bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Transcription of Na,K‐ATPase by G‐Protein Coupled Receptors: Involvement of Alternative Binding Partners
Authors: Taub Mary , Matlhagela Keikantse , Borsick Maryann
Published: June 2021

Transcription of Na,K‐ATPase alpha and beta subunit genes in renal cells is regulated by signaling initiated via G‐protein coupled receptors. Included amongst these receptors are the receptors for PGE2, the major renal prostaglandin. In the MDCK cell line PGE stimulates transcription of Na,K‐ATPase alpha and beta subunit genes. Transient transfection studies conducted in MDCK cells with a human Na,K‐ATPase β1 subunit promoter/luciferase construct, pHβ1–1141 Luc, indicate the PGE stimulation is mediated by EP1 receptors (coupled to phospholipase C) as well as EP2 and EP4 receptors (coupled to adenylate cyclase). A prostaglandin regulatory element (PGRE) within the β1 subunit promoter (−110 to −92, AGTCCCTGC) is sufficient to elicit a PGE1 stimulation, although an alternative PGRE (TGACCTTC, −445 to −438) is also a site for regulation. In addition other G‐protein coupled receptors (e.g. dopamine) as well as ionic stress mediate regulation through PGREs. Evidence for CREB binding to these PGREs has been obtained. In addition, DNA affinity precipitation, electrophoretic mobility shift, and co‐immunoprecipitation studies indicate that in addition to CBP, Sp1 is an alternative binding partner for CREB. Transducers of regulated CREB activity TORCs) are other possible alternative binding partners in mediating regulation of Na,K‐ATPase transcription through PGREs.
Pleiotropic effects of PHLPP on cell signaling
Authors: Reyes Gloria Ximena , Newton Alexandra
Published: June 2021

The PH domain Leucine Repeat Protein Phosphatases PHLPP1 and PHLPP2 were discovered in a directed search for phosphatases that directly dephosphorylate and inactivate Akt, a key controller of many cellular processes such as cell survival, cell death, proliferation, and motility. Given the relative paucity of phosphatases compared to the abundance of phosphorylated sequences in cells, we asked whether the PHLPP phosphatases controlled additional signaling networks to the Akt pathway. To this end, we used siRNA to deplete PHLPPs from diverse cell lines and analyzed substrates phosphorylated downstream of growth factors such as EGF, PDGF, insulin and TGF‐beta. We found that genetic depletion of PHLPP isoforms dramatically increased agonist‐evoked signaling by each of these agonists. The effects of PHLPP knockdown depended on the tumorigenic state of the cell lines and the growth factor used. Importantly, PHLPP knockdown affected not only Akt phosphorylation, but also the phosphorylation of substrates which are modulated by distinct pathways from Akt, such as those involving the MAPK and SMAD proteins. These findings reveal differential and/or synergistic contributions of PHLPP isoforms to suppressing signaling in numerous cellular pathways, revealing an important pleiotropic role of these phosphatases on cell signaling.

This work was funded by NIH GM NIH 067946
Differences in the dimerization properties mediated by the transmembrane domains of Zea mays and Arabidopsis CRINKLY4 AND CRINKLY4‐like receptor kinases
Authors: Rao Gururaj A , Stokes Kevin D
Published: June 2021

CRINKLY4 is a transmembrane (TM) containing serine/threonine receptor kinase that functions in the differentiation of the leaf epidermis and the aleurone cell layer in Z. mays (ZmCR4) and development of the integument and seed coat in Arabidopsis (AtCR4). In addition, the Arabidopsis genome also encodes four CRINKLY4 homologs whose functions are not known. Based on studies of animal receptor kinase proteins, it is likely that CRINKLY4, and its homologs, also function as dimers. The importance of the TM domain in the dimerization of several receptor kinases has been demonstrated by the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment. In this study, we have used the TOXCAT assay to investigate the potential of the CRINKLY4 and CRINKLY4‐like TM domains to homodimerize. Our investigation indicates that the ZmCR4 TM domain and the Arabidopsis homologs have similar homodimerization potentials. In contrast, the dimerization potential of the AtCR4 TM domain is significantly weaker, which suggests that the functional mechanisms of ZmCR4 and AtCR4 may be different. Interestingly, 13 of 24 amino acids are identical between the ZmCR4 and AtCR4 TM domains. We also report on studies aimed at delineating the contribution of specific amino acids to differences in the dimerization potential of CRINKLY4 TM domains of the two organisms.
Murine proteine serine/threonine kinase 38 (MPK38) positively regulates apoptosis signal‐regulating kinase 1 in a kinase‐dependent manner through physical interaction
Authors: Ha Hyunjung , Jung Haiyoung , Seong Hyun‐A.
Published: June 2021

