SIRT6-PAI-1 axis is a promising therapeutic target in aging-related bone metabolic disruption

doi:10.21203/rs.3.rs-1954276/v1

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SIRT6-PAI-1 axis is a promising therapeutic target in aging-related bone metabolic disruption

https://doi.org/10.21203/rs.3.rs-1954276/v1

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published 17 May, 2023

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The mechanistic regulation of bone mass in aged animals is poorly understood. In this study, we examined the role of SIRT6, a longevity-associated factor, in osteocytes, using mice lacking Sirt6 in Dmp-1-expressing cells (cKO mice) and the MLO-Y4 osteocyte-like cell line. cKO mice exhibited increased osteocytic expression of Sost, Fgf23 and senescence inducing gene Pai-1 and the senescence markers p16 and Il-6, decreased serum phosphate levels, and low-turnover osteopenia. The cKO phenotype was reversed in mice that were a cross of PAI-1-null mice with cKO mice. Furthermore, senescence induction in MLO-Y4 cells increased the Fgf23 and Sost mRNA expression. Sirt6 knockout and senescence induction increased HIF-1α binding to the Fgf23 enhancer sequence. Bone mass and serum phosphate levels were higher in PAI-1-null aged mice than in wild-type mice. Therefore, SIRT6 agonists or PAI-1 inhibitors may be promising therapeutic options for aging-related bone metabolism disruptions.

Osteoporosis is a skeletal bone disease characterized by reduced bone mass, which results in increased fracture risk. Aging is a pivotal risk factor for osteoporosis. The yeast sirtuin SIR2 is an NAD⁺-dependent histone deacetylase that is involved in the regulation of aging¹. In mammals, the sirtuin family contains seven proteins (SIRT1-SIRT7), each with different tissue specificity, intracellular localization, and enzyme activity. SIRT6 is expressed in the nucleus and plays a role in transcriptional silencing and maintaining genomic stability². SIRT6 deficient mice die young and display phenotypes of premature aging, including osteoporosis². We previously showed that SIRT6 regulates chondrocyte proliferation and differentiation by regulating Ihh expression³. Furthermore, SIRT6 deficiency aggravates joint cartilage degeneration in a metabolic syndrome-associated osteoarthritis model⁴. SIRT6 is ubiquitously expressed cells, including osteoblasts and osteocytes, and SIRT6-null mice are characterized by low-turnover osteoporosis⁵. SIRT6 regulates bone metabolism in osteoblasts by binding to the promoters of Runx2 and osterix and deacetylating histone H3 at lysine 9 (H3K9)⁵. However, SIRT6-null mice die young, so the precise role of SIRT6 in bone metabolism, especially in osteocytes, is poorly understood⁶.

Osteocytes are derived from osteoblasts and they play a central role in bone remodeling. Osteocytes can regulate bone turnover with the production of nitric acid, prostaglandins, RANKL, and sclerostin^7,8. Furthermore, osteocytes play a pivotal role in the regulation of phosphate homeostasis by secreting FGF23. FGF23 is a potent phosphaturic peptide hormone that is predominantly secreted by osteocytes and osteoblasts⁹. FGF23 suppresses serum phosphate levels by inhibiting the reabsorption of proximal tubular phosphate and the absorption of intestinal phosphate. Serum levels of FGF23 increase with age in men aged over 60¹⁰. FGF23 mRNA levels are significantly higher in older women (mean age of 72.9 years) than in young women (mean age of 30.0 years)¹¹. Patients with chronic kidney dysfunction and aging-related renal disease exhibit higher serum FGF23 levels¹².

Sclerostin, encoded by SOST, is secreted by differentiated osteocytes and inhibits canonical Wnt signaling and bone formation by interacting with LRP5^13–17. Sost-null mice exhibit elevated bone volume as a result of enhanced Wnt signaling, whereas transgenic mice overexpressing Sost display reduced bone volume. The pivotal role of SOST in aging-related osteoporosis has been elucidated in rodent models¹⁸. When senescent cells are genetically depleted in mice, aging-related osteoporosis is prevented, and Sost expression is reduced¹⁸. Additionally, calorie restriction, which extends lifespan by activating the sirtuin family, suppresses Sost mRNA expression¹⁹.

