The present study attempted to identify and characterize the embryonic promoter of Sox6, a determinant regulator of chondrogenic differentiation. A common transcription start region for human and mouse Sox6 was initially identified, which contained a highly conserved sequence, A-box. Tandem repeats of A-box had a strong transcriptional activity both at the basal level and in response to Sox9. Cells carrying the 4xA-box-DsRed2 reporter fluoresced only upon chondrogenic differentiation. The 46-bp core enhancer region (CES6) was then identified in the 3' half of A-box, within which a C/EBP-binding motif was identified. Overexpressed C/EBPbeta activated the Sox6 promoter, and mutant 4xCES6 constructs lacking the C/EBP motif lost their basal activity. CES6 and nuclear extracts formed a specific complex, which was supershifted by anti-C/EBPbeta antibody, and in vitro translated C/EBPbeta specifically bound to CES6. Thus, we successfully identified the Sox6 promoter and its core enhancer and characterized the interactions with regulatory transcription factors.

HIF-1α, an essential transcription factor under hypoxic condition, is indispensable for chondrocytes during skeletal development but its expression and roles in articular chondrocytes are yet to be revealed. We examined HIF-1α protein expression and the hypoxic condition during mouse osteoarthritis (OA) development using state of the art hypoxic probes and found that its expression decreased as OA progressed, coinciding with the change in hypoxic conditions in articular cartilage. Gain- and loss-of-function of HIF-1α in cell culture experiments showed that HIF-1α suppressed catabolic genes such as Mmp13 and Hif2a. We confirmed these anticatabolic effects by measuring glycosaminoglycan release from wild type and conditional knock-out mice femoral heads cultured ex vivo. We went on to surgically induce OA in mice with chondrocyte-specific deletion of Hif1a and found that the development of OA was exacerbated. Increased expression of catabolic factors and activation of NF-κB signalling was clearly evident in the knock-out mice. By microarray analysis, C1qtnf3 was identified as a downstream molecule of HIF-1α, and experiments showed it exerted anti-catabolic effects through suppression of NF-κB. We conclude that HIF-1α has an anti-catabolic function in the maintenance of articular cartilage through suppression of NF-κB signalling.

Cell therapy using adipose-derived mesenchymal stem cells (ASCs) is a promising avenue of regenerative medicine for the treatment of various diseases. It has been considered that ASCs exert their therapeutic effects through the secretion of multiple factors that are critical for tissue remodeling or the suppression of inflammation. Recently, conditioned medium (CM) from ASCs that contains a complex of secreted factors has received attention as a cost-effective alternative to cell therapy.

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.

Exposure of articular cartilage to excessive mechanical loading is deeply involved in the pathogenesis of osteoarthritis. Here, we identify gremlin-1 as a mechanical loading-inducible factor in chondrocytes, detected at high levels in middle and deep layers of cartilage after cyclic strain or hydrostatic pressure loading. Gremlin-1 activates nuclear factor-κB signalling, leading to subsequent induction of catabolic enzymes. In mice intra-articular administration of gremlin-1 antibody or chondrocyte-specific deletion of Gremlin-1 decelerates osteoarthritis development, while intra-articular administration of recombinant gremlin-1 exacerbates this process. Furthermore, ras-related C3 botulinum toxin substrate 1 activation induced by mechanical loading enhances reactive oxygen species (ROS) production. Amongst ROS-activating transcription factors, RelA/p65 induces Gremlin-1 transcription, which antagonizes induction of anabolic genes such as Sox9, Col2a1, and Acan by bone morphogenetic proteins. Thus, gremlin-1 plays essential roles in cartilage degeneration by excessive mechanical loading.

Activation of NF-κB signaling promotes osteoarthritis (OA) through the transcriptional induction of Hif-2α and catabolic enzymes. This study sought to examine whether inhibiting IκBα kinase (IKK) could suppress the development of surgically-induced OA of the knee in a mouse model. We employed BMS-345541 (4(2'-aminoethyl) amino-1, 8-dimethylimidazo (1,2-a) quinoxaline) as a selective inhibitor of the subunits of IKK. OA was created by resecting the medial collateral ligament and the medial meniscus in the knees of mice. The mice were then treated with an intra-articular injection of BMS-345541 (50 nM to 500 µM) or vehicle three times a week for 8 weeks. We found that the intra-articular administration of 500 nM and 5 µM BMS-345541 significantly suppressed OA development. In the BMS-345541-treated cartilage, there was a decrease in the phosphorylation of IκBα and the expression of Hif-2α, Mmp13, and Adamts5. In human articular chondrocytes, the IL-1β-enhanced expression of Hif-2α and catabolic factors were decreased by BMS-345541 treatment in dose-dependent manner. We conclude that the intra-articular administration of BMS-345541 at some concentrations may suppress the development of OA by downregulating signaling through the NF-κB-Hif-2α axis.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.

