Cartilage-specific Sirt6 deficiency represses IGF-1 and enhances osteoarthritis severity in mice

Objectives Prior studies noted that chondrocyte SIRT6 activity is repressed in older chondrocytes rendering cells susceptible to catabolic signalling events implicated in osteoarthritis (OA). This study aimed to define the effect of Sirt6 deficiency on the development of post-traumatic and age-associated OA in mice. Methods Male cartilage-specific Sirt6-deficient mice and Sirt6 intact controls underwent destabilisation of the medial meniscus (DMM) or sham surgery at 16 weeks of age and OA severity was analysed at 6 and 10 weeks postsurgery. Age-associated OA was assessed in mice aged 12 and 18 months of age. OA severity was analysed by micro-CT, histomorphometry and scoring of articular cartilage structure, toluidine blue staining and osteophyte formation. SIRT6-regulated pathways were analysed in human chondrocytes by RNA-sequencing, qRT-PCR and immunoblotting. Results Sirt6-deficient mice displayed enhanced DMM-induced OA severity and accelerated age-associated OA when compared with controls, characterised by increased cartilage damage, osteophyte formation and subchondral bone sclerosis. In chondrocytes, RNA-sequencing revealed that SIRT6 depletion significantly repressed cartilage extracellular matrix (eg, COL2A1) and anabolic growth factor (eg, insulin-like growth factor-1 (IGF-1)) gene expression. Gain-of-function and loss-of-function studies in chondrocytes demonstrated that SIRT6 depletion attenuated, whereas adenoviral overexpression or MDL-800-induced SIRT6 activation promoted IGF-1 signalling by increasing Aktser473 phosphorylation. Conclusions SIRT6 deficiency increases post-traumatic and age-associated OA severity in vivo. SIRT6 profoundly regulated the pro-anabolic and pro-survival IGF-1/Akt signalling pathway and suggests that preserving the SIRT6/IGF-1/Akt axis may be necessary to protect cartilage from injury-associated or age-associated OA. Targeted therapies aimed at increasing SIRT6 function could represent a novel strategy to slow or stop OA.

