Mocetinostat activates Krüppel-like factor 4 and protects against tissue destruction and inflammation in osteoarthritis.

Osteoarthritis (OA) is the most common joint disorder, and disease-modifying OA drugs (DMOADs) represent a major need in OA management. Krüppel-like factor 4 (KLF4) is a central transcription factor upregulating regenerative and protective functions in joint tissues. This study was aimed to identify small molecules activating KLF4 expression and to determine functions and mechanisms of the hit compounds. High-throughput screening (HTS) with 11,948 clinical-stage compounds was performed using a reporter cell line detecting endogenous KLF4 activation. Eighteen compounds were identified through the HTS and confirmed in a secondary screen. After testing in SW1353 chondrosarcoma cells and human chondrocytes, mocetinostat - a class I selective histone deacetylase (HDAC) inhibitor - had the best profile of biological activities. Mocetinostat upregulated cartilage signature genes in human chondrocytes, meniscal cells, and BM-derived mesenchymal stem cells, and it downregulated hypertrophic, inflammatory, and catabolic genes in those cells and synoviocytes. I.p. administration of mocetinostat into mice reduced severity of OA-associated changes and improved pain behaviors. Global gene expression and proteomics analyses revealed that regenerative and protective effects of mocetinostat were dependent on peroxisome proliferator-activated receptor γ coactivator 1-α. These findings show therapeutic and protective activities of mocetinostat against OA, qualifying it as a candidate to be used as a DMOAD.

Human-specific duplicate CHRFAM7A gene is associated with more severe osteoarthritis and amplifies pain behaviours.

OBJECTIVES: CHRFAM7A is a uniquely human fusion gene that functions as a dominant negative regulator of alpha 7 acetylcholine nicotinic receptor (α7nAChR) in vitro. This study determined the impact of CHRFAM7A on α7nAChR agonist responses, osteoarthritis (OA) severity and pain behaviours and investigated mechanisms. METHODS: Transgenic CHRFAM7A (TgCHRFAM7A) mice were used to determine the impact of CHRFAM7A on knee OA histology, pain severity in OA and other pain models, response to nAchR agonist and IL-1ß. Mouse and human cells were used for mechanistic studies. RESULTS: Transgenic (Tg) TgCHRFAM7A mice developed more severe structural damage and increased mechanical allodynia than wild type (WT) mice in the destabilisation of medial meniscus model of OA. This was associated with a decreased suppression of inflammation by α7nAchR agonist. TgCHRFAM7A mice displayed a higher basal sensitivity to pain stimuli and increased pain behaviour in the monoiodoacetate and formalin models. Dorsal root ganglia of TgCHRFAM7A mice showed increased macrophage infiltration and expression of the chemokine fractalkine and also had a compromised antinociceptive response to the α7nAchR agonist nicotine. Both native CHRNA7 and CHRFAM7A subunits were expressed in human joint tissues and the CHRFAM7A/CHRNA7 ratio was increased in OA cartilage. Human chondrocytes with two copies of CHRFAM7A had reduced anti-inflammatory responses to nicotine. CONCLUSION: CHRFAM7A is an aggravating factor for OA-associated inflammation and tissue damage and a novel genetic risk factor and therapeutic target for pain.

Krüppel-like factor-4 and Krüppel-like factor-2 are important regulators of joint tissue cells and protect against tissue destruction and inflammation in osteoarthritis.

OBJECTIVES: Analysing expression patterns of Krüppel-like factor (KLF) transcription factors in normal and osteoarthritis (OA) human cartilage, and determining functions and mechanisms of KLF4 and KLF2 in joint homoeostasis and OA pathogenesis. METHODS: Experimental approaches included human joint tissues cells, transgenic mice and mouse OA model with viral KLF4 gene delivery to demonstrate therapeutic benefit in structure and pain improvement. Mechanistic studies applied global gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq). RESULTS: Several KLF genes were significantly decreased in OA cartilage. Among them, KLF4 and KLF2 were strong inducers of cartilage collagen genes and Proteoglycan-4. Cartilage-specific deletion of Klf2 in mature mice aggravated severity of experimental OA. Transduction of human chondrocytes with Adenovirus (Ad) expressing KLF4 or KLF2 enhanced expression of major cartilage extracellular matrix (ECM) genes and SRY-box transcription factor-9, and suppressed mediators of inflammation and ECM-degrading enzymes. Ad-KLF4 and Ad-KLF2 enhanced similar protective functions in meniscus cells and synoviocytes, and promoted chondrocytic differentiation of human mesenchymal stem cells. Viral KLF4 delivery into mouse knees reduced severity of OA-associated changes in cartilage, meniscus and synovium, and improved pain behaviours. ChIP-seq analysis suggested that KLF4 directly bound cartilage signature genes. Ras-related protein-1 signalling was the most enriched pathway in KLF4-transduced cells, and its signalling axis was involved in upregulating cartilage ECM genes by KLF4 and KLF2. CONCLUSIONS: KLF4 and KLF2 may be central transcription factors that increase protective and regenerative functions in joint tissue cells, suggesting that KLF gene transfer or molecules upregulating KLFs are therapeutic candidates for OA.

