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NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation
Accepted Manuscript NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation Linghao Wang, Ke Chen, Xinxing Wan, Fang Wang, Zi Guo, Zhaohui Mo PII:
S0006-291X(17)30268-1
DOI:
10.1016/j.bbrc.2017.02.007
Reference:
YBBRC 37255
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 30 January 2017 Accepted Date: 2 February 2017
Please cite this article as: L. Wang, K. Chen, X. Wan, F. Wang, Z. Guo, Z. Mo, NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation
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Linghao Wang, Ke Chen, Xinxing Wan, Fang Wang, Zi Guo, Zhaohui Mo* Department of Endocrinology and Metabolism, Third Xiangya Hospital of Central South University
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* Corresponding author.
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Abstract Osteoporosis is one of the most common skeletal disease featured by osteopenia and
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adipose accumulation in bone tissue. NLRP3 inflammasome activation is an essential player in aging-related chronic diseases like osteoporosis, particularly due to the causal caspase-1 activation and its correlation to adipose accumulation in bone tissue. Moreover, the
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expression of anti-aging/senescence SIRT1 was reported to decline along with aging. As the
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major cellular contributor of bone formation, mesenchymal stem cells (MSCs) are multipotent stem cells processing mutually exclusive differentiatability toward osteocytes or adipocytes. Therefore, we hypothesized that NLRP3 inflammasome activation promotes adipogenesis and repress osteogenesis in MSCs via inhibiting SIRT1 expression. We
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activated NLRP3 inflammasome in human MSCs via lipopolysaccharide and palmitic acid (LPS/PA) treatment for self-renewal maintenance, adipogenic differentiation or osteogenic differentiation. LPS/PA treatment significantly increased NLRP3 expression, decreased
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SIRT1 expression and promoted caspase-1 activity in MSCs. LPS/PA treatment also boosted
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adipogenesis of MSCs and suppressed osteogenesis. Moreover, inhibition of caspase-1 activity repressed adipogenic differentiation and partially improved osteogenic differentiation of MSCs with LPS/PA treatment. Our study demonstrated the pivotal roles of NLRP3 inflammasome and downstream mediator caspase-1 for the progress of osteo-differentiation MSCs, and offered novel therapeutic target of treatment for osteoporosis. Key words mesenchymal stem cell, NLRP3 inflammasome, osteoporosis, adipogenesis, osteogenesis
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Introduction
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Aging-related primary osteoporosis is rampant worldwide, which annually causes over 9 million fractures and increases social and economic burden for aging population Error! Reference source not found.. Osteoporosis is characterized by bone fragility, damage of
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microstructure of bone tissue, decreased bone mass and adipose infiltration Error!
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Reference source not found.. Dramatic alteration from osteocytes to adipocytes in bone tissue during osteoporosis is related with the dynamic change from mesenchymal stem cells (MSCs), due to their bi-directional differentiatability toward adipocytes or osteocytes Error! Reference source not found..
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MSCs, the major cellular contributor of bone tissue, demonstrate differentiation potential toward bone/cartilage and adipose tissue. During osteogenesis, expression of osteogenic-specific transcription factor RUNX2 is upregulated in MSCs Error! Reference
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source not found.. In contrast, adipogenesis of MSCs presents high expression of PPAP-γ,
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which triggers expression of adipogenic genes including AP2 and FAFP Error! Reference source not found.. Adipogenesis and osteogenesis in MSCs regulated by RUNX2 or PPAP-γ are mutual exclusive Error! Reference source not found.. During aging process, MSCs exhibit biased fate toward adipogenesis with accumulation of adipose tissue and decline of bone formation in bone marrow Error! Reference source not found.. NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome is reported as the most clinically implicated inflammasome Error! Reference source not
ACCEPTED MANUSCRIPT found.. NLRP3 inflammasome is an intracellular protein complex involved in initiation of innate immune response Error! Reference source not found.. NLRP3 inflammasome contains NLRP3, apoptotic speck protein (ASC) and pro-caspase-1 [10]. NLRP3 plays
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critical roles in multiple chronic diseases Error! Reference source not found.. Activated NLRP3 protein is capable of recruiting ASC and pro-caspase-1 to assemble NLRP3 inflammasome Error! Reference source not found.. As such, NLRP3 inflammasome sets up
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the stage for caspase-1 activation, and triggers secretion of inflammatory interleukin (IL)-1β
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and IL-18 Error! Reference source not found.. Ultimately, NLRP3 inflammasome causes low-grade systemic inflammation and chronic organ failure Error! Reference source not found.. The overt increase of cortical bone in NLRP3-/- aging mice compared with wild type counterparts was observed, indicating NLRP3 may twist round MSC differentiation from
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osteogenesis to adipogenesis Error! Reference source not found.. The high level of NLRP3 was also detected in human with obesity Error! Reference source not found.. Sirtuin1 (SIRT1) is ubiquitously expressed as class III histone deacetylase and regulates
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survival, metabolic rate, and oxygen consumption. SIRT1 interferes with NF-κb signaling,
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counteracts with inflammatory process and impedes bioactivity of PPAR-γ to repress adipogenesis Error! Reference source not found.. SIRT1 expression declines within aged bone tissue along with the increase of adipose components. Knockout of SIRT1 in MSCs in aged mice caused osteopenia and osteogenesis inefficiency Error! Reference source not found.. Nicotinamide phosphoribosyltransferase (NAMPT) positively regulates SIRT1 activity via synthesis of nicotinamide adenine dinucleotide (NAD) and promotes osteogenesis Error! Reference source not found.. Also, NLRP3 inflammasome prevents expression of
ACCEPTED MANUSCRIPT SIRT1, and the caspase-1 within NLRP3 inflammasome induces proteolysis of SIRT1 Error! Reference source not found.. Taking together, we hypothesized that NLRP3 inflammasome promotes adipogenesis
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and represses osteogenesis of MSCs by inhibiting SIRT1. We induced activation of NLRP3 inflammasome in human MSCs by lipopolysaccharide (LPS) and palmitic acid (PA) treatment during self-renewal, adipogenic or osteogenic differentiation. LPS/PA treatment
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enhanced NLRP3 expression, reduced SIRT1 expression and promoted caspase-1 activation
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in MSCs. Moreover, LPS/PA treatment enhanced adipogenesis of MSCs but downregulated the osteogenesis efficiency. We also observed caspase-1 inhibition successfully repressed
Materials and Methods Cell culture
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adipogenesis and modestly reinstalled osteogenesis of MSCs treated with LPS/PA.
