ZBP1(DAI/DLM-1)promotesosteogenic differentiation while inhibiting adipogenic di

时间：2025-01-07

Xuefeng Zhao,Liang Xie,Zhiyong Wang,Jiongke Wang,Hao Xu,Xianglong Han,Ding Bai and Peng Deng

INTRODUCTION

The term Mesenchymal stem or stromal cell(MSC)refers to a group of heterogeneous multipotent stem cells that can selfrenew and further differentiate into several cell types,including osteoblasts,chondrocytes,and adipocytes.1They are originally derived from bone marrow and are also relatively easy to obtain from other tissue types,such as adipose tissue and umbilical cord.2–4In recent years,many completed clinical trials using MSCs from bone marrow have shown the ef ficacy of MSC-based bone regeneration without obvious adverse effects.5–6

The process of MSClineage speci fication is tightly regulated by many signaling pathways,transcription factors,and epigenetic mechanisms.7–9For instance,the evolutionarily conserved Wntsignaling pathway has been long known to play essential roles in MSC commitment and differentiation.10Canonical Wnt signaling triggered by the binding of ligands to receptors leads to the phosphorylation of LDL receptor-related protein 5/6 coreceptors,β-catenin stabilization,and subsequent translocation into nuclei.β-Catenin with T-cell factor/lymphoid enhancer factor then promote the downstream gene expression.11–12In regard to osteogenesis,Wnts promote osteoblastic differentiation,proliferation,and mineralization activity.10,13–14Several osteogenesisrelated transcription factors are reported to be the direct targets of Wnt/β-catenin signaling,such as Runt-related transcription factor 2(Runx2)andosterix(also known as transcription factor 7,Sp7).15–17On the other hand,activation of Wnt/β-catenin signaling also represses the adipogenic and chondrogenic differentiation of MSCs.18–19

Z-DNA binding protein 1(ZBP1)is a member of the Zαfamily,which comprises two Zαdomains at its N-terminus,and~200 amino acidsat its C-terminus.20ZBP1 wasinitially considered to be a tumor-related protein that is dramatically upregulated in the surrounding tissue in contact with a tumor but not in the tumor tissue itself.21HumanZBP1is highly expressed in lymphatic tissues,including the lymph nodes,bone marrow,and spleen,22while the expression of mouseZbp1mRNA has been reported in the lungs,liver,and spleen.21,23Zbp1has also been found to be upregulated upon the activation of mouse peritoneal macrophages.21In addition,Takaoka et al.reported that ZBP1 functions as a sensor of DNA from various sources that rapidly activates innate immune responses,during which the Cterminus of ZBP1 interact with interferon(IFN)regulatory factor 3 and tank-binding kinase 1 to activate the expression of type IIFN.24Arecent study further reported that ZBP1 senses ribonucleoprotein complexes of in fluenza Avirusesand becomesubiquitinated,which iscriticalfor inducing apoptosis,pyroptosis and necroptosis in infected tissues.25

However,the biological function of ZBP1 during the lineage speci fication of MSCs remains largely elusive.In this study,we found that ZBP1 expression was elevated during the osteogenic differentiation but downregulated during the adipogenic differentiation of MSCs.Through gain-and loss-of-function assays,we revealed that ZBP1 promoted osteogenesis but suppressed adipogenesis in MSCs.Furthermore,ZBP1 is a Wnt/β-catenin target and is required for Wnt signaling.

RESULTS

ZBP1 is upregulated during the osteogenic differentiation but downregulated during the adipogenic differentiation of mBMSCs We first sought to examine the potential role of ZBP1 in MSC osteogenesis.As shown in Fig.1a,ZBP1 expression was elevated upon osteogenic stimulation in mBMSCs and peaked at day 4 of induction.In contrast,it was dramatically downregulated during the adipogenesis of mBMSCs(Fig.1b).We then evaluated the expression of ZBP1 in the mouse femurs.The immunostaining of ZBP1 showed that it was highly expressed in macrophages and osteoblasts.ZBP1 was also expressed at a relatively low level in osteocytes and marrow adipocytes(Fig.1c,d).

