Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Mar 31;7(3):e2169.
doi: 10.1038/cddis.2016.75.

Pdcd4 Restrains the Self-Renewal and White-To-Beige Transdifferentiation of Adipose-Derived Stem Cells

Affiliations
Free PMC article

Pdcd4 Restrains the Self-Renewal and White-To-Beige Transdifferentiation of Adipose-Derived Stem Cells

Y Bai et al. Cell Death Dis. .
Free PMC article

Abstract

The stemness maintenance of adipose-derived stem cells (ADSCs) is important for adipose homeostasis and energy balance. Programmed cell death 4 (Pdcd4) has been demonstrated to be involved in the development of obesity, but its possible roles in ADSC function and adipogenic capacity remain unclear. In this study, we demonstrate that Pdcd4 is a key controller that limits the self-renewal and white-to-beige transdifferentiation of ADSCs. Pdcd4 deficiency in mice caused stemness enhancement of ADSCs as evidenced by increased expression of CD105, CD90, Nanog and Oct4 on ADSCs, together with enhanced in situ proliferation in adipose tissues. Pdcd4 deficiency promoted proliferation, colony formation of ADSCs and drove more ADSCs entering the S phase accompanied by AKT activation and cyclinD1 upregulation. Blockade of AKT signaling in Pdcd4-deficient ADSCs led to a marked decline in cyclinD1, S-phase entry and cell proliferation, revealing AKT as a target for repressing ADSC self-renewal by Pdcd4. Intriguingly, depletion of Pdcd4 promoted the transdifferentiation of ADSCs into beige adipocytes. A reduction in lipid contents and expression levels of white adipocyte markers including C/EBPα, PPAR-γ, adiponectin and αP2 was detected in Pdcd4-deficient ADSCs during white adipogenic differentiation, substituted by typical beige adipocyte characteristics including small, multilocular lipid droplets and UCP1 expression. More lactate produced by Pdcd4-deficient ADSCs might be an important contributor to the expression of UCP1 and white-to-beige transdifferentiation. In addition, an elevation of UCP1 expression was confirmed in white adipose tissues from Pdcd4-deficient mice upon high-fat diet, which displayed increased energy expenditure and resistance to obesity as compared with wild-type obese mice. These findings provide evidences that Pdcd4 produces unfavorable influences on ADSC stemness, which contribute to adipose dysfunction, obesity and metabolic syndromes, thereby proposing Pdcd4 as a potential intervening target for regulating ADSC function.

