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LEP O-GlcNAcylation inactivates NF-κB pathway by suppressing LEP protein level and thus mediates cellular senescence and osteogenic differentiation in mouse mesenchymal stem cells

BMC Molecular and Cell Biology volume 25, Article number: 26 (2024) Cite this article

Abstract

Background

Cellular senescence is a key driver of decreased bone formation and osteoporosis. Leptin (LEP) has been implicated in cellular senescence and osteogenic differentiation. The aim of this study was to investigate the mechanisms by which LEP mediates cellular senescence and osteogenic differentiation.

Methods

C3H10T1/2 cells were treated with etoposide to induce cellular senescence, which was assessed by β-galactosidase staining. Quantitative real-time PCR and western blotting were used to measure the levels of senescence markers p21 and p16, as well as osteogenic differentiation-related genes ALP, COL1A1, and RUNX2. Alkaline phosphatase (ALP) staining and alizarin red S staining were performed to evaluate osteogenic differentiation. The NF-κB pathway and O-GlcNAcylation were assessed by western blotting.

Results

Etoposide treatment increased the number of senescent cells and the levels of p21 and p16, along with elevated LEP expression. These effects were reversed by LEP knockdown. Additionally, LEP knockdown increased ALP staining density and osteoblast mineralization nodules, as well as the mRNA and protein levels of ALP, COL1A1, and RUNX2, indicating that LEP knockdown promoted osteogenic differentiation in C3H10T1/2 cells. Mechanistically, LEP knockdown inactivated the NF-κB pathway by inhibiting the nuclear translocation of p65. Furthermore, OGT was found to promote O-GlcNAcylation of LEP at the S50 site.

Conclusion

Our findings demonstrated that O-GlcNAcylation of LEP inactivated the NF-κB pathway by reducing LEP protein levels, thereby inhibiting cellular senescence and promoting osteogenic differentiation in C3H10T1/2 cells. This study may provide a novel therapeutic target for the treatment of osteoporosis.

Peer Review reports

Introduction

The global trend towards population aging has led to an increased incidence of age-related diseases. Cellular senescence, an intrinsic aging mechanism, plays a pivotal role in various age-related conditions, including osteoporosis, atherosclerosis, diabetes, cataracts, and osteoarthritis [1,2,3]. Numerous studies have explored the mechanisms by which cellular senescence contributes to osteoporosis. For instance, a prior study confirmed that bone loss in aging mice results from increased osteoblastic bone formation coupled with a reduction in bone marrow adipose tissue, leading to a shift in bone marrow mesenchymal stem cell (BMSC) differentiation from osteoblasts to adipocytes [4]. Other research has suggested that an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption underlies age-related bone loss [5,6,7]. Furthermore, the removal of senescent osteoblasts has been shown to improve bone microarchitecture and strength [4]. Consequently, targeting cellular senescence represents a promising strategy for mitigating bone loss in osteoporosis.

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells with the potential to differentiate into multiple cell types, including chondrocytes, myocytes, osteoblasts, and adipocytes [8,9,10,11]. Osteoporosis arises from a decline in the osteogenic differentiation capacity of these cells. Therefore, enhancing osteogenic differentiation while inhibiting adipogenic differentiation of MSCs is crucial for treating osteoporosis [12,13,14]. However, senescent MSCs exhibit reduced osteogenic differentiation and enhanced adipogenic differentiation, leading to diminished bone formation and limiting their regenerative potential [15,16,17]. These findings underscore the importance of MSC differentiation potential in bone formation, suggesting that investigating MSC senescence and osteogenic differentiation may provide an effective therapeutic target for alleviating osteoporosis.+.

Leptin (LEP) is a circulating adipokine produced by adipocytes that plays a critical role in the regulation of energy balance and body weight [18,19,20]. Additionally, LEP is involved in numerous physiological activities and disease processes, including diabetes, chronic kidney disease, tumor development, and apoptosis [21,22,23,24]. Several studies have elucidated the mechanisms by which LEP mediates cellular senescence. For example, high doses of leptin have been shown to induce cell cycle arrest and senescence in chondrogenic progenitor cells (CPCs) via the activation of the p53/p21 pathway [25]. Furthermore, LEP has been reported to enhance the osteogenic potential of CPCs [25]. It has also been proposed that LEP serves as a biomarker for aging and age-related diseases [26]. LEP is a regulator of osteogenic differentiation; targeting LEP promotes the osteogenic differentiation of BMSCs [27, 28]. Additionally, the regulation of LEP has been shown to promote osteogenic differentiation while inhibiting adipocyte formation in high-fat-diet-induced models [29]. These findings suggested that LEP plays a role in cellular senescence and osteogenic differentiation, although the specific mechanisms underlying LEP-mediated osteoporosis require further investigation.

