ELF3 modulates type II collagen gene (COL2A1) transcription in chondrocytes by inhibiting SOX9-CBP/p300-driven histone acetyltransferase activity.

AIM: We showed previously that E74-like factor 3 (ELF3) protein levels are increased in osteoarthritic (OA) cartilage, that ELF3 accounts for inflammatory cytokine-driven MMP13 gene expression, and that, upon induction by interleukin-1ß, ELF3 binds to the COL2A1 promoter and suppresses its activity in chondrocytes. Here, we aimed to further investigate the mechanism/s by which ELF3 represses COL2A1 transcription in chondrocytes. METHODS AND RESULTS: We report that ELF3 inhibits Sox9-driven COL2A1 promoter activity by interfering with the activator functions of CBP/300 and Sox9. Co-transfection of the pGL2B-COL2A1 (-577/+3428 bp) reporter construct with Sox9 and with Sox5 and/or Sox6 increased COL2A1 promoter activity, and ELF3 overexpression significantly reduced the promoter transactivation. Co-transfection of ELF3 with the pLuc 4x48 enhancer construct, containing the 89-bp COL2A1 promoter and lacking the previously defined ELF3 binding sites, decreased both basal and Sox9-driven promoter activity. Co-transfection of ELF3 with a Gal4 reporter construct also inhibited Gal4-Sox9-driven transactivation, suggesting that ELF3 directly interacts with Sox9. Using truncated Sox9 fragments, we found that ELF3 interacts directly with the HMG domain of Sox9. Importantly, overexpression of ELF3 significantly decreased Sox9/CBP-dependent HAT activity. Finally, we show evidence that increased ELF3 mRNA expression in OA chondrocytes correlates with hypermethylation of the proximal promoter, suggesting that ELF3 transcription is subjected to epigenetic control in OA disease. CONCLUSION: Our results highlight the contribution of ELF3 to transcriptional regulation of COL2A1 and its potential role in OA disease, and uncover epigenetic mechanisms at play in the regulation of ELF3 and its downstream targets in articular chondrocytes.

Class I histone deacetylase inhibition modulates metalloproteinase expression and blocks cytokine-induced cartilage degradation.

OBJECTIVE: To examine the ability of a broad-spectrum histone deacetylase (HDAC) inhibitor to protect cartilage in vivo, and to explore the effects of class-selective HDAC inhibitors and small interfering RNA (siRNA)-induced knockdown of HDACs on metalloproteinase expression and cartilage degradation in vitro. METHODS: A destabilization of the medial meniscus (DMM) model was used to assess the in vivo activity of the HDAC inhibitor trichostatin A (TSA). Human articular chondrocytes (HACs) and SW-1353 chondrosarcoma cells were treated with cytokines and TSA, valproic acid, MS-275, or siRNA, and quantitative reverse transcription-polymerase chain reaction was performed to determine the effect of treatment on metalloproteinase expression. HDAC inhibitor activity was detected by Western blotting. A bovine nasal cartilage (BNC) explant assay was performed to measure cartilage resorption in vitro. RESULTS: Systemically administered TSA protected cartilage in the DMM model. TSA, valproic acid, and MS-275 repressed cytokine-induced MMP1 and MMP13 expression in HACs. Knockdown of each class I HDAC diminished interleukin-1-induced MMP13 expression. All of the HDAC inhibitors prevented degradation of BNC, in which TSA and MS-275 repressed cytokine-induced MMP expression. CONCLUSION: Inhibition of class I HDACs (HDAC-1, HDAC-2, HDAC-3) by MS-275 or by specific depletion of HDACs is capable of repressing cytokine-induced metalloproteinase expression in cartilage cells and BNC explants, resulting in inhibition of cartilage resorption. These observations indicate that specific inhibition of class I HDACs is a possible therapeutic strategy in the arthritides.

Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo.