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Human MSCs were derived from human umbilical cord, with approval of Ethics Committee
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of Third Xiangya Hospital of Central South University. MSCs were maintained in self-renewal medium (DMEM/F12, 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin and 1% non-essential amino acid (all from ThermoFisher)). The lipopolysaccharide and palmitic acid (Sigma) were supplemented to activate NLRP3 inflammasome. Adipogenic differentiation of MSCs and characterization MSCs were cultured in adipogenesis medium (DMEM/F12, 10% FBS, 1% L-glutamine, 1%
ACCEPTED MANUSCRIPT penicillin/streptomycin, 1µM dexamethasone (Sigma), 0.2µM indomethacin (Sigma), 500µM 3-isobutyl-1-methylxantine (IBMX, Sigma) and 5µg/ml insulin (Sigma)) for 21 days. Adipogenesis was assessed by Oil Red O (Solarbio) staining.
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Osteogenic differentiation of MSCs and characterization MSCs were cultured in osteogenesis medium (DMEM/F12, 10% FBS, 50 µM ascorbic acid (Sigma), 0.1µM dexamethasone (Sigma), 10mM glycerol 2-phosphate (Sigma), 1%
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L-glutamine, and 1% penicillin/streptomycin) for 21 days. Osteogenesis was assessed by
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Alizarin Red (Solarbio) or alkaline phosphatase activity staining (Sigma). Flow cytometric analysis
MSCs maintained in self-renewal medium were dissociated and incubated with antibody against CD90, CD34, CD44 or CD45 (ebioscience) for one hour. All flow cytometric assays
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were performed using Coulter Flow Cytometer system (Beckman). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) RNA was isolated using Trizol (ThermoFisher), and the reverse transcription was completed
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by RevertAid RT Reverse Transcription Kit (ThermoFisher). Quantitative PCR were
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accomplished using SYBR® Green Realtime PCR Master Mix kit (Toyobo). Primers were synthesized by Sangon Biotech Co., Ltd. Table S1 lists the primers. Three biological and two technical replicates were c. Western blotting assay
Protein concentrations of samples were determined by BCA assay. Samples were boiled, loaded into 10% SDS-PAGE for protein electrophoresis, and transferred onto PVDF membranes. Membranes were blocked in 5% non-fat dry milk for 30 minutes and incubated
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of antibodies. Caspase-1 activity detection assay
Activity of caspase-1 were detected by using Caspase-1 activity assay kit (beyotime,
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biological and two technical replicates were completed.
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Shanghai China). The samples were read samples at 405nm in a microtiter plate reader. Three
Statistical analysis
Statistical analyses were completed using GraphPad Prism6. One-way ANOVA was applied
Results
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to comparison for multiple groups. P value < 0.05 was considered significant.
LPS/PA treatment induced the NLRP3 inflammasome activity in MSCs
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To characterize human MSCs, flow cytometric analysis indicated that MSCs were highly
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enriched in expression of positive markers CD90 and CD44, while negative markers CD34 and CD45 were low expressed (Supplementary Fig.1). To activate NLRP3 inflammasome, MSCs were exposed to 0.25mM PA and 0.1µg/ml or 1µg/ml LPS (LPS/PA) for 6 or 24 hours. Gene expression associated with NLRP3 components, adipogenesis, osteogenesis and inflammation was assessed (Fig.1A, Fig.S2). LPS/PA treatment increased expression level of NLRP3 but downregulated expression of SIRT1. Expression of pro-caspase-1 and ASC was not influenced by LPS/PA treatment. No significance was observed in IL-1β and NAMPT
ACCEPTED MANUSCRIPT expression. We subsequently determined the role of LPS/PA treatment in pro-caspase-1 activation in MSCs. MSCs were treated with or without LPS/PA at various concentrations for 24 hours, and caspase-1 activation was assessed (Figure 1B). Caspase-1 was overtly activated
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along with the increase of LPS concentration. These data suggested that treatment favoring inflammation induced NLRP3 inflammasome activity in MSCs and triggered adipogenic differentiation via downregulation of SIRT1 expression.
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LPS/PA treatment enhanced adipogenic differentiatability of MSCs
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MSCs were maintained in self-renewal medium (control) or adipogenic medium with or without LPS/PA for 24 hours. Compared with control and adipogenic differentiation group without LPS/PA, dramatic increase of NLRP3 expression and decreased SIRT1 expression were observed in MSCs for adipogenic differentiation with LPS/PA (Fig.2A). Western
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blotting further confirmed the high expression of NLRP3 and decreased expression of SIRT1 in MSCs in adipogenic medium with LPS/PA (Fig.2B). In addition, caspase-1 protein activation was dramatically heightened when treated with LPS/PA (Fig.2B). These data
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suggested that LPS/PA treatment successfully and promptly induced NLRP3 inflammasome
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activity in MSC in medium favoring adipogenesis. Subsequently we evaluated the effect of NLRP3 inflammasome on adipogenesis in MSCs. MSCs were pre-treated with or without LPS/PA for 72 hours and subjected to adipogenic differentiation or self-renewal medium for 21 days. Lipid accumulation was visualized by Oil Red O assay. MSCs primed by LPS/PA treatment dramatically promoted lipid accumulation compared with adipogenic group without LPS/PA treatment (Fig.2C). In addition, expression of PPARγ and CEBPα were enhanced after adipogenic differentiation
ACCEPTED MANUSCRIPT when primed with LPS/PA treatment (Fig.2D). These data suggested that induction of NLRP3 inflammasome could heighten efficiency of adipogenic differentiation of MSCs. LPS/PA treatment reduced osteogenic differentiation efficiency of MSCs
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MSCs were cultured in self-renewal or osteogenic differentiation medium in the presence or absence of LPS/PA for 24 hours. PCR and western blotting results displayed the remarkably high levels of NLRP3 and caspase-1, as well as the low expression of SIRT1 in
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MSCs exposed to LPS/PA (Fig.3A-B). The activation of caspase-1 was induced by LPS/PA
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treatment (Fig.3B).
We further determined if NLRP3 inflammasome induction affects osteogenesis of MSCs. MSCs were treated with or without 0.1µg/ml LPS/0.25mM PA for 72 hours and subsequently subjected to osteogenic differentiation for 21 days. The efficiency of osteogenic
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differentiation was assessed by visualizing mineral accumulation. Osteogenic medium prompted osteogenesis while LPS/PA pre-treatment significantly declined osteogenesis (Fig.3C). Moreover expression of osteogenic genes RUNX2 and BMP2 was significantly
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enhanced when MSCs maintained in osteogenic medium. However, it was noticed that MSCs
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with LPS/PA treatment demonstrated significantly low expression levels of these genes compared with osteogenic MSCs without treatment (Fig.3D). These data demonstrated that induction of NLRP3 inflammasome significantly ameliorated the efficiency of osteogenesis of MSCs.
