DNMT aberration-incurred GPX4 suppression prompts osteoblast ferroptosis and osteoporosis

doi:10.21203/rs.3.rs-4301039/v1

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DNMT aberration-incurred GPX4 suppression prompts osteoblast ferroptosis and osteoporosis

https://doi.org/10.21203/rs.3.rs-4301039/v1

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published 02 Dec, 2024

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Osteoporosis (OP) is a common and fracture-prone skeletal disease featured by deteriorated trabecular microstructure and pathologically involves various forms of regulated bone cell death. However, the role and regulatory mechanisms of ferroptosis in OP are not fully understood. Our study showed marked iron deposition, ferroptosis, and a core anti-ferroptotic factor GPX4 (glutathione peroxidase 4) suppression in OP femurs of ovariectomized (Ovx) mice, coinciding with Gpx4 promoter hypermethylation and elevated DNMT1/3a/3b levels. In addition, KLF5, along with the transcriptional corepressors NCoR and SnoN, induces binding to the hypermethylated GPX4 promoter in osteoporotic femurs sensitive to DNMT inhibition. Conversely, DNMT inhibition with SGI-1027 reversed hypermethylation and GPX4 suppression, reducing the ferroptotic and osteoporotic damage. In cultured primary bone cells, ferric ammonium citrate (FAC) mimicking iron loading similarly induced GPX4 suppression and ferroptosis in osteoblasts, but not in osteoclasts, which were rescued by siRNA-mediated individual knockdown of DNMT 1/3a/3b respectively. Intriguingly, SGI-1027 relieved the ferroptotic alterations induced by FAC, but not by a GPX4 inactivator RSL3. More importantly, we generated a strain of osteoblast-specific Gpx4 haplo-deficient mice (Gpx4+/-) that developed spontaneous ferroptotic OP alterations and further demonstrated that GPX4 inactivation by RSL3 or osteoblastic GPX4 haplo-deficiency largely abrogated the anti-ferroptotic and osteoprotective effects of SGI-1027. Together, our data suggest that the DNMT aberration-incurred epigenetic GPX4 suppression and the resultant osteoblastic ferroptosis contribute significantly to OP pathogenesis and the strategies preserving GPX4 by DNMT intervention is potentially effective to treat OP and the related bone disorders.

Biological sciences/Physiology/Bone

Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis

Health sciences/Pathogenesis

DNA methylation

Epigenetics

Ferroptosis

GPX4

Osteoporosis

Osteoporosis (OP) is a common and silent skeletal disease with a high risk of fragility fracture that affects approximately 18.3% of populations globally, especially in postmenopausal women (11.6 % of men and 23.1% of women) ¹^,². OP pathogenesis is characterized by reduced bone mass and deteriorated trabecular microstructure attributed directly to imbalanced osteoclastogenesis and osteoblastogenesis ³. Under normal condition, bone undergoes constant turnover mainly controlled by osteoblasts and osteoclasts of opposite functions. Osteoclasts secret hydrochloric acid and proteolytic enzymes that dissolve organic collagen, inorganic calcium and phosphoruse, resulting in bone resorption ⁴^,⁵. In contrast, osteoblasts produce various growth factors, hormones and collagens to ensure proper bone biosynthesis ⁶^,⁷. OP occurs as a result of increased osteoclastogenesis or decreased osteoblastogenesis, or both ⁸^,⁹. Although past researches have established that various forms of regulated cell death, such as apoptosis ¹⁰, autophagic cell death ¹¹, pyroptosis ¹², necroptosis ¹³.and ferroptosis ¹⁴^,¹⁵ contribute to OP pathogenesis, the precise role, cell nature and the regulatory mechanisms of ferroptosis are only partially understood.

Ferroptosis is a unique form of regulated cell death featured by iron-dependent lipid peroxide accumulation and actively involved in various pathological conditions, including cancers, neurodegenerative disorders, cardiovascular and bone diseases ^16-18. Ferroptosis is not prevented by inhibitors of necroptosis, pyroptosis or apoptosis, but inhibited by iron chelators and small lipophilic antioxidants such as ferrostatin¹⁹ and liproxstatin ²⁰ and directly regulated by endogenous glutathione GSH/GPX4 (glutathione peroxidase 4), the core anti-ferroptosis signaling pathway ²¹. GSH tripeptide composed of glutamic acid, cysteine and glycine acts as a scavenger of free radicals and cofactor of GPX4. Insufficient GSH generation reduces the synthesis of GPX4 that is capable of converting the deleterious phospholipid hydroperoxides to the corresponding benign phospholipid alcohols and blocking ferroptosis ²². GPX4 repression due to its reduced synthesis, enzymatic inactivation or protein degradation is a hallmark of ferroptosis ²³. However, GPX4 transcriptional regulation by epigenetic or non-epigenetic regulations might also affect its abundances. GPX4 promoter contains a dense CpG island, a structural feature of DNA methylation modification. GPX4 suppression due to DNA methylation affects its transcription under various ferroptotic conditions ²⁴^,²⁵, suggesting that GPX4 expression is likely subjected to epigenetic DNA methylation controls in OP, which represents a fundamental novel mechanism of OP pathogenesis.

