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Circ-ITCH promotes the ubiquitination degradation of HOXC10 to facilitate osteogenic differentiation in disuse osteoporosis through stabilizing BRCA1 mRNA via IGF2BP2-mediated m6A modification

Journal of Translational Medicine volume 23, Article number: 376 (2025) Cite this article

Abstract

Background

Osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) facilitated by mechanical loading is a promising therapy for disuse osteoporosis (DOP), however, it is difficult to implement mechanical loading for a majority of patients. Our study aims to identify circ-ITCH-mediated novel approach to facilitate osteogenic differentiation in DOP.

Methods

A rat DOP model and human BM-MSCs under microgravity condition were generated as in vivo and in vitro models of DOP, respectively. The bone mineral density (BMD) and bone parameters were examined in rats. The histological changes of bones and mineralization were monitored by H&E, Alcian blue and Alizarin red S staining. Co-IP was employed to examine the ubiquitination of HOXC10 and the interaction between HOXC10 and BRCA1. The direct associations among circ-ITCH, IGFBP2 and BRCA1 mRNA were assessed by RIP, FISH and RNA pull-down assays.

Results

Circ-ITCH was downregulated in rat model of DOP and BM-MSCs under microgravity stimulation. Circ-ITCH overexpression promoted osteogenic differentiation in BM-MSCs under microgravity condition. The altered bone parameters, such as BMD, trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and bone microstructure in DOP rats were rescued by circ-ITCH overexpression. Mechanistically, circ-ITCH enhanced the ubiquitination degradation of HOXC10 through enhancing BRCA1 mRNA stability. Circ-ITCH directly bound to IGF2BP2 protein to stabilize BRCA1 mRNA via m6A modification, thus facilitating osteogenic differentiation in BM-MSCs under microgravity condition.

Conclusion

Circ-ITCH stabilized BRCA1 mRNA via IGF2BP2-mediated m6A modification, thereby facilitating the ubiquitination degradation of HOXC10 to promote osteogenic differentiation in DOP.

Introduction

Disuse osteoporosis (DOP) is defined as a state of bone loss resulting from lack of sufficient mechanical loading on bones [1, 2]. It is common in patients with paralysis, long-term therapeutic bed rest, immobilization after limb fracture, space flights and neurological disorders [1]. Patients with DOP exhibit decreased bone mineral density (BMD) and high fracture risk [2]. Lack of ideal therapeutic strategy and difficulty in recovery are major challenges for DOP treatment [2]. In recent years, emerging evidence supports that promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) ameliorates symptom of DOP [3,4,5]. Mechanical loading is known to facilitate osteogenic differentiation of BM-MSCs [6, 7]. Unfortunately, mechanical loading is difficult to implement for patients with paralysis, long-term limb immobilization and weightlessness. Insufficient mechanical loading remains the obstacle of DOP treatment. Therefore, a novel method facilitating osteogenic differentiation of BM-MSCs is needed to improve the clinical outcomes of DOP.

Circular RNAs (circRNAs) are a class of non-coding RNAs with closed loop which were generated by back splicing [8]. Emerging evidence supports that circRNAs modulate the balance between osteoblasts-induced bone deposition and osteoclasts-mediated bone resorption in bone diseases [9, 10]. Circ-ITCH is derived from itchy E3 ubiquitin protein ligase (ITCH), and it is recognized as a multifunctional regulator in different physiological and pathological processes [11,12,13,14]. For instance, circ-ITCH functions as a tumor suppressor in bladder cancer and ovarian cancer through miR-17/miR-224/p21/PTEN axis and miR-106a/CDH1 axis, respectively [12, 13]. Circ-ITCH also alleviates doxorubicin-induced cardiotoxicity by sponging miR-330-5p to increase SIRT6, survivin and SERCA2a expression in cardiomyocytes [14]. More importantly, circRNA profiling has reported that circ-ITCH modulates osteogenic differentiation of periodontal ligament stem cells (PDLSC) via MAPK signaling [15]. Our previous report has illustrated that circ-ITCH promotes osteogenic differentiation in osteoporosis through modulating miR-214/YAP1 axis [16], while little is known about the role of circ-ITCH in DOP, and if circ-ITCH can replace or compensate the effects of mechanical loading remains undefined.

Interestingly, increasing evidence indicates the role N6-methyladenosine (m6A) modification, the most abundant form of methylation in mRNA, in osteoporosis [17,18,19]. m6A modification regulates various biological or pathological processes to modulate bone homeostasis in osteoporosis, such as osteogenic differentiation [20]. However, little information is available regarding the role of m6A modification in DOP. In addition, IGF2BP2 has been recognized as an m6A modification reader [21]. Our preliminary bioinformatics analysis (ENCORI) predicted the direct binding between circ-ITCH and IGF2BP2 protein. A recent study has illustrated the role of IGF2BP2 in regulating osteogenic differentiation in dental pulp stem cells [22], and IGF2BP2 also inhibits cell growth and facilitates osteogenic differentiation by stabilizing SRF mRNA in MC3T3-E1 cells [21]. Previous studies have demonstrated that non-coding RNAs, such as circRNAs and long non-coding RNAs (lncRNAs), stabilize the mRNAs of downstream targets via IGF2BP2-dependent m6A modification [23, 24]. For instance, circCDK1 modulates the CPPED1 mRNA stability through IGF2BP2-mediated m6A modification in laryngeal squamous cell carcinoma [23]. Moreover, interaction between IGF2BP2 and the breast cancer susceptibility protein 1 (BRCA1) mRNA was predicted by ENCORI, and RMBase database also predicted the m6A sites in BRCA1 mRNA. BRCA1 is a well-studied tumor suppressor in breast and ovarian cancers, and it also serves as an E3 ubiquitin ligase in various biological processes [25, 26]. Notably, bone loss is frequently observed in women with BRCA1 mutation [27]. We therefore hypothesized that circ-ITCH might regulate bone metabolism in DOP through IGF2BP2-mediated m6A modification of BRCA1.

