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Published in final edited form as: Osteoarthritis Cartilage. 2015 Dec 31;24(6):1021–1028. doi: 10.1016/j.joca.2015.12.014

Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis

Elizabeth W Bradley 1, Lomeli R Carpio 2, Meghan E McGee-Lawrence 1,5, Clara Castillejo Becerra 1, Derek F Amanatullah 1, Lauren E Ta 4, Miguel Otero 6, Mary B Goldring 6, Sanjeev Kakar 1, Jennifer J Westendorf 1,3
PMCID: PMC4875839  NIHMSID: NIHMS748856  PMID: 26746148

Abstract

OBJECTIVE

Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability. OA is characterized by articular chondrocyte deterioration, subchondral bone changes and debilitating pain. One strategy to promote cartilage regeneration and repair is to accelerate proliferation and matrix production of articular chondrocytes. We previously reported that the protein phosphatase Phlpp1 controls chondrocyte differentiation by regulating the activities of anabolic kinases. Here we examined the role of Phlpp1 in osteoarthritis progression in a murine model. We also assessed PHLPP1 expression and promoter methylation.

DESIGN

Knee joints of WT and Phlpp1−/− mice were surgically destabilized by transection of the medial meniscal ligament (DMM). Mice were assessed for signs of OA progression via radiographic and histological analyses, and pain assessment for mechanical hypersensitivity using the von Frey assay. Methylation of the PHLPP1 promoter and PHLPP1 expression was evaluated in human articular cartilage and chondrocyte cell lines.

RESULTS

Following DMM surgeries, Phlpp1 deficient mice showed fewer signs of OA and cartilage degeneration. Mechanical allodynia associated with DMM surgeries was also attenuated in Phlpp1−/− mice. PHLPP1 was highly expressed in human articular cartilage from OA patients, but was undetectable in cartilage specimens from femoral neck fractures. Higher PHLPP1 levels correlated with less PHLPP1 promoter CpG methylation in cartilage from OA patients. Blocking cytosine methylation or treatment with inflammatory mediators enhanced PHLPP1 expression in human chondrocyte cell lines.

CONCLUSION

Phlpp1 deficiency protects against OA progression while CpG demethylation and inflammatory responses promote PHLPP1 expression.

Keywords: DNA methylation, articular cartilage, Il6, TNFα, mechanical allodynia, DMM mouse model

INTRODUCTION

Osteoarthritis (OA) is a leading cause of pain and disability. The underlying causes of OA are individualized and heterogeneous, ranging from biomechanical instability, traumatic injury, genetics and systemic inflammation [1]. Regardless of the etiology, OA is characterized by both structural and cellular changes to articular cartilage and subchondral bone. Structural changes to osteoarthritic articular cartilage include decreased matrix content, subchondral bone thickening and surface disruptions that range from fibrillations, clefting and delamination to complete articular surface erosion [1–3]. Cellular changes recapitulate chondrocyte differentiation occurring during endochondral ossification [1–3]. These changes include increased chondrocyte cloning, undesired induction of chondrocyte hypertrophy, decreased cartilage matrix gene expression, increased production of cartilage degrading enzymes (e. g., ADAMTS, MMPs), chondrocyte apoptosis, and excessive ossification/osteophyte formation [1–3]. Transcriptional changes underpin decreased cartilage anabolic gene expression and enhanced catabolic transcript production during the OA disease process.

Recent data indicate that epigenetic modifications, including DNA methylation, post-translational modification of histones and the presence of non-coding RNAs, contribute to gene expression changes during OA progression [4]. Of these modifications, DNA methylation is the most extensively characterized in OA [5–10]. Although sometimes associated with gene activation, methylation of clustered CpG dinucleotides within a gene promoter is most often correlated with gene repression [11]. CpG methylation of promoters suppresses transcription by impeding transcription factor binding and/or by recruiting methyl-binding proteins and chromatin silencing proteins (reviewed in [12]). Conversely, promoter demethylation enhances gene expression. Inflammatory signals present within OA joints promote DNA CpG demethylation [13], and several studies have reported altered DNA methylation of genes affecting cartilage catabolism [14–19] and anabolism [20–23] during OA progression.

Phlpp1 is a Ser/Thr phosphatase that decreases the activity of several kinases that promote cartilage anabolic signaling, including Akt2, p70 S6 kinase, and protein kinase C (PKC) [24–27]. Deficiency in Phlpp1 decreases snout-to-tail body length as well as long bone length [28, 29]. We previously demonstrated that Phlpp1 deletion promotes chondrocyte proliferation and matrix production [28]. Because of its role in chondrocyte differentiation, we sought to determine if Phlpp1 affects OA progression. Here we report that Phlpp1−/− mice are significantly protected against cartilage deterioration associated with surgically-induced OA. Phlpp1 deficiency also increases the cellular content and the thickness of articular cartilage. Furthermore, elevated PHLPP1 levels were found in OA patient specimens. Increased PHLPP1 levels in human OA specimens were attributed to epigenetic changes and inflammation associated with OA, as DNA methylation inhibitors and inflammatory cytokines augmented PHLPP1 levels.