Depletion of ZBP1 suppresses the osteogenic differentiation but promotes the adipogenic differentiation of mBMSCs

Next,two speci fic siRNAs targeting ZBP1 was used to knock down its expression in mBMSCs(Fig.2a).While the depletion of ZBP1 slightly inhibited the proliferation of mBMSCs(Fig.S1a),we found that ZBP1 knockdown strongly inhibited the ALPactivity of mBMSCs(Fig.2b).ECM mineralization was also reduced via the ZBP1 depletion(Fig.2c).The expression of osteogenesis-related genes such asRunx2,Sp7,integrin-binding sialoprotein(Ibsp),andbone gamma-carboxyglutamate protein(Bglap)was downregulated in ZBP1-depleted mBMSCs(Fig.2d).To examine whether ZBP1 is required for in vivo bone formation,mBMSCs were infected with lentiviruses expressing ZBP1 shRNAs(shZBP1)(Fig.S1b)and subcutaneously transplanted with hydroxyapatite/tricalcium phosphate(HA/TCP)scaffolds at dorsal sites in nude mice.Asshown in Fig.2e,ectopic bone formation wasinhibited in ZBP1 knowndown group in comparison with the controls.In addition,the knockdown of ZBP1 increased lipid droplet formation and the expression levels of adipogenesis-related genes in mBMSCs,includingCD36,CCAAT enhancer-binding protein alpha,lipoprotein lipaseandperoxisome proliferator-activated receptor gamma(Fig.2f–h).

Overexpression of OvereZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of mBMSCs

To further examine whether ZBP1 overexpression enhances the osteogenesis,mBMSCs were infected with lentiviruses expressing mouse ZBP1 (Fig. 3a). The ectopic overexpression of ZBP1 signi ficantly increased the ALP activity of mBMSCs in response to osteogenic stimulation(Fig.3b).Accordingly,ECM mineralization was increased in ZBP1-overexpressing mBMSCs compared with the vector control(Fig.3c).The overexpression of ZBP1 also increased the expression levels ofRunx2,sp7,Ibsp,andBglapduring osteogenesis in mBMSCs(Fig.3d).Furthermore,ectopic bone formation was increased in ZBP1-overexpressing mBMSCs in comparison with control cells(Fig.3e).On the other hand,the overexpression of ZBP1 strongly suppressed formation of lipid droplets and the expression of adipogenesis-related genes in mBMSCs(Fig.3f,g).

Overexpression of ZBP1 enhances the osteogenesis while inhibiting the adipogenesis of hMSCs

Unlike mBMSCs,hMSCs express ZBP1 at an extremely low level.Thus,we decided to infect hMSCs with lentiviruses expressing human ZBP1(Fig.4a).As revealed by ALP activity assays and Alizarin Red S (ARS) staining, the overexpression of ZBP1 signi ficantly increased the ALP activity and ECM mineralization of hMSCs(Fig.4b,c).The expression levels of osteogenic markers,includingRUNX2,SP7,collagen type 1 alpha 1andsecreted phosphoprotein 1was also increased in ZBP1-overexpressing hMSCs(Fig.4d).In contrast,we found that the overexpression of ZBP1 in hMSCs inhibited lipid droplet formation and the expression of adipogenic markers after several days of adipogenic induction(Fig.4e,f).

Restoring the expression of ZBP1 rescues the osteogenic potential of ZBP1-depleted mBMSCs

To further con firm the speci fic effectsof ZBP1 on the osteogenesis of MSCs,we reintroduced ZBP1 expression in mBMSCs that harbored a ZBP1 shRNA targeting the 5′untranslated region ofZbp1mRNA(Fig.5a).When mBMSC/shZBP1/ZBP1 cells were treated with osteogenic stimuli,ALPactivity,and ECM mineralization were successfully rescued compared with the vector control(Fig.5b,c).In addition,the suppressed the expression ofsp7,Ibsp,andBglapby ZBP1 depletion was also rescued when ZBP1 expression was restored(Fig.5d).Collectively,these findings further con firmed that ZBP1 is crucial for the MSC osteogenic differentiation.

