Nesprins form a novel class of nuclear envelope-anchored spectrin-repeat proteins. We show that a direct association of their highly conserved C-terminal luminal domain with the inner nuclear membrane protein Sun1 mediates their nuclear envelope localisation. In Nesprin-1 and Nesprin-2 the conserved C-terminal amino acids PPPX are essential for the interaction with a C-terminal region in Sun1. In fact, Sun1 is required for the proper nuclear envelope localisation of Nesprin-2 as shown using dominant-negative mutants and by knockdown of Sun1 expression. Sun1 itself does not require functional A-type lamins for its localisation at the inner nuclear membrane in mammalian cells. Our findings propose a conserved nuclear anchorage mechanism between Caenorhabditis elegans and mammals and suggest a model in which Sun1 serves as a `structural bridge' connecting the nuclear interior with the actin cytoskeleton.

Cyclin-dependent kinase 1 (Cdk1) is an archetypical kinase and a central regulator that drives cells through G2 phase and mitosis. Knockouts of Cdk2, Cdk3, Cdk4, or Cdk6 have resulted in viable mice, but the in vivo functions of Cdk1 have not been fully explored in mammals. Here we have generated a conditional-knockout mouse model to study the functions of Cdk1 in vivo. Ablation of Cdk1 leads to arrest of embryonic development around the blastocyst stage. Interestingly, liver-specific deletion of Cdk1 is well tolerated, and liver regeneration after partial hepatectomy is not impaired, indicating that regeneration can be driven by cell growth without cell division. The loss of Cdk1 does not affect S phase progression but results in DNA re-replication because of an increase in Cdk2/cyclin A2 activity. Unlike other Cdks, loss of Cdk1 in the liver confers complete resistance against tumorigenesis induced by activated Ras and silencing of p53.cancer | knockout mice | cell cycle regulation | polyploidy

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The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.

Giant isoforms, encoded by Nesprin-1 (Syne1) and Nesprin-2 (Syne2), are multifunctional actin-binding and nuclear-envelope-associated proteins belonging to the spectrin superfamily. Here, we investigate the function of Nesprin-2 Giant (NUANCE) in skin by generating mice lacking the actin-binding domain of Nesprin-2 (Nesprin-2ΔABD). This loss results in a slight but significant thickening of the epidermis, which is a consequence of the increased epithelial nuclear size. Nonetheless, epidermal proliferation and differentiation appear normal in the knockout epidermis. Surprisingly, Nesprin-2 C-terminal-isoform expression and nuclear envelope localization were affected in certain tissues. Nuclei of primary dermal knockout fibroblasts and keratinocytes were heavily misshapen, displaying a striking similarity to nuclear deformations characteristic of laminopathies. Furthermore, emerin, the protein involved in the X-linked form of Emery-Dreifuss muscular dystrophy (EDMD), was unevenly distributed along the nuclear envelope in mutant fibroblasts, often forming aggregates in the deformed nuclear envelope areas. Thus, Nesprin-2 is an important scaffold protein implicated in the maintenance of nuclear envelope architecture. Aged knockout fibroblasts readily generated, by alternative splicing and alternative translation initiation, aberrant Nesprin-2 Giant isoforms that lacked an ABD but that were sufficient to restore nuclear shape and emerin localization; this suggests that other regions of Nesprin-2 Giant, potentially including its spectrin repeats, are crucial for these functions.

Cancer stroma has a profound influence on tumor development and progression. The conversion of fibroblasts to activated myofibroblasts is a hallmark of reactive tumor stroma. Among a number of factors involved in this conversion, TGF-β has emerged as a major regulator. CLIC4, an integral protein in TGF-β signaling, is highly upregulated in stroma of multiple human cancers, and overexpression of CLIC4 in stromal cells enhances the growth of cancer xenografts. In this study we show that conditioned media from tumor cell lines induces expression of both CLIC4 and the myofibroblast marker alpha smooth muscle actin (α-SMA) in stromal fibroblasts via TGF-β signaling. Genetic ablation of CLIC4 in primary fibroblasts prevents or reduces constitutive or TGF-β induced expression of α-SMA and extracellular matrix components that are markers of myofibroblasts. CLIC4 is required for the activation of p38 Map Kinase by TGF-β, a pathway that signals myofibroblast conversion in stromal cells. This requirement involves the interaction of CLIC4 with PPM1a, the selective phosphatase of activated p-38. Conditioned media from fibroblasts overexpressing CLIC4 increases tumor cell migration and invasion in a TGF-β dependent manner and promotes epithelial to mesenchymal transition indicating that high stromal CLIC4 serves to enhance tumor invasiveness and progression. Thus, CLIC4 is significantly involved in the development of a nurturing tumor microenvironment by enhancing TGF-β signaling in a positive feedback loop. Targeting CLIC4 in tumor stroma should be considered as a strategy to mitigate some of the tumor enhancing effects of the cancer stroma.

