Epoxyeicosatrienoic acids (EETs) have potent anti-inflammatory properties. Hydrolysis of EETs by soluble epoxide hydrolase (sEH/EPHX2) to less active diols attenuates their anti-inflammatory effects. Macrophage activation is critical to many inflammatory responses; however, the role of EETs and sEH in regulating macrophage function remains unknown. Lung bacterial clearance of S. pneumoniae was impaired in Ephx2-deficient (Ephx2-/-) mice and in mice treated with an sEH inhibitor. The EET receptor antagonist, EEZE, restored lung clearance of S. pneumoniae in Ephx2-/- mice. Ephx2-/- mice had normal lung Il-1β, Il-6 and Tnfα expression and macrophage recruitment to lungs during S. pneumoniae infection; however, Ephx2 disruption attenuated proinflammatory cytokine induction, Tlr2 and Pgylrp1 receptor upregulation and Rac1/2 and Cdc42 activation in PGN-stimulated macrophages. Consistent with these observations, Ephx2-/-macrophages displayed reduced phagocytosis of S. pneumoniae in vivo and in vitro. Heterologous overexpression of TLR2 and PGLYRP1 in Ephx2-/- macrophages restored macrophage activation and phagocytosis. Human macrophage function was similarly regulated by EETs. Together, these results demonstrate that EETs reduce macrophage activation and phagocytosis of S. pneumoniae through down-regulation of TLR2 and PGLYRP1 expression. Defining the role of EETs and sEH in macrophage function may lead to development of new therapeutic approaches for bacterial diseases.
Hong Li, J. Alyce Bradbury, Matthew L. Edin, Joan P. Graves, Artiom Gruzdev, Jennifer Cheng, Samantha L. Hoopes, Laura Miller-Degraff, Michael B. Fessler, Stavros Garantziotis, Shepherd H. Schurman, Darryl Craig Zeldin
In this study, we demonstrate that Forkhead Box F1 (FOXF1), a mesenchymal transcriptional factor essential for lung development, is retained in a topographically distinct mesenchymal stromal cell population along the bronchovascular space in an adult lung and identify this distinct subset of collagen-expressing cells as a key player in lung allograft remodeling and fibrosis. Utilizing Foxf1_tdTomato BAC (Foxf1_tdTomato) and Foxf1_tdTomato;Col1a1_GFP mice, we show that Lin-Foxf1+ cells encompass the Sca1+CD34+ subset of collagen I-expressing mesenchymal cells (MCs) with capacity to generate colony forming units and lung epithelial organoids. Histologically, Foxf1-expressing MCs formed a three-dimensional network along the conducting airways; FOXF1 was noted to be conspicuously absent in MCs in the alveolar compartment. Bulk and single-cell RNA sequencing confirmed distinct transcriptional signatures of Foxf1pos/neg MCs, with Foxf1-expressing cells delineated by their high Gli1 and low Integrin α8 expression, from other collagen-expressing MCs. Foxf1+Gli1+ MCs demonstrated proximity to Sonic hedgehog (Shh) expressing bronchial epithelium, and mesenchymal Foxf1/Gli1 expression was found to be dependent on the paracrine Shh signaling in epithelial organoids. Utilizing a murine lung transplant model, we show dysregulation of the epithelial mesenchymal Shh/Gli1/Foxf1 crosstalk and expansion of this specific peri-bronchial MC population in chronically rejecting fibrotic lung allografts.
Russell R. Braeuer, Natalie M. Walker, Keizo Misumi, Serina Mazzoni-Putman, Yoshiro Aoki, Ruohan Liao, Ragini Vittal, Gabriel G. Kleer, David S. Wheeler, Jonathan Z. Sexton, Carol F. Farver, Joshua D. Welch, Vibha N. Lama
Formation of nitric oxide (NO) by the endothelial NO-synthase (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and result in the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as Ca2+ and PDK1, plays a pivotal role in this process. Active PKN2 promoted phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation while active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone and blood pressure.
