The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to unmask redundant functions of the NR4A family in other immune cells. Here we use a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1–/– Nr4a3–/– (DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cell-extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional, phenotypic, and transcriptional features of anergy accumulate in chimeric mice. Nevertheless, we observe upregulation of genes encoding inflammatory mediators in anergic DKO T cells, and DKO T cells exhibit enhanced capacity for IL-2 production. These studies reveal cell-intrinsic roles for the NR4A family in both central and peripheral T cell tolerance, and demonstrate that each is essential to preserve immune homeostasis.
Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman
G protein-coupled receptors (GPCRs) are highly desirable drug targets for human disease. Although GPCR dysfunction drives development and progression of many tumors, including breast cancer (BC), targeting individual GPCRs has limited efficacy as a cancer therapy because numerous GPCRs are activated. Here, we sought a new way of blocking GPCR activation in HER2+-BC by targeting a subgroup of GPCRs that couple to Gi/o proteins (Gi/o-GPCRs). In mammary epithelial cells of transgenic mouse models, and BC cell lines, HER2 hyperactivation altered GPCR expression, particularly, Gi/o-GPCRs. Gi/o-GPCR stimulation transactivated EGFR and HER2 and activated the PI3K/AKT and Src pathways. If we uncoupled Gi/o-GPCRs from their cognate Gi/o proteins by pertussis toxin (PTx), then BC cell proliferation and migration was inhibited in vitro and HER2-driven tumor formation and metastasis suppressed in vivo. Moreover, targeting Gi/o-GPCR signaling via PTx, PI3K, or Src inhibitors enhanced HER2-targeted therapy. These results indicate that, in BC cells, HER2 hyperactivation drives aberrant Gi/o-GPCR signaling, and Gi/o-GPCR signals converge on PI3K/AKT and Src signaling pathways to promote cancer progression and resistance to HER2-targeted therapy. Our findings point to a new way to pharmacologically deactivate GPCR signaling to block tumor growth and enhance therapeutic efficacy.
Cancan Lyu, Yuanchao Ye, Maddison M. Lensing, Kay-Uwe Wagner, Ronald J. Weigel, Songhai Chen
cGMP-dependent protein kinase 1α (PKG1α) promotes left ventricle (LV) compensation to pressure overload. PKG1-activating drugs improve heart failure (HF) outcomes but are limited by vasodilation-induced hypotension. Signaling molecules which mediate PKG1α cardiac therapeutic effects but do not promote PKG1α-induced hypotension could therefore represent improved therapeutic targets. We investigated roles of mixed lineage kinase 3 (MLK3) in mediating PKG1α effects on LV function after pressure overload, and in regulating blood pressure (BP). In a transaortic constriction HF model PKG activation with sildenafil preserved LV function in MLK3+/+, but not MLK3-/- littermates. MLK3 co-immunoprecipitated with PKG1α. MLK3-PKG1α co-interaction decreased in failing LVs. PKG1a phosphorylated MLK3 on Thr-277/Ser-281 sites required for kinase activation. MLK3-/- mice displayed hypertension and increased arterial stiffness, though PKG stimulation with sildenafil or the sGC stimulator BAY41-2272 still reduced BP in MLK3-/- mice. MLK3 kinase inhibition with URMC-099 did not affect BP, but induced LV dysfunction in mice. These data reveal MLK3 as a PKG1α substrate mediating PKG1α preservation of LV function but not acute PKG1α BP effects. Mechanistically, MLK3 kinase-dependent effects preserve LV function, while MLK3 kinase-independent signaling regulates BP. These findings suggest augmenting MLK3 kinase activity could preserve LV function in HF but avoid hypotension from PKG1α activation.
