Adaptation and Changes in Actin Dynamics and Cell Motility as Early Responses of Cultured Mammalian Cells to Altered Gravitational Vector.

Cultured mammalian cells have been shown to respond to microgravity (µG), but the molecular mechanism is still unknown. The study we report here is focused on molecular and cellular events that occur within a short period of time, which may be related to gravity sensing by cells. Our assumption is that the gravity-sensing mechanism is activated as soon as cells are exposed to any new gravitational environment. To study the molecular events, we exposed cells to simulated µG (SµG) for 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h using a three-dimensional clinostat and made cell lysates, which were then analyzed by reverse phase protein arrays (RPPAs) using a panel of 453 different antibodies. By comparing the RPPA data from cells cultured at 1G with those of cells under SµG, we identified a total of 35 proteomic changes in the SµG samples and found that 20 of these changes took place, mostly transiently, within 30 min. In the 4 h and 8 h samples, there were only two RPPA changes, suggesting that the physiology of these cells is practically indistinguishable from that of cells cultured at 1 G. Among the proteins involved in the early proteomic changes were those that regulate cell motility and cytoskeletal organization. To see whether changes in gravitational environment indeed activate cell motility, we flipped the culture dish upside down (directional change in gravity vector) and studied cell migration and actin cytoskeletal organization. We found that compared with cells grown right-side up, upside-down cells transiently lost stress fibers and rapidly developed lamellipodia, which was supported by increased activity of Ras-related C3 botulinum toxin substrate 1 (Rac1). The upside-down cells also increased their migratory activity. It is possible that these early molecular and cellular events play roles in gravity sensing by mammalian cells. Our study also indicated that these early responses are transient, suggesting that cells appear to adapt physiologically to a new gravitational environment.

Senescent Phenotype Induced by p90RSK-NRF2 Signaling Sensitizes Monocytes and Macrophages to Oxidative Stress in HIV-Positive Individuals.

BACKGROUND: The incidence of cardiovascular disease is higher in HIV-positive (HIV+) patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for cardiovascular disease. However, the molecular mechanisms that link cART and cardiovascular disease are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. METHODS: Peripheral whole blood from cART-treated HIV+ individuals and nontreated HIV-negative individuals was treated with H2O2 (200 µmol/L) for 4 minutes, and p90RSK activity in CD14+ monocytes was measured. Plaque formation in the carotids was also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK-specific inhibitor (FMK-MEA) or dominant-negative p90RSK (DN-p90RSK) and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild-type and DN-p90RSK transgenic mice. RESULTS: Monocytes from HIV+ patients exhibited higher levels of p90RSK activity and were also more sensitive to reactive oxygen species than monocytes from HIV-negative individuals. A multiple linear regression analysis involving cART, Reynolds cardiovascular risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the 2 significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H2O2-induced overactivation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Last, the data obtained from our gain- or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. CONCLUSIONS: cART increased monocyte/macrophage sensitivity to reactive oxygen species- in HIV+ individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and proinflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing cardiovascular disease, especially in HIV+ patients treated with cART.

Mechanotransduction properties of the cytoplasmic tail of PECAM-1.