To study the role of circ-ITCH/IGF2BP2/BRCA1 axis in osteogenic differentiation of BM-MSCs in DOP, bioinformatics analysis (Ubibrowser) also predicted that BRCA1 served as an E3 ligase responsible for homeobox C10 (HOXC10) degradation. HOXC10, a member of homeobox gene family, encodes a highly conserved transcription factor. HOXC10 plays pivotal roles in embryonic development, cell proliferation, differentiation and apoptosis [28, 29]. More importantly, HOXC10 impairs the osteogenic differentiation potential of mesenchymal stem cells (MSCs) [30]. For example, HOXC10 is upregulated in MSCs derived from multiple myeloma patients compared with healthy donors, which is accompanied with suppressed osteogenic differentiation of MM-MSCs [31]. Ubiquitin is a well-studied label for protein degradation [32], while the ubiquitin-proteasome pathway of HOXC10 remains elusive in DOP. The results of bioinformatics analysis raised the possibility that BRCA1-mediated ubiquitination and proteasomal degradation of HOXC10 might contribute to the osteogenic differentiation of BM-MSCs in DOP.

In the current study, we speculated that circ-ITCH was downregulated in a rat model of DOP and BM-MSCs under microgravity treatment. Both in vitro and in vivo findings revealed that circ-ITCH overexpression promoted osteogenic differentiation of BM-MSCs in DOP. Mechanistic studies illustrated that circ-ITCH promoted HOXC10 ubiquitination degradation through enhancing BRCA1 mRNA stability in an IGF2BP2-dependent manner, thus facilitating osteogenic differentiation of BM-MSCs. Our data identify circ-ITCH as a novel therapeutic target and provide insightful insights into the pathogenesis of DOP. Notably, this study first unravels the role of m6A modification and ubiquitination degradation in DOP.

Materials and methods

Animal study

Female Wistar rats (12 ~ 14-week-old, n = 8 per group) were purchased from Hunan SJA Laboratory Animal Co. Ltd. (Changsha, Hunan, China). All animal studies were approved by the Ethics Committee of Xiangya Hospital of Central South University (Changsha, Hunan, China), [2024030385]. The rat DOP model was established as previously described [33]. Briefly, rats were anesthetized with 3% isoflurane by inhalation. Botulinum toxin A (BTX, Agrisera, Sweden) with 4 IU per 100 g body weight was injected into the right quadriceps femoris muscle, hamstring muscles, and posterior calf muscles. Rats in sham group were injected with same volume of saline. Rats were allowed to recover from the anesthetics on the heating pad. The femurs of rats were harvested for bone parameters, histological analyses, qRT-PCR and western blot analyses at 6 weeks post-injection.

Assessment of BMD and micro-CT

BMD of rats was assessed by Dual-energy X-ray absorptiometry (Hologic, Bedford, MA, USA) as described [34]. The femurs were fixed in 4% paraformaldehyde (PFA) and scanned using a high-resolution micro-CT (Bruker microCT, Kontich, Belgium). Quantitative analysis of trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) were conducted as described [35].

Hematoxylin & Eosin (H&E) and alcian blue staining

The femurs were fixed with 4% PFA, followed by the decalcification with 10% EDTA for 3 weeks and paraffin embedding. The sections were stained with H&E (C0105S, Beyotime, Shanghai, China) or Alcian Blue kit (ab150662, Abcam, Cambridge, UK) [36, 37], and images were photographed under a light microscope (Zeiss, Germany).

Rat BM-MSCs isolation and culture

Rat BM-MSCs isolation was conducted as previously described [38]. The rat femurs were dissected and placed in α-MEM containing ribonucleosides and deoxyribonucleosides (Sigma-Aldrich, St. Louis, MO, USA). Bone marrow cells were collected by rinsing of bones with α-MEM containing 20% foetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 100 µM L-ascorbic acid-2-phosphate (Sigma-Aldrich). Cells were then filtered with a 70 μm cell strainer (Sigma-Aldrich) and maintained at 37 °C and 5% CO2. The non-adherent cells were removed by rinsing with PBS after 24 h. On day 3, cells were collected using StemPro Accutase (Gibco) and reseeded in fibronectin-coated dishes. Medium was changed every 2–3 days.

Human BM-MSCs culture and the establishment of in vitro DOP model

Human BM-MSCs were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM containing 10% FBS (Gibco). The in vitro DOP model was generated as previously described [3]. Briefly, 2D-clinostat developed by China Astronaut Research and Training Center was employed to generate a microgravity environment. Cells were maintained in osteogenic differentiation medium that DMEM containing 200 µM ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone (Sigma-Aldrich). The culture flasks were then rotated on 2D-clinostat for 7 or 14 days, and control cells were maintained in the normal incubator.

Lentiviral packaging and transduction

Lentiviral vectors to overexpress circ-ITCH (OE-circ-ITCH) and to knockdown circ-ITCH, BRCA1, or IGF2BP2 (sh-circ-ITCH/BRCA1/IGF2BP2), and corresponding controls were ordered from GenePharma (Shanghai, China). 293T cells were co-transfected with lentiviral vectors and Lenti-Pac HIV Expression Packaging Mix (GeneCopoeia, Guangzhou, China) using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). The culture medium was collected and filtered at 48 h post-transfection. BM-MSCs were transduced with the designated lentiviral as previously described [39]. For in vivo intervention, lentiviral overexpressing circ-ITCH was injected through the tail vein of rats once a week. Rats were sacrificed and subjected to subsequent analyses at 6 weeks post-injection.

