Cylindromatosis lysine 63 deubiquitinase (CYLD) suppress TLR3-mediated CCL5 expression in human renal proximal tubular epithelial cells.

BACKGROUND: One of the causes of tubulointerstitial nephritis is viral infection, with innate immune responses affecting its pathogenesis. Toll-like receptor 3 (TLR3) recognizes viral infections and acts antivirally by activating signaling to produce inflammatory cytokines/chemokines, including C-C motif chemokine ligand 5 (CCL5) and interferon-ß (IFN-ß). Although cylindromatosis lysine 63 deubiquitinase (CYLD) is known to be associated with tubulointerstitial nephritis and renal function, its role in the antiviral innate immune response in tubular epithelial cells remains unknown. In this study, we investigated the association between CYLD and TLR3-mediated CCL5 production in cultured human renal proximal tubular epithelial cells (hRPTECs). METHODS AND RESULTS: Polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand, was used to stimulate hRPTECs. mRNA expression was measured using reverse transcription-quantitative polymerase chain reaction. Protein expression was assayed using western blotting or an enzyme-linked immunosorbent assay. Knockdown of IFN-ß, nuclear factor-kappa B (NF-κB) p65, and CYLD was performed by transfecting cells with specific small interfering RNAs. The intracellular localization of CYLD in hRPTECs was analyzed using immunofluorescence. Poly IC induced CCL5 expression in a time- and concentration-dependent manner, and knockdown of either IFN-ß or p65 reduced poly IC-induced CCL5 expression. CYLD knockdown increased the poly IC-induced CCL5, phosphorylated IκB kinase α/ß (IKK complex), and phosphorylated p65 expression. The CYLD protein was localized in the cytoplasm, and poly IC did not alter its expression. CONCLUSION: CYLD may prevent excessive inflammation due to an antiviral innate immune response by suppressing IKK complex and NF-κB activation downstream of TLR3 in hRPTECs.

CYLD/HDAC6 signaling regulates the interplay between epithelial-mesenchymal transition and ciliary homeostasis during pulmonary fibrosis.

The primary cilium behaves as a platform for sensing and integrating extracellular cues to control a plethora of cellular activities. However, the functional interaction of this sensory organelle with epithelial-mesenchymal transition (EMT) during pulmonary fibrosis remains unclear. Here, we reveal a critical role for cylindromatosis (CYLD) in reciprocally linking the EMT program and ciliary homeostasis during pulmonary fibrosis. A close correlation between the EMT program and primary cilia is observed in bleomycin-induced pulmonary fibrosis as well as TGF-ß-induced EMT model. Mechanistic study reveals that downregulation of CYLD underlies the crosstalk between EMT and ciliary homeostasis by inactivating histone deacetylase 6 (HDAC6) during pulmonary fibrosis. Moreover, manipulation of primary cilia is an effective means to modulate the EMT program. Collectively, these results identify a pivotal role for the CYLD/HDAC6 signaling in regulating the reciprocal interplay between the EMT program and ciliary homeostasis during pulmonary fibrosis.

Cyld restrains the hyperactivation of synovial fibroblasts in inflammatory arthritis by regulating the TAK1/IKK2 signaling axis.

TNF is a potent cytokine known for its involvement in physiology and pathology. In Rheumatoid Arthritis (RA), persistent TNF signals cause aberrant activation of synovial fibroblasts (SFs), the resident cells crucially involved in the inflammatory and destructive responses of the affected synovial membrane. However, the molecular switches that control the pathogenic activation of SFs remain poorly defined. Cyld is a major component of deubiquitination (DUB) machinery regulating the signaling responses towards survival/inflammation and programmed necrosis that induced by cytokines, growth factors and microbial products. Herein, we follow functional genetic approaches to understand how Cyld affects arthritogenic TNF signaling in SFs. We demonstrate that in spontaneous and induced RA models, SF-Cyld DUB deficiency deteriorates arthritic phenotypes due to increased levels of chemokines, adhesion receptors and bone-degrading enzymes generated by mutant SFs. Mechanistically, Cyld serves to restrict the TNF-induced hyperactivation of SFs by limiting Tak1-mediated signaling, and, therefore, leading to supervised NF-κB and JNK activity. However, Cyld is not critically involved in the regulation of TNF-induced death of SFs. Our results identify SF-Cyld as a regulator of TNF-mediated arthritis and inform the signaling landscape underpinning the SF responses.

