Artykuł badawczy

N6-Methyladenosine Modification of Rab27A Promotes Osteoporosis by Influencing Osteoclast Differentiation in the Ovariectomized Mouse Model

30 wyświetleń

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DOI:

10.3791/71964

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11 sierpnia 2026

 ,  ,  ,  ,  ,  ,  , 
1Department of Orthopedics, Affiliated Hospital of Southwest Medical University, 2Department of Orthopedics, The People's Hospital of Pengzhou, 3Department of Clinical Skills Center, Affiliated Hospital of Southwest Medical University, 4Southwest Medical University, 5Stem Cell Immunity and Regeneration Key Laboratory of Luzhou, Affiliated Hospital of Southwest Medical University

* These authors contributed equally

W tym artykule

Podsumowanie

YTHDC1 promotes the cytoplasmic export of m6A-modified Rab27A, whereas IGF2BP2 stabilizes Rab27A in the cytoplasm, thereby enhancing osteoclast differentiation and contributing to osteoporosis.

Streszczenie

The development of novel therapies for osteoporosis has attracted increasing attention. Rab27A is upregulated during osteoclast differentiation, and N6-methyladenosine (m6A) modification has emerged as an important epigenetic regulator in orthopedic diseases. Bioinformatic analyses predicted that Rab27A contains multiple m6A modification sites and interacts with several m6A-associated proteins. Knockdown experiments were performed to evaluate the role of Rab27A in osteoclast differentiation and osteoporosis using an ovariectomized (OVX) mouse model. Rab27A depletion attenuated osteoporosis and increased the bone formation percentage from 14.73% to 28.13% (P < 0.05) in OVX mice. Western blotting and quantitative PCR demonstrated that Rab27A knockdown reduced the protein and mRNA expression of the osteoclast markers TRAP, NFATc1, and c-FOS, thereby suppressing osteoclast differentiation of bone marrow-derived macrophages (BMMs) induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). TRAP staining further showed that Rab27A depletion significantly reduced TRAP activity from 24.1% to 12.9% (P < 0.01) following M-CSF and RANKL induction. RNA immunoprecipitation assays confirmed interactions between Rab27A and the m6A reader proteins YTHDC1 and IGF2BP2. Nuclear and cytoplasmic fractionation demonstrated that YTHDC1 facilitates the cytoplasmic export of m6A-modified Rab27A. In addition, q-PCR and western blot analyses showed that knockdown of either YTHDC1 or IGF2BP2 reduced Rab27A expression and decreased the expression of TRAP, NFATc1, and c-FOS, thereby suppressing osteoclast differentiation induced by M-CSF and RANKL. Collectively, these findings indicate that YTHDC1-mediated cytoplasmic export and IGF2BP2-mediated stabilization of m6A-modified Rab27A promote osteoclast differentiation and contribute to bone loss in the OVX mouse model.

Wprowadzenie

Osteoporosis (OP) is a common skeletal disorder characterized by deterioration of bone microarchitecture and reduced bone mineral density, resulting in an increased risk of fractures worldwide1,2,3. Osteoporotic fractures occur most frequently in the forearm, humerus, hip, and spine and are particularly prevalent among older adults. These fractures substantially impair quality of life and impose considerable physical, psychological, and socioeconomic burdens on patients, their families, and healthcare systems3. Consequently, the prevention, management, and treatment of osteoporosis remain major clinical and public health priorities.

A variety of therapeutic strategies have been developed for osteoporosis and can generally be classified into two categories: bone-forming agents and anti-resorptive therapies4,5. Traditional anti-resorptive drugs include bisphosphonates, raloxifene, and strontium ranelate, whereas newer agents include denosumab, odanacatib, and saracatinib. Bone-forming therapies include parathyroid hormone (PTH 1–84), while several additional candidates, such as MK-5442, AMG 785, and BHQ 880, are undergoing clinical development5. Although these treatments can be effective, many are associated with significant adverse effects, limited long-term efficacy, or restrictions on prolonged use4,6,7,8,9. For example, long-term administration of aminobisphosphonates and denosumab may impair normal bone remodeling5. Therefore, identifying safer and more effective therapeutic targets remains an important objective in osteoporosis research.

Rab proteins are key regulators of intracellular vesicle trafficking and cell signaling and participate in diverse biological processes, including viral infection, autophagy, and cytoskeletal organization10. In recent years, increasing attention has been directed toward the roles of Rab family proteins in bone metabolism and osteoporosis. Rab44 has been reported to regulate osteoclast differentiation by modulating intracellular Ca2⁺ signaling and activating nuclear factor of activated T cells 1 (NFATc1) in receptor activator of nuclear factor-κB ligand (RANKL)-treated macrophages11. Osteoclasts are multinucleated cells derived from macrophage lineage precursors and play essential roles in bone resorption and skeletal homeostasis12,13,14,15,16,17,18,19. Therefore, understanding the molecular mechanisms governing osteoclast differentiation is critical for developing novel therapeutic approaches for osteoporosis.

Rab27A is an important member of the Rab GTPase family and is widely expressed in secretory and immune cells. It plays pivotal roles in vesicle transport, exocytosis, and intracellular signaling20,21,22,23,24,25,26,27. Previous studies have demonstrated that Rab27A regulates eosinophil degranulation during airway inflammation and controls vesicular mobilization associated with NADPH oxidase activity in activated macrophages20.23. Notably, accumulating evidence indicates that Rab27A expression is significantly upregulated during osteoclast differentiation. Shimada-Sugawara et al. reported that Rab27A expression increases during the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts, suggesting a potential role in osteoclastogenesis28. Collectively, these findings suggest that Rab27A may influence osteoclast formation and function by regulating vesicular trafficking and intracellular signaling pathways28,29.

