ObjectivesRecently, necroptosis has attracted increasing attention in arthritis research; however, it remains unclear whether its regulation is involved in osteoarthritis (OA) pathogenesis. Since receptor-interacting protein kinase-3 (RIP3) plays a pivotal role in necroptosis and its dysregulation is involved in various pathological processes, we investigated the role of the RIP3 axis in OA pathogenesis.MethodsExperimental OA was induced in wild-type or Rip3 knockout mice by surgery to destabilise the medial meniscus (DMM) or the intra-articular injection of adenovirus carrying a target gene (Ad-Rip3 and Ad-Trim24 shRNA). RIP3 expression was examined in OA cartilage from human patients; Trim24, a negative regulator of RIP3, was identified by microarray and in silico analysis. Connectivity map (CMap) and in silico binding approaches were used to identify RIP3 inhibitors and to examine their direct regulation of RIP3 activation in OA pathogenesis.ResultsRIP3 expression was markedly higher in damaged cartilage from patients with OA than in undamaged cartilage. In the mouse model, adenoviral RIP3 overexpression accelerated cartilage disruption, whereas Rip3 depletion reduced DMM-induced OA pathogenesis. Additionally, TRIM24 knockdown upregulated RIP3 expression; its downregulation promoted OA pathogenesis in knee joint tissues. The CMap approach and in silico binding assay identified AZ-628 as a potent RIP3 inhibitor and demonstrated that it abolished RIP3-mediated OA pathogenesis by inhibiting RIP3 kinase activity.ConclusionsTRIM24-RIP3 axis perturbation promotes OA chronicity by activating RIP3 kinase, suggesting that the therapeutic manipulation of this pathway could provide new avenues for treating OA.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD + ) salvage pathway and plays a crucial role in the maintenance of the NAD + pool during inflammation. Considering that macrophages are essential for tissue homeostasis and inflammation, we sought to examine the functional impact of NAMPT in inflammatory macrophages, particularly in the context of inflammatory bowel disease (IBD). In this study, we show that mice with NAMPT deletion within the myeloid compartment ( Nampt f/f LysMCre +/- , Nampt mKO) have more pronounced colitis with lower survival rates, as well as numerous uncleared apoptotic corpses within the mucosal layer. Nampt -deficient macrophages exhibit reduced phagocytic activity due to insufficient NAD + abundance, which is required to produce NADPH for the oxidative burst. Nicotinamide mononucleotide (NMN) treatment rescues NADPH levels in Nampt mKO macrophages and sustains superoxide generation via NADPH oxidase. Consequently, Nampt mKO mice fail to clear dead cells during tissue repair, leading to substantially prolonged chronic colitis. Moreover, systemic administration of NMN, to supply NAD + , effectively suppresses the disease severity of DSS-induced colitis. Collectively, our findings suggest that activation of the NAMPT-dependent NAD + biosynthetic pathway, via NMN administration, is a potential therapeutic strategy for managing inflammatory diseases.

Background: The programmed cell death pathway necroptosis may synergize with the DNA damage response (DDR) in opposing tumor progression. While our basic mechanistic understanding of the necroptotic cell death advances rapidly, its prognostic implications have not been thoroughly examined in cancers.Methods: We included 394 patients with stage I non-small-cell lung cancer (NSCLC) who underwent surgical tumor resection between 1 January 1997 and 31 December 2011 and measured expression levels of nine proteins involved in necroptosis and the DDR in primary samples from 394 patients using tissue microarray. Protein expression evaluated by using an H-score method was dichotomized by the median value. The overall survival as the endpoint was calculated from the time of diagnosis to the time of the last follow-up or death.Results: We find that low-level expression of the necroptosis markers RIPK3 and PELI1 is associated with high risk of patient death. High-level expression of the key DDR factor p53 in combination with lowlevel expression of either RIPK3 or PELI1 increases the risk further. These gene expression effects appear to occur specifically in the squamous cell carcinoma (SCC) subtype of stage I NSCLC, while not observed in the non-SCC subtypes.Conclusions: Low-level expression of such necroptosis factors as RIPK3 and PELI1 in combination with high-level expression of the DDR factor p53 can serve as a critical indicator in predicting survival of stage I NSCLC patients with the SCC subtype.

Receptor‐interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4+CD8+ double‐positive (DP) thymocytes. Abnormal proliferation of RIPK3‐deficient DP thymocytes occurs independently of the well‐known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3‐null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N‐ethyl‐N‐nitrosourea (ENU)‐induced tumor model. Moreover, RIPK3 deficiency in p53‐null mice promotes thymic lymphoma development via upregulated extracellular signal‐regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte‐specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper‐activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3‐PP2A‐ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.

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