MATERIALS AND METHODS

Phlpp1 Deficient Mice

Wild-type (WT) and Phlpp1−/− littermates [29] were maintained in an accredited facility with 12-hour light/dark cycle and supplied water and food (PicoLab® Rodent Diet 20, LabDiet) ad libitum. All animal research was conducted according to National Institute of Health and the Institute of Laboratory Animal Resources, National Research Council guidelines. The Mayo Clinic Institutional Animal Care and Use Committee preapproved all animal studies.

Mouse Microsurgeries

Destabilization of the medial meniscal ligament (DMM) surgeries were performed according to Glasson et al. [30]. Briefly, 12 week-old male mice (WT = 5, Phlpp1−/− = 7) were anesthetized with isofluorane and the right hind limb was shaved and sterilized with iodine prior to surgery. An incision was made over the knee and the medial meniscal ligament was transected. The joint capsule was closed with an 8−0 Vicryl suture and the skin was sutured with 7−0 Vicryl. Animals received pre-operative and post-operative analgesics (0.09 mg/kg buprenorphine) every 12 hours for two days. Hard plastic (CPVC) tubes measuring 10 cm long × 6 cm wide × 4.8 cm tall were placed in each cage as activity enrichment devices [31]. Mice were euthanized 12 weeks after surgery.

Faxitron Imaging

Radiographs of the surgically altered, right hind limbs of mice were collected using a Faxitron X-ray imaging cabinet (Faxitron Bioptics, Tucson, AZ) and assessed for structural changes to overall joint architecture and bone density for up to 12 weeks after DMM surgeries.

Histological Assessment

Tibiae from 4-week-old mice (WT = 4, Phlpp1−/− = 3) were fixed in 10% neutral buffered formalin, decalcified in 15% EDTA for 7 days, paraffin embedded, sectioned, and stained with Alcian blue (1% Alcian blue, 3% acetic acid). Hind limbs from 24 week-old mice subjected to DMM surgeries (WT = 5, Phlpp1−/− = 7) were processed similarly except decalcification lasted 14 days. Sections were collected every 100 µM and Safranin O/Fast Green staining was performed. Three reviewers scored deidentified sections following OARSI standardized methods [32].

von Frey Filament Assays

WT (n = 5) and Phlpp1−/− (n = 7) mice were assessed using the von Frey filament assay one week prior to DMM surgeries and 6 and 12 weeks post-surgery as previously reported [33]. Briefly, mice were habituated to the testing chambers (plexiglass cubicles with a mesh floor) two weeks prior to baseline readings, and for one hour before each testing. Mechanical sensitivity was measured by determining the hind paw-withdrawal threshold with calibrated von Frey nylon monofilaments (0.16 g) using the up-down method of Dixon [34]. Mice were assessed three times at each time point and percent change from baseline readings is reported.

Equilibrium Partitioning of an Ionic Contrast Agent with Micro-computed Tomography (EPIC μ-CT)

Articular cartilage thickness of 4 week-old Phlpp1−/− (n = 7) or WT (n = 7) mice was assessed by EPIC μ-CT as previously reported [35–39]. Briefly, right femora were carefully cleaned of soft tissues and submerged in a 20% Hexabrix solution for one hour. The epiphysis of each femur was scanned in air on a μCT35 scanner (Scanco Medical AG, Basserdorf, Switzerland) at 7 µM voxel size (scanner settings: energy = 45 kVp, I=88 μA, integration time = 800 ms). Cartilage was selected for visualization by manual ROI segmentation (sigma = 2.0, support = 4.0, threshold = 90 to 315).

Immunohistochemical Staining

Surgically discarded, de-identified tissues from patients undergoing total knee arthroplasties for OA (n = 6) or patients undergoing a femoral neck fracture (FNFx, n = 4) repair were collected with approval from the Institutional Review Board. Specimens were fixed in 10% neutral buffered formalin, decalcified in 15% EDTA for 7 days, paraffin embedded and sectioned. Immunohistochemical (IHC) staining was performed with antibodies directed to Phlpp1 (Abcam, Cambridge, MA), or with an isotype control IgG. Detection was accomplished using the Mouse and Rabbit Specific HRP (ABC) Detection IHC Kit (Abcam) using the substrate 3,3’-diaminobenzidine (Sigma Aldrich, St. Louis, MO). Sections were counterstained with Fast Green stain. Staining intensity of individual cells (n = 5) within each tissue specimen was quantified using Image J software.

Chondrocyte Cell Culture

ATDC5, C28/I2 and T/C28a2 cells were maintained in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic. For experiments, ATDC5 C28/I2 or T/C28a2 cells were seeded at a density of 2.7×104 cells/cm2 and incubated overnight. To inhibit DNA methylation, 1 µM 5-azacytidine or vehicle (PBS) was added on days 1 and 3 of culture. Cells were collected on day 4 for analyses. Inflammatory cytokines (10 ng/ml IL-1, 20 ng/ml IL-6, 10 ng/ml TNFα), 100 mM H2O2 or vehicle were added for one hour prior to lysis. Primary chondrocytes from 5-day-old mice were collected as previously described [28, 40] and cultured in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic.

