Transcription of NOD1 and NOD2 and their interaction with CARD9 and RIPK2 in IFN signaling in a perciform fish, the Chinese perch, Siniperca chuatsi.

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.

NOD1 cooperates with HAX-1 to promote cell migration in a RIPK2- and NF-ĸB-independent manner.

The human Nod-like receptor protein NOD1 is a well-described pattern-recognition receptor (PRR) with diverse functions. NOD1 associates with F-actin and its protein levels are upregulated in metastatic cancer cells. A hallmark of cancer cells is their ability to migrate, which involves actin remodelling. Using chemotaxis and wound healing assays, we show that NOD1 expression correlated with the migration rate and chemotactic index in the cervical carcinoma cell line HeLa. The effect of NOD1 in cell migration was independent of the downstream kinase RIPK2 and NF-ĸB activity. Additionally, NOD1 negatively regulated the phosphorylation status of cofilin, which inhibits actin turnover. Co-immunoprecipitation assays identified HCLS1-associated protein X-1 (HAX-1) as a previously unknown interaction partner of NOD1. Silencing of HAX-1 expression reduced the migration behaviour to similar levels as NOD1 knockdown, and simultaneous knockdown of NOD1 and HAX-1 showed no additive effect, suggesting that both proteins act in the same pathway. In conclusion, our data revealed an important role of the PRR NOD1 in regulating cell migration as well as chemotaxis in human cervical cancer cells and identified HAX-1 as a protein that interacts with NOD1 and is involved in this signalling pathway.

RIPK2 promotes the progression of colon cancer by regulating BIRC3-mediated ubiquitination of IKBKG.

Colon cancer is a cancer with high morbidity and mortality worldwide. Receptor interacting serine/threonine kinase 2 (RIPK2) has been identified as a proto-oncogene, but its role in colon cancer is largely unknown. Herein, we found that RIPK2 interference could inhibit the proliferation and invasion of colon cancer cells, and promote apoptosis. Baculoviral IAP repeat containing 3 (BIRC3) is an E3 ubiquitin ligase, which was found highly expressed in colon cancer cells. Co-immunoprecipitation (Co-IP) experiments showed that RIPK2 could directly bind with BIRC3. Then, we demonstrated that RIPK2 overexpression promoted the expression of BIRC3, BIRC3 interference could eliminate RIPK2-dependent cell proliferation and invasion, and BIRC3 overexpression rescued the suppressive effect of RIPK2 interference on cell proliferation and invasion. We further identified IKBKG, an inhibitor of nuclear factor kappa B, as a ubiquitination substrate targeted by BIRC3. IKBKG interference could eliminate the inhibitory effect of BIRC3 interference on cell invasion. RIPK2 could promote BIRC3-mediated ubiquitination of IKBKG, inhibit the expression of IKBKG protein, and promote the expression of NF-κB subunits p50 and p65 proteins. In addition, DLD-1 cells transfected with sh-RIPK2 or/and sh-BIRC3 were injected into mice to establish a tumor xenograft model, and we found that administration of sh-RIPK2 or sh-BIRC3 impeded the growth of xenograft tumors in vivo, and co-administration displayed a better inhibitory effect. In general, RIPK2 promotes the progression of colon cancer by promoting BIRC3-mediated ubiquitination of IKBKG and activating the NF-κB signaling pathway.

Paired protein kinases PRKCI-RIPK2 promote pancreatic cancer growth and metastasis via enhancing NF-κB/JNK/ERK phosphorylation.

