Mutations in the nuclear envelope proteins lamins A and C cause a broad variety of human diseases, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. Cells lacking lamins A and C have reduced nuclear stiffness and increased nuclear fragility, leading to increased cell death under mechanical strain and suggesting a potential mechanism for disease. Here, we investigated the contribution of major lamin subtypes (lamins A, C, and B1) to nuclear mechanics by analyzing nuclear shape, nuclear dynamics over time, nuclear deformations under strain, and cell viability under prolonged mechanical stimulation in cells lacking both lamins A and C, cells lacking only lamin A (i.e."lamin C-only" cells), cells lacking wild-type lamin B1, and wild-type cells. Lamin A/C-deficient cells exhibited increased numbers of misshapen nuclei and had severely reduced nuclear stiffness and decreased cell viability under strain. Lamin C-only cells had slightly abnormal nuclear shape and mildly reduced nuclear stiffness but no decrease in cell viability under strain. Interestingly, lamin B1-deficient cells exhibited normal nuclear mechanics despite having a significantly increased frequency of nuclear blebs. Our study indicates that lamins A and C are important contributors to the mechanical stiffness of nuclei, whereas lamin B1 contributes to nuclear integrity but not stiffness.Lamins are type V intermediate filament proteins that form the nuclear lamina, a filamentous network underlying the inner nuclear membrane of eukaryotic cells. Lamins form stable structures in the nuclear lamina and the nucleoplasm, determine nuclear shape and size, resist nuclear deformation, and position nuclear pore complexes (reviewed in Refs. 1-3). In addition, lamins recruit and anchor, either directly or indirectly, several nuclear envelope proteins (e.g. nesprin-1␣, emerin, and the lamin B receptor) to the inner nuclear membrane (3).Mammalian cells express two types of lamins, the A and B types. Both share a common structural organization: a globular N-terminal domain separated from a larger C-terminal globular domain by a central helical rod domain that allows lamins to form parallel coiled-coil dimers, which in turn assemble into stable strings and higher order networks. Lamins A and C, the major A-type lamins, are alternatively spliced isoforms of a single gene, LMNA. The expression of A-type lamins is developmentally regulated, beginning midway through embryonic development (4). A-type lamins are expressed in most but not all differentiated cells (5). B-type lamins (lamins B1 and B2) are encoded by separate genes, LMNB1 and LMNB2, respectively (6). Unlike A-type lamins, B-type lamins are expressed in all cells and throughout development (7,8), although it is not clear if they are always coexpressed at equivalent levels in the same cell.

SummaryHutchinson-Gilford progeria syndrome (HGPS), reportedly a model for normal aging, is a genetic disorder in children marked by dramatic signs suggestive for premature aging. It is usually caused by de novo mutations in the nuclear envelope protein lamin A. Lamins are essential to maintaining nuclear integrity, and loss of lamin A/C results in increased cellular sensitivity to mechanical strain and defective mechanotransduction signaling. Since increased mechanical sensitivity in vascular cells could contribute to loss of smooth muscle cells and the development of arteriosclerosis -the leading cause of death in HGPS patients -we investigated the effect of mechanical stress on cells from HGPS patients. We found that skin fibroblasts from HGPS patients developed progressively stiffer nuclei with increasing passage number. Importantly, fibroblasts from HGPS patients had decreased viability and increased apoptosis under repetitive mechanical strain, as well as attenuated wound healing, and these defects preceded changes in nuclear stiffness. Treating fibroblasts with farnesyltransferase inhibitors restored nuclear stiffness in HGPS cells and accelerated the wound healing response in HGPS and healthy control cells by increasing the directional persistence of migrating cells. However, farnesyltransferase inhibitors did not improve cellular sensitivity to mechanical strain. These data suggest that increased mechanical sensitivity in HGPS cells is unrelated to changes in nuclear stiffness and that increased biomechanical sensitivity could provide a potential mechanism for the progressive loss of vascular smooth muscle cells under physiological strain in HGPS patients.