Mohawk is a transcription factor that promotes meniscus cell phenotype and tissue repair and reduces osteoarthritis severity.

Meniscus tears are common knee injuries and a major osteoarthritis (OA) risk factor. Knowledge gaps that limit the development of therapies for meniscus injury and degeneration concern transcription factors that control the meniscus cell phenotype. Analysis of RNA sequencing data from 37 human tissues in the Genotype-Tissue Expression database and RNA sequencing data from meniscus and articular cartilage showed that transcription factor Mohawk (MKX) is highly enriched in meniscus. In human meniscus cells, MKX regulates the expression of meniscus marker genes, OA-related genes, and other transcription factors, including Scleraxis (SCX), SRY Box 5 (SOX5), and Runt domain-related transcription factor 2 (RUNX2). In mesenchymal stem cells (MSCs), the combination of adenoviral MKX (Ad-MKX) and transforming growth factor-ß3 (TGF-ß3) induced a meniscus cell phenotype. When Ad-MKX-transduced MSCs were seeded on TGF-ß3-conjugated decellularized meniscus scaffold (DMS) and inserted into experimental tears in meniscus explants, they increased glycosaminoglycan content, extracellular matrix interconnectivity, cell infiltration into the DMS, and improved biomechanical properties. Ad-MKX injection into mouse knee joints with experimental OA induced by surgical destabilization of the meniscus suppressed meniscus and cartilage damage, reducing OA severity. Ad-MKX injection into human OA meniscus tissue explants corrected pathogenic gene expression. These results identify MKX as a previously unidentified key transcription factor that regulates the meniscus cell phenotype. The combination of Ad-MKX with TGF-ß3 is effective for differentiation of MSCs to a meniscus cell phenotype and useful for meniscus repair. MKX is a promising therapeutic target for meniscus tissue engineering, repair, and prevention of OA.

FOXO1 and FOXO3 transcription factors have unique functions in meniscus development and homeostasis during aging and osteoarthritis.

The objective of this study was to examine FoxO expression and FoxO function in meniscus. In menisci from human knee joints with osteoarthritis (OA), FoxO1 and 3 expression were significantly reduced compared with normal menisci from young and old normal donors. The expression of FoxO1 and 3 was also significantly reduced in mouse menisci during aging and OA induced by surgical meniscus destabilization or mechanical overuse. Deletion of FoxO1 and combined FoxO1, 3, and 4 deletions induced abnormal postnatal meniscus development in mice and these mutant mice spontaneously displayed meniscus pathology at 6 mo. Mice with Col2Cre-mediated deletion of FoxO3 or FoxO4 had normal meniscus development but had more severe aging-related damage. In mature AcanCreERT2 mice, the deletion of FoxO1, 3, and 4 aggravated meniscus lesions in all experimental OA models. FoxO deletion suppressed autophagy and antioxidant defense genes and altered several meniscus-specific genes. Expression of these genes was modulated by adenoviral FoxO1 in cultured human meniscus cells. These results suggest that FoxO1 plays a key role in meniscus development and maturation, and both FoxO1 and 3 support homeostasis and protect against meniscus damage in response to mechanical overuse and during aging and OA.

TAFI deficiency causes maladaptive vascular remodeling after hemophilic joint bleeding.

Excessive vascular remodeling is characteristic of hemophilic arthropathy (HA) and may contribute to joint bleeding and the progression of HA. Mechanisms for pathological vascular remodeling after hemophilic joint bleeding are unknown. In hemophilia, activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is impaired, which contributes to joint bleeding and may also underlie the aberrant vascular remodeling. Here, hemophilia A (factor VIII-deficient; FVIII-deficient) mice or TAFI-deficient mice with transient (antibody-induced) hemophilia A were used to determine the role of FVIII and TAFI in vascular remodeling after joint bleeding. Excessive vascular remodeling and vessel enlargement persisted in FVIII-deficient and TAFI-deficient mice, but not in transient hemophilia WT mice, after similar joint bleeding. TAFI-overexpression in FVIII-deficient mice prevented abnormal vessel enlargement and vascular leakage. Age-related vascular changes were observed with FVIII or TAFI deficiency and correlated positively with bleeding severity after injury, supporting increased vascularity as a major contributor to joint bleeding. Antibody-mediated inhibition of uPA also prevented abnormal vascular remodeling, suggesting that TAFI's protective effects include inhibition of uPA-mediated plasminogen activation. In conclusion, the functional TAFI deficiency in hemophilia drives maladaptive vascular remodeling in the joints after bleeding. These mechanistic insights allow targeted development of potentially new strategies to normalize vascularity and control rebleeding in HA.

Wwp2 maintains cartilage homeostasis through regulation of Adamts5.

The WW domain-containing protein 2 (Wwp2) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.

FOXO are required for intervertebral disk homeostasis during aging and their deficiency promotes disk degeneration.

Intervertebral disk (IVD) degeneration is a prevalent age-associated musculoskeletal disorder and a major cause of chronic low back pain. Aging is the main risk factor for the disease, but the molecular mechanisms regulating IVD homeostasis during aging are unknown. The aim of this study was to investigate the function of FOXO, a family of transcription factors linked to aging and longevity, in IVD aging and age-related degeneration. Conditional deletion of all FOXO isoforms (FOXO1, 3, and 4) in IVD using the Col2a1Cre and AcanCreER mouse resulted in spontaneous development of IVD degeneration that was driven by severe cell loss in the nucleus pulposus (NP) and cartilaginous endplates (EP). Conditional deletion of individual FOXO in mature mice showed that FOXO1 and FOXO3 are the dominant isoforms and have redundant functions in promoting IVD homeostasis. Gene expression analyses indicated impaired autophagy and reduced antioxidant defenses in the NP of FOXO-deficient IVD. In primary human NP cells, FOXO directly regulated autophagy and adaptation to hypoxia and promoted resistance to oxidative and inflammatory stress. Our findings demonstrate that FOXO are critical regulators of IVD homeostasis during aging and suggest that maintaining or restoring FOXO expression can be a therapeutic strategy to promote healthy IVD aging and delay the onset of IVD degeneration.

Age-related reduction in the expression of FOXO transcription factors and correlations with intervertebral disc degeneration.

Aging is a main risk factor for intervertebral disc (IVD) degeneration, the main cause of low back pain. FOXO transcription factors are important regulators of tissue homeostasis and longevity. Here, we determined the expression pattern of FOXO in healthy and degenerated human IVD and the associations with IVD degeneration during mouse aging. FOXO expression was assessed by immunohistochemistry in normal and degenerated human IVD samples and in cervical and lumbar IVD from 6-, 12-, 24-, and 36-month-old C57BL/6J mice. Mouse spines were graded for key histological features of disc degeneration in all the time points and expression of two key FOXO downstream targets, sestrin 3 (SESN3) and superoxide dismutase (SOD2), was assessed by immunohistochemistry. Histological analysis revealed that FOXO proteins are expressed in all compartments of human and mouse IVD. Expression of FOXO1 and FOXO3, but not FOXO4, was significantly deceased in human degenerated discs. In mice, degenerative changes in the lumbar spine were seen at 24 and 36 months of age whereas cervical IVD showed increased histopathological scores at 36 months. FOXO expression was significantly reduced in lumbar IVD at 12-, 24-, and 36-month-old mice and in cervical IVD at 36-month-old mice when compared with the 6-month-old group. The reduction of FOXO expression in lumbar IVD was concomitant with a decrease in the expression of SESN3 and SOD2. These findings suggest that reduced FOXO expression occurs in lumbar IVD during aging and precedes the major histopathological changes associated with lumbar IVD degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2682-2691, 2017.

TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes.

The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis.

Vascular remodeling underlies rebleeding in hemophilic arthropathy.

Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal, and cartilage changes in response to a single induced hemarthrosis in the FVIII-deficient mouse. We found soft-tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft-tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII-deficient mice involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105), and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α-SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis and osteoarthritis and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees, and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease.

Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis.

OBJECTIVE: Amyloid deposits are prevalent in osteoarthritic (OA) joints. We undertook this study to define the dominant precursor and to determine whether the deposits affect chondrocyte functions. METHODS: Amyloid deposition in human normal and OA knee cartilage was determined by Congo red staining. Transthyretin (TTR) in cartilage and synovial fluid was analyzed by immunohistochemistry and Western blotting. The effects of recombinant amyloidogenic and nonamyloidogenic TTR variants were tested in human chondrocyte cultures. RESULTS: Normal cartilage from young donors did not contain detectable amyloid deposits, but 7 of 12 aged normal cartilage samples (58%) and 12 of 12 OA cartilage samples (100%) had Congo red staining with green birefringence under polarized light. TTR, which is located predominantly at the cartilage surfaces, was detected in all OA cartilage samples and in a majority of aged normal cartilage samples, but not in normal cartilage samples from young donors. Chondrocytes and synoviocytes did not contain significant amounts of TTR messenger RNA. Synovial fluid TTR levels were similar in normal and OA knees. In cultured chondrocytes, only an amyloidogenic TTR variant induced cell death as well as the expression of proinflammatory cytokines and extracellular matrix-degrading enzymes. The effects of amyloidogenic TTR on gene expression were mediated in part by Toll-like receptor 4, receptor for advanced glycation end products, and p38 MAPK. TTR-induced cytotoxicity was inhibited by resveratrol, a plant polyphenol that stabilizes the native tetrameric structure of TTR. CONCLUSION: These findings are the first to suggest that TTR amyloid deposition contributes to cell and extracellular matrix damage in articular cartilage in human OA and that therapies designed to reduce TTR amyloid formation might be useful.