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.

Osteoarthritis (OA) is one of the world's most common degenerative diseases, but there is no disease-modifying treatment available. Previous studies have shown that prostaglandin E2 (PGE2) and PGE2 receptor 4 (EP4) are involved in OA pathogenesis; however, their roles are not fully understood. Here, we examined the efficacy of oral administration of KAG-308, an EP4-selective agonist, in surgically induced mouse knee OA. Cartilage degeneration and synovitis were significantly inhibited by the KAG-308 treatment. Chondrocyte hypertrophy and expression of tumor necrosis factor alpha (TNF) and matrix metalloproteinase 13 (Mmp13) in the synovium were suppressed in the KAG-308-treated mice. In cultured chondrocytes, hypertrophic differentiation was inhibited by KAG-308 and intranuclear translocation of histone deacetylase 4 (Hdac4) was enhanced. In cultured synoviocytes, lipopolysaccharide (LPS)-induced expression of TNF and Mmp13 was also suppressed by KAG-308. KAG-308 was detected in the synovium and cartilage of orally treated mice. TNF secretion from the synovia of KAG-308-treated mice was significantly lower than control mice. Thus, we conclude that oral administration of KAG-308 suppresses OA development through suppression of chondrocyte hypertrophy and synovitis. KAG-308 may be a potent candidate for OA drug development.

Human dental pulp stem cells (DPSCs), which have the ability to differentiate into multiple lineages, were recently identified. DPSCs can be collected readily from extracted teeth and are now considered to be a type of mesenchymal stem cell with higher clonogenic and proliferative potential than bone marrow stem cells (BMSCs). Meanwhile, the treatment of severe bone defects, such as fractures, cancers, and congenital abnormalities, remains a great challenge, and novel bone regenerative techniques are highly anticipated. Several studies have previously shown that 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin derivative, induces osteogenic differentiation of preosteoblastic and mesenchymal cells. However, the osteogenic differentiation activities of TH have only been confirmed in some mouse cell lines. Therefore, in this study, toward the clinical use of TH in humans, we analyzed the effect of TH on the osteogenic differentiation of DPSCs, and the in-vivo osteogenesis ability of TH-induced DPSCs, taking advantage of the simple transplantation system using cell-sheet technology.

Both loss- and gain-of-function of Wnt/β-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/β-catenin signaling in the superficial zone (SFZ) of articular cartilage.

Osteoarthritis (OA) results from an imbalance of the dynamic equilibrium between the breakdown and repair of joint tissues. Previously, we reported that Runx1 enhanced chondrogenic differentiation through transcriptional induction of COL2A1, and suppressed hypertrophic differentiation. Here, we investigated the involvement of Runx1 in OA development as well as its potential underlying molecular mechanism. When we analysed OA development in Col2a1-Cre;Runx1fl/fl and Runx1fl/fl mice by surgically inducing joint instability, Cartilage degradation and osteophyte formation of Col2a1-Cre;Runx1fl/fl joints was accelerated compared with joints in Runx1fl/fl animals 8 weeks after surgery. To investigate chondrocyte regulation by Runx1, we analysed interactions with co-factors and downstream molecules. Runx1 enhanced cartilage matrix production in cooperation with Sox5, Sox6, and Sox9, and co-immunoprecipitation assays showed protein-protein binding between Runx1 and each Sox protein. Knockdown of Runx1 increased expression of a hypertrophic marker, Co10a1, in mouse articular cartilage and primary chondrocytes. This expression was accompanied by decreased expression of Bapx1, a potent suppressor of hypertrophic differentiation. Notably, Runx1-induced suppression of hypertrophic differentiation was diminished by siRNA silencing of Bapx1, whereas chondrogenic markers were unaltered. Thus, Runx1 contributes to articular cartilage maintenance by enhancing matrix production in cooperation with Sox proteins, and suppressing hypertrophic differentiation at least partly via Bapx1 induction.