Young-June Jin, Ramesh Chennupati, Rui Li, Guozheng Liang, ShengPeng Wang, András Iring, Johannes Graumann, Nina Wettschureck, Stefan Offermanns
The transcription factor NFATC2 induces β-cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified ~2,200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle, and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse are less responsive to NFATC2-induced β-cell proliferation, suggesting the FOXP family works to regulate β-cell proliferation in concert with NFATC2. NFATC2 induced β-cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified ~250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle-associated transcripts, and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce β-cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of non-coding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate β-cell proliferation.
Shane P. Simonett, Sunyoung Shin, Jacob A. Herring, Rhonda Bacher, Linsin A. Smith, Chenyang Dong, Mary E. Rabaglia, Donnie S. Stapleton, Kathryn L. Schueler, Jeea Choi, Matthew N. Bernstein, Daniel R. Turkewitz, Carlos Perez-Cervantes, Jason Spaeth, Roland Stein, Jeffery S. Tessem, Christina Kendziorski, Sunduz Keles, Ivan P. Moskowitz, Mark P. Keller, Alan D. Attie
Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, CD146 was found to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the anti-tumor response of T cells. Together, our data reveal a previously unrecognized LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.
Hongxia Duan, Lin Jing, Xiaoqing Jiang, Yanbin Ma, Daji Wang, Jianquan Xiang, Xuehui Chen, Zhenzhen Wu, Huiwen Yan, Junying Jia, Zheng Liu, Jing Feng, Mingzhao Zhu, Xiyun Yan
Hypoxia-induced pulmonary hypertension (PH) is one of the most common and deadliest forms of PH. Fibroblast growth factor receptors 1 and 2 (FGFR1/2) are elevated in patients with PH and in mice exposed to chronic hypoxia. Endothelial FGFR1/2 signaling is important for the adaptive response to several injury types and we hypothesized that endothelial FGFR1/2 signaling would protect against hypoxia-induced PH. Mice lacking endothelial FGFR1/2, mice with activated endothelial FGFR signaling, and human pulmonary artery endothelial cells (HPAECs) were challenged with hypoxia. We assessed the effect of FGFR activation and inhibition on right ventricular pressure, vascular remodeling, and endothelial-mesenchymal transition (EndMT), a known pathologic change seen in patients with PH. Hypoxia-exposed mice lacking endothelial FGFRs developed increased PH, while mice overexpressing a constitutively active FGFR in endothelial cells did not develop PH. Mechanistically, lack of endothelial FGFRs or inhibition of FGFRs in HPAECs led to increased TGF-β signaling and increased EndMT in response to hypoxia. These phenotypes were reversed in mice with activated endothelial FGFR signaling, suggesting that FGFR signaling inhibits TGF-β pathway–mediated EndMT during chronic hypoxia. Consistent with these observations, lung tissue from patients with PH showed activation of FGFR and TGF-β signaling. Collectively, these data suggest that activation of endothelial FGFR signaling could be therapeutic for hypoxia-induced PH.
Kel Vin Woo, Isabel Y. Shen, Carla J. Weinheimer, Attila Kovacs, Jessica Nigro, Chieh-Yu Lin, Murali Chakinala, Derek E. Byers, David M. Ornitz
Pancreatic β cell failure in type 2 diabetes mellitus (T2DM) is attributed to perturbations of the β cell’s transcriptional landscape resulting in impaired glucose-stimulated insulin secretion. Recent studies identified SLC4A4 (a gene encoding an electrogenic Na+-coupled HCO3– cotransporter and intracellular pH regulator, NBCe1) as one of the misexpressed genes in β cells of patients with T2DM. Thus, in the current study, we set out to test the hypothesis that misexpression of SLC4A4/NBCe1 in T2DM β cells contributes to β cell dysfunction and impaired glucose homeostasis. To address this hypothesis, we first confirmed induction of SLC4A4/NBCe1 expression in β cells of patients with T2DM and demonstrated that its expression was associated with loss of β cell transcriptional identity, intracellular alkalinization, and β cell dysfunction. In addition, we generated a β cell–selective Slc4a4/NBCe1-KO mouse model and found that these mice were protected from diet-induced metabolic stress and β cell dysfunction. Importantly, improved glucose tolerance and enhanced β cell function in Slc4a4/NBCe1-deficient mice were due to augmented mitochondrial function and increased expression of genes regulating β cell identity and function. These results suggest that increased β cell expression of SLC4A4/NBCe1 in T2DM plays a contributory role in promotion of β cell failure and should be considered as a potential therapeutic target.