Mutations of the nuclear lamins cause a wide range of human diseases, including Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome. Defects in A-type lamins reduce nuclear structural integrity and affect transcriptional regulation, but few data exist on the biological role of B-type lamins. To assess the functional importance of lamin B1, we examined nuclear dynamics in fibroblasts from Lmnb1 ⌬/⌬ and wild-type littermate embryos by time-lapse videomicroscopy. Here, we report that Lmnb1 ⌬/⌬ cells displayed striking nuclear rotation, with ϳ90% of Lmnb1 ⌬/⌬ nuclei rotating at least 90°during an 8-h period. The rotation involved the nuclear interior as well as the nuclear envelope. The rotation of nuclei required an intact cytoskeletal network and was eliminated by expressing lamin B1 in cells. Nuclear rotation could also be abolished by expressing larger nesprin isoforms with long spectrin repeats. These findings demonstrate that lamin B1 serves a fundamental role within the nuclear envelope: anchoring the nucleus to the cytoskeleton.Mutations in nuclear lamins and lamin-associated proteins cause a panoply of human diseases (laminopathies) including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction system disease, Dunnigan-type familial partial lipodystrophy, limb-girdle muscular dystrophy, Charcot-Marie tooth disorder type 2, and Hutchinson-Gilford progeria syndrome (1-7). The mechanism by which these ubiquitously expressed proteins cause such diverse phenotypes is unclear. This is not particularly surprising, though, because the functions of the nuclear lamins themselves are currently incompletely understood.Lamins are the principal components of the nuclear lamina, an intermediate filament meshwork that lines the inner nuclear membrane (8, 9). Lamins are associated with chromatin, other integral proteins of the inner nuclear membrane, inner portions of the nuclear pore complexes (NPCs), 2 and several transcription factors such as SREBP1, retinoblastoma protein, and MOK (10). Thus, lamins are critical for the structural integrity of the nucleus and also play a role in DNA replication, chromatin organization, and transcriptional regulation (11).In mammalian cells, the major A-type lamins, lamins A and C, are alternatively spliced products of LMNA, whereas the major B-type lamins, lamin B1 and lamin B2, are encoded by two distinct genes, LMNB1 and LMNB2, respectively (12). Although the A-and B-type lamins share a similar structure, they differ in their behavior during cell division and their patterns of expression. B-type lamins are found in all cell types and are expressed throughout development, whereas A-type lamins are not present in early embryos (13). Within the nucleus, lamin B1 binds directly to chromatin and histones (14) and interacts with several chromatin-binding inner nuclear membrane proteins (e.g. lamina-associated proteins, lamin B receptor (LBR), and the nuclear pore protein nucleoporin 153) (15). Following mitosis, B-type lamins assemble first into t...

Abstract-We tested the hypothesis that steady laminar shear stress activates the glucocorticoid receptor (GR) and its transcriptional signaling pathway in an effort to investigate the potential involvement of GR in shear stress-induced antiatherosclerosis actions in the vasculature. In both bovine aortic endothelial cells (BAECs) and NIH3T3 cells expressing GFP-GR chimeric protein, wall shear stress of 10 or 25 dynes/cm 2 caused a marked nuclear localization of GFP-GR within 1 hour to an extent comparable to induction with 25 mol/L dexamethasone. The shear mediated nuclear localization of GFP-GR was significantly reduced by 25 mol/L of the MEK1 inhibitor (PD098059) or the PI 3-kinase inhibitor (LY294002). Also, Western blots demonstrated translocation of endogenous GR into nucleus of sheared BAECs. Promoter construct studies using glucocorticoid response element (GRE)-driven expression of secreted alkaline phosphatase (SEAP) indicated that BAECs exposed to shear stress of 10 and 25 dynes/cm 2 for 8 hours produced Ͼ9-fold more SEAP (nϭ6; PϽ0.005) than control cells, a level comparable to that observed with dexamethasone. Shear stress enhanced SEAP expression at 6 hours was reduced 50% (nϭ5; PϽ0.005) by MEK1/2 or PI 3-kinase inhibitors, but not by the NO inhibitor, L-NAME. Finally, in human internal mammary artery, endothelial GR is found to be highly nuclear localized. We report a new shear responsive transcriptional element, GRE. The finding that hemodynamic forces can be as potent as high dose glucocorticoid steroid in activating GR and GRE-regulated expression correlates with the atheroprotective responses of endothelial cells to unidirectional arterial shear stress.