DNA methylation on cytosine of CpG dinucleotide (5-methylcytosine, 5mC) is one of the core epigenetic machineries that potentially regulate the transcription of more than 60% of genes ²⁶^,²⁷. DNA methylation is catalyzed by maintaining DNA methyltransferases DNMT1 and de novo DNMT3a and DNMT3b, while the demethylation is processed by three ten-eleven translocation enzymes TET1, TET2 and TET3 ²⁸. DNA methylation can occur on any CpG site along the genome, however its preferential modifications of CpG islands in gene promoters/enhancers attract DNA methylation readers, transcriptional repressors/cofactors and histone deacetylases to form a transcriptional repressive complex, resulting in silencing of the downstream gene transcription ²⁸. DNA methylation-mediated suppressions of tumor suppressors, anti-aging and cellular protective proteins are common epigenetic features of tumorgenesis, aging and various degenerative and chronic diseases. Recent DNA methylation profiling investigations of OP bone tissues from animal models and OP patients detect a large number of genomic loci/genes that are modified by DNA methylations ^29-31. More pertinently, DNA methylation-incurred suppression of ferroptosis-associated genes GPX4 and CDH1 increases the ferrotosis sensitivity ²⁴^,³², strongly suggesting that epigenetic GPX4 suppression due to DNA methylation aberration might mechanistically affect OP.

In this study, we aimed to investigate the role, nature and regulatory network of ferroptosis in a mouse OP model of ovariectomy (Ovx). We discovered that GPX4 suppression and ferroptotic alterations in both Ovx mouse femurs and ferroptotic osteoblasts occurred substantially at mRNA levels that correlated with aberrant elevations of bioactive DNMT1/3a/3b. We then employed both pharmacological and genetic approaches to determine the critical role of the GPX4 suppression and ferroptosis in OP. Our study might provide molecular insights into the epigenetic mechanisms of ferroptosis in the OP pathologies with clinical prophylactic and therapeutic implications.

1．Mouse osteoporotic femurs induced by ovariectomy displays marked ferroptosis and GPX4 suppression

To gain insight into the possible epigenetic GPX4 suppression and ferroptosis in OP, we employed a well-established mouse OP model of ovariectomy ³³. As anticipated, mice receiving Ovx surgery for 6 weeks demonstrated thinned trabeculae with lost continuity and enlarged areolae in distal femurs as stained by H&E (Fig.1a). Microcomputed tomography (μCT) scanning revealed that the OP femurs had reduced trabecular bone volume versus tissue volume (BV/TV), trabecular bone number (Tb.N), trabecular thickness (Tb,Th) and increased trabecular bone separation (Tb.Sp) (Fig.1a and b). Perls’ Prussian blue staining recognizing the hemosiderin-associated Fe³⁺ showed an increase of iron deposition (Fig.1c), and TUNEL assay, a sensitive way to catch both apoptotic and ferroptotic cells ²⁰, detected an increased numbers of positively-stained cells (Fig.1c). We further performed western blotting assays and detected reduced osteoblast marker type 1 collagen and enhanced osteoclast marker NFATc1 (Nuclear Factor Of Activated T Cells 1), the typical osteoporotic signs of reduced osteoblastogenesis and enhanced osteoclatsogenesis. Notably, GPX4, a core anti-ferroptosis enzyme, was repressed with concomitant elevation of a lipid peroxidation marker 4-hydroxynonenal (4-HNE), while the expression of another anti-ferroptosis protein FSP-1 (ferroptosis suppressor protein 1) whose reduced form ubiquinol supposedly traps lipid peroxyl radicals independent of GSH/GPX4 signaling ³⁴ was not affected (Fig. 1d). We also analyzed a RNA-seq data from CNCB(China National Center for Bioinformation) database³⁵which confirmed Gpx4 down-regulation in tibia of Ovx mice ((Log2 (FC)=-2.49906, Fig.1e). We further performed immunohistochemistry (IHC) staining of femur sections and found that GPX4 was broadly expressed in almost all visible cells around femoral trabecular, where both osteoblasts and osteoclasts resided, but drastically decreased in Ovx mice (Fig. 1f). Finally, we confirmed by electron microscopy that the mouse OP femurs displayed distinct mitochondrial characters of ferroptotic alterations, such as smaller mitochondria and diminished mitochondrial crista (Fig.1g). These results indicate that OP pathogenesis is associated with marked GPX4 suppression and ferroptosis.

2. The GPX4 suppression was mainly due to aberrant elevations of all three bioactive DNMT isoforms

To explore the potential role of DNA methylation that might account for the GPX4 suppression, we analyzed the human and mouse GPX4/Gpx4 promoters online (http://www.urogene.org/methprimer), and found that both contained conserved CpG islands at -253/47 (human, relative to the transcriptional starting site) and -210/170 loci (mouse) as depicted in Fig.2a, suggesting that GPX4 is sensitive to DNA methylation modification. We then assayed the expression of all three bioactive DNA methyltransferases DNMT1, DNMT3a and DNMT3b by western blotting and found that all three isoforms were relatively low in control femurs, but significantly upregulated in femurs of Ovx mice and OP patients (Fig.2b). We further confirmed by RT-PCR that the femoral Gpx4/GPX4 mRNA was substantially reduced in Ovx mice and patients with OP (Fig.2c). Since DNMTs negatively affect gene transcription via increasing the gene promoter methylation, We subsequently examined the femoral DNA methylation status of the promoters by MSP. The results showed that the OP bone tissues from both OP patients and Ovx mice exhibited Gpx4/GPX4 promoter hypermethylation comparing to the controls (77.45 ± 4.32 % of Ovx vs 20.15 ± 2.74 % of Sham and 72.53 ± 2.81 % of OP vs 21.89 ± 2.63 % of Normal, respectively, P < 0.05, Fig. 3d). However, administration of a DNMT inhibitor SGI-1027 to Ovx mice significantly reduced the methylation levels (43.07 ± 3.44 % of SGI/Ovx vs 73.03 ± 2.99 % of Ovx, P < 0.05, Fig. 3e). To confirm the results, we performed BSP, the gold standard of DNA methylation measurement that detects individual CpG site. The results showed that Ovx caused an increase of Gpx4 promoter methylation from 3.19 ± 1.57 % to 31.23 ± 1.26 %, but SGI-1027 treatment lowered the level to 14.74 ± 1.05 %, P < 0.05 (Fig. 3f). These results indicate that the GPX4 suppression was mainly caused by DNMT aberrations-incurred promoter hypermethylation.

