lncRNA和干细胞-Long noncoding RNA Bmncr regulates mesenchymal stem cell fate during skeletal aging.pdf

Long noncoding RNA Bmncr regulatesmesenchymal stem cell fate during skeletalagingChang-Jun Li,Yan Huang,Xiang-Hang LuoJ Clin Invest.2018;128(12):5251-5266.https:/doi.org/10.1172/JCI99044.Bone marrow mesenchymal stem cells(BMSCs)exhibit an age-related lineage switchbetween osteogenic and adipogenic fates,which contributes to bone loss and adiposity.Here we identified a long noncoding RNA,Bmncr,which regulated the fate of BMSCsduring aging.Mice depleted of Bmncr(Bmncr-KO)showed decreased bone mass andincreased bone marrow adiposity,whereas transgenic overexpression of Bmncr(Bmncr-Tg)alleviated bone loss and bone marrow fat accumulation.Bmncr regulated the osteogenicniche of BMSCs by maintaining extracellular matrix protein fibromodulin(FMOD)andactivation of the BMP2 pathway.Bmncr affected local 3D chromatin structure andtranscription of Fmod.The absence of Fmod modified the bone phenotype of Bmncr-Tgmice.Further analysis revealed that Bmncr would serve as a scaffold to facilitate theinteraction of TAZ and ABL,and thus facilitate the assembly of the TAZ andRUNX2/PPARG transcriptional complex,promoting osteogenesis and inhibitingadipogenesis.Adeno-associated viral-mediated overexpression of Taz in osteoprogenitorsalleviated bone loss and marrow fat accumulation in Bmncr-KO mice.Furthermore,restoringBMNCR levels in human BMSCs reversed the age-related switch between osteoblast andadipocyte differentiation.Our findings indicate that Bmncr is a key regulator of the age-related osteogenic niche alteration and cell fate switch of BMSCs.Research ArticleEndocrinologyFind the latest version:http:/jci.me/99044/pdfThe Journal of Clinical Investigation R ES E ARC H ARTICLE5 251jci.org Volume 128 Number 12 December 2018IntroductionGuided by genetic and molecular mediators,as well as the local microenvironment,bone marrow mesenchymal stem cells(BMSCs)differentiate into various mature cell types,including adipocytes and osteoblasts(13).Age-related osteoporosis is characterized by reduced bone formation and the accumulation of fat in the bone marrow compartment.With age,BMSCs are more inclined to under-go differentiation into adipocytes rather than osteoblasts,resulting in an increased number of adipocytes and a decreased number of osteoblasts,causing osteoporosis(46);however,this mechanism is not fully understood.Long noncoding RNAs(lncRNAs),nonprotein coding tran-scripts longer than 200 nucleotides,have been reported to play key roles in the regulation of various diseases and biological pro-cesses,including cellular differentiation,proliferation,apoptosis,gene regulation,and cancer development(710).Although the biological functions of most lncRNAs are not wholly comprehend-ed,lncRNAs have been shown to regulate gene expression pat-terns by helping bind key transcription factors to promoters and through the physical association of transcription factors them-selves(1114).Yet,there is little research on the role of lncRNAs in the age-related lineage fate switch of BMSCs in the skeleton.In the present study,we identified a lncRNA,Bmncr,that regulates the fate of BMSCs during aging.Mice with Bmncr defi-ciency exhibited an osteoporosis-like phenotype characterized by reduced bone mass and accumulated bone marrow fat.Genetic overexpression of Bmncr in mice stimulated bone formation and decreased bone marrow adiposity.After analyzing the lineage fate switch mechanisms of the mesenchymal stem cell(MSC),it revealed that Bmncr upregulated extracellular matrix molecu-lar fibromodulin to anchor BMSCs in trabecular-rich metaphysis regions and also activated the BMP2 pathway in BMSCs.Bmncr also served as a scaffold to assemble TAZ and ABL