OBJECTIVE: Sulforaphane (SFN) has been reported to regulate signaling pathways relevant to chronic diseases. The aim of this study was to investigate the impact of SFN treatment on signaling pathways in chondrocytes and to determine whether sulforaphane could block cartilage destruction in osteoarthritis. METHODS: Gene expression, histone acetylation, and signaling of the transcription factors NF-E2-related factor 2 (Nrf2) and NF-κB were examined in vitro. The bovine nasal cartilage explant model and the destabilization of the medial meniscus (DMM) model of osteoarthritis in the mouse were used to assess chondroprotection at the tissue and whole-animal levels. RESULTS: SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes and in fibroblast-like synovial cells. SFN acted independently of Nrf2 and histone deacetylase activity to regulate metalloproteinase expression in human articular chondrocytes but did mediate prolonged activation of JNK and p38 MAPK. SFN attenuated NF-κB signaling at least through inhibition of DNA binding in human articular chondrocytes, with decreased expression of several NF-κB-dependent genes. Compared with cytokines alone, SFN (10 µM) abrogated cytokine-induced destruction of bovine nasal cartilage at both the proteoglycan and collagen breakdown levels. An SFN-rich diet (3 µmoles/day SFN versus control chow) decreased the arthritis score in the DMM model of osteoarthritis in the mouse, with a concurrent block of early DMM-induced gene expression changes. CONCLUSION: SFN inhibits the expression of key metalloproteinases implicated in osteoarthritis, independently of Nrf2, and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo.

HDAC6 regulates NF-κB signalling to control chondrocyte IL-1-induced MMP and inflammatory gene expression.

Elevated pro-inflammatory signalling coupled with catabolic metalloproteinase expression is a common feature of arthritis, leading to cartilage damage, deterioration of the joint architecture and the associated pain and immobility. Countering these processes, histone deacetylase inhibitors (HDACi) have been shown to suppress matrix metalloproteinase (MMP) expression, block cytokine-induced signalling and reduce the cartilage degradation in animal models of the arthritis. In order to establish which specific HDACs account for these chondro-protective effects an HDAC1-11 RNAi screen was performed. HDAC6 was required for both the interleukin (IL)-1 induction of MMP expression and pro-inflammatory interleukin expression in chondrocytes, implicating an effect on NF-κB signalling. Depletion of HDAC6 post-transcriptionally up-regulated inhibitor of κB (IκB), prevented the nuclear translocation of NF-κB subunits and down-regulated NF-κB reporter activation. The pharmacological inhibition of HDAC6 reduced MMP expression in chondrocytes and cartilage collagen release. This work highlights the important role of HDAC6 in pro-inflammatory signalling and metalloproteinase gene expression, and identifies a part for HDAC6 in the NF-κB signalling pathway. By confirming the protection of cartilage this work supports the inhibition of HDAC6 as a possible therapeutic strategy in arthritis.

MMP28 gene expression is regulated by Sp1 transcription factor acetylation.

MMP-28 (epilysin) is a recently cloned member of the MMP (matrix metalloproteinase) family. It is highly expressed in the skin by keratinocytes, the developing and regenerating nervous system and a number of other normal human tissues, as well as a number of carcinomas. The MMP28 promoter has previously been cloned and characterized identifying a conserved GT-box that binds Sp1/Sp3 (specificity proteins 1 and 3) proteins and is essential for the basal expression of the gene. The present study demonstrates that MMP28 expression is induced by HDAC (histone deacetylase) inhibitors and that this effect is mediated through the GT-box. Transient transfection assays have shown that the induction of MMP28 expression by the HDAC inhibitior TSA (trichostatin A) is mediated via Sp1 at the GT-box. Immunoprecipitation experiments have shown that the acetylation of Sp1 and Sp3 is increased by TSA treatment; however, no effect on DNA binding was observed. Histone acetyltransferases such as p300 and P/CAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] increased induction of the MMP28 promoter by Sp1. Knockdown of HDAC1 using siRNA (small interfering RNA) also induces the MMP28 promoter. Oligonucleotide pulldown identified STRAP (serine/threonine kinase receptor-associated protein) as a further protein recruited to the MMP28 promoter and acting functionally with Sp1.

Mouse Models of Osteoarthritis: Surgical Model of Post-traumatic Osteoarthritis Induced by Destabilization of the Medial Meniscus.

The surgical model of destabilization of the medial meniscus (DMM) has become a gold standard for studying the onset and progression of post-traumatic osteoarthritis (OA). The DMM model mimics clinical meniscal injury, a known predisposing factor for the development of human OA, and permits the study of structural and biological changes over the course of the disease. In addition, when applied to genetically modified or engineered mouse models, this surgical procedure permits dissection of the relative contribution of a given gene to OA initiation and/or progression. This chapter describes the requirements for the surgical induction of OA in mouse models, and provides guidelines and tools for the subsequent histological, immunohistochemical, and molecular analyses. Methods for the assessment of the contributions of selected genes in genetically modified strains are also provided.