Murine protein serine/threonine kinase 38 (MPK38)/maternal embryonic leucine zipper kinase (Melk) is a member of the AMP‐activated protein kinase (AMPK)‐related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self‐renewal of stem cells. Here we show a functional association between MPK38 and apoptosis signal‐regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 is mediated through their C‐terminal regulatory domains. The use of kinase‐dead MPK38 and ASK1 mutants reveals that the complex formation is dependent on the activities of both kinases. Ectopic expression of wild‐type MPK38, but not kinase‐dead MPK38, stimulates ASK1 activity by phosphorylating ASK1 on threonine 838 and enhances ASK1‐mediated signaling to both JNK and p38 kinases. In addition, MPK38‐mediated activation of ASK1 is induced through the dissociation of 14‐3‐3 from ASK1‐14‐3‐3 complex and the increase of interaction between ASK1 and its substrate MKK3. We also demonstrate that MPK38 stimulates ASK1‐mediated apoptosis by upregulating the ASK1‐induced capase‐3 activity. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive regulator of ASK1.
The role of MEK in TBT‐induced activation of p44/42 in human natural killer cells
Authors: Abraha Abraham B , Whalen Margaret M
Published: June 2021

Natural killer cells are lymphocytes capable of killing tumor cells, virally infected cells and antibody‐coated cells. Tributyltin (TBT) is a toxic chemical that was used in large scale in wood preservation, marine antifouling paints, and slime control in paper mills. TBT has been detected in human foods such as fish and detectable levels have been found in human blood. Studies have shown that tributyltin (TBT) can significantly reduce the cytotoxic function of the human NK cells with accompanying changes in the phosphorylation (activation) state of the mitogen activated protein kinase (MAPK), p44/42. The current studies are designed to examine the role of MAPK pathway activation in the TBT‐induced loss of NK cytotoxic function, by using MAPK kinase (MEK) inhibitors. MAPK pathway activation was inhibited by compounds that specifically block the activation of MEK. Several MEK inhibitors were examined. There was about a 30% decrease in NK cell cytotoxic function when NK cells were exposed for 1h to the MEK inhibitor, PD98059. Western blot analysis using an antibody to phospo‐p44/42 showed that PD98059 was able to completely block TBT‐induced activation of p44/42. These results indicate that activation of p44/42 pathway is needed for the cytotoxic function of freshly isolated human NK cells and that TBT‐induced activation of p44/42 occurs via the activation of its upstream activator, MEK.
Activation of p44/42 by Phorbol 12‐Myristate 13‐Acetate (PMA) and Tributyltin (TBT) in Human Natural killer (NK) Cells Results in Loss of Cytotoxic Function
Authors: Dudimah Fred Duafalia , Whalen Margaret
Published: June 2021

Natural Killer (NK) cells destroy (lyse) tumor cells, virally infected cells and antibody‐coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates the MAPK, p44/42. If activation of p44/42 is required for TBT‐induced decreases of lytic function, then pharmacological agents such as Phorbol 12‐myristate 13‐acetate (PMA) that activate p44/42 should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 100 and 0.25 nM for 10 min and 1 h before determining the lytic function (51Cr release assay) and phosphorylation state of MAPKs (Western blot). 1h exposure of NK cells to PMA (100‐10 nM) resulted in significant loss of lytic function (between 66‐58%). Additionally, a 1 h exposure to TBT (500‐200 nM) caused decreases in lytic function of 96‐61%. Western blot analysis showed that a 10 min exposure to 2.5 nM PMA caused a 10 fold increase in phospho(P)‐p44/42 levels. Previous studies showed an 8.8 fold increase in P‐p44/42 in response to a 10 min exposure to 300 nM TBT. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) precedes a loss of NK‐cell lytic function. Supported by NIH grant 2S06GM08092‐28.
Ligand‐induced activation of preformed inactive EGF/ErbB receptor homo‐ and heterodimers: a model for EGF/ErbB receptors
Authors: Tao Rong‐Hua , Maruyama Ichi N.
Published: June 2021