3. KLF5 is a potential co-regulator of the Gpx4 transcriptional inhibition

To understand the regulatory mechanisms of the DNMT-incurred Gpx4 transcriptional inhibition, we analyzed the mouse Gpx4 promoter by AnimalTFBD (http://bioinfo.life.hust.edu.cn/AnimalTFDB#!) that predicts the potential binding sites for both transcriptional and co-transcriptional regulators. The results showed that mouse Gpx4 proximate promoter contained a numerous potential binding motifs for various transcriptional factors, including Sp1, C/EBPβ and NF-1C, and transcriptional co-repressors known to participate in epigenetic gene transcriptional inhibition. Among them a Kruppel- like factor 5 (KLF5) motif was close to the transcriptional stating site with a very high binding score (-43/gccccgccca, score 19.303, Fig.3a). KLF5 plays critical roles in various cancers ³⁶ and is capable of either positively or negatively regulate the downstream gene transcription ³⁷^,³⁸. However its regulations of ferroptosis or bone metabolisms are largely unknown. We found that the expressions of KLF5, but not KLF2 of similar binding specificity, as well as transcriptional co-repressor NCoR, SMRT and SnoN, were upregulated in Ovx femurs (Fig.3b). Subsequently, we performed chromatin immunoprecipitation assay (ChIP) and found that KLF5 together with NCoR and SnoN, but not SMRT, inducibly bound to the KLF5 motif region of the Gpx4 promoter in Ovx femurs, but SGI-1027 treatment significantly reduced the bindings (Fig.3c and d). Further, primary osteoblasts treated with ferric ammonium citrate (FAC) known to mimic iron overloading and induce ferroptosis ³⁹ also exhibited KLF5 upregulation, GPX4 suppression and 4-HNE induction. A KLF5-selective inhibitor ML264, however, significantly reduced the alterations (Fig.3e and f). In addition, we also constructed a Gpx4 promoter/luciferase reporter plasmid (Fig.3a) and found that the reporter transfected into osteoblastics displayed reduced luciferase activity upon FAC treatment, which was relieved partially by SGI-1027, coincidentally a KLF5-selective inhibitor ML264 partially blocked the reduction (Fig. 3g). Taken together, these results suggest that the DNMT-incurred Gpx4 transcriptional inhibition is at least partially mediated by KLF5 with NCoR and SnoN participations.

4. DNMT inhibition by SGI-1027 derepresses GPX4 and protects Ovx mice from ferroptotic osteoporosis

To gain further insight into the functional relevance of the GPX4 suppressionand ferroptosis in OP pathogenesis, we treated the control and Ovx mice with or without SGI-1027 (6 weeks, 6 animals in each group), and found that Ovx-incurred osteoporotic alterations of trabecular bone volume versus tissue volume (BV/TV), trabecular bone number (Tb.N), thickness (Tb,Th) and bone separation (Tb.Sp), as well as the TUNEL assays, were effectively corrected by SGI-1027 (Fig. 4a and b). Consistently, SGI-1027 also significantly reduced the Ovx-induced adverse expressions of GPX4 and the lipid peroxidation marker MDA (malondialdehyde), the osteoblast marker collagen 1 (Col1) and osteopontin (OPN) and the osteoclast marker NFATc1 and ACP5 (acid phosphatase 5, Fig. 4c and d). In addition, ferropstatin-1 (Fer-1), a lipophilic antioxidant that specifically inhibits ferroptosis by preventing lipid peroxidation without affecting other forms of regulated cell death ¹⁹, also achieved similar, although less effective, anti-ferroptotic and osteoprotective effects (Fig. 4a-d). Together, these results indicate that the DNMT aberration and the GPX4 suppression-associated ferroptosis are central to the pathologies of OP.

5. The GPX4 suppression and ferroptosis ocurr mainly in osteoblasts

To explore the cell nature of the GPX4 suppression and ferroptosis, we performed immunofluorescent double-staining of femurs with GPX4 plus osteoblast marker OCN (osteocalcin) or osteoclast marker CTSK, respectively. The results showed that GPX4 was co-expressed with OCN in osteoblasts, and both reduced in Ovx femurs (Fig. 5a, the left panel). In contrast, CTSK was barely detectable with discernable GPX4 co-expression in normal femurs but increased in Ovx femurs, in which GPX4 expression remained relatively high (Fig. 5a, the right panel), suggesting that GPX4 suppression mainly occurred in osteoblasts. We further isolated and cultured primary osteoblasts and osteoclasts (differentiated from bone marrow monocytes/macrophages induced by RANKL) and treated both cells with FAC, which induced the alkaline phosphatase activities in osteobalsts and increased TRAP activities in osteoclasts (Fig.5b). We found that FAC induced GPX4 suppression, 4-HNE induction and OPN reduction accompanied by the increases of all three DNMT isoforms in osteoblasts, but only DNMT1 elevation and NFATc1 induction in osteoclasts (Fig.5c and d). We also transfected the osteoblasts with individual siRNAs specifically knocking down DNMT1, DNMT3a and DNMT3b, respectively (Fig. 5e) and found that knockdown of DNMT1, DNMT3a and DNMT3b each reversed the FAC-induced GPX4 suppression (Fig.5f), suggesting all three DNMT isoforms contribute to the ferroptotic GPX4 suppression in osteoblasts.