Thus, although the involvement of FGF23 and SOST in aging-related skeletal disorders has been reported, the mechanisms by which the aging process affects FGF23 and SOST expression are poorly understood. Thus, we examined the role of SIRT6 in osteocytes by analyzing Dmp-1-expressing cells in Sirt6-deficient mice (Dmp-1cre::Sirt6 ^f/f mice; cKO). Furthermore, as Sirt6 deficiency enhances Pai-1 expression, PAI-1-null mice were crossed with cKO mice, generating cPKO mice (Dmp-1cre::Sirt6 ^f/f::Pai-1-/- mice); the function of the Sirt6-Pai-1 axis in osteocyte-related skeletal metabolism was examined.

Evaluation of bone parameters in cKO mice

The cKO mice grew normally, and the body weights of the cKO mice and littermates were similar. Bone volume was evaluated in the lumbar and femoral bones. At 20 weeks after birth, male cKO mice exhibited lower trabecular bone volume in the femur and lumbar spine than the control mice (Fig. 1a–d). Micro-computed tomography (micro-CT) analysis of the femoral bone indicated higher trabecular separation (Tb.Sp) and trabecular bone spacing (Tb.Spac) in cKO mice compared to the control mice, whereas the trabecular number (Tb.N) was lower in cKO mice than in the control mice (Fig. 1b). Bone histomorphometry of the lumbar spine indicated that the mineralizing surface to bone surface (MS/BS) ratio, bone formation rate (BFR)/BS, and mineral apposition rate (MAR) tended to be lower in cKO mice than in the control mice; however, the differences were not significant (Fig. 1e, f). The number of osteoclasts (osteoclast number to bone perimeter, osteoclast number to total area) and osteoclast surface (osteoclast surface to BS) increased in cKO mice (Fig. 1g, h). These data indicate that bone resorption activity was higher in cKO mice than in the control mice.

Evaluation of bone parameters and serum phosphate levels in cPKO mice

To elucidate the molecular targets of SIRT6 in osteocytes, we assessed the mRNA expression of potent regulators of bone formation (Sost), bone resorption (Opg and Rankl), and Fgf23 in osteocyte-enriched cortical bones. Quantitative polymerase chain reaction (qPCR) analysis indicated that the mRNA expression of Sost and Fgf23 was significantly increased in the tibial cortical bone of cKO mice (Fig. 2a), whereas differences between Opg and Rankl mRNA expression were not significant between cKO and the control mice (Supplementary Fig. 1). We previously revealed that SIRT6 deficiency enhanced the expression of Pai-1, a potent senescence marker, in chondrocytes²⁰, so the mRNA level of Pai-1 was also assessed. As expected, Pai-1 mRNA expression also increased in cKO osteocytes (Fig. 2a). We then evaluated the expression of senescence marker genes, such as interleukin-6 (Il-6), C-X-C motif chemokine ligand 1 (Cxcl1), p53, and p16. qPCR analysis indicated that the expression of these genes was increased in cKO mice (Fig. 2a). In humans, FGF23 is a potent regulator of phosphate metabolism⁹. Therefore, serum phosphate levels were evaluated in cKO mice. As expected, Serum phosphorus levels were significantly lower in cKO mice compared to control mice, whereas, serum calcium levels were comparable in control and cKO mice (Fig. 2b).