and facilitat-ed the formation of the TAZ and RUNX2/PPARG transcriptional complex,which promoted osteogenesis and repressed adipogene-sis.Our study reveals a new mechanism of lineage shift of BMSCs during aging and may represent a therapeutic strategy against age-related bone loss.ResultsBmncr expression in BMSCs decreased with aging.To identify dys-regulated lncRNAs in BMSCs during aging,we sorted the Sca-1+CD29+CD45CD11b BMSCs from young and aged mice using FACS(15),and conducted microarray analysis(Figure 1A).A total of 92 differentially expressed lncRNAs with at least 3-fold change were identified.Among these lncRNAs,83 were decreased and 9 were increased in BMSCs isolated from aged mice.We selected 5 lncRNAs with the following properties:(a)expression is high in BMSCs and dramatically decreases during aging;(b)evolution-ary conservation score greater than 70%;(c)expression in human BMSCs is negatively correlated with age;and(d)no splicing or overlap with any coding gene present in the National Center for Biotechnology Informations RefSeq database,University of Cali-fornia Santa Cruz Genome Browser,or Ensembl genome browser annotations.Among them,NONMMUT002667 has the best evo-Bone marrow mesenchymal stem cells(BMSCs)exhibit an age-related lineage switch between osteogenic and adipogenic fates,which contributes to bone loss and adiposity.Here we identified a long noncoding RNA,Bmncr,which regulated the fate of BMSCs during aging.Mice depleted of Bmncr(Bmncr-KO)showed decreased bone mass and increased bone marrow adiposity,whereas transgenic overexpression of Bmncr(Bmncr-Tg)alleviated bone loss and bone marrow fat accumulation.Bmncr regulated the osteogenic niche of BMSCs by maintaining extracellular matrix protein fibromodulin(FMOD)and activation of the BMP2 pathway.Bmncr affected local 3D chromatin structure and transcription of Fmod.The absence of Fmod modified the bone phenotype of Bmncr-Tg mice.Further analysis revealed that Bmncr would serve as a scaffold to facilitate the interaction of TAZ and ABL,and thus facilitate the assembly of the TAZ and RUNX2/PPARG transcriptional complex,promoting osteogenesis and inhibiting adipogenesis.Adeno-associated viral-mediated overexpression of Taz in osteoprogenitors alleviated bone loss and marrow fat accumulation in Bmncr-KO mice.Furthermore,restoring BMNCR levels in human BMSCs reversed the age-related switch between osteoblast and adipocyte differentiation.Our findings indicate that Bmncr is a key regulator of the age-related osteogenic niche alteration and cell fate switch of BMSCs.Long noncoding RNA Bmncr regulates mesenchymal stem cell fate during skeletal agingChang-Jun Li,1,2 Ye Xiao,1 Mi Yang,1,2,3 Tian Su,1 Xi Sun,1 Qi Guo,1 Yan Huang,1 and Xiang-Hang Luo1,21Department of Endocrinology,Endocrinology Research Center,Xiangya Hospital of Central South University,Changsha,Hunan,China.2Key Laboratory of Organ Injury,Aging and Regenerative Medicine of Hunan Province,Hunan,China.3Department of Orthopaedic Surgery,Johns Hopkins University School of Medicine,Baltimore,Maryland,USA.Authorship note:CJL,YX,and MY contributed equally to this work.Conflict of interest:The authors have declared that no conflict of interest exists.License:Copyright 2018,American Society for Clinical Investigation.Submitted:December 5,2017;Accepted:September 11,2018.Reference information:J Clin Invest.2018;128(12):52515266.https:/doi.org/10.1172/JCI99044.The Journal of Clinical Investigation R ESE A RCH A RTI CL E5 2 5 2jci.org Volume 128 Number 12 December 2018D).These data suggest that Bmncr may play an important role in reg-ulating BMSC functions along with aging in both mice and humans.Bone marrow microenvironment undergoes age-related changes during aging.Among them,plenty of