Influenza A virus infection activates caspase-8 to enhance innate antiviral immunity by cleaving CYLD and blocking TAK1 and RIG-I deubiquitination.

Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.

Associations of CYLD, JAK2 and TLR4 Genotypes with PSA Levels and Immunophenotype in Benign Prostatic Hyperplasia and Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is one of the most common malignant tumors of the male urinary system, and its incidence and mortality rates have been increasing worldwide. Benign prostatic hyperplasia (BPH) represents stromal and epithelial cell proliferation in the prostate in elderly males. Abnormal activation of inflammation-related signalling molecules, such as toll-like receptor 4 (TLR4) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) has been linked to the initiation and progression of various human diseases including PCa and BPH. Cylindromatosis (CYLD) gene alterations are associated with PCa progression. In this study, the contribution of CYLD, JAK2, and TLR4 gene variants to PCa and BPH risks and their associations with prostate-specific antigen (PSA) levels, immunophenotype, and clinical features in Vietnamese men were determined. METHODS: A total of 102 patients with PCa, 65 with BPH, and 114 healthy controls were enrolled. The immunophenotype was analyzed by flow cytometry, cytokine secretion by enzyme-linked immunosorbent assay (ELISA), and gene variants by DNA sequencing. RESULTS: Lower levels of transforming growth factor ß (TGF-ß) and higher numbers of CD13+CD117- and CD56+CD25+ cells were observed in the PCa group than in the BPH group. Genetic analysis of the CYLD gene identified five single nucleotide polymorphisms (SNPs), of which c.2351-47 C>T, c.2351-46A>T, and rs1971432171 T>G had significantly higher frequencies in PCa patients than in the control and BPH groups. Sequencing of the TLR4 gene revealed five nucleotide changes, in which the rs2149356 SNP showed an increased risk for both PCa and BPH and the c.331-206 SNP had a reduced risk for PCa. Importantly, the expansion of activated natural killer (NK) cells and higher levels of PSA were found in PCa patients carrying the CT genotype of the CYLD c.2351-47 compared to those with the wild-type genotype. CONCLUSION: Activation of NK cells in CYLD-sensitive PCa patients was associated with serum PSA release and the CYLD c.2351-47 variant may be a significant risk factor for prostatitis in PCa patients.

Grading Challenges and Prognostic Insights in Chromophobe Renal Cell Carcinoma: A Retrospective Study of 72 Patients.

Chromophobe RCC (ChRCC) carries the best prognosis among all RCC subtypes, yet it lacks a proper grading system. Various systems have been suggested in the past, causing much controversy, and Avulova et al. recently proposed a promising four-tier grading system that takes into consideration tumor necrosis. Dysregulation of the mammalian target of the rapamycin (mTOR) pathway plays a key role in ChRCC pathogenesis, highlighting its molecular complexity. The present retrospective study aimed to evaluate the prognostic factors associated with a more aggressive ChRCC phenotype. Materials and Methods: Seventy-two patients diagnosed with ChRCC between 2004 and 2017 were included in our study. Pathology reports and tissue blocks were reviewed, and immunohistochemistry (IHC) was performed in order to assess the expressions of CYLD (tumor-suppressor gene) and mTOR, among other markers. Univariate analysis was performed, and OS was assessed using the Kaplan-Meier method. Results: In our study, 74% of patients were male, with a mean age of 60 years, and the mean tumor size was 63 mm (±44). The majority (54%) were followed for more than 10 years at intervals ranging between 44 and 222 months. The risk of death was significantly higher for patients that were classified as Grade 4 in the Avulova system (HR: 5.83; 95% CI, 1.37-24.7; p: = 0.017). As far as the IHC is concerned, mTOR expression was associated with an HR of 8.57 (95% CI, 1.91-38.5; p = 0.005), and CYLD expression was associated with an HR of 17.3 (95% CI, 1.57-192; p = 0.02). Conclusions: In our study, the Avulova grading system seems to be positively correlated with OS in patients diagnosed with ChRCC. Furthermore, an elevated mTOR expression also shows a negative correlation with OS, whereas an elevated CYLD expression does not seem to exert a protective role. However, because only a small proportion (4.2%) of our patients died due to ChRCC, despite the long follow-up period, the results must be interpreted with caution. Further research is needed to validate our findings.