RNA Isolation and Real-Time PCR

Total RNA was extracted from cell lines and primary chondrocytes cells using TRIzol (Invitrogen) and chloroform, and 2 µg was reverse transcribed using the SuperScript III first-strand synthesis system (Invitrogen). The resulting cDNAs were used to assay gene expression via real-time PCR using the following gene-specific primers: PHLPP1 (5′-AGCTGAAAGCCATCCCCAACA-3′, 5′-GCTCAGGTCCACACACTTGA-3′), GAPDH (5′-GACCTGACCTGCCGTCCTAGAAA-3′, 5′-CCTGCTTCACCACCTTCTTGA-3′) [41]. Fold changes in gene expression for each sample were calculated using the 2−ΔΔCt method relative to control after normalization of gene-specific Ct values to GAPDH Ct values [41]. Each experiment was performed in triplicate and repeated at least three times. Results from a representative experiment are shown.

Phlpp1 Promoter Methylation, Methylome Sequencing and Bioinformatics Analysis

Genomic DNA was isolated from regions of damaged and undamaged articular cartilage of patients undergoing total knee arthroplasties for OA (n = 5) or patients undergoing total hip arthroplasty to repair a femoral neck fracture (FNFx, n = 9). Overall percent methylation of the PHLPP1 promoter was estimated using the EpiTect II DNA Methylation Enzyme Kit (Qiagen, Valencia, CA). Briefly, this kit includes methylation-sensitive and methylation-dependent restriction enzymes that differentially digest methylated and non-methylated DNA. After digestion, samples were amplified by qPCR using supplied primers and overall promoter methylation was estimated as recommended by the manufacturer. To identify specific CpG sites altered in OA, reduced representation bisulfite sequencing (RRBS) was performed on genomic DNA from damaged OA (n = 14) cartilage or FNFx (n = 6) cartilage using the Illumina HiSeq 2000 Genome Analyzer (San Diego, CA) as previously described [42]. Percent methylation at each CpG site within the PHLPP1 proximal promoter (<2.0 kB of the transcriptional start site) was evaluated using Integrative Genomics Viewer (IGV) software. Putative transcription factor binding sites were identified using JASPAR.

PHLPP1 Promoter Cloning and Transcriptional Assays

A portion (−1589 to +202 bp) of the PHLPP1 promoter was constructed by GenScript. This sequence was subcloned into a CpG-free luciferase reporter [13]. Plasmids were left untreated or treated in vitro with the CpG methyltransferase M. Sssl and transfected into T/C28a2 cells. After 48 hours, luciferase activity was measured with the Dual Luciferase Reporter Assay System (Promega, Madison, WI).

Image Quantitation

Images were digitally scanned or collected using phase contrast microscopy. The average number of articular chondrocytes per tissue area was evaluated using NIH Image J software.

Statistical Analysis

Data obtained are the mean ± standard error of the mean (SEM). p values were determined with the student's t-test.

RESULTS

Phlpp1 deficiency protects against OA progression

We recently showed that Phlpp1 deficiency promotes anabolic signaling in immature chondrocytes [28]. Here we sought to determine if Phlpp1 deficiency protected articular cartilage from degeneration induced by surgical destabilization of the medial meniscal tibial ligament [30]. Surgeries were performed on skeletally mature 12-week-old male WT and Phlpp1−/− mice. All animals resumed weight-bearing after surgery. We evaluated both groups radiologically and histologically 12 weeks post-surgeries. Similar to previous reports, articular cartilage damage induced by DMM surgery was most pronounced at the medial tibial plateau (Figure 1A) [43–45]. WT mice had clear signs of OA, including increased chondrocyte hypertrophy, reduced Safranin O staining, articular cartilage erosion and delamination of the articular surface (Figure 1A). Phlpp1−/− mice were resistant to these changes and had an OARSI score 1.7 points lower than WT mice. The score reduction was driven primarily by decreased OA grade (Figure 1B). Thus, Phlpp1 deficiency reduced the severity of OA induced by DMM surgery, but the area of affected tissue was mostly unchanged. Phlpp1 deficient mice also demonstrated reduced subchondral bone thickness compared to WT mice after DMM surgery (Figure 2).

Figure 1. Phlpp1 deletion protects against post-traumatic osteoarthritis.

Figure 1

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Twelve week-old male WT (n = 5) or Phlpp1−/− (n = 7) mice were subjected to DMM surgeries and evaluated 12 weeks post-surgery. (A) Safranin O/Fast Green staining from WT and Phlpp1−/− mice subjected to DMM surgeries. The left panels are 4X magnifications of the knee joint and the right panels are 40X magnifications of the medial knee surfaces. (B) OARSI scores from WT and Phlpp1−/− mice, * p < 0.05.

Figure 2. Phlpp1 deletion minimizes subchondral bone thickening associated with post-traumatic OA.