BACKGROUND: Protein kinases play a pivotal role in the malignant evolution of pancreatic cancer (PC) through mediating phosphorylation. Many kinase inhibitors have been developed and translated into clinical use, while the complex pathology of PC confounds their clinical efficacy and warrants the discovery of more effective therapeutic targets. METHODS: Here, we used the Gene Expression Omnibus (GEO) database and protein kinase datasets to map the PC-related protein kinase-encoding genes. Then, applying Gene Expression and Profiling Interactive Analysis (GEPIA), GEO and Human Protein Atlas, we evaluated gene correlation, gene expression at protein and mRNA levels, as well as survival significance. In addition, we performed protein kinase RIPK2 knockout and overexpression to observe effects of its expression on PC cell proliferation, migration and invasion in vitro, as well as cell apoptosis, reactive oxygen species (ROS) production and autophagy. We established PC subcutaneous xenograft and liver metastasis models to investigate the effects of RIPK2 knockout on PC growth and metastasis. Co-immunoprecipitation and immunofluorescence were utilized to explore the interaction between protein kinases RIPK2 and PRKCI. Polymerase chain reaction and immunoblotting were used to evaluate gene expression and protein phosphorylation level. RESULTS: We found fourteen kinases aberrantly expressed in human PC and nine kinases with prognosis significance. Among them, RIPK2 with both serine/threonine and tyrosine activities were validated to promote PC cells proliferation, migration and invasion. RIPK2 knockout could inhibit subcutaneous tumor growth and liver metastasis of PC. In addition, RIPK2 knockout suppressed autophagosome formation, increased ROS production and PC cell apoptosis. Importantly, another oncogenic kinase PRKCI could interact with RIPK2 to enhance the phosphorylation of downstream NF-κB, JNK and ERK. CONCLUSION: Paired protein kinases PRKCI-RIPK2 with multiple phosphorylation activities represent a new pathological mechanism in PC and could provide potential targets for PC therapy.

Mechanism of RIP2 enhancing stemness of glioma cells induces temozolomide resistance.

AIMS: We aimed to investigate the role of receptor-interacting protein 2 (RIP2) in regulation of stemness of glioma cells and chemotherapy resistance. METHODS: Plasmid transfection was used to overexpress RIP2. Chemical inhibitors were used to inhibit RIP2 or NF-κB activity. Cancer stemness of glioma cells was investigated by sphere formation assays, clone formation assays, and xenograft tumor formation assays. The expression of RIP2, p-NF-κB, IκBα, CD133, or SOX-2 was detected by Western blotting and immunofluorescence. Apoptosis was detected by flow cytometry. Immunohistochemical staining was used to detect the expression of RIP2, CD133, and SOX-2 in xenograft tumor tissue. The effect of the RIP2/NF-κB pathway on temozolomide (TMZ) resistance was evaluated by xenograft tumor assay. RESULTS: Transfection with RIP2 plasmid enhanced the sphere formation capability of U251 cells, clone formation capability, and xenograft tumor formation capability. RIP2 could mediate TMZ resistance by upregulating the expression of CD133 and SOX-2 by activating the NF-κB pathway. Both RIP2 inhibitor GSK583 and the NF-κB inhibitor SC75741 could reverse the resistance of U251 cells to TMZ. CONCLUSION: RIP2 mediates TMZ resistance by regulating the maintenance of stemness in glioma cells through NF-κB. Interventions targeting the RIP2/NF-κB pathway may be a new strategy for TMZ-resistant gliomas.

NOD/RIPK2 signalling pathway contributes to osteoarthritis susceptibility.

OBJECTIVES: How inflammatory signalling contributes to osteoarthritis (OA) susceptibility is undetermined. An allele encoding a hyperactive form of the Receptor Interacting Protein Kinase 2 (RIPK2) proinflammatory signalling intermediate has been associated with familial OA. To test whether altered nucleotide-binding oligomerisation domain (NOD)/RIPK2 pathway activity causes heightened OA susceptibility, we investigated whether variants affecting additional pathway components are associated with familial OA. To determine whether the Ripk2104Asp disease allele is sufficient to account for the familial phenotype, we determined the effect of the allele on mice. METHODS: Genomic analysis of 150 independent families with dominant inheritance of OA affecting diverse joints was used to identify coding variants that segregated strictly with occurrence of OA. Genome editing was used to introduce the OA-associated RIPK2 (p.Asn104Asp) allele into the genome of inbred mice. The consequences of the Ripk2104Asp disease allele on physiology and OA susceptibility in mice were measured by histology, immunohistochemistry, serum cytokine levels and gene expression. RESULTS: We identified six novel variants affecting components of the NOD/RIPK2 inflammatory signalling pathway that are associated with familial OA affecting the hand, shoulder or foot. The Ripk2104Asp allele acts dominantly to alter basal physiology and response to trauma in the mouse knee. Whereas the knees of uninjured Ripk2Asp104 mice appear normal histologically, the joints exhibit a set of marked gene expression changes reminiscent of overt OA. Although the Ripk2104Asp mice lack evidence of chronically elevated systemic inflammation, they do exhibit significantly increased susceptibility to post-traumatic OA (PTOA). CONCLUSIONS: Two types of data support the hypothesis that altered NOD/RIPK2 signalling confers susceptibility to OA.

