Potential Involvement Of Protein Ubiquitination And Ubiquitin E3 Itch In Bone Fracture Healing

In this study,the role of ubiquitin E3 ligase,Itch,in the process of fracture repair was studied using real-time quantitative PCR?qPCR?,Western blot and chromatin immunoprecipitation?Ch Ip?.Tibia fracture model is essential for this experiment.Mouse will be anesthetized by katamine and xylazine by intraperitoneal injection.After induction and confirmation of deep anesthesia,the skin overlying the left knee and leg will be shaved and prepped with povidone iodine.An incision?6 mm?will be made using sterile scissors.Both knees will be flexed to its full extent?approximately 120 degree?.Insert 27 G Precision Glide Needle into medullary canal of tibia through the patellar tendon between the medial and lateral condyles of the tibia until the sharp end of the pin has reached the end of the bone marrow cavity.The open mid-diaphyseal fracture will be created using Dremel 7700 drill.The skin will be closed using 5-0 sutures.X-ray imaging will be performed when the mice are still in deep sleep to confirm needle placement and induction of fracture.Total RNA was extracted from callus tissues using TRIzol Reagent at day 3,7,10,14,and 21 post-fracture.c DNAs were synthesised using an i SCRIPT c DNA Synthesis Kit.Quantitative real-time RT-PCR amplifications were performed in the i Cycler real-time PCR machine using i Q SYBR Green Supermix according to themanufacturer's instruction.Glyceraldehyde 3-phosphate dehydrogenase?GAPDH?was amplified on the same plates and used to normalise the data.Each sample was prepared in triplicate and each experiment was repeated at least once.The relative abundance of each gene was calculated by subtracting the Cycle Threshold?CT?value of each sample for an individual gene from the corresponding CT value of GAPDH??CT?.??CT were obtained by subtracting the ?CT of the reference point.These values were then raised to the power of 2?2??CT?to yield fold expression relative to the reference point.To determine if these E3 ligases are involved in fracture repair,we examined their expression levels in callus tissues from WT C57/B6 mice which had undergone surgical tibia bone fracture.The expression levels of Wwp1,Smurf1,Smurf2 and Itch were all significantly increased,starting at seven days post-fracture and reached maximum expression at ten days post-fracture?Itch expression reaching the maximum at 14 days?.We also examined expression of NF-?B members in RNA extracted from calluses at different time points post-fracture.The expression levels of all NF-?B members,including p50,Rel A,p52 and Rel B,were significantly increased,starting at seven days after fracture and reaching maximum expression at 14 days,a similar time point to when that of E3 ligase levels were elevated.We sought to determine if the total number of ubiquitinated?Ub?-proteins in callus tissues is increased in the presence of proteasome inhibitor MG132.For examining the expression levels of total ubiquitinated proteins,mice were treated with the proteasome inhibitor MG132?1mg/kg by intraperitoneal injection?24 hours before they were sacrificed at day 7 post-fracture.Total proteins were examined from callustissue and subjected to Western blot analysis.The cortical bone samples from the non-fractured legs were used as control.Whole-cell lysates were prepared from fracture calluses.Homogenized fracture calluses with liquid nitrogen were lysed with mammalian protein extraction reagent containing a protease inhibitor mixture.Whole cell lysates were loaded in 10% Sodium Dodecyl Sulfate?SDS?-PAGE gels,transferred to a nitrocellulose membrane,and immunoblotted with antibodies to?-actin and ubiquitin.Bands were visualized using ECL chemiluminescence.The total amount of Ub-proteins in callus tissues from the fractured legs was increased compared with cortical bone tissues from non-fractured leg.We have demonstrated that NF-?B Rel A mediated Receptor Activator of Nuclear Factor kappa-B?RANKL?induces Itch expression in osteoclasts by directly binding to NF-?B sites in the itch promoter.We wanted to know if this also occurs in osteoblasts.We have also previously identified two putative NF-?B binding sites at the-3473/-3465 and-3183/-3175 location of the murine Itch promoter {NCBI?National Center for Biotechnology Information?Ref Seq accession number NC₀00068} using TFSEARCH