Cell cycle inhibition has yet to offer a generally effective approach to cancer treatment, but a full evaluation of different combinations of cell cycle inhibitors has yet to be evaluated. Cyclin A2, a core component of the cell cycle, is often aberrantly expressed in cancer where it may impact cell proliferation. In this study, we investigated the role of cyclin A2 in tumorigenesis using a conditional genetic knockout mouse model. Cyclin A2 deletion in oncogene-transformed MEFs suppressed tumor formation in immunocompromised mice. These findings were confirmed in mice with cyclin A2-deficient hepatocytes, where a delay in liver tumor formation was observed. Since cyclin A2 acts in complex with Cdk2 in the cell cycle, we explored a hypothesized role for Cdk2 dysregulation in this effect through conditional deletions of both genes. In oncogene-transformed MEFs lacking both genes, tumor formation was strongly suppressed in a manner associated with decreased proliferation, premature senescence and error-prone recovery from serum deprivation after immortalization. Whereas loss of cyclin A2 led to a compensatory increase in Cdk1 activity, this did not occur with loss of both Cdk2 and cyclin A2. Our work offers a rationale to explore combinations of Cdk1 and Cdk2 inhibitors as a general approach in cancer therapy.

Nuclear translocation of cytosolic CLIC4 is an essential feature of its proapoptotic and prodifferentiation functions. Here we demonstrate that CLIC4 is induced concurrently with inducible nitric oxide synthase (iNOS) and S-nitrosylated in proinflammatory peritoneal macrophages. Chemical inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4. In macrophages, iNOS-induced nuclear CLIC4 coincides with the proto anti-inflammatory transition of the cells because IL-1β and CXCL1 mRNA remain elevated in CLIC4 and iNOS knockout macrophages at late time points, whereas TNFα mRNA is elevated only in the iNOS knockout macrophages. Active IL-1β remains elevated in CLIC4 knockout macrophages and in macrophages in which CLIC4 nuclear translocation is prevented by the NOS inhibitor L-NAME. Moreover, overexpression of nuclear-targeted CLIC4 down-regulates IL-1β in stimulated macrophages. In mice, genetically null for CLIC4, the number of phagocytosing macrophages stimulated by LPS is reduced. Thus, iNOS-induced nuclear CLIC4 is an essential part of the macrophage deactivation program.protein nitrosylation | IL-1beta | phagocytosis C hloride intracellular channel 4 (CLIC4) is the most wellcharacterized member of a family of channel proteins conserved from Caenorhabditis elegans to humans. Although the chloride-selective channel activity of various members has long been established by multiple groups (1-3), the signaling and adaptor functions of soluble CLIC4 and other members have only recently come to the fore. Soluble CLIC4, originally identified as a p53-and c-myc-responsive proapoptotic protein (4, 5), is important for PKCdependent keratinocyte differentiation (6) and a modulator of TGFβ signaling in multiple cell types (7-9). Many of these proapoptotic and differentiation functions of CLIC4 are dependent on its translocation to the nucleus (6,8,10). CLIC4 exhibits redox sensitivity in both its soluble and membrane-associated states (11,12). Indeed, a central mechanism for induction of nuclear CLIC4 is nitric oxide (NO)-dependent modification (S-nitrosylation) of the protein that induces a conformational change, enhancing its association with nuclear transporters and thus its nuclear levels (13).Recently, a role for CLIC4 has been implicated in innate immunity. CLIC4 is an IRF3-dependent early response gene in LPSstimulated macrophages, transrepressed by the anti-inflammatory activity of the glucocorticoid receptor (14). Moreover, CLIC4 knockout macrophages exhibit dysregulation of multiple inflammatory mediators during the early response to LPS, in part as a consequence of altered IRF3 activity (15). These studies suggest that CLIC4 is important in macrophage early functions in response to stimulation but do not address later aspects of macrophage biology related to deactivation and phagocytosis. Furthermore, information is not available to reveal if the CLIC4 channel or nuclear activity is involved in these functions. Therefore, we investigated the role of CLIC4 activity in infl...