Involvement of Lamin B1 Reduction in Accelerated Cellular Senescence during Chronic Obstructive Pulmonary Disease Pathogenesis.

Downregulation of lamin B1 has been recognized as a crucial step for development of full senescence. Accelerated cellular senescence linked to mechanistic target of rapamycin kinase (MTOR) signaling and accumulation of mitochondrial damage has been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. We hypothesized that lamin B1 protein levels are reduced in COPD lungs, contributing to the process of cigarette smoke (CS)-induced cellular senescence via dysregulation of MTOR and mitochondrial integrity. To illuminate the role of lamin B1 in COPD pathogenesis, lamin B1 protein levels, MTOR activation, mitochondrial mass, and cellular senescence were evaluated in CS extract (CSE)-treated human bronchial epithelial cells (HBEC), CS-exposed mice, and COPD lungs. We showed that lamin B1 was reduced by exposure to CSE and that autophagy was responsible for lamin B1 degradation in HBEC. Lamin B1 reduction was linked to MTOR activation through DEP domain-containing MTOR-interacting protein (DEPTOR) downregulation, resulting in accelerated cellular senescence. Aberrant MTOR activation was associated with increased mitochondrial mass, which can be attributed to peroxisome proliferator-activated receptor γ coactivator-1ß-mediated mitochondrial biogenesis. CS-exposed mouse lungs and COPD lungs also showed reduced lamin B1 and DEPTOR protein levels, along with MTOR activation accompanied by increased mitochondrial mass and cellular senescence. Antidiabetic metformin prevented CSE-induced HBEC senescence and mitochondrial accumulation via increased DEPTOR expression. These findings suggest that lamin B1 reduction is not only a hallmark of lung aging but is also involved in the progression of cellular senescence during COPD pathogenesis through aberrant MTOR signaling.

Photoexcited calphostin C selectively destroys nuclear lamin B1 in neoplastic human and rat cells - a novel mechanism of action of a photodynamic tumor therapy agent.

Lamin B1, a major component of the nuclear lamina, anchors the nucleus to the cytoskeletal cage, and controls nuclear orientation, chromosome positioning and, alongside several enzymes, fundamental nuclear functions. Exposing polyomavirus-transformed rat pyF111 fibroblasts and human cervical carcinoma (HCC) C4-I cells for 30 min to photoexcited perylenequinone calphostin C, i.e. Cal C(phiE), an established reactive oxygen species (ROS)-generator and protein kinase C (PKC) inhibitor, caused the cells to selectively oxidize and then totally destroy their nuclear lamin B1 by only 60 min after starting the treatment, i.e. when apoptotic caspases' activities had not yet increased. However, while the oxidized lamin B1 was being destroyed, lamins A/C, the lamin A-associated nuclear envelope protein emerin, and the nucleoplasmic protein cyclin E were neither oxidized nor destroyed. The oxidized lamin B was ubiquitinated and demolished in the proteasome probably by an enhanced peptidyl-glutaminase-like activity. Hence, the Cal C(phiE)-induced rapid and selective lamin B1 oxidation and proteasomal destruction ahead of the activation of apoptotic caspases was by itself a most severe molecular lesion impairing vital nuclear functions. Conversely, Cal C directly added to the cells kept in the dark damaged neither nuclear lamin B1 nor cell viability. Thus, our findings reveal a novel cell-damaging mechanism of a photodynamic tumor therapeutic agent.

Cell surface expression of intermediate filament proteins vimentin and lamin B1 in human neutrophil spontaneous apoptosis.

Neutrophils represent an important source of autoantigens for antineutrophil cytoplasmic antibody associated with vasculitis. To date, two cytoskeletal proteins, vinculin and vimentin, have been reported to be expressed on the cell surfaces of activated macrophages, platelets, and apoptotic T lymphocytes. However, such cell surface expression has never been studied in human neutrophils. As we recently demonstrated that different cytoskeletal proteins were cleaved in apoptotic neutrophils, we hypothesized that some of these were expressed on the cell surface of apoptotic neutrophils. Herein, we found that among vinculin, paxillin, gelsolin, vimentin, lamin B1, alpha-tubulin, and beta-tubulin, only the two intermediate filament (INFIL) proteins, vimentin and lamin B1, are expressed on the cell surface of 24-h aged neutrophils [spontaneous apoptosis (SA)]. By monitoring intracellular expression of vimentin and lamin B1 during SA, we found that these two proteins were cleaved and that such cleavage was reversed by the pan caspase inhibitor N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD-fmk). When neutrophil apoptosis was delayed or suppressed by lipopolysaccharide or the cytokines granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, or interleukin-4, the loss of intracellular expression of vimentin and lamin B1 was prevented. The INFIL proteins were absent from the cell surface when neutrophil apoptosis was delayed. Addition of z-VAD-fmk significantly decreased the cell surface expression of vimentin and lamin B1 during SA. This study provides the first evidence that apoptotic neutrophils express cytoskeletal proteins on their surface, opening the possibility that these cells may participate in the development of autoantibodies directed against cytoskeletal proteins, a condition frequently reported in several inflammatory diseases.