Figure 2

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(A) Radiographs from WT and Phlpp1−/− mice 12 weeks-post DMM surgery. The arrow denotes an area of increased subchondral thickness. (B) Mean gray values of the medial subchondral bone radiographs shown in (A) from WT (n = 5) and Phlpp1−/− (n = 7) mice were determined using Image J software, *p < 0.05.

Phlpp1 null mice are protected from secondary allodynia associated with OA

Pain associated with OA is the primary reason patients seek medical care. Therefore, we assessed the effects of Phlpp1 deficiency on pain following DMM surgeries using the von Frey filament assay. WT mice showed increased secondary allodynia at 6 and 12 weeks post-surgery (Figure 3). However, pain levels did not change in Phlpp1−/− mice following DMM surgery (Figure 3). These data support the conclusion that Phlpp1 deficiency reduces OA severity.

Figure 3. Pain following OA surgery is attenuated by Phlpp1 deficiency.

Figure 3

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Percent change in paw withdrawal frequency from baseline using a 0.16 g filament observed at 6 and 12 weeks post-surgery, *p < 0.05, #p < 0.1.

Articular cartilage of Phlpp1 deficient mice has greater cellular content

To determine if Phlpp1 facilitates articular cartilage maintenance, we histologically assessed the articular cartilage of 4-week-old WT or Phlpp1−/− mice (Figure 4A). The cellular content of articular cartilage was increased approximately 20% on the proximal tibiae of Phlpp1−/− mice (Figure 4B). Increased cell density was particularly evident at the superficial layer (Figure 4A). Phlpp1−/− mice also displayed a strong trend towards thicker articular cartilage as measured by EPIC μ-CT (Figure 4C). These results suggest that loss of Phlpp1 may promote articular cartilage maintenance.

Figure 4. Phlpp1 deficiency increases cellular content and articular thickness.

Figure 4

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(A) Sections from paraffin embedded tibiae of 4 week-old WT (n = 3) or Phlpp1−/− (n = 4) mice were stained with Alcian blue and (B) the number of articular chondrocytes per tissue area was determined, *p < 0.05. (C) Articular cartilage thickness (mm) of 4 week-old WT (n = 7) and Phlpp1−/− (n = 7) mice was measured by EPIC μCT, #p = 0.07.

PHLPP1 expression and promoter CpG methylation are altered in human OA samples

Since Phlpp1 deficiency protected joints from cartilage deterioration in a surgically-induced, murine model of OA, we determined if PHLPP1 levels were altered in human OA cartilage specimens using IHC. Articular cartilage specimens were collected from patients undergoing a joint arthroplasty surgery due to OA or a femoral neck fracture (FNFx) where the articular cartilage showed minimal degeneration. Whereas minimal PHLPP1 immunostaining was observed in FNFx articular cartilage, OA articular cartilage chondrocytes displayed robust PHLPP1 immunostaining (Figure 5).

Figure 5. PHLPP1 expression is elevated in human OA tissue.

Figure 5

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Surgically discarded tissue from total joint arthroplasties due to OA or femoral neck fracture (FNFx) repair was collected. (A) Sections from paraffin embedded tissues were incubated with an isotype control IgG (left column) or an anti-PHLPP1 antibody (right columns). Shown are representative 10X and 40X images. (B) Average PHLPP1 staining intensity from OA (n = 6) and FNFx (n = 4) tissues was determined using Image J software, *p < 0.05.

The PHLPP1 promoter contains several CpG dinucleotides within the proximal promoter and CpG demethylation is frequently associated with increased expression of OA-linked genes. We next sought to determine if PHLPP1 DNA methylation was altered in OA specimens and could be a mechanism responsible for increased PHLPP1 levels. Overall methylation of the PHLPP1 promoter region was less than 2.5% in cartilage collected from either damaged or undamaged articular cartilage, but ranged from 1–31% in genomic DNA isolated from FNFx specimens (Figure 6A). Methylome sequencing identified six clustered CpG dinucleotides (−1532, −1616, −1514, −1491 and −1462) within the PHLPP1 proximal promoter that were differentially demethylated in OA samples as compared to FNFx specimens (Figure 6BC) [42]. To determine if DNA methylation controls PHLPP1 mRNA levels, we treated the human chondrocytic cell lines, C28/I2 and T/C28a2, with the DNA methyltransferase inhibitor, 5-azacytidine. PHLPP1 transcript levels increased by approximately two-fold following 5-azacytidine treatment (Figure 6D). To determine if the effects of PHLPP1 promoter demethylation directly affected PHLPP1 expression, a portion of the PHLPP1 promoter was cloned into a CpG-free luciferase reporter [13]. The PHLPP1-luciferase reporter or empty vectors were then treated in vitro with the M. Sssl CpG methyltransferase. Methylation of the PHLPP1 reporter completely abrogated transcriptional activity of this reporter in T/C28a2 cells (Figure 6E). Finally, since chronic exposure to inflammatory cytokines can induce DNA demethylation, we next assessed whether or not inflammatory mediators induced PHLPP1 expression. IL-6, TNFα and H2O2, an inducer of hypoxia inducible factor (HIF), all increased PHLPP1 expression in T/C28a2 cells (3-, 17- and 12-fold respectively, Figure 6F). IL1 did not drastically change PHLPP1 expression. Similar results were observed in ATDC5 cells and mouse primary chondrocytes; however, the increases in Phlpp1 expression were only 2-fold (data not shown). Together, these results indicate that the PHLPP1 promoter is hypomethylated in OA articular cartilage and correlates with increased PHLPP1 levels.

