Endothelial-Specific Overexpression of Caveolin-1 Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice

Caveolin-1 (Cav-1) is the major structural protein essential to the formation of the caveolae in endothelial cells. Genetic ablation of Cav-1 on an apolipoprotein E knockout background inhibits the progression of atherosclerosis, whereas re-expression of Cav-1 in the endothelium promotes lesion expansion. Although Cav-1-null mice are useful to delineate the importance of caveolae in atherosclerosis, there are additional problems that are difficult to dissect because loss of Cav-1 abolishes both the caveolae organelle as well as the Cav-1-mediated signaling pathways. To study how Cav-1 influences the progression of atherosclerosis in mice with caveolae, we generated a transgenic mouse that overexpresses Cav-1 in the endothelial cells in an apolipoprotein E-deficient background. We found that endothelial-specific overexpression of Cav-1 enhanced the progression of atherosclerosis in mice. Mechanistically, overexpression of Cav-1 reduced endothelial cell proliferation, migration, and nitric oxide production in vitro and increased expression of vascular cell adhesion molecule-1 in vivo.Atherosclerosis is a progressive disease characterized by the accumulation of lipids, inflammatory cells, and fibrous elements in the large arteries.^1,2 In Western societies, it is the underlying cause of approximately 50% of all deaths.² Traditional hypotheses of atherogenesis have suggested that the dysfunction or injury of vascular endothelial cells (ECs) is critical for the development of atherosclerosis.^3,4 The endothelium functions as a selectively permeable barrier between blood and tissues as it can regulate transcytosis and generate effector molecules such us nitric oxide (NO) that regulate thrombosis, inflammation, vascular tone, and vascular remodeling.⁵ The anatomic sites where the atheromas develop are associated with perturbed flow and increased EC turnover rate and these athero-prone areas show increased permeability to macromolecules such as low-density lipoproteins (LDLs) at preferential sites for lesion formation.⁶Caveolae are 50- to 100-nm cell surface flask-shaped invaginated structures observed in many cell types including endothelium.⁷ Among the three isoforms of caveolin (Cav) that have been described (Cav-1, Cav-2, and Cav-3), Cav-1 is the major coat protein of endothelial caveolae and is necessary for caveolae assembly.^8,9 Indeed, several studies have shown a complete loss of caveolae organelles in blood vessels, adipocytes, and fibroblasts obtained from Cav-1-deficient mice.^8,9 The putative functions of caveolae include cholesterol transport, endocytosis, transcytosis, and signal transduction. Physiologically, an absence of caveolae results in impairment of cholesterol homeostasis, insulin resistance, nitric oxide production, and calcium signaling.^10,11,12 In the cardiovascular system, caveolae and Cav-1 show an important role in the proliferation and migration of smooth muscle cells,¹³ regulation of NO production and vascular permeability,^8,14 angiogenesis,^15,16,17 and atherosclerosis.^18,19 Cav-1-deficient mice develop cardiac hypertrophy, hypercholesterolemia, and hypertriglyceridemia and have increased tumor microvascular permeability, angiogenesis, and growth.¹⁷ Interestingly, the absence of Cav-1 reduces the progression of atherosclerosis.¹⁹ Very recently, we showed, using a genetic model, that endothelial caveolae are essential for the progression of atherosclerosis.¹⁸ Mechanistically, endothelial Cav-1 and caveolae are critical for LDL infiltration in the artery wall, NO production, and macrophage accumulation, all events necessary for atherogenesis.¹⁸ Although Cav-1-null mice are useful to delineate the importance of caveolae in atherosclerosis, there are additional problems that are difficult to dissect, because loss of Cav-1 abolishes both caveolae organelles as well as Cav-1-mediated signaling pathways. To study how Cav-1 influences the progression of atherosclerosis in mice with caveolae, we generated a transgenic mice that overexpress Cav-1 in the endothelial cells in an apolipoprotein E-deficient (ApoE^−/−) background. We found that endothelial-specific overexpression of Cav-1 enhanced the progression of atherosclerosis in mice. Mechanistically, overexpression of Cav-1 reduces EC proliferation, migration, and NO production and increases vascular cell adhesion molecule 1 (VCAM-1) expression in vivo.     

Greb1 Transiently Accelerates Pancreatic β-Cell Proliferation in Diabetic Mice Exposed to Estradiol

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LKB1-PTEN axis controls Th1 and Th17 cell differentiation via regulating mTORC1

Journal of Molecular Medicine - Immuno-environmental change triggers CD4+ T cell differentiation. T cell specialization activates metabolic signal pathways to meet energy requirements. Defective T...     