A carboxyl-terminal interaction of lamin B1 is dependent on the CAAX endoprotease Rce1 and carboxymethylation.

The mammalian nuclear lamina protein lamin B1 is posttranslationally modified by farnesylation, endoproteolysis, and carboxymethylation at a carboxyl-terminal CAAX motif. In this work, we demonstrate that the CAAX endoprotease Rce1 is required for lamin B1 endoproteolysis, demonstrate an independent pool of proteolyzed but nonmethylated lamin B1, as well as fully processed lamin B1, in interphase nuclei, and show a role for methylation in the organization of lamin B1 into domains of the nuclear lamina. Deficiency in the endoproteolysis or methylation of lamin B1 results in loss of integrity and deformity of the nuclear lamina. These data show that the organization of the nuclear envelope and lamina is dependent on a mechanism involving the methylation of lamin B1, and they identify a potential mechanism of laminopathy involving a B-type lamin.

GFAP and nuclear lamins share an epitope recognized by monoclonal antibody J1-31.

Monoclonal antibody J1-31 was raised against plaque materials taken from brains of patients who had suffered from multiple sclerosis (MS). Preliminary characterization of the antigen revealed it to be a protein of M(w) 68-70 kDa with both a cytoplasmic and nuclear localization. Here we report the results of isolation and peptide sequencing of the antigen from human brains, and immunocytochemical analysis of the antigen in F98 glioma cells. Purification and peptide sequencing indicate that the antibody recognizes a form of glial fibrillary acidic protein, possibly a phosphorylated variant. However, confocal immunocytochemistry and western analysis of F98 glioma cells raise the possibility that it also recognizes a phosphorylated epitope found in nuclear lamins. Analysis of the expression of the J1-31 epitope in F98 cells with respect to time in culture, cell density, and DNA synthesis showed a developmental relationship: cells that were engaged in rapid growth and DNA synthesis exhibited strong J1-31 staining in nuclei, whereas quiescent cells did not. We conclude that mAB J1-31 remains a useful antibody for studying multiple sclerosis, and is likely to prove useful in studies of the dynamics of nuclear lamins, particularly in models for wound-healing.

Association of autoantibodies to nuclear lamin B1 with thromboprotection in systemic lupus erythematosus: lack of evidence for a direct role of lamin B1 in apoptotic blebs.

OBJECTIVE: To demonstrate the association between autoantibodies to nuclear lamin B1 (aLB1) and protection against thrombosis ("thromboprotection") in patients with systemic lupus erythematosus (SLE), and to elucidate the mechanism by which aLB1 cause thromboprotection in vivo. Since a number of autoantigens in SLE have been localized specifically to the external surface of apoptotic blebs, it was hypothesized that circulating aLB1 may block the procoagulant effect of apoptotic blebs by binding to LB1 displayed at the external bleb surface. METHODS: A cross-sectional study was performed using serum samples obtained at first evaluation of 259 English Canadian and French Canadian patients from SLE registries at 3 hospitals. A case-control study was performed to analyze the relationship between aLB1 and lupus anticoagulant (LAC) status and thrombotic manifestations between onset of disease and last followup. Reactivity of aLB1 with Jurkat or endothelial cells, which had been induced to undergo apoptosis, was determined by indirect immunofluorescence. Localization of LB1 in apoptotic cells and blebs was analyzed by confocal microscopy and surface labeling of cell membrane proteins. RESULTS: High-titer aLB1 was restricted to a subset of SLE patients (46 patients), with an overall frequency of 17.8% (range 11.6-24.3% in the 3 centers). LB1 antibodies were significantly associated with LAC but not with antibodies to cardiolipin (aCL) or beta(2)-glycoprotein I (anti-beta(2)GPI). The frequency of thrombosis differed markedly depending on aLB1 and LAC status, as follows: presence of LAC and absence of aLB1 50%, presence of both LAC and aLB1 22.7%, absence of both LAC and aLB1 25.5%, absence of LAC and presence of aLB1, 20.8%. Further subclassification of patients based on aCL and anti-beta(2)GPI status revealed that, in the presence of LAC but in the absence of aCL, anti-beta(2)GPI, and aLB1, the frequency of thrombosis was 40%, whereas in the presence of aLB1, it decreased strikingly, to 9.1%. LB1 was found to be translocated into surface membrane blebs during apoptosis and to be entirely enclosed within the apoptotic bleb plasma membrane of Jurkat and endothelial cells. CONCLUSION: The presence of aLB1 in SLE patients with LAC essentially nullifies the strong prothrombotic risk associated with LAC. Hence, aLB1 is associated with thromboprotection. Reactivity of aLB1 with apoptotic blebs does not seem to play a direct role in mediating this protection, since LB1 is buried within apoptotic blebs and inaccessible to circulating aLB1. The mechanism by which aLB1 confers thromboprotection in SLE remains to be elucidated.