In this study, we aimed to investigate the effects and underlying mechanisms of LEP on cellular senescence and osteogenic differentiation in MSCs, which may provide a new therapeutic target for the treatment of osteoporosis.

Methods

Cell culture and treatment

Mouse mesenchymal stem cells (MSCs), specifically C3H10T1/2 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 100 µg/mL penicillin, 100 U/mL streptomycin, and 10% fetal bovine serum (FBS, Gibco) in a humidified incubator at 37 °C with 5% CO2. To establish cellular senescence models, cells were treated with 20 µM etoposide for 24 h. For inducing osteogenic differentiation in C3H10T1/2 cells, they were cultured in osteogenic induction medium containing DMEM, 10% FBS, 10 mM β-glycerophosphate, 50 µg/L L-ascorbic acid, and 100 nM dexamethasone for 14 days.

Cell transfection

Short hairpin RNA (shRNA) targeting leptin (shLEP), shRNA negative control (shNC), empty vector (pcDNA3.1), OGT-overexpressing plasmids (pcDNA3.1-OGT), and OGA-overexpressing plasmids (pcDNA3.1-OGA) were obtained from Genechem (Shanghai, China). C3H10T1/2 cells, either treated with etoposide or untreated, were seeded into 6-well plates the day before transfection to achieve approximately 70% confluence. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, the transfection was complete, and the cells were harvested for further analysis.

Bioinformatics analysis

The GSE35956 dataset was obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). Differentially expressed genes (DEGs) were identified based on criteria of P < 0.05 and |log(fold change)| > 2. Data analysis was conducted using GEO2R, and visualization was performed using the Sangerbox platform (http://vip.sangerbox.com/). The STRING database (https://cn.string-db.org/) was utilized to construct a protein-protein interaction (PPI) network of the DEGs relevant to osteoporosis. DEGs were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify enriched pathways. An enrichment threshold of P < 0.05 was applied. O-GlcNAcylation sites in LEP were predicted using the DictyOGlyc-1.1 database (https://services.healthtech.dtu.dk/services/DictyOGlyc-1.1/).

β-galactosidase staining

Cellular senescence was detected using a senescence β-galactosidase staining kit (Beyotime, Beijing, China). Cells were washed once with phosphate-buffered saline (PBS, Gibco), fixed with β-galactosidase fixation solution for 15 min at room temperature, and then washed three times with PBS (3 min per wash). Subsequently, cells were stained with the staining solution overnight at 37 °C. Senescent cells were visualized and quantified using a light microscope.

Quantitative real-time PCR (qPCR)

Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and quantified using a NanoDrop spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). RNA was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Tokyo, Japan) following the manufacturer’s protocol. The cDNA was diluted tenfold, and qPCR was performed using SYBR Green Master Mix (Yeasen, Shanghai, China). The reaction mixture consisted of 10 µL SYBR Green Master Mix, 9.2 µL diluted cDNA, and 0.8 µL of each primer. Amplification was carried out on a QuantStudio 6 (ThermoFisher Scientific). Relative expression was calculated using the 2−ΔΔCt method and GAPDH was used as a endogenous reference. The qPCR primers for LEP were as follows: forward, 5’-GTGGCTTTGGTCCTATCTGTC-3’; reverse, 5’-CGTGTGTGAAATGTCATTGATCC-3’.

Nuclear and cytoplasmic protein isolation

Nuclear and cytoplasmic proteins were extracted from C3H10T1/2 cells using a nuclear and cytoplasmic protein extraction kit (Beyotime). Cells were washed with PBS, and the supernatant was removed by centrifugation to collect the cell pellet. Cytoplasmic protein extraction reagent A was added to the cell pellet, and the mixture was vortexed vigorously for 5 s, followed by incubation on ice for 10 min. Cytoplasmic protein extraction reagent B was then added, and the mixture was vortexed vigorously for 5 s and incubated on ice for 1 min. The mixture was centrifuged at 12,000 × g for 5 min at 4 °C. The supernatant, containing the cytoplasmic protein, was transferred to a pre-chilled tube. Nuclear protein extraction reagent was added to the remaining pellet, which was vortexed vigorously for 30 s and incubated on ice for 30 min with intermittent vortexing every 2 min for 30 s. The mixture was then centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant, containing the nuclear protein, was collected.

