bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integration of GPCR‐induced endothelial cytoprotection signaling by β‐arrestin‐2
Authors: Molinar-Inglis Olivia , Zhang Lili , Nicholas Dequina , Chen Buxin , Cisneros Metztli , Grimsey Neil J. , Coronel Luisa J. , Lin Ying , Rada Cara , Chi Neil , Lawson Mark A. , Trejo JoAnn 
Published: April 2020

Vascular dysfunction is a hallmark of inflammation and results in endothelial barrier disruption, vascular leakage and apoptosis. G protein‐coupled receptors (GPCRs) are highly druggable and mediate inflammatory responses associated with endothelial dysfunction. Specifically, two GPCRs, protease‐activated receptor‐1 (PAR1) and sphingosine‐1‐phosphate receptor‐1 (S1PR1) promote endothelial cytoprotection including barrier protection, Akt‐mediated cell survival, and anti‐inflammatory responses. Activated protein C (APC) activates PAR1 to enhance endothelial barrier stability, whereas S1PR1 contributes to basal barrier stability, whether the two receptors coordinate signaling to promote endothelial cytoprotection is not known. Our previous work showed that APC/PAR1 signaling via the β‐arrestin‐2 and Dishevelled‐2 (Dvl2) scaffolds promote Rac1 signaling and endothelial barrier protection. However, whether β‐arrestin‐2 and Dvl2 are required for integration of S1PR1 into APC/PAR1‐mediated cytoprotection is not known. Here, we report that APC/PAR1 protection against TNF‐α‐induced apoptosis requires S1PR1, whereas S1PR1 is not necessary for APC/PAR1‐mediated barrier protection. We further show that APC/PAR1 transactivation of S1PR1 is mediated by β‐arrestin‐2 dependent activation of sphingosine kinase 1 (Sphk1) and is pivotal for anti‐apoptotic Akt signaling. S1PR1, is activated by sphingosine‐1‐phosphate generated by Sphk1 and we hypothesized that β‐arrestin‐2 and Dvl2 were required for Sphk1 activity. We found that APC promotes Sphk1 phosphorylation and translocation to the membrane. In addition, loss of β‐arrestin‐2 but not Dvl2 abolished APC‐mediated Sphk1 activation. Pharmacological antagonism of S1PR1, inhibition of Sphk1, and β‐arrestin‐2 depletion abolished APC/PAR1‐mediated Akt signaling. Moreover, inhibition of Sphk1, and β‐arrestin‐2 depletion also resulted in loss of APC/PAR1‐mediated cell survival suggesting that Sphk1 and β‐arrestin‐2 are involved in APC/PAR1‐mediated cytoprotection. Our mechanistic studies were corroborated in vivo using a Sphk1 gene knockout zebrafish model. Loss of Sphk1 ablated APC/PAR1‐mediated cell survival and we are currently in the process of validating a β‐arrestin‐2 knockout zebrafish line to test its role in vivo in cytoprotection. In summary, our work demonstrates that APC/PAR1 signaling via the β‐arrestin‐2 multi‐functional scaffold segregates into two distinct pathways: 1) β‐arrestin‐2‐Sphk1 activation induces transactivation of S1PR1 to promote Akt‐mediated cell survival and 2) β‐arrestin‐2/Dvl2‐Rac1 activation promotes endothelial barrier protection.Support or Funding InformationSupported by NIH/ NIGMS R35 GM127121 and NIGMS IRACDA K12 GM068524.
Lactobacillus reuteri alleviates Glucocorticoid induced Avascular Necrosis of Femoral Head.
Authors: Kang Ho Jun , Schepper Jonathan , Vidal-Franco Monica , Parameswaran Narayanan , McCabe Laura R. 