Inhibition of caspase-1 activation suppressed adipogenic differentiation and modestly improved osteogenic differentiation of MSCs after NLRP3 inflammasome induction Since caspase-1 is a critical executor from NLRP3 inflammasome, we assessed the
ACCEPTED MANUSCRIPT effect of caspase-1 inhibition on osteogenic and adipogenic differentiation. First, MSCs were cultivated in self-renewal or adipose differentiation medium, with or without LPS/PA and/or Ac-YVAD-cmk. Results from Western blotting and caspase-1 activity detection assay
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demonstrated Ac-YVAD-cmk suppressed caspase-1 induced activation by LPS/PA treatment (Fig.4A-B). When subjected adipogenic differentiation, Ac-YVAD-cmk significantly reduced lipid accumulation in MSCs previously exposed to LPS/PA treatment (Fig.4C). In addition,
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CEBPα and PPARγ expression was significantly reduced by treatment of Ac-YVAD-cmk
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(Fig.4E). In comparison, Ac-YVAD-cmk treatment modestly enhanced osteogenesis of MSCs pre-treated by LPS/PA indicated by mineral accumulation and alkaline phosphatase activity (Fig.4D). The expression of osteogenic markers including BMP2 and RUNX2 was also dramatically enhanced with Ac-YVAD-cmk treatment (Fig.4F). These results demonstrated
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that inhibition of caspase-1 could modestly reinforce the osteogenesis and significantly
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Discussion
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repress adipogenesis of MSCs after NLRP3 inflammasome induction.
As most of the aging related chronic diseases, osteoporosis, featured by loss of bone mass and increased adipose infiltration, is associated with the escalating inflammation, especially the role of NLRP3 inflammasome. Our NLRP3 inflammasome activation can be induced by LPS and PA. LPS presents in outer membrane of gram-negative bacteria, and can activate Toll-like receptor (TLR) 4 and downstream inflammatory signaling Error! Reference source not found.. LPS plays essential role in periodontal disease by triggering
ACCEPTED MANUSCRIPT chronic inflammatory response Error! Reference source not found.. Moreover, saturated fatty acids such as PA are known to increase host inflammatory response, and markedly boosts TLR4-mediated pro-inflammatory signaling triggered by LPS in macrophages Error!
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Reference source not found.. In our study, LPS/PA treatment promoted generation of NLRP3 inflammasome, as well as rising the expression of pro-adipogenesis gene PPARγ. During adipogenic or osteogenic differentiation, we witnessed distinct effects by
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LPS/PA-induced NLRP3 inflammasome: LPS/PA upregulated adipogenesis efficiency but
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decreased osteogenesis of MSCs. Our data also suggested that decrease of SIRT1 expression is associated with NLRP3 inflammasome-triggered adipocyte generation from MSCs. SIRT1 is a nuclear NAD+-dependent class III histone deacetylase class III Error! Reference source not found.. SIRT1 activity is modulated by cellular NAD+ levels and controls metabolic
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homeostasis through the regulation of multiple actions across variant organ or tissues Error! Reference source not found.. In adipose tissue, SIRT1 controls lipolysis and impedes inflammation by inhibiting PPARγ and NFκB activity Error! Reference source not found..
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We noticed that SIRT1 decreased along with LPS/PA treatment, while inhibition of caspase-1
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failed to reinstall protein level of SIRT1. Recent studies on endothelial cells and MSCs have posed that SIRT1 plays an upstream role in inhibiting NLRP3 inflammasome mediated by CD40 or inhibition of transactivation potential of NF-κb Error! Reference source not found.. Corresponding to our results, the first hit of NLRP3 inflammasome activation may be LPS/PA treatment-induced SIRT1 decreased expression or activity inhibition, which further promoted adipogenesis. We found that inhibition of caspapse-1 played a critical role in ameliorating the
ACCEPTED MANUSCRIPT adipogenesis of MSCs with pro-inflammation treatment. Metabolic effects during obesity-induced inflammation can be ascribed to IL-1β and IL-18 Error! Reference source not found.. IL-1β and IL-18 can be activated via caspase-1's protease function Error!
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Reference source not found.. Activation of caspase-1 can be achieved by conformational transformation within NLRP3 inflammasome. Also, caspase-1 is upregulated during adipocyte differentiation and leads adipocytes to insulin-resistant phenotype Error!
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Reference source not found.. In addition, we noticed that inhibition of caspase-1 with
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adipogenic differentiation and LPS/PA induction failed to reinstall the protein level of SIRT1. This indicated that caspase-1 activation from NLRP3 inflammasome is downstream to downregulation of SIRT1. Our results demonstrated the significant inhibition of adipogenesis of MSCs when caspase-1 cleavage was inactivated. In comparison, the function of caspase-1
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on dynamics of osteo-tissue remodeling was seldom reported. Our results indicated that under the LPS/PA-induced activation of NLRP3 inflammasome, caspase-1 inhibition modestly upregulated osteodifferentiation. This suggested that the loss of osteogenic capability during
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NLRP3 inflammasome-associated osteoporosis may be a secondary effect due to strong
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activation of adipogenic-promoting signaling in MSCs. The chronic bone remodeling is regulated by the behaviors of osteoblasts, osteoclasts, and osteocytes Error! Reference source not found.. Bone resorbing osteoclasts originate from hematopoietic precursors while bone-forming osteoblasts are differentiated from MSCs. Along with aging and generation of NLRP3 inflammasome, the milieu of bone marrow shifts to favor osteoclast activity, which may contributes to bone loss Error! Reference source not found.. The relation between NLRP3 inflammasome and MSC-mediated osteoclast activation deserves attention and
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Acknowledgements
2013sk5073.
Reference
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Error! Reference source not found.
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The study was funded by Science and Technology Project of Hunan Province, No.
ACCEPTED MANUSCRIPT Figure legend
Figure 1. LPS/PA treatment induced NLRP3 inflammasome activity and pro-adipogenic
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gene expression in MSCs. (A) Expression of genes related to NLRP3 inflammasome activity in MSCs with LPS/PA treatment at variant concentrations and time. Y-axis represents gene expression fold change relative to GAPDH. X-axis represent treatment groups: 1.
