CYLD: a multifunctional deubiquitinase

The nuclear factor-kappaB (NF-kappaB) and c-Jun NH2-terminal kinase (JNK) signaling pathways regulate diverse biological processes, including the immune and inflammatory response, cell growth, apoptosis, and tumour formation. Not surprisingly therefore defects to either pathway contributes to the progression of numerous human disorders. Enhancing our understanding of the mechanisms that control signaling through these pathways is therefore significant as it may enable development of specific treatments. In this regard, CYLD was recently identified as a negative regulator of NF-kappaB and JNK signaling. CYLD has a C-terminal catalytic domain characteristic of deubiquitinating enzymes, and this is essential for CYLD to remove ubiquitin from certain proteins that positively mediate signaling through the NF-kappaB and JNK pathways. Recent studies have revealed a requirement for CYLD in many different processes and have provided some insight into the underlying mechanisms.     

CYLD: A Multifunctional Deubiquitinase

The nuclear factor-kappaB (NF-κB) and c-Jun NH2-terminal kinase (JNK) signaling pathways regulate diverse biological processes, including the immune and inflammatory response, cell growth, apoptosis, and tumor formation. Not surprisingly therefore defects to either pathway contribute to the progression of numerous human disorders. Enhancing our understanding of the mechanisms that control signaling through these pathways is therefore significant as it may enable development of specific treatments. In this regard, CYLD was recently identified as a negative regulator of NF-κB and JNK signaling. CYLD has a C-terminal catalytic domain characteristic of deubiquitinating enzymes, and this is essential for CYLD to remove ubiquitin from certain proteins that positively mediate signaling through the NF-κB and JNK pathways. Recent studies have revealed a requirement for CYLD in many different processes and have provided some insight into the underlying mechanisms.     

Pathophysiological significance of cylindromatosis in the vascular endothelium and macrophages for the initiation of age-related atherogenesis

Cardiovascular disease is one of the leading causes of death in the elderly, and novel therapeutic targets against atherogenesis are urgent. The initiation of atherosclerotic changes of monocyte adhesion on the vascular endothelium and subsequent foam cell formation are noteworthy pathophysiologies when searching for strategies to prevent the progression of age-related atherosclerosis. We report the significance of the deubiquitinating enzyme cylindromatosis (CYLD) in vascular remodeling by interference with inflammatory responses regulated by NF-κB signaling. The purpose of this study was to elucidate the pathological functions of CYLD in the early phase of atherogenesis associated with aging.Treatment with inflammatory cytokines induced endogenous CYLD in aortic endothelial cells (HAECs) and THP-1?cells. siRNA-mediated CYLD silencing led to enhanced monocyte adhesion along with increased adhesion molecules in HAECs treated with TNFα. In siRNA-mediated CYLD silenced RAW 264.7 macrophages treated with oxidized LDL (oxLDL), augmented lipid accumulation was observed, along with increased expression of the class A macrophage scavenger receptor (SR-A), lectin-like oxidized LDL receptor-1 (LOX-1), CD36, fatty acid binding protein 4 (FABP4), the cholesterol ester synthase acyl-CoA cholesterol acyltransferase (ACAT1), MCP-1, and IL-1β and decreased expression of scavenger receptor class B type I (SR-BI). Intriguingly, CYLD gene expression was significantly reduced in bone marrow-derived macrophages of aged mice compared that of young mice, as well as in senescent HAECs compared with young cells.These findings suggest that age-related attenuation of CYLD expression in endothelial cells (ECs) and macrophages triggers the initiation of age-related atherogenesis by exacerbating monocyte adhesion on the endothelium and foam cell formation. CYLD in the vasculature may be a novel therapeutic target, especially in the early preventive intervention against the initiation of age-related atherogenesis.     

CYLD is a crucial negative regulator of innate immune response in Escherichia coli pneumonia

Bacteraemic pneumonia is a common cause of sepsis in critically ill patients today and is characterized by dysregulation of inflammation. The genetic factors predisposing to bacteraemic pneumonia are not yet fully understood. Innate immunity is pivotal for host defence against invading bacteria, and nuclear factor-kappa B (NF-kappaB) is central to bacteria-induced inflammation and immune responses. The deubiquitinating enzyme CYLD has been identified as a key negative regulator for NF-kappaB. In the present study, we investigated the role of CYLD in innate immune response in Escherichia coli pneumonia. Upon E. coli inoculation, Cyld(-/-) mice were hypersusceptible to E. coli pneumonia with higher mortality. Innate immune response to E. coli was enhanced in Cyld(-/-) cells and mice. Cyld(-/-) cells exhibited enhanced NF-kappaB activation upon E. coli inoculation, and the enhanced NF-kappaB activation by E. coli was abolished by perturbing IkappaB kinase (IKK) signalling. Furthermore, IKK inhibitor rescued Cyld(-/-) mice from lethal infection during E. coli pneumonia along with reduced inflammation. Taken together, these data showed that CYLD acts as a crucial negative regulator for E. coli pneumonia by negatively regulating NF-kappaB. These findings provide novel insight into the regulation of bacteraemic pneumonia and related diseases and may help develop novel therapeutic strategies for these diseases.     