Western blot

Total protein was extracted using RIPA lysis buffer (Beyotime) and quantified using a BCA protein assay kit (Beyotime). Equal amounts of protein were loaded onto 10% SDS-PAGE gels and subsequently transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in TBS-T for 1 h at room temperature. After blocking, the membranes were washed and incubated with primary antibodies overnight at 4 °C. The following day, the membranes were washed again and incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000, ab205718, Abcam, Cambridge, UK) for 2 h at room temperature. Protein bands were visualized using an ECL chemiluminescence reagent (Yeasen) and quantified using ImageJ software. The primary antibodies used in this study were as follows: anti-p21 (1/1000, ab109520, Abcam), anti-p16 (1/1000, #80772, CellSignaling, Danvers, MA, USA), anti-ALP (1/1000, ab307726, Abcam), anti-COL1A1 (1/1000, ab34710, Abcam), anti-RUNX2 (1/1000, ab236639, Abcam), anti-p65 (1/1000, ab32536, Abcam), anti-p-p65 (1/1000, ab76302, Abcam), anti-OGT (1/1000, ab96718), anti-OGA (1/5000, ab124807), anti-LEP (1/1000, ab16227, Abcam), anti-GAPDH (1/10000, ab181602, Abcam), anti-Lamin B (1/10000, ab133741, Abcam) and anti-O-GlcNAc (1: 1000, MA1-072, Thermo Scientific, Waltham, MA, USA).

Identification of osteogenic differentiation

To identify osteogenic differentiation of C3H10T1/2 cells after 14 days of osteogenic induction, alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining were performed. ALP staining was conducted using a BCIP/NBT ALP Color Development Kit (Beyotime) following the manufacturer’s protocol. Briefly, cells were washed three times with PBS and stained with BCIP/NBT stain solution for 30 min in the dark. For ARS staining, an ARS staining kit (Beyotime) was used according to the manufacturer’s instructions. Cells were washed with PBS, fixed with the provided fixative for 20 min, and then stained with ARS solution for 30 min at room temperature. Images were captured using a microscope.

Protein stability assay

To assess the stability of LEP protein, C3H10T1/2 cells transfected with pcDNA3.1 or pcDNA3.1-OGT were treated with 10 µM cycloheximide (MKBio, Shanghai, China). The stability of LEP protein was evaluated by Western blot analysis at baseline and 8, 16, and 24 h post-treatment.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 7.0. Data are presented as the mean ± standard deviation. Each experimental group included at least three replicates. Comparisons between two groups were made using Student’s t-test, while comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05.

Results

LEP expression is upregulated in elderly patients with osteoporosis

Osteoporosis is a chronic condition with a high prevalence among older individuals. To further explore the mechanisms underlying osteoporosis, we performed microarray analysis to identify DEGs in human MSCs derived from elderly patients with osteoporosis compared to middle-aged non-osteoporotic donors. Our results indicated that the expression of LEP was significantly upregulated in elderly patients with osteoporosis (Fig. 1A, B). Protein-protein interaction (PPI) network analysis revealed several proteins that interact with LEP (Fig. 1C). These findings suggested that LEP was upregulated in elderly patients with osteoporosis, indicating its potential as a therapeutic target for osteoporosis.

Fig. 1
figure 1

LEP expression was upregulated in elderly patients with osteoporosis (A and B) Differentially expressed genes in elderly patients with osteoporosis and middle-aged non-osteoporotic donors were analyzed by microarray analysis. (C) PPI network analysis of differentially expressed genes

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Etoposide increases LEP expression and promotes cellular senescence in C3H10T1/2 cells

To investigate the effects of cellular senescence, C3H10T1/2 cells were treated with etoposide to induce a senescence model. Our results showed that the expression of LEP was significantly elevated in etoposide-treated C3H10T1/2 cells (Fig. 2A). Furthermore, etoposide treatment increased the number of senescent cells (Fig. 2B, C). We also measured the expression of cellular senescence markers in C3H10T1/2 cells and confirmed that the expression of p21 and p16 was significantly upregulated by etoposide (Fig. 2D, E). Western blot analysis corroborated these findings, showing increased protein levels of p21 and p16 in etoposide-induced C3H10T1/2 cells (Fig. 2F). Collectively, these data demonstrated that etoposide increased LEP expression and promoted cellular senescence in C3H10T1/2 cells.