ZBP1 interacts withβ-catenin to facilitate its binding to the promoters ofRunx2andSp7

To reveal the molecular mechanisms by which ZBP1 regulates the lineage speci fication of mBMSCs,RNA-sequencing(RNA-seq)was performed in ZBP1-depleted mBMSCs using scrambled siRNA(siScr)-transfected mBMSCs as a control.A total of 1548 upregulated genes and 2149 downregulated genes were revealed by comparing the gene expression pro files of these cells.Gene ontology(GO)analysis revealed the downregulated genes were highly associated with ECM organization,Wnt signaling,and osteoblast differentiation(Fig.6a,Supplementary Table S1).26Gene set enrichment analysis(GSEA)further con firmed the inhibition of Wnt signaling in ZBP1-depleted mBMSCs(Fig.6b,Supplementary Table S2).27

Previous studies have highlighted the essential role of ZBP1 in IFN induction by associating with TBK1 and the transcription factor IRF3.24We found that ZBP1 knockdown suppressed the β-catenin-mediated transcription induced by Wnt3a treatment,as demonstrated by TOP flash luciferase reporter assays(Fig.6c).Accordingly,the induction ofAxin2andCcnd1expression by Wnt3a treatment was also reduced in ZBP1-depleted mBMSCs(Fig.6d,e).To examine the potential interaction between ZBP1 andβ-catenin,we next performed immunoprecipitation in ZBP1-overexpressing mBMSCs.As shown in Figs.6f,g,ZBP1 and β-catenin were coprecipitated with each other.We then obtained nuclear and cytoplasmic extracts of mBMSCs in the presence and absence of Wnt3a treatment and found that the depletion of ZBP1 signi ficantly suppressed the nuclear translocation of β-catenin(Fig.6h,Supplementary Fig.S2a).β-Catenin is reported to induce osteogenic differentiation through the upregulation ofRunx2andSp7expression.17We con firmed that ZBP1 depletion inhibited the expression ofRunx2andSp7in mBMSCs(Supplementary Fig.S2b,c).We then designed ChIP primers that encompassed the Wnt/β-catenin response element(WRE)in the promoters ofRunx2andSp7and found that the occupancy of β-catenin at the promoters ofRunx2andSp7was signi ficantly reduced by the depletion of ZBP1(Figs.6i,j).

Zbp1is a direct target of Wnt/β-catenin signaling

Next,we showed ZBP1 expression was induced by Wnt3a treatment(Figs.7a,b),while the induction ofZbp1was almost abolished by pretreatment with Dickkopf-1(DKK1),a Wnt antagonist(Fig.7c).Three putative WREs with the core sequence“CTTTG(A/T)(A/T)”were found within ±2kb of the transcription start site of the mouseZbp1gene.28ChIPassay revealedβ-catenin was recruited to theZbp1promoter in Wnt3a-treated mBMSCs(Fig.7d).Collectively,the results suggested the existence of positive feedback between ZBP1 and Wnt/β-catenin signaling that enhances osteogenic differentiation at the expense of adipogenic differentiation in MSCs.

DISCUSSION

Understanding MSClineage commitment is critical for harnessing MSC differentiation into the required cell types for regenerative medicine.29–30In this study,we found that ZBP1 was expressed in osteoblasts and was dramatically upregulated upon osteogenic stimulation in mBMSCs.In contrast,the expression of ZBP1 in marrow adipocytes was relatively low compared with that in osteoblasts,and ZBP1 was downregulated during the adipogenic differentiation of mBMSCs.Although ZBP1 expression in hMSCs was extremely low,the ectopic overexpression of ZBP1 strongly promoted the osteogenic but inhibited the adipogenic differentiation of hMSCs.

Our results also indicated that ZBP1 and Wnt/β-catenin signaling formed a positive feedback loop that promoted the osteogenesis while inhibiting the adipogenesis of MSCs.