Activation of mTORC1 Signaling Pathway in AIDS-Related Lymphomas

Using immunohistochemistry with antibodies against the phosphoserine residues in both S6rp and 4E binding protein 1, we identified the activation of the mammalian target of rapamycin (mTORC)1 pathway in 29 cases of AIDS-related lymphoma. These cases represented a diverse spectrum of histological types of non-Hodgkin lymphoma (24 cases) and classic Hodgkin lymphoma (five cases). mTORC1 was also activated in the hyperplastic but not involuted follicles of HIV-associated lymphadenopathy in eight cases, supporting the notion that mTORC1 activation is a common feature of transformed lymphocytes irrespective of either their reactive or malignant phenotype. We also found that in B-cell lines that represent diffuse large B-cell lymphoma, Burkitt lymphoma, Epstein-Barr virus-infected lymphocytes, and human herpesvirus 8-positive primary effusion lymphoma, inhibitors of Syk, MEK, and, seemingly, phosphoinositide 3 kinases suppressed mTORC1 activation, in particular when these inhibitors were used in combination. These findings indicate that AIDS-related lymphoma and other histologically similar types of lymphomas that are derived from transformed B lymphocytes may display clinical responses to inhibitors that directly target mTORC1 or, possibly, upstream activators of the mTORC1 pathway.The incidence of lymphomas in HIV-positive patients is nearly 200 times higher than in those uninfected by the virus. Lymphomas accounts for an increasing percentage of AIDS-defining illness, particularly from the advent of highly active antiretroviral therapy therapy.^1,2 These AIDS-related lymphomas (ARLs) typically represent the proliferation of enlarged, transformed B lymphocytes, which usually fall into the category of diffuse large cell lymphoma (DLBCL), with morphology ranging from centroblasts to immunoblasts. Other histological types such as Burkitt lymphoma (BL), Hodgkin lymphoma, and T/null cell anaplastic large cell lymphoma are also overrepresented among ARLs.^1,2,3,4 The pathogenesis of ARL is poorly understood. It has been postulated that cell proliferation occurring in the setting of severe immunosuppression and driven by chronic antigenemia resulting at first in the polyclonal and ultimately in the monoclonal lymphoproliferation plays a key role in lymphomagenesis in HIV patients. In addition, cell infection by the Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) most likely contributes to the malignant cell phenotype in some subtypes of ARL, with the association of primary effusion lymphoma with HHV8 being essentially universal.^1,2,3,4 Regardless of the histological type of ARL, chemotherapy is typically ineffective and new treatment approaches are clearly needed to combat this group of lymphomas. In addition to ARL, HIV patients develop a benign reactive lymphadenopathy, particularly early after the infection as an overall ineffective response to the virus. This immune response is characterized by florid follicular hyperplasia that over time may lead to follicular involution and lymphocyte depletion.Mammalian target of rapamycin (mTOR) is a ubiquitously expressed serine/threonine kinase involved in key cellular functions including protein synthesis and proliferation.^5,6 mTOR associates with several proteins including either raptor or rictor to form the mTORC1 and mTORC2 complexes, respectively, with the signaling pathways activated by mTORC1 being much better characterized.^4,5,6,7 Accordingly, it is well established that mTORC1 activates p70S6 kinase 1 and inhibits 4E binding protein 1 (4E-BP1). In turn, p70S6 kinase 1 phosphorylates an S6 protein of the 40S ribosomal subunit (S6rp) at several sites including serines 235 and 236. The exact mechanisms of mTORC1 activation are less understood but both phosphoinositide 3 kinases (PI3K)/Akt^8,9,10 and extracellular regulated (ERK)/mitogen-activated kinase (MEK) kinases^11,12 signaling pathways have been found to activate mTORC1 with members of the insulin growth factor family providing the primary signal, at least in some instances. The highly potent and specific inhibitors from the rapamycin family can functionally inactivate mTORC1. In addition to being used as immunosuppressants, mTORC1 inhibitors are evaluated as therapeutic agents in various types of cancer,^5,6 with high efficacy already documented in renal cell carcinoma.¹³Syk is a protein tyrosine kinase expressed in B lymphocytes,¹⁴ monocytes/macrophages,¹⁵ mast cells,¹⁶ and other cell types. Syk has been found to be involved in signal transduction through several types of receptors including the antigen B-cell receptor¹⁷ and at least three different receptors for the Fc component of immunoglobulins G and E.^{14,15,16,18,19,20} A recent report suggests that Syk may be involved in mTORC1 activation in a follicular and possibly other types of B-cell lymphoma.²¹ Although inhibitors of either PI3K/Akt or MEK/ERK signaling pathways did fully inhibit mTORC1 activation in transformed B lymphocytes, at least when applied alone,²² these pathways have been found to contribute to mTORC1 stimulation in two types of T-cell lymphoma.^23,24In this study, we identified the activation of the mTORC1 pathway in all ARL cases examined, regardless of their specific histological classification and immunophenotype. mTORC1 was also activated in the hyperplastic follicles of the HIV-associated lymphadenopathy. Furthermore, we found that in the different types of transformed B-lymphocytes cell lines, inhibition of Syk, MEK, and, seemingly, PI3K resulted in suppression of the mTORC1 activation, in particular when the combination of the inhibitors was used. These findings indicate that ARL and histologically similar types of lymphoma may benefit from targeted therapy with inhibitors of mTORC1 or, possibly, its upstream activators.     