Fig. 2
figure 2

Etoposide increased LEP expression and promotes cellular senescence in C3H10T1/2 cells (A) The expression of LEP was measured using qPCR. (B and C) Cellular senescence was assessed by β-galactosidase staining. (D and E) qPCR was performed to measure the expression of aging markers p21 and p16. (F) The protein levels of p21 and p16 were evaluated by western blot

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LEP knockdown inhibits etoposide-induced cellular senescence in C3H10T1/2 cells

To further elucidate the role of LEP, C3H10T1/2 cells were transfected with shLEP, resulting in a significant decrease in LEP expression (Fig. 3A). Subsequently, the number of senescent cells induced by etoposide was notably reduced upon LEP knockdown (Fig. 3B, C). Additionally, LEP knockdown inhibited the etoposide-induced increase in the expression of p21 and p16 (Fig. 3D, E). Consistent with these observations, Western blot analysis revealed that the protein levels of p21 and p16, which were upregulated by etoposide, were restored to lower levels by LEP knockdown (Fig. 3F). Collectively, these results indicated that LEP knockdown inhibited etoposide-induced cellular senescence in C3H10T1/2 cells.

Fig. 3
figure 3

LEP knockdown inhibited etoposide-induced cellular senescence in C3H10T1/2 cells (A) The expression of LEP was measured using qPCR. (B and C) Cellular senescence was assessed by β-galactosidase staining. (D and E) qPCR was performed to measure the expression of aging markers p21 and p16. (F) The protein levels of p21 and p16 were evaluated by western blot

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LEP knockdown promotes osteogenic differentiation in C3H10T1/2 cells

To further explore the relationship between LEP and osteogenic differentiation, C3H10T1/2 cells were transfected with shLEP. Compared to the control group, LEP knockdown significantly increased the ALP staining density in C3H10T1/2 cells (Fig. 4A, B). ARS staining further confirmed that LEP knockdown enhanced the formation of osteoblastic mineralization nodules in C3H10T1/2 cells (Fig. 4C, D). Moreover, the expression levels of osteogenic differentiation-related genes, including ALP, COL1A1, and RUNX2, were markedly elevated by LEP knockdown (Fig. 4E). Western blot analysis corroborated these findings, demonstrating that the protein levels of ALP, COL1A1, and RUNX2 were also upregulated upon LEP knockdown (Fig. 4F). Moreover, LEP knockdown promoted osteogenic differentiation in vivo (Figure S5A-H). Conversely, we found that LEP overexpression inhibited osteogenic differentiation in C3H10T1/2 cells (Figure S1A-G). In summary, these results demonstrated that LEP knockdown promoted osteogenic differentiation in C3H10T1/2 cells.

Fig. 4
figure 4

LEP knockdown promoted osteogenic differentiation in C3H10T1/2 cells (A and B) An ALP staining kit was applied to evaluate the ALP staining density. (C and D) ARS staining was performed to assess the osteoblast mineralization nodules of C3H10T1/2 cells. (E) The expression of osteogenic differentiation-related genes ALP, COL1A1 and RUNX2 was measured by qPCR. (F) The protein levels of ALP, COL1A1 and RUNX2 were evaluated by western blot

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LEP promotes osteoporosis through the activation of NF-κB signaling pathway

To identify DEGs affected by LEP knockdown in C3H10T1/2 cells, we performed transcriptomic analysis. The top ten upregulated and downregulated genes are displayed in a heatmap (Fig. 5A). KEGG pathway enrichment analysis of these DEGs revealed significant enrichment in the NF-κB signaling pathway (Fig. 5B). Given that the NF-κB pathway is a key regulatory pathway in osteoporosis [30], we further investigated whether LEP modulates this pathway in C3H10T1/2 cells. Our results showed that LEP knockdown inhibited the phosphorylation of p65 (Fig. 5C). Additionally, LEP knockdown suppressed the nuclear translocation of p65 (Fig. 5D). These findings suggested that LEP knockdown inactivated the NF-κB pathway in C3H10T1/2 cells. Moreover, activation of NF-κB pathway in C3H10T1/2 cells inhibited osteogenic differentiation increased by LEP knockdown (Figure S2A-F). In conclusion, our data confirmed that LEP promoted osteoporosis through the activation of the NF-κB signaling pathway.