Figures

Figure 1
Figure 1
Pdcd4 deficiency increases the expression levels of stem cell-related phenotypes on ADSCs. ADSCs were isolated from epididymal fat pads of WT and Pdcd4−/− mice (n⩾8 per group) and expanded in DMEM containing 10% FBS and bFGF (5 ng/ml). (a) The morphology of WT and Pdcd4−/− ADSCs was examined by light microscope. Scale bar=200 μm. (b) The expression of stem cell-related positive markers CD90 and CD105 and negative makers CD11b and CD11c was detected by flow cytometry. Data are representative of three independent experiments
Figure 2
Figure 2
Pdcd4 deficiency increases the stemness of ADSCs in WAT. (a) ADSCs from WT and Pdcd4−/− mice were isolated and expanded as described in Figure 1, the gene expression of stemness markers on ADSCs were measured by qPCR. Bars represent mean±S.E.M. *P<0.05, ***P<0.001. (b) The primary stromal vascular fraction was isolated from epididymal fat pads of WT and Pdcd4−/− mice (n=4 per group), the gene expression of stemness markers were examined by qPCR. Bars represent mean±S.E.M. **P<0.01, ***P<0.001. (c) WT or Pdcd4−/− mice (n=4) was intraperitoneally injected with 100 μg EdU. After 24 h, the epididymal fat pads were collected and paraffin-embedded sections were prepared. The incorporation of EdU in dividing cells was examined by fluorescence signals and visualized with fluorescence microscope. The original magnification is 100. Scale bar=100 μm
Figure 3
Figure 3
Pdcd4 deficiency increases the proliferation of ADSCs through promoting S-phase entry. (a) The cell proliferation of WT and Pdcd4−/− ADSCs was tested using CCK-8 assay at indicated time points. Data are representative of three independent experiments. (b, c) Colony formation developed by WT and Pdcd4−/− ADSCs was measured by Crystal Violet staining after 7 days' culture in vitro. Representative (b) and statistic (c) data are from two independent experiments. Bars represent mean±S.E.M. *P<0.05. (d, e) WT and Pdcd4−/−ADSCs entering S phase were determined by EdU incorporation assay. Fluorescence signals were examined by fluorescence microscope. Typical (d) and statistic (e) data are representative of three independent experiments. The original magnification is 100. Scale bar=100 μm. Bars represent mean±S.E.M. **P<0.01. (f, g) Cell cycle profile of WT and Pdcd4−/−ADSCs was stained with propidium iodide and analyzed by flow cytometry, typical (f) and statistic (g) data are from three independent experiments. Bars represent mean±S.E.M. **P<0.01, ***P<0.001
Figure 4
Figure 4
AKT activation is responsible for the S-phase elevation caused by Pdcd4 deficiency in ADSCs. WT and Pdcd4−/−ADSCs were cultured in the presence or absence of MK2206 (2 μM) overnight, then the cells were collected for the following experiments. (af) Protein levels of p-AKT, AKT and cyclinD1 were detected by western blot assay. Representative (a, d) and statistical (b, c, e, f) data are shown. Bars represent mean±S.E.M. *P<0.05, **P<0.01, ***P<0.001. (g, h) ADSCs entering S phase were determined by EdU incorporation assay, fluorescence signals were examined by fluorescence microscope. The original magnification is 100. Scale bar=100 μm. Typical (g) and statistic (h) data are representative of three independent experiments. Bars represent mean±S.E.M. **P<0.01, ***P<0.001
Figure 5
Figure 5
Pdcd4 deficiency drives the transdifferentiation of ADSCs from white adipocytes into beige adipocytes. Adipogenic induction was performed using white adipogenic differentiation medium for 12–18 days. (a) The gene expression of white adipocyte markers was measured by qPCR at day 0, 4, 8 and 12 during the induction. Bars represent mean±S.E.M. ***P<0.001. (b, c) Lipid contents were visualized using Oil Red O staining (b) and quantified by elution of Oil Red O (c). The original magnification is 200. Scale bar=50 μm. Bars represent mean±S.E.M. ***P<0.001. (d, e) The expression of UCP1 was examined by qPCR (d) and immunocytochemistry (e). Original magnification is 100. Scale bar=100 μm. Data are representative of three independent experiments. Bars represent mean±S.E.M. **P<0.01
Figure 6
Figure 6
Pdcd4 deficiency induces UCP1-expressing beige adipocytes in WAT of mice in response to HFD. WT and Pdcd4−/− male mice (n=4 per group) were fed on ND or HFD for 24 weeks, then the epididymal fat pads were collected for assay. (ac) Protein (a, b) and mRNA (c) levels of UCP1 expression in WAT from indicated mice. Bars represent mean±S.E.M. *P<0.05. (d) In situ expression of UCP1 was examined by immunofluorescence staining on WAT sections from indicated mice. Original magnification is 100. Scale bar=100 μm
Figure 7
Figure 7
Pdcd4 deficiency increases the levels of lactate produced by ADSCs. (a) WT and Pdcd4−/−ADSCs were cultured in DMEM containing 10% FBS and bFGF (5 ng/ml) for 3 days; the supernatants (n=4 per group) were collected for lactate assay. (b) WT and Pdcd4−/−ADSCs were subjected to adipogenic induction under adipogenic differentiation medium. At 12 days post induction, the supernatants (n=4 per group) from one day of cell culture were collected for lactate detection. Bars represent mean±S.E.M. ***P<0.001. (c) Working model of Pdcd4 on ADSC function. Pdcd4 targeting AKT activation restrains ADSC self-renewal by suppressing G1/S transition dependent on cyclinD1. Upon adipogenic differentiation, the restriction of proliferation by Pdcd4 causes reduction in lactate levels and suppression in UCP1 expression, thus impeding the transdifferentiation of ADSCs into beige adipocytes

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Baglioni S, Francalanci M, Squecco R, Lombardi A, Cantini G, Angeli R et al. Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J 2009; 23: 3494–3505. - PubMed
    1. De Ugarte DA, Morizono K, Elbarbary A, Alfonso Z, Zuk PA, Zhu M et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 2003; 174: 101–109. - PubMed
    1. Strioga M, Viswanathan S, Darinskas A, Slaby O, Michalek J. Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells. Stem Cells Dev 2012; 21: 2724–2752. - PubMed
    1. Mizuno H, Tobita M, Uysal AC. Concise review: Adipose-derived stem cells as a novel tool for future regenerative medicine. Stem Cells 2012; 30: 804–810. - PubMed
    1. Taha MF, Hedayati V. Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells. Tissue Cell 2010; 42: 211–216. - PubMed

Publication types

MeSH terms

Feedback