ROR1/STAT3 positive feedback loop facilitates cartilage degeneration in Osteoarthritis through activation of NF-κB signaling pathway.

BACKGROUND: Osteoarthritis (OA) is a chronic joint disorder with a serious impact on society. The main pathological change in OA is articular cartilage degeneration, which is directly associated with imbalance of anabolic and catabolic activities in chondrocytes. OBJECTIVE: To evaluate the expression and biological effects of ROR1 in OA cartilage and determine whether knockdown of ROR1 attenuates cartilage degeneration. METHODS: ROR1 expression in OA clinical specimens was evaluated by western blotting and immunohistochemistry. The effects of ROR1 on anabolic and catabolic activities were evaluated in Wnt5a-treated human primary chondrocytes by western blotting, immunofluorescence, and luciferase assay. The effects of ROR1 knockdown on cartilage degeneration in a surgical OA mouse model were examined by X-ray imaging and Safranin O-Fast Green histological staining. RESULTS: ROR1 was considerably upregulated in cartilage tissues of OA patients. ROR1 knockdown alleviated the activation of the NF-κB signaling pathway and reversed the suppression of collagen II and aggrecan by Wnt5a, as well as upregulation of ADAMTS-5 and MMP-13 in chondrocytes. In addition, ROR1 knockdown significantly reduced Wnt5a-induced STAT3 nuclear translocation. STAT3 binding to the ROR1 promoter indicated a positive feedback loop between ROR1 and STAT3. ROR1 knockdown was confirmed to dramatically alleviate cartilage degradation in the DMM induced-OA mouse model. CONCLUSION: Increased expression of ROR1 in OA cartilage tissues leads to a positive feedback loop with STAT3, which activates the NF-κB signaling pathway, resulting in an imbalance between chondrocyte anabolism and catabolism. These results indicate a potential new therapeutic target for the treatment of OA.

AdipoAI suppresses osteoclastogenesis by activating AdipoR1/APPL1: An in vivo experimental study in diabetes-associated peri-implantitis.

AIM: Diabetics experience severe peri-implant inflammatory bone damage. We aimed to provide powerful evidence supporting the novel adiponectin receptor agonist AdipoAI in treating diabetes-associated peri-implantitis. MATERIALS AND METHODS: Twenty-four ZDF-Leprfa/Crl rats were randomly allocated to three groups (N = 8). After feeding with a high-fat diet to establish diabetic rats, experimental peri-implantitis was induced by implanting titanium rods (1.5 mm diameter and 20 mm length) contaminated with Staphylococcus aureus into the femurs. Radiographic evaluation, microCT, histological analyses and qRT-PCR were used to detect inflammatory infiltration and bone destruction. In vitro, the inhibition by AdipoAI of osteoclastogenesis, including the number and function of osteoclasts, was investigated by TRAP staining, immunofluorescence, qRT-PCR and Western blotting. Immunofluorescence, qRT-PCR and Western blotting were also utilized to explore AdipoR1, APPL1, NF-κB and Wnt5a-Ror2 signalling molecules in this process. One-way ANOVA with Tukey's post hoc test was used to compare the data. RESULTS: AdipoAI reduced inflammation and bone destruction caused by peri-implantitis in diabetic rats, which were manifested by a reduction in F4/80-positive macrophage infiltration by 72%, the number of osteoclasts by 58% and the levels of cytokines (p < .05) in disease group. In vitro, 1 µM AdipoAI decreased the number of osteoclasts to 51%, inhibited F-actin ring formation and reduced the levels of related markers (p < .05). Mechanistically, AdipoAI activated AdipoR1/APPL1 and conversely suppressed the phosphorylation of IκB-α, nuclear translocation of P65 and the Wnt5a-Ror2 signalling pathway (p < .05). CONCLUSIONS: AdipoAI suppressed osteoclastogenesis in diabetes-associated peri-implantitis by inhibiting the NF-κB and Wnt5a-Ror2 pathways via the AdipoR1/APPL1 axis.

Design, synthesis, and biological evaluation of 6-(imidazo[1,2-a] pyridin-6-yl) quinazolin-4(3H)-one derivatives as potent anticancer agents by dual targeting Aurora kinase and ROR1.

ROR1 and Aurora kinase were overexpressed in various cancers and essential for cell proliferation, survive and metastasis. Pharmaceutical inhibition of ROR1 and Aurora kinase abrogated the activation of downstream signaling and induced cancer cell apoptosis. Hence, ROR1 and Aurora kinase considered as attractive therapeutic targets for the development of anticancer drugs. In the present work, three series of novel 6-(imidazo[1,2-a] pyridin-6-yl)-quinazolin-4(3H)-one derivatives were designed and synthesized via bioisosterism and scaffold-hopping strategies guided by FLF-13, an Aurora kinase inhibitor we discovered earlier. Most of compounds in series 2 and series 3 showed submicromolar to nanomolar inhibitory activity against multiple cancer cell lines. More importantly, compounds 12d and 12f in series 3 showed nanomolar inhibitory activity against all test cancer cells. The most promising compound 12d exhibited potent inhibitory activity against Aurora A and Aurora B with IC50 values of 84.41 nM and 14.09 nM, respectively. Accordingly, compounds 12d induced G2/M phase cell cycle arrest at 24 h and polyploidy at 48 h. It's worth noting that 12d also displayed inhibitory activity against ROR1 and induce cell apoptosis. Furthermore, 12d could significantly inhibit the tumor growth in SH-SY5Y xenograft model with tumor growth inhibitory rate (IR) up to 46.31 % at 10 mg/kg and 52.66 % at 20 mg/kg. Overall, our data suggested that 12d might serve as a promising candidate for the development of therapeutic agents for cancers with aberrant expression of ROR1 and Aurora kinases by simultaneously targeting ROR1 and Aurora kinase.

