A vast body of literature has established GRK2 as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function post injury in numerous animal models. In recent years, discovery of several non-canonical GRK2 targets has expanded our view of this kinase. Here, we describe the novel and exciting finding that cardiac GRK2 activity can regulate whole body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgβARKct) display an enhanced obesogenic phenotype when fed a high fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD fed TgβARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics we found that cardiac GRK2 signaling altered myocardial BCAA and endocannabinoid metabolism and modulated circulating BCAA and endocannabinoid metabolite profiles on a HFD, and one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. Taken together, these results suggest that metabolic changes in the heart due to GRK2 signaling on a HFD control whole body metabolism.
Benjamin P. Woodall, Kenneth S. Gresham, Meryl A. Woodall, Mesele-Christina Valenti, Alessandro Cannavo, Jessica Pfleger, J. Kurt Chuprun, Konstantinos Drosatos, Walter J. Koch
Systemic lupus erythematosus (SLE) is an autoimmune disorder that predominantly affects women and is driven by autoreactive T cell–mediated inflammation. It is known that individuals with multiple X-chromosomes are at increased risk for developing SLE; however, the mechanisms underlying this genetic basis are unclear. Here, we use single cell imaging to determine the epigenetic features of the inactive X (Xi) in developing thymocytes, mature T cell subsets, and T cells from SLE patients and mice. We show that Xist RNA and heterochromatin modifications transiently reappear at the Xi and are missing in mature single positive T cells. Activation of mature T cells restores Xist RNA and heterochromatin marks simultaneously back to the Xi. Notably, X-chromosome inactivation (XCI) maintenance is altered in T cells of SLE patients and late-stage–disease NZB/W F1 female mice, and we show that X-linked genes are abnormally upregulated in SLE patient T cells. SLE T cells also have altered expression of XIST RNA interactome genes, accounting for perturbations of Xi epigenetic features. Thus, abnormal XCI maintenance is a feature of SLE disease, and we propose that Xist RNA localization at the Xi could be an important factor for maintaining dosage compensation of X-linked genes in T cells.
Camille M. Syrett, Bam Paneru, Donavon Sandoval-Heglund, Jianle Wang, Sarmistha Banerjee, Vishal Sindhava, Edward M. Behrens, Michael Atchison, Montserrat C. Anguera
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.
Jun-ichi Abe, Kyung Ae Ko, Sivareddy Kotla, Yin Wang, Jesus Paez-Mayorga, Ik Jae Shin, Masaki Imanishi, Hang Thi Vu, Yunting Tao, Miguel M. Leiva-Juarez, Tamlyn N. Thomas, Jan L. Medina, Jong Hak Won, Yuka Fujii, Carolyn J. Giancursio, Elena McBeath, Ji-Hyun Shin, Liliana Guzman, Rei J. Abe, Jack Taunton, Naoki Mochizuki, William Faubion, John P. Cooke, Keigi Fujiwara, Scott E. Evans, Nhat-Tu Le
Tregs require IL-2 signaling for signal transducer and activator of transcription 5 (STAT5)-mediated induction of Foxp3. While phosphatase 2A (PP2A) is a negative regulator of IL-2 production in effector T cells and Tregs do not produce IL-2, it is not known whether PP2A controls IL-2 signaling in Tregs. To address the role of PP2A in IL-2 signaling in Tregs we studied mice engineered to lack PP2A in all Foxp3-expressing cells. We report that PP2A is required to enable Foxp3 expression and to maintain sufficient numbers of Tregs in the thymus. We show for the first time that PP2A prevents the selective loss of surface IL-2Rβ and preserves IL-2R signaling potency in Tregs. The loss of IL-2Rβ in thymus- and spleen-derived Tregs that lack PP2A is due to increased sheddase activity. Pan-sheddase or selective A disintegrin and metalloproteinase 10 (ADAM10) inhibition, like forced expression of IL-2Rβ in PP2A-deficient Tregs restored IL-2Rβ expression and signaling. Thus, PP2A restrains the sheddase activity of ADAM10 in Treg cells to prevent the cleavage of IL-2Rβ from the cell surface to enable competent IL-2R signaling which is essential for Tregs development and homeostasis.
Amir Sharabi, Hao Li, Isaac R. Kasper, Wenliang Pan, Esra Meidan, Maria G. Tsokos, Vaishali R. Moulton, George C. Tsokos
The mTOR pathway is central to most cells. How mTOR is activated in macrophages and modulates macrophage physiology remain poorly understood. The tumor suppressor Folliculin (FLCN) is a GAP for RagC/D, a regulator of mTOR. We show here that LPS potently suppresses FLCN in macrophages, allowing nuclear translocation of the transcription factor TFE3, leading to lysosome biogenesis, cytokine production, and hypersensitivity to inflammatory signals. Nuclear TFE3 additionally activates a transcriptional RagD positive feedback loop that stimulates FLCN-independent canonical mTOR signaling to S6K and increases cellular proliferation. LPS thus simultaneously suppresses the TFE3 arm and activates the S6K arm of mTOR. In vivo, mice lacking myeloid FLCN reveal chronic macrophage activation, leading to profound histiocytic infiltration and tissue disruption, with hallmarks of human histiocytic syndromes like Erdheim-Chester Disease. Our data thus identify a critical FLCN-mTOR-TFE3 axis in myeloid cells, modulated by LPS, that balances mTOR activation and curbs innate immune responses.
