Runx2 and Runx3 differentially regulate articular chondrocytes during surgically induced osteoarthritis development.

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.

Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling.

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.

Wnt/ß-catenin signaling contributes to articular cartilage homeostasis through lubricin induction in the superficial zone.

BACKGROUND: Both loss- and gain-of-function of Wnt/ß-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/ß-catenin signaling in the superficial zone (SFZ) of articular cartilage. METHODS: Wnt/ß-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice for loss- and gain-of-function, respectively, of Wnt/ß-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/ß-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/ß-catenin signaling in SFZ cells. RESULTS: Wnt/ß-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific ß-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific ß-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by ß-catenin knockout, while it was upregulated by ß-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/ß-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4. CONCLUSIONS: We demonstrated that Wnt/ß-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.

Intra-articular administration of IκBα kinase inhibitor suppresses mouse knee osteoarthritis via downregulation of the NF-κB/HIF-2α axis.

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-1ß-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.

Alteration of gait parameters in a mouse model of surgically induced knee osteoarthritis.

PURPOSE: Joint pain is the most common symptom of osteoarthritis (OA); however, its mechanism remains unclarified. The present study investigated hindlimb motion during locomotion on the treadmill using a three-dimensional (3D) motion analysis system with high-speed cameras to evaluate whether this method can be used as an indication of joint pain in a mouse model of surgically induced OA. METHODS: We resected the medial meniscus and medial collateral ligament in 8-week old C57BL/6 male mice and performed locomotion recording 6 months post-operatively. Additionally, we performed the same recording after oral administration of the selective cyclooxygenase-2 inhibitor to determine whether alteration of the parameters were associated with joint pain. RESULTS: OA development, characterized by cartilage degeneration and osteophyte formation, was markedly enhanced in the OA group. There was no significant difference between the sham and OA groups in basic gait parameters, including stance duration, swing duration and gait cycle. However, when we divided the gait cycle into four phases and calculated the joint ranges of motion in each phase, the range of motion of the knee joint during the stepping-in phase and the swing duration were significantly decreased in the OA group. These significant differences between the sham and OA groups were diminished by the oral administration of a selective cyclooxygenase-2 inhibitor to the OA group. CONCLUSION: The present method may be useful to evaluate joint pain in experimental mice and contribute to elucidating the molecular mechanisms of pain in the OA knee joint in combination with genetically modified mice.

Loss of Mob1a/b in mice results in chondrodysplasia due to YAP1/TAZ-TEAD-dependent repression of SOX9.

Hippo signaling is modulated in response to cell density, external mechanical forces, and rigidity of the extracellular matrix (ECM). The Mps one binder kinase activator (MOB) adaptor proteins are core components of Hippo signaling and influence Yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ), which are potent transcriptional regulators. YAP1/TAZ are key contributors to cartilage and bone development but the molecular mechanisms by which the Hippo pathway controls chondrogenesis are largely unknown. Cartilage is rich in ECM and also subject to strong external forces - two upstream factors regulating Hippo signaling. Chondrogenesis and endochondral ossification are tightly controlled by growth factors, morphogens, hormones, and transcriptional factors that engage in crosstalk with Hippo-YAP1/TAZ signaling. Here, we generated tamoxifen-inducible, chondrocyte-specific Mob1a/b-deficient mice and show that hyperactivation of endogenous YAP1/TAZ impairs chondrocyte proliferation and differentiation/maturation, leading to chondrodysplasia. These defects were linked to suppression of SOX9, a master regulator of chondrogenesis, the expression of which is mediated by TEAD transcription factors. Our data indicate that a MOB1-dependent YAP1/TAZ-TEAD complex functions as a transcriptional repressor of SOX9 and thereby negatively regulates chondrogenesis.