The TRIF-RIPK1-Caspase-8 signalling in the regulation of TLR4-driven gene expression.

The inflammatory response is tightly regulated to eliminate invading pathogens and avoid excessive production of inflammatory mediators and tissue damage. Caspase-8 is a cysteine protease that is involved in programmed cell death. Here we show the TRIF-RIPK1-Caspase-8 is required for LPS-induced CYLD degradation in macrophages. TRIF functions in the upstream of RIPK1. The homotypic interaction motif of TRIF and the death domain of RIPK1 are essential for Caspase-8 activation. Caspase-8 cleaves CYLD and the D235A mutant is resistant to the protease activity of Caspase-8. TRIF and RIPK1 serve as substrates of Capase-8 in vitro. cFLIP interacts with Caspase-8 to modulate its protease activity on CYLD and cell death. Deficiency in TRIF, Caspase-8 or CYLD can lead to a decrease or increase in the expression of genes encoding inflammatory cytokines. Together, the TRIF-Caspase-8 and CYLD play opposite roles in the regulation of TLR4 signalling.

CYLD alleviates NLRP3 inflammasome-mediated pyroptosis in osteoporosis by deubiquitinating WNK1.

BACKGROUND: Osteoporosis (OP) is the result of bone mass reduction and bone structure disorder. Bone marrow mesenchymal stem cells (BMSCs) are the main source of osteogenic precursor cells involved in adult bone remodeling. The involvement of the deubiquitinating enzyme CYLD in OP has recently been discovered. However, the detailed role and mechanism of CYLD remain unknown. METHODS: The OP mouse model was established by performing ovariectomy (OVX) on mice. Hematoxylin and eosin staining, Masson and Immunohistochemical staining were used to assess pathologic changes. Real-time quantitative PCR, Western blot, and immunofluorescence were employed to assess the expression levels of CYLD, WNK1, NLRP3 and osteogenesis-related molecules. The binding relationship between CYLD and WNK1 was validated through a co-immunoprecipitation assay. The osteogenic capacity of BMSCs was determined using Alkaline phosphatase (ALP) and alizarin red staining (ARS). Protein ubiquitination was evaluated by a ubiquitination assay. RESULTS: The levels of both CYLD and WNK1 were decreased in bone tissues and BMSCs of OVX mice. Overexpression of CYLD or WNK1 induced osteogenic differentiation in BMSCs. Additionally, NLRP3 inflammation was activated in OVX mice, but its activation was attenuated upon overexpression of CYLD or WNK1. CYLD was observed to reduce the ubiquitination of WNK1, thereby enhancing its protein stability and leading to the inactivation of NLRP3 inflammation. However, the protective effects of CYLD on osteogenic differentiation and NLRP3 inflammation inactivation were diminished upon silencing of WNK1. CONCLUSION: CYLD mitigates NLRP3 inflammasome-triggered pyroptosis in osteoporosis through its deubiquitination of WNK1.

Inactivation of the Tumor Suppressor CYLD Sensitizes Mice to Breast Cancer Development.

BACKGROUND/AIM: Breast cancer is a leading cause of cancer-related deaths among women. Down-regulation of the tumor suppressor gene Cyld in breast cancer has been linked to a poor prognosis. This study investigated the role of Cyld in breast cancer using conditional mutant mouse models carrying a Cyld mutation, which inactivates the deubiquitinating activity of its protein product CYLD in mammary epithelial cells. MATERIALS AND METHODS: We examined the potential of CYLD inactivation to induce mammary tumors spontaneously or modify the susceptibility of mice to mammary tumorigenesis by DMBA treatment or ErbB2 over-expression. RESULTS: CYLD inactivation significantly increased susceptibility to breast cancer induced by either DMBA treatment or ErbB2 over-expression. Moreover, while CYLD inactivation alone did not lead to spontaneous mammary tumorigenesis, it did contribute to the formation of multifocal hyperplastic lesions in virgin mice of predominantly FVB/NJ background. CONCLUSION: Our study demonstrates the tumor enhancing potential of CYLD inactivation in mammary tumorigenesis in vivo and establishes novel relevant mouse models that can be exploited for developing prognostic and therapeutic protocols.