Alizarin red S (ARS) staining

After fixation with 70% ethanol, BM-MSCs were with 2% ARS solution (Sigma-Aldrich) at room temperature for 15 min. After rinsing with PBS, images were acquired under a microscope (Zeiss).

Alkaline phosphatase (ALP) activity assay

BM-MSCs were lysed with 1% Triton X-100 (Sigma-Aldrich) for 15–20 min and the cell supernatant was obtained through centrifugation. The ALP activity of BM-MSCs was assessed using an ALP Colorimetric Assay Kit (BioVision, Milpitas, CA, USA) following the manufacturer’s instruction.

qRT-PCR and mRNA stability assay

Total RNA was extracted from BM-MSCs using Trizol (Invitrogen), and reversely transcribed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed using SYBR Green quantitative RT-qPCR Kit (Sigma-Aldrich), and calculated using 2 –ΔΔCT method with expression normalized to that of GAPDH. For BRCA1 mRNA stability assay, BM-MSCs were treated with actinomycin D (5 µg/mL, Sigma-Aldrich) for 0, 4, 8, 12 and 16 h. The mRNA level of BRCA1 was detected by qRT-PCR.

Western blot and protein stability assay

BM-MSCs were lysed with RIPA lysis buffer (Beyotime) and protein quantification was conducted using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated by gel electrophoresis and transferred onto PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated with anti-ALP (1:500, ab65834, Abcam), anti-osteopontin (OPN, 1:1000, ab63856, Abcam), anti-osteocalcin (OCN, 1:1000, MA1-20786, Invitrogen), anti-HOXC10 (1:1000, PA5-31078, Invitrogen), anti-BRCA1 (1:1000, ab238983, Abcam), anti-IGF2BP2 (1:1000, ab124930, Abcam), anti-ubiquitin (1:1000, ab140601, Abcam) or anti-GAPDH (1:2000, ab8245, Abcam) antibody at 4 °C overnight. This was followed by the incubation with secondary antibodies (1:2000, 31460/31430, Invitrogen) for 1 h at room temperature. Signals were visualized using SuperSignal West Pico PLUS substrate (Thermo Fisher Scientific). For protein stability assay, BM-MSCs were treated with cycloheximide (CHX, 20 nM, Sigma-Aldrich) for 0, 1, 3 and 6 h. The protein level of HOXC10 was detected by western blot.

Co-immunoprecipitation (Co-IP) assay

The ubiquitination level of HOXC10 and the association between BRCA1 and HOXC10 were detected using Pierce Crosslink Magnetic IP/Co-IP Kit (88805, Thermo Fisher Scientific). Briefly, BM-MSCs were lysed with IP lysis buffer and protein lysates were incubated with anti-BRCA1 (1:500, ab9141, Abcam), anti-HOXC10 (1:500, A303-177 A, Thermo Fisher Scientific) or normal IgG (1:500, ab172730, Abcam) antibody at 4 °C overnight. The protein complexes were then enriched by Protein A/G magnetic beads, and the immunoprecipitate was analyzed by western blot.

RNA immunoprecipitation (RIP) assay

RIP assay was employed to detect the interaction between circ-ITCH and BRCA1/IGF2BP2 protein, as well as between IGF2BP2 protein and BRCA1 mRNA. RIP assay was conducted using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17–700, Sigma-Aldrich). Briefly, BM-MSCs were lysed with RIP lysis buffer. Anti-BRCA1 (1:1000, ab245330, Abcam), anti-IGF2BP2 (1:1000, ab128175, Abcam) or normal IgG were conjugated to Protein A/G beads, and incubated with cell lysates. RNA was then purified and analyzed by qRT-PCR.

RNA pull-down assay

The interaction between circ-ITCH and IGF2BP2 protein was assessed by RNA pull-down assay. RNA pull-down assay was performed using Pierce Magnetic RNA-Protein Pull-Down Kit (20164, Thermo Fisher Scientific). In brief, circ-ITCH sense or anti-sense probe was labeled with desthiobiotin and conjugated to streptavidin beads, and then incubated with BM-MSCs cell lysates. The RNA-protein complexes were then eluted, and the protein level of IGF2BP2 was analyzed by western blot.

Fluorescence in situ hybridization (FISH)/immunofluorescence staining

FITC-labeled circ-ITCH was synthesized by Ribobio (Guangzhou, China). BM-MSCs were fixed with 4% PFA and permeabilized with 0.3% Triton X-100. Cells were incubated with pre-hybrid solution at 42 °C for 1 h, followed by the incubation with hybrid solution containing FITC-labeled circ-ITCH probe at 42 °C overnight. Cells were then stained with anti-IGF2BP2 (1:100, ab124930, Abcam) antibody at 4 °C overnight, followed by the incubation with Alexa Fluor 594-conjugated secondary antibody (1:500, A78956, Invitrogen). Nuclei were visualized by Hoechst 33,342. Images were acquired using confocal microscopy (Zeiss).

Statistical analysis

All experiments were repeated at least three times, and data were expressed as mean ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism 8.0 (San Diago, CA, USA). One-way analysis of variance (ANOVA) followed by the Tukey post hoc test or Student’s t-test was conducted to assess the differences among multiple groups or between two groups, respectively. P < 0.05 was considered statistically significant.

Results

Circ-ITCH is downregulated in rat BM-MSCs derived from DOP model

To assess the biological role of circ-ITCH, a rat model of DOP was generated. As presented in Fig. 1A, the BMD of DOP rats was much lower than that of sham rats as detected by DXA. Micro-CT showed that DOP rats exhibited decreased Tb.Th, and Tb.N, as well as increased Tb.Sp, compared with that of sham rats (Fig. 1B). Consistently, H&E and Alcian blue stainings revealed the impaired bone microstructure in DOP rats (Fig. 1C). The above results indicated the successful establishment of DOP rat model. In addition, the mRNA (Fig. 1D) and protein (Fig. 1E) levels of osteogenic differentiation markers ALP, OPN and OCN were markedly downregulated in BM-MSCs derived from DOP rats. This was accompanied with the reduction of circ-ITCH in BM-MSCs derived from DOP rats (Fig. 1F). These findings suggest that a rat model of DOP is successfully generated and circ-ITCH is decreased in BM-MSCs derived from DOP rats.