Figure 6. The human PHLPP1 promoter is demethylated in OA.

Figure 6

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(A) Genomic DNA was isolated from FNFx articular cartilage (n = 9). Damaged and undamaged portions of articular cartilage from total knee arthroplasty specimens (n = 5) were also collected. Percent methylation of the CpG island within the Phlpp1 promoter using the Epitect II DNA Methylation kit for each sample was determined. (B,C) Genomic DNA was isolated from FNFx (n = 6) or OA (n = 14) specimens and reduced representation bisulfite sequencing was performed. (B) Differentially methylated CpG sites and putative transcription factor binding sites in the PHLPP1 promoter. Percent methylation at each site for each specimen (FnFx: F, OA: O) is noted by shading intensity. (C) Average percent methylation at each CpG site was determined in all OA or FNFx specimens from (B), *p < 0.05, #p < 0.06. (D) C28/I2 and T/C28a2 human chondrocytes were treated with 5-azacytidine (AzaC) or PBS. Expression of PHLPP1 was determined by qPCR, *p < 0.05. (E) The PHLPP1-LUC reporter or empty vector were treated in vitro with the CpG methyltrasferasae M. Sssl or left untreated. Plasmids were then transfected into T/C28a2 cells and luciferase activity was measured. (F) T/C 28a2 chondrocytes were treated with the indicated inflammatory mediators for one hour and expression of PHLPP1 was evaluated by qPCR, *p < 0.05. The dotted line denotes a 1.0 fold change.

DISCUSSION

In this report we examined the effects of Phlpp1 deficiency on articular cartilage and explored the ability of Phlpp1 to repress OA progression in a surgically-induced model. Phlpp1 deficiency increased the cellular content of articular cartilage and limited OA progression induced by destabilization of the medial meniscal tibial ligament. In human OA, PHLPP1 was minimally expressed by chondrocytes from healthy articular cartilage, but was easily detected in chondrocytes from human OA articular cartilage. Elevated levels in OA cartilage were correlated with decreased CpG methylation within the PHLPP1 promoter and inhibition of DNA methylation augmented PHLPP1 expression. Inflammatory mediators and HIF induction also enhanced expression of PHLPP1 by human chondrocytes. Thus, PHLPP1 expression may be induced by inflammatory and oxidative stress associated with OA and facilitate disease progression.

Our previous studies showed that Phlpp1 deficient mice have higher numbers of proliferating chondrocytes in the growth plate, resulting in more matrix production [28, 46]. These effects were associated with more Akt2 and PKC phosphorylation, as well as more Fgf18 expression [28]. Thus, Phlpp1 suppresses growth plate development. Here we demonstrate the Phlpp1 deficiency also increases articular chondrocyte cellular content and limits cartilage matrix loss associated with OA. We also noted decreased chondrocyte hypertrophy associated with OA in Phlpp1 deficient animals. Our previous work demonstrated that Hdac3 represses chondrocyte hypertrophy and Phlpp1 expression; thus, Phlpp1 may be a positive regulator of chondrocyte hypertrophy [46]. Establishing a direct effect of Phlpp1 on chondrocyte hypertrophy will be an area for future study.

DNA methylation reduces gene transcription by blocking transcription factor binding [12]. Since PHLPP1 expression was inversely associated with decreased CpG methylation, we assessed potential transcription factor consensus binding sites located at affected CpG sites (Figure 6B). The most distal CpG site (−1532) encompasses a c-Rel/NF-κB consensus sequence followed by two E2F binding sites (−1516, −1514). Also located within the demethylated CpG sequence are two HIF consensus sites (−1504, −1491) and a THAP1 binding site (−1462). NF-κB and HIF signaling are frequently associated with inflammatory conditions, such as OA, and Thap1 mediates the effects of TNFα [47]. Changes to articular chondrocyte DNA methylation can be induced by repeated and chronic exposure to inflammatory cytokines [13]. Although we do not know if demethylation is a cause or consequence of OA, we hypothesize that PHLPP1 induction within an inflammatory environment will require demethylation of the CpG sites that normally block binding of transcription factors associated with inflammation, such as the NF-κB, THAP1 and HIF consensus sequences we identified in the PHLPP1 promoter. Taylor et al. also recently noted a differentially methylated region within the PHLPP1 promoter in primary OA chondrocytes [48]. One limitation of our study is that we compared cartilage from femoral neck fracture repair surgeries, where the cartilage showed minimal degeneration, to OA articular cartilage from the knee. Future studies will examine changes in PHLPP1 expression and transcription in hip OA specimens.