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic messenger RNAs and non-coding RNAs and serves as a critical regulator of RNA metabolism and gene expression. As the first identified mRNA modification in mammals, m6A has been implicated in numerous biological processes, including RNA stability, splicing, transport, and translation. Wang et al. demonstrated that m6A modifications, commonly occurring within conserved sequence motifs, are recognized by YTH domain-containing proteins and contribute to the regulation of mRNA stability and metabolism30. Furthermore, m6A modification has been implicated in diverse biological functions, including genome stability, transcriptional regulation, chromatin organization, cancer progression, and immune responses31. Recent studies have also highlighted important roles for m6A signaling in bone-related diseases and osteoporosis32.

Among m6A reader proteins, YTHDC1 functions as a nuclear reader that regulates RNA processing and nuclear export, whereas insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) stabilizes m6A-modified transcripts in the cytoplasm. Chen et al. demonstrated that YTHDC1 promotes the nuclear export of m6A-modified circNSUN2, which subsequently interacts with IGF2BP2 to stabilize HMGA2 mRNA and promote colorectal cancer metastasis33. These findings suggest that coordinated regulation by YTHDC1 and IGF2BP2 may represent a general mechanism controlling the fate of m6A-modified transcripts.

The present study investigated predicted m6A modification sites within Rab27A mRNA and examined its interactions with YTHDC1 and IGF2BP2. YTHDC1 was found to facilitate the nuclear export of m6A-modified Rab27A transcripts, whereas IGF2BP2 interacted with Rab27A in the cytoplasm and enhanced its stability. Through this regulatory mechanism, Rab27A promoted osteoclast differentiation and contributed to the progression of osteoporosis. Collectively, the findings identify a previously unrecognized m6A-dependent regulatory pathway involving YTHDC1, IGF2BP2, and Rab27A in osteoclast differentiation. These results provide new insights into the molecular pathogenesis of osteoporosis and may facilitate the development of novel therapeutic strategies targeting m6A-mediated regulation of Rab27A.

Protokół

All animal procedures were approved by the Animal Protection and Utilization Committee of the Affiliated Hospital of Southwest Medical University (Approval No. 2024-Ethics-042). All experiments were conducted in accordance with the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act 1986 and associated guidelines, EU Directive 2010/63/EU, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978). Mice were randomly assigned to experimental groups, and the allocation sequence was concealed from the investigator. All data acquisition and analyses, including histological evaluation, micro-computed tomography (micro-CT) image analysis, and statistical analyses, were performed by investigators blinded to group allocation. No animals or samples were excluded from the analyses. All reagents, equipment, software, and other resources used in this protocol are listed in the Table of Materials.

1. Construction of the Ovariectomy (OVX) model

Twenty-four female wild-type C57BL/6 mice aged 8 weeks were used to establish an osteoporosis model. Following a 1-week acclimatization period, mice were randomly assigned to either the Sham or OVX group. The OVX model was established by bilateral ovariectomy as previously described34,35. Sham-operated mice underwent surgical exposure of the ovaries without removal.

For intra-femoral gene delivery, lentiviral vectors expressing sh-Rab27A or sh-NC were injected 7 days after OVX surgery as previously reported36,37. Briefly, concentrated lentiviral particles (1 × 106 transducing units/mL) were injected into the femur through the patellar route using a 22-gauge needle under anesthesia. Each mouse received 20 µL of lentiviral suspension. The incision was subsequently sutured, and the animals were monitored throughout recovery.

All animal experiments were performed under specific pathogen-free conditions. Femoral specimens were collected 8 weeks after surgery. At least six mice per group were used for histological analyses and RNA or protein extraction.

2. In-vivo micro-CT imaging

Micro-computed tomography (micro-CT) was performed to evaluate trabecular bone morphology. The right tibia of each mouse was scanned in vivo before OVX surgery at 14 weeks of age. Follow-up scans were performed weekly until 22 weeks of age. At least six mice per group were included in the micro-CT analysis.

Mice were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg body weight). Adequate anesthesia was confirmed by the absence of the toe-pinch reflex and by continuous monitoring of respiratory rate, and it was maintained throughout the imaging procedure. All euthanasia procedures were conducted in accordance with the approved animal protocol, institutional animal welfare guidelines, and national regulations for laboratory animal euthanasia.

At 24 weeks of age, mice were euthanized by cervical dislocation following deep anesthesia, and both tibiae were harvested for ex vivo scanning using the same imaging protocol. Scanning parameters included a voxel size of 10.4 µm, a voltage of 55 kV, a current of 145 µA, a field of view of 32 mm, 1500/750 samples per projection, and an integration time of 100 ms.

Images were processed using a third-order polynomial beam-hardening correction algorithm calibrated against a 1200 mg HA/cm3 phantom. Bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) were quantified in the distal femoral metaphysis located 1.0 mm proximal to the growth plate.

3. Plasmid transfection

sh-Rab27A and sh-NC plasmids were generated and used for lentivirus production. Lentiviral packaging plasmids and shRNA constructs were co-transfected into HEK293T cells according to the manufacturer's instructions. Viral titers were determined before intra-femoral administration as previously described36. Three independent biological replicates were performed.

4. Hematoxylin and eosin (HE) staining

Femoral specimens were fixed in 4% paraformaldehyde for 24 h, decalcified in 10% EDTA for 4 weeks, cryoprotected in 30% sucrose/PBS, embedded in paraffin, and sectioned at a thickness of 5 µm. Tissue sections were stained with hematoxylin and eosin and examined using a light microscope. At least six mice per group were analyzed.