Cytoplasmic and nuclear distribution of the protein complexes mTORC1 and mTORC2: rapamycin triggers dephosphorylation and delocalization of the mTORC2 components rictor and sin1

The mammalian target of rapamycin (mTOR) is part of two distinct complexes, mTORC1, containing raptor and mLST8, and mTORC2, containing rictor, mLST8 and sin1. Although great endeavors have already been made to elucidate the function and regulation of mTOR, the cytoplasmic nuclear distribution of the mTOR complexes is unknown. Upon establishment of the proper experimental conditions, we found mTOR, mLST8, rictor and sin1 to be less abundant in the nucleus than in the cytoplasm of non-transformed, non-immortalized, diploid human primary fibroblasts. Although raptor is also high abundant in the nucleus, the mTOR/raptor complex is predominantly cytoplasmic, whereas the mTOR/rictor complex is abundant in both compartments. Rapamycin negatively regulates the formation of both mTOR complexes, but the molecular mechanism of its effects on mTORC2 remained elusive. We describe that in primary cells short-term treatment with rapamycin triggers dephosphorylation of rictor and sin1 exclusively in the cytoplasm, but does not affect mTORC2 assembly. Prolonged drug treatment leads to complete dephosphorylation and cytoplasmic translocation of nuclear rictor and sin1 accompanied by inhibition of mTORC2 assembly. The distinct cytoplasmic and nuclear upstream and downstream effectors of mTOR are involved in many cancers and human genetic diseases, such as tuberous sclerosis, Peutz-Jeghers syndrome, von Hippel-Lindau disease, neurofibromatosis type 1, polycystic kidney disease, Alzheimer's disease, cardiac hypertrophy, obesity and diabetes. Accordingly, analogs of rapamycin are currently tested in many different clinical trials. Our data allow new insights into the molecular consequences of mTOR dysregulation under pathophysiological conditions and should help to optimize rapamycin treatment of human diseases.     

Overexpression of the SNQ3/YAP1 gene confers hyper-resistance to nitrosoguanidine in Saccharomyces cerevisiae via a glutathione-independent mechanism

The MNNG hyper-resistance of yeast transformants containing multiple copies of the SNQ3/YAP1 yeast gene is not caused by lowered MNNG activation due to depleted pools of glutathione. On the contrary, the SNQ3/YAP1-encoded protein stimulates production of GSH, apparently by promoter activation due to the AP-1 recognition element. Expression of at least one further gene, encoding a protein with a strong detoxifying activity, must also be stimulated to explain the MNNG hyper-resistance phenotype.     

Rapamycin Reverses Elevated mTORC1 Signaling in Lamin A/C-Deficient Mice, Rescues Cardiac and Skeletal Muscle Function, and Extends Survival

Mutations in LMNA, the gene that encodes A-type lamins, cause multiple diseases including dystrophies of the skeletal muscle and fat, dilated cardiomyopathy, and progeria-like syndromes (collectively termed laminopathies). Reduced A-type lamin function, however, is most commonly associated with skeletal muscle dystrophy and dilated cardiomyopathy rather than lipodystrophy or progeria. The mechanisms underlying these diseases are only beginning to be unraveled. We report that mice deficient in Lmna, which corresponds to the human gene LMNA, have enhanced mTORC1 (mammalian target of rapamycin complex 1) signaling specifically in tissues linked to pathology, namely, cardiac and skeletal muscle. Pharmacologic reversal of elevated mTORC1 signaling by rapamycin improves cardiac and skeletal muscle function and enhances survival in mice lacking A-type lamins. At the cellular level, rapamycin decreases the number of myocytes with abnormal desmin accumulation and decreases the amount of desmin in both muscle and cardiac tissue of Lmna(-/-) mice. In addition, inhibition of mTORC1 signaling with rapamycin improves defective autophagic-mediated degradation in Lmna(-/-) mice. Together, these findings point to aberrant mTORC1 signaling as a mechanistic component of laminopathies associated with reduced A-type lamin function and offer a potential therapeutic approach, namely, the use of rapamycin-related mTORC1 inhibitors.     