bone-related hor-mones(e.g.,estrogen),growth factor signaling(e.g.,TGF-sig-naling pathway),and senescence-associated stress(e.g.,oxidative stress)changed.To further investigate what is regulating Bmncr expression in BMSCs during aging,we first tested whether estro-gen or TGF-pathway would affect Bmncr expression in BMSCs.Bmncr expression level was detected in BMSCs with the admin-istration of 17-estradiol and TGF-1.Somewhat unexpectedly,none of the 2 factors affected Bmncr expression in BMSCs(Supple-mental Figure 4,A and B).We subsequently measured the effects of oxidative stress on Bmncr expression in BMSCs.Mouse and human BMSCs were treated with buthionine-sulfoximine(BSO),and Bmncr expression was detected.Surprisingly,BSO downreg-ulated Bmncr expression in a dosage-dependent manner(Sup-plemental Figure 4,C and D).These data indicate that elevated oxidative stress at least partially contributed to the reduced Bmncr expression in BMSCs during aging.Bmncr knockout mice showed accelerated bone loss and bone marrow fat accumulation.To investigate the role of Bmncr in osteogenic differentiation in adulthood and the aging process,we lutionary conservation.Mammalian sequence conservation scores of NONMMUT002667 were lower than protein-coding exons but higher than random intergenic regions,in keeping with previous reports(16).For nearly 75%of human lncRNA-LOC107985249,we identified orthologous mouse genomic regions of greater than 200 bp,compared with 58%of random human intergenic regions(Sup-plemental Figure 1 and 2;supplemental material available online with this article;https:/doi.org/10.1172/JCI99044DS1).Therefore,NONMMUT002667 was selected for further in-depth experiments.To begin,we measured the NONMMUT002667 tissue expres-sion pattern in the femur,brain,kidney,lung,liver,heart,and muscle in young mice.A high level of NONMMUT002667 was detected in the femur and white adipocyte tissue(WAT),but not in other tissues(Supplemental Figure 3A).Furthermore,BMSCs and osteoblasts showed a higher level of NONMMUT002667 expression relative to monocytes and preosteoclasts(Supplemen-tal Figure 3B).We named the candidate Bmncr(bone marrow stem cellrelated lncRNA).Bmncr displayed high expression in BMSCs of young mice and significantly decreased during aging(Figure 1B),as confirmed by quantitative real-time PCR(qRT-PCR).We isolated human BMSCs(defined as STRO-1+CD146+CD45)from bone marrow by FACS(17).Similarly,the BMNCR level in human BMSCs was negatively correlated with age(Figure 1,C and Figure 1.Bmncr expression in BMSCs decreased during aging.(A)Heat map of microarray profiling results of deregulated lncRNAs in BMSCs from young and aged mice(fold change 3,false-discovery rate 0.20).Red arrow indicates the lncRNA(Bmncr)selected for study.(B)qRT-PCR analysis of the levels of Bmncr expression in BMSCs derived from the mice at different ages(n=5 per group).(C and D)Age-associated changes of BMNCR levels in human BMSCs from 32 males(C)and 32 females(D).Data are mean SD.*P 0.01(1-way ANOVA).The Journal of Clinical Investigation R ES E ARC H ARTICLE5 25 3jci.org Volume 128 Number 12 December 2018nificantly lower bone formation rates(BFRs)compared with their WT littermates(Figure 2,E and F).Immunohistochemical analysis revealed that the number of osteocalcin-positive mature osteoblasts in the bone surface of Bmncr-KO mice was decreased compared with their WT litter-mates(Figure 2,G and H).Moreover,the marrow fat was signifi-cantly increased in the bone marrow of Bmncr-KO mice compared with the WT controls as evidenced by a much higher number of adipocytes and fat droplets in decalcified femurs stained with osmium tetroxide(OsO4)(Figure 2,IK).Histological analysis showed that the number and surface of osteoblasts normalized to