In summary, Phlpp1 deficiency limited OA progression in a surgically-induced model and increased the cellular content of murine articular cartilage. Whereas PHLPP1 immunostaining was restricted in control specimens, OA chondrocytes displayed robust PHLPP1 immunoreactivity. Increased PHLPP1 levels were correlated with decreased DNA methylation in situ at CpG dinucleotides located within consensus binding sites for inflammatory mediators (e.g. NF-κB, THAP1 and HIF). Further, PHLPP1 was induced by inflammatory cytokines such as IL-6 and TNFα and reactive oxygen species production. Together, these results indicate that PHLPP1 could be a therapeutic target for disease modifying OA drugs. Future work will be aimed at refining the transcriptional mechanisms that control PHLPP1 expression in response to inflammatory agents and defining the cell type-specific roles of Phlpp1 in limiting OA progression.

Acknowledgments

We thank Xiaodong Li, David Razidlo, Bridget Stensgard and the Mayo Division of Biomedical Statistics and Informatics for technical assistance. This work was made possible by research and training grants from the National Institutes of Health (AR065397, GM055252 and AR68103), the State of Minnesota Regenerative Medicine Initiative, the Mayo Clinic Center for Individualized Medicine, and the Mayo Graduate School. These contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Footnotes

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Authors’ Contributions

Study design: JJW, EWB, MBG, LET, MO

Data collection: EWB, LRC, MML, DA, SK

Interpretation of data: JJW, EWB, MML, LRC, MGB, SK, LET, MO

Drafting of article: EWB, JJW

Final approval: JJW

Conflict of Interest

The authors have no conflicts of interest to disclose.

