CYLD negatively regulates cell-cycle progression by inactivating HDAC6 and increasing the levels of acetylated tubulin.
ABSTRACT: CYLD is a tumour-suppressor gene that is mutated in a benign skin tumour syndrome called cylindromatosis. The CYLD gene product is a deubiquitinating enzyme that was shown to regulate cell proliferation, cell survival and inflammatory responses, mainly through inhibiting NF-kappaB signalling. Here we show that CYLD controls cell growth and division at the G(1)/S-phase as well as cytokinesis by associating with alpha-tubulin and microtubules through its CAP-Gly domains. Translocation of activated CYLD to the perinuclear region of the cell is achieved by an inhibitory interaction of CYLD with histone deacetylase-6 (HDAC6) leading to an increase in the levels of acetylated alpha-tubulin around the nucleus. This facilitates the interaction of CYLD with Bcl-3, leading to a significant delay in the G(1)-to-S-phase transition. Finally, CYLD also interacts with HDAC6 in the midbody where it regulates the rate of cytokinesis in a deubiquitinase-independent manner. Altogether these results identify a mechanism by which CYLD regulates cell proliferation at distinct cell-cycle phases.
Project description:Cilia are hair-like organelles extending from the cell surface with important sensory and motility functions. Ciliary defects can result in a wide range of human diseases known as ciliopathies. However, the molecular mechanisms controlling ciliogenesis remain poorly defined. Here we show that cylindromatosis (CYLD), a tumor suppressor protein harboring deubiquitinase activity, plays a critical role in the assembly of both primary and motile cilia in multiple organs. CYLD knockout mice exhibit polydactyly and various ciliary defects, such as failure in basal body anchorage and disorganization of basal bodies and axenomes. The ciliary function of CYLD is partially attributed to its deconjugation of the polyubiquitin chain from centrosomal protein of 70 kDa (Cep70), a requirement for Cep70 to interact with ?-tubulin and localize at the centrosome. In addition, CYLD-mediated inhibition of histone deacetylase 6 (HDAC6), which promotes tubulin acetylation, constitutes another mechanism for the ciliary function of CYLD. Small-molecule inhibitors of HDAC6 could partially rescue the ciliary defects in CYLD knockout mice. These findings highlight the importance of protein ubiquitination in the modulation of ciliogenesis, identify CYLD as a crucial regulator of this process, and suggest the involvement of CYLD deficiency in ciliopathies.
Project description:On detecting viral RNAs, the RNA helicase retinoic acid-inducible gene I (RIG-I) activates the interferon regulatory factor 3 (IRF3) signalling pathway to induce type I interferon (IFN) gene transcription. How this antiviral signalling pathway might be negatively regulated is poorly understood. Microarray and bioinformatic analysis indicated that the expression of RIG-I and that of the tumour suppressor CYLD (cylindromatosis), a deubiquitinating enzyme that removes Lys 63-linked polyubiquitin chains, are closely correlated, suggesting a functional association between the two molecules. Ectopic expression of CYLD inhibits the IRF3 signalling pathway and IFN production triggered by RIG-I; conversely, CYLD knockdown enhances the response. CYLD removes polyubiquitin chains from RIG-I as well as from TANK binding kinase 1 (TBK1), the kinase that phosphorylates IRF3, coincident with an inhibition of the IRF3 signalling pathway. Furthermore, CYLD protein level is reduced in the presence of tumour necrosis factor and viral infection, concomitant with enhanced IFN production. These findings show that CYLD is a negative regulator of RIG-I-mediated innate antiviral response.
Project description:The clinical presentation of multiple, rare, skin appendage tumours called cylindromas has been attributed to germline mutations in the tumour suppressor gene CYLD (OMIM 605018). Brooke-Spiegler Syndrome (BSS), familial cylindromatosis (FC) and multiple familial trichoepitheliomas (MFT) (OMIM #605041, #132700, #601606 respectively) differ due to the types of other skin appendage tumour seen together with cylindroma, such as spiradenoma and trichoepithelioma. Previously thought to be separate entities, they are now viewed as allelic variants with overlapping phenotypes, supported by mutation analysis of CYLD . The conditions display autosomal dominant inheritance and affected individuals develop multiple benign skin tumours most commonly on the head and neck. CYLD testing can be performed using PCR and Sanger sequencing for patients with: 1. Multiple cylindromas, spiradenomas or trichoepitheliomas. 2. A single cylindroma, spiradenoma or trichoepithelioma and an affected first-degree relative with any of these tumours. 3. An asymptomatic family member at 50% risk with a known mutation in the family.