Megalosplenia as an initial manifestation of multiple myeloma with a novel CYLD gene mutation: A case report and literature review.

INTRODUCTION: Megalosplenia in newly diagnosed multiple myeloma (MM) is extremely rare, posing diagnostic and therapeutic challenges due to its unusual location and clinical manifestations and lack of optimal therapeutic strategies. CASE PRESENTATION: A 65-year-old female who was previously healthy presented with a history of ecchymosis on her right leg accompanied by progressive fatigue for 2 weeks. She was admitted to our center in July 2019 due to thrombocytopenia. The patient presented with megalosplenia, anemia, monoclonal protein (λ-light chain type) in the serum and urine, and 45.6% malignant plasma cells in the bone marrow. Splenectomy was performed due to persistent splenomegaly after 3 cycles of the bortezomib plus dexamethasone regimen, and immunohistochemistry results indicated λ-plasmacytoma of the spleen. The same cytogenetic and molecular abnormalities, including t(14;16), 14q32 amplification, 16q32 amplification, 20q12 amplification, and a novel CYLD gene mutation, were identified using fluorescence in situ hybridization and next-generation sequencing in both bone marrow and spleen samples. Therefore, a diagnosis of MM (λ-light chain type, DS III, ISS III, R-ISS III, high-risk) with spleen infiltration was proposed. The patient did not achieve remission after induction treatment with bortezomib plus lenalidomide and dexamethasone or salvage therapy with daratumumab plus ixazomib and dexamethasone. However, she ultimately did achieve very good partial remission with a regimen of bendamustine plus lenalidomide and dexamethasone. Unfortunately, she died of pneumonia associated with chemotherapy. CONCLUSION: To our knowledge, only 8 cases of spleen plasmacytoma at MM diagnosis have been described previously. Extramedullary myeloma patients with spleen involvement at diagnosis are younger and that the condition is usually accompanied by splenic rupture with aggressive clinical features and poor prognosis. Further studies are needed to explore pathogenesis and effective therapies to prolong the survival of such patients.

TNF and IFNγ-induced cell death requires IRF1 and ELAVL1 to promote CASP8 expression.

TNFα and IFNγ (TNF/IFNγ) synergistically induce caspase-8 activation and cancer cell death. However, the mechanism of IFNγ in promoting TNF-initiated caspase-8 activation in cancer cells is poorly understood. Here, we found that in addition to CASP8, CYLD is transcriptionally upregulated by IFNγ-induced transcription factor IRF1. IRF1-mediated CASP8 and CYLD upregulation additively mediates TNF/IFNγ-induced cancer cell death. Clinically, the expression levels of TNF, IFNγ, CYLD, and CASP8 in melanoma tumors are increased in patients responsive to immune checkpoint blockade (ICB) therapy after anti-PD-1 treatment. Accordingly, our genetic screen revealed that ELAVL1 (HuR) is required for TNF/IFNγ-induced caspase-8 activation. Mechanistically, ELAVL1 binds CASP8 mRNA and extends its stability to sustain caspase-8 expression both in IFNγ-stimulated and in basal conditions. Consequently, ELAVL1 determines death receptors-initiated caspase-8-dependent cell death triggered from stimuli including TNF and TRAIL by regulating basal/stimulated caspase-8 levels. As caspase-8 is a master regulator in cell death and inflammation, these results provide valuable clues for tumor immunotherapy and inflammatory diseases.

CYLD regulates cell ferroptosis through Hippo/YAP signaling in prostate cancer progression.

Prostate cancer (PCa) is one of the most common malignancy in men. However, the molecular mechanism of its pathogenesis has not yet been elucidated. In this study, we demonstrated that CYLD, a novel deubiquitinating enzyme, impeded PCa development and progression via tumor suppression. First, we found that CYLD was downregulated in PCa tissues, and its expression was inversely correlated with pathological grade and clinical stage. Moreover, we discovered that CYLD inhibited tumor cell proliferation and enhanced the sensitivity to cell ferroptosis in PCa in vitro and in vivo, respectively. Mechanistically, we demonstrated that CYLD suppressed the ubiquitination of YAP protein, then promoted ACSL4 and TFRC mRNA transcription. Then, we demonstrated that CYLD could enhance the sensitivity of PCa xenografts to ferroptosis in vivo. Furthermore, we discovered for the first time that there was a positive correlation between CYLD expression and ACSL4 or TFRC expression in human PCa specimens. The results of this study suggested that CYLD acted as a tumor suppressor gene in PCa and promoted cell ferroptosis through Hippo/YAP signaling.