Fig. 1
figure 1

Circ-ITCH is downregulated in rat BM-MSCs derived from DOP model. Rats were randomly divided into two groups: sham and DOP groups (n = 8 per group). (A) The BMD of rats was detected by DXA. (B) The bone parameters including Tb.Th, Tb.N and Tb.Sp were examined by micro-CT. (C) The histological changes of bones were assessed by H&E and Alcian blue stainings. Scale bar, 100 μm. (D) The mRNA levels of osteogenic differentiation markers ALP, OPN and OCN in rat BM-MSCs were detected by qRT-PCR. (E) The protein levels of osteogenic differentiation markers ALP, OPN and OCN in rat BM-MSCs were measured by western blot. (F) The expression level of circ-ITCH in rat BM-MSCs was tested by qRT-PCR. *P < 0.05, **P < 0.01, and ***P < 0.001

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Circ-ITCH is decreased in BM-MSCs under microgravity simulation

The expression of circ-ITCH was next evaluated in the in vitro model of DOP. Human BM-MSCs were cultured in osteogenic differentiation medium under microgravity simulation for 7 or 14 days. ARS staining revealed that the process of mineralization in human BM-MSCs was slowed down by microgravity treatment on day 7 and 14 (Fig. 2A). Moreover, ALP, OPN and OCN mRNA (Fig. 2B) and protein (Fig. 2C) levels were increased time-dependently under osteogenic differentiation induction, while microgravity negatively regulated these three osteogenic molecules on day 7 and 14, compared with corresponding controls. Furthermore, the time-dependent increase of ALP activity in human BM-MSCs was also attenuated by microgravity on day 7 and 14 (Fig. 2D). As expected, circ-ITCH was elevated during osteogenic differentiation of human BM-MSCs, and the level of circ-ITCH in the same day was decreased under microgravity treatment (Fig. 2E). These data suggest that circ-ITCH is downregulated in the in vitro DOP model.

Fig. 2
figure 2

Circ-ITCH is decreased in BM-MSCs under microgravity simulation. Human BM-MSCs were cultured in osteogenic differentiation medium under microgravity simulation for 7 or 14 days. (A) The mineralization process in BM-MSCs was monitored by ARS staining. (B) The mRNA levels of osteogenic differentiation markers ALP, OPN and OCN in BM-MSCs were detected by qRT-PCR. (C) The protein levels of osteogenic differentiation markers ALP, OPN and OCN in BM-MSCs were measured by western blot. (D) The ALP activity in BM-MSCs was analysed using commercial kit. (E) The expression level of circ-ITCH in BM-MSCs was detected by qRT-PCR. *P < 0.05 and **P < 0.01

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Circ-ITCH promotes osteogenic differentiation of BM-MSCs under microgravity simulation

We next sought to delineate the function of circ-ITCH on osteogenic differentiation in the in vitro DOP model on day 14. As shown in Fig. 3A, lentiviral-mediated overexpression of circ-ITCH successfully increased circ-ITCH level in human BM-MSCs under microgravity treatment. ARS staining revealed that microgravity-decreased the mineralized deposits in BM-MSCs were reversed by circ-ITCH overexpression (Fig. 3B). In line with these findings, overexpression of circ-ITCH led to a rebound of ALP activity (Fig. 3C), as well as the upregulation of ALP, OPN and OCN in the in vitro DOP model (Fig. 3D-E). It is worth noting that microgravity induced HOXC10 mRNA (Fig. 3D) and protein (Fig. 3E) expression in BM-MSCs. Circ-ITCH overexpression had no effect on HOXC10 mRNA level, whereas it markedly attenuated microgravity-induced HOXC10 protein level in BM-MSCs (Fig. 3D-E). These findings suggest that circ-ITCH might regulate HOXC10 expression via post-translational modification, and circ-ITCH facilitates osteogenic differentiation of BM-MSCs in the in vitro DOP model.

Fig. 3
figure 3

Circ-ITCH promotes osteogenic differentiation of BM-MSCs under microgravity simulation. Human BM-MSCs were overexpressed circ-ITCH and transfected BM-MSCs were cultured in osteogenic differentiation medium under microgravity simulation for 14 days. (A) The expression level of circ-ITCH was detected by qRT-PCR. (B) The mineralization process in BM-MSCs was monitored by ARS staining. (C) The ALP activity in BM-MSCs was detected using commercial kit. (D) The mRNA levels of ALP, OPN, OCN and HOXC10 in BM-MSCs were analysed by qRT-PCR. (E) The protein levels of ALP, OPN, OCN and HOXC10 in BM-MSCs were detected by western blot. *P < 0.05, **P < 0.01, and ***P < 0.001

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Circ-ITCH promotes the ubiquitination of HOXC10 in a BRCA1-dependent manner