Genotyping was carried out using PCR protocols provided by Jackson Laboratories.At 12 weeks of age, mice received daily intraperitoneal injections of tamoxifen (40 mg/kg diluted to 10 mg/ml in corn oil) for 5 days to activate cre recombinase activity of Aggrecan-Cre ERT2 and induce Sirt6 deficiency.To control for the effects of tamoxifen, Sirt6 intact mice (without Aggrecan-Cre ERT2 ) also received tamoxifen dosing.Our prior data demonstrates that treatment with tamoxifen alone or the Aggrecan-Cre ERT2 allele alone has no effect on OA outcomes in the DMM model (2).
Deletion of Sirt6 in this model has been previously established ex vivo by our group (3) and was confirmed in the present study using IHC and an antibody to SIRT6 on mouse joint tissue sections (Supplementary Figure 1).
To assess the effect of Sirt6 deficiency on surgery-induced OA, mice from each genotype (Sirt6 intact or Sirt6 cKO) were randomized to surgical groups and DMM or sham surgery was performed on the right knees of mice at 16 weeks of age, as we (2,4) and others (1, 5) have described.Mice were sacrificed at 6-or 10-weeks after surgery as these are time points that have been shown to elicit moderate and moderate-severe OA (6,7).We used n=13-15 mice for DMM groups and an n=6-9 for sham control groups.To assess the effects of Sirt6 deficiency on ageassociated OA, male Sirt6 intact and Sirt6 cKO mice received injections of tamoxifen at 12 weeks of age and were then aged to 12 or 18 months (n=15 per age group and genotype).Mice assigned to the 18-month-old groups received a second round of tamoxifen dosing (daily for 3 days) at 12 months of age.Mouse numbers were chosen based on power analyses from our previously published studies (2,4).We did not observe significant changes in body weights or gross morphological differences between mice in our study.
Micro computed tomography (µCT) analysis.Mice were sacrificed and hindlimbs were harvested, surrounding muscles were removed, and hindlimbs were fixed in 10% formalin (48 hours) prior to µCT analysis using a Bruker SkyScan 1275 scanner with 7μm 3 voxel size and Xray energies of 70 kVp and 142 μA, as previously described (8).Scans comprised of approximately 1300 slices spanning the femur, tibia, and knee joint.µCT reconstructions and quantitative analyses were performed using the SkyScan CT Analyzer (CTan) from regions of interest (medial tibia) that spanned approximately 160 coronal slices.The following structural parameters were analyzed; tibial subchondral bone volume fraction (BV/TV), Trabecular Thickness (Tb.Th), Trabecular Separation (Tb.Sp), Subchondral Bone Plate thickness (SCBP), BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) osteophyte volume, and max.osteophyte area.Osteophytes were observed as outgrowing bone protrusions compared with sham limbs, as described in (9).Histological analysis of OA.After µCT analysis, hindlimbs were decalcified (EDTA, 19%, 21 days), processed, embedded in paraffin, and sectioned (5 μm thickness) along the coronal plane, as described previously (10)(11)(12)(13).Approximately 12 midcoronal sections were collected per limb (surgical limb only) for histological and histomorphometric analysis.Mid-coronal sections were stained with hematoxylin and eosin (H&E) or toluidine blue.Articular Cartilage Structure (ACS) score (0-12 scale) and toluidine blue score (0-12 scale) were analyzed on medial tibial plateaus (MTP), lateral tibial plateaus (LTP), medial femoral condyles (MFC), and lateral femoral condyles (LFC), as previously described (13).Osteophytes (0-3 scale) were scored on the MTP and LTP and synovial hyperplasia (0-3 scale) was analyzed in the medial compartment (13) from H&E stained sections.All sections were scored by two individuals blinded to experimental groups who had been trained to use the grading schemes.Histomorphometric measurements of articular cartilage thickness and area, calcified cartilage thickness and area, and subchondral bone thickness and area were analyzed on the MTP and LTP, as previously described (12,13), using ImageJ software.
Cell culture of human chondrocytes and mouse femoral cap explants.Normal (nonosteoarthritic) human primary human articular chondrocytes were isolated from tali cartilage of deceased human tissue donors provided by the National Disease Research Interchange and a collaboration between The Gift of Hope Organ and Tissue Donor Network (Itasca, IL) and Rush University Medical Center (Chicago, IL), with IRB approval.Cartilage was macroscopically inspected prior to dissection and scored for degeneration using a modified version of the 5-point Collins grading system (14).Only normal appearing cartilage was used (grade 0-2).Normal cartilage was obtained from a total of 23 donors (male, 17; female, 6; age range 39-84 years, av.age; 60.3±12.6 years).Chondrocytes were isolated by sequential digestion of cartilage with Pronase and Collagenase P and maintained in monolayer culture in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics.
Chondrocytes (passage 0) were cultured at high density and grown to experimental confluence (80-90%) and cultured in serum-free conditions prior to experimental incubations.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Femoral head cartilage from the hip joints of 8-week-old Sirt6 intact and Sirt6 cKO mice (n=3) were dissected and cultured as explants as we have previously described (3,15).Femoral cap explants were treated with 5 μM 4-Hydroxytamoxifen daily for 4 days to activate Cre recombinase activity of Aggrecan-Cre ERT2 and induce Sirt6 deficiency.Cartilage explants were maintained in DMEM supplemented with 10% FBS and antibiotics.
Knockdown of SIRT6 by Amaxa nucleofection of siRNA.Knockdown of SIRT6 was achieved by electroporation of SIRT6 siRNA (SignalChem) using Amaxa Nucleofection (Lonza).Human chondrocytes were cultured in monolayer in 10 cm dishes until 80% confluency was achieved.Chondrocytes were then isolated by incubation in a Collagenase P and Pronase solution (1mg/ml in serum containing media) until chondrocytes were in a single cell suspension (60-120 mins).Cells were centrifuged (90 xg, 8 mins) and washed twice in 1X PBS.
Chondrocytes were then maintained in serum containing DMEM for 48 hours prior to lysis.For SIRT6 activation studies, confluent human chondrocytes were treated with the small molecule activator of SIRT6, MDL-800 (12.5 µM), for 0-24 hours and then total cell lysates were prepared.SIRT6 activity was analyzed in human chondrocyte lysates by isolation of histones and analysis of H3K9 acetylation (H3K9ac) as we have previously described (3).Analysis of H3K9ac is routinely performed by our lab, and others, as an inverse marker of SIRT6 activity (3,(17)(18)(19)(20)(21)(22).
Histones were isolated from total cell lysates using the Histone Extraction Kit (Abcam).
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

