Maintaining adequate proteasomal proteolytic activity is essential for eukaryotic cells. For metazoan cells, little is known about the composition of genes that are regulated in the proteasome network or the mechanisms that modulate the levels of proteasome genes. Previously, two distinct treatments have been observed to induce 26S proteasome levels in Drosophila melanogaster cell lines, RNA interference (RNAi)-mediated inhibition of the 26S proteasome subunit Rpn10/S5a and suppression of proteasome activity through treatment with active-site inhibitors. We have carried out genome array profiles from cells with decreased Rpn10/S5a levels using RNAi or from cells treated with proteasome inhibitor MG132 and have thereby identified candidate genes that are regulated as part of a metazoan proteasome network. The profiles reveal that the majority of genes that were identified to be under the control of the regulatory network consisted of 26S proteasome subunits. The 26S proteasome genes, including three new subunits, Ubp6p, Uch-L3, and Sem1p, were found to be up-regulated. A number of genes known to have proteasome-related functions, including Rad23, isopeptidase T, sequestosome, and the genes for the segregase complex TER94/VCP-Ufd1-Npl4 were also found to be up-regulated. RNAi-mediated inhibition against the segregase complex genes demonstrated pronounced stabilization of proteasome substrates throughout the Drosophila cell. Finally, transcriptional reporter assays and deletion mapping studies in Drosophila demonstrate that proteasome mRNA induction is dependent upon the 5 untranslated regions (UTRs). Transfer of the 5 UTR from the proteasome subunit Rpn1/S2 to a noninducible promoter was sufficient to confer transcriptional upregulation of the reporter mRNA after proteasome inhibition.Proteasome-dependent degradation serves an essential role in the removal of a wide variety of key nuclear and cytosolic proteins (35,42,45,52). This pathway also carries out an important housekeeping function by clearing cells from potentially harmful abnormal proteins that arise as the result of mutations, translational errors, misfolding, or postsynthetic damage and functions in the cytoplasm as a part of the protein quality control system for the endoplasmic reticulum (25).Structurally, the 26S proteasome consists of a 20S catalytic core and a 19S regulatory complex that associates with the ends of the 20S proteasome in an ATP-dependent manner (3,20,46). The eukaryotic 20S proteasome is composed of 14 different subunits arranged in four stacked, seven-membered rings that form the barrel-shaped complex (18, 43). The 19S regulatory complex is itself composed of two distinct subcomplexes, the base and the lid (15). Six distinct ATPase subunits proposed to function in substrate unfolding and gating of the 20S pore have been localized to the base along with two additional subunits (7,12,16,36). At least eight subunits form a lid subcomplex that is thought to be necessary for the processing of polyubiquitinated proteins and exhibit high...

Valve stiffening is a hallmark of aortic valve stenosis caused by excess extracellular matrix accumulation by myofibroblasts. We aimed to elucidate whether matrix stiffness regulates endothelial-to-mesenchymal transition (EndMT) of adult valvular endothelial cells (VECs) to myofibroblasts as a mechanism to further promote valve fibrosis. In addition, we specifically examined the role of the Wnt/β-catenin signaling pathway in the development of myofibroblasts during EndMT, as Wnt/β-catenin signaling has been implicated in EndMT during heart development, is reactivated in valve disease, and is required for mechanically-regulated myofibrogenesis of valve interstitial cells. Clonally derived porcine VECs were cultured on soft (5 kPa) or stiff (50 kPa) silicone Sylgard 527 substrates and treated with transforming growth factor (TGF)-β1 to induce EndMT. Immunofluorescent staining revealed that TGF-β1 preferentially promoted EndMT in VECs on stiffer substrates, evidenced by a decrease in the endothelial marker VE-cadherin and an increase in the myofibroblast marker α-smooth muscle actin (α-SMA). These changes were accompanied by β-catenin nuclear localization both in vitro and in vivo, assessed by immunostaining. Degradation of β-catenin with endostatin reduced VEC myofibroblast transition, as indicated by decreased α-SMA fiber expression. We conclude that TGF-β1-induced EndMT in aortic VECs is dependent on matrix stiffness and Wnt/β-catenin signaling promotes myofibrogenesis during EndMT.