Immunohistochemistry analysis of cortical bone in cKO mice

Immunohistochemistry of the cortical bone was performed to detect the presence of SOST, FGF23, and PAI-1. The percentage of SOST-, FGF23-, and PAI-1-positive cells relative to total osteocytes was higher in cKO mice than in the control mice (Fig. 3a, b). These results indicate that SIRT6 deficiency stimulates the expression of SOST, FGF23, and PAI-1 in osteocytes. SIRT6 suppresses cellular senescence through multiple factors^21-24, and PAI-1 is not only a marker of cellular senescence but is also sufficient for the induction of senescence²⁵. Thus, cKO mice were crossed with PAI-1-null mice (DMP-1cre::Sirt6^f/f::Pai-1-/- mice, cPKO mice) to investigate the role of PAI-1 in the phenotype of cKO mice. The cPKO mice grew normally, and their body weights were comparable with those of cKO and control mice.

qPCR analysis of cortical bone in cPKO mice

We used qPCR analysis to investigate the expression of Sost and Fgf23 in cortical bones from cKO and cPKO mice and found that Sost and Fgf23 mRNA levels were significantly reduced in cPKO compared to cKO mice (Fig. 4a). Thus, PAI-1 deletion clearly reduced the upregulation of these genes cause by SIRT6 deficiency. Similarly, the expression of Il-6, Cxcl1, p53, and p16 was decreased in cPKO mice compared to cKO mice (Fig. 4a),

Evaluation of bone parameters and serum phosphate levels in cPKO mice

Histological analyses showed higher bone volume in cPKO mice than in cKO mice in the lumber bone (Fig. 4b, c). In contrast, micro-CT analyses of distal femur showed BV/TV in cPKO mice was lower than in cKO mice. Reduced phosphate levels in cKO mice were mitigated by the deletion of PAI-1 in cPKO mice (Fig. 4d), whereas serum Ca²⁺ levels were comparable between cKO mice and cPKO mice (Fig. 4d).

qPCR analysis of Sirt6 knockout in MLO-Y4 cells

To investigate whether SIRT6 directly regulates Sost and Fgf23 in osteocytes, Sirt6 was knocked out by siRNA in osteocyte-like MLO-Y4 cells. qPCR analysis indicated that Sost and Fgf23 expression increased due to SIRT6 deficiency (Fig. 5a). This suggests that SIRT6 regulates Fgf23 and Sost expression.

qPCR analysis of senescent MLO-Y4 cells

To address the role of cell senescence in the expression of Sost and Fgf23, senescence was induced in MLO-Y4 cells using two different methods: long-term (21 days) culturing after confluency (Long-Term Confluent; LTC)^26,27, and culturing under doxorubicin administration²⁸. qPCR analysis revealed that Sost and Fgf23 mRNA expression was stimulated in MLO-Y4 cells by both LTC and doxorubicin treatments, and the increase in Sost and Fgf23 mRNA levels occurred concurrently with the upregulation of senescence marker genes, such as p16 and Il-6 (Fig. 5b, c). MLO-Y4 cells exhibited an increase in senescence-associated β-galactosidase (SAβ-gal) expression after induction (Fig. 5d).

HIF-1α activation enhances Fgf23 expression

We investigated the mechanisms underlying the regulation of Fgf23 transcription. There is a novel enhancer of Fgf23 at -16 kb upstream of the transcriptional start site²⁹. Additionally, the transcription factor HIF-1α activates FGF23 transcription in osteoblast-like cell lines³⁰. SIRT6 directly binds to HIF-1α, preventing its activity³¹. Thus, we focused on the role of SIRT6 and cell senescence in HIF-1α-mediated Fgf23 transcription. We examined the effect of SIRT6 deletion on the binding of HIF-1α to the Fgf23 enhancer with a chromatin immunoprecipitation (ChIP) assay using the enhancer as bait (Fig. 6a, b). HIF-1α protein was detected at low levels with the Fgf23 enhancer DNA under basal conditions (Fig. 6a). However, when Sirt6 was knocked out using the CRISPR/Cas9 system in MLO-Y4 cells, HIF-1α binding to the Fgf23 enhancer sequence increased significantly (Fig. 6a). This finding suggests that SIRT6 regulates the binding of HIF-1α to the enhancer region of Fgf23 in a cell-senescence-independent manner. We then investigated the binding of HIF-1α to the Fgf23 enhancer in senescent cells. When senescence was induced in MLO-Y4 cells after 21 days of culture without passage, HIF-1α binding to the Fgf23 enhancer increased significantly compared with that in MLO-Y4 cells under basal conditions (Fig. 6b). These data indicate that HIF-1α binds to the Fgf23 enhancer region of MLO-Y4 cells in a senescence-dependent manner as well.