Project description:<h4>Background</h4>Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase that promotes many cellular processes that lead to cell transformation and tumour development. We previously documented an interaction between Ran-Binding Protein M (RanBPM) and HDAC6 and found that RanBPM expression inhibits HDAC6 activity. RanBPM is part of a putative E3 ubiquitin ligase complex, termed the C-terminal to LisH (CTLH) complex. Here, we investigated the involvement of the CTLH complex on HDAC6 inhibition and assessed the outcome of this regulation on the cellular motility induced by HDAC6.<h4>Methods</h4>Cell lines (Hela, HEK293 and immortalized mouse embryonic fibroblasts) stably or transiently downregulated for several components of the CTLH complex were employed for the assays used in this study. Interactions of HDAC6, RanBPM and muskelin were assessed by co-immunoprecipitations. Quantifications of western blot analyses were employed to evaluate acetylated ?-tubulin levels. Confocal microscopy analyses were used to determine microtubule association of HDAC6 and CTLH complex members. Cell migration was evaluated using wound healing assays.<h4>Results</h4>We demonstrate that RanBPM-mediated inhibition of HDAC6 is dependent on its association with HDAC6. We show that, while HDAC6 does not require RanBPM to associate with microtubules, RanBPM association with microtubules requires HDAC6. Additionally, we show that Twa1 (Two-hybrid-associated protein 1 with RanBPM) and MAEA (Macrophage Erythroblast Attacher), two CTLH complex members, also associate with ?-tubulin and that muskelin, another component of the CTLH complex, is able to associate with HDAC6. Downregulation of CTLH complex members muskelin and Rmnd5A (Required for meiotic nuclear division homolog A) resulted in decreased acetylation of HDAC6 substrate ?-tubulin. Finally, we demonstrate that the increased cell migration resulting from downregulation of RanBPM is due to the relief in inhibition of HDAC6 ?-tubulin deacetylase activity.<h4>Conclusions</h4>Our work shows that RanBPM, together with the CTLH complex, associates with HDAC6 and restricts cell migration through inhibition of HDAC6 activity. This study uncovers a novel function for the CTLH complex and suggests that it could have a tumour suppressive role in restricting HDAC6 oncogenic properties.
Project description:Abstract CYLD cutaneous syndrome (CCS) is an inclusive label for the inherited skin adnexal tumour syndromes Brooke–Spiegler Syndrome (BSS-OMIM 605041), familial cylindromatosis (FC – OMIM 132700) and multiple familial trichoepitheliomas (MFT-OMIM 601606). All three syndromes arise due to germline pathogenic variants in CYLD, a tumour suppressor gene (OMIM 605018). CCS is transmitted in an autosomal dominant pattern, and has variable expressivity, both of the three syndromic phenotypes, and of the severity of tumour burden. Age-related penetrance figures are not precisely reported. The first tumours typically appear during puberty and progressively accumulate through adulthood. Penetrance is typically high, with equal numbers of males and females affected. Genetic testing is important for confirmation of the clinical diagnosis, genetic counselling and family planning, including preimplantation diagnosis. Additionally, identified CCS patients may be eligible for future clinical trials of non-surgical pre-emptive interventions that aim to prevent tumour growth. In this update, we review the clinical presentations of germline and mosaic CCS. An overview of the germline pathogenic variant spectrum of patients with CCS reveals more than 100 single nucleotide variants and small insertions and deletions in coding exons, most frequently resulting in predicted truncation. In addition, a minority of patients have large deletions involving the CYLD gene, intronic pathogenic variants that affect splicing, or inversions. We discuss germline and somatic testing approaches. Somatic testing of tumour tissue, relevant in mosaic CCS, can reveal recurrently detected pathogenic variants when two or more tumours are tested. This can influence genetic testing of children, who may inherit this as a germline variant, and inform genetic counselling and prenatal diagnosis. Finally, we discuss testing technologies that are currently used, their benefits and limitations, and future directions for genetic testing in CCS.
Project description:Cylindromatosis (CYLD) is a deubiquitinating enzyme that is altered in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. However, the regulatory function of CYLD remains unsettled. Here we show that the development of B cells, T cells, and myeloid cells was unaffected in CYLD-deficient mice, but that the activation of these cells with mediators of innate and adaptive immunity resulted in enhanced NF-kappaB and JNK activity associated with increased TNF receptor-associated factor 2 (TRAF2) and NF-kappaB essential modulator (NEMO) ubiquitination. CYLD-deficient mice were more susceptible to induced colonic inflammation and showed a dramatic increase in the incidence of tumors compared with controls in a colitis-associated cancer model. These results suggest that CYLD limits inflammation and tumorigenesis by regulating ubiquitination in vivo.