Death associated protein kinase (DAPK) is a positive regulator in tumor necrosis factor α (TNFα)-induced apoptotic pathway, and DAPK expression is lost in cancer cells. In the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation and physiological shear stress is protective against apoptosis. Using bovine aortic endothelial cells, we found that DAPK expression increased, while the auto-inhibitory phosphorylation of serine 308 decreased with shear stress at 12 dynes/cm(2) for 6 h. Quantitative RT-PCR revealed a corresponding increase in DAPK mRNA [P < 0.01]. We found that after 18-h TNFα induction, shearing cells for another 6 h significantly reduced apoptosis based on TUNEL staining [P < 0.05], although cell necrosis was not affected. Under the same conditions, we observed significantly decreased overall DAPK, as well as phospho-serine 308 DAPK [P < 0.05] compared to TNFα treatment alone. Caspase-3 and -7 activities downstream of DAPK were also attenuated. Shearing cells alone resulted in enhanced apoptosis, likely due to increased DAPK activity. Our findings were further supported by DAPK siRNA, which yielded contrary results. We present conclusive evidence for the first time that shear stress of up to 6 h up-regulates DAPK expression and activation. However, in the presence of apoptotic stimuli such as TNFα, shear stress caused decrease in DAPK activity. In fact, long-term shear stress of 24 h significantly reduced overall DAPK expression. Our findings strongly support a novel role for DAPK in mediating effects of shear stress in suppressing cytokine-activated apoptosis.

The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/␣-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.The insulin-like growth factor 1 (IGF-1) 5 biological actions are mediated by the IGF-1 receptor, a member of the tyrosine kinase family of growth factor receptors, composed of two extracellular ␣-subunits and two membrane-spanning ␤-subunits, encoding an intracellular tyrosine kinase (1). Upon IGF-1 binding, the activated IGF-1 receptor is generally thought to proceed through phosphorylation of specific cytosolic substrates, such as the adaptor proteins Src homology 2/␣-collagen (Shc). The IGF-1 signal is then transmitted to two downstream pathways: ERK of the MAPK family and phosphatidylinositol 3-kinase (PI3K), which potentially leads to two distinct events: cell proliferation or cell differentiation (1). How IGF-1 signaling bifurcates at some point from proliferation to differentiation is not known. IGF-I is a critical regulator of adipose tissue mass through its regulation of adipogenesis (2). During adipogenesis, dividing preadipocytes shift to growth-arrested cells allowing full differentiation of adipocytes. IGF-1 mediates both proliferation and differentiation of preadipocytes in vivo (3) and in vitro, including 3T3-L1 cells (2) and primary cultures of mouse (4) and human cells (5). IGF-1 stimulation of mitogen-activated protein kinase (MAPK) activity is lost as preadipocytes differentiate, and this is paralleled by a loss in Shc tyrosine phosphorylation. Inhibition of Shc tyrosine phosphorylation inhibits DNA synthesis and increases expression of the master regulator of adipogenesis, the nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) and its...

Arterial shear stress can regulate endothelial phenotype. The potential for antiinflammatory effects of shear stress on TNFα-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm 2 blocked by > 80% the induction by 5 ng/ml TNFα of interleukin-8 (IL-8) and IL-6 secretion (50% and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 μM nitro-L-arginine methylester, L-NAME).Exposure of TNFα-stimulated HAEC to arterial shear stress for 5 hr also reduced by 60% (P < 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm 2 . Also, neutrophil rolling lengths at 1 dyne/cm 2 were longer when TNFα-stimulated HAEC were presheared for 5 hr at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFκB, shear stress caused a marked 40-fold induction of luciferase in TNFα-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress.Hemodynamic force exerts anti-inflammatory effects on cytokine activated endothelium by attenuation of cytokine expression and neutrophil firm arrest.

We report the first measurement of the neutron cross section on argon in the energy range of 100-800 MeV. The measurement was obtained with a 4.3-hour exposure of the Mini-CAPTAIN detector to the WNR/LANSCE beam at LANL. The total cross section is measured from the attenuation coefficient of the neutron flux as it traverses the liquid argon volume. A set of 2,631 candidate interactions is divided in bins of the neutron kinetic energy calculated from time-offlight measurements. These interactions are reconstructed with custom-made algorithms specifically designed for the data in a time projection chamber the size of the Mini-CAPTAIN detector. The energy averaged cross section is 0.91 ± 0.10 (stat.) ± 0.09 (sys.) barns. A comparison of the measured cross section is made to the GEANT4 and FLUKA event generator packages, where the energy averaged cross sections in this range are 0.60 and 0.68 barns respectively.