Chondrocyte -based tissue engineering has been a promising option for the treatment of cartilage lesions. In previous literature, TD198946 has been shown to promote chondrogenic differentiation which could prove useful in cartilage regeneration therapies. Our study aimed to investigate the effects of TD198946 in generating engineered cartilage using dedifferentiated chondrocyte-seeded collagen scaffolds treated with TD198946.

Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) in chondrocytes during endochondral ossification.

Ectopic endochondral ossification in the tendon/ligament is caused by repetitive mechanical overload or inflammation. Tendon stem/progenitor cells (TSPCs) contribute to tissue repair, and some express lubricin [proteoglycan 4 (PRG4)]. However, the mechanisms of ectopic ossification and association of TSPCs are not yet known. Here, we investigated the characteristics of Prg4-positive (+) cells and identified that R-spondin 2 (RSPO2), a WNT activator, is specifically expressed in a distinct Prg4+ TSPC cluster. The Rspo2+ cluster was characterized as mostly undifferentiated, and RSPO2 overexpression suppressed ectopic ossification in a mouse Achilles tendon puncture model via chondrogenic differentiation suppression. RSPO2 expression levels in patients with ossification of the posterior longitudinal ligament were lower than those in spondylosis patients, and RSPO2 protein suppressed chondrogenic differentiation of human ligament cells. RSPO2 was induced by inflammatory stimulation and mechanical loading via nuclear factor κB. Rspo2+ cells may contribute to tendon/ligament homeostasis under pathogenic conditions.

The Runt-related transcription factor (Runx) family plays various roles in the homeostasis of cartilage. Here, we examined the role of Runx2 and Runx3 for osteoarthritis development in vivo and in vitro. Runx3-knockout mice exhibited accelerated osteoarthritis following surgical induction, accompanied by decreased expression of lubricin and aggrecan. Meanwhile, Runx2 conditional knockout mice showed biphasic phenotypes: heterozygous knockout inhibited osteoarthritis and decreased matrix metallopeptidase 13 (Mmp13) expression, while homozygous knockout of Runx2 accelerated osteoarthritis and reduced type II collagen (Col2a1) expression. Comprehensive transcriptional analyses revealed lubricin and aggrecan as transcriptional target genes of Runx3, and indicated that Runx2 sustained Col2a1 expression through an intron 6 enhancer when Sox9 was decreased. Intra-articular administration of Runx3 adenovirus ameliorated development of surgically induced osteoarthritis. Runx3 protects adult articular cartilage through extracellular matrix protein production under normal conditions, while Runx2 exerts both catabolic and anabolic effects under the inflammatory condition.

Subarachnoid hemorrhage (SAH) frequently results in several complications, including cerebral vasospasm, associated with high mortality. Although cerebral vasospasm is a major cause of brain damages after SAH, other factors such as inflammatory responses and oxidative stress also contribute to high mortality after SAH. Trehalose is a non-reducing disaccharide in which two glucose units are linked by α,α-1,1-glycosidic bond, and has been shown to induce tolerance to a variety of stressors in numerous organisms. In the present study, we investigated the effect of trehalose on cerebral vasospasm, inflammatory responses, and oxidative stress induced by blood in vitro and in vivo.

Degeneration of the nucleus pulposus (NP) might serve as a trigger for intervertebral disc degeneration (IDD). A recent drug screening study revealed that the thienoindazole derivative, TD-198946, is a novel drug for the treatment of osteoarthritis. Because of the environmental and functional similarities between articular cartilage and intervertebral disc, TD-198946 is expected to prevent IDD. Herein, we sought to evaluate the effects of TD-198946 on IDD. TD-198946 enhanced glycosaminoglycan (GAG) production and the related genes in mouse NP cells and human NP cells (hNPCs). Further, Kyoto Encyclopedia of Genes and Genomes pathway analysis using the mRNA sequence of hNPCs suggested that the mechanism of action of TD-198946 primarily occurred via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. The Akt inhibitor suppressed the enhancement of GAG production induced by TD-198946. The effects of TD-198946 on IDD at two different time points (immediate treatment model, immediately after the puncture; latent treatment model, 2 weeks after the puncture) were investigated using a mouse tail-disc puncture model. At both time points, TD-198946 prevented a loss in disc height. Histological analysis also demonstrated the preservation of the NP structures. TD-198946 exhibited therapeutic effects on IDD by enhancing GAG production via PI3K/Akt signaling.