Published: April 2020

Glucocorticoids (GC) are well‐known anti‐inflammatory drugs that are widely used to manage many diseases including autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease. Chronic GC use can have significant side effects including 1) dysbiosis (an imbalance of gut microbiota composition), 2) increased intestinal permeability which is linked to increased serum endotoxin levels, 3) trabecular bone loss and 4) osteonecrosis (i.e., avascular necrosis of the femur head). Especially, patients with GC‐induced osteonecrosis of femur head suffer from pain and stiffness, resulting in long‐term disability. Our lab and others have shown that the gut (microbiome and barrier function) regulates bone density. Recently, we have also shown that the probiotic Lactobacillus reuteri 6475 (LR) prevented GC‐induced trabecular bone loss in male and female mice. Based on these findings, we examined if LR treatment can prevent GC‐induced osteonecrosis (GCON) of the femur head. Male C57BL/6J (16‐weeks‐old) mice were treated for 8‐weeks with GC (2.5 mg/kg/day, subcutaneously slow release pellet) and supplemented with or without 3.3×108 cfu/ml LR in their drinking water. Femur immunohistochemistry examining von Willebrand factor positive blood vessels revealed that GC‐induced avascularization of the femur head was prevented by LR supplementation. Specifically, compared to controls, GC treatment caused a 50% reduction in the number of blood vessels in the femur head – this was completely prevented by LR treatment. Similarly, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses determined that LR treatment prevented GC‐induced apoptosis of trabecular osteoblast and osteocytes. In addition, LR treatment prevented GC‐induced marrow adiposity. Consistent with these findings, microcomputed tomography indicated that femoral head bone loss was also prevented by LR treatment. Gene expression analysis of the femur head identified that GC‐induction of tumor necrosis factor alpha and Bcl‐2‐associated x protein (pro‐apoptotic) was prevented by LR. Together, these data indicate that probiotic LR supplementation prevents the pathological bone effects of GCs (avascularization, cell apoptosis, adiposity, bone loss) and may be a straightforward cost‐effective way to mitigate avascular necrosis of femoral head.Support or Funding InformationThese studies were supported by funding from the National Institute of Health, grants RO1 DK101050, AT007695 and by Michigan State University Foundation.
Impact of Redox Modification on MAPK Global Substrate Selection
Authors: Hampton Laquaundra , Postiglione Anthony , Keyes Jeremiah , Poole Leslie B. , Newman Robert H. 
Published: April 2020

Mitogen activated protein kinases (MAPKs) are activated by various stimuli, ranging from growth factors to environmental stress factors. However, given this diverse set of stimuli, it is not currently clear how specificity is achieved in MAPK signaling pathways. We are exploring the hypothesis that, by modulating the substrate selectivity of MAPK family members, cells are able to control which “arm” of a branched pathway is activated in response to a given signal. Specifically, we are investigating the impact of redox modification on the global substrate selection of the canonical MAPKs, extracellular regulated kinase 2 (ERK2) and p38α, which play important roles in regulating a variety of cellular outcomes, including cellular migration, differentiation, apoptosis, proliferation and survival. MAPK family members recognize the majority of their substrates via one of two binding surfaces, termed the D‐recognition site (DRS) and the F‐recognition site (FRS). Interestingly, MAPKs contain redox‐sensitive cysteine residues within or in close proximity to their substrate recognition sites. Therefore, to explore the effects of oxidation on MAPK substrate selection, we first investigated the ability of ERK2 and p38α to phosphorylate model DRS and FRS peptide substrates following pre‐treatment of the kinases with various concentrations of H2O2. While their activity toward FRS substrates was not changed by H2O2 pre‐treatment, both ERK2 and p38α exhibited an increase in their relative activity toward the DRS substrate, Sub‐D, at H2O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2O2 leads to substantial changes in Km and, to a lesser extent, kcat for both kinases. Together, these data suggest that redox modification of p38α and ERK2 may alter their ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter the activity of p38α and ERK2 toward some, but not all, of their downstream substrates. To explore this possibility further, we investigated the impact of redox modification on the global substrate selectivity of these MAPKs using functional protein microarrays. Consistent with our hypothesis, phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2‐dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). These findings suggest that redox modification of the cysteines located within the MAPK DRS may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states.Support or Funding InformationThis research was supported by the NIH National Institute of General Medical Sciences through grants SC2GM113784 and SC1GM130545 (to RHN) and R01GM119227 (to LBP). Likewise, LM is supported through a Title III HBGI grant from the U.S. Department of Education.