Pan-cancer analysis reveals RIPK2 predicts prognosis and promotes immune therapy resistance via triggering cytotoxic T lymphocytes dysfunction.

BACKGROUND: Receptor-interacting protein kinase 2 (RIPK2, also known as RIP2) was reported to be associated with bacterial infections as well as inflammatory responses. However, the role of RIPK2 in prognosis and immunotherapy response is yet to be elucidated in human pan-cancer. METHODS: In this study, we investigated the expression, gene alteration landscape and prognostic value of RIPK2 in 33 cancers through various databases including Ualcan, cBioportal and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Then, the correlation between RIPK2 and immune infiltration, immune score, stromal score, and ESTIMATE score was investigated in the Cancer Genome Atlas (TCGA) and tumor immune estimation resource (TIMER) databases. Independent cohorts were utilized to explore the role of RIPK2 in tumor immunotherapy response. Furthermore, Gene set enrichment analysis (GSEA) was conducted to explore the mechanisms by which RIPK2 regulates immune therapy resistance. Single-cell RNA-seq datasets were used to analyze the expression level of RIPK2 on different immune cells. Moreover, CellMiner database was used to explore the relationship between RIPK2 expression with drug response. RESULT: Compared with normal tissue, tumor tissue had a higher expression level of RIPK2 in various cancers. Survival analysis showed that high expression of RIPK2 associated with poor prognosis in numerous cancers. RIPK2 was found to promote a series of immune cell infiltration and B cells, macrophages, and neutrophils were significantly positively correlated with the expression of RIPK2. Moreover, RIPK2 affected immune score, stromal score and ESTIMATE score for a wide range of cancers. In the vast majority of 33 cancers, gene co-expression analysis showed that RIPK2 was positively correlated with the expression of immune checkpoint markers, such as PDCD1 (PD-1), CD274 (PD-L1), CTLA4 and TIGIT. RIPK2 aggravated cytotoxic T lymphocyte (CTL) dysfunction and related to the poor efficacy of immune checkpoint blockade in skin cutaneous melanoma (SKCM) and kidney renal clear cell carcinoma (KIRC). High expression of RIPK2 promoted innate immunotherapy resistance and adaptive immunotherapy resistance through IL-6/JAK/STAT3 signaling, interferon-gamma response, and interferon-alpha response pathway. CONCLUSIONS: These results confirmed that RIPK2 could serve as a prognostic biomarker and promoted immune therapy resistance via triggering cytotoxic T lymphocytes dysfunction.

Transcriptional Regulation of RIP2 Gene by NFIB Is Associated with Cellular Immune and Inflammatory Response to APEC Infection.