BACKGROUND INFORMATION: Vascular endothelial cells (ECs) are a well-known cell system used in the study of mechanobiology. Using cultured ECs, we found that platelet EC adhesion molecule 1 (PECAM-1, CD31), a cell adhesion protein localised to regions of EC-EC contact, was rapidly tyrosine phosphorylated in ECs exposed to shear or cyclic stretch. Src-homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) binds phosphorylated PECAM-1 and activates the extracellular signal-regulated kinase1/2 (ERK1/2) signalling cascade, a known flow-activated signalling pathway. RESULTS: Although PECAM-1 tyrosine phosphorylation is characterised in ECs exposed to fluid shear stress, it is less well demonstrated in the cells stretched cyclically. Thus, we first show that PECAM-1 is tyrosine-phosphorylated in ECs cyclically stretched. We hypothesise that when an external force is applied to a monolayer of ECs, the force is directly transmitted to PECAM-1 which is then stretched and phosphorylation sites in its cytoplasmic domain are exposed and phosphorylated. This hypothesis requires the presence of any stretchable structure within the PECAM-1 cytoplasmic domain. Force spectroscopy measurements were performed with a construct containing cytoplasmic PECAM-1 domains inserted between I27 motifs, a recombinant string of the structural elements from titin. This strategy allowed us to identify the events in which a single molecule is being pulled and to detect the unravelling of the cytoplasmic domain of PECAM-1 by force. The response by PECAM-1 to mechanical loading was heterogeneous but with magnitudes as high as or higher than the naturally force bearing I27 domains. CONCLUSIONS: The PECAM-1 cytoplasmic domain has a structure that can be unfolded by externally applied force and this unfolding of PECAM-1 may be necessary for its phosphorylation, the first step of PECAM-1 mechanosignalling. SIGNIFICANCE: When EC monolayers are mechanically stimulated, the PECAM-1 found at EC contacts is phosphorylated. We have proposed that under these conditions, the cytoplasmic domain of PECAM-1 is unfolded, which then exposes a phosphorylation site, allowing it to be accessed. The stretch induced unfolding is essential to this model of PECAM-1 mechanosignalling. In this study, we investigate whether the cytoplasmic domain of PECAM-1 has a stretchable structure, and the results are in line with our hypothesis.

MK2 SUMOylation regulates actin filament remodeling and subsequent migration in endothelial cells by inhibiting MK2 kinase and HSP27 phosphorylation.

Actin filament remodeling regulates several endothelial cell (EC) processes such as contraction, migration, adhesion, and shape determination. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2)-mediated phosphorylation of heat-shock protein 27 kDa (HSP27) promotes actin filament remodeling, but little is known about the regulation of this event in ECs. We found that tumor necrosis factor-α (TNF-α) SUMOylated MK2 at lysine (K)-339 affected EC actin filament organization and migration. Loss of the MK2 SUMOylation site (MK2-K339R) increased MK2 kinase activity and prolonged HSP27 phosphorylation, enhancing its effects on actin filament-dependent events. Both TNF-α-mediated EC elongation and steady laminar shear stress-mediated EC alignment were increased by MK2-K339R. Moreover, kinase-dead dominant-negative MK2 (DN-MK2) inhibited these effects. Cell migration is a dynamic process regulated by actin filament remodeling. Both wild-type MK2 (WT-MK2) and DN-MK2 significantly enhanced TNF-mediated inhibition of EC migration, and MK2-K339R further augmented this effect. Interestingly, the p160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 reversed this effect by MK2-K339R, which strongly suggests that both excessive and insufficient levels of actin filament remodeling can block EC migration. Our study shows that MK2 SUMOylation is a new mechanism for regulating actin filament dynamics in ECs.

MAGI1 inhibits interferon signaling to promote influenza A infection.

We have shown that membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1), a scaffold protein with six PSD95/DiscLarge/ZO-1 (PDZ) domains, is involved in the regulation of endothelial cell (EC) activation and atherogenesis in mice. In addition to causing acute respiratory disease, influenza A virus (IAV) infection plays an important role in atherogenesis and triggers acute coronary syndromes and fatal myocardial infarction. Therefore, the aim of this study is to investigate the function and regulation of MAGI1 in IAV-induced EC activation. Whereas, EC infection by IAV increases MAGI1 expression, MAGI1 depletion suppresses IAV infection, suggesting that the induction of MAGI1 may promote IAV infection. Treatment of ECs with oxidized low-density lipoprotein (OxLDL) increases MAGI1 expression and IAV infection, suggesting that MAGI1 is part of the mechanistic link between serum lipid levels and patient prognosis following IAV infection. Our microarray studies suggest that MAGI1-depleted ECs increase protein expression and signaling networks involve in interferon (IFN) production. Specifically, infection of MAGI1-null ECs with IAV upregulates expression of signal transducer and activator of transcription 1 (STAT1), interferon b1 (IFNb1), myxovirus resistance protein 1 (MX1) and 2'-5'-oligoadenylate synthetase 2 (OAS2), and activate STAT5. By contrast, MAGI1 overexpression inhibits Ifnb1 mRNA and MX1 expression, again supporting the pro-viral response mediated by MAGI1. MAGI1 depletion induces the expression of MX1 and virus suppression. The data suggests that IAV suppression by MAGI1 depletion may, in part, be due to MX1 induction. Lastly, interferon regulatory factor 3 (IRF3) translocates to the nucleus in the absence of IRF3 phosphorylation, and IRF3 SUMOylation is abolished in MAGI1-depleted ECs. The data suggests that MAGI1 inhibits IRF3 activation by maintaining IRF3 SUMOylation. In summary, IAV infection occurs in ECs in a MAGI1 expression-dependent manner by inhibiting anti-viral responses including STATs and IRF3 activation and subsequent MX1 induction, and MAGI1 plays a role in EC activation, and in upregulating a pro-viral response. Therefore, the inhibition of MAGI1 is a potential therapeutic target for IAV-induced cardiovascular disease.