Timothy D. Calamaras, Suchita Pande, Robert A.U. Baumgartner, Seung Kyum Kim, Joseph C. McCarthy, Gregory L. Martin, Kelly Tam, Angela L. McLaughlin, Guang-rong Wang, Mark J. Aronovitz, Weiyu Lin, Jonathan I. Aguirre, Paulina Baca, Peiwen Liu, Daniel A. Richards, Roger J. Davis, Richard H. Karas, Iris Z. Jaffe, Robert M. Blanton
Cardiac inflammation and fibrosis contribute significantly to hypertension-related adverse cardiac remodeling. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been demonstrated as a key molecular link between inflammation and cardiovascular disease. However, the cell-specific contribution of IKKβ signaling towards adverse cardiac remodeling remains elusive. Cardiac fibroblasts are one of the most populous non-myocyte cell types in the heart that play a key role in mediating cardiac fibrosis and remodeling. To investigate the function of fibroblast IKKβ, we generated inducible fibroblast-specific IKKβ-deficient mice. Here we report an important role of IKKβ in the regulation of fibroblast functions and cardiac remodeling. Fibroblast-specific IKKβ deficient male mice were protected from angiotensin II (Ang II)-induced cardiac hypertrophy, fibrosis, and macrophage infiltration. Ablation of fibroblast IKKβ inhibited Ang II-stimulated fibroblast proinflammatory and profibrogenic responses, leading to ameliorated cardiac remodeling and improved cardiac function in IKKβ-deficient mice. Findings from this study establish fibroblast IKKβ as a key factor regulating cardiac fibrosis and function in hypertension-related cardiac remodeling.
Weiwei Lu, Zhaojie Meng, Rebecca Hernandez, Changcheng Zhou
Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies and surgical specimens of abdominal aortic aneurysms (AAA) from EH patients were used. Cell-based assays, NETs/human aortic endothelial cells co-cultures and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly-diagnosed, EH patients. Stimulation of control neutrophils with plasma from untreated EH patients generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via a reactive oxygen species (ROS)/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced significantly in EH patients starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants were accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAA. These data reveal the important pathogenic role of Ang II/ROS/NETs/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.
Akrivi Chrysanthopoulou, Eugenia Gkaliagkousi, Antonios Lazaridis, Stella Arelaki, Panagiotis Pateinakis, Maria Ntinopoulou, Alexandros Mitsios, Christina Antoniadou, Christos Argyriou, George S. Georgiadis, Vasileios Papadopoulos, Alexandra Giatromanolaki, Konstantinos Ritis, Panagiotis Skendros
Creatine transporter (CrT) upholds the brain creatine (Cr) levels, but the impacts of its deficiency on energetics adaptation under stress remain unclear. There are also no effective treatments of CrT-deficiency, the second most common cause of X-linked intellectual disabilities. Herein we examined the consequences of CrT-deficiency in brain energetics and stress-adaptation responses plus the effects of intranasal Cr supplement. We found that CrT-deficient (CrT-/y) mice harbored dendritic spine and synaptic dysgenesis. Nurtured newborn CrT-/y mice maintained the baseline brain ATP level with a tendency towards the pAMPK/autophagy from mTOR signaling activity. Starvation elevated the signaling imbalance and reduced the brain ATP level in P3 CrT-/y mice. Similarly, CrT-/y neurons and P10 CrT-/y mice showed an imbalance between autophagy/mTOR signaling pathways and greater susceptibility to cerebral hypoxia-ischemia and ischemic insults. Notably, intranasal administration of Cr after cerebral ischemia increased the brain Cr/NAA (N-acetylaspartate) ratio, partially averted the signaling imbalance, and reduced the infarct size more potently than intraperitoneal Cr injection. These findings suggest important functions of CrT and Cr in preserving the homeostasis of brain energetics in stress conditions. Moreover, intranasal Cr supplement may be an effective treatment of congenital CrT-deficiency and acute brain injury.
Hong-Ru Chen, Xiaohui Zhang-Brotzge, Yury M. Morozov, Yuancheng Li, Siming Wang, Helen Zhang, Irena S. Kuan, Elizabeth M. Fugate, Hui Mao, Yu-Yo Sun, Pasko Rakic, Diana M. Lindquist, Ton DeGrauw, Chia-Yi Kuan
Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than one thousand nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here we show that histone demethylase LSD1 knockout from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in nucleus. Lsd1 knockout reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPβ and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.