6. The GPX4 inactivation by RSL3 blunts the anti-ferroptosis effects of DNMT inhibition

To determine the critical role of GPX4 preservation by SGI-1027 in osteoblastic ferroptosis, we treated osteoblasts with FAC or RSL3 (RAS-selective lethal 3), a small molecule that induces ferroptosis via inactivating and degrading GPX4²⁰, and observed drastic GPX4 suppression and 4-HNE induction (Fig.6a). However, SGI-1027 significantly corrected the abnormal GPX4 and 4-HNE alterations induced by FAC, but not by RSL3 (Fig.6a and 6b). To confirm the osteoblast ferroptosis, we performed TUNEL assay and found that FAC and RSL3 induced significant increase of TUNEL-positive osteoblasts, but not osteoclasts, and SGI-1027 treatment reduced the positive cell numbers of osteoblasts induced by FAC, but not that by RSL3 (Fig.6c, the upper two panels, and 6d). We further assessed the lipid peroxidation status of osteoblasts with C11-BODIPY, a fluorescent radio-probe that monitors lipid peroxidation in live cells ⁴⁰. The results showed that the resting osteoblasts displayed prominent non-oxidized BODIPY (N-BOD), but barely detectable oxidized BODIPY (O-BOD). FAC or RSL3 treatment caused an inversion of the O-BODIPY/N-BODIPY distributions; however, SGI-1027 effectively corrected the inversion conferred by FAC, but not that by RSL3 (Fig.6c, the middle three panels). Furthermore, we examined the osteoblasts by EM and found that FAC and RSL3 induced the typical ferroptotic alterations, such as smaller mitochondria and demolished crista; however, SGI-1027 lightened the alterations induced by FAC, but not by RSL3 (Fig.6c, the bottom panel). Collectively, these results suggest that osteoblasts, but not osteoclasts, undergo ferroptosis under OP condition that are regulated by DNMT1/3a/3b aberration and the GPX4 suppression.

7. Osteoblast Gpx4 deficiency potentiates ferroptotic osteoporosis

To directly test the role of osteoblast Gpx4 suppression in the ferroptotic osteoporosis, we generated a strain of osteoblast-specific Gpx4 knockout mice by crossing Gpx4^fl/fl mice with collagen 1 promoter-driven Cre mice (Col1a1-Cre), in which the exons 2-4 of Gpx4 gene were deleted in osteoblasts (Fig.7a). Previous study indicated that globe Gpx4 gene knockout was embryonically lethal ⁴¹. We found that the offsprings (more than 70 tested) were either wild type or heterozygous Gpx4^+/- mice, but none was homozygous Gpx4^-/- by genotyping (Fig.7b), indicating that osteoblast-specific Gpx4 depletion is also lethal. These Gpx4^+/- mice at 8 weeks appeared smaller, displayed lighter weights, shorter femurs (Fig.7c), reduced femoral GPX4 and adverse expressions of osteoblastic markers OPN and collagen1, but not osteoclast markers NFATc1 and ACP5 comparing to Gpx4^fl/flcontrols (Fig.7d). IHC staining of osteoblast marker OCN and TRAP staining confirmed the reduced OCN intensities and unchanged the numbers of TRAP-positive cells, respectively (Fig.7e). In addition, these mice displayed altered osteoporotic parameters of BV/TV, Tb.N, Tb,Th and Tb.Sp (Fig.7e and f) and increased TUNEL-positive cells (Fig.6e, the lower panel), supporting that GPX4 deficiency potentiates osteoblastic ferroptosis and OP pathologies.

8. GPX4 derepression is essential for the anti-ferroptotic and anti-osteoporotic effects of SGI-1027

DNMT aberrations and the SGI-1027 intervention might affect the transcription of numerous genes. We assumed that if the GPX4 derepression by SGI-1027 was critical for its anti-ferroptotic and anti-osteoporotic effects, blocking the GPX4 reactivation would eliminate the protective effects. To test the idea, we compared the anti-ferroptotic and anti-osteoporotic effects of SGI-1017 between Gpx4^fl/fl and Gpx4^+/-mice as well as RSL3-treated Gpx4^fl/fl mice. Both Gpx4^+/-mice and RSL3-treated Gpx4^fl/fl mice displayed the adverse femoral osteoporotic alterations of trabecular bone volume versus tissue volume (BV/TV), trabecular bone number (Tb.N), trabecular thickness (Tb,Th) and trabecular bone separation (Tb.Sp), as well as increased TUNEL positive cell ratio under basal and Ovx-conditions (Fig.8a and b). SGI-1027 treatment significantly improved the bone microstructural and TUNE staining abnormalities in Gpx4^fl/fl mice; however the protective effects were largely abrogated in Gpx4^+/- and RSL3-Gpx4^fl/fl mice (Fig.8a and b). Similarly, SGI-1027 significantly reduced the abnormal protein levels of GPX4, MDA, collagen I, OPN, NFATc1 and ACP5 in Gpx4^fl/fl mice, but the beneficial effects were largely diminished in in Gpx4^+/- and RSL3-Gpx4^fl/fl mice (Fig.8c and d). Together, these results indicate that the GPX4 preservation is essential for the anti-ferroptotic and osteoprotective effects of SGI-1027.

Study of epigenetic ferroptosis in OP represents a novel approach to better understand the OP pathogenesis and potentially leads to novel therapeutic strategies. In this study, we have discovered that (1) GPX4, a key anti-ferroptotic factor, was transcriptionally suppressed in Ovx mouse femurs due to DNMT1/3a/3b elevations and subsequent GPX4 promoter hypermethylation; (2) The GPX4 suppression was co-regulated by repressive KLF5 and likely by transcriptional co-repressor NCoR and SnoN; (3) A DNMT inhibitor SGI-1027 effectively reduced the GPX4 suppression and mitigated the ferroptotic bone loss in Ovx mice; (4) The GPX4 suppression and ferroptosis occurred mainly in osteoblasts and osteoblast-specific GPX4 semi-knockout (Gpx4^-/+) incurred spontaneous and exacerbated ferroptotic OP alterations in Ovx mice, and finally (5) GPX4 pharmacoligical inactivation or semi-knockout in osteoblasts significantly diminished the protective effect of SGI-1027 against DNMT-mediated ferroptotic bone loss. Thus, our data indicate that the DNMT1/3a/3b aberration-incurred GPX4 suppression and the consequential osteoblastic ferroptosis causally impact the development of OP, which can be effectively targeted by DNA demethylating agents for therapeutic benefits.