Late‐In‐Life Treadmill‐Training Improves Autophagy, Protein Clearance, and Contractile Function in Mouse Hearts
Authors: Ly Kellsey , Cho Jae Min , Hansen Michele , Ramous Caroline , Thompson Lauren , Pires Karla Maria , Ferhat Maroua , Whitehead Kevin , Carter Kandis , Buffolo Marcio , Ghosh Rajeshwary , Park Seul-Ki , Boudina Sihem , Symons J. David 
Published: April 2020

Evidence exists that a progressive decline of the cellular quality control mechanism called macroautophagy occurs during the process of primary aging. This leads to the accumulation of protein aggregates (i.e., proteotoxicity) that would otherwise be transported to and degraded in the lysosome. Post‐mitotic cells like cardiomyocytes rely heavily on macroautophagy because of their inability to clear defective proteins via cell division. While voluntary wheel running improves cardiac autophagy, lessens cardiac proteotoxicity, and attenuates myocardial dysfunction in mice with desmin‐related cardiomyopathy (Bhuiyan et al., JCI, 2013), the ability for a physiological intervention to improve these indexes in aged mice is unknown. We hypothesized that late‐in‐life exercise training improves autophagy, protein aggregate clearance, and function that is otherwise dysregulated in hearts from old vs adult mice. As expected, 24‐month old male C57Bl6 mice (old) exhibited repressed autophagosome formation in the heart, myocardial protein aggregate accumulation, systolic and diastolic dysfunction, and reduced exercise capacity vs. 6‐month old (adult) mice (all p< 0.05; n=10 per group) Separate cohorts of 21 month old mice completed a 10‐week progressive resistance / duration treadmill‐running program (old‐ETR) that improved (all p<0.05): (i) body composition; (ii) maximal workload capacity; and (iii) soleus muscle citrate synthase activity vs. age‐matched sedentary mice (old‐SED). Of note, (iv) mRNA and protein expression of autophagy markers indicated trafficking of the autophagosome to the lysosome increased, (v) protein aggregate clearance improved, and (vi) contractile function was enhanced (all p<0.05), in hearts from old‐ETR vs. old‐SED mice. Dietary maneuvers (e.g., caloric restriction, nutraceutical supplementation) and pharmacological interventions (e.g., rapamycin) are reported to elevate myocardial autophagy and mitigate / reverse age‐associated cardiac dysfunction. Here we show the first evidence that a physiological intervention i.e., late‐in‐life exercise training, improves autophagic flux, protein aggregate clearance, and contractile function in hearts from aged mice.Support or Funding InformationKL [APS STRIDE, UU Undergraduate Research Program (UROP)], JMC (UU Research Fellowship), SKP (AHA 17POST33670663), CR (APS UGRF, UROP), LT (UROP), SB (NIDDK R01‐DK‐098646‐01A1, R01‐DK‐099110, AHA 16GRNT30990018), JDS (AHA16GRNT31050004, NIH RO3AGO52848, NIHRO1HL141540).
The novel high‐frequency variant of TRPV3 p.A628T in East Asians showing faster sensitization in response to chemical agonists
Authors: Choi Si Won , Choi Seong Woo , Chae Jeesoo , Yoo Hae Young , Kim Jong-il , Kim Sung Joon 
Published: April 2020

TRPV3, a member of the thermosensitive Ca2+‐permeable TRPV channel subfamily expressed in skin and sensory nerves, is also activated by chemical agonists such as 2‐aminoethyl diphenylborinate (2‐APB). Repetitive stimuli induce sensitization of TRPV3 activation, characterized by the cumulative increase in current amplitude and linearization of current‐voltage relation (I/V curve). Through genomic analysis of various populations, we found non‐rare TRPV3 mutation (p.A628T) in East Asian people with an allele frequency of 0.249 while 0.007 in Caucasian. Slope‐conductance of unitary channel was not different between WT and p.A628T. Whole‐cell patch clamp study of wildtype TRPV3 (WT) and p.A628T overexpressed in HEK293T cells showed similar sensitization by the repetitive increase in temperature from 23 to 37 °C, while slightly higher sensitization to 43 °C in p.A628T. In contrast, the repetitive application of 2‐APB (10 μM) or carvacrol (100 μM) induced faster sensitization in p.A628T than WT. However, 1 μM farnesyl pyrophosphate, an intrinsic lipid metabolite agonist induced similar level of slow activations in WT and p.A628T. In Fura‐2 microspectrofluorimetry, the 2‐APB pulses induced a faster increase of [Ca2+]c in p.A628T than WT. In terms of ionic selectivity of channels, WT and p.A628T showed similar Ca2+ permeability (PCa/PNa) calculated from the reversal potential of I/V curves. Taken together, p.A628T shows faster sensitization to chemical agonists that are reflected as higher [Ca2+]c signaling. Based on the intriguing pharmacological sensitivity, the physiological implications of p.A628T in the East Asian population require further investigation. (Published in Pflugers Archiv (2019) 471:1273–1289)Support or Funding InformationThis work was supported in part by the National Research Foundation of Korea (NRF) Grants funded by the Korean government (NRF‐2018R1A5A2025964 and NRF‐2018R1D1A1B07048998).