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Objective-Calcific aortic valve disease is associated with the differentiation of valvular interstitial cells (VICs) to myofibroblast and osteoblast-like cells, particularly in the fibrosa layer of the valve. Previous studies suggested that C-type natriuretic peptide (CNP) protects against calcific aortic valve disease to maintain homeostasis. We aimed to determine whether CNP inhibits VIC pathological differentiation as a mechanism to explain its protective effects. Methods and Results-CNP expression was prominent in normal porcine aortic valves, particularly on the ventricular side, but reduced in sclerotic valves concomitant with the appearance of pathological VIC phenotypes in the fibrosa. In vitro, CNP inhibited calcified aggregate formation and bone-related transcript and protein expression by VICs grown in osteogenic conditions. Under myofibrogenic culture conditions, CNP reduced ␣-smooth muscle actin expression and cell-mediated gel contraction, indicating inhibition of myofibroblast differentiation. Similar to CNP, simvastatin inhibited VIC osteoblast and myofibroblast differentiation in vitro. Strikingly, simvastatin upregulated CNP expression in VICs cultured under myofibrogenic conditions, and small interfering RNA knockdown of natriuretic peptide receptor-b (a CNP receptor) significantly reduced the antifibrotic effect of simvastatin, suggesting that it acts in part via CNP/NPR-B autocrine/paracrine signaling. 1 The normal VIC population is heterogeneous, consisting primarily of fibroblasts, a subpopulation of which consists of mesenchymal progenitor cells with multilineage differentiation potential, 2 and a small population (Ϸ1% to 5%) of smooth muscle cells and myofibroblasts. [3][4][5][6] VICs undergo myofibroblast and osteoblast differentiation during the progression of CAVD, contributing to fibrosis and calcification. 5,7 Myofibroblasts, which are up to 30% of the total VIC population in sclerotic valves, 3,5 are identified by prominent ␣-smooth muscle actin (␣-SMA) stress fibers and are associated with increased collagen synthesis and cellular contractility. 8 Osteoblast-like cells are also often found in calcified aortic valves, 7,9 accompanied by an increase in the expression of bone-related transcripts and proteins. Conclusion-CNPPathological differentiation of VICs has been shown to be inhibited by statins in vitro 10 -12 ; however, clinical trials with statins have yielded conflicting results. [13][14][15][16][17] Retrospective and prospective cohort studies show that statins delay progression of CAVD, 16,18 whereas randomized, controlled trials such as Simvastatin and Ezetimibe for Aortic Stenosis and Scottish Aortic Stenosis and Lipid Lowering Trial, Impact on Regression found no benefit with statin therapy. 14,15,19 An improved understanding of the molecular determinants leading to pathological differentiation of VICs may provide insights into the potential of statins or other pharmacological therapies to arrest CAVD, which will eventually aid in refining treatment regimens and pat...

Genetic susceptibility for psoriasis is regulated to the greatest extent by the PSORS1 locus. Three psoriasis-associated susceptibility alleles have been identified within it, namely, HLACw6, HCR*WWCC and CDSN*5, but strong linkage disequilibrium between them has made it difficult to distinguish their individual genetic effects, and animal models to study their effects are not known. To study the function of HCR, we engineered transgenic mice with either a non-risk allele of HCR or the HCR*WWCC risk allele under the control of the cytokeratin-14 promoter. These choices were motivated by the apparently dominant effect of PSORS1 on psoriasis susceptibility and the physiological expression of HCR in basal keratinocytes. Transgenic mice appeared phenotypically normal and histologically their skin was indistinguishable from wild-type mice. Expression studies using Affymetrix arrays suggested that the HCR risk allele has specific functional consequences relevant to the pathogenesis of psoriasis. Comparison of gene expression changes between non-risk and risk allele mice revealed similarities to previous observations in human psoriatic skin, including upregulation of cytokeratins 6, 16 and 17 in risk allele mice. We also observed changes in the expression of genes associated with terminal differentiation and formation of the cornified cell envelope. Our results support the concept that HCR may constitute an essential gene in the PSORS1 locus. These observations are also compatible with a model that a susceptibility gene for psoriasis induces changes that are contributory but not sufficient by itself to produce the clinical phenotype.

Our data establish CNP/NPR2 signaling as a novel regulator of aortic valve development and disease and elucidate the therapeutic potential of targeting this pathway to arrest disease progression.

The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, α-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of α-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over cross-links during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actin-binding proteins, deformability and mechanosensing.

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