Self-assembled monolayers of phosphonates promote primary chondrocyte adhesion to silicon dioxide and polyvinyl alcohol materials.

The optimal solution for articular cartilage repair has not yet been identified, in part because of the challenges in achieving integration with the host. Coatings have the potential to transform the adhesive features of surfaces, but their application to cartilage repair has been limited. Self-assembled monolayer of phosphonates (SAMPs) have been demonstrated to increase the adhesion of various immortalized cell types to metal and polymer surfaces, but their effect on primary chondrocyte adhesion has not been studied. The objective of this study was to investigate the response of primary chondrocytes to SAMP coatings. We hypothesized a SAMP terminated with an α,ω-bisphosphonic acid, in particular butane-1,4-diphosphonic acid, would increase the number of adherent primary chondrocytes to polyvinyl alcohol (PVA). To test our hypothesis, we first established our ability to successfully modify silicon dioxide (SiO2) surfaces to enable chondrocytes to attach to the surface, without substantial changes in gene expression. Secondly, we applied identical chemistry to PVA, and quantified chondrocyte adhesion. SAMP modification to SiO2 increased chondrocyte adhesion by ×3 after 4 hr and ×4.5 after 24 hr. PVA modification with SAMPs increased chondrocyte adhesion by at least ×31 after 4 and 24 hours. Changes in cell morphology indicated that SAMP modification led to improved chondrocyte adhesion and spreading, without changes in gene expression. In summary, we modified SiO2 and PVA with SAMPs and observed an increase in the number of adherent primary bovine chondrocytes at 4 and 24 hr post-seeding. Mechanisms of chondrocyte interaction with SAMP-modified surfaces require further investigation.

Inducible knockout of CHUK/IKKα in adult chondrocytes reduces progression of cartilage degradation in a surgical model of osteoarthritis.

CHUK/IKKα contributes to collagenase-driven extracellular matrix remodeling and chondrocyte hypertrophic differentiation in vitro, in a kinase-independent manner. These processes contribute to osteoarthritis (OA), where chondrocytes experience a phenotypic shift towards hypertrophy concomitant with abnormal matrix remodeling. Here we investigated the contribution of IKKα to OA in vivo. To this end, we induced specific IKKα knockout in adult chondrocytes in AcanCreERT2/+; IKKαf/f mice treated with tamoxifen (cKO). Vehicle-treated littermates were used as wild type controls (WT). At 12 weeks of age, WT and cKO mice were subjected to the destabilization of medial meniscus (DMM) model of post-traumatic OA. The cKO mice showed reduced cartilage degradation and collagenase activity and fewer hypertrophy-like features at 12 weeks after DMM. Interestingly, in spite of the protection from structural articular cartilage damage, the postnatal growth plates of IKKα cKO mice after DMM displayed abnormal architecture and composition associated with increased chondrocyte apoptosis, which were not as evident in the articular chondrocytes of the same animals. Together, our results provide evidence of a novel in vivo functional role for IKKα in cartilage degradation in post-traumatic OA, and also suggest intrinsic, cell-autonomous effects of IKKα in chondrocytes that control chondrocyte phenotype and impact on cell survival, matrix homeostasis, and remodeling.

Phlpp inhibitors block pain and cartilage degradation associated with osteoarthritis.

Phlpp protein phosphatases are abnormally abundant within human osteoarthritic articular chondrocytes and may contribute to the development of osteoarthritis. Mice lacking Phlpp1 were previously shown to be resistant to post-traumatic osteoarthritis. Here a small molecule with therapeutic properties that inhibits Phlpp1 and Phlpp2 was tested for its ability to slow post-traumatic OA in mice and to stimulate anabolic pathways in human articular cartilage from OA joints. PTOA was induced in male C57Bl/6 mice by surgically destabilizing the meniscus. Seven weeks after surgery, mice received a single intra-articular injection of the Phlpp inhibitor NSC117079 or saline. Mechanical allodynia was measured with von Frey assays, mobility was tracked in an open field system, and cartilage damage was assessed histologically. A single intra-articular injection of the Phlpp inhibitor NSC117079 attenuated mechanical allodynia and slowed articular cartilage degradation in joints with a destabilized meniscus. Animals treated with the Phlpp inhibitor 7 weeks after injury maintained normal activity levels, while those in the control group traveled shorter distances and were less active 3 months after the joint injury. NSC117079 also increased production of cartilage extracellular matrix components (glycosaminoglycans and aggrecan) in over 90% of human articular cartilage explants from OA patients and increased phosphorylation of Phlpp1 substrates (AKT2, ERK1/2, and PKC) in human articular chondrocytes. Our results indicate that Phlpp inhibitor NSC117079 is a novel osteoarthritis disease modifying drug candidate that may have palliative affects. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1487-1497, 2018.