To evaluate the role of HIF-1α in the regulation of Fgf23 expression, MLO-Y4 cells were cultured with an HIF-1α activator or inhibitor. The expression of Fgf23, along with that of Pai-1, was significantly increased in MLO-Y4 cells following the administration of N-(2-methoxy-2-oxoacetyl) glycine methyl ester (DMOG), a prolyl 4-hydroxylase (P4H) inhibitor, which increases HIF-1α levels by inhibiting HIF-1α prolyl hydroxylase (HIF-PH) (Fig. 6c). The expression of Sost was also increased by DMOC administration (Fig. 6c). In contrast, when MLO-Y4 cells were cultured with CAY10585, an HIF-1α inhibitor that suppresses the transcription of HIF-1α target genes, Fgf23 expression was not enhanced even when Sirt6 was knocked out (Fig. 6d). These data indicate that HIF-1α activation is indispensable for Fgf23 upregulation due to SIRT6 deficiency or senescence.

Evaluation of bone parameters in aged PAI-1-/- mice

Finally, to verify whether PAI-1 deficiency prevents age-related changes in bone tissues and mRNA transcripts, we compared bone tissues from aged PAI-1-deficient (PAI-1KO) and wild-type mice. At 6 months of age, the BV / TV of PAI-1KO was lower than that of WT and the trabecular number, trabecular separation, and trabecular spacing were higher in both the femur and lumbar spine. BMD was comparable in the lumbar spine, but PAI-1KO was lower in the femur. Conversely, at 18 months after birth, PAI-1KO was higher for BV / TV and trabecule number in both femur and lumbar spine, and PAI-1KO was lower for trabecular separation and trabecular spacing. BMD was also higher in PAI-1KO (Fig. 6e). Analysis of the cortical bone tissue by qPCR indicated that the mRNA expression of Fgf23 and Sost decreased in PAI-1-KO mice compared with those in littermates at 18 months after birth (Fig. 6f). Serum phosphate levels were elevated in 18 months-old PAI-1-KO mice compared with those in littermates (Fig. 6g).

The mechanistic regulation of bone mass in aged animals is poorly understood. We aimed to investigate the role of SIRT6, a longevity-associated factor, in osteocytes. We showed that SIRT6-deficient osteocytes express increased levels of Sost, Fgf23, and Pai-1. We proposed two signaling pathways downstream of SIRT6: a senescence-dependent and a senescence-independent pathway for the regulation of Sost and Fgf23 (Fig. 7).

SIRT6 deletion induces cell senescence^{6, 24} SIRT6 binds to telomeres and prevents cell senescence by preserving telomere function through H3K9 deacetylation^24,32. Furthermore, SIRT6 deficiency enhances Pai-1 expression, which induces replicative senescence downstream of p53^4,20,21,25. cKO mice showed increased Fgf23 expression and osteocyte senescence, and Pai-1 depletion in cKO mice led to the normalization of Fgf23 expression. Similarly, two different methods for the induction of cell senescence (LTC and doxorubicin treatment) stimulated Fgf23 expression. Senescent cells express inflammatory cytokines, such as IL-6, PAI-1, and CXCL1, as part of the senescence-associated secretory phenotype (SASP) response³². IL-6 stimulates FGF23 promoter activity through STAT3 in osteoblast-like UMR106 cells³³. NF-κB signaling plays a critical role in the induction of SASP³⁴. NF-κB activation results in FGF23 upregulation downstream of PKC or p38 MAPK^35,36. These data indicate that senescence plays a role in FGF23 regulation. In hepatic cells, SIRT6 directly binds to HIF-1α and inhibits the transcription of its target gene, Glut-1³¹. Through the deletion of Sirt6 in MLO-Y4 cells, we showed that HIF-1α was able to bind to the Fgf23 enhancer via a consensus motif within its sequence, which promotes Fgf23 transcription (Fig. 6a).