To unravel the mechanism underlying circ-ITCH-suppressed HOXC10 protein expression, protein stability assay was conducted in human BM-MSCs. Overexpression of circ-ITCH accelerated the degradation of HOXC10 protein in the presence of protein synthesis inhibitor CHX, while knockdown of circ-ITCH exerted opposite effects (Fig. 4A), suggesting that post-translational modification is implicated in the process of circ-ITCH-downregulated HOXC10 expression. Co-IP results further revealed that circ-ITCH overexpression increased the ubiquitination of HOXC10, whereas decreased ubiquitination of HOXC10 was observed in circ-ITCH knockdown cells (Fig. 4B). Additionally, bioinformatics analysis (Ubibrowser) predicted the putative E3 ubiquitin ligases responsible for HOXC10 degradation (Fig. 4C). We next examined the mRNA levels of Top 5 E3 ubiquitin ligases including NEDD4, BRCA1, MDM2, SYVN1 and SMURF1 in circ-ITCH overexpression or knockdown BM-MSCs. Intriguingly, qRT-PCR showed that E3 ubiquitin ligase BRCA1 was positively regulated by circ-ITCH in human BM-MSCs, whereas no significant changes were observed in other four E3 ubiquitin ligases (Fig. 4D). Co-IP results further showed that circ-ITCH overexpression enhanced the association between BRCA1 and HOXC10, while lack of circ-ITCH suppressed this interaction (Fig. 4E). Moreover, the decreased expression of BRCA1 was also found in DOP rats and human BM-MSCs under microgravity simulation (Fig. 4F). These findings indicate that circ-ITCH promotes BRCA1-mediated ubiquitination degradation of HOXC10 in BM-MSCs.

Fig. 4
figure 4

Circ-ITCH promotes the ubiquitination of HOXC10 in a BRCA1-dependent manner. Human BM-MSCs were overexpressed or silenced circ-ITCH for 48 h. (A) The protein level of HOXC10 in BM-MSCs was detected by western blot with treatment of CHX for 0, 1, 3 and 6 h. (B) The ubiquitination of HOXC10 was assessed by Co-IP assay in the presence of MG-132 treatment. Whole lysates served as input. (C) The putative E3 ubiquitin ligases of HOXC10 were predicted by Ubibrowser bioinformatics analysis. (D) The mRNA levels of NEDD4, BRCA1, MDM2, SYVN1 and SMURF1 were detected by qRT-PCR. (E) The interaction between BRCA1 and HOXC10 was detected by Co-IP assay. Whole lysates or normal IgG served as an input or negative control, respectively. (F) The mRNA levels of BRCA1 in BM-MSCs from sham or DOP rats, as well as from human BM-MSCs under microgravity simulation, were detected by qRT-PCR. *P < 0.05, **P < 0.01, and ***P < 0.001

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Circ-ITCH promotes the ubiquitination degradation of HOXC10 through enhancing BRCA1 mRNA stability

To further decipher the mechanism by which circ-ITCH regulated BRCA1 expression, RIP assay was performed and revealed that no direct interaction between BRCA1 protein and circ-ITCH in human BM-MSCs (Fig. 5A). This finding promoted us to investigate if circ-ITCH regulated BRCA mRNA level in BM-MSCs. To test this hypothesis, RNA stability assay was performed to examine the effect of circ-ITCH on BRCA1 mRNA stability. In the presence of transcription inhibitor actinomycin D, circ-ITCH overexpression improved the mRNA stability of BRCA1, while circ-ITCH knockdown exerted the opposite effect in BM-MSCs (Fig. 5B). To eliminate the possibility that circ-ITCH regulated osteogenic differentiation by modulating BRCA1 expression, circ-ITCH overexpression and BRCA1 knockdown were conducted in BM-MSCs simultaneously. Circ-ITCH overexpression resulted in the elevated expression of circ-ITCH and BRCA1 in BM-MSCs under microgravity simulation as anticipated, while silencing of BRCA1 had no effect on circ-ITCH level (Fig. 5C-D). Results of Co-IP further revealed that circ-ITCH overexpression promoted the ubiquitination of HOXC10, as well as the association between BRCA1 and HOXC10, in human BM-MSCs under microgravity simulation. By contrast, BRCA1 knockdown attenuated these effects (Fig. 5E-F). Functional studies further showed that circ-ITCH overexpression caused rebound of mineralization (Fig. 5G) and ALP, OPN and OCN expression (Fig. 5H) in human BM-MSCs under microgravity simulation. These effects of circ-ITCH were reversed by BRCA1 knockdown (Fig. 5G-H). Together, these findings suggest that circ-ITCH stabilizes BRCA1 mRNA to promote the ubiquitination of HOXC10, thereby facilitating osteogenic differentiation in DOP.

Fig. 5
figure 5

Circ-ITCH promotes the ubiquitination degradation of HOXC10 through enhancing BRCA1 mRNA stability. (A) The interaction between BRCA1 protein and circ-ITCH was detected by RIP assay. Normal IgG served as a negative control. (B) The mRNA stability of BRCA1 was monitored after overexpressing or silencing circ-ITCH in the presence of transcription inhibitor actinomycin D. Human BM-MSCs were overexpressed circ-ITCH and silenced BRCA1, and then cultured in osteogenic differentiation medium under microgravity simulation for 14 days. (C) The levels of circ-ITCH and BRCA1 mRNA were detected by qRT-PCR. (D) The protein level of BRCA1 was detected by western blot. (E) The ubiquitination of HOXC10 in BM-MSCs were detected by Co-IP assay. (F) The interaction between BRCA1 and HOXC10 in BM-MSCs were assessed by Co-IP assay. Whole lysates or normal IgG served as an input or negative control, respectively. (G) The mineralization process in BM-MSCs was monitored by ARS staining. (H) The protein levels of osteogenic differentiation markers ALP, OPN and OCN in BM-MSCs were detected by western blot. *P < 0.05, **P < 0.01, and ***P < 0.001

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Circ-ITCH regulates BRCA1 mRNA stability via IGF2BP2-mediated m6A modification