Wnt5a-Ror2 signaling mediates root resorption.

INTRODUCTION: This study aimed to investigate the role of wingless-type MMTV integration site family member 5a (Wnt5a)-receptor tyrosine kinase-like orphan receptor 2 (Ror2) signaling in root resorption. METHODS: The messenger RNA (mRNA) expression of Wnt5a, Ror2, and RANKL in periodontal ligament cells (PDLCs) under compression force (CF) with or without Ror2 small interfering RNA (siRNA) were measured by quantitative reverse transcription-polymerase chain reaction, and these proteins released into culture supernatants were measured using enzyme-linked immunosorbent assay. Then these PDLC-conditioned media under CF with or without Ror2 siRNA were used to culture osteoclast precursors to detect osteoclastogenesis effects via tartrate-resistant acid phosphatase staining. In in vivo studies, the odontoclast number and the root resorption volume under excessive CF with or without Ror2 siRNA were investigated by tartrate-resistant acid phosphatase immunohistochemical staining and microcomputed tomography. The protein levels for Wnt5a, Ror2, and receptor activator of nuclear factor-kappa B ligand (RANKL) in the periodontal ligament tissues were also detected using immunohistochemical staining. Finally, the odontoclast number, root resorption volume, and the mRNA and protein expressions were compared between immature and mature teeth. RESULTS: The mRNA production and protein release level of Wnt5a, Ror2, and RANKL increased after CF, whereas they were significantly downregulated with Ror2 siRNA. The osteoclast number increased treating with culture medium from PDLC applying CF, but the increase was inhibited after adding Ror2 siRNA. In the animal model, the odontoclast number and root resorption volume significantly increased in the CF group but decreased in the CF with the Ror2 siRNA group. The protein levels of Wnt5a, Ror2, and RANKL in periodontal ligament were upregulated under excessive CF, and the pathway was inhibited with Ror2 siRNA. In the immature tooth group, the odontoclast number, root resorption volume, and the mRNA and protein expressions of Wnt5a-Ror2 signaling were reduced. CONCLUSIONS: Wnt5a-Ror2 signaling in PDLCs enhanced by excessive CF could promote RANKL release and induce precursor differentiation, partly leading to increased odontoclast activity and ultimate root resorption. The less resorption of the immature tooth may be due to odontoclastogenesis inhibition by decreased expression of Wnt5a-Ror2 signaling.

ROR2 modulates neuropathic pain via phosphorylation of NMDA receptor subunit GluN2B in rats.

BACKGROUND: Neuropathic pain, a type of chronic pain as a result of direct central or peripheral nerve damage, is associated with significant quality of life and functional impairment. Its underlying mechanisms remain unclear. We investigated whether ROR2, a member of the receptor tyrosine kinase-like orphan receptor (ROR) family, participates in modulation of neuropathic pain. METHODS: Thermal hyperalgesia and mechanical allodynia were measured using radiant heat and von Frey filament testing. Immunofluorescence staining was used to detect expression of ROR2 in neuronal nuclei. Fos expression was determined by immunocytochemistry. Phosphorylation status was detected by western blot and immunoprecipitation. Small interfering RNA was used to knock down ROR2 expression. RESULTS: ROR2 was upregulated and activated in spinal neurones after chronic constriction injury (CCI) in mice [1.3 (0.1) to 2.1 (0.1)-fold of sham, P<0.01] from Day 1-21. CCI induced significant demethylation of the CpG island in the ROR2 gene promoter [0.37 (0.06) vs 0.12 (0.03)% CpG methylation, P<0.001]. Knockdown of ROR2 in the spinal cord prevented and reversed CCI-induced pain behaviours and spinal neuronal sensitisation [Fos expression: 130 (12) vs 81 (8) cells, P<0.05; 120 (11) vs 70 (7) cells, P<0.05]. In contrast, activation of spinal ROR2 by intrathecal injection of Wnt5a induced pain behaviours and spinal neuronal sensitisation [Fos expression: 11 (1) vs 100 (12) cells, P<0.001] in wild-type mice. Furthermore, ROR2-mediated pain modulation required phosphorylation of N-methyl-D-aspartate receptor 2B subunit (GluN2B) at Ser 1303 and Tyr1472 by pathways involving protein kinase C (PKC) and Src family kinases. Intrathecal injection of GluN2B, PKC, or Src family kinase-specific inhibitors significantly attenuated Wnt5a-induced pain behaviours. CONCLUSIONS: ROR2 in the spinal cord regulates neuropathic pain via phosphorylation of GluN2B, suggesting a potential target for prevention and relief of neuropathic pain.