Previous studies on ZBP1 have mainly focused on its structure and function in immune responses.20,24,31–32To our knowledge,this is the first time that ZBP1 has been reported to be involved in the MSClineage speci fication.In this study,we found that ZBP1 was also expressed in mBMSCs and their sublineages in addition to lymphatic tissues.Our results also showed that ZBP1 was essential for the osteogenic differentiation but suppressed the adipogenic differentiation of mBMSCs and hMSCs.Interestingly,while ZBP1 was highly expressed in osteoblasts,it was detected in only a small portion of osteocytes at a relatively low level.It is likely that the role of ZBP1 in regulating osteogenesis is more important in the early and middle stages compared with the late stages of osteogenesis.Unlike the pattern observed in osteogenesis,ZBP1 expression was consistently downregulated during mBMSC adipogenesis.Several previous studies have suggested a negative correlation between the osteogenesis and adipogenesisof MSCs.8,33It isvery likely that ZBP1 playsa key role in the early fate decision of MSCs.

We further found thatZbp1was a target of Wnt/β-catenin signaling and that ZBP1 was required for Wnt/β-catenin signaling.Speci fically,β-catenin bound to theZbp1promoter to activate its transcription,and Zbp1 in turn associated withβ-catenin to facilitate its nuclear translocation and the expression of its downstream genes.As an important viral DNA sensor in innate immune responses,ZBP1 has been reported to activate IRF and nuclear factor-kappa B,promoting the production of type-I IFN.22,34This study has further extended the function of ZBP1 to the facilitation of translocation ofβ-catenin in mBMSCs.As noted above,Wnt/β-catenin signaling may promote MSC lineage speci fication through multiple mechanisms.Although we focused on the binding ofβ-catenin to the promoters ofRunx2andSp7in this study,there might be other factors involved that maintainRunx2expression independent of Wnt/β-catenin signaling in untreated mBMSCs.16,35On the other hand,sinceSp7is a downstream gene ofRunx2,these results cannot exclude the possibility that ZBP1 depletion may decrease the transcription activity of RUNX2,leading to the downregulation ofSp7expression.36More importantly,ZBP1 may serve as a transcription factor or co-factor,since a possible association between transcriptional activity and Z-DNA formation has been suggested.37–39

There are several limitations of this study.For example,it has been reported that more than 2000 different transcripts might be generated from the humanZBP1gene by alternative splicing and/or the use of distinct 5′and 3′ends.22Two variants are expressed from the mouseZbp1gene.The siRNAsused in thisstudy targeted both variants.However,whether different mRNAs produced by the mouseZbp1gene are also involved in osteogenesis still requires further investigation.In addition,the biological function of Z-DNA is still elusive to us.Interestingly,double-stranded RNA adenosine deaminase 1,another member of the Z-DNA-binding protein family,may act asa cis-element in regulating gene expression in yeast.40We also noticed that ZBP1 was expressed in both nuclei and the cytoplasm.However,additional efforts will be required to explore whether ZBP1 may directly bind to Z-DNA in promoter regions and function as a transcription factor or coactivator.

In conclusion,our findings revealed a positive feedback loop involving ZBP1 and Wnt/β-catenin signaling that promoted the osteogenesis but inhibited the adipogenesis of MSCs,indicating that ZBP1 might be a key regulator of transdifferentiation between bone and fat.

MATERIALSAND METHODS

MSCisolation and differentiation

Primary mBMSCs were from the femoral bone marrow of wild-type C57BL/6Jmice at 3 months of age as previously described.41The isolated cells were maintained in Dulbecco’s modi fied Eagle’s medium,containing 15%heat-inactivated fetal bovine serum,100 U·mL-1of penicillin and 100 μg·mL-1of streptomycin sulfate for 3 days(all from Invitrogen).The nonadherent hematopoietic cells were then removed by vigorous washing with phosphatebuffered saline(PBS)three times.The growing colonies were collected by trypsinization after 2 weeks for further passaging and differentiation.The in vivo and in vitro animal procedures were approved by the Committee for the Care and Use of Laboratory Animals of the State Key Laboratory of Oral Diseases,West China Hospital of Stomatology,Sichuan University.

hMSCs were purchased from the ATCCand were maintained in Dulbecco’s modi fied Eagle’s medium supplemented with 15%FBS plus 100 U·mL-1penicillin and 100 μg·mL-1streptomycin sulfate(all from Invitrogen).