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Self-renewal medium for 6h; 2. 0.1µg/ml LPS/ 0.25mM PA for 6h; 3. 1µg/ml LPS/
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0.25mmol/L PA for 6h; 4. Self-renewal medium for 24h; 5. 0.1µg/ml LPS/ 0.25mM PA for 24h; 6. 1µg/ml LPS/ 0.25mmol/L PA for 24h. *: p<0.05 vs. group 1. (B) Western blotting evaluating protein level of cleaved caspase-1 in MSCs with or without treatment of LPS/PA
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in variant concentrations.
Figure 2. NLRP3 inflammasome induction in MSCs enhanced the adipogenic differentiation. (A) Expression of NLRP3 and SIRT in MSCs treated with or without
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LPS/PA in self-renewal or adipogenic medium. Y-axis represents gene expression fold change
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relative to GAPDH. X-axis represents treatment groups: 1. Self-renewal control; 2. Adipogenic differentiation; 3. Adipogenic differentiation with LPS/PA. *: p<0.05 vs. group 1. (B) Western blotting for protein levels of NLRP3, cleaved caspase-1 and SIRT1 in MSCs in self-renewal or adipogenic medium with or without 0.1µg/ml LPS/0.25mM PA for 24 hours. (C) Oil Red O staining in MSCs for 21-day self-renewal maintenance or adipogenic differentiation with or without 72 hours (day 0 to day 3) of treatment by 0.1µg/ml LPS/0.25mM PA (magnification: 40x). (D) Expression levels of pro-adipogenic genes in
ACCEPTED MANUSCRIPT MSCs when treated with or without LPS/PA in self-renewal or adipogenic medium. Y-axis represents fold change relative to GAPDH. X-axis represent treatment groups: 1. Self-renewal control; 2. Adipogenic differentiation; 3. Adipogenic differentiation with
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LPS/PA. *: p<0.05 vs. group 1. #: p<0.05 vs. group 2.
Figure 3. NLRP3 inflammasome induction in MSCs impeded osteogenic differentiation.
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(A) Expression levels of NLRP3 activity-related genes in MSCs when treated with or without
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LPS/PA in self-renewal or osteogenic medium. Y-axis represents the expression fold change relative to GAPDH. X-axis represent different groups: 1. Self-renewal control; 2. Osteogenic differentiation; 3. Osteogenic differentiation with LPS/PA. *: p<0.05 vs. group 1. #: p<0.05 vs. group 2. (B) Western blotting evaluating levels of NLRP3, cleaved caspase-1 and SIRT1
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in MSCs in self-renewal or osteogenic medium with or without 0.1µg/ml LPS/0.25mM PA for 24 hours. (C) Alizarin Red staining in MSCs for 21-day self-renewal or osteogenic culture with or without 72 hours (day 0 to day 3) of treatment by 0.1µg/ml LPS/0.25mM PA
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(magnification: 40x). (D) Expression levels of pro-osteogenic genes in MSCs when treated
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with or without LPS/PA in self-renewal or osteogenic medium. Y-axis represents fold change relative to GAPDH. X-axis represent different groups: 1. Self-renewal control; 2. Osteogenic differentiation; 3. Osteogenic differentiation with LPS/PA. *: p<0.05 vs. group 1. #: p<0.05 vs. group 2.
Figure 4. Inhibition of caspase-1 activation ameliorated adipogenic differentiation in MSCs and modestly enhanced the osteogenic differentiation in the presence of NLRP3
ACCEPTED MANUSCRIPT induction. (A) Western blotting assay evaluating the protein levels of cleaved caspase-1 and SIRT1 in MSCs in self-renewal medium or adipogenic medium with or without treatment by 0.1µg/ml LPS/0.25mM PA and/or Ac-YVAD-cmk. (B) Caspase-1 activities of MSCs in
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self-renewal medium or adipogenic medium with or without treatment by 0.1µg/ml LPS/0.25mM PA and/or caspase-1 specific inhibitor Ac-YVAD-cmk: 1. Self-renewal control ; 2. Self-renewal control treated with LPS/PA; 3. Adipogenic differentiation; 4. Adipogenic
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differentiation with LPS/PA treatment; 5. Adipogenic differentiation with LPS/PA plus
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Ac-YVAD-cmk treatment. (C) Oil Red O staining in MSCs for 21-day self-renewal maintenance or adipogenic differentiation with or without treatment by 0.1µg/ml LPS/0.25mM PA and/or Ac-YVAD-cmk (magnification: 40x). (D) Alizarin Red staining and alkaline phosphatase activity staining in MSCs for 21-day self-renewal maintenance or
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osteogenic differentiation with or without treatment by 0.1µg/ml LPS/0.25mM PA and/or Ac-YVAD-cmk (magnification: 40x). (E) Expression of pro-adipogenic genes in MSCs when treated with or without LPS/PA or Ac-YVAD-cmk in self-renewal or adipogenic medium.
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Y-axis represents fold change relative to GAPDH. X-axis represent different treatment groups:
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1. Self-renewal control ; 2. Self-renewal control treated with LPS/PA; 3. Adipogenic differentiation; 4. Adipogenic differentiation with LPS/PA; 5. Adipogenic differentiation with LPS/PA plus Ac-YVAD-cmk. *: p<0.05 vs. group 1. #: p<0.05 vs. group 2. (F) Expression levels of pro-osteogenic genes in MSCs when treated with or without LPS/PA or Ac-YVAD-cmk in self-renewal or osteogenic medium. Y-axis represents fold change relative to GAPDH. The numbers in x-axis represent different treatment groups: 1. Self-renewal control ; 2. Self-renewal control treated with LPS/PA; 3. Osteogenic differentiation; 4.
ACCEPTED MANUSCRIPT Osteogenic differentiation with LPS/PA; 5. Osteogenic differentiation with LPS/PA plus
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Ac-YVAD-cmk. *: p<0.05 vs. group 1. #: p<0.05 vs. group 4.
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Figures
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Figure 1
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1. NLRP3 inflammasome and downstream mediator caspase-1 can be activated
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in mesenchymal stem cells.
2. NLRP3 inflammasome cascade activation is associated with increased
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adipogenesis and decreased osteogenesis of mesenchymal stem cells.
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3. The inhibition of capsase-1 activation suppressed adipogenic differentiation and modestly improved osteogenic differentiation of MSCs after NLRP3 inflammasome activation.