YAP1调控间质成纤维细胞活化的研究进展

间质成纤维细胞位于实质细胞周围,参与细胞外基质(extracellular matrix,ECM)分泌.该细胞通常处于静止状态,但在炎症和肿瘤等疾病发生时异常活化.成纤维细胞活化受多种基因调控,这些基因通过促进细胞增殖、促进胶原蛋白和纤维蛋白等ECM分泌,导致局部组织过度纤维化,促进疾病进展.Yes相关蛋白1(Yes-associated protein 1,YAP1)是一种转录因子共刺激分子,同时也是多种信号通路的效应分子.YAP1在活化状态成纤维细胞内表达水平较高.且该蛋白通过调控包含生长因子在内的活化相关的基因表达,可促进成纤维细胞活化.因此,YAP1可能是成纤维细胞活化的关键调控因子,下调YAP1表达有望成为纤维化疾病的潜在治疗方式.     

NLK phosphorylates Raptor to mediate stress-induced mTORC1 inhibition

The mechanistic target of rapamycin (mTOR) is a central cell growth controller and forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates a wide range of upstream signals, both positive and negative, to regulate cell growth. Although mTORC1 activation by positive signals, such as growth factors and nutrients, has been extensively investigated, the mechanism of mTORC1 regulation by stress signals is less understood. In this study, we identified the Nemo-like kinase (NLK) as an mTORC1 regulator in mediating the osmotic and oxidative stress signals. NLK inhibits mTORC1 lysosomal localization and thereby suppresses mTORC1 activation. Mechanistically, NLK phosphorylates Raptor on S863 to disrupt its interaction with the Rag GTPase, which is important for mTORC1 lysosomal recruitment. Cells with Nlk deletion or knock-in of the Raptor S863 phosphorylation mutants are defective in the rapid mTORC1 inhibition upon osmotic stress. Our study reveals a function of NLK in stress-induced mTORC1 modulation and the underlying biochemical mechanism of NLK in mTORC1 inhibition in stress response.     

Pregnancy restores the regenerative capacity of the aged liver via activation of an mTORC1-controlled hyperplasia/hypertrophy switch

Regenerative capacity is progressively lost with age. Here we show that pregnancy markedly improved liver regeneration in aged mice concomitantly with inducing a switch from proliferation-based liver regeneration to a regenerative process mediated by cell growth. We found that the key mediator of this switch was the Akt/mTORC1 pathway; its inhibition blocked hypertrophy, while increasing proliferation. Moreover, pharmacological activation of this pathway sufficed to induce the hypertrophy module, mimicking pregnancy. This treatment dramatically improved hepatic regenerative capacity and survival of old mice. Thus, cell growth-mediated mass reconstitution, which is relatively resistant to the detrimental effects of aging, is employed in a physiological situation and holds potential as a therapeutic strategy for ameliorating age-related functional deterioration.     

Inhibition of mTORC1 renders cardiac protection against lipopolysaccharide

Sepsis-induced cardiac dysfunction is a severe clinical problem. It is evident that rapamycin can protect heart from pathological injuries. However, there are no data demonstrating rapamycin reverse cardiac dysfunction induced by sepsis. In this study, Lipopolysaccharide (LPS) was administrated to mice and H9c2 cells. After treatment, we further determined cardiac function by echocardiography, ANP, BNP and inflammatory markers by qPCR and apoptosis by TUNEL staining. Moreover, mTORC1 signaling pathway and Akt activity were measured by Western blots. We found that rapamycin attenuated cardiac dysfunction, increase in ANP and BNP as well as apoptosis induced by LPS both in mice and in H9c2 cells. Unexpectedly, LPS did not significantly affect the mRNA levels of TNF-α and IL-6. Furthermore, rapamycin further reduced the decrease in mTORC1 signaling and Akt activity induced by LPS. In conclusion, rapamycin can protect heart from LPS induced damages by inhibition mTORC1 signaling and elevation of Akt activity.     

Lipopolysaccharide Accelerates Neuropilin-1 Protein Degradation by Activating the Large GTPase Dynamin-1 in Macrophages

Inflammation - Neuropilin-1 (Nrp1) is highly expressed in macrophages and plays a critical role in acute and chronic inflammation-associated diseases, such as sepsis, type II diabetes, and...     

癌蛋白YAP1在宫颈癌中的研究现状与进展

Yes相关蛋白1(Yes-associated protein 1,YAP1)是Hippo信号通路下游中的一个转录共激活因子,既可参与细胞生长、发育及DNA修复,也可作为癌基因促进细胞异常增殖及恶性转化.YAP1表达与宫颈癌临床分期和淋巴结转移关系密切,可作为宫颈癌预后的独立影响因素.本文对YA P 1在宫颈癌中的研究...