Matthew R. Brown, Heather Holmes, Kuntol Rakshit, Naureen Javeed, Tracy K. Her, Alison A. Stiller, Satish Sen, Gary E. Shull, Y.S. Prakash, Michael F. Romero, Aleksey V. Matveyenko
ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. We herein detected a de novo heterozygous point mutation in ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally or soon after birth with signs of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamic simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. MALDI-imaging mass-spectrometry showed a marked increase of SM levels in the brains of Q84E knock-in mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.
Katsumori Segawa, Atsuo Kikuchi, Tomoyasu Noji, Yuki Sugiura, Keita Hiraga, Chigure Suzuki, Kazuhiro Haginoya, Yasuko Kobayashi, Mitsuhiro Matsunaga, Yuki Ochiai, Kyoko Yamada, Takuo Nishimura, Shinya Iwasawa, Wataru Shoji, Fuminori Sugihara, Kohei Nishino, Hidetaka Kosako, Masahito Ikawa, Yasuo Uchiyama, Makoto Suematsu, Hiroshi Ishikita, Shigeo Kure, Shigekazu Nagata
Liver tumor-initiating cells (TICs) are involved in liver tumorigenesis, metastasis, drug resistance and relapse, but the regulatory mechanisms of liver TICs are largely unknown. Here, we have identified a functional circular RNA, termed circRNA activating MAFF (cia-MAF), that is robustly expressed in liver cancer and liver TICs. cia-MAF knockout primary cells and cia-maf knockout liver tumors harbor decreased ratios of TICs, and display impaired liver tumorigenesis, self-renewal and metastatic capacities. In contrast, cia-MAF overexpression drives liver TIC propagation, self-renewal and metastasis. Mechanistically, cia-MAF binds to the MAFF promoter, recruits the TIP60 complex to the MAFF promoter, and finally promotes MAFF expression. Loss of cia-MAF function attenuates the combination between the TIP60 complex and the MAFF promoter. MAFF is highly expressed in liver tumors and liver TICs, and its antisense oligo (ASO) has therapeutic potential in treating liver cancer without MAFA/MAFG gene copy number alterations (CNAs). This study reveal an additional layer for liver TIC regulation as well as circRNA function, and also provide an additional target for eliminating liver TICs, especially for liver tumor without MAFA/MAFG gene CNAs.
Zhenzhen Chen, Tiankun Lu, Lan Huang, Zhiwei Wang, Zhongyi Yan, Yubo Guan, Wenjing Hu, Zusen Fan, Pingping Zhu
PFKP (phosphofructokinase, platelet), the major isoform of PFK1 expressed in T cell acute lymphoblastic leukemia (T-ALL), is predominantly expressed in the cytoplasm to carry out its glycolytic function. Our study showed PFKP was a cyto-nuclear shuttling protein with functional nuclear export and nuclear localization sequences. Cyclin D3/CDK6 facilitated PFKP nuclear translocation by dimerization and by exposing the NLS of PFKP to induce the interaction between PFKP and importin 9. Nuclear PFKP stimulated the expression of C-X-C chemokine receptor type 4 (CXCR4), a chemokine receptor regulating leukemia homing/infiltration, to promote T-ALL cell invasion, which depended on the activity of c-Myc. In vivo experiments showed that nuclear PFKP promoted leukemia homing/infiltration into the bone marrow, spleen and liver, which could be blocked with CXCR4 antagonists. Immunohistochemistry staining of tissues from a clinically well-annotated cohort of T cell lymphoma/leukemia patients showed nuclear PFKP localization only in invasive cancers, but not in non-malignant T lymph node or reactive hyperplasia. The presence of nuclear PFKP in these specimens correlated with poor survival in patients with T cell malignancy, suggesting the potential utility of nuclear PFKP as a diagnostic marker.
Xueliang Gao, Shenghui Qin, Yongxia Wu, Chen Chu, Baishan Jiang, Roger H. Johnson, Dong Kuang, Jie Zhang, Xi Wang, Anand Mehta, Kenneth D. Tew, Gustavo W. Leone, Xue-Zhong Yu, Haizhen Wang
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