Volume 8, Issue 2, Pages (February 2017)

Slides:



Advertisements
Similar presentations
Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2  Ke Liu,
Advertisements

Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells  Tao Zhang, Xiao-Hang Li, Dian-Bao.
Molecular Therapy - Nucleic Acids
SiRNA Knockdown of Tissue Inhibitor of Metalloproteinase-1 in Keloid Fibroblasts Leads to Degradation of Collagen Type I  Masayo Aoki, Koichi Miyake,
Mesenchymal Stem Cells Ameliorate Podocyte Injury and Proteinuria in a Type 1 Diabetic Nephropathy Rat Model  Shuai Wang, Yi Li, Jinghong Zhao, Jingbo.
Volume 15, Issue 6, Pages (June 2009)
Tumor-Derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells  Nilay Sethi, Xudong Dai, Christopher.
MicroRNA-30b is a multifunctional regulator of aortic valve interstitial cells  Mi Zhang, MD, Xiaohong Liu, MD, Xiwu Zhang, MD, Zhigang Song, MD, Lin Han,
Mesenchymal Stem Cells Ameliorate Podocyte Injury and Proteinuria in a Type 1 Diabetic Nephropathy Rat Model  Shuai Wang, Yi Li, Jinghong Zhao, Jingbo.
MicroRNA-92a-3p regulates the expression of cartilage-specific genes by directly targeting histone deacetylase 2 in chondrogenesis and degradation  G.
SNEVhPrp19/hPso4 Regulates Adipogenesis of Human Adipose Stromal Cells
MicroRNA-92a-3p regulates the expression of cartilage-specific genes by directly targeting histone deacetylase 2 in chondrogenesis and degradation  G.
Volume 7, Issue 2, Pages (August 2016)
Volume 55, Issue 1, Pages (July 2014)
Identification of Bone Marrow-Derived Soluble Factors Regulating Human Mesenchymal Stem Cells for Bone Regeneration  Tsung-Lin Tsai, Wan-Ju Li  Stem Cell.
Restoration of Corneal Transparency by Mesenchymal Stem Cells
M. Wang, H. Jin, D. Tang, S. Huang, M.J. Zuscik, D. Chen 
X. Zhang, I. Prasadam, W. Fang, R. Crawford, Y. Xiao 
Modulation of K-Ras-Dependent Lung Tumorigenesis by MicroRNA-21
Volume 5, Issue 6, Pages (December 2015)
M. Wang, H. Jin, D. Tang, S. Huang, M.J. Zuscik, D. Chen 
Volume 76, Issue 1, Pages (July 2009)
Volume 4, Issue 3, Pages (March 2015)
Volume 6, Issue 4, Pages (April 2016)
An Essential Role of Hrs/Vps27 in Endosomal Cholesterol Trafficking
Volume 44, Issue 3, Pages (November 2011)
Volume 6, Issue 4, Pages (April 2016)
Bmi-1 Regulates Extensive Erythroid Self-Renewal
Volume 11, Issue 2, Pages (August 2018)
Molecular Therapy - Nucleic Acids
Volume 18, Issue 13, Pages (March 2017)
Enxuan Jing, Stephane Gesta, C. Ronald Kahn  Cell Metabolism 
Inhibition of KLF4 by Statins Reverses Adriamycin-Induced Metastasis and Cancer Stemness in Osteosarcoma Cells  Yangling Li, Miao Xian, Bo Yang, Meidan.
Volume 9, Issue 3, Pages (September 2017)
Volume 23, Issue 10, Pages (October 2016)
Volume 6, Issue 6, Pages (June 2016)
Jungmook Lyu, Vicky Yamamoto, Wange Lu  Developmental Cell 
Volume 13, Issue 6, Pages (November 2015)
Volume 11, Issue 1, Pages (July 2012)
Volume 4, Issue 6, Pages (June 2015)
Robust Self-Renewal of Rat Embryonic Stem Cells Requires Fine-Tuning of Glycogen Synthase Kinase-3 Inhibition  Yaoyao Chen, Kathryn Blair, Austin Smith 
14-3-3σ Regulates Keratinocyte Proliferation and Differentiation by Modulating Yap1 Cellular Localization  Sumitha A.T. Sambandam, Ramesh B. Kasetti,
Volume 24, Issue 2, Pages (February 2016)
Promotion Effects of miR-375 on the Osteogenic Differentiation of Human Adipose- Derived Mesenchymal Stem Cells  Si Chen, Yunfei Zheng, Shan Zhang, Lingfei.