Avian pathogenic E. coli (APEC) can cause localized or systemic infection, resulting in large economic losses per year, and impact health of humans. Previous studies showed that RIP2 (receptor interacting serine/threonine kinase 2) and its signaling pathway played an important role in immune response against APEC infection. In this study, chicken HD11 cells were used as an in vitro model to investigate the function of chicken RIP2 and the transcription factor binding to the RIP2 core promoter region via gene overexpression, RNA interference, RT-qPCR, Western blotting, dual luciferase reporter assay, CHIP-PCR, CCK-8, and flow cytometry assay following APEC stimulation. Results showed that APEC stimulation promoted RIP2 expression and cells apoptosis, and inhibited cells viability. Knockdown of RIP2 significantly improved cell viability and suppressed the apoptosis of APEC-stimulated cells. Transcription factor NFIB (Nuclear factor I B) and GATA1 (globin transcription factor 1) binding site was identified in the core promoter region of RIP2 from -2300 bp to -1839 bp. However, only NFIB was confirmed to be bound to the core promoter of RIP2. Overexpression of NFIB exacerbated cell injuries with significant reduction in cell viability and increased cell apoptosis and inflammatory cytokines levels, whereas opposite results were observed in NFIB inhibition treatment group. Moreover, RIP2 was up-regulated by NFIB overexpression, and RIP2 silence mitigated the effect of NFIB overexpression in cell apoptosis, inflammation, and activation of NFκB signaling pathways. This study demonstrated that NFIB overexpression accelerated APEC-induced apoptosis and inflammation via up-regulation of RIP2 mediated downstream pathways in chicken HD11 cells.

High expression of RIPK2 is associated with Taxol resistance in serous ovarian cancer.

BACKGROUND: Taxol resistance in serous ovarian cancer is responsible for its poor prognosis, yet the underlying mechanism is still poorly understood. Thus, we probed the mechanism of Taxol resistance in serous ovarian cancer with multiple bioinformatic methods to provide novel insights into potential therapies. METHODS: The differentially expressed genes (DEGs) in Taxol-sensitive and Taxol-resistant cell lines and their relationship with the overall survival (OS) and progression-free interval (PFI) of ovarian cancer patients were analyzed using gene expression datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The role of receptor interacting serine/threonine kinase 2 (RIPK2) was validated via identification of its coexpressed genes, functional analysis and generation of a protein-protein interaction (PPI) network. The single sample gene set enrichment analysis (ssGSEA) was used to explore immune infiltration, and genomic alterations of RIPK2 were also analyzed via cBio Cancer Genomics Portal (cBioProtal). RESULTS: RIPK2 was highly expressed in Taxol resistant ovarian cancer cell lines, and its high expression was also linked with shorter OS and PFI in serous ovarian cancer patients. The PPI network analysis and pathway analysis demonstrated that RIPK2 might participate in the positive regulation of NF-κB transcription factor activity. RIPK2 expression was related to tumor microenvironment alterations, which might participate in the formation of Taxol resistance. CONCLUSIONS: Our studies suggested that high expression of RIPK2 is related to Taxol resistance in serous ovarian cancer, and that RIPK2 induces Taxol resistance through NOD1/RIPK2/NF-κB inflammatory pathway activation and tumor microenvironment changes.

A pancancer analysis of the carcinogenic role of receptor-interacting serine/threonine protein kinase-2 (RIPK2) in human tumours.

BACKGROUND: To explore the expression and carcinogenic mechanism of RIPK2 in human tumours, and to provide the theoretical basis for the further study of RIPK2. METHODS: We used the TCGA, CPTAC, HPA databases to analyse the expression, mutation, and prognosis of RIPK2 in human tumours. Through the Cbioportal, Ualcan, TIMER2.0, and STRING websites, We understand the genetic variation, immune infiltration and enrichment analysis of RIPK2 related genes. RESULTS: RIPK2 was highly expressed in most tumours (such as BRCA, COAD and LUSC, etc.), and the high expression of RIPK2 was correlated with tumour stage and prognosis. In addition, Amplification was the main type of RIPK2 in tumour mutation state, and the amplification rate was about 8.5%. In addition, RIPK2 was positively associated with tumour-infiltrating immune cells (such as CD8+ T, Tregs, and cancer-associated fibroblasts). According to the KEGG analysis, RIPK2 may play a role in tumour mainly through NOD-like signaling pathway and NF-kappaB signaling pathway. GO enrichment analysis showed that the RIPK2 is mainly related to I-kappaB kinase/NF-kappaB signaling, Ribonucleoprotein granule and Ubiquitin-like protein ligase binding. CONCLUSION: RIPK2 plays an important role in the occurrence, development and prognosis of malignant tumours. Our pancancer study provided a relatively comprehensive description of the carcinogenic effects of RIPK2 in different tumours, and provided useful information for further study of RIPK2.

Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis.

Despite progress in prostate cancer (PC) therapeutics, distant metastasis remains a major cause of morbidity and mortality from PC. Thus, there is growing recognition that preventing or delaying PC metastasis holds great potential for substantially improving patient outcomes. Here we show receptor-interacting protein kinase 2 (RIPK2) is a clinically actionable target for inhibiting PC metastasis. RIPK2 is amplified/gained in ~65% of lethal metastatic castration-resistant PC. Its overexpression is associated with disease progression and poor prognosis, and its genetic knockout substantially reduces PC metastasis. Multi-level proteomics analyses reveal that RIPK2 strongly regulates the stability and activity of c-Myc (a driver of metastasis), largely via binding to and activating mitogen-activated protein kinase kinase 7 (MKK7), which we identify as a direct c-Myc-S62 kinase. RIPK2 inhibition by preclinical and clinical drugs inactivates the noncanonical RIPK2/MKK7/c-Myc pathway and effectively impairs PC metastatic outgrowth. These results support targeting RIPK2 signaling to extend metastasis-free and overall survival.

RIP2 Knockdown Attenuates Vascular Smooth Muscle Cells Activation via Negative Regulating Myocardin Expression.

BACKGROUND: RIP2 is an adaptor protein contributing to the activation of nuclear factor-κB induced by TNF receptor-associated factor (TRAF) and nucleotide oligomerization domain (NOD)-dependent signaling implicated in innate and adaptive immune response. Beyond regulation of immunity, we aimed to elucidate the role of RIP2 in vascular smooth muscle cell (VSMC) phenotypic modulation. METHODS AND RESULTS: In the current study, we observed that RIP2 showed an increased expression in VSMCs with PDGF-BB stimulation in a dose-dependent manner. Knockdown of RIP2 expression mediated by adenovirus dramatically accelerated the expression of VSMC-specific differentiation genes induced by PDGF-BB. Silencing of RIP2 inhibited proliferative and migratory ability of VSMCs. Additionally, we demonstrated that RIP2 knockdown can promoted myocardin expression. Furthermore, RIP2 inhibition also can attenuate the formation of intimal hyperplasia. CONCLUSIONS: These findings suggested that RIP2 played an important role in regulation of VSMCs differentiation, migration, and proliferation that may due to affect myocardin expression. Our results indicated that RIP2 may be a novel therapeutic target for intimal hyperplasia.

Identification of hub genes associated with cognition in the hippocampus of Alzheimer's Disease.

Alzheimer's Disease (AD) is a neurodegenerative disease featured by cognitive impairment. This bioinformatic analysis was used to identify hub genes related to cognitive dysfunction in AD. The gene expression profile GSE48350 in the hippocampus of AD patients aged >70 years was obtained from the Gene Expression Omnibus (GEO) database. A total of 96 differentially expressed genes (DEGs) were identified, and subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses; a protein-protein interaction (PPI) network was constructed. The DEGs were enriched in synapse-related changes. A protein cluster was teased out of PPI. Furthermore, the cognition ranked the first among all the terms of biological process (BP). Next, 4 of 10 hub genes enriched in cognition were identified. The function of these genes was validated using APP/PS1 mice. Cognitive performance was validated by Morris Water Maze (MWM), and gene expression by RT-qPCR, Cholecystokinin (CCK), Tachykinin precursor 1 (TAC1), Calbindin 1 (CALB1) were downregulated in the hippocampus. These genes can provide new directions in the research of the molecular mechanism of AD.