RNA-sequencing analysis.
Total RNA Extraction, Library Preparation and RNA-sequencing SIRT6 was depleted in human chondrocytes by Amaxa nucleofection of siRNA.Chondrocytes nucleofected with a scrambled siRNA were used as controls as described above.Total RNA from cells was extracted using the NucleoSpin RNA mini kit for RNA purification (Macherey-Nagel) according to manufacturer's instructions.Library preparation, RNA-sequencing, and initial data analysis was carried out by Novogene.In brief, RNA quality was assessed using an Agilent 2100 bioanalyzer.Messenger RNA was purified from total RNA using poly-T oligoattached magnetic beads.Following fragmentation, the first strand cDNA was synthesized using random hexamer primers.Second strand cDNA synthesis was undertaken using either dUTP for directional library or dTTP for non-directional library.For non-stranded library preparation polyA enrichment was utilized to capture mRNA fragments.The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.Quantified resulting pooled libraries were sequenced on Novaseq 6000 (Illumina) with 270-300bp pairedend reads.RNA-sequencing data has been submitted to the National Centre for Biotechnology Information Gene Expression Omnibus (NCBI GEO) under Array Express accession number GSE235082.

RNA-sequencing Data Analysis
Overall quality of the next-generation sequencing data was evaluated using in-house perl scripts.
Clean reads were obtained following removal of reads containing adapters, reads containing ploy-N and low-quality reads from raw data.All the downstream analyses were based on the clean data with high quality.Reads were mapped against the reference genome Homo Sapiens (GRCh38/hg38) and gene model annotation files were downloaded from genome website directly (http://ftp.ensembl.org/pub/).Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean 2 reads were aligned to the reference genome using Hisat2 v2.0.5.FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene (23)(24)(25).
Parameters for mismatches were 2-6 max and min penalties for mismatch; lower qual = lower penalty <2,6>. of each gene was calculated based on the length of the gene and reads count mapped to this gene.Differential expression analysis of two conditions/groups (two biological BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) replicates per condition) was performed using the DESeq2 R package (1.20.0)(26).The resulting P-values were adjusted using the Benjamini and Hochberg's approach to control the false discovery rate.Genes with an adjusted P-value <=0.05 found by DESeq2 were assigned as differentially expressed.Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor.Differential expression analysis of two conditions was performed using the edgeR R package (3.22.5).Corrected P-value of 0.05 and absolute fold change of 2 were set as the threshold for significantly differential expression.

Metaboanalyst Analysis
RNA expression was normalized by median and pareto scaled prior to multivariate analysis.All principal component analysis (PCA) and heat map analysis was conducted using MetaboAnalyst 4.0 (http://www.metaboanalyst.ca).