Serum response factor controls CYLD expression via MAPK signaling pathway

Tumor suppressor gene CYLD is a deubiquitinating enzyme which negatively regulates various signaling pathways by removing the lysine 63-linked polyubiquitin chains from several specific substrates. Loss of CYLD in different types of tumors leads to either cell survival or proliferation. In this study we demonstrate that lack of CYLD expression in CYLD-/- MEFs increases proliferation rate of these cells compared to CYLD+/+ in a serum concentration dependent manner without affecting cell survival. The reduced proliferation rate in CYLD+/+ in the presence of serum was due to the binding of serum response factor (SRF) to the serum response element identified in the CYLD promoter for the up-regulation of CYLD levels. The serum regulated recruitment of SRF to the CYLD promoter was dependent on p38 mitogen-activated protein kinase (MAPK) activity. Elimination of SRF by siRNA or inhibition of p38 MAPK reduced the expression level of CYLD and increased cell proliferation. These results show that SRF acts as a positive regulator of CYLD expression, which in turn reduces the mitogenic activation of serum for aberrant proliferation of MEF cells.     

CYLD: a DUB with many talents

The deubiquitinating enzyme CYLD is a tumor suppressor protein known for its role in repression of generally pro-oncogenic NF-kappaB activation pathways. Two new studies published in this and the September issue of Developmental Cell show that CYLD dismantles distinct types of polyubiquitin chains formed on select signaling proteins and is thereby required for normal vertebrate and invertebrate development.     

CYLD negatively regulates cell‐cycle progression by inactivating HDAC6 and increasing the levels of acetylated tubulin

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‐κB signalling. Here we show that CYLD controls cell growth and division at the G₁/S‐phase as well as cytokinesis by associating with α‐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 α‐tubulin around the nucleus. This facilitates the interaction of CYLD with Bcl‐3, leading to a significant delay in the G₁‐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.     

Reduced expression of CYLD promotes cell survival and inflammation in gefitinib‐treated NSCLC PC‐9 cells: Targeting CYLD may be beneficial for acquired resistance to gefitinib therapy

The application of tyrosine kinase inhibitors (TKIs) to the epidermal growth factor receptor (EGFR) has been proven to be highly effective for non‐small‐cell lung cancer (NSCLC). However, patients often evolve into acquired resistance. The secondary mutations in EGFR account for nearly half of the acquired resistance. While the remaining 50% of patients exhibit tolerance to EGFR‐TKIs with unclear mechanism(s). Cylindromatosis (CYLD), a deubiquitinase, functions as a tumor suppressor to regulate cell apoptosis, proliferation, and immune response, and so on. The role of CYLD in NSCLC EGFR‐TKI resistance remains elusive. Here, we found CYLD was upregulated in PC‐9 cells, whereas downregulated in PC‐9 acquired gefitinib‐resistant (PC‐9/GR) cells in response to the treatment of gefitinib, which is consistent with the results in the Gene Expression Omnibus database. Overexpression of CYLD promoted a more apoptotic death ratio in PC‐9/GR cells than that in PC‐9 cells. In addition, silencing the expression of CYLD resulted in an increase of the expression level of interleukin‐6, transforming growth factor‐β and tumor necrosis factor‐α, which may contribute to acquired resistance of PC‐9 cells to gefitinib. Taken together, our data in vitro demonstrate that PC‐9/GR cells downregulated CYLD expression, enhanced subsequent CYLD‐dependent antiapoptotic capacity and inflammatory response, which may provide a possible target for acquired gefitinib‐resistant treatment in NSCLC.     

Regulation of the deubiquitinating enzyme CYLD by IkappaB kinase gamma-dependent phosphorylation

Tumor suppressor CYLD is a deubiquitinating enzyme (DUB) that inhibits the ubiquitination of key signaling molecules, including tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). However, how the function of CYLD is regulated remains unknown. Here we provide evidence that inducible phosphorylation of CYLD is an important mechanism of its regulation. Under normal conditions, CYLD dominantly suppresses the ubiquitination of TRAF2. In response to cellular stimuli, CYLD undergoes rapid and transient phosphorylation, which is required for signal-induced TRAF2 ubiquitination and activation of downstream signaling events. Interestingly, the CYLD phosphorylation requires IκB kinase gamma (IKKγ) and can be induced by IKK catalytic subunits. These findings suggest that CYLD serves as a novel target of IKK and that the site-specific phosphorylation of CYLD regulates its signaling function.     

Regulation of inflammatory and antiviral signaling by A20

The deubiquitinating enzyme A20 (also known as TNFAIP3) is essential for maintaining immune homeostasis. A20 is a key regulator of inflammatory, antiviral and apoptotic signaling pathways. Here, we review recent advances illustrating the role of A20 as an essential negative regulator of inflammatory and antiviral signaling.     