Myeloma cell–derived Runx2 promotes myeloma progression in bone
Volume 9, Issue 5, Pages (November 2017)
Volume 70, Issue 10, Pages (November 2006)
Volume 5, Issue 5, Pages (November 2015)
Volume 3, Issue 6, Pages (December 2014)
Volume 9, Issue 3, Pages (September 2017)
Volume 26, Issue 2, Pages (February 2018)
Volume 7, Issue 2, Pages (August 2016)
Volume 5, Issue 5, Pages (December 2013)
Volume 4, Issue 3, Pages (March 2015)
Volume 10, Issue 5, Pages (May 2018)
GRM7 Regulates Embryonic Neurogenesis via CREB and YAP
Increased Expression of Wnt2 and SFRP4 in Tsk Mouse Skin: Role of Wnt Signaling in Altered Dermal Fibrillin Deposition and Systemic Sclerosis  Julie Bayle,
Activin Signals through SMAD2/3 to Increase Photoreceptor Precursor Yield during Embryonic Stem Cell Differentiation  Amy Q. Lu, Evgenya Y. Popova, Colin.
Volume 4, Issue 3, Pages (March 2015)
Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells  Tao Zhang, Xiao-Hang Li, Dian-Bao.
Volume 22, Issue 5, Pages (January 2018)
Fig. 6. CBX7 expression in adipocyte differentiation of adipose-derived stem cells.(A) The adipose-derived stem cells, ADS1, were analyzed for the capability.
Volume 12, Issue 1, Pages (January 2019)
Volume 9, Issue 4, Pages (October 2017)
Molecular Therapy - Nucleic Acids
Volume 21, Issue 2, Pages (February 2013)
Volume 15, Issue 11, Pages (June 2016)
Volume 8, Issue 6, Pages (June 2017)
Volume 55, Issue 1, Pages (July 2014)
Volume 25, Issue 6, Pages (June 2017)
Presentation transcript:

Volume 8, Issue 2, Pages 373-386 (February 2017) Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis  Abbas Jafari, Diyako Qanie, Thomas L. Andersen, Yuxi Zhang, Li Chen, Benno Postert, Stuart Parsons, Nicholas Ditzel, Sundeep Khosla, Harald Thidemann Johansen, Per Kjærsgaard-Andersen, Jean-Marie Delaisse, Basem M. Abdallah, Daniel Hesselson, Rigmor Solberg, Moustapha Kassem  Stem Cell Reports  Volume 8, Issue 2, Pages 373-386 (February 2017) DOI: 10.1016/j.stemcr.2017.01.003 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Regulation of Legumain Expression during In Vitro and In Vivo Differentiation of Human Bone Marrow Stromal Cells (A and B) Immunohistochemical (A) and RNA in situ hybridization (B) analysis of legumain expression and localization in normal human iliac crest bone biopsies. n = 11 donors. Scale bar, 50 μm. Red arrows, canopy cells; black arrows, reversal cells; arrow heads, osteoclasts; v, vessel. (C) qRT-PCR analysis of LGMN expression during osteoblast (OB) differentiation of hBMSCs at 6, 12, and 18 days (D6–D18) after start of differentiation (day 0, D0). Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (D) Western blot analysis of legumain expression in cell lysates from hBMSC cultures during OB differentiation. (E) Quantification of the mature legumain (36 kDa) band intensity. Arbitrary units (ARBU). Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (F) Quantification of legumain activity in cell lysates from hBMSCs during OB differentiation. Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, two-tailed unpaired Student’s t test. (G) qRT-PCR analysis of LGMN expression during adipocyte (AD) differentiation of hBMSCs. Data represent mean ± SD from three independent experiments. ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (H) Western blot analysis of legumain expression in cell lysates from hBMSC cultures during AD differentiation. (I) Quantification of the mature legumain (36 kDa) band intensity. Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, two-tailed unpaired Student’s t test. Stem Cell Reports 2017 8, 373-386DOI: (10.1016/j.stemcr.2017.01.003) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Legumain Knockdown Enhanced Osteoblast Differentiation and In Vivo Bone Formation and Inhibited Adipocyte Differentiation of Human Bone Marrow Stromal Cells hBMSCs were stably transfected with control (shCtrl) or LGMN shRNA (shLGMN). (A) qRT-PCR analysis of LGMN expression. Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, two-tailed unpaired Student’s t test. (B) Western blot analysis of legumain and GAPDH control. Data represent three independent experiments. (C) Quantification of legumain activity. Data represent mean ± SD from three independent experiments. ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (D) Quantification of alkaline phosphatase (ALP) activity in the presence of standard culture medium (SCM) or osteoblast induction medium (OIM) (day 6). Data represent mean ± SD from three independent experiments. p > 0.05, two-tailed unpaired Student’s t test. (E) qRT-PCR gene expression analysis of the early (ALP, Col1a1) and late (BGLAP, IBSP) OB marker genes. Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (F) Quantification of alizarin red staining at day 12. Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, two-tailed unpaired Student’s t test. (G and H) Quantification of accumulated lipid droplets in the presence of AD induction medium using oil red O staining (day 12). Scale bar, 150 μm, Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, two-tailed unpaired Student’s t test. (I) qRT-PCR gene expression analysis of the AD marker genes PPARG2, FABP4, LPL, and ADIPOQ. Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, two-tailed unpaired Student’s t test. (J) Histological analysis of in vivo bone formation by hBMSCs stably transfected with non-targeting control shRNA (shCtrl) or LGMN shRNA (shLGMN), 8 weeks after implantation in immune-deficient mice. Arrows, hydroxyapatite; arrow heads, bone. Scale bars: top panels, 500 μm; bottom panels, 250 μm. (K) Quantification of the heterotopic bone formation, n = 4 implants for each cell type, Data represent mean ± SEM. ∗p ≤ 0.05, Mann-Whitney test. (L) Human-specific vimentin staining of shLGMN implants. Arrows, hydroxyapatite; arrow heads, bone. Scale bars: top panels, 500 μm; bottom panels, 250 μm. ab, antibody. See also Figure S1. Stem Cell Reports 2017 8, 373-386DOI: (10.1016/j.stemcr.2017.01.003) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Legumain Overexpression Inhibited Osteoblast and Enhanced Adipocyte Differentiation of Human Bone Marrow Stromal Cells See also Figure S1. (A–C) Legumain (LGMN)-transduced hBMSCs was established using a retroviral transduction system and the successful overexpression of legumain was confirmed using (A) qRT-PCR analysis of LGMN mRNA expression, (B) western blot analysis of legumain in cell lysate, and (C) quantification of legumain activity. Data represent mean ± SD from three independent experiments. ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (D–G) Secretion of legumain in the conditioned medium (CM) was evaluated using (D) ELISA measurement (data represent mean from three technical replicates) and (E) western blot analysis of legumain in the CM from hBMSC-LGMN-overexpressing cell line (LGMN-CM) (data represent three independent experiments). To assess the effects of legumain on OB differentiation, control hBMSCs containing empty vector (E.V.) and hBMSC-LGMN cell lines were cultured in OB induction medium, and expressions of OB marker genes were analyzed using qRT-PCR (F) and mineralized matrix formation was determined by quantification of eluted alizarin red staining (G). Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (H and I) To assess the effect of legumain on AD differentiation, control hBMSCs and hBMSC-LGMN cell lines were cultured in AD induction medium, and (H), (I) accumulation of lipid droplets was measured by quantification of the eluted oil red O staining, ∗p ≤ 0.05, two-tailed unpaired Student’s t test. Scale bar, 150 μm. (J) Expressions of AD marker genes were measured by qRT-PCR (day 7). Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, two-tailed unpaired Student’s t test. E.V., empty vector; LGMN, legumain-overexpressing hBMSCs. Stem Cell Reports 2017 8, 373-386DOI: (10.1016/j.stemcr.2017.01.003) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Legumain Degrades Fibronectin in Human Bone Marrow Stromal Cell cultures (A) Western blot analysis of legumain and fibronectin in cell lysates of hBMSC lines with stable knockdown of legumain (shLGMN) during ex vivo OB differentiation (day 0–7). (B and C) Quantification of protein band intensities in (A). ∗p ≤ 0.05, ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (D) Western blot analysis of legumain and fibronectin in cell lysates of hBMSC lines with stable overexpression of legumain (LGMN) during ex vivo OB differentiation (day 0–7). (E and F) Quantification of protein band intensities in (D). Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (G) Western blot analysis of human fibronectin degradation by purified legumain from bovine kidneys (bLeg; 10:1 w/w; control) and the cell lysates from hBMSCs stably transfected with E.V. or legumain (LGMN; legumain overexpression). (H) Quantitation of mineralized matrix formation on day 15 of OB differentiation, in the presence of siRNA against fibronectin (siFN) and non-targeting control siRNA (siCtrl). Data represent mean ± SD from three independent experiments. ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. (I and J) Effect of different ECM proteins (gelatin, collagen 1, or fibronectin) on mineralized matrix formation by hBMSCs visualized by alizarin red staining and quantification. Data represent mean ± SD from three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, two-tailed unpaired Student’s t test. See also Figure S2. Stem Cell Reports 2017 8, 373-386DOI: (10.1016/j.stemcr.2017.01.003) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Legumain Inhibits OB Differentiation and Bone Mineralization In Vivo (A) Conservation of zebrafish lgmn. I, amino acid identity; S, amino acid similarity. (B) High-resolution melting analysis of pooled lgmn-TALEN and control-injected animals at 3 days post-fertilization (dpf). (C) qRT-PCR analysis of OB and AD marker genes at 5 dpf. Data represent mean ± SEM of three pools of ten animals. ∗p ≤ 0.05, two-tailed unpaired Student’s t test. (D and E) Control and lgmn mutant animals at 7 dpf. Scale bars, 500 μM. (D) Bright field and (E) fluorescent alizarin red staining. (E′ and E″) Higher-magnification images of boxed regions. Arrowheads, whole vertebrae stained; arrows, partial vertebrae stained. Scale bars, 100 μM. (F) Number of alizarin-red-stained vertebrae in lgmn and control animals at 7 dpf. Data represent mean ± SEM, n > 30 for each group. ∗p ≤ 0.05, ∗∗∗p ≤ 0.005 two-tailed unpaired Student’s t test. (G) Animals were treated with SD-134 (500 μM) or 1% DMSO from 3 to 7 dpf. Number of alizarin-red-stained vertebrae at 7 dpf. Data represent mean ± SEM, n = 12 for each group. ∗∗∗p ≤ 0.005 two-tailed unpaired Student’s t test. Stem Cell Reports 2017 8, 373-386DOI: (10.1016/j.stemcr.2017.01.003) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Effect of Aging and Osteoporosis on Legumain Protein Levels in Human Serum and Bone Microenvironment (A) Serum legumain levels in 89 women, aged 48–87 years, p < 0.0001 using Spearman correlation analysis. (B) Western blot analysis of mature legumain (36 kDa) and GAPDH in cell lysates from primary hBMSC cultures established from bone marrow aspirates of osteoporotic patients (n = 5) and age-matched controls (n = 3). (C) Quantification of western blot band intensities normalized to GAPDH. Data represent mean ± SD from three independent experiments. ∗∗∗p ≤ 0.005, two-tailed unpaired Student’s t test. (D) Upper panel: legumain staining of bone biopsies from postmenopausal osteoporotic patients (n = 11) and age-matched control individuals (n = 13). Scale bars, 1 mm. Lower panel: higher-magnification images of the boxed regions. Scale bars, 50 μm. (E) Correlation of trabecular bone area with legumain expression by adipocytes in biopsies from postmenopausal osteoporotic patients (n = 80 random regions of interest [ROI] = 1 mm2), p = 0.004 using Spearman correlation analysis. (F) Proposed mode-of-action of legumain for regulation of hBMSC lineage commitment. Stem Cell Reports 2017 8, 373-386DOI: (10.1016/j.stemcr.2017.01.003) Copyright © 2017 The Authors Terms and Conditions