5. Tartrate-resistant acid phosphatase (TRAP) staining

TRAP staining of femoral sections was performed according to the manufacturer's instructions38. TRAP activity in bone marrow-derived macrophages (BMMs) was evaluated using a TRAP staining assay. Mature osteoclasts were defined as TRAP-positive multinucleated cells containing at least three nuclei. Three independent biological replicates were performed, each with three technical replicates.

6. q-PCR

Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions. Complementary DNA was synthesized using reverse transcriptase and subjected to quantitative real-time PCR using SYBR Green chemistry. Three independent biological replicates were performed, each with three technical replicates.

Primer sequences were as follows:

Rab27A: F, TTCCTGCTTCTGTTCGACCT; R, GGCAGCACTGGTTTCAAAAT

TRAP: F, TGTTGACAGCGGTCCATCTA; R, CCTCCTTCTTAACCCGAAGC

NFATc1: F, GGTGCTGTCTGGCCATAACT; R, GCGGAAAGGTGGTATCTCAA

c-FOS: F, TACTACCATTCCCCAGCCGA; R, GCTGTCACCGTGGGGATAAA

YTHDC1: F, GAAGTGGAAGCTCTGCATCAG; R, TTGATCTTTTCGGACAGCACG

IGF2BP2: F, GACTACCCCGACCAGAACTG; R, GAGGCGGGATGTTCCGAATC

β-actin: F, GGGAAATCGTGCGTGACATTAAG; R, TGTGTTGGCGTACAGGTCTTTG

7. Western blotting

Cells or tissues were lysed using RIPA buffer supplemented with protease inhibitors. Protein concentrations were determined using a Bradford assay. Equal amounts of protein (20 µg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) or nitrocellulose membranes. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against TRAP, NFATc1, c-FOS, Rab27A, YTHDC1, IGF2BP2, or β-actin. After washing, membranes were incubated with the appropriate secondary antibodies for 1 h at room temperature. Protein bands were visualized using standard detection procedures. Three independent biological replicates were performed.

8. Cell culture

Bone marrow-derived macrophages (BMMs) were isolated as previously described39. Bone marrow cells harvested from mouse femurs were cultured overnight in α-minimum essential medium supplemented with 10% fetal bovine serum, 30 ng/mL macrophage colony-stimulating factor (M-CSF), and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. Non-adherent cells were collected and cultured in a stromal cell-free system containing 30 ng/mL M-CSF. After 3 days, adherent cells were harvested as BMMs and further cultured in the presence of 30 ng/mL M-CSF and 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) for 72 h to induce osteoclast differentiation.

9. RNA interference (RNAi)

Small interfering RNAs (siRNAs) were synthesized and transfected using a lipid-based transfection reagent according to the manufacturer's instructions. Cells were seeded at a density of 2 × 104 cells per well in six-well plates before transfection. Three independent biological replicates were performed.

Target sequences were as follows:

si-Rab27A: GGACUUAAUCAUCTAAGAGAAUGGAA

si-YTHDC1: GCAAGGAGUGUUAUCUUAATT

si-IGF2BP2: CCGUUAACCAACAAGCCAATT

10. Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 10 min. Following blocking with 5% bovine serum albumin for 30 min, cells were incubated with primary antibodies overnight at 4 °C.

After washing, fluorophore-conjugated secondary antibodies were applied for 30 min at room temperature. Nuclei were counterstained with DAPI, and images were acquired using confocal microscopy. Three independent biological replicates were performed.

11. Nuclear and cytoplasmic separation

Nuclear and cytoplasmic fractions were isolated according to the manufacturer's instructions. Cells were lysed on ice for 10 min and centrifuged at 500 × g for 3 min at 4 °C. The supernatant containing cytoplasmic components was collected, whereas the pellet containing nuclei was washed and processed separately.

12. RNA-binding protein immunoprecipitation (RIP)

RNA immunoprecipitation (RIP) assays were performed according to the manufacturer's instructions. Briefly, cells were lysed on ice for 30 min. After centrifugation, the supernatants were incubated overnight with antibodies and Protein A/G agarose beads. Following washing, immunoprecipitated proteins were analyzed by western blotting, and co-immunoprecipitated RNA was analyzed by quantitative real-time PCR. Three independent biological replicates were performed.

13. Rab27A mRNA stability assay

Total RNA was extracted at the indicated time points following IGF2BP2 knockdown. Reverse transcription and quantitative real-time PCR were subsequently performed. Rab27A mRNA levels were normalized to the corresponding si-NC group at 0 h. Three independent biological replicates were performed.

14. Statistical analysis

All experiments were independently performed using at least three biological replicates. For q-PCR, RNA interference, TRAP staining, western blotting, and other cell-based assays, each biological replicate included three technical replicates unless otherwise specified. Data are presented as the mean ± standard deviation (SD).

Statistical analyses were performed using GraphPad Prism version 9.0 and SPSS software. Comparisons between two groups were conducted using a two-tailed Student's t-test. Comparisons among multiple groups were analyzed using two-way analysis of variance (ANOVA) followed by Tukey's multiple-comparison post hoc test to adjust for multiple comparisons. A P value < 0.05 was considered statistically significant.

Wyniki

Wyciszenie genu Rab27A zmniejszyło stopień osteoporozy in vivo.