Fig. 5
figure 5

LEP promoted osteoporosis through the activation of NF-κB signaling pathway (A) The DEGs in C3H10T1/2 cells with or without LEP knockdown were shown in a heatmap. (B) Bubble diagram of KEGG pathway enrichment analysis. (C) The protein levels of p65 were assessed by western blot. (D) Western blot was performed to measure the protein levels of p65 in cytoplasm and nucleus

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OGT promotes O-GlcNAcylation of LEP at S50 site

Previous studies have indicated that O-GlcNAcylation plays a role in osteogenic differentiation [31]. To investigate whether O-GlcNAcylation is involved in LEP-mediated osteogenic differentiation in C3H10T1/2 cells, we first assessed the O-GlcNAcylation level in these cells. Western blot analysis revealed that etoposide treatment inhibited O-GlcNAcylation in C3H10T1/2 cells (Fig. 6A). Since O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), we measured the protein levels of both enzymes. Etoposide treatment specifically downregulated OGT levels without affecting OGA (Fig. 6A). We then overexpressed OGT to examine its impact on LEP. Overexpression of OGT reduced the protein level of LEP but increased its O-GlcNAcylation (Fig. 6B). Conversely, overexpression of OGA did not affect the protein level or O-GlcNAcylation status of LEP (Fig. 6C). To identify specific O-GlcNAcylation sites on LEP, we predicted several potential sites (Fig. 6D). The top three predicted sites were illustrated in Fig. 6E. To validate which site was indeed modified by O-GlcNAcylation, we mutated these sites to alanine (A) using site-directed mutagenesis. Western blot analysis showed that mutating S50 to alanine (S50A) increased the protein level of LEP and decreased its O-GlcNAcylation, whereas mutations at S91 and S164 did not alter the protein level or O-GlcNAcylation status of LEP (Fig. 6F). Finally, we found that OGT overexpression accelerated the degradation of LEP and decreased its protein stability (Fig. 6G). Collectively, these results indicated that OGT promoted O-GlcNAcylation of LEP at the S50 site. Moreover, OGT overexpression inhibited etoposide-induced cellular senescence and osteogenic differentiation in C3H10T1/2 cells (Figure S3 A-F and S4 A-F).

Fig. 6
figure 6

OGT promoted O-GlcNAcylation of LEP at S50 site. (A) The O-GlcNAcylation level and the protein levels of OGT and OGA were measured by western blot. (B and C) The protein levels of OGT, OGA and LEP and the O-GlcNAcylation levels of LEP in C3H10T1/2 cells with OGT or OGA overexpression were measured by western blot. (D and E) The potential O-GlcNAcylation sites in LEP was predicted using DictyOGlyc-1.1 database. (F) After mutating the potential O-GlcNAcylation sites in LEP, the protein and O-GlcNAcylation levels of LEP was measured by western blot. (G) After C3H10T1/2 cells were treated with 10 µM cycloheximide for 0, 8, 16, and 24 h, the stability of LEP protein was measured by western blot

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Discussion

Osteoporosis is a chronic metabolic bone disorder predominantly affecting the elderly population. Cellular senescence is a key driver of age-related osteoporosis, and inhibiting cellular senescence has been shown to be an effective strategy for improving osteoporotic conditions. Previous research has highlighted the protective effects of Sirt3 overexpression against bone marrow stromal cell (BMSC) senescence, thereby ameliorating senile osteoporosis [32]. Similarly, Liu et al. [33] demonstrated that reducing bone loss through the inhibition of BMSC senescence can improve osteoporosis in mice.