A Single Amino Acid in ABCG2 is Essential for Clamping Substrates and Inhibitors into the Binding Pocket
Authors: Gose Tomoka , Shafi Talha , Fukuda Yu , Das Sourav , Wang Yao , Lynch John , McHarg Ailsa Gavan , Shelat Anang , Ford Robert C. , Schuetz John D. 
Published: April 2020

ABCG2 is an ATP‐binding cassette transporter that, by exporting diverse structurally unrelated anticancer drugs, was identified as a strong contributor to cancer cell multidrug resistance. Subsequently, it was revealed to have important physiological roles in restricting the penetration of multiple xeno‐ and endobiotics into cells and playing a protective role in vivo against cytotoxins for susceptible host tissues. Although ligand binding is the obligate initial step in the transport cycle, it is unknown if any one residue has an indispensable role in binding either substrates and/or inhibitors. The ABCG2 structural studies indicate that substrates interact with fewer residues in the binding pocket than do inhibitors. This finding suggests that a single residue that strongly impacts the binding of both substrates and inhibitors (we will refer to as ligands) would not be found.To evaluate ABCG2 ligand binding, we performed a thermal shift assay using isolated plasma membranes. Among the tyrosine kinase inhibitors (ABCG2 ligands) tested, lapatinib showed the strongest stabilization of ABCG2 against thermal denaturation. Structure‐based docking identified potential lapatinib binding residues. With this knowledge as a guide, mutagenesis and biochemical studies (thermal shift, viability, and transport assays), revealed that a single highly conserved phenylalanine residue was critical for binding, not just lapatinib, but all tested substrates and inhibitors. The alanine substitution for phenylalanine resulted in the loss of ABCG2 interaction with all ligands tested. Conversion of the phenylalanine to amino acids with similar planar, aromatic side‐chain rings, i.e., tryptophan or tyrosine, restored binding and transport activity comparable to wildtype ABCG2. Importantly, amino acid substitution at other individual binding pocket residues did not result in a complete loss of ABCG2 binding and function. Structural modeling using ABCG2 (PDB: 6ETI) showed that the π‐π interactions from the phenylalanine of each monomer were capable of mediating the binding of a surprisingly diverse array of structurally unrelated therapeutic substrates and inhibitors. Apparently, this symmetrical π‐π interaction “clamps” the ligand into the binding pocket. From the molecular features of ABCG2 ligands that use the π‐π clamp, along with structural studies, we created a “global” ABCG2 pharmacophore model that will be invaluable in predicting ABCG2 interacting drugs and xenobiotics.These findings have important therapeutic implications by revealing a key structural feature that ABCG2 uses to engage and initiate transport of multiple drugs and xenobiotics.Support or Funding InformationThis work was supported by NIH and by ALSAC.