Aby zbadać rolę Rab27A w osteoporozie, przeprowadzono knockdown Rab27A w modelu myszy po owariektomii (OVX), opracowanym zgodnie z wcześniej opisanymi metodami34,40. Ogólny schemat badania przedstawiono na Rysunku 1. Analiza mikrotomografii komputerowej wykazała, że gęstość mineralna kości, frakcja objętości kości (BV/TV), grubość beleczek (Tb.Th) oraz liczba beleczek (Tb.N) były znacznie obniżone u myszy OVX w porównaniu z grupą kontrolną Sham (Rysunek 2A). Wyniki te potwierdziły pomyślne ustanowienie modelu osteoporozy. Knockdown Rab27A znacząco przywrócił wszystkie mierzone parametry strukturalne kości, co wskazuje na złagodzenie utraty masy kostnej i pogorszenia mikroarchitektury wywołanej przez OVX (Rysunek 2A).

Badania histologiczne dodatkowo potwierdziły te wyniki. Barwienie hematoksyliną i eozyną wykazało wyraźną utratę kości beleczkowej oraz zaburzenie architektury beleczkowej u myszy OVX w porównaniu z grupą Sham (Rysunek 2B). Z kolei deplecja Rab27A złagodziła pogorszenie stanu kości beleczkowej i częściowo przywróciła morfologię kości (Rysunek 2B). Ponieważ Rab27A jest zaangażowany w różnicowanie osteoklastów28, aktywność osteoklastów oceniono za pomocą barwienia na fosfatazę kwaśną odporną na TRAP (TRAP). Aktywność TRAP była wyraźnie podwyższona u myszy OVX w stosunku do kontroli Sham i uległa znacznemu obniżeniu po wyciszeniu Rab27A (Rysunek 2C).

Następnie zbadano ekspresję markerów związanych z osteoklastami. Analiza Western blot wykazała, że poziomy białek Rab27A, TRAP, NFATc1 i c-FOS były znacząco zwiększone u myszy OVX i uległy obniżeniu po usunięciu Rab27A (Rysunek 2D). Spójne wyniki uzyskano na poziomie transkrypcyjnym, a ilościowa reakcja łańcuchowa polimerazy w czasie rzeczywistym (q-PCR) wykazała podobne wzorce ekspresji dla mRNA Rab27A, TRAP, NFATc1 i c-FOS (Rysunek 2E).

figure-results-1
Rysunek 1: Schematyczny przegląd projektu eksperymentalnego i proponowanego mechanizmu regulacyjnego leżącego u podstaw regulacji zmodyfikowanego m6A Rab27A za pośrednictwem YTHDC1 i IGF2BP2 podczas różnicowania osteoklastów i progresji osteoporozy. W celu zbadania roli Rab27A w osteoklastogenezie wykorzystano model myszy po owariektomii (OVX) oraz model różnicowania osteoklastów indukowanego ligandem receptora aktywatora czynnika jądrowego-κB (RANKL) oraz czynnikiem stymulującym wzrost kolonii makrofagów (M-CSF). Przeprowadzono wyciszenie Rab27A, frakcjonowanie jądrowe i cytoplazmatyczne oraz analizy immunoprecypitacji RNA w celu oceny markerów różnicowania osteoklastów (TRAP, NFATc1 i c-FOS), ekspresji Rab27A oraz lokalizacji subkomórkowej. YTHDC1 ułatwiał eksport zmodyfikowanego m6A Rab27A do cytoplazmy, podczas gdy IGF2BP2 stabilizował Rab27A w cytoplazmie. Wspólnie te skoordynowane mechanizmy regulacyjne promowały różnicowanie osteoklastów i przyczyniły się do utraty masy kostnej w modelu OVX. Aby zobaczyć większą wersję tego rysunku, kliknij tutaj.

figure-results-2
Rycina 2: Knockdown Rab27A zmniejszył osteoporozę in vivo. (A) Analiza morfometryczna kości beleczkowej. Ilościowo określono gęstość mineralną kości, frakcję objętości kości (BV/TV), grubość beleczek (Tb.Th) oraz liczbę beleczek (Tb.N). **P < 0,01, ***P < 0,001. (B) Ocena histologiczna beleczkowej tkanki kostnej kości udowej po wyciszeniu Rab27A. Wykonano barwienie hematoksyliną i eozyną (HE) w celu oceny zmian strukturalnych w dalszej części kości udowej. Grupy OVX oraz OVX + sh-NC służyły jako kontrole. Pasek skali = 50 µm. ***P < 0,001. (C) Barwienie TRAP w przekrojach kości udowej. Aktywność TRAP oceniono w celu zbadania aktywności osteoklastów. Grupy OVX oraz OVX + sh-NC służyły jako kontrole. Pasek skali = 50 µm. ***P < 0,001. (D–E) Ekspresja Rab27A, TRAP, NFATc1 i c-FOS po wyciszeniu Rab27A. Ekspresję białek określono metodą western blotting (D), a ekspresję mRNA metodą q-PCR (E). β-aktyna służyła jako kontrola ładunku. **P < 0,01, ***P < 0,001. Grupa Sham została poddana chirurgicznemu odsłonięciu jajników bez owariektomii, natomiast grupa OVX poddana była obustronnej owariektomii. sh-NC, negatywna kontrola krótkiego zapinek RNA (shRNA). W każdej grupie eksperymentalnej uwzględniono sześć myszy. Dane przedstawiono jako średnia ± SD. Kliknij tutaj, aby wyświetlić powiększoną wersję tej ryciny.

Wyciszenie Rab27A zahamowało różnicowanie osteoklastów z BMM indukowane przez M-CSF i RANKL

Aby dalej ocenić rolę Rab27A podczas osteoklastogenezy, makrofagi pochodzące z szpiku kostnego (BMMs) stymulowano czynnikiem stymulującym kolonie makrofagów (M-CSF) oraz ligandem receptora aktywatora czynnika jądrowego κB (RANKL) w celu indukcji różnicowania osteoklastów28.

