Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

Abstract

Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.

Keywords: adipocyte; bone marrow stromal cells; differentiation; extracellular matrix; fibronectin; legumain; mesenchymal stem cell; osteoblast; osteoporosis; proliferation.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

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.

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.

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.

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.

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.

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 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.