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Tissue-Specific Stem Cells
Article first published online: 5 JUL 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 7, pages 1383–1395, July 2013
How to Cite
Wang, L., Zhao, Y., Liu, Y., Akiyama, K., Chen, C., Qu, C., Jin, Y. and Shi, S. (2013), IFN-γ and TNF-α Synergistically Induce Mesenchymal Stem Cell Impairment and Tumorigenesis via NFκB Signaling. STEM CELLS, 31: 1383–1395. doi: 10.1002/stem.1388
Author contributions: S.S., Y.J. and L.W.: conception and design, writing manuscript, and final approval of manuscript; L.W., Y.Z., Y.L., K.A., C.C. and C.Q.: collection and assembly of data; S.S., L.W., Y.Z. and Y.L.: data analysis and interpretation.
Disclosure of potential conflicts of interest is found at the end of this article.
first published online in STEM CELLS EXPRESS April 4, 2013.
- Issue published online: 5 JUL 2013
- Article first published online: 5 JUL 2013
- Accepted manuscript online: 4 APR 2013 01:12AM EST
- Manuscript Accepted: 4 MAR 2013
- Manuscript Revised: 18 FEB 2013
- Manuscript Received: 7 DEC 2012
- National Institute of Dental and Craniofacial Research
- National Institutes of Health
- Department of Health and Human Services. Grant Numbers: R01DE017449, R01DE019932, R01DE019413
- California Institute for Regenerative Medicine. Grant Number: RN1-00572
- National Natural Science Foundation of China. Grant Number: 81020108019
- National Basic Research Program (973 Program) of China. Grant Number: 2011CB964700
Additional Supporting Information may be found in the online version of this article.
|sc-12-1149_sm_SupplFigure1.TIF||2074K||Figure S1. Elevated levels of IFN-γ and TNF-α in OVX mice. Immunostaining showed increased IFN-γ and TNF-α positive cells (white arrows) in bone marrow and derma, but not in subcutaneous fat, in OVX mice when compared with sham-operated mice. Scale bars, 50 μm.|
|sc-12-1149_sm_SupplFigure2.pdf||266K||Figure S2. The number of dermal MSCs is reduced in OVX mice. Immunohistochemical staining of MSC markers CD105, CD44 and CD73 showed markedly reduced number of dermal MSCs in OVX mice when compared with sham-operated mice. Scale bars, 50 μm.|
|sc-12-1149_sm_SupplFigure3.TIF||1019K||Figure S3. OVX results in trabecular bone deterioration and derma atrophy in mice. (A, B) H&E staining showed markedly thinner trabecular bone and reduced dermal thickness, respectively, in OVX mice when compared with sham-operated mice. n = 8 animals per group. *, P < 0.05. Scale bars, 50 μm.|
|sc-12-1149_sm_SupplFigure4.TIF||1905K||Figure S4. IFN-γ and TNF-α synergistically cause MSC deficiency in nude mice. (A, B) H&E staining showed that only IFN-γ/TNF-α treatment group showed marked loss of trabecular bone and derma atrophy. n = 8 animals per group. Scale bars, 100 μm.|
|sc-12-1149_sm_SupplFigure5.TIF||1227K||Figure S5. Local administration of IFN-γ/TNF-α causes MSC deficiency in nude mice. When IFN-γ and TNF-α, either alone or in combination, were subcutaneously transplanted with hydrogel as a controlled release system to nude mice, only IFN-γ–/TNF-α–infused group showed significantly reduced population doublings (A) and capacity to form mineralized nodules (B) in DMSCs. (C) H&E staining demonstrated that only IFN-γ/TNF-α locally transplanted with hydrogel showed significant derma atrophy. n = 5 animals per group. *, P < 0.05. Scale bars, 100 μm.|