Identification of DNMT1/3a/3b as the critical initiators of GPX4 suppression and ferroptosis critical for OP is an important discovery of study. It is well known that both ferroptosis and OP are affected by epigenetic DNA methylation aberration ²⁹^,⁴², however the mechanistic linker of these two events has been missing. Recent studies suggest that GPX4 suppression due to aberrant DNA methylation might be a decisive event of ferroptosis under ferroptotic conditions ²⁴^,²⁵. The actual methylation status of a particular gene promoter is regulated by a number of regulators, including methyl donors, DNA insulators, DNA methylation “writer” NDMTs, “reader” methyl-CpG-binding domain (MBD) proteins and DNA demethylating TET enzymes ^43-45, and the initiative “drivers” of the DNA methylation alterations in OP are unclear. Previous studies from our and other labs have reported that aberrant DNMT elevations causally affected OP ^46-49. In this study, we detected that the GPX4 suppression occurred substantially at transcriptional level that correlated with increased expression of all three bioactive DNMT isoforms and GPX4 promoter hypermethylations in the distal femurs of both OP patients and Ovx mice. We further demonstrated that individual knockdown of DNMT1/3a/3b significantly reduced the GPX4 suppression and lipid peroxidation in osteoblasts, and a pan-DNMT inhibitor SGI-1027 effectively corrected the GPX4 promoter hypermethylation, GPX4 suppression and the ferroptotic OP alterations in Ovx mice, which were abrogated when GPX4 was genetically-deficient or pharmacologically-inactivated (Fig.8). These are strong evidence that DNMT1/3a/3b aberration-driven GPX4 suppression and ferroptosis contribute substantially to OP pathologies.

Osteo-ferroptosis has been reported in OP animals of various etiologies, including estrogen deficiency such as postmenopausal OP modeled by Ovx mice, glucocorticoid and diabetic OP, which affects bone marrow mesenchymal stem cells ⁵⁰, osteocytes ⁵¹ osteoblasts⁵² and osteoclasts ⁵³. However the cell nature of ferroptosis involved in estrogen-deficient OP has been controversial. In this study, we showed that GPX4 suppression and ferroptotic alterations mainly occurred in osteoblasts as demonstrated by immunofluorescent double-staining of femoral sections and in primarily-cultured osteoblasts, which also displayed typical ferroptotic alterations such as GPX4 suppression, abnormal lipid peroxidation and mitochondrial morphological changes that were absent from osteoclasts. This is further confirmed by osteoblast-specific Gpx4 deficient mice that developed spontaneous and worse ferroptotic OP alterations in Ovx mice. These are consistent with the consensus that the reduced osteoblastic cell numbers and activities break the balanced of osteoblastogenesis and osteoclastogenesis, leading to OP.

Revealing of KLF5 as a transcriptional co-regulator critically involved in DNA methylation-associated Gpx4 suppression has provided meaningful information regarding the DNA methuylation-incurred transcriptional silencing of the downstream genes. DNA methylation-incurred gene transcriptional inhibition is mediated by a transcriptional repressive complex containing multiple transcriptional repressors and co-regulators that might confer the gene specificity ²⁸. KLF5 is well documented for its regulation of multiple cellular processes, including the cell cycle and proliferation, apoptosis and autophagy, migration and invasion, and stemness and differentiation ³⁶, via either positively or negatively regulating the transcription of target genes ⁵⁴^,⁵⁵. We found that Gpx4 promoter contains a strong KLF5 motif and KLF5 together with transcriptional co-repressor NCoR and SnoN inducibly bound to the hypermethylated Gpx4 promoter in OP femurs that is sensitive to DNMT inhibition (Fig.3). We further showed that KLF5 inhibition by ML264 partially relieved the GPX4 suppression in osteoblasts. These data support that KLF5, NCoR and SnoN might be the core components of the transcriptional repressive complex that mediates the DNA methylation-incurred Gpx4 transcriptional suppression in OP.

It is noteworthy that our study has revealed an important feature of epigenetic osteoblast ferroptosis in OP pathogenesis, but some important questions remain to be answered. For example, GPX4 suppression is affected by many known and unkonwn ferroptotic stimulations and it is unclear which upstream network actually induced the DNMT aberration. It is also unclear how epigenetic DNMT alterations differentially affect ferroptosis of osteoblasts versus osteocytes and osteoclasts under OP conditions and whether the DNMT aberration-incurred ferroptosis is unique to Ovx mice or a general OP character of all etiologies, which warrant further investigation.

In conclusion, the results from our study demonstrate that aberrant DNMT1/3a/3b elevations and subsequent GPX4 suppression play a decisive role in osteoblastic ferroptosis that contributes significantly to OP. Since epigenetic modifications are reversible and DNA demethylating drugs such as 5-azacytidine and 5-aza-2-deoxycytidine (decitabine) have been approved for clinical treatment of myelodysplastic syndromes and chronic myelomonocytic leukemia ⁵⁶, our results also suggest that targeting epigenetic GPX4 suppression and ferroptosis by DNMT intervention might be a feasible and effective strategy to treat patients with OP and the related bone disorders.