Elf3 Contributes to Cartilage Degradation in vivo in a Surgical Model of Post-Traumatic Osteoarthritis.

The E-74 like factor 3 (ELF3) is a transcription factor induced by inflammatory factors in various cell types, including chondrocytes. ELF3 levels are elevated in human cartilage from patients with osteoarthritis (OA), and ELF3 contributes to the IL-1ß-induced expression of genes encoding Mmp13, Nos2, and Ptgs2/Cox2 in chondrocytes in vitro. Here, we investigated the contribution of ELF3 to cartilage degradation in vivo, using a mouse model of OA. To this end, we generated mouse strains with cartilage-specific Elf3 knockout (Col2Cre:Elf3f/f) and Comp-driven Tet-off-inducible Elf3 overexpression (TRE-Elf3:Comp-tTA). To evaluate the contribution of ELF3 to OA, we induced OA in 12-week-old Col2Cre:Elf3f/f and 6-month-old TRE-Elf3:Comp-tTA male mice using the destabilization of the medial meniscus (DMM) model. The chondrocyte-specific deletion of Elf3 led to decreased levels of IL-1ß- and DMM-induced Mmp13 and Nos2 mRNA in vitro and in vivo, respectively. Histological grading showed attenuation of cartilage loss in Elf3 knockout mice compared to wild type (WT) littermates at 8 and 12 weeks following DMM surgery that correlated with reduced collagenase activity. Accordingly, Elf3 overexpression led to increased cartilage degradation post-surgery compared to WT counterparts. Our results provide evidence that ELF3 is a central contributing factor for cartilage degradation in post-traumatic OA in vivo.

Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression.

We previously reported that perlecan, a heparan-sulfate proteoglycan (Hspg2), expressed in the synovium at the cartilage-synovial junction, is required for osteophyte formation in knee osteoarthritis. To examine the mechanism underlying this process, we examined the role of perlecan in the proliferation and differentiation of synovial mesenchymal cells (SMCs), using a recently established mouse synovial cell culture method. Primary SMCs isolated from Hspg2-/- -Tg (Hspg2-/- ;Col2a1-Hspg2Tg/- ) mice, in which the perlecan-knockout was rescued from perinatal lethality, lack perlecan. The chondrogenic-, osteogenic-, and adipogenic-potentials were examined in the Hspg2-/- -Tg SMCs compared to the control SMCs prepared from wild-type Hspg2+/+ -Tg (Hspg2+/+ ;Col2a1-Hspg2Tg/- ) littermates. In a culture condition permitting proliferation, both control and Hspg2-/- -Tg SMCs showed similar rates of proliferation and expression of cell surface markers. However, in micromass cultures, the cartilage matrix production and Sox9 and Col2a1 mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs, compared with control SMCs. The reduced level of Sox9 mRNA was restored by the supplementation with exogenous perlecan protein. There was no difference in osteogenic differentiation between the control and Hspg2-/- -Tg SMCs, as measured by the levels of Runx2 and Col1a1 mRNA. The adipogenic induction and PPARγ mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs compared to control SMCs. The reduction of PPARγ mRNA levels in Hspg2-/- -Tg SMCs was restored by supplementation of perlecan. Perlecan is required for the chondrogenic and adipogenic differentiation from SMCs via its regulation of the Sox9 and PPARγ gene expression, but not for osteogenic differentiation via Runx2. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:837-846, 2017.

Mouse models of osteoarthritis: surgical model of posttraumatic osteoarthritis induced by destabilization of the medial meniscus.

The surgical model of destabilization of the medial meniscus (DMM) has become a gold standard for studying the onset and progression of posttraumatic osteoarthritis (OA). The DMM model mimics clinical meniscal injury, a known predisposing factor for the development of human OA, and permits the study of structural and biological changes over the course of the disease. In addition, when applied to genetically modified or engineered mouse models, this surgical procedure permits dissection of the relative contribution of a given gene to OA initiation and/or progression. This chapter describes the requirements for the surgical induction of OA in mouse models, and provides guidelines and tools for the subsequent histological, immunohistochemical, and molecular analyses. Methods for the assessment of the contributions of selected genes in genetically modified strains are also provided.