CYLD induces high oxidative stress and DNA damage through class I HDACs to promote radiosensitivity in nasopharyngeal carcinoma.

Abnormal expression of Cylindromatosis (CYLD), a tumor suppressor molecule, plays an important role in tumor development and treatment. In this work, we found that CYLD binds to class I histone deacetylases (HDAC1 and HDAC2) through its N-terminal domain and inhibits HDAC1 activity. RNA sequencing showed that CYLD-HDAC axis regulates cellular antioxidant response via Nrf2 and its target genes. Then we revealed a mechanism that class I HDACs mediate redox abnormalities in CYLD low-expressing tumors. HDACs are central players in the DNA damage signaling. We further confirmed that CYLD regulates radiation-induced DNA damage and repair response through inhibiting class I HDACs. Furthermore, CYLD mediates nasopharyngeal carcinoma cell radiosensitivity through class I HDACs. Thus, we identified the function of the CYLD-HDAC axis in radiotherapy and blocking HDACs by Chidamide can increase the sensitivity of cancer cells and tumors to radiation therapy both in vitro and in vivo. In addition, ChIP and luciferase reporter assays revealed that CYLD could be transcriptionally regulated by zinc finger protein 202 (ZNF202). Our findings offer novel insight into the function of CYLD in tumor and uncover important roles for CYLD-HDAC axis in radiosensitivity, which provide new molecular target and therapeutic strategy for tumor radiotherapy.

miR-130b regulates B cell proliferation via CYLD-mediated NF-κB signaling.

Previous studies have revealed that B cell activation is regulated by various microRNAs(miRNAs). However, the role of microRNA-130b regulating B cell activation and apoptosis is still unclear. In the present study, we first found that the expression of miR-130b was the lowest in Pro/Pre-B cells and the highest in immature B cells. Besides, the expression of miR-130b decreased after activation in B cells. Through the immuno-phenotypic analysis of miR-130b transgenic and knockout mice, we found that miR-130b mainly promoted the proliferation of B cells and inhibited B cell apoptosis. Furthermore, we identified that Cyld, a tumor suppressor gene was the target gene of miR-130b in B cells. Besides, the Cyld-mediated NF-κB signaling was increased in miR-130b overexpressed B cells, which further explains the enhanced proliferation of B cells. In conclusion, we propose that miR-130b promotes B cell proliferation via Cyld-mediated NF-κB signaling, which provides a new theoretical basis for the molecular regulation of B cell activation.

YTHDC2 Retards Cell Proliferation and Triggers Apoptosis in Papillary Thyroid Cancer by Regulating CYLD-Mediated Inactivation of Akt Signaling.

N6-Methyladenosine (m6A) mRNA methylation modification is regarded as an important mechanism involved in diverse physiological processes. YT521-B homology (YTH) domain family members are associated with the tumorigenesis of several cancers. However, the role of YTHDC2 in papillary thyroid cancer (PTC) progression remains unknown. Results showed that YTHDC1, YTHDF1, YTHDF2, and YTHDF3 showed no observable difference in thyroid cancer samples. YTHDC2 was significantly downregulated in thyroid cancer samples and cells. YTHDC2 inhibited cell proliferation in PTC cells. YTHDC2 elicited apoptosis in PTC cells, as demonstrated by the elevated expression of pro-apoptotic factors cl-caspase-3/caspase-3 and Bcl-2-associated (Bax), and the reduced anti-apoptotic B cell lymphoma-2 (Bcl-2) expression. There was a positive correlation between YTHDC2 and cylindromatosis (CYLD) expression based on GEPIA database. YTHDC2 increased CYLD expression in PTC cells. CYLD knockdown abolished the effects of YTHDC2 on PTC cell proliferation and apoptosis. Additionally, YTHDC2 inactivated the protein kinase B (Akt) pathway by increasing CYLD in PTC cells. Overall, YTHDC2 inhibited cell proliferation and induced apoptosis in PTC cells by regulating CYLD-mediated inactivation of Akt pathway.