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4.The inhibition of capsase-1 activation could be a promising biomarker and
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target for anti-osteoporosis treatment in future.
S0006-291X(17)30268-1
DOI:
10.1016/j.bbrc.2017.02.007
Reference:
YBBRC 37255
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 30 January 2017 Accepted Date: 2 February 2017
Please cite this article as: L. Wang, K. Chen, X. Wan, F. Wang, Z. Guo, Z. Mo, NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation
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Linghao Wang, Ke Chen, Xinxing Wan, Fang Wang, Zi Guo, Zhaohui Mo* Department of Endocrinology and Metabolism, Third Xiangya Hospital of Central South University
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* Corresponding author.
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Abstract Osteoporosis is one of the most common skeletal disease featured by osteopenia and
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adipose accumulation in bone tissue. NLRP3 inflammasome activation is an essential player in aging-related chronic diseases like osteoporosis, particularly due to the causal caspase-1 activation and its correlation to adipose accumulation in bone tissue. Moreover, the
SC
expression of anti-aging/senescence SIRT1 was reported to decline along with aging. As the
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major cellular contributor of bone formation, mesenchymal stem cells (MSCs) are multipotent stem cells processing mutually exclusive differentiatability toward osteocytes or adipocytes. Therefore, we hypothesized that NLRP3 inflammasome activation promotes adipogenesis and repress osteogenesis in MSCs via inhibiting SIRT1 expression. We
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activated NLRP3 inflammasome in human MSCs via lipopolysaccharide and palmitic acid (LPS/PA) treatment for self-renewal maintenance, adipogenic differentiation or osteogenic differentiation. LPS/PA treatment significantly increased NLRP3 expression, decreased
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SIRT1 expression and promoted caspase-1 activity in MSCs. LPS/PA treatment also boosted
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adipogenesis of MSCs and suppressed osteogenesis. Moreover, inhibition of caspase-1 activity repressed adipogenic differentiation and partially improved osteogenic differentiation of MSCs with LPS/PA treatment. Our study demonstrated the pivotal roles of NLRP3 inflammasome and downstream mediator caspase-1 for the progress of osteo-differentiation MSCs, and offered novel therapeutic target of treatment for osteoporosis. Key words mesenchymal stem cell, NLRP3 inflammasome, osteoporosis, adipogenesis, osteogenesis
ACCEPTED MANUSCRIPT
Introduction
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Aging-related primary osteoporosis is rampant worldwide, which annually causes over 9 million fractures and increases social and economic burden for aging population Error! Reference source not found.. Osteoporosis is characterized by bone fragility, damage of
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microstructure of bone tissue, decreased bone mass and adipose infiltration Error!
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Reference source not found.. Dramatic alteration from osteocytes to adipocytes in bone tissue during osteoporosis is related with the dynamic change from mesenchymal stem cells (MSCs), due to their bi-directional differentiatability toward adipocytes or osteocytes Error! Reference source not found..
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MSCs, the major cellular contributor of bone tissue, demonstrate differentiation potential toward bone/cartilage and adipose tissue. During osteogenesis, expression of osteogenic-specific transcription factor RUNX2 is upregulated in MSCs Error! Reference
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source not found.. In contrast, adipogenesis of MSCs presents high expression of PPAP-γ,
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which triggers expression of adipogenic genes including AP2 and FAFP Error! Reference source not found.. Adipogenesis and osteogenesis in MSCs regulated by RUNX2 or PPAP-γ are mutual exclusive Error! Reference source not found.. During aging process, MSCs exhibit biased fate toward adipogenesis with accumulation of adipose tissue and decline of bone formation in bone marrow Error! Reference source not found.. NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome is reported as the most clinically implicated inflammasome Error! Reference source not
ACCEPTED MANUSCRIPT found.. NLRP3 inflammasome is an intracellular protein complex involved in initiation of innate immune response Error! Reference source not found.. NLRP3 inflammasome contains NLRP3, apoptotic speck protein (ASC) and pro-caspase-1 [10]. NLRP3 plays
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critical roles in multiple chronic diseases Error! Reference source not found.. Activated NLRP3 protein is capable of recruiting ASC and pro-caspase-1 to assemble NLRP3 inflammasome Error! Reference source not found.. As such, NLRP3 inflammasome sets up
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the stage for caspase-1 activation, and triggers secretion of inflammatory interleukin (IL)-1β
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and IL-18 Error! Reference source not found.. Ultimately, NLRP3 inflammasome causes low-grade systemic inflammation and chronic organ failure Error! Reference source not found.. The overt increase of cortical bone in NLRP3-/- aging mice compared with wild type counterparts was observed, indicating NLRP3 may twist round MSC differentiation from
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osteogenesis to adipogenesis Error! Reference source not found.. The high level of NLRP3 was also detected in human with obesity Error! Reference source not found.. Sirtuin1 (SIRT1) is ubiquitously expressed as class III histone deacetylase and regulates
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survival, metabolic rate, and oxygen consumption. SIRT1 interferes with NF-κb signaling,
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counteracts with inflammatory process and impedes bioactivity of PPAR-γ to repress adipogenesis Error! Reference source not found.. SIRT1 expression declines within aged bone tissue along with the increase of adipose components. Knockout of SIRT1 in MSCs in aged mice caused osteopenia and osteogenesis inefficiency Error! Reference source not found.. Nicotinamide phosphoribosyltransferase (NAMPT) positively regulates SIRT1 activity via synthesis of nicotinamide adenine dinucleotide (NAD) and promotes osteogenesis Error! Reference source not found.. Also, NLRP3 inflammasome prevents expression of
ACCEPTED MANUSCRIPT SIRT1, and the caspase-1 within NLRP3 inflammasome induces proteolysis of SIRT1 Error! Reference source not found.. Taking together, we hypothesized that NLRP3 inflammasome promotes adipogenesis
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and represses osteogenesis of MSCs by inhibiting SIRT1. We induced activation of NLRP3 inflammasome in human MSCs by lipopolysaccharide (LPS) and palmitic acid (PA) treatment during self-renewal, adipogenic or osteogenic differentiation. LPS/PA treatment
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enhanced NLRP3 expression, reduced SIRT1 expression and promoted caspase-1 activation
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in MSCs. Moreover, LPS/PA treatment enhanced adipogenesis of MSCs but downregulated the osteogenesis efficiency. We also observed caspase-1 inhibition successfully repressed
Materials and Methods Cell culture
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adipogenesis and modestly reinstalled osteogenesis of MSCs treated with LPS/PA.