Jia Li, Shogo Wada, Lehn K. Weaver, Chhanda Biswas, Edward M. Behrens, Zoltan Arany
Human adipose cells cannot secrete endogenous PPARγ ligands and are dependent on unknown exogenous sources. We postulated that the adipose tissue microvascular endothelial cells (aMVECs) cross-talk with the adipose cells for fatty acid (FA) transport and storage and also may secrete PPARγ ligands. We isolated aMVECs from human subcutaneous adipose tissue and showed that in these cells, but not in (pre)adipocytes from the same donors, exogenous FAs increased cellular PPARγ activation and markedly increased FA transport and the transporters FABP4 and CD36. Importantly, aMVECs only accumulated small lipid droplets and could not be differentiated to adipose cells and are not adipose precursor cells. FA exchange between aMVECs and adipose cells was bidirectional, and FA-induced PPARγ activation in aMVECs was dependent on functional adipose triglyceride lipase (ATGL) protein while deleting hormone-sensitive lipase in aMVECs had no effect. aMVECs also released lipids to the medium, which activated PPARγ in reporter cells as well as in adipose cells in coculture experiments, and this positive cross-talk was also dependent on functional ATGL in aMVECs. In sum, aMVECs are highly specialized endothelial cells, cannot be differentiated to adipose cells, are adapted to regulating lipid transport and secreting lipids that activate PPARγ, and thus, regulate adipose cell function.
Silvia Gogg, Annika Nerstedt, Jan Boren, Ulf Smith
The satiety effects and metabolic actions of cholecystokinin (CCK) have been recognized as potential therapeutic targets in obesity for decades. We identified a potentially novel Ca2+-activated chloride (Cl–) current (CaCC) that is induced by CCK in intestinal vagal afferents of nodose neurons. The CaCC subunit Anoctamin 2 (Ano2/TMEM16B) is the dominant contributor to this current. Its expression is reduced, as is CCK current activity in obese mice on a high-fat diet (HFD). Reduced expression of TMEM16B in the heterozygote KO of the channel in sensory neurons results in an obese phenotype with a loss of CCK sensitivity in intestinal nodose neurons, a loss of CCK-induced satiety, and metabolic changes, including decreased energy expenditure. The effect on energy expenditure is further supported by evidence in rats showing that CCK enhances sympathetic nerve activity and thermogenesis in brown adipose tissue, and these effects are abrogated by a HFD and vagotomy. Our findings reveal that Ano2/TMEM16B is a Ca2+-activated chloride channel in vagal afferents of nodose neurons and a major determinant of CCK-induced satiety, body weight control, and energy expenditure, making it a potential therapeutic target in obesity.
Runping Wang, Yongjun Lu, Michael Z. Cicha, Madhu V. Singh, Christopher J. Benson, Christopher J. Madden, Mark W. Chapleau, François M. Abboud
Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.
Adrian P. Wiegmans, Jodi M. Saunus, Sunyoung Ham, Richard J. Lobb, Jamie R. Kutasovic, Andrew J. Dalley, Mariska Miranda, Caroline Atkinson, Simote T. Foliaki, Kaltin Ferguson, Colleen Niland, Cameron N. Johnstone, Victoria Lewis, Steven Collins, Sunil R. Lakhani, Fares Al-Ejeh, Andreas Möller
Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens, but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all three Munc18 isoforms. Using conditional airway epithelial deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane, and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.
Ana M. Jaramillo, Lucia Piccotti, Walter V. Velasco, Anna Sofia Huerta Delgado, Zoulikha Azzegagh, Felicity S. Chung, Usman I. Nazeer, Junaid Farooq, Joshua M. Brenner, Jan Parker-Thornburg, Brenton L. Scott, Christopher M. Evans, Roberto Adachi, Alan R. Burns, Silvia M. Kreda, Michael J. Tuvim, Burton F. Dickey
RNA binding proteins represent an emerging class of proteins with a role in cardiac dysfunction. We show that activation of the RNA binding protein Human antigen R (HuR) is increased in the failing human heart. To determine the functional role of HuR in pathological cardiac hypertrophy, we created an inducible cardiomyocyte-specific HuR deletion mouse, and showed that HuR deletion reduces left ventricular hypertrophy, dilation, and fibrosis while preserving cardiac function in a transverse aortic constriction (TAC) model of pressure-overload-induced hypertrophy. Assessment of HuR-dependent changes in global gene expression suggests that the mechanistic basis for this protection occurs through a reduction in fibrotic signaling, specifically through a reduction in transforming growth factor beta (Tgfb) expression. Finally, pharmacological inhibition of HuR at a clinically relevant time point following the initial development of pathological hypertrophy post-TAC also yielded a significant reduction in pathological progression, as marked by a reduction in hypertrophy, dilation, and fibrosis, and preserved function. In summary, this study demonstrates a functional role for HuR in the progression of pressure overload-induced cardiac hypertrophy and establishes HuR inhibition as a viable therapeutic approach for pathological cardiac hypertrophy and heart failure.
Lisa C. Green, Sarah R. Anthony, Samuel Slone, Lindsey Lanzillotta, Michelle L. Nieman, Xiaoqing Wu, Nathan Robbins, Shannon M. Jones, Sudeshna Roy, A. Phillip Owens III, Jeffrey Aube, Liang Xu, John N. Lorenz, Burns C. Blaxall, Jack Rubinstein, Joshua B. Benoit, Michael Tranter
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