Associations of A20, CYLD, Cezanne and JAK2 Genes and Immunophenotype with Psoriasis Susceptibility.

Background and Objectives: Psoriasis is an immune-mediated chronic inflammatory skin disorder and commonly associated with highly noticeable erythematous, thickened and scaly plaques. Deubiquitinase genes, such as tumor necrosis factor-alpha protein 3 (TNFAIP3, A20), the cylindromatosis (CYLD) and Cezanne, function as negative regulators of inflammatory response through the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways. In this study, polymorphisms and expressions of A20, CYLD and Cezanne genes as well as immunophenotype in psoriatic patients were determined. Materials and Methods: In total, 82 patients with psoriasis and 147 healthy individuals with well-characterized clinical profiles were enrolled. Gene polymorphisms were determined by direct DNA sequencing, gene expression profile by quantitative real time-polymerase chain reaction (PCR), immunophenotype by flow cytometry, and the secretion of cytokines and cancer antigen (CA) 125 by enzyme-linked Immunosorbent assay (ELISA). Results: The inactivation of A20, CYLD and Cezanne and increased levels of TNF-α, IFN-γ and CA 125 was observed in psoriatic patients. Importantly, patients with low A20 expression had significant elevations of triglyceride and total cholesterol concentrations and higher numbers of CD13+CD117- and CD19+CD23+ (activated B) cells than those with high A20 expression. Genetic analysis indicated that all rs4495487 SNPs in the JAK2 gene, rs200878487 SNPs in the A20 gene and four SNPs (c.1584-375, c.1584-374, rs1230581026 and p.W433R) in the Cezanne gene were associated with significant risks, while the rs10974947 variant in the JAK2 gene was at reduced risk of psoriasis. Moreover, in the Cezanne gene, p.W433R was predicted to be probably damaging by the Polyphen-2 prediction tool and an AA/CC haplotype was associated with a high risk of psoriasis. In addition, patients with higher CA 125 levels than the clinical cutoff 35 U/mL showed increased levels of IFN-γ than those with normal CA 125 levels. Conclusions: A20 expression was associated with lipid metabolism and the recruitment of CD13+ CD117- and activated B cells into circulation in psoriatic patients. Besides this, the deleterious effect of the p.W433R variant in the Cezanne gene may contribute to the risk of psoriasis.

An siRNA library screen identifies CYLD and USP34 as deubiquitinases that regulate GPCR-p38 MAPK signaling and distinct inflammatory responses.

G protein-coupled receptors (GPCRs) are highly druggable and implicated in numerous diseases, including vascular inflammation. GPCR signals are transduced from the plasma membrane as well as from endosomes and controlled by posttranslational modifications. The thrombin-activated GPCR protease-activated receptor-1 is modified by ubiquitin. Ubiquitination of protease-activated receptor-1 drives recruitment of transforming growth factor-ß-activated kinase-1-binding protein 2 (TAB2) and coassociation of TAB1 on endosomes, which triggers p38 mitogen-activated protein kinase-dependent inflammatory responses in endothelial cells. Other endothelial GPCRs also induce p38 activation via a noncanonical TAB1-TAB2-dependent pathway. However, the regulatory processes that control GPCR ubiquitin-driven p38 inflammatory signaling remains poorly understood. We discovered mechanisms that turn on GPCR ubiquitin-dependent p38 signaling, however, the mechanisms that turn off the pathway are not known. We hypothesize that deubiquitination is an important step in regulating ubiquitin-driven p38 signaling. To identify specific deubiquitinating enzymes (DUBs) that control GPCR-p38 mitogen-activated protein kinase signaling, we conducted a siRNA library screen targeting 96 DUBs in endothelial cells and HeLa cells. We identified nine DUBs and validated the function two DUBs including cylindromatosis and ubiquitin-specific protease-34 that specifically regulate thrombin-induced p38 phosphorylation. Depletion of cylindromatosis expression by siRNA enhanced thrombin-stimulated p38 signaling, endothelial barrier permeability, and increased interleukin-6 cytokine expression. Conversely, siRNA knockdown of ubiquitin-specific protease-34 expression decreased thrombin-promoted interleukin-6 expression and had no effect on thrombin-induced endothelial barrier permeability. These studies suggest that specific DUBs distinctly regulate GPCR-induced p38-mediated inflammatory responses.