To unravel the mechanism underlying circ-ITCH regulated BRCA1 mRNA stability, ENCORI database predicted the potential associations between circ-ITCH and m6A modification reader IGF2BP2, as well as between IGF2BP2 and BRCA1 mRNA (Fig. 6A). FISH combined with immunofluorescence staining showed the co-localization of circ-ITCH and IGF2BP2 in cytoplasm of human BM-MSCs (Fig. 6B). Moreover, the association between circ-ITCH and IGF2BP2 was also validated by RNA pull-down (Fig. 6C) and RIP (Fig. 6D) assays, and RNA pull-down assay further revealed that circ-ITCH overexpression increased the binding between circ-ITCH and IGF2BP2 in human BM-MSCs (Fig. 6C). Additionally, antibody against IGF2BP2 successfully enriched circ-ITCH and BRCA1 mRNA as detected by RIP assay (Fig. 6D). Furthermore, overexpression of circ-ITCH increased the interaction between IGF2BP2 and BRCA1 mRNA in human BM-MSCs (Fig. 6E). qRT-PCR assay further revealed that silencing of IGF2BP2 remarkably decreased the BRCA1 mRNA level in human BM-MSCs (Fig. 6F). These data suggest that circ-ITCH enhances BRCA1 mRNA stability by recruiting m6A modification reader IGF2BP2.

Fig. 6
figure 6

Circ-ITCH promotes the ubiquitination degradation of HOXC10 through enhancing BRCA1 mRNA stability. (A) The associations among circ-ITCH, IGF2BP2 protein and BRCA1 mRNA were predicted by ENCORI database. (B) The co-localization of circ-ITCH and IGF2BP2 protein in BM-MSCs was detected by FISH combined with immunofluorescence staining. Scale bar, 50 μm. Green, circ-ITCH. Red, IGF2BP2. Blue, Hoechest. (C) BM-MSCs were overexpressed circ-ITCH, and the interaction between circ-ITCH and IGF2BP2 was detected by RNA pull-down assay. Anti-sense probes acted as a negative control. (D) The associations among circ-ITCH, IGF2BP2 protein and BRCA1 mRNA were validated by RIP assay. Normal IgG served as a negative control. (E) The interaction between IGF2BP2 protein and BRCA1 mRNA was detected by RIP assay under circ-ITCH overexpression. Normal IgG served as a negative control. (F) BM-MSCs were silenced IGF2BP2, and the mRNA level of BRCA1 was detected by qRT-PCR. **P < 0.01 and ***P < 0.001

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Circ-ITCH promotes osteogenic differentiation via recruiting IGF2BP2 in BM-MSCs under microgravity simulation

Rescued studies were next conducted to investigate the role of circ-ITCH/IGF2BP2 axis in the in vitro DOP model. Human BM-MSCs were overexpressed circ-ITCH and silenced IGF2BP2 under microgravity condition. Western blot results showed that microgravity-induced the decrease of BRCA1 expression was reversed by circ-ITCH overexpression, while this effect was abrogated by IGF2BP2 knockdown in BM-MSCs (Fig. 7A). To test the effect of circ-ITCH/IGF2BP2 axis on BRCA1 mRNA stability, BM-MSCs were treated with actinomycin D and the results showed that overexpression of circ-ITCH inhibited microgravity-accelerated degradation of BRCA1 mRNA, whereas silencing of IGF2BP2 partially attenuated this effect (Fig. 7B). Also, microgravity inhibited the ubiquitination degradation of HOXC10 in BM-MSCs. Circ-ITCH overexpression reversed this effect on HOXC10 ubiquitination, whereas IGF2BP2 knockdown further abrogated the effect of circ-ITCH (Fig. 7C). Additionally, microgravity impaired the association between BRCA1 and HOXC10 in BM-MSCs, which was rescued by circ-ITCH overexpression (Fig. 7D). By contrast, IGF2BP2 knockdown reversed the promotive effect of circ-ITCH on BRCA1/HOXC10 interaction (Fig. 7D). Moreover, overexpression of circ-ITCH counteracted the suppressed mineralization (Fig. 7E), the downregulated ALP, OPN and OCN caused by microgravity (Fig. 7F). These effects of circ-ITCH on mineralization and osteogenic differentiation markers were abolished by IGF2BP2 knockdown (Fig. 7E-F). Collectively, these findings suggest that IGF2BP2 functions as a critical downstream effector of circ-ITCH in osteogenic differentiation of DOP.

Fig. 7
figure 7

Circ-ITCH promotes osteogenic differentiation via recruiting IGF2BP2 in BM-MSCs under microgravity simulation. Human BM-MSCs were overexpressed circ-ITCH and silenced IGF2BP, and transfected BM-MSCs were cultured in osteogenic differentiation medium under microgravity simulation for 14 days. (A) The protein level of BRCA1 was detected by western blot. (B) The mRNA stability of BRCA1 was monitored in the presence of transcription inhibitor actinomycin D. (C) The ubiquitination of HOXC10 in BM-MSCs were assessed by Co-IP assay. (D) The interaction between BRCA1 and HOXC10 were detected by Co-IP assay. Whole lysates or normal IgG served as an input or negative control, respectively. (E) The mineralization process was monitored by ARS staining in BM-MSCs. (F) The protein levels of osteogenic differentiation markers ALP, OPN and OCN in BM-MSCs were detected by western blot. *P < 0.05, **P < 0.01, and ***P < 0.001

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Circ-ITCH promotes osteogenic differentiation in DOP rats

The in vitro data were further validated in DOP rats. Rats were randomly divided into four groups: sham, DOP, DOP + OE-NC and DOP + OE-circ-ITCH groups. The reduced BMD in DOP rats was rescued by circ-ITCH overexpression (Fig. 8A). Consistently, the decreased Tb.Th and Tb.N, and the increased Tb.Sp in DOP rats were reversed by circ-ITCH overexpression (Fig. 8B). H&E and Alcian blue stainings revealed that the impairment on bone microstructure was alleviated by circ-ITCH overexpression in DOP rats (Fig. 8C). We next examined the expression of key molecules in BM-MSCs derived from these rats. As expected, the levels of circ-ITCH and BRCA1 mRNA were downregulated in DOP group, while overexpression of circ-ITCH led to rebounds of circ-ITCH and BRCA1 in rat BM-MSCs (Fig. 8D). Western blot further showed that DOP reduced BRCA1, ALP, OPN and OCN protein levels, while it induced HOXC10 expression. These effects of DOP group were abrogated by circ-ITCH overexpression in rat BM-MSCs (Fig. 8E). These data suggest that circ-ITCH overexpression promotes osteogenic differentiation and alleviated DOP in vivo.

