Roles And Underlying Mechanisms Of CUL4B In Regulating Osteo-adipogenic Differentiation From Mesenchymal Stem Cells

Background and Objectives:Mesenchymal stem cells(MSCs)are a kind of pluripotent stem cells,which are common progenitors of various cells such as osteoblasts,chondrocytes and adipocytes.The fate of MSCs is regulated by transcription factors.Runt-related transcription factor 2(RUNX2)and OSTERIX(also known as Sp7)are major regulators of osteogenic differentiation,while Peroxisome proliferator-activated receptor γ(PPARγ)and CCAAT/enhancer-binding protein(C/EBP)family members(C/EBPα,C/EBPβ and C/EBPδ)are essential for adipogenic differentiation.Kruppel-like factor 4(KLF4)can attenuate osteoblast formation by transcriptionally suppressing Runx2 and is also an important early regulator of adipogenesis.C/EBPδ is a recognized marker of early osteogenic differentiation.In the bone marrow microenvironment,the differentiation of MSCs into adipocytes(adipogenic differentiation)and osteoblasts(osteogenic differentiation)is inversely related,and increased adipogenic differentiation inhibits osteogenic differentiation,leading to insufficient bone formation.Conversely,increased osteogenic differentiation inhibits adipogenic differentiation.Under normal conditions,MSCs differentiate into osteoblasts and adipocytes is delicately balanced to maintain normal bone homeostasis and metabolism.However,under pathological conditions such as aging or hormone disorders,bone marrow MSCs(BMSCs)favor adipogenic to osteogenic differentiation,resulting in decreased bone mass and increased marrow adipose tissue(MAT),leading to osteoporosis.Therefore,identification of the key factors that regulate the balance of osteogenic/adipogenic differentiation of MSCs will not only help to understand the pathogenesis of metabolic diseases such as osteoporosis,but also help to discover new therapeutic targets.CUL4B(Cullin 4B),as a scaffold protein,forms Cullin 4B ubiquitin ligase complex(Cullin 4B-RING E3 ligases,CRL4B)together with DNA damage binding protein 1(DDB1),RING box protein(RBX1)and DDB1-CUL4B associated factors(DCAFs).CRL4B regulates various physiological and developmental processes by catalyzing the polyubiquitination of substrate proteins for proteasomal degradation,or catalyzing the monoubiquitination of histone H2AK119.In 2007,a British research group and our group found that loss of function mutations in CUL4B leads to human X-linked mental retardation syndrome.In addition to mental retardation,patients with CUL4B mutation also showed short stature,brachydactyly and central obesity,suggesting a potential role of CUL4B in the regulation of adipogenesis and osteogenesis and their balance.Therefore,this study will investigate the roles and underlying mechanisms of CUL4B in orchestrating osteogenic or adipogenic differentiation of MSCs using mesenchymal stem cell-specific Cul4b gene knockout mouse model.Methods and Results:1.CUL4B expresses in BMSCs,and its expression level decreases during aging.In order to investigate the role of CUL4B in regulating MSCs differentiation,the BMSCs of mice were obtained by bone stick method.The surface markers were detected by flow cytometry and osteogenic/chondrogenic/adipogenic differentiation was induced,which proved that the obtained cells were MSCs.Immunofluorescence analysis showed that CUL4B was colocalized with Nestin.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting analysis showed that CUL4B was expressed in mouse BMSCs,and the expression level decreased significantly during aging.The expression level of CUL4B in BMSCs from healthy people of different ages was analyzed using data from public databases,and confirmed that the expression level of CUL4B in human BMSCs is negatively corelated to age,suggesting that CUL4B plays an important role in regulating the osteogenic/adipogenic differentiation of BMSCs.2.Depletion of CUL4B in MSCs impairs postnatal skeletal development.1)Generate mesenchymal stem cell specific Cul4b knockout mice.Prx1-Cre mice were crossed with Cul4b floxed mice,and the offspring Prx1Cre+/-;Cul4bflox/Ymale mice were backcrossed with Cul4b floxed female mice to generate mice with Cul4b conditionally knocked out in MSCs(Prx1-Cre+/-;Cul4bflox/Y and Prx1Cre+/-;Cul4bflox/flox referred to MKO hereafter).The Prx1-Cre-/-;Cul4bflox/Y and Prx1-Cre/-;Cul4bflox/flox littermates were used as controls(referred to WT hereafter).The knockout efficiency of Cul4b was confirmed by RT-qPCR and Western blotting assays,indicating that the mouse model was successfully generated.2)Deletion of CUL4B in MSCs did not affect bone development in neonatal mice.Alizarin red-Alcian blue double staining analysis of newborn