Studies have also reported a decline in osteogenic differentiation potential in MSCs derived from older individuals [34]. Additionally, Zhang et al. [35] observed that aged BMSCs preferentially differentiate into adipocytes rather than osteoblasts, indicating an imbalance between bone formation and resorption in senescent MSCs. This imbalance is attributed to reduced osteogenic differentiation potential, and enhancing the osteogenic differentiation capacity of senescent cells can aid in restoring bone formation. For instance, Liu et al. [36] found that UBE2E3 positively correlates with osteogenesis-related genes and that its overexpression attenuates cellular senescence in BMSCs while promoting osteogenic differentiation. Chen et al. [37] also demonstrated that overexpression of YBX1 enhances bone formation and reduces fat accumulation in aged mice. Collectively, these findings underscore the interplay between cellular senescence and osteogenic differentiation, highlighting their critical roles in bone metabolism. In our study, we observed that LEP was highly expressed in etoposide-induced senescent cells, and knockdown of LEP inhibited cellular senescence while promoting osteogenic differentiation in senescent MSCs. LEP, known as a regulator of lipid metabolism, has been implicated in various diseases such as diabetes and cardiovascular disorders [38, 39]. Recent studies have begun to elucidate the role of LEP in bone metabolism, suggesting a multifaceted involvement in skeletal health. For instance, Lindenmaier et al. [40] demonstrated that LEP treatment reduces marrow adipose tissue and enhances bone formation in leptin-deficient mice. Mei et al. [27] confirmed that miR-874-3p promotes the proliferation and differentiation of human BMSCs by downregulating LEP expression, thereby inhibiting osteoporosis. Additionally, Zhao et al. [25] suggested that LEP-mediated cellular senescence acts as a modulator of obesity-related osteoarthritis. These findings collectively suggested that LEP may serve as a regulator of osteogenic differentiation and osteoporosis. Compared to these previous studies, our results confirmed that LEP knockdown inhibited cellular senescence while promoting osteogenic differentiation, indicating that LEP played a regulatory role in the development of osteoporosis.

Furthermore, we demonstrated that LEP knockdown inactivated the NF-κB signaling pathway and inhibitd the nuclear translocation of p65. NF-κB is a key mediator of inflammatory responses and plays a crucial role in immune regulation, being involved in numerous diseases [41]. Multiple studies have highlighted the role of the NF-κB pathway in osteoporosis. For example, Li et al. [42] found that urolithin B attenuates bone loss in ovariectomized (OVX) mice by inhibiting osteoclast formation and activation via down-regulation of the ERK/NF-κB signaling pathway. Sun et al. [30] revealed that inhibition of NF-κB signaling attenuates osteoclastogenesis and bone loss in OVX-induced mice. Moreover, certain drugs improve osteoporosis by inhibiting osteoclast differentiation and bone resorption through the suppression of the NF-κB signaling pathway [43, 44]. Additionally, NF-κB is considered a therapeutic target for aging-related osteoporosis, and its inhibition mitigates cellular senescence and rescues osteoporosis in premature aging mice [45]. Hu et al. [17] confirmed that NAP1L2 inhibits etoposide-induced senescence in MSCs and improves impaired osteogenesis. Collectively, these findings demonstrate that NF-κB is a potential regulator of aging-related osteoporosis. In this study, we confirmed that LEP knockdown inactivated the NF-κB pathway, revealing for the first time the mechanism by which LEP regulated osteoporosis through the NF-κB pathway.

Finally, we confirmed that OGT mediated O-GlcNAcylation in C3H10T1/2 cells, and that OGT promotes LEP O-GlcNAcylation at the S50 site, leading to a reduction in the protein level of LEP. O-GlcNAcylation is a post-translational modification whose role in cancer, cardiovascular diseases, and immune system disorders has been extensively studied [46]. This modification is regulated by two enzymes: OGT, which adds O-GlcNAc to proteins, and OGA, which removes O-GlcNAc [47]. Recent studies have highlighted the functions of O-GlcNAcylation in osteogenic differentiation. Nagel and Ball [31] initially found that suppression of OGA enhances ALP activity in BMSCs. Zhang et al. [48] also revealed that O-GlcNAcylation promotes osteogenic differentiation of BMSCs by modifying and activating RUNX2. Consistent with these findings, our study demonstrated that increased O-GlcNAcylation of LEP promoted osteogenic differentiation in mouse mesenchymal stem cells. However, contrasting results have been reported in periodontal ligament stem cells (PDLCs), where suppression of OGT or O-GlcNAcylation levels promoted osteogenic differentiation [49, 50]. We hypothesize that these differences may be due to distinct regulatory mechanisms of O-GlcNAcylation on osteogenic differentiation in mesenchymal stem cells versus periodontal ligament stem cells, warranting further investigation.