Senescent cells have a causal role in aging-related osteoporosis, and the depletion of senescent cells in the bone ameliorates aging-related osteoporosis¹⁸. Senescent cell-depleted mice present a bone tissue phenotype characterized by a reduced number of osteoclasts and an increased number of osteoblasts, both of which have been linked to the suppression of Sost expression¹⁸. These data, together with the findings of the present study, indicate that cell senescence, regulation of Sost expression, and possibly SASP, play important roles in aging-related osteoporosis. Epigenetic regulation of Sost controls sclerostin levels, which correlates with bone density and fracture rates³⁷. The promoter region of Sost has a CpG-rich domain, which contributes to the transcriptional regulation of Sost³⁸. A study of postmenopausal women showed lower Sost mRNA levels in patients with osteoporosis and significantly increased CpG methylation in the Sost promotor region³⁸. SIRT6 has histone deacetylase (HDAC) activity, and so it may regulate the expression of Sost through deacetylation of the promoter region; however, the role of the HDAC activity of SIRT6 was not elucidated in this study.

PAI-1 is a novel regulator of FGF23 metabolism³⁹. HIF-1 promotes PAI-1 transcription, thereby increasing its expression. In osteocytes, hypoxia or age-related decrease in SIRT6 activity may promote Pai-1 expression through HIF-1 activation, leading to osteocyte senescence. In fact, there was an increase in Pai-1 expression in cKO mice. (Fig. 6f, g).

The candidate senescence-independent factors for the regulation of Sost downstream of SIRT6 are HIF-1 and NF-κB. Sost expression is induced by hypoxia through HIF-1α activation in MC3T3-E1 osteoblastic cells⁴⁰. HIF-1α activates Sost transcription by binding to its promoter in MC3T3-E1 cells⁴⁰. Additionally, SOST levels are elevated by hypoxia in human Saos-2 osteogenic sarcoma cells⁴¹. Consistently, we reported that Sost expression was induced by SIRT6 deletion or cell senescence in the MLO-Y4 osteocyte-like cell line in a HIF-1α-dependent manner. Together, these results suggest that Sirt6 may bind to HIF-1α and regulate Sost expression via its HDAC activity. TNFα stimulates Sost expression through an NF-κB-dependent mechanism in MLO-Y4 cells. NF-κB directly binds to NF-κB binding elements on the Sost promoter to induce Sost transcription⁴². SIRT6 is also a negative regulator of NF-κB signaling²³. Thus, NF-κB activation may be involved in Sost upregulation in cKO mice; however, the role of NF-κB was not elucidated in this study.

A limitation of this study was that we did not obtain human data. By analyzing the correlation between SIRT6 expression and PAI-1, SOST, and FGF23 expression in bone specimens from healthy young individuals and the elderly, the mechanism of human bone aging would become more apparent.

In conclusion, we showed that osteocyte SIRT6 regulates bone volume and phosphate metabolism by modulating Sost and Fgf23 expression. This novel pathway explains the association between Fgf23 and Sost and aging-related disorders. Furthermore, PAI-1 deficiency, along with the normalization of Sost expression, in cKO mice abolished the effects of SIRT6 deletion in bone tissues. A study of the Berne Amish community members, including those that are carriers of the null SERPINE1 gene that encodes PAI-1, revealed that a rare loss-of-function mutation in SERPINE1 prolongs human life and prevents age-related metabolic abnormalities⁴³. Moreover, a small molecule PAI-1 inhibitor has been developed, and clinical trials are underway in humans⁴⁴. We previously revealed that PAI-1 inhibition via a small molecule PAI-1 inhibitor prevented ovariectomy-induced bone loss in mice⁴⁵. Nicotinamide mononucleotide (NMN) activates sirtuins, including SIRT1 and SIRT6, and its long-term administration corrects age-related metabolic disorders⁴⁶. NMN administration promotes bone formation by preventing bone marrow mesenchymal stem cells from differentiating into adipocytes in aged mice⁴⁷. NMN is confirmed to be safe in humans, and clinical studies regarding their use are being conducted⁴⁸. Overall, these results support the potential therapeutic application of a SIRT6 agonist, such as NMN or a PAI-1 inhibitor, against aging-related disruptions of bone metabolism.