Project description:Mutations in the cylindromatosis (CYLD) gene cause benign tumors of skin appendages, referred to as cylindromas. The CYLD gene encodes a deubiquitinating enzyme that removes Lys-63-linked ubiquitin chains from I kappa B kinase signaling components and thereby inhibits NF-kappaB pathway activation. The dysregulation of NF-kappaB activity has been proposed to promote cell transformation in part by increasing apoptosis resistance, but it is not clear whether this is CYLD's only or predominant tumor-suppressing function. Here, we show that CYLD is also required for timely entry into mitosis. Consistent with a cell-cycle regulatory function, CYLD localizes to microtubules in interphase and the midbody during telophase, and its protein levels decrease as cells exit from mitosis. We identified the protein kinase Plk1 as a potential target of CYLD in the regulation of mitotic entry, based on their physical interaction and similar loss-of-function and overexpression phenotypes. Our findings raise the possibility that, as with other genes regulating tumorigenesis, CYLD has not only tumor-suppressing (apoptosis regulation) but also tumor-promoting activities (enhancer of mitotic entry). We propose that this additional function of CYLD could provide an explanation for the benign nature of most cylindroma lesions.
Project description:CYLD is a gene mutated in familial cylindromatosis and related diseases, leading to the development of skin appendages tumors. Although the deubiquitinase CYLD is a skin tumor suppressor, its role in skin physiology is unknown. Using skin organotypic cultures as experimental model to mimic human skin, we have found that CYLD acts as a regulator of epidermal differentiation in humans through the JNK signaling pathway. We have determined the requirement of CYLD for the maintenance of epidermal polarity, keratinocyte differentiation and apoptosis. We show that CYLD overexpression increases keratinocyte differentiation while CYLD loss of function impairs epidermal differentiation. In addition, we describe the important role of CYLD in the control of human non-melanoma skin cancer progression. Our results show the reversion of the malignancy of human squamous cell carcinomas that express increased levels of CYLD, while its functional inhibition enhances the aggressiveness of these tumors which progress toward spindle cell carcinomas. We have found that the mechanisms through which CYLD regulates skin cancer progression include the control of tumor differentiation, angiogenesis and cell survival. These findings of the role of CYLD in human skin cancer prognosis make our results relevant from a therapeutic point of view, and open new avenues for exploring novel cancer therapies.
Project description:Pathogenic mutations in CYLD can be identified in patients affected with Brooke-Spiegler syndrome, (Familial) Cylindromatosis or multiple familial trichoepithelioma. To date, only technologies which are able to identify small point mutations in CYLD, such as sequence and WAVE analysis, were used. Here we describe the identification of a larger rearrangement identified by Quantitative PCR analysis of CYLD, indicating that a combination of these technologies is necessary when searching for pathogenic mutations in CYLD.
Project description:Sporadic inclusion body myositis (sIBM) is the most commonly acquired myopathy in middle-aged and elderly people. The muscle histology is characterized by both inflammation and degeneration, including sarcoplasmic aggregation of TDP-43. Cylindromatosis (CYLD) is a deubiquitinating enzyme that targets Lys63-linked ubiquitin chains and negatively regulates signal transduction pathways, such as NF-?B signalling pathways. We examined localization of CYLD as well as phosphorylated TDP-43, phosphorylated p62, and Lys63-linked ubiquitin in muscle tissues of sIBM patients and muscle-specific wild-type TDP-43 transgenic (TDP-43 TG) mice. We investigated whether overexpression of CYLD can affect muscle toxicity in the cell models treated by endoplasmic reticulum (ER) stress inducers tunicamycin and thapsigargin. CYLD expressed with phosphorylated TDP-43, phosphorylated p62, and Lys63-linked ubiquitin in the nuclear and perinuclear regions of muscle fibres of wild-type TDP-43 TG mice and the degenerative myofibres of sIBM patients with rimmed vacuoles and endomysial cellular infiltration. Although expression levels of CYLD decreased and cell viability was reduced in cells treated with ER stress inducers, wild-type CYLD, but not the catalytic mutant, substantially improved cell viability based on the deubiquitinase activity. Dysregulation of CYLD may reinforce myodegeneration in the pathophysiology of sIBM by attenuating autophagic clearance of protein aggregates. Regulating CYLD in muscle fibres might serve as a novel therapeutic strategy for sIBM treatment.