Defective immune responses in mice lacking LUBAC-mediated linear ubiquitination in B cells.

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF-κB pathway, which is important for B-cell development and function. Here, we describe a mouse model (B-HOIP(Δlinear)) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF-κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B-HOIP(Δlinear) mice due to defective activation of the IKK complex; however, B-cell receptor (BCR)-mediated activation of the NF-κB and ERK pathways was unaffected. B-HOIP(Δlinear) mice show impaired B1-cell development and defective antibody responses to thymus-dependent and thymus-independent II antigens. Taken together, these data suggest that LUBAC-mediated linear polyubiquitination is essential for B-cell development and activation, possibly via canonical NF-κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.

Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIß (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.

SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex.

Cpdm (chronic proliferative dermatitis) mice develop chronic dermatitis and an immunodeficiency with increased serum IgM, symptoms that resemble those of patients with X-linked hyper-IgM syndrome and hypohydrotic ectodermal dysplasia (XHM-ED), which is caused by mutations in NEMO (NF-κB essential modulator; also known as IKBKG). Spontaneous null mutations in the Sharpin (SHANK-associated RH domain interacting protein in postsynaptic density) gene are responsible for the cpdm phenotype in mice. SHARPIN shows significant similarity to HOIL-1L (also known as RBCK1), a component of linear ubiquitin chain assembly complex (LUBAC), which induces NF-κB activation through conjugation of linear polyubiquitin chains to NEMO. Here, we identify SHARPIN as an additional component of LUBAC. SHARPIN-containing complexes can linearly ubiquitinate NEMO and activated NF-κB. Thus, we re-define LUBAC as a complex containing SHARPIN, HOIL-1L, and HOIP (also known as RNF31). Deletion of SHARPIN drastically reduced the amount of LUBAC, which resulted in attenuated TNF-α- and CD40-mediated activation of NF-κB in mouse embryonic fibroblasts (MEFs) or B cells from cpdm mice. Considering the pleomorphic phenotype of cpdm mice, these results confirm the predicted role of LUBAC-mediated linear polyubiquitination in NF-κB activation induced by various stimuli, and strongly suggest the involvement of LUBAC-induced NF-κB activation in various disorders.

IFN-γ or IFN-α ameliorates chronic proliferative dermatitis by inducing expression of linear ubiquitin chain assembly complex.

The linear ubiquitin chain assembly complex (LUBAC) ubiquitin ligase complex, composed of HOIL-1L-interacting protein (HOIP), heme-oxidized IRP2 ubiquitin ligase-1L (HOIL-1L), and SHANK-associated RH domain protein, specifically generates linear polyubiquitin chains and is involved in NF-κB activation. Lack of SHANK-associated RH domain protein, which drastically reduces the amount of HOIP and HOIL-1L, causes chronic proliferative dermatitis (cpdm) in mice. Impaired NF-κB activation and augmented apoptosis have been implicated in the pathogenesis of cpdm in mice. In this study, we found that IFN-γ increased the amount of LUBAC by inducing HOIP and HOIL-1L mRNA transcription and enhanced the signal-induced NF-κB activation in embryonic fibroblasts, keratinocytes, and bone marrow-derived macrophages from wild-type and/or cpdm mice; however, IFN-γ failed to augment NF-κB activation in mouse embryonic fibroblasts lacking linear polyubiquitination activity of LUBAC. Moreover, s.c. injection of IFN-γ for 3 wk into the skin of cpdm mice increased the amount of HOIP, suppressed apoptosis, and ameliorated the dermatitis. Inhibition of keratinocyte apoptosis by IFN-γ injection suppressed neutrophil, macrophage, and mast cell infiltration and the amount of TNF-α in the skin of cpdm mice. Similarly, IFN-α also enhanced the amount of HOIP as well as NF-κB activation, inhibited apoptosis, and ameliorated cpdm dermatitis. These results indicate that the IFNs enhance NF-κB activation and ameliorate cpdm dermatitis by augmenting expression of HOIP and HOIL-1L and linear polyubiquitination activity of LUBAC.

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens.

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.

Effect of Subcultivation of Human Bone Marrow Mesenchymal Stem on their Capacities for Chondrogenesis, Supporting Hematopoiesis, and Telomea Length.

Effects of subcultivation of human bone marrow mesenchymal stem cells on their capacities for chondrogenesis and supporting hematopoiesis, and telomea length were investigated. Mesenchymal stem cells were isolated from human bone marrow aspirates and subcultivated several times at 37 degrees C under a 5% CO(2) atmosphere employing DMEM medium containing 10% FCS up to the 20th population doubling level (PDL). The ratio of CD45(-) CD105(+) cells among these cells slightly increased as PDL increased. However, there was no marked change in the chondrogenic capacity of these cells, which was confirmed by expression assay of aggrecan mRNA and Safranin O staining after pellet cell cultivation. The change in capacity to support hematopoiesis of cord blood cells was not observed among cells with various PDLs. On the other hand, telomere length markedly decreased as PDL increased at a higher rate than that at which telomere length of primary mesenchymal stem cells decreased as the age of donor increased.