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Human MSCs were derived from human umbilical cord, with approval of Ethics Committee
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of Third Xiangya Hospital of Central South University. MSCs were maintained in self-renewal medium (DMEM/F12, 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin and 1% non-essential amino acid (all from ThermoFisher)). The lipopolysaccharide and palmitic acid (Sigma) were supplemented to activate NLRP3 inflammasome. Adipogenic differentiation of MSCs and characterization MSCs were cultured in adipogenesis medium (DMEM/F12, 10% FBS, 1% L-glutamine, 1%
ACCEPTED MANUSCRIPT penicillin/streptomycin, 1µM dexamethasone (Sigma), 0.2µM indomethacin (Sigma), 500µM 3-isobutyl-1-methylxantine (IBMX, Sigma) and 5µg/ml insulin (Sigma)) for 21 days. Adipogenesis was assessed by Oil Red O (Solarbio) staining.
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Osteogenic differentiation of MSCs and characterization MSCs were cultured in osteogenesis medium (DMEM/F12, 10% FBS, 50 µM ascorbic acid (Sigma), 0.1µM dexamethasone (Sigma), 10mM glycerol 2-phosphate (Sigma), 1%
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L-glutamine, and 1% penicillin/streptomycin) for 21 days. Osteogenesis was assessed by
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Alizarin Red (Solarbio) or alkaline phosphatase activity staining (Sigma). Flow cytometric analysis
MSCs maintained in self-renewal medium were dissociated and incubated with antibody against CD90, CD34, CD44 or CD45 (ebioscience) for one hour. All flow cytometric assays
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were performed using Coulter Flow Cytometer system (Beckman). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) RNA was isolated using Trizol (ThermoFisher), and the reverse transcription was completed
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by RevertAid RT Reverse Transcription Kit (ThermoFisher). Quantitative PCR were
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accomplished using SYBR® Green Realtime PCR Master Mix kit (Toyobo). Primers were synthesized by Sangon Biotech Co., Ltd. Table S1 lists the primers. Three biological and two technical replicates were c. Western blotting assay
Protein concentrations of samples were determined by BCA assay. Samples were boiled, loaded into 10% SDS-PAGE for protein electrophoresis, and transferred onto PVDF membranes. Membranes were blocked in 5% non-fat dry milk for 30 minutes and incubated
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of antibodies. Caspase-1 activity detection assay
Activity of caspase-1 were detected by using Caspase-1 activity assay kit (beyotime,
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biological and two technical replicates were completed.
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Shanghai China). The samples were read samples at 405nm in a microtiter plate reader. Three
Statistical analysis
Statistical analyses were completed using GraphPad Prism6. One-way ANOVA was applied
Results
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to comparison for multiple groups. P value < 0.05 was considered significant.
LPS/PA treatment induced the NLRP3 inflammasome activity in MSCs
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To characterize human MSCs, flow cytometric analysis indicated that MSCs were highly
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enriched in expression of positive markers CD90 and CD44, while negative markers CD34 and CD45 were low expressed (Supplementary Fig.1). To activate NLRP3 inflammasome, MSCs were exposed to 0.25mM PA and 0.1µg/ml or 1µg/ml LPS (LPS/PA) for 6 or 24 hours. Gene expression associated with NLRP3 components, adipogenesis, osteogenesis and inflammation was assessed (Fig.1A, Fig.S2). LPS/PA treatment increased expression level of NLRP3 but downregulated expression of SIRT1. Expression of pro-caspase-1 and ASC was not influenced by LPS/PA treatment. No significance was observed in IL-1β and NAMPT
ACCEPTED MANUSCRIPT expression. We subsequently determined the role of LPS/PA treatment in pro-caspase-1 activation in MSCs. MSCs were treated with or without LPS/PA at various concentrations for 24 hours, and caspase-1 activation was assessed (Figure 1B). Caspase-1 was overtly activated
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along with the increase of LPS concentration. These data suggested that treatment favoring inflammation induced NLRP3 inflammasome activity in MSCs and triggered adipogenic differentiation via downregulation of SIRT1 expression.
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LPS/PA treatment enhanced adipogenic differentiatability of MSCs
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MSCs were maintained in self-renewal medium (control) or adipogenic medium with or without LPS/PA for 24 hours. Compared with control and adipogenic differentiation group without LPS/PA, dramatic increase of NLRP3 expression and decreased SIRT1 expression were observed in MSCs for adipogenic differentiation with LPS/PA (Fig.2A). Western
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blotting further confirmed the high expression of NLRP3 and decreased expression of SIRT1 in MSCs in adipogenic medium with LPS/PA (Fig.2B). In addition, caspase-1 protein activation was dramatically heightened when treated with LPS/PA (Fig.2B). These data
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suggested that LPS/PA treatment successfully and promptly induced NLRP3 inflammasome
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activity in MSC in medium favoring adipogenesis. Subsequently we evaluated the effect of NLRP3 inflammasome on adipogenesis in MSCs. MSCs were pre-treated with or without LPS/PA for 72 hours and subjected to adipogenic differentiation or self-renewal medium for 21 days. Lipid accumulation was visualized by Oil Red O assay. MSCs primed by LPS/PA treatment dramatically promoted lipid accumulation compared with adipogenic group without LPS/PA treatment (Fig.2C). In addition, expression of PPARγ and CEBPα were enhanced after adipogenic differentiation
ACCEPTED MANUSCRIPT when primed with LPS/PA treatment (Fig.2D). These data suggested that induction of NLRP3 inflammasome could heighten efficiency of adipogenic differentiation of MSCs. LPS/PA treatment reduced osteogenic differentiation efficiency of MSCs
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MSCs were cultured in self-renewal or osteogenic differentiation medium in the presence or absence of LPS/PA for 24 hours. PCR and western blotting results displayed the remarkably high levels of NLRP3 and caspase-1, as well as the low expression of SIRT1 in
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MSCs exposed to LPS/PA (Fig.3A-B). The activation of caspase-1 was induced by LPS/PA
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treatment (Fig.3B).
We further determined if NLRP3 inflammasome induction affects osteogenesis of MSCs. MSCs were treated with or without 0.1µg/ml LPS/0.25mM PA for 72 hours and subsequently subjected to osteogenic differentiation for 21 days. The efficiency of osteogenic
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differentiation was assessed by visualizing mineral accumulation. Osteogenic medium prompted osteogenesis while LPS/PA pre-treatment significantly declined osteogenesis (Fig.3C). Moreover expression of osteogenic genes RUNX2 and BMP2 was significantly
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enhanced when MSCs maintained in osteogenic medium. However, it was noticed that MSCs
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with LPS/PA treatment demonstrated significantly low expression levels of these genes compared with osteogenic MSCs without treatment (Fig.3D). These data demonstrated that induction of NLRP3 inflammasome significantly ameliorated the efficiency of osteogenesis of MSCs.