Animal and treatment

The use of animals and the experimental protocols were approved by the Animal Care Committee of Nanjing University in accordance with the Institutional Animal Care and Use guidelines (2020AE01113). C57BL/6J mice were from Gempharmatech Co., Ltd., Nanjing, China. We intended to generate osteoblastic Gpx4 knockout mice by crossing Gpx4-flox mice (Gpx4^fl/fl, Strain NO. T050827) and Col1a1-Cre mice (Strain NO. T004734, both were of C57BL/6J background, GemPharmatech, Nanjing, China), but only obtained Gpx4 haplo-deficient (Gpx4^-/+) progenies. The PCR genotyping was performed on mouse toe DNAs with primers F1: GTACTGCAACAGCTCCGAGTTC; R1: ACTTATCCAGGCAGACCATGTG; R2: AACTCCAATTCCCAGGACTCAC as depicted in figure 7A. All mice were housed under specific pathogen-free and standard 25 ± 2 °C, 50 ± 5% humidity and a 12 h/12 h light/dark cycle conditions.

A mouse OP model was established with a bilateral ovariectomy protocol ³³. Briefly, the experimental mice were anesthetized with isoflurane and the bilateral ovaries of Ovx mice were removed through a midline incision of the skin and flank incisions of the peritoneum. The skin incision was then closed with metallic clips. Sham operation was processed similarly without ovary removal. For intervention study, female C57BL/6J mice of around 10-12 weeks old were divided into four groups: (1) Sham surgery group; (2) SGI-1027 (2.5 mg/kg, HY-13962) or Ferrostatin-1 (Fer-1, 5 mg/kg, HY-100579) from MCE, USA, dissolved in 2% DMSO, 30% PEG 300 and 2% Tween 80 administered by daily intraperitoneal injection; (3) The Ovx group; (4) SGI-1027/Fer-1 intervention group. For assay to determine the role of GPX4 in SGI-1027 intervention, C57BL/6J mice treated with or without RSL3 (100 mg/kg, HY-100218A, MCE, USA), or Gpx4^-/+ mice and the control Gpx4^fl/flwere subjected to sham, Ovx and SGI-1027 intervention described above. The experiments went for six weeks, and then mice were sacrificed by excessive isoflurane and the mouse femurs were collected and stored at -80^oC or treated with paraformaldehyde for further analysis.

Human samples

Human samples were collected from the Northern Jiangsu People's Hospital, the Teaching Hospital of Nanjing University Medical School. OP is defined as a T score of ≤−2.5, and a T score of ≥−1 is considered normal bone density according to the National Osteoporosis Foundation. Osteoporotic lumbar were obtained from 6 female patients with lumbar fracture who received percutaneous vertebroplasty (62–82 years old) with an average bone densitometry T score of −3.9 (−3.6, −4.8, −4.4, −4.5, −3.8 and −2.8). The control non-OP lumbar were from six age-matched patients receiving internal fixation treatment (58–79 years old) with an average T score of −0.5 (−0.3, −0.6, −0.2, −0.6, -0.3 and −0.9). The T scores were determined by dual energy X-ray absorptiometry. The samples were stored at −80 °C before further protein, histology and MSP analyses. The study was approved by the ethics committee of the Northern Jiangsu People's Hospital (2023ky232), and written informed consent was received from all subjects. Patients or members of the public are not involved in the design, conduct, reporting, or dissemination plans of the research.

Bone micro-CT analysis

Trabecular microstructure analysis was performed as described previously ⁵⁷ with freshly-removed mouse right femurs fixed in 4% paraformaldehyde for 24 h. The micro-CT scanner (Scanco Medical, Bruettisellen, Switzerland) was set at 55 kV, 145 μA and 15.6 μm voxel with 250 ms integration time. Femoral mid-diaphysis above the growth plate and distal metaphysis were selected as the region of interest (ROI). For each sample, a total of 100 slices were evaluated to generate the three dimensional (3D) trabecular images, and ratio of the sectional trabecular volume to total bone tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were calculated.

Histological, immunohistochemical (IHC), Perl's Prussian blue, TUNEL and immunofluorescent staining

The mouse femural sections were processed with hematoxylin and eosin (H&E), Perl's Prussian blue or IHC staining essentially as described before ⁵⁸. For IHC staining, the sectioned slides were incubated overnight at 4 °C with primary antibodies against GPX4 (A11243, Abclonal, China), osteocalcin (OCN, 23418-1-AP, proteintech, China), and then with HRP-conjugated secondary antibodies. Afterward, the slides were processed with a DAB horseradish peroxidase color development kit (PR30010, proteintech, china) and counterstained with hematin, the IHC Profiler plug-in in Image J was used to automatically score the staining status of samples (High positive (3), Positive (2), Low Positive (1) and Negative (0)). Perl's Prussian blue staining (G1029, Servicebio, china) and TUNEL (TdT-mediated dUTP Nick-End Labeling, A111-01, Vazyme, China) followed the instructions in the kit. The percentages of TUNEL-positively-stained cells over total cells from 10 randomly-selected fields were counted in a double-blinded manner.

Immunofluorescent double-staining of murine femural sections were performed essentially as before ⁵⁹. The sections were first incubated overnight with primary antibody mouse anti-GPX4 (67763-1-Ig, proteintech, China) plus rabbit anti-OCN or rabbit anti-CTSK (11239-1-AP, proteintech, China), respectively. Next day, the sections were incubated with secondary antibody CoraLite488-conjugated Goat Anti-Rabbit IgG (SA00013-2, Proteintech, China) and CoraLite594-conjugated goat anti-mouse IgG (SA00013-3, Proteintech, China) followed by nuclear DAPI (C1005, Beyotime, China) staining. A laser confocal microscope (Olympus, Tokyo, Japan) was used for image capturing.