References

  • 1.Lories RJ, Luyten FP. Osteoarthritis, a disease bridging development and regeneration. Bonekey Rep. 2012;1:136. doi: 10.1038/bonekey.2012.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 2012;64:1697–1707. doi: 10.1002/art.34453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Suri S, Walsh DA. Osteochondral alterations in osteoarthritis. Bone. 2012;51:204–211. doi: 10.1016/j.bone.2011.10.010. [DOI] [PubMed] [Google Scholar]
  • 4.Goldring MB, Marcu KB. Epigenomic and microRNA-mediated regulation in cartilage development, homeostasis, and osteoarthritis. Trends Mol Med. 2012;18:109–118. doi: 10.1016/j.molmed.2011.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.den Hollander W, Ramos YF, Bos SD, Bomer N, van der Breggen R, Lakenberg N, et al. Knee and hip articular cartilage have distinct epigenomic landscapes: implications for future cartilage regeneration approaches. Ann Rheum Dis. 2014;73:2208–2212. doi: 10.1136/annrheumdis-2014-205980. [DOI] [PubMed] [Google Scholar]
  • 6.Fernandez-Tajes J, Soto-Hermida A, Vazquez-Mosquera ME, Cortes-Pereira E, Mosquera A, Fernandez-Moreno M, et al. Genome-wide DNA methylation analysis of articular chondrocytes reveals a cluster of osteoarthritic patients. Ann Rheum Dis. 2014;73:668–677. doi: 10.1136/annrheumdis-2012-202783. [DOI] [PubMed] [Google Scholar]
  • 7.Jeffries MA, Donica M, Baker LW, Stevenson ME, Annan AC, Humphrey MB, et al. Genome-wide DNA methylation study identifies significant epigenomic changes in osteoarthritic cartilage. Arthritis Rheumatol. 2014;66:2804–2815. doi: 10.1002/art.38762. [DOI] [PubMed] [Google Scholar]
  • 8.Moazedi-Fuerst FC, Hofner M, Gruber G, Weinhaeusel A, Stradner MH, Angerer H, et al. Epigenetic differences in human cartilage between mild and severe OA. J Orthop Res. 2014;32:1636–1645. doi: 10.1002/jor.22722. [DOI] [PubMed] [Google Scholar]
  • 9.Rushton MD, Reynard LN, Barter MJ, Refaie R, Rankin KS, Young DA, et al. Characterization of the cartilage DNA methylome in knee and hip osteoarthritis. Arthritis Rheumatol. 2014;66:2450–2460. doi: 10.1002/art.38713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Bomer N, den Hollander W, Ramos YF, Bos SD, van der Breggen R, Lakenberg N, et al. Underlying molecular mechanisms of DIO2 susceptibility in symptomatic osteoarthritis. Ann Rheum Dis. 2015;74:1571–1579. doi: 10.1136/annrheumdis-2013-204739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Deaton AM, Bird A. CpG islands and the regulation of transcription. Genes Dev. 2011;25:1010–1022. doi: 10.1101/gad.2037511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci. 2006;31:89–97. doi: 10.1016/j.tibs.2005.12.008. [DOI] [PubMed] [Google Scholar]
  • 13.Hashimoto K, Oreffo RO, Gibson MB, Goldring MB, Roach HI. DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytes. Arthritis Rheum. 2009;60:3303–3313. doi: 10.1002/art.24882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Taylor SE, Smeriglio P, Dhulipala L, Rath M, Bhutani N. A global increase in 5-hydroxymethylcytosine levels marks osteoarthritic chondrocytes. Arthritis Rheumatol. 2014;66:90–100. doi: 10.1002/art.38200. [DOI] [PubMed] [Google Scholar]
  • 15.Hashimoto K, Otero M, Imagawa K, de Andres MC, Coico JM, Roach HI, et al. Regulated transcription of human matrix metalloproteinase 13 (MMP13) and interleukin-1beta (IL1B) genes in chondrocytes depends on methylation of specific proximal promoter CpG sites. J Biol Chem. 2013;288:10061–10072. doi: 10.1074/jbc.M112.421156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.de Andres MC, Imagawa K, Hashimoto K, Gonzalez A, Roach HI, Goldring MB, et al. Loss of methylation in CpG sites in the NF-kappaB enhancer elements of inducible nitric oxide synthase 16 is responsible for gene induction in human articular chondrocytes. Arthritis Rheum. 2013;65:732–742. doi: 10.1002/art.37806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Cheung KS, Hashimoto K, Yamada N, Roach HI. Expression of ADAMTS-4 by chondrocytes in the surface zone of human osteoarthritic cartilage is regulated by epigenetic DNA de-methylation. Rheumatol Int. 2009;29:525–534. doi: 10.1007/s00296-008-0744-z. [DOI] [PubMed] [Google Scholar]
  • 18.Iliopoulos D, Malizos KN, Tsezou A. Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention. Ann Rheum Dis. 2007;66:1616–1621. doi: 10.1136/ard.2007.069377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Roach HI, Yamada N, Cheung KS, Tilley S, Clarke NM, Oreffo RO, et al. Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions. Arthritis Rheum. 2005;52:3110–3124. doi: 10.1002/art.21300. [DOI] [PubMed] [Google Scholar]
  • 20.Kim KI, Park YS, Im GI. Changes in the epigenetic status of the SOX-9 promoter in human osteoarthritic cartilage. J Bone Miner Res. 2013;28:1050–1060. doi: 10.1002/jbmr.1843. [DOI] [PubMed] [Google Scholar]
  • 21.Reynard LN, Bui C, Canty-Laird EG, Young DA, Loughlin J. Expression of the osteoarthritis-associated gene GDF5 is modulated epigenetically by DNA methylation. Hum Mol Genet. 2011;20:3450–3460. doi: 10.1093/hmg/ddr253. [DOI] [PubMed] [Google Scholar]
  • 22.Loeser RF, Im HJ, Richardson B, Lu Q, Chubinskaya S. Methylation of the OP-1 promoter: potential role in the age-related decline in OP-1 expression in cartilage. Osteoarthritis Cartilage. 2009;17:513–517. doi: 10.1016/j.joca.2008.08.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Imagawa K, de Andres MC, Hashimoto K, Itoi E, Otero M, Roach HI, et al. Association of reduced type IX collagen gene expression in human osteoarthritic chondrocytes with epigenetic silencing by DNA hypermethylation. Arthritis Rheumatol. 2014;66:3040–3051. doi: 10.1002/art.38774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Liu J, Stevens PD, Li X, Schmidt MD, Gao T. PHLPP-mediated dephosphorylation of S6K1 inhibits protein translation and cell growth. Mol Cell Biol. 2011;31:4917–4927. doi: 10.1128/MCB.05799-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gao T, Brognard J, Newton AC. The phosphatase PHLPP controls the cellular levels of protein kinase C. J Biol Chem. 2008;283:6300–6311. doi: 10.1074/jbc.M707319200. [DOI] [PubMed] [Google Scholar]
