Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins

Animals

A total of ten 12-week-old wild-type C57BL/6J male mice were purchased from Jackson Laboratory (Bar Harbour, Maine). A total of five Itch KO mice on a C57BL/6J background were generated by breeding heterozygous female with heterozygous male mice. Homozygous mice for Itch deficiency (Itch KO mice) were genotyped using polymerase chain reaction (PCR) as we previously reported.¹¹ The University Committee on Animal Resources at the University of Rochester has approved all procedures performed on the animals, as well as the housing conditions and the diet in this study.

Tibial fracture model

Open tibial fractures were created in WT and Itch KO mice (five pairs). In brief, an incision of 6 mm in length was made in the skin on the anterior side of the tibia after anaesthesia. A sterile 27 G x 1.25 inch needle was inserted into the marrow cavity of the tibia from the proximal end, temporarily withdrawn to facilitate transection of the tibia using a scalpel at midshaft, and then reinserted to stabilise the fracture. The incision was closed with 5-0 nylon sutures. Fractures were confirmed by radiograph. Callus tissues were harvested at days 3, 7, 10, 14, and 21 following fracture for RNA extraction. For examining the expression levels of total ubiquitinated proteins, the mice were treated with the proteasome inhibitor MG132 (1 mg/kg by intraperitoneal injection, EMD Millipore, Billerica, Massachusetts) 24 hours before they were killed at day 7 following fracture. Total ubiquitinated proteins were examined in callus tissues by Western blot analysis. The cortical bone tissues from the non-fractured legs were used as control.

Quantitative-PCR

Total RNA was extracted from callus tissues using TRIzol Reagent (Invitrogen, Carlsbad, California). cDNAs were synthesised using an iSCRIPT cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, California). Quantitative real-time (RT)-PCR amplifications were performed in the iCycler (Bio-Rad) real-time PCR machine using iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instruction. The expression levels of ubiquitin E3 ligases (Itch, Wwp1, Smurf1 and Smur2), NF-κB members (RelA, RelB, p50, p52), osteoblast-related genes (Runx2 and alkaline phosphatase, ALP) and osteoclast-related genes (Receptor Activator of Nuclear Factor kappa-B, RANKL and osteoprotegerin, OPG) were examined with sequence specific primers (Table I).

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 (ΔCT). ΔΔCT were obtained by subtracting the ΔCT of the reference point. These values were then raised to the power of 2 (2ΔΔCT) to yield fold expression relative to the reference point.

Western blot analysis

Whole-cell lysates were prepared from fracture callus tissues or C2C12 cells, an osteoblast/myoblast precursor cell line that was purchased from ATCC (Manassas, Virginia). Homogenised fracture callus tissues with liquid nitrogen or cells were lysed with mammalian protein extraction reagent (Pierce Chemical, Dallas, Texas) containing a protease inhibitor mixture (Roche Applied Science, Indianapolis, Indiana). Whole cell lysates (10 μg protein/lane) were loaded in 10% Sodium Dodecyl Sulfate (SDS)-PAGE gels, transferred to a nitrocellulose membrane, and immunoblotted with antibodies to β-actin, ubiquitin (Santa Cruz Biotechnology) and Itch (BD Biosciences, San Jose, California). Bands were visualized using ECL chemiluminescence (Amersham Biosciences, Piscataway, New Jersey).

Chromatin immunoprecipitation (ChIP) assay

The C2C12 cells were infected with retroviral virus encoding green fluorescent protein (GFP) control, NF-κB RelA or RelB for 24 hours. Infection efficiency was confirmed by a fluorescent microscopy. ChIP assay was performed with the MAGnify Chromatin Immunoprecipitation System (Invitrogen) according to the manufacturer’s instructions. 1x10⁶ cells in 500 µl PBS were fixed with 1% formaldehyde for 15 minutes. Cells were then lysed in 50 µl 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 (Diagenode, Denville, New Jersey) to shear chromatin into 200 bp to 500 bp fragments. 10 µl of chromatin was diluted to 100 µl with dilution buffer. 10 µl was used as an Input. Antibodies against RelA and RelB or control rabbit IgG (Invitrogen) were used in the immunoprecipitation step. Antibodies were coupled to the Dynabeads (ThermoFisher Scientific, Waltham, Massachusetts) and then incubated with 100 µl diluted chromatin for two 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 55°C. The purified DNA fragments were determined by quantitative PCR using the primers localised at the binding sites (Table II). Control primers localised at irrelevant down-streaming sites were used as a negative control. Amplification efficiency was calculated by 100x2e (Average Input - Average IP), where the Input Control was named as Input recovery.

Statistical analysis

All results are given as mean and standard deviation (sd). Comparisons between the two groups were analysed using the two-tailed unpaired Student’s t-test. One-way analysis of variance (ANOVA) and Dunnett’s post hoc multiple comparisons were used for comparisons among three or more groups. A p-value of less than 0.05 was considered statistically significant.