the trabecular bone surface were signifi-cantly decreased in Bmncr-KO mice,in contrast with age-matched generated Bmncr knockout mice(Bmncr-KO)via gene targeting mediated by the TetraOne technique.qRT-PCR confirmed that the expression level of Bmncr was rarely detected in Bmncr-KO mice relative to wild-type(WT)controls(Supplemental Figure 5A).We analyzed the bone phenotypes of femurs from young(3-month-old),adult(6-month-old),and aged(12-month-old)mice.The Bmncr-KO mice showed reduced bone mineral den-sity(BMD)in comparison with age-matched WT controls as measured by dual-energy x-ray absorptiometry(DEXA)analysis(Figure 2A).Micro-CT analysis showed that Bmncr-KO mice had lower trabecular bone volume than WT controls.Cortical bone thickness was also reduced in Bmncr-KO mice(Figure 2,BD).Calcein double-labeling confirmed that Bmncr-KO mice had sig-Figure 2.Bmncr-KO mice exhibited lower bone loss and higher bone marrow fat accumulation.(A)BMD in femurs from 3-,6-,and 12-month-old WT and Bmncr-KO mice were measured by dual-energy x-ray absorptiometry scan.Representative micro-CT images(B)and quantitative micro-CT analysis(CD)of trabecular bone volume and cortical bone thickness in femurs(n=10 per group).(EF)Calcein double-labelingbased quantification of bone formation rate per bone surface(BFR/BS)(n=5 per group).Scale bar:50 M.(GH)Representative images of osteocalcin immunohistochemical staining and quantification of number of osteoblasts in distal femurs.Red arrows represent osteocalcin-positivestaining cells.Scale bar:100 M.n=5 per group.(IK)OsO4 staining of decalcified tibiae by micro-CT analysis(I)and quantification of number and volume of adipocytes in femurs(JK).n=5 per group.Data are mean SD.*P 0.05(Students t test).The Journal of Clinical Investigation R ESE A RCH A RTI CL E5 2 5 4jci.org Volume 128 Number 12 December 2018fibromodulin,which belongs to the family of small interstitial leu-cine-rich repeat proteoglycans(SLRPs)(20).Fibromodulin plays an important role in regulating the tendon stem cell niche in the extracellular matrix(21,22).To assess the function of fibromod-ulin on cell adherence in vivo,Fmod-deficient(Fmod-KO)mice were generated by CRISPR/Cas-mediated genome engineering with Exon 2 as the target(23,24).qPCR analysis showed that Fmod was deleted in Fmod-KO mice(Supplemental Figure 6A).The expression of the Fmod gene in different tissues showed an expres-sion pattern similar to the Bmncr gene,which displayed a high lev-el in the femur and WAT(Supplemental Figure 6B).We first tested whether fibromodulin might modulate BMSC adherent potential to the matrix.BMSCs isolated from Fmod-KO mice and WT mice were subjected to an adhesion assay in matrigel with and without the addition of fibromodulin.We found that Fmod-KO BMSCs showed a reduced ability to adhere to matrigel with and without fibromodulin compared with WT BMSCs.Meanwhile,the number of WT and Fmod-KO BMSCs adherent to matrigel with the addi-tion of fibromodulin was much higher than the number of BMSCs that adhered to matrigel without fibromodulin(Figure 4,B and C).Fmod-KO mice showed significantly decreased LepR+BMSCs in the trabecular bone surface and increased LepR+BMSCs in bone marrow as measured by immunofluorescence staining analysis(Supplemental Figure 7,AC).We also confirmed this result by flow cytometry analysis(Supplemental Figure 7,D and E).These results indicate that Bmncr may regulate the ability of BMSCs to adhere to bone surface matrix through fibromodulin.We also found that an array of osteogenesis-related genes,including Bmp2,Alpl,Runx2,Sp7,and Bglap,were downregulat-ed in Bmncr-KO BMSCs by gene expression microarray analysis(Figure 4A and Supplemental Figure 8A).Bone morphogenetic protein 2(BMP2)belongs to the BMP f