All methods were carried out in accordance with ARRIVE guidelines.

Reagents

The following reagents were purchased from the indicated sources: doxorubicin (Fujifilm, Tokyo, Japan), N-(2-methoxy-2-oxoacetyl)glycine methyl ester (R&D Systems, Minneapolis, MN, USA), and CAY10585 (Abcam Biochemicals, Cambridge, UK).

Animals

All animal experiments were approved by the Animal Care and Use Committee of Tokyo Medical and Dental University and were carried out in accordance with the approved guidelines (approval number: A2018316). All mice were allowed unrestricted activity and given access to standard rodent food pellets (Labo MR Stock, Nosan, Tokyo, Japan) and tap water ad libitum. Sirt6^f/+mice (FVB.129S6(Cg)-Sirt6tm1.1Cxd/J) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Dmp1-Cre (B6N.FVB-Tg(Dmp-1-Cre)1Jqfe/BwdJ) mice were kindly supplied by Dr. Shu Takeda from the Endocrine Center, Toranomon Hospital. Sirt6^f/f mice were backcrossed for at least ten generations with C57/BL6J mice (CLEA Japan Inc., Tokyo, Japan) and then crossed with Dmp-1-Cre mice to generate Dmp-1-Cre::Sirt6^f/+ mice, and their progeny were intercrossed to obtain cKO mice. PAI-1+/- mice (C57BL6/J background) were obtained from the Jackson Laboratory. PAI-1+/- mice were crossed with Dmp-1-Cre::Sirt6^f/? mice to obtain Dmp-1-Cre Sirt6^f/+::Pai-1+/- mice. cPKO mice were obtained by crossing Dmp-1-Cre::Sirt6^f/+::PAI-1+/- mice. Male mice were euthanized at 20 weeks of age using an overdose of isoflurane inhalation followed by cervical dislocation. Male PAI-1-/- mice and their littermates were grown for 72 weeks, and their samples were analyzed by micro-CT and qPCR analysis following euthanasia. None of the mice died during the experimental period.

Histological and histomorphometric analysis

Calcein (Sigma-Aldrich, St. Louis, MO, USA) was injected subcutaneously for bone labeling five and two days prior to euthanasia. Blood and bone samples were collected at the time of euthanasia. The undecalcified samples of the spinal bone were sectioned, and the third and fourth lumbar vertebrae were stained using von Kossa and tartrate-resistant acid phosphatase (TRAP) staining, as previously described⁴⁹. The OsteoMeasure Analysis System (OsteoMetrics, Decatur, GA, USA) was used for static and dynamic histomorphometric analyses following the nomenclature defined by the American Society for Bone and Mineral Research, as previously described⁵⁰.

Immunohistological analysis

Protein expression of FGF23, sclerostin, and PAI-1 was determined by immunohistochemistry using anti-FGF23 (R&D systems; catalog Number: MAB26291), anti-sclerostin (R&D systems; catalog Number: AF1589), and anti-PAI-1 (Abcam Biochemicals; catalog number: ab66705) antibodies, according to the manufacturer’s instructions. The signals were visualized using secondary antibodies conjugated with fluorescent dyes, such as Goat Anti-Rabbit IgG (H+L), Mouse/Human ads-FITC (Southern Biotech, Birmingham, AL, USA; Catalog Number: AB_2795952), donkey anti-goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, MA, USA, Catalog Number: A-11058), and anti-Rat IgG2a Antibody Conjugate FITC (Bethyl Laboratories Inc., Montgomery, AL, USA, Catalog Number: A110-109F) according to the manufacturer’s instructions.