Persistent Changes in Salt Intake by Estrogen‐Treated Rats after Repeated Episodes of Dietary Sodium Deficiency
Authors: Ehresman Emily , Curtis Kath 
Published: April 2020

AimsPrevious research from our lab and others demonstrated sex differences in sodium intake, though estrogen’s role in these differences is unclear. Further, previous studies analyzing salt intake with repeated episodes of sodium depletion using furosemide showed that stimulated salt intake was similar between males and females; however, there were clear sex differences in sodium intake after rats were no longer sodium depleted. We aimed to further elucidate the long‐term effects of estrogen on sodium intake using repeated episodes on a low salt diet, a more physiological challenge.MethodsOvariectomized rats were placed on two‐week periods of access to regular chow (NaR) or sodium deficient chow (NaD), which we alternated for a total of two exposures to each diet. Estrogen benzoate (EB) or oil injections were given twice weekly throughout the experiment. At the end of each diet period, rats were given a 2‐hour two bottle test (water and 0.5 M NaCl). At the end of the experiment, rats were sacrificed and trunk blood was collected and centrifuged to collect plasma. Plasma protein levels were measured. Uteri were collected and 1 cm segments of the uterine horns were weighed.ResultsWe confirmed the efficacy of EB treatment on body weight, uterine weight, and plasma proteins. As expected, EB‐treated rats maintained body weight throughout the experiment, with transient decreases, while oil‐treated rats gained weight. Both plasma proteins and uterine weights were greatly increased in EB‐treated compared to oil rats. During two‐bottle tests, both EB‐ and oil‐treated rats had similar baseline intakes of 0.5 M NaCl and water. When salt intake was stimulated by dietary sodium deficiency, both EB‐ and oil‐treated rats drank similarly increased amounts of 0.5 M NaCl. Interestingly, EB‐treated rats drank more 0.5 M NaCl and water than oil‐treated rats after returning to regular chow. The period of dietary sodium deficiency again elicited similar intakes of 0.5 M NaCl and water by EB‐ and oil‐treated rats.ConclusionsOur study demonstrated that estrogen did not alter induced salt intake induced by the physiological challenge, dietary sodium deficiency. However, the low salt diet led to persistent changes in behavior, as demonstrated by the continued consumption of salt and water by EB‐treated rats even after lost dietary sodium was replenished.
Lipidomic Profiling of Host‐Pathogen Interactions Using Untargeted and Targeted Lipidomics Approaches in a Sepsis Cell Culture Model
Authors: Amunugama Kaushalya , Ford David A. 
Published: April 2020

Sepsis is a life‐threatening organ dysfunction caused by a dysregulated host response to an infection. When pathogens disseminate into the systemic blood circulation from a localized infection, the body initiates a complex series of immune and biochemical reactions. Neutrophils are the main leukocytes that are recruited to an infection site to eliminate bacteria. In sepsis, systemic neutrophil activation is also evident. One of the main pathologies of sepsis is the damage of the endothelial cell integrity by immune stimuli leading to leaky blood vessels. Although numerous research has been conducted on sepsis, the characteristic heterogeneity of sepsis has challenged the identification of specific biomarkers and treatments. We have designed an in vitro co‐culture model of endothelial cells, human neutrophils and, E.coli to identify lipid biomarkers of sepsis. The three cell types in the model allow us to dissect lipidome of individual cell systems under a cell culture sepsis‐like state. EA.hy926 cells were initially incubated with urosepsis E.Coli strain CFT073 and further incubated after adding neutrophils. We compared the lipidome of co‐culture samples with non‐co‐culture samples to identify altered lipid profiles. In addition, we have performed similar experiments with non‐pathogenic JM109 E.Coli strain. We first used an untargeted lipidomics platform with high‐resolution mass spectrometry in both positive and negative ion modes. Potential lipid biomarkers identified in untargeted lipidomics were furthered confirmed by a targeted lipidomics approach using shotgun lipidomics. In CFT073 co‐cultures, lipids were changed mainly in 4 lipid classes; phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine and triglycerides. Minor changes were observed in sphingomyelin, phosphatidylserine and phosphatidylglycerol classes. Several of the changes in molecular species in each lipid class included significant increases in PE 32:1, PE 34:1, PE 32:2, PC 36:0, PC 40:8 and PC 32:0. Lipid profiles were also altered in non‐pathogenic JM109 co‐cultures. Interestingly, there are marked species variations depending on E.coli strain used in co‐cultures such as PC 32:1, PC 36:1 are only elevated in CFT073 co‐cultures. Collectively, our studies demonstrate that lipid profiles are altered during inflammation initiated by bacterial exposure and subsequent neutrophil activation. We have also shown that pathogenicity of bacteria may lead to distinct alterations in lipidomes. These studies represent initial insights into the complex lipidomic alterations during host‐pathogen interactions and may provide new targets to examine as biomarkers and mediators during in vivo sepsis.Support or Funding InformationR01 GM115553, R01 GM129508, U01 ES026458, U01 ES027697, U01 ES028182