Fig. 8
figure 8

Circ-ITCH promotes osteogenic differentiation in DOP rats. Rats were randomly divided into four groups: sham, DOP, DOP + OE-NC and DOP + OE-circ-ITCH (n = 8 per group). (A) The BMD of rats was detected by DXA. (B) The bone parameters including Tb.Th, Tb.N and Tb.Sp were examined by micro-CT. (C) The histological changes of bones were assessed by H&E and Alcian blue stainings. Scale bar, 100 μm. (D) The expression levels of circ-ITCH and BRCA1 mRNA in rat BM-MSCs were detected by qRT-PCR. (E) The protein levels of BRCA1, HOXC10, ALP, OPN and OCN in rat BM-MSCs were detected by western blot. **P < 0.01 and ***P < 0.001

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Discussion

The skeleton is recognized as the dynamic tissue undergoing bone remodeling which entails bone resorption and deposition [40]. Clinical studies have illustrated that the bone loss in DOP induced by mechanical unloading or immobilization is accompanied with downregulated bone formation and upregulated bone resorption [1, 2]. For instance, mechanical unloading triggers apoptosis of osteocytes and increases RANKL production, thus facilitating bone resorption [41]. It is inconvenient to receive mechanical loading for the majority of DOP patients, therefore, an alternative approach which facilitates osteogenic differentiation is urgently needed for DOP treatment. Our findings illustrated that circ-ITCH stabilized BRCA1 mRNA via IGF2BP2-mediated m6A modification. BRCA1 further enhanced the ubiquitination degradation of HOXC10, thereby promoting osteogenic differentiation of BM-MSCs in DOP. This study deciphered a novel regulatory mechanism underlying osteogenic differentiation in DOP, and shed light on the targeted therapeutic strategy for DOP.

It is well-accepted that circRNAs function as critical molecules in the progression of human diseases by regulating gene transcription, sponging microRNA (miRNA) and interacting with RNA-binding protein (RBP) [11]. Compelling evidence indicates that circ-ITCH serves as a key regulator in different diseases. For instance, circ-ITCH facilitates cell growth and migration in Hirschsprung disease via miR-146b-5p/RET axis [42]. Additionally, circ-ITCH protests against sepsis-induced acute kidney injury through miR-579-3p/ZEB2 axis [43]. In recent years, the crucial role of circ-ITCH has emerged in bone diseases, including osteoporosis, osteosarcoma and intervertebral disc degeneration [11]. More importantly, recent reports have revealed the important function of circ-ITCH in osteogenic differentiation. For instance, circ-ITCH is differentially expressed in PDLSC and it is predicted to regulate PDLSC osteogenic differentiation by sponging miR-34a and miR-146a via MAPK pathway [15]. Moreover, circ-ITCH is time-dependently increased in BM-MSCs during osteogenic differentiation, and lack of circ-ITCH suppresses ALP activity, mineralization and expression of osteogenic differentiation markers [16], supporting that circ-ITCH functions as a positive regulator of osteogenic differentiation. In accordance with these findings, we found that circ-ITCH was decreased in BM-MSCs derived from DOP rats and human BM-MSCs under mechanical unloading condition, indicating that downregulated circ-ITCH is associated with impaired osteogenic differentiation in DOP. We firstly demonstrated that circ-ITCH overexpression promoted osteogenic differentiation in the in vitro and in vivo DOP models, suggesting its clinical significance in DOP. Besides mechanical loading, circ-ITCH also acted as a key player in osteogenic differentiation in DOP, indicating its potential in the replacement or compensation for insufficient mechanical loading to alleviate DOP.

HOXC10 is recognized as negative regulator in white adipose tissue browning [44], and a recent study has also illustrated that HOXC10 promotes cell proliferative capacity and abrogates lipid accumulation in sheep BM-MSCs [45]. HOXC10 acts as a negative regulator of osteogenic differentiation in MSCs [30, 31]. For instance, HOXC10 suppresses osteogenic differentiation of BM-MSCs via binding to lncRNA HOXC-AS3 [31]. Intriguingly, we found that circ-ITCH did not alter the mRNA level of HOXC10, but downregulated its protein level, supporting the implication of post-translational modification in this process. Bioinformatics analysis coupled with Co-IP results further showed that BRCA1, an E3 ubiquitin ligase, was responsible for circ-ITCH-enhanced ubiquitination of HOXC10. Previous studies have illustrated that the inherited mutations in BRCA1 and BRCA2 confer increased susceptibility to breast and ovarian cancers [46, 47]. Besides its well-known tumor suppressive role, recent studies have illustrated women with BRCA1/2 mutation exhibit high risk of bone loss after risk-reducing bilateral salpingo-oophorectomy (RRSO) [48, 49]. Knockout study has reported that lack of BRCA1 leads to bone marrow failure and hematopoietic defects in mice [50]. In addition, BRCA1/2 in neural crest cells is required for craniofacial bone development [51]. These reports indicate the potential role of BRCA1 in bone metabolism. Mechanistic studies have reported the crucial roles of BRCA1 in DNA damage repair, ubiquitination and transcription [52,53,54]. In this study, we found that BRCA1 was decreased in BM-MSCs derived from DOP rats and human BM-MSCs under mechanical unloading condition, and it was positively regulated by circ-ITCH. Circ-ITCH enhanced the association between BRCA1 and HOXC10 to regulate HOXC10 ubiquitination degradation, suggesting the indispensable role of BRCA1 in circ-ITCH-regulated HOXC10 expression. Functional studies also showed that circ-ITCH facilitated osteogenic differentiation through promoting BRCA1-mediated ubiquitination modification of HOXC10. Our findings demonstrated a novel mechanism among circ-ITCH, BRCA1 and HOXC10 in DOP, and first illustrated a regulatory role of ubiquitin-proteasome system in DOP pathogenesis.