Induction of osteogenesis and characterization of osteogenic phenotypes

Atotal of 1×105cells per well were seeded into 12-well plates and cultured in osteogenic induction medium,which is consisted of 90%α-MEM(Invitrogen)supplemented with 10%FBS(Invitrogen),100 μmol·L-1vitamin C(Sigma-Aldrich),10 nmol·L-1dexamethasone (Sigma-Aldrich),and 10 mmol·L-1β-glycerophosphate(Sigma-Aldrich).

After several days of osteogenic stimulation,ALP staining,ALP activity assay,and ARS staining was carried out as previously described.29

Induction of adipogenesis and characterization of adipogenic phenotypes

At 95%–100%cell con fluence,the cells were treated with adipogenic induction medium,and Oil Red Ostaining was carried out after 2–4 weeks of stimulation to measure the formation of lipid droplets in the cells using a kit from Diagnostic Biosystems.42

Immunohistochemistry(IHC)

Femurs from 3-month-old mice were collected and fixed with 10%neutral buffered formalin(Fisher)for 48h.After decalci fication with a 10%ethylenediaminetetraacetic acid solution(p H=7.4)for 2 weeks,paraf fin sections were obtained,and the specimens were then dewaxed with xylene twice and rehydrated through a gradient from ethanol to PBS.Immunostaining was performed using an IHC kit(Gene Tech)according to the manufacturers’instructions.A rabbit polyclonal anti-ZBP1 antibody(Invitrogen)was used to detect the expression of ZBP1 in bone marrow and trabecular bones.The semiquantitative quanti fication of immunostaining intensities(0,negative;1,weak staining;2,moderate staining;3,intense staining)wasindependently carried out by two pathologists as previously reported.43–44

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay

A total of 3×103cells were seeded into each well of 96-well plates in at least triplicate and cultured until the indicated time points posttransfection with siRNAs.Overall,10μL of MTT(Sigma-Aldrich)at a concentration of 5 mg·mL-1was added,followed by incubation for additional 3 h.The medium was then replaced with dimethyl sulfoxide,and the optical absorbance was read at 570 nm.

Reverse transcription-quantitative PCR(RT-qPCR)

RNA was isolated using the TRIzol reagent(Invitrogen).For each sample,complementary DNA was generated from 1 to 2μg of total RNA using PrimeScript RTReagent Kit(TAKARA BIO).Please refer to Supplementary Table S3 for the primers used for RT-qPCR.

Western blotting

The cells were rinsed with cold PBS and lysed in RIPA buffer(Boster Biological Technology,Wuhan,China)for 30 min with constant agitation.A 10–40μg aliquot of the supernatant from each sample mixed with loading dye was boiled at 95°Cfor 4 min and then separated on sodium dodecyl sulfate-polyacrylamide gels at 80–120 V.Western blotting was carried out as previously described.42The primary antibodies used included a rabbit anti-ZBP1 antibody(Invitrogen),a mouse anti-β-catenin antibody(BD Biosciences),a mouse anti-TATA-binding protein TBP antibody(Abcam),and a mouse anti-α-tubulin antibody(Sigma-Aldrich).

Transfection and viral infection

All the siRNAs used in this study were obtained from Origene Technologies,including a siScr and two siRNAs targeting mouse ZBP1(siZBP1–1,siZBP1–2).More speci fically,siZBP1–1 targeted the open reading frame ofZbp1mRNA,and its sequence was“GGGAAUGACGACAGCCAAA”.siZBP1–2 targeted the 5′untranslated region(UTR)ofZbp1mRNA,and its sequence was“GGGUAUUUGUUUCCGGGAU”.Transfection with the siRNAs was conducted by using Lipofectamine RNAiMAX reagent(Invitrogen).For the longterm knockdown of ZBP1 and rescue experiments,lentiviruses expressing ZBP1 shRNA(shZBP1)were generated by Fulengen Inc.(Guangzhou,China).For the ectopic overexpression of ZBP1,lentivirus particles expressing mouse ZBP1 and human ZBP1 were obtained from OriGene Technologies.mBMSCs and hMSCs were infected and then selected with 1 μg·mL-1puromycin for 72h.