Nucleus-mitochondria positive feedback loop formed by ERK5 S496 phosphorylation-mediated poly (ADP-ribose) polymerase activation provokes persistent pro-inflammatory senescent phenotype and accelerates coronary atherosclerosis after chemo-radiation.

The incidence of cardiovascular disease (CVD) is higher in cancer survivors than in the general population. Several cancer treatments are recognized as risk factors for CVD, but specific therapies are unavailable. Many cancer treatments activate shared signaling events, which reprogram myeloid cells (MCs) towards persistent senescence-associated secretory phenotype (SASP) and consequently CVD, but the exact mechanisms remain unclear. This study aimed to provide mechanistic insights and potential treatments by investigating how chemo-radiation can induce persistent SASP. We generated ERK5 S496A knock-in mice and determined SASP in myeloid cells (MCs) by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. Candidate SASP inducers were identified by high-throughput screening, using the ERK5 transcriptional activity reporter cell system. Various chemotherapy agents and ionizing radiation (IR) up-regulated p90RSK-mediated ERK5 S496 phosphorylation. Doxorubicin and IR caused metabolic changes with nicotinamide adenine dinucleotide depletion and ensuing mitochondrial stunning (reversible mitochondria dysfunction without showing any cell death under ATP depletion) via p90RSK-ERK5 modulation and poly (ADP-ribose) polymerase (PARP) activation, which formed a nucleus-mitochondria positive feedback loop. This feedback loop reprogramed MCs to induce a sustained SASP state, and ultimately primed MCs to be more sensitive to reactive oxygen species. This priming was also detected in circulating monocytes from cancer patients after IR. When PARP activity was transiently inhibited at the time of IR, mitochondrial stunning, priming, macrophage infiltration, and coronary atherosclerosis were all eradicated. The p90RSK-ERK5 module plays a crucial role in SASP-mediated mitochondrial stunning via regulating PARP activation. Our data show for the first time that the nucleus-mitochondria positive feedback loop formed by p90RSK-ERK5 S496 phosphorylation-mediated PARP activation plays a crucial role of persistent SASP state, and also provide preclinical evidence supporting that transient inhibition of PARP activation only at the time of radiation therapy can prevent future CVD in cancer survivors.

Topoisomerase 2B Decrease Results in Diastolic Dysfunction via p53 and Akt: A Novel Pathway.

Diastolic dysfunction is condition of a stiff ventricle and a function of aging. It causes significant cardiovascular mortality and morbidity, and in fact, three million Americans are currently suffering from this condition. To date, all the pharmacological clinical trials have been negative. The lack of success in attenuating/ameliorating diastolic dysfunction stems from lack of duplication of myriads of clinical manifestation in pre-clinical settings. Here we report, a novel genetically engineered mice which may represents a preclinical model of human diastolic dysfunction to some extent. Topoisomerase 2 beta (Top2b) is an important enzyme in transcriptional activation of some inducible genes through transient double-stranded DNA breakage events around promoter regions. We created a conditional, tissue-specific, inducible Top2b knockout mice in the heart. Serendipitously, echocardiographic parameters and more invasive analysis of left ventricular function with pressure-volume loops show features of diastolic dysfunction. This was also confirmed histologically. At the cellular level, the Top2b knockdown showed morphological changes and molecular signaling akin to human diastolic dysfunction. Reverse phase protein analysis showed activation of p53 and inhibition of, Akt, as the possible mediators of diastolic dysfunction. Finally, activation of p53 and inhibition of Akt were confirmed in myocardial biopsy samples obtained from human diastolic dysfunctional hearts. Thus, we report for the first time, a Top2b downregulated preclinical mice model for diastolic dysfunction which demonstrates that Akt and p53 are the possible mediators of the pathology, hence representing novel and viable targets for future therapeutic interventions in diastolic dysfunction.

Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase.

BACKGROUND: Disturbed flow (d-flow)-induced senescence and activation of endothelial cells (ECs) have been suggested to have critical roles in promoting atherosclerosis. Telomeric repeat-binding factor 2 (TERF2)-interacting protein (TERF2IP), a member of the shelterin complex at the telomere, regulates the senescence-associated secretory phenotype (SASP), in which EC activation and senescence are engendered simultaneously by p90RSK-induced phosphorylation of TERF2IP S205 and subsequent nuclear export of the TERF2IP-TERF2 complex. In this study, we investigated TERF2IP-dependent gene expression and its role in regulating d-flow-induced SASP. METHODS: A principal component analysis and hierarchical clustering were used to identify genes whose expression is regulated by TERF2IP in ECs under d-flow conditions. Senescence was determined by reduced telomere length, increased p53 and p21 expression, and increased apoptosis; EC activation was detected by NF-κB activation and the expression of adhesion molecules. The involvement of TERF2IP S205 phosphorylation in d-flow-induced SASP was assessed by depletion of TERF2IP and mutation of the phosphorylation site. RESULTS: Our unbiased transcriptome analysis showed that TERF2IP caused alteration in the expression of a distinct set of genes, including rapamycin-insensitive companion of mTOR (RICTOR) and makorin-1 (MKRN1) ubiquitin E3 ligase, under d-flow conditions. In particular, both depletion of TERF2IP and overexpression of the TERF2IP S205A phosphorylation site mutant in ECs increased the d-flow and p90RSK-induced MKRN1 expression and subsequently inhibited apoptosis, telomere shortening, and NF-κB activation in ECs via suppression of p53, p21, and telomerase (TERT) induction. CONCLUSIONS: MKRN1 and RICTOR belong to a distinct reciprocal gene set that is both negatively and positively regulated by p90RSK. TERF2IP S205 phosphorylation, a downstream event of p90RSK activation, uniquely inhibits MKRN1 expression and contributes to EC activation and senescence, which are key events for atherogenesis.

Endothelial senescence is induced by phosphorylation and nuclear export of telomeric repeat binding factor 2-interacting protein.

The interplay among signaling events for endothelial cell (EC) senescence, apoptosis, and activation and how these pathological conditions promote atherosclerosis in the area exposed to disturbed flow (d-flow) in concert remain unclear. The aim of this study was to determine whether telomeric repeat-binding factor 2-interacting protein (TERF2IP), a member of the shelterin complex at the telomere, can regulate EC senescence, apoptosis, and activation simultaneously, and if so, by what molecular mechanisms. We found that d-flow induced p90RSK and TERF2IP interaction in a p90RSK kinase activity-dependent manner. An in vitro kinase assay revealed that p90RSK directly phosphorylated TERF2IP at the serine 205 (S205) residue, and d-flow increased TERF2IP S205 phosphorylation as well as EC senescence, apoptosis, and activation by activating p90RSK. TERF2IP phosphorylation was crucial for nuclear export of the TERF2IP-TRF2 complex, which led to EC activation by cytosolic TERF2IP-mediated NF-κB activation and also to senescence and apoptosis of ECs by depleting TRF2 from the nucleus. Lastly, using EC-specific TERF2IP-knockout (TERF2IP-KO) mice, we found that the depletion of TERF2IP inhibited d-flow-induced EC senescence, apoptosis, and activation, as well as atherosclerotic plaque formation. These findings demonstrate that TERF2IP is an important molecular switch that simultaneously accelerates EC senescence, apoptosis, and activation by S205 phosphorylation.

MAGI1 as a link between endothelial activation and ER stress drives atherosclerosis.

