP2, CD36 expression and lipid accumulation (P < 0.05). Suppression of HO-1 gene by siRNA decreased Wnt10b, pGSK3β and β-catenin expression, and increased lipid accumulation. The canonical Wnt responsive genes, IL-8 and SFRP1, were upregulated by CoPP and their expression was decreased by the concurrent administration of tin mesoporphyrin (SnMP), an inhibitor of HO activity. Furthermore, knockdown of Wnt10b gene expression by using siRNA showed increased lipid accumulation, and this effect was not decreased by concurrent treatment with CoPP. Also our results show that blocking the Wnt 10b antagonist, Dickkopf 1 (Dkk-1), by siRNA decreased lipid accumulation and this effect was further enhanced by concurrent administration of CoPP.

Conclusions: This is the first study to demonstrate that HO-1 acts upstream of canonical Wnt signaling cascade and decreases lipogenesis and adipocyte differentiation suggesting that the HO-1 mediated increase in Wnt10b can modulate the adipocyte phenotype by regulating the transcriptional factors that play a role in adipogenesis. This is evidenced by a decrease in lipid accumulation and inflammatory cytokine levels, increased adiponectin levels and elevation of the expression of genes of the canonical Wnt signaling cascade.

Figure 1

Expression of heme oxygenase-1 (HO-1), with time, on adipogenesis. (A) Pictures of lipid droplets of a representative experiment at days 3, 5, 10, and 14. Adipogenesis was measured as the relative absorbance of Oil Red O as described in Materials and methods (mean ± SD, *P < 0.05 vs control). (B) Western blot of HO-1 and actin in mesenchymal stem cell (MSC)-derived adipocytes from day 0 to day 14. Representative immunoblots and densitometry analysis are shown. Data are expressed as means ± SD; n = 4; *P < 0.05. (C) Lactic dehydrogenate (LDH) assay done to study the cytotoxicity at increasing concentrations of cobalt-protoporphyrin IX (CoPP). Data are expressed as means ± SD; n = 8, *P < 0.05 vs MSC control, **P < 0.05 vs adipocytes control.

Figure 2

Effect of cobalt-protoporphyrin IX (CoPP) on lipid accumulation, heme oxygenase-1 (HO-1) expression and activity, and cytokine levels. (A) Pictures of lipid droplets of a representative sample in the presence of increasing CoPP concentrations. Adipogenesis was measured as the relative absorbance of Oil Red O at day 14 after inducing adipogenesis as described in Materials and methods (mean ± SD, *P < 0.05 vs control). (B) Western blot analysis of HO-1protein expression in response to increasing concentration of CoPP treatment (0.5, 1.0, 2.0, 5.0, 10.0 μm). Data are expressed as means ± SD; n = 4 (*P < 0.05 vs control). (C) HO activity measured by CO formation (**P < 0.05 vs control). (D-E) Effect of CoPP on cytokine levels in control and cells treated with CoPP. CoPP was added every 2 days for 2 weeks, and cultured media samples were obtained immediately before the media was changed. Results are calculated as pg/ml of cultured media for TNF-α (D) and μg/ml for adiponectin (E) (*P < 0.05 versus control).

Figure 3

Effect of CoPP and SnMP on MSC-derived adipocyte cell differentiation. (A-B) Effect of CoPP and SnMP on adipogenesis. Adipogenesis was measured as the relative absorbance of Oil Red O at day 14 after inducing adipogenesis as described in Materials and Methods. (C-E) Effect of CoPP and SnMP on lipid droplet size. Lipid droplets were stained with boron-dipyrromethene (BODIPY) and the sizes of droplets were measured using Image Pro Analyzer (ver. 6.2, Media Cybernetics, Inc., MD, USA), *P < 0.05 vs control).

Figure 4

Effect of cobalt-protoporphyrin IX (CoPP) and tin (stannic)-mesophorphyrin IX (SnMP) on the surface marker CD36. Membrane antigen expression of CD36 on h-mesenchymal stem cells (MSCs) and MSC-derived adipocytes was analyzed by a fluorescence-activated cell sorter (FACS). Cells were treated with adipogenic media and collected after 14 days. Data are expressed as means ± SD (*P < 0.05, **P < 0.05).