Micro-CT analysis

Two-dimensional images of the distal femur and lumbar spine were obtained through micro-CT analysis (Comscan, Yokohama, Japan). The following three-dimensional morphometric parameters were determined using TRI/3D-BON software (RATOC, Tokyo, Japan): bone morphometric analysis of femoral bones performed at a region of 0.2 to 1 mm above the distal growth plates of the femora, bone volume/tissue volume (BV/TV), trabecular bone thickness (Tb.Th), Tb.N, Tb.Sp, and Tb.Spac for trabecular bone analyses⁵¹. The mineralized tissue volumes were measured using a calibration curve obtained from the BMD phantom.

Cell culture and gene knockout with the CRISPR/Cas9 system and small interfering RNA (siRNA)

The mouse osteocyte-like MLO-Y4 cell line was purchased from RIKEN Cell Bank (Tsukuba, Japan). MLO-Y4 cells were cultured in alpha minimal essential medium (αMEM) containing 2.5% fetal bovine serum (FBS) and 2.5% calf serum (CS; Gibco Bovine Serum, Thermofisher scientific, Massachusetts, USA) with medium change every 3 days as described previously⁵². MLO-Y4 cells were transferred to 6-well plates and, at 70% confluency, were transfected with 50 nM Sirt6 siRNA or scrambled siRNA with HiPerFect (Qiagen, Valencia, CA, USA) in medium supplemented with 10% FBS for 24 h. The target sequence of Sirt6 siRNA was 5′-GAAGCUCCCAAUGCAAUAAAU-3′ (forward) and 5′-UUAUUGCAUUGGGAGCUUCUG-3′ (reverse).

The CRISPR/Cas9 knockout (catalog number: sc-424467-KO-2), HDR (catalog number: sc-424467-HDR-2), and control (catalog number: sc-418922) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Sirt6 knockout was performed according to the manufacturer’s instructions.

Senescence induction

For senescence induction by long-term confluency, MLO-Y4 cells were grown at confluency for 21 days without subculture^53,54. αMEM medium supplemented with 2.5% FBS, 2.5% CS (Gibco Bovine Serum), and 1% antibiotics (Gibco Antibiotic-Antimycotic (100X); catalog number: 15240062) was used to grow MLO-Y4 cells. For DNA damage-induced cellular senescence, MLO-Y4 cells were treated with 50 nM doxorubicin, and 48 h later, mRNA was harvested for qPCR⁵⁵. SA-β-gal activity was detected using the Senescence Detection Kit according to the manufacturer’s instructions (BioVision, CA, USA, catalog number: K320-250).

Chromatin immunoprecipitation assay

The EpiQuik ChIP kit (Epigentek Group Inc., NY, USA) was used for the ChIP assay, and it was performed according to the manufacturer’s instructions. To evaluate the binding of HIF-1α to the Fgf23 enhancer²⁹, PCR was carried out using a Thermal Cycler 9700 (Applied Biosystems, Foster City, CA, USA) according to a standard procedure. The primer sequences used were

5′-GTCAAGTGAGTCCGGCTTCA-3′ (forward) and

5′-CCGAGCCAGGACTTTCCTTT-3′ (reverse).

Statistical analysis

Data are presented as mean ± standard deviation. Statistical analyses of the quantitative measures among the three groups were performed using Dunnett’s test. To assess the significance of differences between groups, a two-tailed Student’s t-test was employed. Statistical significance was set at P < 0.05.

Acknowledgments

This study was supported by JSPS KAKENHI grants (grant numbers: JP18H02920 and JP18K19604).

Author Contributions

Y.A. designed and supervised the study as the corresponding author. A.A. and L.T. conducted animal studies and data acquisition. J.P., H.O., and K.T. contributed to the animal studies and statistical analyses. S.S., H.O., and A.O. contributed reagents, materials, and analysis tools. Y.A. and L.T. critically revised the manuscript. All authors approved the final manuscript.

Competing Interests

The authors declare no competing interests.

Materials requests & Correspondence

Yoshinori ASOU, [email protected]

Data Availability

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

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SIRT6-PAI-1 axis is a promising therapeutic target in aging-related bone metabolic disruption

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Evaluation of bone parameters in cKO mice

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