|sc-12-1149_sm_SupplFigure6.TIF||557K||Figure S6. IFN-γ and TNF-α impair MSC differentiation via NFκB/SMAD7 pathway. (A) Western blot showed upregulation of p-NFκBp65, p-IκB, SMAD7 and downregulation of p- SMAD1, RUNX2 and ALP in OVX-derived DMSCs. (B) TNF-α treatment showed a dosedependent NFκB activation. (C, D) Alizarin red staining showed that IFN-γ and TNF-α in combination but not alone caused a markedly reduction in mineralized nodule formation in DMSCs, with upregulated p-IκB, SMAD7 and down-regulated p-SMAD1, RUNX2 and ALP. (E, F) With knockdown of NFκB by IKKα siRNA, IFN-γ-/TNF-α-induced upregulation of SMAD7 and downregulation of p-SMAD1, RUNX2 and ALP were abolished, leading to the rescue of IFN-γ-/TNF-α–induced reduction of mineralized nodule formation in DMSCs. All experiments were performed in triplicate. *, P < 0.05.|
|sc-12-1149_sm_SupplFigure7.TIF||329K||Figure S7. IFNGRα and TNFR1 are required for IFN-γ/TNF-α to snergistically induce activation of NFκB. (A) Combined knockdown of IFNGRα and TNFR1 resulted in markedly inhibition of IFN-γ–/TNF-α–induced activation of NFκBp65 and p-IκB when compared with TNFR1 knockdown group. (B) Knockdown of STAT-1 abolished TNF-α-/IFN-γ–induced activation of NFκB pathway. (C, D) Western blot analysis confirmed efficacy of siRNA in inhibiting IKKα, IFNGRα, TNFR1, STAT-1 and SMAD7.|
|sc-12-1149_sm_SupplFigure8.tif||443K||Figure S8. SMAD7 knockdown rescues impaired differentiation of BMMSCs and DMSCs. (A) Western blot showed that knockdown of SMAD7 downregulated p-SMAD1, RUNX2 and ALP in BMMSCs without downregulating p-IκB. (B) Alizarin red staining showed that knockdown of SMAD7 resulted in rescuing of IFN-γ-/TNF-α–induced reduction of mineralized nodule formation in BMMSCs. (C) Western blot showed that knockdown of SMAD7 downregulated RUNX2 and ALP in DMSCs without downregulating p-IκB. (D) Alizarin red staining showed that knockdown of SMAD7 resulted in rescuing of IFN-γ–/TNF-α–induced reduction of mineralized nodule formation in DMSCs. All experiments were performed in triplicate. *, P < 0.05.|
|sc-12-1149_sm_SupplFigure9.TIF||2332K||Figure S9. Long-term IFN-γ/TNF-α exposure increases susceptibility to MSC malignant transformation via NFκB–induced oncogenes. (A) Immunostaining of pan-CK showed negative cells in MCA–induced tumor in OVX mice. (B) Immunostaining of the tumors derived from s.c. inoculation of IFN-γ–/TNF-α–treated BMMSCs showed elevated expressions of VIM and PCNA, but negative expression of pan-CK. (C) Immunostaining of p-NFκB, c-FOS and c- MYC in bone marrow showed more positive cells in aging OVX mice. Scale bars, 50 μm.|
|sc-12-1149_sm_SupplFigure10.TIF||1455K||Figure S10. Aspirin rescues osteoporosis and derma atrophy in OVX mice. H&E staining showed that systemically applied aspirin rescued the deteriorated trabecular bone structures (A) and derma atrophy (B) in OVX mice. Scale bars, 100 μm. *, P < 0.05.|
|sc-12-1149_sm_SupplFigure11.TIF||313K||Figure S11. Schematic diagram illustrating the mechanism in IFN-γ– and TNF-α–induced MSC deficiency and MSC tumorigenesis. Elevated levels of IFN-γ and TNF-α together, but not independently, synergistically cause MSC deficiency via NFκB–mediated activation of SMAD7, whereas long-term elevated levels of IFN-γ and TNF-α synergistically result in increased tendency toward MSC malignant transformation through NFκB–mediated upregulation of the oncogenes c-Fos and c-Myc.|
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