The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH2-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419–3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation.

Abstract. Patients with ESRD have a high circulating calcium (Ca) ϫ phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vascular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesicles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhibitor fetuin-A and calcified minimally. Importantly, MV released under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pretreatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.Patients with ESRD develop extensive medial calcification, or Monckeberg's sclerosis, that causes increased arterial stiffness and contributes to the high cardiovascular mortality (1,2). Calciphylaxis is an increasingly common and life-threatening form of calcification characterized by destructive calcification in the media of subcutaneous arterioles, leading to occlusion and subsequent widespread tissue necrosis (2,3). The precise pathophysiology of vascular calcification in ESRD is unknown, but risk factors include age, hypertension, time on dialysis, and, most significant, abnormalities in calcium (Ca) and phosphate (P) metabolism (4,5). Normal serum concentrations of Ca and inorganic P ions are metastable with respect to basic calcium phosphate (BCP; a mixture of octacalcium phosphate, dicalcium phosphate dihydrate, and apatite) precipitation but can support growth of nascent crystals. In ESRD, systemic Ca and inorganic P concentrations typically exceed 2.4 and 2.0 mM, respectively (4). Consequently, calcification in ESRD has traditionally been ascribed to supersaturation and subsequent precipitation of mineral ions. This has led to therapeutic measures to reduce the Ca/P product aimed mostly at reduction of P.Recent studies have shown that vascular calcification is a regulated process similar to bone formation (6,7). VSMC in the normal artery wall constitutively express potent inhibitors of calcification, such as matrix Gla ...

Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease (CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients, and drives vascular calcification. In this article, we review the physiological regulatory mechanisms for Ca and P homeostasis and the basis for their dysregulation in CKD. In addition, we highlight recent findings indicating that elevated Ca and P have direct effects on vascular smooth muscle cells (VSMCs) that promote vascular calcification, including stimulation of osteo/chondrogenic differentiation, vesicle release, apoptosis, loss of inhibitors, and ECM matrix degradation. These studies suggest a major role for elevated P in promoting osteo/chondrogenic differentiation of VSMC, whereas elevated Ca has a predominant role in promoting VSMC apoptosis and vesicle release. Furthermore, the effects of elevated Ca and P are synergistic providing a major stimulus for vascular calcification in CKD. Unravelling the complex regulatory pathways that mediate the effects of both Ca and P on VSMCs will ultimately provide novel targets and therapies to limit the destructive effects of vascular calcification in CKD patients.

Vascular calcification is associated with a significant increase in all-cause mortality and atherosclerotic plaque rupture. Calcification has been determined to be an active process driven in part by vascular smooth muscle cell (VSMC) transdifferentiation within the vascular wall. Historically, VSMC phenotype switching has been viewed as binary, with the cells able to adopt a physiological contractile phenotype or an alternate ‘synthetic’ phenotype in response to injury. More recent work, including lineage tracing has however revealed that VSMCs are able to adopt a number of phenotypes, including calcific (osteogenic, chondrocytic, and osteoclastic), adipogenic, and macrophagic phenotypes. Whilst the mechanisms that drive VSMC differentiation are still being elucidated it is becoming clear that medial calcification may differ in several ways from the intimal calcification seen in atherosclerotic lesions, including risk factors and specific drivers for VSMC phenotype changes and calcification. This article aims to compare and contrast the role of VSMCs in driving calcification in both atherosclerosis and in the vessel media focusing on the major drivers of calcification, including aging, uraemia, mechanical stress, oxidative stress, and inflammation. The review also discusses novel findings that have also brought attention to specific pro- and anti-calcifying proteins, extracellular vesicles, mitochondrial dysfunction, and a uraemic milieu as major determinants of vascular calcification.