To further investigate the relationship between circ-ITCH and BRCA1, RIP assay was conducted and failed to detect the interaction between circ-ITCH and BRCA1 protein, but circ-ITCH enhanced BRCA1 mRNA stability. These findings promoted us to speculate that post-transcriptional modification, such as m6A modification, might be implicated in circ-ITCH-mediated regulation of BRCA1 mRNA. IGF2BP2, a member of IGF2BP family, is a m6A reader which enhances mRNA stability and translation [55]. For instance, lncRNA PCAT6 stabilizes IGF1R mRNA to promote bone metastasis in prostate cancer through IGF2BP2-mediated m6A modification [56]. Additionally, IGF2BP2 is implicated in osteoimmunomodulation via direct interaction with CD5L and CD36 mRNAs in periodontitis [57]. A recent study has reported that IGF2BP2 is differentially expressed in osteoporosis, and it promotes osteogenic differentiation via stabilizing SRF mRNA [21]. Circ-Plod2 binds to IGF2BP2 to modulate the mRNA stability of Mpo, thereby facilitating osteogenic differentiation of BM-MSCs [58]. In the current study, IGF2BP2 interacted with circ-ITCH and BRCA1 mRNA directly, and it was required for the m6A modification of BRCA1. Consistently, lack of IGF2BP2 reversed circ-ITCH-mediated BRCA1 mRNA stability, as well as circ-ITCH-facilitated HOXC10 ubiquitination, indicating that IGF2BP2 contributes to circ-ITCH-promoted osteogenic differentiation via modulating BRCA1 m6A modification in DOP. This regulatory mechanism merits further in-depth investigation in DOP rats in the future study. The present study first demonstrated the novel role of m6A modification of BRCA1 in DOP.

In conclusion, our findings illustrated that circ-ITCH stabilized BRCA1 mRNA via IGF2BP2-mediated m6A modification, thereby enhancing the ubiquitination degradation of HOXC10 to promote osteogenic differentiation of BM-MSCs in DOP. Circ-ITCH targeted therapy might be used in combination with physical exercise for DOP treatment in the future.

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Funding

This work was supported by National Natural Science Foundation of China (82472609), Natural Science Foundation of Hunan Province (2023JJ30941 and 2022JJ30941), the Fundamental Research Funds for the Central Universities of Central South University, Horizontal Research Funding of Central South University (JXDY-ZD-20231101).

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Authors and Affiliations

  1. Department of Orthopaedics, Xiangya Hospital of Central South University, Changsha, China

    Da Zhong, Xi Li, Zhen Yin, Peng Chen, Yusheng Li, Jian Tian, Long Wang, Hua Liu & Chenggong Wang

  2. Hunan Key Laboratory of Aging Biology, Xiangya Hospital, Central South University, Changsha, China

    Da Zhong & Chenggong Wang

  3. National Clinical Research Center of Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China

    Xi Li, Yusheng Li & Chenggong Wang

  4. The School of Medicine, Nankai University, Tianjin, China

    Long Wang

  5. The First Affiliated Hospital, Department of Orthopedics, Hengyang Medical School, University of South China, Hengyang, China

    Ke Yin

  6. School of Public Health, Changsha Medical University, Changsha, China

    Lemei Zhu

  7. Department of Radiology, Xiangya Hospital, Central South University, Changsha, China

    Lingyu Kong

  8. Department of Rehabilitation Medicine, Xiangya Hospital, Central South University, Changsha, China

    Kunli Chen & Yaochun Li

  9. Department of Orthopedics, Movement System Injury and Repair Research Center, Xiangya Hospital, Central South University, Changsha, China

    Chungu Hong

  10. Department of Orthopaedics, Xiangya Hospital of Central South University, No. 87 Xiangya Road, Changsha, Hunan Province, 410008, China

    Chenggong Wang

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  1. Da Zhong
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Contributions

Da Zhong: Methodology, Validation, Formal analysis, Investigation, Resources & Data curation, Writing original draft. Xi Li Conceptualization, Methodology, Writing-review & editing, Funding acquisition. Zhen Yin Methodology, Writing-review & editing. Peng Chen Methodology, Visualization. Yusheng Li Investigation, Data curation. Jian Tian Methodology, Visualization. Long Wang Writing-review & editing, Supervision. Hua Liu: Investigation, Data curation. Ke Yin Investigation, Data curation. Lemei Zhu Methodology, Investigation & Formal analysis. Lingyu Kong Investigation, Data curation. Kunli Chen Methodology, Supervision, Validation & Formal analysis. Yaochun Li Writing-review & editing, Supervision. Chungu Hong Supervision, Data curation. Chenggong Wang: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing-original draft, Writing-review & editing, Visualization, Project administration, Funding acquisition.

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Correspondence to Chenggong Wang.

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All animal studies were approved by the Ethics Committee of Xiangya Hospital of Central South University (Changsha, Hunan, China), [2024030385].

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The data that support the findings of this study are available from the corresponding author, [CW], upon reasonable request.

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Zhong, D., Li, X., Yin, Z. et al. Circ-ITCH promotes the ubiquitination degradation of HOXC10 to facilitate osteogenic differentiation in disuse osteoporosis through stabilizing BRCA1 mRNA via IGF2BP2-mediated m6A modification. J Transl Med 23, 376 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-06050-5

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Journal of Translational Medicine

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