Analizy ilościowe w czasie rzeczywistym metodą PCR oraz western blot wykazały, że stymulacja M-CSF i RANKL znacząco zwiększyła ekspresję mRNA i białka Rab27A, podczas gdy transfekcja si-Rab27A skutecznie zredukowała ekspresję Rab27A (Rycina 3A, B). Barwienie TRAP wykazało wyraźny wzrost aktywności osteoklastów po zastosowaniu M-CSF i RANKL. Wyciszenie Rab27A znacząco zredukowało aktywność TRAP w porównaniu z grupą indukowaną (Rycina 3C).

Następnie zbadano ekspresję markerów różnicowania osteoklastów. Analiza ilościowej RT-PCR wykazała, że poziomy mRNA dla TRAP, NFATc1 i c-FOS były znacząco podwyższone po indukcji osteoklastów, a ich poziom uległ obniżeniu po deplecji Rab27A (Rycyna 3D). Podobne wyniki zaobserwowano na poziomie białka w analizie western blotting (Rycyna 3E).

figure-results-3
Rysunek 3: Wyciszenie Rab27A zahamowało różnicowanie osteoklastów z makrofagów pochodzących z szpiku kostnego (BMMs) indukowane przez M-CSF i RANKL. (A–B) Ekspresja Rab27A po indukcji osteoklastów. Poziomy mRNA Rab27A (A) i białka (B) określono odpowiednio za pomocą q-PCR i western blotting. β-aktyna służyła jako kontrola ładunku. ***P < 0,001. (C) Barwienie TRAP osteoklastów pochodzących z BMM. Zwiększona aktywność TRAP indukowana przez M-CSF i RANKL została zahamowana po wyciszeniu Rab27A. Pasek skali = 50 µm. ***P < 0,001. (D–E) Ekspresja markerów różnicowania osteoklastów po usunięciu Rab27A. Poziomy mRNA TRAP, NFATc1 i c-FOS określono za pomocą q-PCR (D), a poziomy białek za pomocą western blotting (E). β-aktyna służyła jako kontrola ładunku. **P < 0,01, ***P < 0,001. Control, komórki nieleczone; si-NC, negatywna kontrolna siRNA. Wykonano trzy niezależne powtórzenia biologiczne. Dane przedstawiono jako średnia ± SD. Kliknij tutaj, aby wyświetlić powiększoną wersję tego rysunku.

Eksport cytoplazmatyczny zmodyfikowanego m6A białka Rab27A był wspomagany przez YTHDC1

W celu zbadania potencjalnego udziału modyfikacji m6A w regulacji Rab27A przeprowadzono analizy bioinformatyczne. W obrębie transkryptu Rab27A zidentyfikowano wiele przewidywanych miejsc modyfikacji m6A (Rysunek 4A). Ponadto analiza przewidywania interakcji zasugerowała potencjalne powiązania między Rab27A a białkami czytającymi m6A: YTHDC1 i IGF2BP2 (Rysunek 4B).

Aby potwierdzić interakcję między YTHDC1 a mRNA Rab27A, przeprowadzono testy immunoprecypitacji RNA (RIP). mRNA Rab27A było znacząco wzbogacone w kompleksach immunoprecypitowanych przeciwciałem anty-YTHDC1 w porównaniu z kontrolą immunoglobuliną G (IgG) (Rycina 4C).

Ponieważ YTHDC1 pełni funkcję jądrowego czytnika m6A uczestniczącego w eksporcie RNA41, przeprowadzono analizy frakcjonowania jądrowego i cytoplazmatycznego. Wyciszenie ekspresji YTHDC1 znacząco zwiększyło retencję jądrową mRNA Rab27A, jednocześnie zmniejszając jego zawartość w cytoplazmie (Rysunek 4D).

Dalszej ocenie poddano wpływ YTHDC1 na ekspresję Rab27A. Analiza ilościowej RT-PCR wykazała, że usunięcie YTHDC1 zmniejszyło ekspresję mRNA Rab27A (Rysunek 4E). Zgodnie z tym, analiza western blot wykazała obniżoną ekspresję białka Rab27A po wyciszeniu YTHDC1 (Rysunek 4F).

figure-results-4
Rycina 4: Eksport cytoplazmatyczny zmodyfikowanego m6A białka Rab27A był wspomagany przez YTHDC1. (A) Przewidywane miejsca modyfikacji m6A w Rab27A zidentyfikowane za pomocą narzędzia predykcyjnego SRAMP. (B) Przewidywane interakcje między Rab27A a białkami odczytującymi m6A: YTHDC1 i IGF2BP2, zidentyfikowane za pomocą StarBase. (C) Analiza immunoprecypitacji RNA (RIP) wykazująca interakcję między YTHDC1 a mRNA Rab27A. **P < 0.01. Jako kontrolę negatywną zastosowano IgG. Wzbogacenie Rab27A znormalizowano do procentowej wartości wkładu (input). Prążek immunoblotu dla YTHDC1 odzwierciedla wydajność immunoprecypitacji. (D) Analiza frakcjonowania jądrowego i cytoplazmatycznego wykazująca zwiększoną retencję jądrową Rab27A po wyciszeniu YTHDC1. **P < 0.01, ***P < 0.001. (E–F) Ekspresja YTHDC1 i Rab27A po wyciszeniu YTHDC1. Ekspresję mRNA określono za pomocą q-PCR (E), a ekspresję białka za pomocą western blotingu (F). β-actin służyła jako kontrola ładunku. **P < 0.01. si-NC, siRNA kontrolne negatywne. Wykonano trzy niezależne powtórzenia biologiczne. Dane przedstawiono jako średnia ± SD. Kliknij tutaj, aby wyświetlić powiększoną wersję tej ryciny.