Ingenuity Pathway Analysis
Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood City, California, US), was used to analyze the RNASeq data for canonical pathways, upstream regulators, and disease and function analysis using the list of differentially expressed genes (log2FC cutoff of ±2 and FDR<0.05).All molecules were overlaid onto a global molecular network contained in the Ingenuity Knowledge base.Networks of network-eligible molecules were algorithmically generated based on their connectivity.The functional analyses identified the canonical pathways, upstream regulators and biological functions and diseases that were most significant to the data set.A right-tailed Fisher's exact test was used to calculate the raw p-values.The z-score was used to predict the activation or inhibition state of the molecules in our datasets.Canonical pathways, upstream regulators and biological functions and diseases which were likely activated (based on the pattern of differentially abundant proteins or metabolites) were presented in orange (positive z-score), those that were likely inhibited were presented in blue (negative z-score), and those with a z-score which is zero (or close to zero) or ineligible for prediction were presented in white or grey, respectively.
RT-qPCR from human chondrocytes.RNA isolation from human chondrocytes was performed using the NucleoSpin RNA mini kit for RNA purification (Macherey-Nagel) according to BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) manufacturer's instructions.Reverse transcription (500ng of RNA) was performed using the RNA to cDNA EcoDry Premix (Takara) and PCR was performed using Power SYBR ® Green PCR mastermix (Applied Biosystems) and gene-specific oligos purchased from Integrated DNA Technologies (IDT) on a QuantStudio™ 3 real-time PCR system (Applied Biosystems).Primer sequences used in the study are presented in Supplementary Table 1.Gene expression was normalized to HPRT1 as a housekeeping control and analysis of gene expression was conducted using the 2 -ΔΔCq method (27,28).Data is presented as fold change comparing Sirt6 depleted cells to controls.
Immunoblotting.After experimental stimulations, human chondrocytes were incubated in standard lysis buffer for 30 mins under gentle agitation at 4 • C. Mouse femoral cap cartilage explants were lysed and homogenized using a Precellys homogenizer (Bertin Technologies) as previously described (3).Cell lysates were centrifuged at 12,000 rpm (10 mins) to remove nonsoluble proteins and protein concentrations were determined using the Pierce™ micro-BCA assay (Thermo Fisher Scientific).Immunoblotting was performed on cell lysates under reducing conditions (10% β-mercaptoethanol) using specified antibodies as we have previously described (15).Immunoblots were stripped and reprobed with total antibodies or β-tubulin/β-actin as loading controls.All antibodies were used at a 1:1000 dilution (in Tris-buffered saline with 0.1% Tween (TBST) and 5% blotting grade blocker (Bio-Rad)).Densitometric analysis was performed using ImageJ software.

Immunohistochemistry (IHC).
IHC was performed on mouse joint tissue sections using the VECTASTAIN™ Elite™ ABC Kit as we have previously described (3).Joint tissue sections were incubated in 3% H2O2 (10 min) prior to sodium citrate antigen retrieval (10 mM sodium citrate buffer, pH 6.0) at 90ºC for 30 minutes.Sections were rinsed in PBS, blocked in 5% donkey serum, and incubated overnight at room temperature in primary antibodies to SIRT6, IGF-1, or p16 ink4a .Sections were rinsed in PBS and then incubated in biotinylated secondary antibody for 1 hour.The secondary antibody was removed and sections were incubated in VECTASTAIN™ Elite™ Avidin-Biotin Complex solution for 30 min and then incubated in Vector™ DAB peroxidase substrate.Slides were counterstained with hematoxylin and imaged using an EVOS M5000 imaging system.Percent positive cells were analyzed using ImageJ.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Gene
Sequence (5' -3') Amplicon (bp) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) supplemental material which has been supplied by the author(s) supplemental material which has been supplied by the author(s) supplemental material which has been supplied by the author(s) Statistical analysis was performed in GraphPad Prism version 9.All data are presented as mean values ± SD and individual data points are presented in graphs.Exact biological replicates are presented in figure legends.Normal distribution of data was tested using the D'Agostino and Pearson normality test and normally distributed data was analyzed using paired or unpaired students t-tests when comparing two groups, or one-way ANOVA as indicated in figure legends.Data that was not normally distributed was analyzed by Mann-Whitney tests comparing experimental groups to controls.Significant p-values are presented in graphs with a p-value of <0.05 deemed significant.BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Collins JA and percentage SIRT6 positively stained cells were quantified.Individual data points are presented with mean ± standard deviation (SD).Significant differences between groups were detected by unpaired t-test.Exact p-values are presented.BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)