Dishevelled proteins and CYLD reciprocally regulate each other in CML cell lines

Dishevelled (Dvl) proteins are activated by Wnt pathway stimulation and have crucial roles in the regulation of β-catenin destruction complex. CYLD is a tumor suppressor and a deubiquitination enzyme. CYLD negatively regulates the Wnt/β-catenin signaling pathway by deubiquitinating Dvl proteins. Loss of function and mutations of CYLD were linked to different types of solid tumors. Loss of function in CYLD is associated with Dvl hyper ubiquitination, resulting in the transmission of Wnt signaling to downstream effectors. β-catenin upregulation is observed during disease progression in chronic myeloid leukemia (CML). Deregulated Dvl signaling may be a reason for β-catenin activation in CML; and CYLD may contribute to Dvl deregulation. First, we evaluated mRNA expression in three CML cell lines and mRNA expression of the CYLD gene was found to be present in all (K562, MEG01, KU812). Unlike solid tumors sequencing revealed no mutations in the coding sequences of the CYLD gene. DVL genes were silenced by using a pool of siRNA oligonucleotides and gene expression differences in CYLD was determined by RT-PCR and western blot. CYLD protein expression decreased after Dvl silencing. An opposite approach of overexpressing Dvl proteins resulted in upregulated CYLD expression. While previous reports have described CYLD as a regulator of DVL proteins; our data suggests the presence of a more complicated reciprocal regulatory mechanism in CML cell lines.     

肿瘤抑制因子CYLD对细胞功能的调控作用

肿瘤抑制因子（cylindromatosis,CYLD）是一种在体内广泛分布的去泛素酶,其包含去泛素化酶结构域和富含甘氨酸细胞骨架相关蛋白结构域,前者可通过去泛素化信号分子,调控细胞信号传导途径,后者主要通过对微管的调节,改变多聚化和乙酰化过程,进而调控其组装和排列。CYLD主要通过对信号传导和细胞骨架的调节,从而调控细胞增殖、细胞凋亡、细胞运动和细胞分化等细胞功能。该文对近年来肿瘤抑制因子CYLD对细胞功能调控的研究进行概述。     

An emerging role of deubiquitinating enzyme cylindromatosis (CYLD) in the tubulointerstitial inflammation of IgA nephropathy

Immunoglobulin A (IgA) nephropathy is an important cause of end-stage kidney disease (ESKD). Tubulointerstitial inflammation and subsequent fibrosis appear to be a major contributor of the disease progression to ESKD; however, the underlying mechanism is poorly understood. Herein, we report that a unique feature of CYLD expression in kidneys of patients with IgA nephropathy and a CYLD-mediated negative regulation of inflammatory responses in human tubular epithelial cells. Immunochemical staining revealed that CYLD was predominantly expressed in renal tubular epithelial cells in 81% of the patients (37 cases) with proteinuric IgA nephropathy. Patients with positive CYLD had significantly less tubulointerstitial lesions and higher estimated glomerular filtration rate (eGFR) levels when compared with those negative. Logistic regression analysis indicated that eGFR was a predictor for the CYLD expression. In cultured human tubular epithelial HK-2 cells, tumor necrosis factor-alpha (TNFα) up-regulated CYLD expression. Adenoviral knockdown of CYLD did not affect albumin-, hydrogen peroxide (H₂O₂)-, tunicamycin- or thapsigargin-induced cell death; however, it enhanced TNFα-induced expression of intracellular adhesion molecule (ICAM)-1 as well as activation of c-Jun N-terminal kinase (JNK). Moreover, monocyte adhesion to the TNFα-inflamed HK-2 cells was significantly increased by the CYLD shRNA approach. Taken together, our results suggest that CYLD negatively regulates tubulointertitial inflammatory responses via suppressing activation of JNK in tubular epithelial cells, putatively attenuating the progressive tubulointerstitial lesions in IgA nephropathy.     

Tumor suppressor CYLD regulates JNK-induced cell death in Drosophila

CYLD encodes a tumor suppressor that is mutated in familial cylindromatosis. Despite biochemical and cell culture studies, the physiological functions of CYLD in animal development and tumorigenesis remain poorly understood. To address these questions, we generated Drosophila CYLD (dCYLD) mutant and transgenic flies expressing wild-type and mutant dCYLD proteins. Here we show that dCYLD is essential for JNK-dependent oxidative stress resistance and normal lifespan. Furthermore, dCYLD regulates TNF-induced JNK activation and cell death through dTRAF2, which acts downstream of the TNF receptor Wengen and upstream of the JNKK kinase dTAK1. We show that dCYLD encodes a deubiquitinating enzyme that deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic degradation. These data provide a molecular mechanism for the tumor suppressor function of this evolutionary conserved molecule by indicating that dCYLD plays a critical role in modulating TNF-JNK-mediated cell death.