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1-/+ mice inhibited d-flow-induced atherogenesis. In sum, EC activation and ER stress-mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.

Developing a Reliable Mouse Model for Cancer Therapy-Induced Cardiovascular Toxicity in Cancer Patients and Survivors.

BACKGROUND: The high incidence of cardiovascular events in cancer survivors has long been noted, but the mechanistic insights of cardiovascular toxicity of cancer treatments, especially for vessel diseases, remain unclear. It is well known that atherosclerotic plaque formation begins in the area exposed to disturbed blood flow, but the relationship between cancer therapy and disturbed flow in regulating plaque formation has not been well studied. Therefore, we had two goals for this study; (1) Generate an affordable, reliable, and reproducible mouse model to recapitulate the cancer therapy-induced cardiovascular events in cancer survivors, and (2) Establish a mouse model to investigate the interplay between disturbed flow and various cancer therapies in the process of atherosclerotic plaque formation. METHODS AND RESULTS: We examined the effects of two cancer drugs and ionizing radiation (IR) on disturbed blood flow-induced plaque formation using a mouse carotid artery partial ligation (PCL) model of atherosclerosis. We found that doxorubicin and cisplatin, which are commonly used anti-cancer drugs, had no effect on plaque formation in partially ligated carotid arteries. Similarly, PCL-induced plaque formation was not affected in mice that received IR (2 Gy) and PCL surgery performed one week later. In contrast, when PCL surgery was performed 26 days after IR treatment, not only the atherosclerotic plaque formation but also the necrotic core formation was significantly enhanced. Lastly, we found a significant increase in p90RSK phosphorylation in the plaques from the IR-treated group compared to those from the non-IR treated group. CONCLUSIONS: Our results demonstrate that IR not only increases atherosclerotic events but also vulnerable plaque formation. These increases were a somewhat delayed effect of IR as they were observed in mice with PCL surgery performed 26 days, but not 10 days, after IR exposure. A proper animal model must be developed to study how to minimize the cardiovascular toxicity due to cancer treatment.

Endothelial cell-cell adhesion and mechanosignal transduction.

Recent reports indicate that in addition to proteins that form various types of intercellular junctions, a considerable number of proteins are localized to the area of endothelial cell-cell association. Many of these are signaling proteins, suggesting that this is an area of active signaling. In this article, we have attempted to compile a list of proteins that have been localized to the area of interendothelial association and to briefly discuss what is known about each. Since various investigators including ourselves have proposed that the region of interendothelial cell association is an important site for mechanosignaling, we will focus our discussion on the possible role of these proteins in mechanosignal transduction. We will also review the available evidence for PECAM-1 as a mechanotransducing molecule in endothelial cells.

Phosphorylation and localization of protein-zero related (PZR) in cultured endothelial cells.

Protein-zero related (PZR) is an immunoglobulin V (IgV)-type immunoreceptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PZR interacts with Src homology 2 domain-containing tyrosine phosphatase (SHP-2) via its tyrosine-phosphorylated ITIMs, for which c-Src is a putative kinase. Towards elucidating PZR function in endothelial cells (ECs), the authors cloned PZR from bovine aortic endothelial cells (BAECs) and characterized it. Mature bovine PZR had 94.8% and 92.7% sequence identity with canine and human proteins, respectively, and the two ITIM sequences were conserved among higher vertebrates. PZR was expressed in many cell types and was localized to cell contacts and intracellular granules in BAECs and mesothelioma (REN) cells. Coimmunoprecipitation revealed that PZR, Grb-2-associated binder-1 (Gab1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) were three major SHP-2-binding proteins in BAECs. H(2)O(2) enhanced PZR tyrosine phosphorylation and PZR/SHP-2 interaction in ECs in a dose-and time-dependent manner. To see if tyrosine kinases other than Src are also capable of phosphorylating PZR, the authors cotransfected HEK293 cells with PZR and one of several tyrosine kinases and found that c-Src, c-Fyn, c-Lyn, Csk, and c-Abl, but not c-Fes, phosphorylated PZR and increased PZR/SHP-2 interaction. These results suggest that PZR is a cell adhesion protein that may be involved in SHP-2-dependent signaling at interendothelial cell contacts.