Mutations in the CYLD gene cause tumors of hair-follicle keratinocytes. The CYLD gene encodes a deubiquitinase that removes lysine 63-linked ubiquitin chains from TRAF2 and inhibits p65/p50 NF-kappaB activation. Here we show that mice lacking Cyld are highly susceptible to chemically induced skin tumors. Cyld-/- tumors and keratinocytes treated with 12-O-tetradecanoylphorbol-13 acetate (TPA) or UV light are hyperproliferative and have elevated cyclin D1 levels. The cyclin D1 elevation is caused not by increased p65/p50 action but rather by increased nuclear activity of Bcl-3-associated NF-kappaB p50 and p52. In Cyld+/+ keratinocytes, TPA or UV light triggers the translocation of Cyld from the cytoplasm to the perinuclear region, where Cyld binds and deubiquitinates Bcl-3, thereby preventing nuclear accumulation of Bcl-3 and p50/Bcl-3- or p52/Bcl-3-dependent proliferation. These data indicate that, depending on the external signals, Cyld can negatively regulate different NF-kappaB pathways; inactivation of TRAF2 controls survival and inflammation, while inhibition of Bcl-3 controls proliferation and tumor growth.

CASPASE 8 initiates apoptosis downstream of TNF death receptors by undergoing autocleavage and processing the executioner CASPASE 31. However, the dominant function of CASPASE 8 is to transmit a pro-survival signal that suppresses programmed necrosis (or necroptosis) mediated by RIPK1 and RIPK32–6 during embryogenesis and hematopoiesis7–9. Suppression of necrotic cell death by CASPASE 8 requires its catalytic activity but not the autocleavage essential for apoptosis10, however, the key substrate processed by CASPASE 8 to block necrosis has been elusive. A key substrate must meet three criteria: (1) it must be essential for programmed necrosis; (2) it must be cleaved by CASPASE 8 in situations where CASPASE 8 is blocking necrosis; and (3) mutation of the CASPASE 8 processing site on the substrate should convert a pro-survival response to necrotic death without the need for CASPASE 8 inhibition. We now identify CYLD as a novel substrate for CASPASE 8 that satisfies these criteria. Upon TNF stimulation, CASPASE 8 cleaves CYLD to generate a survival signal. In contrast, loss of CASPASE 8 prevented CYLD degradation resulting in necrotic death. A CYLD substitution mutation at D215 that cannot be cleaved by CASPASE 8 switches cell survival to necrotic cell death in response to TNF.

High malignancy and early metastasis are hallmarks of melanoma. Here, we report that the transcription factor Snail1 inhibits expression of the tumor suppressor CYLD in melanoma. As a direct consequence of CYLD repression, the protooncogene BCL-3 translocates into the nucleus and activates Cyclin D1 and N-cadherin promoters, resulting in proliferation and invasion of melanoma cells. Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo. Analysis of a tissue microarray with primary melanomas from patients revealed an inverse correlation of Snail1 induction and loss of CYLD expression. Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression. Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

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-jB signalling. Here we show that CYLD controls cell growth and division at the G 1 /S-phase as well as cytokinesis by associating with a-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 a-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.

Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld form a complex via the interaction through ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved K63-linked ubiquitin chains and catalyzed K48-linked ubiquitination on the kinase Tak1 to terminate inflammatory tumor necrosis factor signaling. Reconstitution of wild-type Cyld but not mutant Cyld(Y485A), which cannot associate with Itch, blocked the sustained Tak1 activation and proinflammatory cytokine production by Cyld−/− bone marrow-derived macrophages. Itch or Cyld deficiency resulted in chronic production of tumor-promoting cytokines by the tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have uncovered an Itch-Cyld mediated regulatory mechanism in innate inflammatory cells.

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B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-κB (NF-κB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLDex7/8 mice), which is a deubiquitinating enzyme that is integral to NF-κB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occurs in this strain. The B lymphocytes themselves exhibit prolonged survival and manifest a variety of signaling disarrangements that do not occur in mice with a complete deletion of CYLD. Although both the full-length and the mutant CYLD are able to interact with Bcl-3, a predominant nuclear accumulation of Bcl-3 occurs in the CYLD mutant B cells. More dramatic, however, is the accumulation of the NF-κB proteins p100 and RelB in CYLDex7/8 B cells, which, presumably in combination with nuclear Bcl-3, results in increased levels of Bcl-2 expression. These findings suggest that CYLD can both positively and negatively regulate signal transduction and homeostasis of B cells in vivo, depending on the expression of CYLD splice variants.

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