  • 26.Gao T, Furnari F, Newton AC. PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell. 2005;18:13–24. doi: 10.1016/j.molcel.2005.03.008. [DOI] [PubMed] [Google Scholar]
  • 27.Shimizu K, Okada M, Nagai K, Fukada Y. Suprachiasmatic nucleus circadian oscillatory protein, a novel binding partner of K-Ras in the membrane rafts, negatively regulates MAPK pathway. J Biol Chem. 2003;278:14920–14925. doi: 10.1074/jbc.M213214200. [DOI] [PubMed] [Google Scholar]
  • 28.Bradley EW, Carpio LR, Newton AC, Westendorf JJ. Deletion of the PH-domain and leucine rich repeat protein phosphatase 1 (Phlpp1) increases fibroblast growth factor (Fgf) 18 expression and promotes chondrocyte proliferation. J Biol Chem. 2015 doi: 10.1074/jbc.M114.612937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Masubuchi S, Gao T, O'Neill A, Eckel-Mahan K, Newton AC, Sassone-Corsi P. Protein phosphatase PHLPP1 controls the light-induced resetting of the circadian clock. Proc Natl Acad Sci U S A. 2010;107:1642–1647. doi: 10.1073/pnas.0910292107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Glasson SS, Blanchet TJ, Morris EA. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage. 2007;15:1061–1069. doi: 10.1016/j.joca.2007.03.006. [DOI] [PubMed] [Google Scholar]
  • 31.Salvarrey-Strati A, Watson L, Blanchet T, Lu N, Glasson SS. The influence of enrichment devices on development of osteoarthritis in a surgically induced murine model. ILAR J. 2008;49:23–30. doi: 10.1093/ilar.49.3.e23. [DOI] [PubMed] [Google Scholar]
  • 32.Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage. 2006;14:13–29. doi: 10.1016/j.joca.2005.07.014. [DOI] [PubMed] [Google Scholar]
  • 33.Ta LE, Low PA, Windebank AJ. Mice with cisplatin and oxaliplatin-induced painful neuropathy develop distinct early responses to thermal stimuli. Mol Pain. 2009;5:9. doi: 10.1186/1744-8069-5-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53:55–63. doi: 10.1016/0165-0270(94)90144-9. [DOI] [PubMed] [Google Scholar]
  • 35.Xie L, Lin AS, Guldberg RE, Levenston ME. Nondestructive assessment of sGAG content and distribution in normal and degraded rat articular cartilage via EPIC-microCT. Osteoarthritis Cartilage. 2010;18:65–72. doi: 10.1016/j.joca.2009.07.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Xie L, Lin AS, Levenston ME, Guldberg RE. Quantitative assessment of articular cartilage morphology via EPIC-microCT. Osteoarthritis Cartilage. 2009;17:313–320. doi: 10.1016/j.joca.2008.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Liang G, Vanhouten J, Macica CM. An atypical degenerative osteoarthropathy in Hyp mice is characterized by a loss in the mineralized zone of articular cartilage. Calcif Tissue Int. 2011;89:151–162. doi: 10.1007/s00223-011-9502-4. [DOI] [PubMed] [Google Scholar]
  • 38.Kotwal N, Li J, Sandy J, Plaas A, Sumner DR. Initial application of EPIC-muCT to assess mouse articular cartilage morphology and composition: effects of aging and treadmill running. Osteoarthritis Cartilage. 2012;20:887–895. doi: 10.1016/j.joca.2012.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Palmer AW, Guldberg RE, Levenston ME. Analysis of cartilage matrix fixed charge density and three-dimensional morphology via contrast-enhanced microcomputed tomography. Proc Natl Acad Sci U S A. 2006;103:19255–19260. doi: 10.1073/pnas.0606406103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gosset M, Berenbaum F, Thirion S, Jacques C. Primary culture and phenotyping of murine chondrocytes. Nat Protoc. 2008;3:1253–1260. doi: 10.1038/nprot.2008.95. [DOI] [PubMed] [Google Scholar]
  • 41.Razidlo DF, Whitney TJ, Casper ME, McGee-Lawrence ME, Stensgard BA, Li X, et al. Histone deacetylase 3 depletion in osteo/chondroprogenitor cells decreases bone density and increases marrow fat. PLoS One. 5:e11492. doi: 10.1371/journal.pone.0011492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Bonin CA, Lewallen EA, Baheti S, Bradley EW, Stuart MJ, Berry DJ, et al. Identification of differentially methylated regions in new genes associated with knee osteoarthritis. Gene. 2015 doi: 10.1016/j.gene.2015.10.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Moore EE, Bendele AM, Thompson DL, Littau A, Waggie KS, Reardon B, et al. Fibroblast growth factor-18 stimulates chondrogenesis and cartilage repair in a rat model of injury-induced osteoarthritis. Osteoarthritis Cartilage. 2005;13:623–631. doi: 10.1016/j.joca.2005.03.003. [DOI] [PubMed] [Google Scholar]
  • 44.Janusz MJ, Bendele AM, Brown KK, Taiwo YO, Hsieh L, Heitmeyer SA. Induction of osteoarthritis in the rat by surgical tear of the meniscus: Inhibition of joint damage by a matrix metalloproteinase inhibitor. Osteoarthritis Cartilage. 2002;10:785–791. doi: 10.1053/joca.2002.0823. [DOI] [PubMed] [Google Scholar]
  • 45.Bendele A, McComb J, Gould T, McAbee T, Sennello G, Chlipala E, et al. Animal models of arthritis: relevance to human disease. Toxicol Pathol. 1999;27:134–142. doi: 10.1177/019262339902700125. [DOI] [PubMed] [Google Scholar]
  • 46.Bradley EW, Carpio LR, Westendorf JJ. Histone deacetylase 3 suppression increases PH domain and leucine-rich repeat phosphatase (Phlpp)1 expression in chondrocytes to suppress Akt signaling and matrix secretion. J Biol Chem. 2013;288:9572–9582. doi: 10.1074/jbc.M112.423723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Roussigne M, Cayrol C, Clouaire T, Amalric F, Girard JP. THAP1 is a nuclear proapoptotic factor that links prostate-apoptosis-response-4 (Par-4) to PML nuclear bodies. Oncogene. 2003;22:2432–2442. doi: 10.1038/sj.onc.1206271. [DOI] [PubMed] [Google Scholar]
  • 48.Taylor SE, Li YH, Wong WH, Bhutani N. Genome-wide mapping of DNA hydroxymethylation in osteoarthritic chondrocytes. Arthritis Rheumatol. 2015;67:2129–2140. doi: 10.1002/art.39179. [DOI] [PMC free article] [PubMed] [Google Scholar]

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