Increased expression of E3 ligases and NF-κB members in callus tissues during bone fracture repair in WT mice

The WW domain-containing ubiquitin E3 ligases, including Wwp1, Smurf1 and Itch, regulates the stability of the positive osteoblast regulators Runx2, JunB and CXCR4.^4,12,13 To determine if these E3 ligases are involved in fracture repair, we examined their expression levels in callus tissues from WT C57BL/6J mice which had undergone surgical tibia bone fracture. The expression levels of Smurf1, Smurf2 and Itch were all significantly increased, starting at day 7 following fracture, Wwp1 expression increased significantly, starting at day 10, and reached maximum expression at ten days following fracture (Itch expression reaching the maximum at 14 days) (Fig. 1).

Fig. 1

Increased expression of multiple Nedd4 subclass of E3 ligases in fracture callus. Three-month-old WT C57 BL/6J male mice underwent a surgical tibia fracture and were killed at different time points post-fracture. The expression levels of E3 ligases in callus tissue were examined by qPCR. Values are mean and standard deviation of three mice. The relative expression was calculated as fold change using the expression level at day 3 as 1. *p<0.05 versus samples from day three.

We have previously reported that NF-κB members are positive regulars of E3 ligase Itch in osteoclast lineage cells.¹⁴ Thus, we examined expression of NF-κB members at mRNA level extracted from callus tissues at different time points post-fracture. The expression levels of all NF-κB members, including p50, RelA, p52 and RelB, were significantly increased, starting at 7 days after fracture and reaching maximum expression at 14 days (Fig. 2), a similar time point to that of when E3 ligase levels were elevated.

Fig. 2

Increased expression of NF-κB members in fracture callus. Samples were obtained from the callus tissue as in Figure 1. The expression levels of NF-κB members were determined by quantitative polymerase chain reaction (qPCR). Values are mean and standard deviation of three mice. The relative expression was calculated as fold change using the expression level at day 3 as 1. *p<0.05 versus samples from day 3.

Increased amount of total ubiquitinated proteins in fracture callus tissues

We sought to determine if the total number of ubiquitinated (Ub)-proteins in callus tissues is increased in the presence of proteasome inhibitor MG132. The total amount of Ub-proteins in callus tissues from the fractured legs was increased compared with cortical bone tissues from non-fractured legs (Fig. 3).

Fig. 3

Increased total ubiquitinated proteins in fracture callus. WT mice received bone fracture surgery as in Figure 1. Callus tissue from fractured legs was harvested at day 7 after mice received MG132 for 24 hours. Total ubiquitinated (Ub) proteins were determined by Western blot analysis using anti-ubiquitin antibody. The cortical bone samples from the non-fractured legs were used as a control (Ctl).

NF-κB upregulates Itch expression in osteoblast progenitor cells

We have demonstrated that NF-κB RelA mediated RANKL induces Itch expression in osteoclasts by directly binding to NF-κB sites in the itch promoter.¹⁴ We wanted to know if this also occurs in osteoblasts. We have also previously identified two putative NF-κB binding sites at the -3473/-3465 and -3183/-3175 location of the murine Itch promoter (National Centre for Biotechnology Information RefSeq accession number NC_000068) using TFSEARCH software (version 1.3, National Institute of Advanced Industrial Science and Technology, Tokyo).¹⁴ We overexpressed RelA and RelB in C2C12 cells and found that both of them could increase Itch expression (Fig. 4a) and then performed ChIP assays using an anti- NF-κB RelA or RelB antibody to pull down protein-chromatin complexes and two primer sets around these NF-κB binding sites. Results showed RelA and RelB bindings to the NF-κB binding sites of the Itch promoter (Figs 4b and 4c).

Fig. 4

NF-κB upregulates Itch expression in osteoblasts. C2C12, an osteoblast/myoblast cell line, was infected with GFP control, NF-κB RelA or RelB virus for 24 hours. a) Expression levels of Itch protein were determined by Western blot analysis. b) Chromatin immunoprecipitation (ChIP) assays were performed on immunocomplexes that were pulled down with anti-RelA, anti-RelB antibody or Immunoglobulin G (IgG). Precipitated DNA was measured by polymerase chain reaction (PCR) and quantitative PCR using sequence-specific primers. Values are mean (sd) of determinates in triplicate. * p < 0.05 versus green fluorescent protein (GFP)-infected samples.

Increased expression of osteoblast-related genes in callus tissues from Itch KO mice following facture

Itch functions as an E3 ligase to promote ubiquitination of targeting proteins, including the osteoblast positive regulators, thereby negatively regulates osteoblast differentiation.¹¹ Thus, in the absence of Itch, osteoblast differentiation should be increased. To determine if this is the case at the bone fracture site, we examined the expression levels of osteoblast-associated genes (Runx2, ALP) and genes associated with bone remodelling, RANKL and 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.^7,15 It was seen that 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 compared to WT, 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 (Fig. 5).

Fig.

Altered expression patterns for genes associated with bone formation and remodelling in fracture callus of Itch KO mice. Itch KO and WT control mice underwent a surgical fracture and were killed at different time points post-fracture. The expression levels of genes related to osteoblast a) and osteoclast b) differentiation were examined by quantitative polymerase chain reaction (qPCR). Values are mean and standard deviation of triplicate experiments. The relative expression was calculated as fold change using the expression level at day 3 as 1.