RNA sequencing data analysis

We downloaded the transcriptome data of bone tissue from Sham and Ovx mice (https://ngdc.cncb.ac.cn/gsa/browse/CRA007214). The reads from mice data were aligned against mm10 genome assembly with hisat 2.1.0. SAM files were sorted and converted to BAM with samtools v1.4. Reads with QS < 20 were excluded. For each sample, unique map reads with map quality score ≥20 were reserved for subsequent analyses. HT Seq Python package (version 0.9.1) was used to count the number of reads of a unique map for each gene. The DESeq2 R package was used to perform differential expression analysis. Differentially expressed genes (DEGs) were assessed by |log2FC|≥1 and P value < 0.05.

Transmission Electron microscopy (TEM) examination

Fresh murine femurs were placed in a fixative containing 2% PFA and 2.5% glutaraldehyde (G5882, Sigma-Aldrich, USA), rinsed sequentially according to a conventional TEM sample preparation protocol, and fixed again in 1% osmium tetroxide. After dehydration and embedding in Epon812 (45345, Sigma-Aldrich,USA), the ultrathin sections were stained with lead citrate and uranyl acetate and observed under JEOL-1200EX microscope (Japan) at Shandong Weiya Laboratory, China.

Primary Cell culture

Primary osteoblasts were extracted as previously described ⁶⁰ from 3-day-young mice. Briefly, calvarial bones were dissected and digested with 0.1% collagenase type I (SCR103, Sigma, USA) for three rounds after periosteum removal, and the final cell pellets were collected and cultured in fresh α-MEM media (SH30265.01B, HyClone, USA) containing 10% FBS(FSD500, ExCell, China) and 1% penicillin/ streptomycin(15140122, Gibco, USA). For osteoblast differentiation, the cells were changed the medium to osteogenic medium (PD-003; Procell, Wuhan, China)which contain 50 μg/mL ascorbic acid, 5 mM β-glycerophosphate, and 10 nM dexamethasone.

For primary osteoclast culture, bone marrow monocytes (BMMs) were isolated from 3-week-old mice by flushing the bone marrow of long bones and cultured in complete α-MEM medium containing 10% FBS and M-CSF (Macrophage colony-stimulating factor, 30 ng/mL, CB34, Novoprotein, China) as before ⁶¹. Three days later, RANKL (Receptor activator for nuclear factor-κB ligand, 50 ng/mL, CR06, Novoprotein, China) was added to induce osteoclast differentiation.

ALP or TRAP activity staining

For osteoblast ALP activity assay, FAC was added for 48 hours, washed with phosphate-buffered saline (PBS) and then fixed with 4% formaldehyde for 15 min. After rinsing with PBS again, cells were stained for ALP activity with the BCIP/NBT ALP color development kit (Beyotime, Nanjing, China). Image J software was used to evaluate the positively stained areas over the total areas from 5 predetermined fixed locations.

For osteoclast TRAP activity assay, the differentiated primary osteoclast treated with FAC for 48 h or mouse femoral sections were fixed with 4% paraformaldehyde and stained for TRAP activities with a commercial kit (Sigma, 387A-1 KT, USA) according to the manufacturer's instructions. For primary osteoclast staining, the positively stained cell areas over the whole fields from 5 pre-set fixed locations were quantified using Image J software. For mouse femural staining, the sections were counterstained with methyl green and the average numbers of TRAP-positively stained cells in 10 randomly selected fields were calculated in a double-blind manner.

C11-BODIPY staining

A fluorescent radioprobe C11-BODIPY(581/591, D3861, Thermo Fisher, USA) was used to assess lipid peroxidation in osteoblasts. Briefly, the primary osteoblasts inoculated in 6-well plates under various treatments were treated with C11-BODIPY dye (10 μM dissolved in DMSO) for 1 hour at 37^oC in dark. After excess C11-BODIPY were removed by washing, the cells were observed under a confocal fluorescence microscope with excitation/emission wavelengths of 488/510 nm for oxidized BODIPY (green) and 581/591 nm for non-oxidized BODIPY (red). The average fluorescence intensities were based on six randomly-selected view fields, adjusted for the number of cells of view and.calculated using ImageJ software.

Western blotting

Western blot assays of mouse hind limb bones or cell homogenates were performed essentially as before ⁶². The primary antibodies used were: NFATc1(AF06823) and DNMT3b (AF300068) from AiFangbiological, China; Col1 (Collagen I, GB114197) and OPN (Osteopontin, GB112328) from Servicebio, China; GPX4 (A1933), MeCP2 (A0707) and β-actin (AC026) from ABclonal, China; 4-HNE (4 Hydroxynonenal, ab46545, Abcam, Cambridge, UK); MDA (Malondialdehyde, abx445120, Abbexa, Cambridge, UK); CTSK (DF6614), KLF5 (AF7542), KFL2 (DF13602), NCoR (AF0270) SMRT (DF8896), SnoN (DF3088) from Affinity Biosciences, China. The horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from proteintech, China. Western blots were visualized with fully automated chemiluminescence image analysis system (5200, Tanon, China) and the protein quantities were analyzed by Image J software.

Methylated specific PCR (MSP) and bisulfite-sequencing PCR (BSP)

Prediction of CpG islands in Gpx4 promoters and primer design for methylation-specific PCR (MSP) and bisulfite-sequencing PCR (BSP) were performed with online software MethPrimer (www.urogene.org/methprimer). DNeasy Blood & Tissue Kit (69504, QIAGEN, Germany) was used to isolated total DNA, and DNA Bisulfite Conversion Kit (DP215, TIANGEN Biotech, China) was used to convert unmethylated cytosine to uracil according to manufacturer’s instructions. MSP and BSP were performed following previously-established protocols ⁶³.

The mouse Gpx4 promoter methylation was assayed by MSP with methylated forward primer 5’-TTTTTTAAGGGGATGATTTTGATAC (-247/-223) and reverse primer 5’- ATACCCAATAATAAAAACGCGA A (-78/-100); unmethylated forward primer 5’-TTTTAAGGGGATGATTTTGATATGT (-245/-221) and reverse primer 5’-CATACC CAATAATAAAAACACAAA (-77/-100); and input DNA control forward primer 5’-CTCTTTAAGGGGATGACTTTGACAC and reverse primer 5’-ATGCCCAGTGAT AGGGACGCGGG. The human GPX4 promoter methylation was assayed by MSP with methylated forward primer 5’-AGTATTTTTAGGTTGTTTGGTTTGC (7/33) and reverse primer 5’-CGAACGTACGAACTTATTATTAACGA (152/179); unmethylated forward primer 5’-GTATTTTTAGGTTGTTTGGTTTGTG (8/34) and reverse primer 5’- CAAA CATACAAACTTATTATTAACAAC (152/180); and input DNA control forward primer 5’-TAGACACAAGCGA GCATGCGCAGTC and reverse primer 5’- CCAGAGCGCTCATTGGTCAGACG. The PCR products were analyzed on a 2% agarose gel and visualized under ultraviolet light, and densitometric analysis was performed using ImageJ software. The methylation status was assayed by BSP with forward primer 5’- GTTTTTTAAGGGGATGATTTTGATA (-248/-224) and reverse primer 5’- CCCTACAA CCAATAAA AAACTAAATA (5/-22). The PCR products were separated by electrophoresis, and the target DNA fragments were purified and cloned into pGEM T Easy Vector (A1360; Promega). Five colonies from each mouse/PCR reaction were randomly chosen for sequencing, and the percentages of methylated cytosines over total cytosines within the cloned fragment were calculated.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA from mouse and human bone tissues was extracted essentially as before ⁶⁴. After cDNA synthesis, PCR was performed with following mouse Gpx4 primers Gpx4F: CCCATTCCTGAACCTTTCAA and Gpx4R: GCACACGAAACCCCT GTACT; ActbF: GATCATTGCTCC TCCTGAGC and ActbR: TGCACCGCAAGTGCT TCTA as internal control and human GPX4 primers Gpx4F: GAAGCAGGAGCCAG GGAGTA and Gpx4R: ATGGCATTTCCCAGGATGCC; Actb primers ActbF: GCCTT CCTTCCTGGGCAT and ActbR: CTTCATTGTGCTGGGTGCC, respectively. PCR products were resolved on a 1.5% agarose gel and visualized under UV light.

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed with mouse bone tissues as before ⁶⁵. The immunoprecipitation was performed with ChIP quality antibodies to KLF5, NCoR , SMRT and SnoN. The starting (input) and immunoprecipitated DNAs were analyzed by PCR and quantitative real-time PCR (qRT-PCR) using primer sets for Gpx4 promoter (Forward, 5’-GGGGATGACTTTGACACGC and Reverse, 5’-GCCTGAATGAAGGGA CGG, which covered the -239 to -14 locus containing a putative KLF5 binding motif (-43/gccccgccca). Regular PCR products were separated on 1.5% agarose gels and analysis of PCR product densitometry were performed with Image J Software.

Luciferase assay

Primary osteoblasts were transiently transfected (FuGENE® HD Transfection Reagent, Promega, USA) with Gpx4 promoter reporter plasmid Gpx4p-luc (containing 2000 bp of the Gpx4 proximal promoter, costumer-constructed by Genechem, China) plus a renilla luciferase reporter (Genechem, China) as internal control. After the cells received various treatments, the luciferase activities from cell lysates were assayed using a dual luciferase reporter assay kit (Promega, USA). The luciferase activities of Gpx4p-luc were normalized to renilla luciferase levels and expressed as relative fold changes.

Statistical analysis

The data normal distribution and assumption of homogeneity of variances were assessed by Shapiro-Wilk test and Levene’s test, respectively. The data quantitation and graphic plotting were accomplished with GraphpadPrism. Main effect P and effect size η² (large effect size, η² ≥0.1379; medium effect size, 0.0588 ≤ η² < 0.1379; small effect size, 0.0099 ≤ η² < 0.0588) were calculated by SPSS V22.0 software. Data were expressed as means ± SEM for animal studies or ± SD for cell assays. The Box-and-whisker plots were defined as follows: midline represents median, box is the 25^th-75^th percentiles, and whiskers are minimum and maximum. The group differences were analyzed by Student’s t test, two-way ANOVA, or two-way ANOVA followed by Tukey’s post-hoc test. The thresholds of P < 0.05 were set as statistically significant.

Contributions: BR, JD and FW conducted the assays, acquired and analyzed the data and drafted the manuscript. ZH, LZ and BY provided technical support and assistance. CL and HD contributed reagents and provided scientific insight and discussion. WC conceived, designed and supervised the study, arranged and interpreted the results, and wrote the manuscript. HW and YW provided supervision and research resources. All authors approved the final version of the manuscript.

Data availability statements

RNA-seq data: China National Center for Bioinformation CRA007214 (https://ngdc.cncb.ac.cn/gsa/browse/CRA007214)

Acknowledgements: This work was supported by the National Key Research and Development Program of China (2023YFB3810200, 2023YFB3810204), the National Natural Science Foundation of China (82272502, 82072423, 81970577)

Conflict of interests: The authors have declared that no conflict of interest exists.

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DNMT aberration-incurred GPX4 suppression prompts osteoblast ferroptosis and osteoporosis

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