The potent and selective RIPK2 inhibitor BI 706039 improves intestinal inflammation in the TRUC mouse model of inflammatory bowel disease.

Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.

Receptor-Interacting Serine/Threonine-Protein Kinase-2 as a Potential Prognostic Factor in Colorectal Cancer.

Background and objectives: Receptor-interacting serine/threonine-protein kinase-2 (RIPK2) is an important mediator in different pathways in the immune and inflammatory response system. RIPK2 was also shown to play different roles in different cancer types; however, in colorectal cancer (CRC), its role is not well established. This study aims at identifying the role of RIPK2 in CRC progression and survival. Materials and methods: Data of patients and mRNA protein expression level of genes associated with CRC (RIPK2, tumor necrosis factor (TNF), TRAF1, TRAF7, KLF6, interlukin-6 (Il6), interlukin-8 (Il8), vascular-endothelial growth factor A (VEGFA), MKI67, TP53, nuclear factor-kappa B (NFKB), NFKB2, BCL2, XIAP, and RELA) were downloaded from the PrognoScan online public database. Patients were divided between low and high RIPK2 expression and different CRC characteristics were studied between the two groups. Survival curves were evaluated using a Kaplan-Meier estimator. The Pearson correlation was used to study the correlation between RIPK2 and the other factors. Statistical analysis was carried out using SPSS version 25.0. The Human Protein Atlas was also used for the relationship between RIPK2 expression in CRC tissues and survival. Differences were considered statistically significant at p < 0.05. Results: A total of 520 patients were downloaded from the PrognoScan database, and RIPK2 was found to correlate with MKI67, TRAF1, KLF6, TNF, Il6, Il8, VEGFA, NFKB2, BCL2, and RELA. High expression of RIPK2 was associated with high expression of VEGFA (p < 0.01) and increased mortality (p < 0.01). Conclusions: In this study, RIPK2 is shown to be a potential prognostic factor in CRC; however, more studies are needed to assess and verify its potential role as a prognostic marker and in targeted therapy.

Optimization of a Series of RIPK2 PROTACs.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.

14-3-3 and erlin proteins differentially interact with RIPK2 complexes.

The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for signal transduction induced by the pattern recognition receptors NOD1 and NOD2 (referred to collectively as NOD1/2). Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells. Here, we identified the molecular composition of these complexes. Infection with Shigella flexneri to activate NOD1-RIPK2 revealed that RIPK2 formed dynamic interactions with several cellular proteins, including A20 (also known as TNFAIP3), erlin-1, erlin-2 and 14-3-3. Whereas interaction of RIPK2 with 14-3-3 proteins was strongly reduced upon infection with Shigella, erlin-1 and erlin-2 (erlin-1/2) specifically bound to RIPK2 complexes. The interaction of these proteins with RIPK2 was validated using protein binding assays and immunofluorescence staining. Beside bacterial activation of NOD1/2, depletion of the E3 ubiquitin ligase XIAP and treatment with RIPK2 inhibitors also led to the formation of RIPK2 cytosolic complexes. Although erlin-1/2 were recruited to RIPK2 complexes following XIAP inhibition, these proteins did not associate with RIPK2 structures induced by RIPK2 inhibitors. While the specific recruitment of erlin-1/2 to RIPK2 suggests a role in innate immune signaling, the biological response regulated by the erlin-1/2-RIPK2 association remains to be determined.

Anti-LINGO-1 antibody ameliorates cognitive impairment, promotes adult hippocampal neurogenesis, and increases the abundance of CB1R-rich CCK-GABAergic interneurons in AD mice.