In conclusion, our study demonstrates that O-GlcNAcylation of LEP at the S50 site inactivates the NF-κB pathway by suppressing LEP protein levels, thereby inhibiting cellular senescence and promoting osteogenic differentiation in C3H10T1/2 cells. This research provides a novel theoretical foundation for understanding the mechanisms underlying osteoporosis.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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  1. Macau University of Science and Technology, Faculty of Chinese Medicine, E205, Avenida Wai Long, Taipa, Macau, 999078, China

    Zhuang Zhang & Lili Yu

  2. The 2nd People’s Hospital of Zhuhai, Zhuhai, China

    Zhuang Zhang

  3. Department of Traumatology, The 2nd People’s Hospital of Zhuhai, No.208 Yuehua Road, Zhuhai, Guangdong, 519020, China

    Chaoqing Zhou

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All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript. Z Z drafted the work and revised it critically for important intellectual content and was responsible for the acquisition, analysis and interpretation of data for the work; C Z and L Y made substantial contributions to the conception or design of the work. All authors read and approved the final manuscript.

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Supplementary Material 1

Supplementary Material 2

12860_2024_523_MOESM3_ESM.jpg

Supplementary Material 3: Figure S1 LEP overexpression inhibited osteogenic differentiation in C3H10T1/2 cells. (A) LEP expression in C3H10T1/2 cells was measured by qPCR. (B and C) An ALP staining kit was applied to evaluate the ALP staining density. (D and E) ARS staining was performed to assess the osteoblast mineralization nodules of C3H10T1/2 cells. (F) The expression of osteogenic differentiation-related genes ALP, COL1A1 and RUNX2 was measured by qPCR. (G) The protein levels of ALP, COL1A1 and RUNX2 were evaluated by western blot.

12860_2024_523_MOESM4_ESM.jpg

Supplementary Material 4: Figure S2 Activation of NF-?B pathway inhibited osteogenic differentiation in C3H10T1/2 cells enhanced by LEP knockdown. (A and B) An ALP staining kit was applied to evaluate the ALP staining density. (C and D) ARS staining was performed to assess the osteoblast mineralization nodules of C3H10T1/2 cells. (E) The expression of osteogenic differentiation-related genes ALP, COL1A1 and RUNX2 was measured by qPCR. (F) The protein levels of ALP, COL1A1 and RUNX2 were evaluated by western blot.

12860_2024_523_MOESM5_ESM.jpg

Supplementary Material 5: Figure S3 OGT overexpression inhibited etoposide-induced cellular senescence in C3H10T1/2 cells.(A) The expression of OGT was measured using qPCR. (B and C) Cellular senescence was assessed by ?-galactosidase staining. (D and E) qPCR was performed to measure the expression of aging markers p21 and p16. (F) The protein levels of p21 and p16 were evaluated by western blot.

12860_2024_523_MOESM6_ESM.jpg

Supplementary Material 6: Figure S4 OGT overexpression promoted osteogenic differentiation in C3H10T1/2 cells. (A and B) An ALP staining kit was applied to evaluate the ALP staining density. (C and D) ARS staining was performed to assess the osteoblast mineralization nodules of C3H10T1/2 cells. (E) The expression of osteogenic differentiation-related genes ALP, COL1A1 and RUNX2 was measured by qPCR. (F) The protein levels of ALP, COL1A1 and RUNX2 were evaluated by western blot.

12860_2024_523_MOESM7_ESM.jpg

Supplementary Material 7: Figure S5 LEP knockdown inhibited osteoporosis in vivo of mice. (A) Representative images from micro-CT analysis of 3D image reconstruction of the femoral midshaft corticoid bone of 6-month-old and 20-month-old mice transfected with shNC or shLEP. (B) The vBMD of trabecular quantified by micro-CT. (C) Trabecular BV/TV, (D) Tb.Th, (E) Tb.Sp were measured by micro-CT to analyze trabecular bone. (F) The vBMD of cortical was quantified by micro-CT. (G) cortical BV/TV and (H) Ct. Th of corticoid bone was analyzed by micro-CT.

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Zhang, Z., Zhou, C. & Yu, L. LEP O-GlcNAcylation inactivates NF-κB pathway by suppressing LEP protein level and thus mediates cellular senescence and osteogenic differentiation in mouse mesenchymal stem cells. BMC Mol and Cell Biol 25, 26 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12860-024-00523-7

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BMC Molecular and Cell Biology

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