Stabilizacja Rab27A poprzez oddziaływanie z IGF2BP2

Interakcję między IGF2BP2 a Rab27A badano za pomocą testów RIP. mRNA Rab27A było znacznie wzbogacone w kompleksach immunoprecypitowanych przeciwciałem anty-IGF2BP2 w porównaniu z kontrolą IgG, co wskazuje na powiązanie między IGF2BP2 a mRNA Rab27A (Rysunek 5A).

Analiza immunofluorescencji wykazała kooperatywną lokalizację IGF2BP2 i Rab27A głównie w cytoplazmie (Rysunek 5B). Aby określić wpływ funkcjonalny IGF2BP2 na ekspresję Rab27A, zastosowano wyciszenie IGF2BP2 za pomocą siRNA. Analiza ilościowej RT-PCR wykazała znaczną redukcję poziomów mRNA Rab27A po usunięciu IGF2BP2 (Rysunek 5C). Zgodnie z tymi wynikami, analiza western blot wykazała obniżoną ekspresję białka Rab27A po wyciszeniu IGF2BP2 (Rysunek 5D).

Następnie oceniono wpływ IGF2BP2 na stabilność mRNA Rab27A. Deplecja IGF2BP2 znacząco skróciła okres półtrwania mRNA Rab27A i zmniejszyła stabilność transkrypcyjną (Rycina 5E).

figure-results-5
Rysunek 5: Stabilizacja Rab27A poprzez oddziaływanie z IGF2BP2. (A) Analiza immunoprecypitacji RNA wykazująca asocjację między IGF2BP2 a mRNA Rab27A. Jako kontrola negatywna posłużyło IgG. Wzbogacenie Rab27A znormalizowano do procentowej wartości materiału wejściowego (input). ***P < 0.001. (B) Analiza immunofluorescencji wykazująca ko-lokalizację Rab27A i IGF2BP2 w cytoplazmie. Jądra komórkowe przeciwbarwiono DAPI. Pasek skali = 20 µm. (C–D) Spadek ekspresji Rab27A po usunięciu IGF2BP2. Poziomy mRNA Rab27A określono za pomocą q-PCR (C), a poziomy białka określono za pomocą western blossomingu (D). Jako kontrola ładunku posłużyła β-aktyna. **P < 0.01, ***P < 0.001. (E) Stabilność mRNA Rab27A po wyciszeniu IGF2BP2. Poziomy mRNA Rab27A znormalizowano do odpowiadającej grupy si-NC w czasie 0 h. **P < 0.01. si-NC, siRNA kontrolne negatywne. Przeprowadzono trzy niezależne powtórzenia biologiczne. Dane przedstawiono jako średnia ± SD. Kliknij tutaj, aby wyświetlić powiększoną wersję tego rysunku.

Wyciszenie YTHDC1 lub IGF2BP2 zahamowało różnicowanie osteoklastów z BMM indukowane przez M-CSF i RANKL

Aby dalej zbadać funkcjonalny związek między YTHDC1, IGF2BP2 a Rab27A podczas różnicowania osteoklastów, w komórkach BMM przeprowadzono eksperymenty z wyciszeniem genów.

Stymulacja M-CSF i RANKL znacząco zwiększyła ekspresję mRNA Rab27A, YTHDC1 i IGF2BP2 (Rycina 6A). Odpowiednio, ekspresja białek tych cząsteczek była również podwyższona po indukcji osteoklastów (Rycina 6B). Wyciszenie YTHDC1 lub IGF2BP2 znacząco zredukowało ekspresję Rab27A, podczas gdy knockdown jednego z regulatorów nie wpłynął w istotny sposób na ekspresję drugiego.

Barwienie na TRAP wykazało, że usunięcie YTHDC1 lub IGF2BP2 znacząco hamuje aktywność osteoklastów indukowaną przez M-CSF i RANKL (Rycina 6C). Co więcej, analiza ilościowej PCR w czasie rzeczywistym ujawniła obniżoną ekspresję TRAP, NFATc1 oraz c-FOS po wyciszeniu YTHDC1 lub IGF2BP2 (Rycina 6D). Analiza Western blot potwierdziła odpowiadające temu spadki na poziomie białka (Rycina 6E).

Wszystkie te wyniki wskazują, że YTHDC1 i IGF2BP2 promują różnicowanie osteoklastów poprzez regulację ekspresji Rab27A.

figure-results-6
Rycina 6: Wyciszenie YTHDC1 lub IGF2BP2 zahamowało różnicowanie osteoklastów z BMM indukowane przez M-CSF i RANKL. (A–B) Ekspresja Rab27A po wyciszeniu YTHDC1 lub IGF2BP2. Poziomy mRNA (A) i białka (B) Rab27A określono odpowiednio za pomocą q-PCR i western blossomingu. β-aktyna służyła jako kontrola ładowania. ***P < 0,001. (C) Barwienie TRAP wykazujące zahamowanie aktywności osteoklastów po usunięciu YTHDC1 lub IGF2BP2. Pasek skali = 50 µm. **P < 0,01, ***P < 0,001. (D–E) Ekspresja markerów różnicowania osteoklastów po usunięciu YTHDC1 lub IGF2BP2. Poziomy mRNA TRAP, NFATc1 i c-FOS określono za pomocą q-PCR (D), a poziomy białek za pomocą western blossomingu (E). β-aktyna służyła jako kontrola ładowania. *P < 0,05, **P < 0,01, ***P < 0,001. Control, komórki nieleczone; si-NC, negatywna kontrolna siRNA. Wykonano trzy niezależne powtórzenia biologiczne. Dane przedstawiono jako średnia ± SD. Kliknij tutaj, aby zobaczyć powiększoną wersję tej ryciny.