Wnt/ß­catenin signaling is a novel therapeutic target for tumor suppressor CYLD­silenced glioblastoma cells.

Tumor suppressor cylindromatosis (CYLD) dysfunction by its downregulation is significantly associated with poor prognosis in patients with glioblastoma (GBM), the most aggressive and malignant type of glioma. However, no effective treatment is currently available for patients with CYLD­downregulated GBM. The aim of the present study was to identify the crucial cell signaling pathways and novel therapeutic targets for CYLD downregulation in GBM cells. CYLD knockdown in GBM cells induced GBM malignant characteristics, such as proliferation, metastasis, and GBM stem­like cell (GSC) formation. Comprehensive proteomic analysis and RNA sequencing data from the tissues of patients with GBM revealed that Wnt/ß­catenin signaling was significantly activated by CYLD knockdown in patients with GBM. Furthermore, a Wnt/ß­catenin signaling inhibitor suppressed all CYLD knockdown­induced malignant characteristics of GBM. Taken together, the results of the present study revealed that Wnt/ß­catenin signaling is responsible for CYLD silencing­induced GBM malignancy; therefore, targeting Wnt/ß­catenin may be effective for the treatment of CYLD­negative patients with GBM with poor prognosis.

Expanding CYLD protein in NF-κß/TNF-α signaling pathway in response to Lactobacillus acidophilus in non-metastatic rectal cancer patients.

The CYLD gene is a tumor suppressor, reduced in many cancers. Here, we aimed to investigate CYLD protein level and NF-κß/TNF-α signaling pathway in rectal cancer patients with Lactobacillus acidophilus (L. acidophilus) consumption. One hundred ten patients with non-metastatic rectal cancer were randomly divided into L. acidophilus probiotic (500 mg, three times daily) and placebo groups for 13 weeks. The expression of CYLD, TNF-α, and NF-κB proteins and the genes involved in the NF-κß/TNF-α pathway were evaluated using ELISA and qPCR techniques. The survival rate was measured after five years. Unlike the placebo group, the results showed a significant increase in the expression of CYLD protein and tumor suppressor genes, including FOXP3, ROR-γ, Caspase3, GATA3, T-bet, and a considerable decrease in the expression of NF-Òß and TNF-α proteins and oncogenes, including STAT3, 4, 5, 6, and SMAD 3, in the probiotic group. A higher overall survival rate was seen after L. acidophilus consumption compared to the placebo group (P < 0.05). L. acidophilus consumption can reduce inflammation factors by affecting CYLD protein and its downstream signaling pathways. A schematic plot of probiotic consumption Effects on the CYLD protein in regulating the NF-Ä¸ß signaling pathway in colorectal cancer. NF-Ä¸ß can be activated by canonical and noncanonical pathways, which rely on IκB degradation and p100 processing, respectively. In the canonical NF-κß pathway, dimmers, such as p65/p50, are maintained in the cytoplasm by interacting with an IκBα protein. The binding of a ligand to a cell-surface receptor activates TRAF2, which triggers an IKK complex, containing -α, -ß, -g, which phosphorylates IKK-ß. It then phosphorylates IκB-α, leading to K48-ubiquitination and degradation of this protein. The p65/p50 protein freely enters the nucleus to turn on target genes. The non-canonical pathway is primarily involved in p100/RelB activation. It differs from the classical pathway in that only certain receptor signals activate this pathway. It proceeds through an IKK complex that contains two IKK-α subunits but not NEMO. Several materials including peptidoglycan, phorbol, myristate, acetate, and gram-positive bacteria such as probiotics inhibit NF-κB by inducing CYLD. This protein can block the canonical and noncanonical NF-κß pathways by removing Lys-63 ubiquitinated chains from activated TRAFs, RIP, NEMO, and IKK (α, ß, and γ). Moreover, TNF-α induces apoptosis by binding caspase-3 to FADD.