Luciferase assay

mBMSCs were seeded into 12-well plates and infected with lentiviruses carrying TOP flash luciferase reporters.After treatment with Wnt3a at 100 ng·mL-1for 1 day,luciferase activity was assessed using Luc-Screen kits(Tropix)as previously described.45

Immunoprecipitation(IP)assay

Protein was isolated from ZBP1-overexpressing mBMSCs.Aliquots of 50–100μg of protein were incubated with 1μg of antibodies overnight at 4°C with gentle rotation.The antigen-antibody complex was then precipitated with Dynabeads Protein A/G(Thermo).The beads carrying the antigen-antibody complexes were washed at least three times to remove nonspeci fic binding and were then subjected to western blotting.The antibodies used for the IP experiments were as follows:mouse monoclonal antiβ-catenin(BD Biosciences),polyclonal anti-mouse normal IgG(Millipore),rabbit polyclonal anti-ZBP1(LifeSpan BioSciences),and polyclonal anti-rabbit normal IgG(Cell Signaling Technology).

ChIPassay

ChIP experiments were carried out according to the manufacturer’s protocol(Zymo Research,USA).More speci fically,cells were collected and crosslinked with 1%formaldehyde for 15min with gentle rotation.Nuclei were then extracted using Nuclei Prep buffer.The isolated nuclei were sheared via 3–5 cycles of sonication.After centrifugation,the supernatant containing the sheared chromatin was incubated with the antibodies overnight at 4°Cunder rotation.ZymoMag Protein A4 beads were added to the reaction.Finally,the precipitated DNA was eluted from the beads and puri fied for quanti fication by qPCR.The amount of ChIP-ed DNA was normalized to the input DNA of each sample.The following antibodies were used:mouse monoclonal antiβ-catenin(BD Biosciences,1:200)and anti-mouse IgG(Millipore,1:500).The primers used in ChIP assay are also shown in Supplementary Table S3.

Transplantation in nude mice

mBMSCs(1×106)expressing shZBP1 and shScr were incubated with 40 mg of HA/TCP scaffolds at 37°C for 6 h and then subcutaneously transplanted into female nude mice(n=6).46The transplants were collected at 6 weeks post transplantation.At least three fields of each H&E-stained sample were randomly chosen(Olympus),and the area of bone tissue versus the total area was measured using SPOT 4.0 software.

RNA-seq

RNA was collected from duplicated mBMSCs transfected with siScrs and siRNAs targeting ZBP1 at 72 h posttransfection.RNA-seq libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit.All the sequenced reads were mapped to the NCBI build 10mm genome using Tophat v2.0.12,and fragments per kilobase of transcript per million mapped reads(FPKM)expression values were extracted using HTSeq.GSEA and GO analysis were performed to identify the genes that were downregulated by 1.5-fold in ZBP1-depleted mBMSCs.26–27The accession number is NCBIGEO:GSE117334.

Statistical analysis

Data are presented as the mean±SD.*P＜0.05;**P＜0.01;***P＜ 0.001.Student’sttest was used for single comparisons;one-way analysis of variance(ANOVA)and two-way ANOVA was performed for multiple comparisons.The chi-square test was performed to examine the signi ficant differences between the immunostaining intensities of different cell types in the mouse femur.

DATA AVAILABILITY

All the data is included in the published files.

ACKNOWLEDGEMENTS

This study was supported by the Foundation of the National Natural Science Foundation of China(No.81671024,81371171,81571009,and 81600877)and the China Postdoctoral Science Foundation(2016M600745).

AUTHORCONTRIBUTIONS

X.Z.,L.X.,Z.W.,J.W.and H.X.performed experiments and analyzed data.X.H.helped supervise the project and provided financial support.D.B.and P.D.conceived the original idea,designed experiments,prepared the manuscript,and provided financial support.