In view of the negative regulatory effect of leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 1 (LINGO-1) on neurons, an antibody against LINGO-1 (anti-LINGO-1 antibody) was herein administered to 10-month-old APP/PS1 transgenic Alzheimer's disease (AD) mice for 2 months as an experimental intervention. Behavioral, stereology, immunohistochemistry and immunofluorescence analyses revealed that the anti-LINGO-1 antibody significantly improved the cognitive abilities, promoted adult hippocampal neurogenesis (AHN), decreased the amyloid beta (Aß) deposition, enlarged the hippocampal volume, and increased the numbers of total neurons and GABAergic interneurons, including GABAergic and CCK-GABAergic interneurons rich in cannabinoid type 1 receptor (CB1R), in the hippocampus of AD mice. In contrast, this intervention significantly reduced the number of GABAergic interneurons expressing LINGO-1 and CB1R in the hippocampus of AD mice. More importantly, we also found a negative correlation between LINGO-1 and CB1R on GABAergic interneurons in the hippocampus of AD mice, while the anti-LINGO-1 antibody reversed this relationship. These results indicated that LINGO-1 plays an important role in the process of hippocampal neuron loss in AD mice and that antagonizing LINGO-1 can effectively prevent hippocampal neuron loss and promote AHN. The improvement in cognitive abilities may be attributed to the improvement in AHN, and in the numbers of GABAergic interneurons and CCK-GABAergic interneurons rich in CB1Rs in the hippocampus of AD mice induced by the anti-LINGO-1 antibody. Collectively, the double target effect (LINGO-1 and CB1R) initiated by the anti-LINGO-1 antibody may provide an important basis for the study of drugs for the prevention and treatment of AD in the future.

microRNA-210 and microRNA-3570 Negatively Regulate NF-κB-Mediated Inflammatory Responses by Targeting RIPK2 in Teleost Fish.

Pathogen infection can cause the production of inflammatory cytokines, which are key mediators that cause the host's innate immune response. Therefore, proper regulation of immune genes associated with inflammation is essential for immune response. Among them, microRNAs (miRNAs) as gene regulator have been widely reported to be involved in the innate immune response of mammals. However, the regulatory network in which miRNAs are involved in the development of inflammation is largely unknown in lower vertebrates. Here, we identified two miRNAs from miiuy croaker (Miichthys miiuy), miR-210 and miR-3570, which play a negative regulatory role in host antibacterial immunity. We found that the expressions of miR-210 and miR-3570 were significantly upregulated under the stimulation of Gram-negative bacterium vibrio harveyi and LPS (lipopolysaccharide). Induced miR-210 and miR-3570 inhibit inflammatory cytokine production by targeting RIPK2, thereby avoiding excessive inflammation. In particular, we found that miR-210 and miR-3570 negatively regulate antimicrobial immunity by regulating the RIPK2-mediated NF-κB signaling pathway. The collective results indicated that both miRNAs are used as negative feedback regulators to regulate RIPK2-mediated NF-κB signaling pathway and thus play a regulatory role in bacteria-induced inflammatory response.

MicroRNA-146a limits tumorigenic inflammation in colorectal cancer.

Chronic inflammation can drive tumor development. Here, we have identified microRNA-146a (miR-146a) as a major negative regulator of colonic inflammation and associated tumorigenesis by modulating IL-17 responses. MiR-146a-deficient mice are susceptible to both colitis-associated and sporadic colorectal cancer (CRC), presenting with enhanced tumorigenic IL-17 signaling. Within myeloid cells, miR-146a targets RIPK2, a NOD2 signaling intermediate, to limit myeloid cell-derived IL-17-inducing cytokines and restrict colonic IL-17. Accordingly, myeloid-specific miR-146a deletion promotes CRC. Moreover, within intestinal epithelial cells (IECs), miR-146a targets TRAF6, an IL-17R signaling intermediate, to restrict IEC responsiveness to IL-17. MiR-146a within IECs further suppresses CRC by targeting PTGES2, a PGE2 synthesis enzyme. IEC-specific miR-146a deletion therefore promotes CRC. Importantly, preclinical administration of miR-146a mimic, or small molecule inhibition of the miR-146a targets, TRAF6 and RIPK2, ameliorates colonic inflammation and CRC. MiR-146a overexpression or miR-146a target inhibition represent therapeutic approaches that limit pathways converging on tumorigenic IL-17 signaling in CRC.