software?version 1.3?which is a free software for transcription factor binding site prediction?http://www.cbrc.jp/htbin/bget_tfmatrix?M00022?.We overexpressed Rel A and Rel B in C2C12 cells and found that both of them could increase Itch expression.To examine whether NF-?B promotes Itch expressions in osteoblasts by binding to the Itch promoter,we used an osteoblast/myoblast precursor cell line,C2C12 cells.C2C12 cells were infected with retroviral virus encoding green fluorescent protein?GFP?control,NF-?B Rel A or Rel B for 24 hours.Infectionefficiency was confirmed by a fluorescent microscopy.Ch IP assay was performed with the MAGnify Chromatin Immunoprecipitation System according to the manufacturer's instructions.1x106 cells were fixed with 1% formaldehyde for 15 minutes.Cells were then lyzed in 50 ?l lysis buffer containing protease inhibitor and sonicated on ice eight times with a 20-seconds-on,20-seconds-off cycle at high power using the Bioruptor UCD-200 sonicator to shear chromatin into 200-500 bp fragments.10 ?l of chromatin was diluted to 100 ?l with dilution buffer.10 ?l was used as an Input.Antibodies against Rel A and Rel B or control rabbit Ig G were used in the immunoprecipitation step.Antibodies were coupled to the Dynabeads and then incubated with diluted chromatin for 2 hours at 40 C.After adequate washing,the immuoprecipiated?IP?chromatin was reversed for the crosslinking with crosslinking buffer containing Proteinase K for 15 minutes at 550 C.The purified DNA fragments were determined by quantitative PCR using the primers localized at the binding sites.Control primers localized at irrelevant down-streaming sites were used as a negative control.Then we performed Ch IP assays using an anti-NF-?B Rel A and Rel B antibody to pull down protein-chromatin complexes and two primer sets around these NF-?B binding sites.Results showed Rel A and Rel B binding to the NF-?B binding sites of the Itch promoter.Itch functions as an E3 ligase to promote ubiquitination of targeting proteins,including the osteoblast positive regulators,thereby negatively regulates osteoblast differentiation.Thus,in the absence of Itch,osteoblast differentiation should be increased.Itch KO mice on a C57BL/6J background were generated by breedingheterozygous female with heterozygous male mice for Itch deficiency.Homozygous mice for Itch deficiency?Itch KO mice?were genotyped by polymerase chain reaction?PCR?.Total RNA was extracted from callus tissues using TRIzol Reagent at day 3,7,10,14,and 21 post-fracture.Glyceraldehyde 3-phosphate dehydrogenase?GAPDH?was amplified on the same plates and used to normalise the data.Each sample was prepared in triplicate and each experiment was repeated at least once.To determine if this is the case at the bone fracture site,we examined the levels of osteoblast-associated genes {Runx2,Alkaline Phosphatase?ALP?} and genes associated with bone remodelling,receptor activator of NF-?B ligand?RANKL?and osteoprotegerin?OPG?in fracture callus of Itch KO mice at different time points post-fracture.Runx2 and ALP are two positive osteoblast regulators which are regulated partially through ubiquitination and proteasome degradation in osteoblasts.RANKL and OPG play critical roles in osteoclast formation and bone remodelling.The expression levels of ALP and Runx2 were significantly increased in fracture callus of Itch KO mice,starting from day 7 post-fracture.The ratio of RANKL/OPG was not changed,although their expression levels were also elevated in Itch KO callus tissues.In this study,we examined the potential involvement of E3 ligase Itch in fracture repair using WT and global Itch knockout?KO?mice.We demonstrated elevated expression levels of WW domain-containing ubiquitin E3 ligases including Itch at early stages of fracture repair,which is accompanied by increased amount of Ub-proteins.Elevated Itch expression was associated with increased expression ofNF-?B members.NF-?B upregulated Itch expression by binding to the NF-?B binding site in the Itch promoter in an osteoblast progenitor cell line.Callus tissues from Itch KO mice expressed high levels of osteoblast-positive regulator.These data suggest for the first time that the ubiquitin–proteasome pathway may participate in the bone fracture repair process.Our findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus.Thus,ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing.