Yoko Hosaka

Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development

Here we examined the involvement of Notch signaling in the endochondral ossification process, which is crucial for osteoarthritis (OA) development. Intracellular domains of Notch1 and -2 were translocated into the nucleus of chondrocytes with their differentiation in mouse limb cartilage and in mouse and human OA articular cartilage. A tissue-specific inactivation of the Notch transcriptional effector recombination signal binding protein for Ig kappa J (RBPjκ) in chondroprogenitor cells of SRY-box containing gene 9 (Sox9)-Cre;Rbpjfl/fl mouse embryos caused an impaired terminal stage of endochondral ossification in the limb cartilage. The RBPjκ inactivation in adult articular cartilage after normal skeletal growth using type II collagen (Col2a1)-CreERT;Rbpjfl/fl mice by tamoxifen injection caused resistance to OA development in the knee joint. Notch intracellular domain with the effector RBPjκ stimulated endochondral ossification through induction of the target gene Hes1 in chondrocytes. Among the Notch ligands, Jagged1 was strongly induced during OA development. Finally, intraarticular injection of N-[N-(3,5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), a small compound Notch inhibitor, to the mouse knee joint prevented OA development. The RBPjκ-dependent Notch signaling in chondrocytes modulates the terminal stage of endochondral ossification and OA development, representing an extracellular therapeutic target of OA.

A novel disease-modifying osteoarthritis drug candidate targeting Runx1

Objectives To identify a new disease-modifying osteoarthritis drug (DMOAD) candidate that can effectively repair cartilage by promoting chondrogenic differentiation and halt osteoarthritis (OA) progression by suppressing aberrant hypertrophy. Methods We screened 2500 natural and synthetic small compounds for chondrogenic agents via four steps using the Col2GFP-ATDC5 system and identified a small thienoindazole derivative compound, TD-198946, as a novel DMOAD candidate. We tested its efficacy as a DMOAD via intra-articular injections directly into the joint space in a surgically-induced mouse model of OA both at the onset (prevention model) and 4   weeks after (repair model) OA induction. The downstream molecules were screened by microarray analysis. We further investigated the mechanism of the drug action and its molecular target using in vitro and in vivo assays. Results TD-198946 strongly induced chondrogenic differentiation without promoting hypertrophy in cell and metatarsal organ cultures. When administered directly into the joint space, TD-198946 successfully prevented and repaired degeneration of the articular cartilage. TD-198946 exerted its effect through the regulation of Runx1 expression, which was downregulated in both mouse and human OA cartilage compared with normal tissue. Conclusions Our data suggest that TD-198946 is a novel class of DMOAD candidate, and that targeting Runx1 will provide a promising new approach in the development of disease-modifying drugs against OA.

Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2

Significance Here we demonstrate that Hes1, an important target of Notch signaling, modulated pathogenesis of osteoarthritis by using Col2a1-CreERT;Hes1fl/fl mice. Adamts5 and Mmp13, catabolic enzymes that break down cartilage matrix, were induced by Hes1. Additionally, microarray analysis and ChIP-seq revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. CaMK2δ was activated during osteoarthritis development. CaMK2δ formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Notch signaling modulates skeletal formation and pathogenesis of osteoarthritis (OA) through induction of catabolic factors. Here we examined roles of Hes1, a transcription factor and important target of Notch signaling, in these processes. SRY-box containing gene 9 (Sox9)-Cre mice were mated with Hes1fl/fl mice to generate tissue-specific deletion of Hes1 from chondroprogenitor cells; this deletion caused no obvious abnormality in the perinatal period. Notably, OA development was suppressed when Hes1 was deleted from articular cartilage after skeletal growth in type II collagen (Col2a1)-CreERT;Hes1fl/fl mice. In cultured chondrocytes, Hes1 induced metallopeptidase with thrombospondin type 1 motif, 5 (Adamts5) and matrix metalloproteinase-13 (Mmp13), which are catabolic enzymes that break down cartilage matrix. ChIP-seq and luciferase assays identified Hes1-responsive regions in intronic sites of both genes; the region in the ADAMTS5 gene contained a typical consensus sequence for Hes1 binding, whereas that in the MMP13 gene did not. Additionally, microarray analysis, together with the ChIP-seq, revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. We further identified calcium/calmodulin-dependent protein kinase 2δ (CaMK2δ) as a cofactor of Hes1; CaMK2δ was activated during OA development, formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Therefore, Hes1 cooperated with CaMK2δ to modulate OA pathogenesis through induction of catabolic factors, including Adamts5, Mmp13, Il6, and Il1rl1. Our findings have contributed to further understanding of the molecular pathophysiology of OA, and may provide the basis for development of novel treatments for joint disorders.

Lack of a chondroprotective effect of cyclooxygenase 2 inhibition in a surgically induced model of osteoarthritis in mice

OBJECTIVE To investigate the chondroprotective effect of cyclooxygenase 2 (COX-2) inhibition in experimental osteoarthritis (OA). METHODS The expression of prostaglandin E2 synthetic enzymes was examined by immunostaining of tibial cartilage from mice with surgically induced knee joint instability and from OA patients undergoing total knee arthroplasty. The effect of orally administered celecoxib (10 mg/kg/day and 30 mg/kg/day) or vehicle alone in mice was examined 12 weeks after the induction of OA. To investigate the involvement of COX-1 and COX-2 in OA development, we also created the model in COX-1-homozygous-knockout (Ptgs1-/-) mice and COX-2-homozygous-knockout (Ptgs2-/-) mice. OA severity was assessed using a grading system developed by our group and by the Osteoarthritis Research Society International scoring system. RESULTS In mouse and human OA cartilage, the expression of the inducible enzymes COX-2 and microsomal prostaglandin E synthase 1 (mPGES-1) was enhanced, while that of the constitutive enzymes COX-1, cytosolic PGES, and mPGES-2 was suppressed. Daily celecoxib treatment did not prevent cartilage degradation or osteophyte formation during OA development in the mouse model. Furthermore, neither Ptgs1-/- mice nor Ptgs2-/- mice exhibited any significant difference in OA development as compared to wild-type littermates. CONCLUSION The two COX enzymes differ in terms of regulation of their expression during OA development. Nevertheless, experiments using inhibitor and genetic deficiency demonstrated a lack of chondroprotective effect of COX-2 inhibition in the mouse surgical OA model.

Biphasic regulation of chondrocytes by Rela through induction of anti-apoptotic and catabolic target genes

In vitro studies have shown that Rela/p65, a key subunit mediating NF-κB signalling, is involved in chondrogenic differentiation, cell survival and catabolic enzyme production. Here, we analyse in vivo functions of Rela in embryonic limbs and adult articular cartilage, and find that Rela protects chondrocytes from apoptosis through induction of anti-apoptotic genes including Pik3r1. During skeletal development, homozygous knockout of Rela leads to impaired growth through enhanced chondrocyte apoptosis, whereas heterozygous knockout of Rela does not alter growth. In articular cartilage, homozygous knockout of Rela at 7 weeks leads to marked acceleration of osteoarthritis through enhanced chondrocyte apoptosis, whereas heterozygous knockout of Rela results in suppression of osteoarthritis development through inhibition of catabolic gene expression. Haploinsufficiency or a low dose of an IKK inhibitor suppresses catabolic gene expression, but does not alter anti-apoptotic gene expression. The biphasic regulation of chondrocytes by Rela contributes to understanding the pathophysiology of osteoarthritis.

Comparison of mouse and human ankles and establishment of mouse ankle osteoarthritis models by surgically-induced instability

OBJECTIVE Prevalence of ankle osteoarthritis (OA) is lower than that of knee OA, however, the molecular mechanisms underlying the difference remain unrevealed. In the present study, we developed mouse ankle OA models for use as tools to investigate pathophysiology of ankle OA and molecular characteristics of ankle cartilage. DESIGN We anatomically and histologically examined ankle and knee joints of C57BL/6 mice, and compared them with human samples. We examined joints of 8-week-old and 25-month-old mice. For experimental models, we developed three different ankle OA models: a medial model, a lateral model, and a bilateral model, by resection of respective structures. OA severity was evaluated 8 weeks after the surgery by safranin O staining, and cartilage degradation in the medial model was sequentially examined. RESULTS Anatomical and histological features of human and mouse ankle joints were comparable. Additionally, the mouse ankle joint was more resistant to cartilage degeneration with aging than the mouse knee joint. In the medial model, the tibiotalar joint was markedly affected while the subtalar joint was less degenerated. In the lateral model, the subtalar joint was mainly affected while the tibiotalar joint was less altered. In the bilateral model, both joints were markedly degenerated. In the time course of the medial model, TdT-mediated dUTP nick end labeling (TUNEL) staining and Adamts5 expression were enhanced at early and middle stages, while Mmp13 expression was gradually increased during the OA development. CONCLUSION Since human and mouse ankles are comparable, the present models will contribute to ankle OA pathophysiology and general cartilage research in future.

Regulation of Chondrocyte Survival in Mouse Articular Cartilage by p63

Transcription factor p63, of the p53 family, regulates cell proliferation, survival, and apoptosis in various cells and tissues. This study was undertaken to examine the expression and roles of p63 transcript variants in the mouse growth plate and articular chondrocytes.

p63 Regulates Chondrocyte Survival in Articular Cartilage

Transcription factor p63, of the p53 family, regulates cell proliferation, survival, and apoptosis in various cells and tissues. This study was undertaken to examine the expression and roles of p63 transcript variants in the mouse growth plate and articular chondrocytes.

Identification of SCAN Domain Zinc-Finger Gene ZNF449 as a Novel Factor of Chondrogenesis

Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5′RACE analysis, and identified the 46-bp core enhancer region (CES6). We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown.

Disease-modifying effects of TD-198946 on progressed osteoarthritis in a mouse model

We previously reported that a thienoindazole derivative TD-198946 (TD) induced chondrogenic differentiation in vitro without promoting hypertrophy, and prevented and repaired the degeneration of articular cartilage observed in a surgically induced mouse model of osteoarthritis (OA).1 In that study, the TD injections into mouse knee joints began on the day of the OA surgery (prevention model) or 4 weeks after the surgery (repair model), and continued periodically until 8 weeks after the surgery.1 Here we report the disease-modifying effects of TD injections on progressed OA in mice. We examined 4-week and 8-week injection protocols that began 8 weeks after the OA surgery (figure 1A). Eight mice in each group (n=8) were then subjected to histopathological assessments of knee joint OA. The severity of OA was quantified using the Osteoarthritis Research Society International histopathology scoring system (scale: 0–6)2 on histological sections stained with Safranin O/fast green. The OA surgery, preparation of TD, injection of TD into mouse knee joints and preparation of paraffin sections were performed as …