DOSTĘPNOŚĆ DANYCH:

Zbiory danych wykorzystane i/lub analizowane w niniejszym badaniu (w tym dane z western blotu, zestawy obrazowe, analizy ilościowe oraz pliki źródłowe) zostały udostępnione w Pliku uzupełniającym 1.

Plik uzupełniający 1: Zbiory danych i dane surowe z niniejszego badania. Prosimy kliknąć tutaj, aby pobrać ten plik.

Dyskusja

Osteoclast differentiation plays essential roles in bone remodeling, bone resorption, fracture repair, and skeletal homeostasis12˒15˒17˒18˒42˒43. Excessive osteoclast activity is a major contributor to osteoporosis, making the identification of regulatory pathways controlling osteoclast differentiation an important research objective. Several anti-resorptive therapies targeting osteoclast function have demonstrated clinical efficacy6,44,45. For example, denosumab has been associated with reduced fracture incidence and a favorable safety profile6, whereas the cathepsin K inhibitor ONO-5334 has shown beneficial effects on bone mineral density and bone strength in ovariectomized non-human primates44. The present study identified a previously unrecognized m6A-dependent regulatory mechanism involving YTHDC1, Rab27A, and IGF2BP2 that promotes osteoclast differentiation and the progression of osteoporosis. These findings provide new insights into the molecular regulation of osteoclastogenesis and suggest potential therapeutic targets for osteoporosis.

Rab27A has previously been implicated in osteoclast differentiation and bone resorption through regulation of vesicular trafficking and intracellular signaling and may also influence RANKL-induced signaling cascades through endosomal recycling of key receptors28,29. Consistent with these reports, Rab27A expression was markedly increased during osteoclast differentiation, whereas Rab27A depletion suppressed osteoclast differentiation in vitro and alleviated osteoporosis-associated bone loss in vivo. These findings support a critical role for Rab27A in osteoclast biology and further extend previous observations by demonstrating that Rab27A activity is regulated through m6A-dependent mechanisms. Collectively, the findings suggest that m6A-modified Rab27A may regulate these processes at the post-transcriptional level and consequently influence bone-resorptive activity.

Accumulating evidence indicates that m6A modification contributes to the development and progression of bone-related diseases32. Bioinformatic analyses identified multiple putative m6A modification sites within Rab27A mRNA (Figure 4A), and functional studies demonstrated that YTHDC1 and IGF2BP2 regulate Rab27A expression and osteoclast differentiation (Figures 4–6). These findings suggest that m6A modification is an important upstream regulatory mechanism controlling Rab27A function during osteoclastogenesis. Because multiple predicted m6A sites were identified within Rab27A, future studies should determine whether individual sites or combinations of sites are required for Rab27A regulation. Construction of m6A-deficient Rab27A mutants and subsequent rescue experiments would provide direct evidence for the functional importance of m6A modification in this pathway.

Mechanistically, the present study demonstrated that YTHDC1 interacts with Rab27A mRNA and facilitates its nuclear export. Following cytoplasmic translocation, IGF2BP2 directly associates with Rab27A mRNA and enhances transcript stability. Consequently, depletion of either YTHDC1 or IGF2BP2 reduced Rab27A expression and suppressed osteoclast differentiation. These findings support a model in which YTHDC1 and IGF2BP2 cooperate to maintain Rab27A expression by coordinating mRNA transport and stability. This regulatory mechanism expands the current understanding of m6A-mediated regulation of osteoclast differentiation and identifies multiple components of this pathway as potential therapeutic targets.

Several limitations should be acknowledged. First, although Rab27A has been shown to regulate osteoclast differentiation, its potential effects on osteoblast function have not been investigated. Because bone homeostasis depends on the balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation, future studies should evaluate whether Rab27A also influences osteoblast differentiation or activity. Second, the present study primarily relied on loss-of-function approaches. Complementary overexpression of Rab27A, YTHDC1, and IGF2BP2, together with rescue experiments using m6A-deficient Rab27A mutants, would further strengthen the proposed regulatory model. Third, although several putative m6A sites within Rab27A mRNA were predicted using bioinformatic approaches, the specific sites responsible for regulating Rab27A function remain unidentified and require experimental validation. Finally, additional studies are needed to determine whether other m6A reader proteins, RNA-binding proteins, or signaling pathways participate in Rab27A regulation during osteoclast differentiation.

Immune regulation has emerged as an important component of osteoporosis pathogenesis46,47. Rab27A has established functions in immune-cell secretion, cytotoxic granule release, neutrophil degranulation, and regulation of the tissue microenvironment48,49,50,51,52,53,54,55. Further investigation of the relationship between Rab27A, immune regulation, and bone homeostasis may provide additional insight into the pathogenesis of osteoporosis.

Future investigations should prioritize several key areas. First, systematic mutagenesis of the predicted m6A sites within Rab27A is required to identify the functional binding sites involved in YTHDC1- and IGF2BP2-mediated regulation. Second, overexpression of Rab27A, YTHDC1, and IGF2BP2, together with rescue experiments using m6A-deficient Rab27A mutants, will be essential to establish the direct contribution of the YTHDC1/Rab27A/IGF2BP2 regulatory axis. Third, validation of this pathway in additional osteoporosis models and evaluation of Rab27A function in osteoblasts will be important to further substantiate the present findings.