Yang Cao, Lingyi Tang, Kang Du, Kitt Paraiso, Qiushi Sun, Zhengxia Liu, Xiaolong Ye, Yuan Fang, Fang Yuan, Yu-Han Chen, Yumay Chen, Xiaorong Wang, Clinton Yu, Ira L. Blitz, Ping H. Wang, Lan Huang, Haibo Cheng, Xiang Lu, Ken W.Y Cho, Marcus Seldin, Zhuyuan Fang, Qin Yang
Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbate disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in COVID-19 patients and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, two signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in COVID-19 patients. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation leading to production of pathological HA fragments which are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.
Kimberly A. Queisser, Rebecca A. Mellema, Elizabeth A. Middleton, Irina Portier, Bhanu Kanth Manne, Frederik Denorme, Ellen J. Beswick, Matthew T. Rondina, Robert A. Campbell, Aaron C. Petrey
Macrophage activation syndrome (MAS) is a life-threatening cytokine storm complicating systemic juvenile idiopathic arthritis (SJIA) driven by IFNγ. SJIA and MAS are associated with an unexplained emerging lung disease (SJIA-LD), with our recent work supporting pulmonary activation of IFNγ pathways pathologically linking SJIA-LD and MAS. Our objective was to mechanistically define the novel observation of pulmonary inflammation in the TLR9 mouse model of MAS. In acute MAS, lungs exhibit mild but diffuse CD4-predominant, perivascular interstitial inflammation with elevated IFNγ, IFN-induced chemokines, and AMΦ expression of IFNγ-induced genes. Single-cell RNA-sequencing confirmed IFN-driven transcriptional changes across lung cell types with myeloid expansion and detection of MAS-specific macrophage populations. Systemic MAS resolution was associated with increased AMΦ and interstitial lymphocytic infiltration. AMΦ transcriptomic analysis confirmed IFNγ-induced proinflammatory polarization during acute MAS, which switches towards an anti-inflammatory phenotype after systemic MAS resolution. Interestingly, recurrent MAS led to increased alveolar inflammation and lung injury, and reset AMΦ polarization towards a proinflammatory state. Furthermore, in mice bearing macrophages insensitive to IFNγ, both systemic feature of MAS and pulmonary inflammation were attenuated. These findings demonstrate that experimental MAS induces IFNγ-driven pulmonary inflammation replicating key features of SJIA-LD, and provides a model system for testing novel treatments directed towards SJIA-LD.
Denny K. Gao, Nathan Salomonis, Maggie Henderlight, Christopher Woods, Kairavee Thakkar, Alexei A. Grom, Sherry Thornton, Michael B. Jordan, Kathryn A. Wikenheiser-Brokamp, Grant S. Schulert
The mechanism controlling long-chain fatty acid (LCFA) mobilization from adipose tissue (AT) is not well understood. Here, we investigated how the LCFA transporter CD36 regulates this process. By using tissue-specific knockout mouse models, we show that CD36 in both adipocytes and endothelial cells mediates both LCFA deposition into and release from AT. We demonstrate the role of adipocytic and endothelial CD36 in promoting tumor growth and chemoresistance conferred by AT-derived LCFA. We show that dynamic cysteine S-acylation of CD36 in adipocytes, endothelial cells, and cancer cells mediates intercellular LCFA transport. We demonstrate that lipolysis induction in adipocytes triggers CD36 de-acylation and deglycosylation, as well as its dissociation from interacting proteins, prohibitin-1 (PHB), and annexin 2 (ANX2). Our data indicate that lipolysis triggers caveolar endocytosis and translocation of CD36 from the cell membrane to lipid droplets. This study suggests a mechanism for both outside-in and inside-out cellular LCFA transport regulated by CD36 S- acylation and its interactions with PHB and ANX2.
Alexes C. Daquinag, Zhanguo Gao, Cale Fussell, Linnet Immaraj, Renata Pasqualini, Wadih Arap, Askar M. Akimzhanov, Maria Febbraio, Mikhail G. Kolonin
No posts were found with this tag.