Inhibition of caspase-1 activation suppressed adipogenic differentiation and modestly improved osteogenic differentiation of MSCs after NLRP3 inflammasome induction Since caspase-1 is a critical executor from NLRP3 inflammasome, we assessed the
ACCEPTED MANUSCRIPT effect of caspase-1 inhibition on osteogenic and adipogenic differentiation. First, MSCs were cultivated in self-renewal or adipose differentiation medium, with or without LPS/PA and/or Ac-YVAD-cmk. Results from Western blotting and caspase-1 activity detection assay
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demonstrated Ac-YVAD-cmk suppressed caspase-1 induced activation by LPS/PA treatment (Fig.4A-B). When subjected adipogenic differentiation, Ac-YVAD-cmk significantly reduced lipid accumulation in MSCs previously exposed to LPS/PA treatment (Fig.4C). In addition,
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CEBPα and PPARγ expression was significantly reduced by treatment of Ac-YVAD-cmk
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(Fig.4E). In comparison, Ac-YVAD-cmk treatment modestly enhanced osteogenesis of MSCs pre-treated by LPS/PA indicated by mineral accumulation and alkaline phosphatase activity (Fig.4D). The expression of osteogenic markers including BMP2 and RUNX2 was also dramatically enhanced with Ac-YVAD-cmk treatment (Fig.4F). These results demonstrated
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that inhibition of caspase-1 could modestly reinforce the osteogenesis and significantly
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Discussion
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repress adipogenesis of MSCs after NLRP3 inflammasome induction.
As most of the aging related chronic diseases, osteoporosis, featured by loss of bone mass and increased adipose infiltration, is associated with the escalating inflammation, especially the role of NLRP3 inflammasome. Our NLRP3 inflammasome activation can be induced by LPS and PA. LPS presents in outer membrane of gram-negative bacteria, and can activate Toll-like receptor (TLR) 4 and downstream inflammatory signaling Error! Reference source not found.. LPS plays essential role in periodontal disease by triggering
ACCEPTED MANUSCRIPT chronic inflammatory response Error! Reference source not found.. Moreover, saturated fatty acids such as PA are known to increase host inflammatory response, and markedly boosts TLR4-mediated pro-inflammatory signaling triggered by LPS in macrophages Error!
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Reference source not found.. In our study, LPS/PA treatment promoted generation of NLRP3 inflammasome, as well as rising the expression of pro-adipogenesis gene PPARγ. During adipogenic or osteogenic differentiation, we witnessed distinct effects by
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LPS/PA-induced NLRP3 inflammasome: LPS/PA upregulated adipogenesis efficiency but
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decreased osteogenesis of MSCs. Our data also suggested that decrease of SIRT1 expression is associated with NLRP3 inflammasome-triggered adipocyte generation from MSCs. SIRT1 is a nuclear NAD+-dependent class III histone deacetylase class III Error! Reference source not found.. SIRT1 activity is modulated by cellular NAD+ levels and controls metabolic
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homeostasis through the regulation of multiple actions across variant organ or tissues Error! Reference source not found.. In adipose tissue, SIRT1 controls lipolysis and impedes inflammation by inhibiting PPARγ and NFκB activity Error! Reference source not found..
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We noticed that SIRT1 decreased along with LPS/PA treatment, while inhibition of caspase-1
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failed to reinstall protein level of SIRT1. Recent studies on endothelial cells and MSCs have posed that SIRT1 plays an upstream role in inhibiting NLRP3 inflammasome mediated by CD40 or inhibition of transactivation potential of NF-κb Error! Reference source not found.. Corresponding to our results, the first hit of NLRP3 inflammasome activation may be LPS/PA treatment-induced SIRT1 decreased expression or activity inhibition, which further promoted adipogenesis. We found that inhibition of caspapse-1 played a critical role in ameliorating the
ACCEPTED MANUSCRIPT adipogenesis of MSCs with pro-inflammation treatment. Metabolic effects during obesity-induced inflammation can be ascribed to IL-1β and IL-18 Error! Reference source not found.. IL-1β and IL-18 can be activated via caspase-1's protease function Error!
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Reference source not found.. Activation of caspase-1 can be achieved by conformational transformation within NLRP3 inflammasome. Also, caspase-1 is upregulated during adipocyte differentiation and leads adipocytes to insulin-resistant phenotype Error!
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Reference source not found.. In addition, we noticed that inhibition of caspase-1 with
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adipogenic differentiation and LPS/PA induction failed to reinstall the protein level of SIRT1. This indicated that caspase-1 activation from NLRP3 inflammasome is downstream to downregulation of SIRT1. Our results demonstrated the significant inhibition of adipogenesis of MSCs when caspase-1 cleavage was inactivated. In comparison, the function of caspase-1
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on dynamics of osteo-tissue remodeling was seldom reported. Our results indicated that under the LPS/PA-induced activation of NLRP3 inflammasome, caspase-1 inhibition modestly upregulated osteodifferentiation. This suggested that the loss of osteogenic capability during
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NLRP3 inflammasome-associated osteoporosis may be a secondary effect due to strong
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activation of adipogenic-promoting signaling in MSCs. The chronic bone remodeling is regulated by the behaviors of osteoblasts, osteoclasts, and osteocytes Error! Reference source not found.. Bone resorbing osteoclasts originate from hematopoietic precursors while bone-forming osteoblasts are differentiated from MSCs. Along with aging and generation of NLRP3 inflammasome, the milieu of bone marrow shifts to favor osteoclast activity, which may contributes to bone loss Error! Reference source not found.. The relation between NLRP3 inflammasome and MSC-mediated osteoclast activation deserves attention and
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Acknowledgements
2013sk5073.
Reference
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Error! Reference source not found.
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The study was funded by Science and Technology Project of Hunan Province, No.
ACCEPTED MANUSCRIPT Figure legend
Figure 1. LPS/PA treatment induced NLRP3 inflammasome activity and pro-adipogenic
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gene expression in MSCs. (A) Expression of genes related to NLRP3 inflammasome activity in MSCs with LPS/PA treatment at variant concentrations and time. Y-axis represents gene expression fold change relative to GAPDH. X-axis represent treatment groups: 1.