In conclusion, the present study identified a novel YTHDC1/Rab27A/IGF2BP2 regulatory axis that promotes osteoclast differentiation and the progression of osteoporosis. YTHDC1 facilitated the nuclear export of m6A-modified Rab27A mRNA, whereas IGF2BP2 enhanced Rab27A mRNA stability in the cytoplasm. These coordinated regulatory events increased Rab27A expression and promoted osteoclastogenesis. Collectively, the findings expand the current understanding of m6A-mediated regulation in osteoporosis and provide a foundation for developing therapeutic strategies targeting the YTHDC1/Rab27A/IGF2BP2 pathway.

Oświadczenia

The authors declare no competing financial interests or conflicts of interest.

Podziękowania

This work was financially supported by The Scientific and Technological Strategic Cooperation Project between Pengzhou People’s Hospital and Southwest Medical University (Grant No. 2023PZXNYD01) to Lian Tang.

Materiały

Lista materiałów użytych w tym artykule
NazwaFirmaNumer katalogowyKomentarze
4% ParaformaldehydeThermo Fisher ScientificA5818101Tissue and cell fixation
Alexa Fluor 488 secondary antibodyThermo Fisher ScientificA-11001Fluorescent secondary antibody for immunofluorescence; RRID: AB_2534069
Alexa Fluor 546 secondary antibodyThermo Fisher ScientificA-11035Fluorescent secondary antibody for immunofluorescence; RRID: AB_2534093
Bovine serum albumin (BSA)MerckA2153Blocking reagent
Bradford Protein AssayMerckB6916Protein quantification
C57BL/6JSlac miceShanghai SLAC Laboratory Animal Co., Ltd.—Female, 8 weeks old; RRID: MGI:2159769
c-FOS antibodyAbcamab222699Primary antibody for western blotting; RRID: AB_2891049
Confocal laser scanning microscope (SP8)Leica MicrosystemsSP8Immunofluorescence image acquisition; RRID: SCR_018169
DAPIThermo Fisher ScientificD3571Nuclear counterstain for immunofluorescence; RRID: AB_2307445
EDTAMerckV900081-500GTissue decalcification reagent
EosinSolarbioG1100Histological cytoplasmic stain
Fetal bovine serum (FBS)Thermo Fisher ScientificA5669701Cell culture supplement
GraphPad PrismGraphPad SoftwareVersion 9.0Statistical analysis software; RRID: SCR_002798
HematoxylinCell Signaling Technology#14166Histological nuclear stain
IBM SPSS StatisticsIBMWindows versionStatistical analysis software; RRID: SCR_016479
IGF2BP2 antibodyProteintech11601-1-APPrimary antibody for western blotting and immunofluorescence; RRID: AB_2122672
Inverted light microscope (DMi8)Leica MicrosystemsDMi8Histological image acquisition; RRID: SCR_026672
Lentiviral titer quantification kitTakara Bio631235Lentiviral titer determination
Lipofectamine 3000Thermo Fisher ScientificL3000015Plasmid transfection reagent
Lipofectamine RNAiMAXThermo Fisher Scientific13778150siRNA transfection reagent
M-CSFMedChemExpressHY-P7085Osteoclast differentiation factor
Micro-computed tomography systemScanco MedicalVivaCT80Bone morphometric analysis
M-MLV Reverse TranscriptasePromegaM1701cDNA synthesis
NFATc1 antibodySanta Cruz Biotechnologysc-7294Primary antibody for western blotting; RRID: AB_2152503
PARIS KitThermo Fisher ScientificAM1556Nuclear and cytoplasmic fractionation
Penicillin/StreptomycinThermo Fisher Scientific15140122Antibiotic supplement for cell culture
Power SYBR Green Master MixThermo Fisher Scientific4367659Quantitative real-time PCR
Protease inhibitor cocktailThermo Fisher Scientific78429Prevention of protein degradation
Protein A/G agarose beadsMerckIP05Immunoprecipitation
PVDF membraneThermo Fisher Scientific88520Protein transfer membrane for western blotting
Rab27A antibodyAbcamab55667Primary antibody for western blotting and immunofluorescence; RRID: AB_945112
RANKLMedChemExpressHY-P7425Osteoclast differentiation factor
Real-time PCR systemApplied BiosystemsABI7900HTQuantitative real-time PCR instrument
RIPA bufferThermo Fisher Scientific78501Protein extraction buffer
RNA Immunoprecipitation (RIP) KitMilliporeRIP-12RXNRNA immunoprecipitation assay
SDS-PAGE reagentsThermo Fisher ScientificBT041210BOXProtein electrophoresis
shRNA plasmidsRiboBio—Gene silencing by RNA interference
Small interfering RNAs (siRNAs)GenePharma—Gene silencing by RNA interference
SRAMPOnline resource—Prediction of m6A modification sites; RRID: SCR_024500
StarBase (ENCORI)Online resource—RNA interaction prediction database; RRID: SCR_016303
TRAP antibodyAbcamab2391Primary antibody for western blotting; RRID: AB_303034
TRAP staining kitMerck387ADetection of osteoclast activity
Triton X-100Thermo Fisher Scientific85111Cell permeabilization reagent
TRIzol ReagentThermo Fisher Scientific15596026CNTotal RNA isolation
YTHDC1 antibodyAbcamab122340Primary antibody for western blotting; RRID: AB_11128253
α-MEMFUJIFILM Wako Pure Chemical Corporation139-15651Cell culture medium
β-Actin antibodyMerckA5441Loading control for western blotting; RRID: AB_476744

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