Inhibition of adipocytogenesis by canonical WNT signaling in human mesenchymal stem cells

Abstract

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPARγ2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of β-catenin protein, inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast, knockdown of β-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.

Figure 1

Modulation of expression of adipocyte marker genes (PPARγ2, adipsin, and lipoprotein lipase, LPL) and WNT genes in representative hMSCs (60-year-old woman) at intervals after transfer to adipocytogenic medium. Agarose gel electrophoretograms show effects of time on RT-PCR products.

Figure 2

Effects of SB-216763 on β-catenin levels in hMSCs. (A) There was a dose-dependent effect of SB-216763 on β-catenin in KM101 cells. (B) SB-216763 (5 μM) increased β-catenin in hMSCs (61-year-old man). Protein was extracted from cells after 6 hours incubation with or without SB-216763 and subjected to immunoblot for β-catenin and β-actin.

Figure 3

Effects of SB-216763 on adipocyte differentiation and expression of WNT4 and WNT11 in hMSCs. (A) Expression of adipocyte signature genes in hMSCs was inhibited by SB-216763 after transfer to adipocytogenic medium. Agarose gel electrophoretogram shows PCR products for hMSCs (36-year-old man) after indicated days in adipocytogenic medium in the absence or presence of 5 μM SB-216763 (B) Expression of WNT4, WNT11 and adipocyte signature genes was inhibited by 5 μM SB-216763 in hMSCs from 6 subjects, evaluated 7 days after transfer to adipocytogenic medium. There was little or no effect on WNT5A. Each pair of lanes indicates the age in years and the gender (M, male; F, female) of the subject from whom the cells were obtained. (C) Expression of adipocyte signature genes was significantly inhibited by 5 μM SB-216763. Data are shown for 6 samples as the mean and standard error (SE) for each gene (* p<0.05, paired t-test). (D) Expression of WNT4 relative to GAPDH is shown (left panel) for each specimen in DMSO vehicle control and 5 μM SB-216763, and (right panel) as the group means and standard error (*p=0.031, Wilcoxon matched-pairs signed-ranks test). (E) Expression of WNT11 relative to GAPDH is shown (left panel) for each specimen in DMSO vehicle control and 5 μM SB-216763, and (right panel) as the group means and standard error (*p=0.031, Wilcoxon matched-pairs signed-ranks test).

Figure 4

Effects of SB-216763 and β-catenin siRNA on adipocytogenesis in hMSCs. (A) Photomicrographs of cultures of representative hMSCs (47-year-old man) without and with 5μM SB-216763 for 18 days show oil red-O-positive cells (arrow) in control, but not in treated cultures. (B) Cultures of hMSCs (47-year-old man) were treated with different concentrations of SB-216763 for 18 days. Bars indicate the numbers of oil red-O-positive cells per mm², presented as mean ± S.D. of 4 replicate wells. (C) Cultures of hMSCs (60-year-old man) were treated with 5 μM SB-216763 for the first 1, 2, 3, 5, 7, days or the entire 18 days. Bars indicate the numbers of oil red-O-positive cells per mm², enumerated at day 18, presented as mean ± S.D. of triplicate wells. Asterisks indicate significance (**p<0.01) of treatments compared with control (no exposure to SB-216763). (D) Western immunoblot shows that β-catenin protein in hMSCs was absent in cells transfected with β-catenin siRNA, but not control siRNA. (E) Phase-contrast photomicrograph shows that there was spontaneous generation of lipid-containing adipocytes (arrows) in control medium in hMSCs transfected with β-catenin siRNA, but not in control hMSCs.