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Self-renewal medium for 6h; 2. 0.1µg/ml LPS/ 0.25mM PA for 6h; 3. 1µg/ml LPS/
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0.25mmol/L PA for 6h; 4. Self-renewal medium for 24h; 5. 0.1µg/ml LPS/ 0.25mM PA for 24h; 6. 1µg/ml LPS/ 0.25mmol/L PA for 24h. *: p<0.05 vs. group 1. (B) Western blotting evaluating protein level of cleaved caspase-1 in MSCs with or without treatment of LPS/PA
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in variant concentrations.
Figure 2. NLRP3 inflammasome induction in MSCs enhanced the adipogenic differentiation. (A) Expression of NLRP3 and SIRT in MSCs treated with or without
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LPS/PA in self-renewal or adipogenic medium. Y-axis represents gene expression fold change
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relative to GAPDH. X-axis represents treatment groups: 1. Self-renewal control; 2. Adipogenic differentiation; 3. Adipogenic differentiation with LPS/PA. *: p<0.05 vs. group 1. (B) Western blotting for protein levels of NLRP3, cleaved caspase-1 and SIRT1 in MSCs in self-renewal or adipogenic medium with or without 0.1µg/ml LPS/0.25mM PA for 24 hours. (C) Oil Red O staining in MSCs for 21-day self-renewal maintenance or adipogenic differentiation with or without 72 hours (day 0 to day 3) of treatment by 0.1µg/ml LPS/0.25mM PA (magnification: 40x). (D) Expression levels of pro-adipogenic genes in
ACCEPTED MANUSCRIPT MSCs when treated with or without LPS/PA in self-renewal or adipogenic medium. Y-axis represents fold change relative to GAPDH. X-axis represent treatment groups: 1. Self-renewal control; 2. Adipogenic differentiation; 3. Adipogenic differentiation with
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LPS/PA. *: p<0.05 vs. group 1. #: p<0.05 vs. group 2.
Figure 3. NLRP3 inflammasome induction in MSCs impeded osteogenic differentiation.
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(A) Expression levels of NLRP3 activity-related genes in MSCs when treated with or without
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LPS/PA in self-renewal or osteogenic medium. Y-axis represents the expression fold change relative to GAPDH. X-axis represent different groups: 1. Self-renewal control; 2. Osteogenic differentiation; 3. Osteogenic differentiation with LPS/PA. *: p<0.05 vs. group 1. #: p<0.05 vs. group 2. (B) Western blotting evaluating levels of NLRP3, cleaved caspase-1 and SIRT1
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in MSCs in self-renewal or osteogenic medium with or without 0.1µg/ml LPS/0.25mM PA for 24 hours. (C) Alizarin Red staining in MSCs for 21-day self-renewal or osteogenic culture with or without 72 hours (day 0 to day 3) of treatment by 0.1µg/ml LPS/0.25mM PA
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(magnification: 40x). (D) Expression levels of pro-osteogenic genes in MSCs when treated
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with or without LPS/PA in self-renewal or osteogenic medium. Y-axis represents fold change relative to GAPDH. X-axis represent different groups: 1. Self-renewal control; 2. Osteogenic differentiation; 3. Osteogenic differentiation with LPS/PA. *: p<0.05 vs. group 1. #: p<0.05 vs. group 2.
Figure 4. Inhibition of caspase-1 activation ameliorated adipogenic differentiation in MSCs and modestly enhanced the osteogenic differentiation in the presence of NLRP3
ACCEPTED MANUSCRIPT induction. (A) Western blotting assay evaluating the protein levels of cleaved caspase-1 and SIRT1 in MSCs in self-renewal medium or adipogenic medium with or without treatment by 0.1µg/ml LPS/0.25mM PA and/or Ac-YVAD-cmk. (B) Caspase-1 activities of MSCs in
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self-renewal medium or adipogenic medium with or without treatment by 0.1µg/ml LPS/0.25mM PA and/or caspase-1 specific inhibitor Ac-YVAD-cmk: 1. Self-renewal control ; 2. Self-renewal control treated with LPS/PA; 3. Adipogenic differentiation; 4. Adipogenic
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differentiation with LPS/PA treatment; 5. Adipogenic differentiation with LPS/PA plus
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Ac-YVAD-cmk treatment. (C) Oil Red O staining in MSCs for 21-day self-renewal maintenance or adipogenic differentiation with or without treatment by 0.1µg/ml LPS/0.25mM PA and/or Ac-YVAD-cmk (magnification: 40x). (D) Alizarin Red staining and alkaline phosphatase activity staining in MSCs for 21-day self-renewal maintenance or
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osteogenic differentiation with or without treatment by 0.1µg/ml LPS/0.25mM PA and/or Ac-YVAD-cmk (magnification: 40x). (E) Expression of pro-adipogenic genes in MSCs when treated with or without LPS/PA or Ac-YVAD-cmk in self-renewal or adipogenic medium.
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Y-axis represents fold change relative to GAPDH. X-axis represent different treatment groups:
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1. Self-renewal control ; 2. Self-renewal control treated with LPS/PA; 3. Adipogenic differentiation; 4. Adipogenic differentiation with LPS/PA; 5. Adipogenic differentiation with LPS/PA plus Ac-YVAD-cmk. *: p<0.05 vs. group 1. #: p<0.05 vs. group 2. (F) Expression levels of pro-osteogenic genes in MSCs when treated with or without LPS/PA or Ac-YVAD-cmk in self-renewal or osteogenic medium. Y-axis represents fold change relative to GAPDH. The numbers in x-axis represent different treatment groups: 1. Self-renewal control ; 2. Self-renewal control treated with LPS/PA; 3. Osteogenic differentiation; 4.
ACCEPTED MANUSCRIPT Osteogenic differentiation with LPS/PA; 5. Osteogenic differentiation with LPS/PA plus
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Ac-YVAD-cmk. *: p<0.05 vs. group 1. #: p<0.05 vs. group 4.
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Figures
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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1. NLRP3 inflammasome and downstream mediator caspase-1 can be activated
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in mesenchymal stem cells.
2. NLRP3 inflammasome cascade activation is associated with increased
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adipogenesis and decreased osteogenesis of mesenchymal stem cells.
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3. The inhibition of capsase-1 activation suppressed adipogenic differentiation and modestly improved osteogenic differentiation of MSCs after NLRP3 inflammasome activation.
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4.The inhibition of capsase-1 activation could be a promising biomarker and
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target for anti-osteoporosis treatment in future.