mice showed that the MKO and control newborns exhibited comparable skeletal pattern with both cartilaginous and calcified skeletal elements.3)Deletion of CUL4B in MSCs impaired postnatal bone development.By measuring the length of body and long bone of 1-and 2-month-old mice,the body length,femur length and tibia length of the MKO mice were shorter than those of the control mice,which recaptures the short stature phenotype in human patients with CUL4B mutations.Safranin-O staining showed a striking expansion of tibia growth plate in MKO mice compared to that in controls at the age of 2 months.Micro-computed tomography(Micro-CT)showed that the trabecular bone mineral density(Tb.BMD),bone volume/tissue volume ratio(Tb.BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th)and the BMD of cortical bone of 1-month-old and 2-month-old MKO mice were decreased compared to control mice,while bone trabecular separation(Tb.Sp)was increased.H&E staining revealed reduced trabecular bones in the femurs and tibia of 1-and 2-month-old MKO mice compared to agematched control mice.Immunohistochemical staining of osteocalcin(OCN)and tartrate resistant acid phosphatase(TRAP)as well as enzyme-linked immunosorbent assay(ELISA)analysis of OCN and Trap5b in serum showed decreased osteoblasts and increased osteoclasts in MKO mice.Double fluorochrome labeling experiments showed that the mineral apposition rate(MAR)and bone formation rate(BFR/BS)of MKO mice were lower than those in control mice.These results suggest that depletion of CUL4B in MSCs impairs postnatal skeletal development.3.Deletion of CUL4B in MSCs exacerbates age-related osteoporosis.We next evaluated whether depletion of CUL4B in MSCs affects aging-related bone loss in male mice aged 6/12/18 months.Micro-CT showed that the trabecular bone mineral density(BMD),bone volume/tissue volume ratio(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th)and the BMD of cortical bone of MKO mice were decreased compared to control mice,while bone trabecular separation(Tb.Sp)and cortical total porosity were increased.H&E and Von Kossa staining of bone sections showed a significant reduction in bone trabecular structure and mineralized tissue.The loss of bone mass was not only detected in the long bones,but also in the vertebral body(Lumbar vertebra 4,L4)of MKO mice.Immunostaining and ELISA analysis showed that MKO mice had reduced osteoblasts and increased osteoclasts.Consistent with reduced osteogenesis,H&E staining of bone sections showed increased MAT accumulation in MKO mice,and immunohistochemical experiments for FABP4 confirmed increased MAT in MKO mice.The reduced BMD in MKO suggests that the mice are susceptible to fracture.To this end,the bone strength was analyzed using a three-point bending test of mouse femurs.It was found that the yield load,maximum load,stiffness,elasticity modulus,bending strength,and yield strength in the femur of MKO mice were significantly decreased.These results suggest that deletion of CUL4B in MSCs accelerates the development of age-related osteoporosis.4.Deletion of CUL4B in MSCs exacerbates osteoporosis caused by estrogen deficiency.In order to confirm the above conclusions,we established a mouse model of postmenopausal osteoporosis induced by ovariectomy(OVX).The analysis of micro-CT,H&E staining,immunohistochemistry,ELISA,and double fluorochrome labeling showed that the MKO mice with OVX exhibited lower bone mass,lower bone formation,but higher osteoclasts and MAT accumulation than the control mice with OVX,indicating that depletion of CUL4B in MSCs aggravates osteoporosis and MAT accumulation caused by estrogen deficiency.5.Mechanism of CUL4B in regulating osteogenic/adipogenic differentiation.1)CUL4B promotes osteogenic differentiation and inhibits adipogenic differentiation of MSCs.Primary BMSCs from MKO and control mice were induced to osteogenic differentiation in vitro.Alkaline phosphatase(ALP)staining and alizarin red staining were used to detect ALP expression and osteogenic calcification nodules.It was found that depletion of CUL4B in BMSCs impaired osteogenic differentiation of MSCs.The expression of osteogenic differentiation related proteins(RUNX2 and OSTERIX)and related genes(Runx2,Osterix,Alpl,Ibsp and Bglap)during the induction confirmed the above results.Furthermore,the effect of CUL4B on osteogenic differentiation was analyzed by overexpressing CUL4B in primary BMSCs and knockdown or overexpression of CUL4B in mouse bone marrow stromal cell line ST2 cells,and the results further confirmed that CUL4B promoted osteogenic differentiation.On the other hand,when primary BMSCs and ST2 cells were induced to differentiate into adipocytes,oil red O staining was used to monitor lipid droplet formation.Knockout or knockdown of CUL4B in MSCs enhanced adipogenesis as well as the expression of adipogenic differentiation-related proteins(PPARγ and C/EBPα)and related genes(Pparg,Cebpa,Fabp4,Adipoq and Plin1),while overexpression of CUL4B inhibited adipogenic differentiation.These results indicated that CUL4B promoted osteogenic differentiation and inhibited adipogenic differentiation of MSCs.2)CUL4B complex regulates osteogenic/adipogenic differentiation of BMSCs by epigenetically suppressing the transcription of Klf4 and Cebpd.