Figure 5

Effect of heme oxygenase-1(HO-1) induction and suppression on adipogenic markers. (A) Effect of cobalt-protoporphyrin IX (CoPP), tin (stannic)-mesophorphyrin IX (SnMP) and siRNA HO-1 on lipid accumulation. Lipogenesis was measured as the relative absorbance of Oil Red O at day 14 after inducing adipogenesis as described in Materials and methods (mean ± SD, *P < 0.05 vs control, **P < 0.05 vs control, ^#P < 0.05 vs control). (B-H) Effect of CoPP, SnMP and siRNA HO-1 on HO-1 expression, and adipogenic marker expression in mesenchymal stem cell (MSC)-derived adipocytes. (B) Densitometry analysis of HO-1expression (*P < 0.01 vs control, **P < 0.05 vs siRNA HO-1). (C-H) Densitometry analysis of Sonic hedgehog (shh), mesoderm-specific transcript (Mest), fatty acid binding protein (aP2), Pre-adipocyte factor-1 (Pref-1), adipogenic transcription factors CCAAT/enhancer binding protein a (C/EBPα), peroxisome proliferator-activated receptor (PPAR)γ levels, respectively (*P < 0.01 vs control, **P < 0.05 vs siRNA HO-1).

Figure 6

Effect of heme oxygenase-1 (HO-1) induction and suppression on adipogenic marker expression in mesenchymal stem cell (MSC)-derived adipocytes. (A-C) Representative immunoblots and densitometry analysis of Wnt10b, p-Glycogen synthase kinase (GSK), β-catenin levels, respectively. Data are expressed as means ± SD (*P < 0.01 vs control, **P < 0.05 vs siRNA HO-1). (D-E) Effect of HO-1 induction and inhibition on Wnt -responsive genes IL-8 and Secreted frizzled-related protein 1 (SFRP1). Data are expressed as means ± SD (*P < 0.05 vs control, ^#P < 0.05 vs cobalt-protoporphyrin IX (CoPP)).

Figure 7

Effect of heme oxygenase-1 (HO-1) on the Wnt canonical pathway during adipogenesis. (A) Effect of inhibition of HO-1, Wnt10b and Dickkopf 1 (Dkk1) using respective siRNA on adipocyte hypertrophy. Lipogenesis was measured as the relative absorbance of Oil Red O at day 14 after inducing adipogenesis as described in Materials and methods (mean ± SD, *P < 0.05 vs vehicle, ^#P < 0.05 vs siRNA Wnt10b, **P < 0.05 vs siRNA HO-1, ^‡P < 0.05 vs cobalt-protoporphyrin IX (CoPP), ^&P < 0.05 vs siRNA Dkk1). (B-C) Effect of inhibition of HO-1, Wnt10b and Dkk1 using respective siRNA on β-catenin and p- glycogen synthase kinase (GSK) levels respectively. Data are expressed as means ± SD. (*P < 0.05 vs scramble, ^#P < 0.05 vs siRNA Wnt10b, **P < 0.05 vs siRNA HO-1, ^‡P < 0.05 vs CoPP, ^&P < 0.05 vs siRNA Dkk1).

Figure 8

Proposed mechanism demonstrating the interplay of heme oxygenase 1 (HO-1) and the Wnt canonical pathway in the modulation of adipogenesis in mesenchymal stem cells (MSC)-derived adipocytes. HO-1 suppresses adipocyte differentiation by increasing expression of key regulators including Wnt10b, p-glycogen synthase kinase (GSK), β-catenin and Sonic hedgehog (shh) and decreasing expression of adipogenic transcription factors CCAAT/enhancer binding protein a (CEBP)α, peroxisome proliferator-activated receptor (PPAR)γ and fatty-acid-binding protein (aP2). These effects led to decrease in lipid accumulation and increase of pre-adipocytes and healthy adipocytes that produce cytoprotective adipokines, such as adiponectin.