① Transcriptome sequencing analysis.To explore the molecular mechanisms by which CUL4B regulates MSCs fate decisions,we performed RNA sequencing(RNA-seq)of BMSCs from MKO mice and control mice.The results showed that 662 differentially expressed genes(DEGs)were identified,of which 457 genes were up-regulated and 205 genes were down-regulated in MKO BMSCs compared to control BMSCs.Cluster analysis of DEGs showed that the expression of cartilage and bone matrix components(Col2a1,Acan,Ucma,Mmp13,Ibsp),cartilage and bone development related genes(Sox5,Fzd9,Cnmd,Tgfb2,Fgfr2),stem cell characteristics related factor(Salll)and fat formation inhibitor(Ptprq)was down-regulated,while the expression of osteogenic pathways inhibitors(Dlx1,Klf4,Hes1,Sfrp1,Bmper,Grem2,Pmepa1,Ecm1)and adipose differentiation related factors(Medag,Klf4,Cebpd)was up-regulated,consistent with the phenotype observed.Based on the functions of these differentially expressed genes.Klf4 and C’ebpd were selected as candidate genes for further analysis.② CUL4B complex negatively regulates Klf4 and Cebpd expression.Western blotting and RT-qPCR analyses showed that both the protein and mRNA levels of KLF4 and C/EBPδ were increased in MKO-BMSCs.Klf4 mRNA level was higher in MKOBMSCs during osteogenic differentiation.Cebpd mRNA level was higher in MKO-BMSCs during adipogenic differentiation.In addition,overexpression of CUL4B in BMSCs downregulated KLF4 and C/EBPδ expression.During osteogenic differentiation,Klf4 expression was decreased in BMSCs overexpressing CUL4B,while Cebpd expression was decreased in BMSCs overexpressing CUL4B during adipogenic differentiation.These results indicate that CUL4B negatively regulates Klf4 and Cebpd expression.③Decreased osteogenic differentiation and enhanced adipogenic differentiation of BMSCs led by CUL4B depletion are mediated by up-regulated KLF4 and C/EBPδ expression.Using small interfering RNA(si-RNA)to knock down the expression of Klf4 and Cebpd in BMSCs,followed by osteogenic/adipogenic induction,we found that knockdown of Klf4 expression could rescue the decreased osteogenic differentiation caused by CUL4B deletion.Knockdown of Cebpd rescued the increased adipogenic differentiation caused by CUL4B deletion.The above results indicate that CUL4B promoted osteogenic differentiation and inhibited adipogenic differentiation of MSC s by inhibiting KLF4 and C/EBPδ expression.3)The CUL4B complex epigenetically represses Klf4 and Cebpd transcription.Quantitative Chromatin Immunoprecipitation(qChIP)assay was used to detect the binding of CUL4B upstream of Klf4 and Cebpd transcription start sites.It was found that CUL4B could directly bind to the region between-8255bp to-8083 bp upstream of Klf4 transcription start site and the region between-5838 to-5645 bp upstream of Cebpd transcription start site.DDB1,EZH2,H2AK119ub1,H3K27me3,HDAC1 and HDAC3 were also enriched in the same regions.Q-ChIP assay showed that deletion of CUL4B reduced the enrichment of the above complex components and their catalytic products in the promoter regions of Klf4 and Cebpd,suggesting that CUL4B complex coordinates with PCR2 and HDAC complexes to suppress the transcription of Klf4 and Cebpd.Further analysis revealed that both HDAC inhibitor TSA and PRC2 inhibitor DZNep rescued the transcriptional down-regulation of Klf4 and Cebpd caused by CUL4B overexpression.Taken together,these results reveal that CRL4B complex cooperates with PRC2 complex and HDACs to repress transcription of Klf4 and Cebpd by promoting H2AK119 monoubiquitination,H3K27 trimethylation.and H3 and H4 deacetylation in the promoter regions.Conclusions:1.CUL4B expresses in BMSCs,and its expression level decreases during aging.2.Depletion of CUL4B in MSCs impairs postnatal skeletal development.3.Deletion of CUL4B in MSCs exacerbates age-related osteoporosis.4.Deletion of CUL4B in MSCs exacerbates osteoporosis caused by estrogen deficiency.5.CRL4B complex cooperates with PRC2 complex and HDACs to repress transcription of Klf4 and Cebpd by promoting H2AK119 monoubiquitination,H3K27 trimethylation,and H3 and H4 deacetylation in the promoter regions.