The epidermal growth factor receptor (EGFR) family, also known as ErbB or HER, mediates a variety of cellular responses in normal biological processes and in pathological states. It is widely considered that ErbB receptors are located on the cell membrane, and that ligand binding to the monomeric form of the receptors induces its dimeric form for activation. However, using bimolecular fluorescence complementation assay in the present study, we demonstrate that in the absence of bound ligand, the four ErbB family members have preformed homo‐ and heterodimers on living cell surface, while ErbB3 homodimers and heterodimers with ErbB4 exist primarily in the nuclei. Binding of ligands such as EGF and HRGbeta1 had no effect on the amount of preformed dimers, but activated the preformed inactive dimers and their downstream molecules, and also induced the preformed dimers endocytosis. These ligand‐independent dimerizations of ErbB receptors occurred via interactions between their intracellular domains. Together, the results indicate that ErbB family receptors may preform inactive dimers before ligand binding. These findings provide new insights into an understanding of a molecular mechanism by which the inactive receptors switch into active form, and can explain at the molecular level for the activated EGFR mutants found in lung and breast cancers, and hence are relevant to the development of anti‐cancer drugs.
Profiling Kinase Pathway Activation and Inhibitor Activity Using a Novel Antibody Array
Authors: Radding Jeffrey Alan , Rauh‐Adelmann Christine , Hall Amy B. , Murphy Cheryl , Yen Lucy G. , Graham James R. , Nadler Timothy K. , Gordon Neal F.
Published: June 2021

Broad screening of protein phosphorylation has wide‐ranging utility for understanding the impact of disease or drug treatment on cell signaling pathways. To address this need, we have developed an antibody array for measuring tyrosine signaling across 45 phosphotyrosine sites across multiple signaling pathways (Ti‐Tyr ™ Profiling Chip). The approach utilizes a novel strategy of measuring peptide fragments liberated by protease digestion of the sample prior to analysis, and allows for both multi‐site profiling and absolute quantification using synthetic peptide standards. The utility of the Ti‐Tyr™ chip is demonstrated by measuring specific modulation of pathway phosphorylation in stimulated cells in the presence and absence of distinct kinase inhibitors. Phosphorylation changes detected in chip targets correlate with known biological effects using comparable conditions. The use of peptide standards on the chip allows for an absolute quantitative assessment of the same stimulation conditions or inhibitor treatments across different cell lines, for example EGF stimulation in A431 and PC‐3 cancer lines. The Ti‐Tyr ™ Tyrosine Chip should improve the ability to understand the effects of cell stimuli, the underlying basis of disease and the impact of drug treatments across multiple cell pathways and cell types.
Investigating the role of the C‐terminal domain in McsB
Authors: Graham James , Fraga Dean
Published: June 2021

McsB is a recently described prokaryotic protein tyrosine kinase found in Bacillus subtilis. In addition to an autophosphorylation activity, McsB can be further induced to phosphorylate other targets upon binding a second protein, McsA, encoded in the same operon as McsB. McsB is a structurally unique tyrosine kinase in that its N‐terminus corresponds to a guanidine‐phosphotransferase domain and is critical for kinase activity. However, the adjacent C‐terminal domain is not homologous to other protein domains and its role in McsB protein kinase activity is unknown. We used a series of deletion mutants to determine what potions of the C‐terminal domain were important for kinase activity and/or its regulation by McsA. Our hypothesis was that the C‐terminal domain regulated activity by either blocking access to the active site by acting as a pseudo substrate, or by holding the enzyme in a conformationally inactive state until bound by McsA. Preliminary results indicate that the deletion mutations greatly reduce or abolish the inducible enzymatic activity thus supporting a model that this region has an important role to play in the inducible catalytic activity but not in autophosphorylation activty.
Monitoring the effect of glyoxal and methylglyoxal glycation on the secondary structure of human serum albumin: an analysis based on CD, ESI‐MALDI‐TOF/MS, thermal melting profiles and UV fluorescence spectroscopy
Authors: Li Yuyuan , Cohenford Menashi A , Dutta Udayan , Dain Joel A
Published: June 2021

Glyoxal and methylglyoxal (MG) are reactive dicarbonyl compounds, which modify amino groups of proteins to form advanced glycation endproducts (AGEs), that are involved in diabetes complications. The objective of this investigation was to determine the effects of nonenzymatic glycation of glyoxal and MG on the secondary structure of human serum albumin (HSA) and to evaluate the factors that influence the modification of HSA with glyoxal and MG. The formation of AGEs was monitored by HPLC, MALDI‐TOF/MS, UV, and fluorescence spectroscopy. The change in the secondary structure of HSA was determined by circular dichroism (CD) and thermal melting (Tm) profiles of the native and glycated proteins. UV and fluorescence demonstrate that the extent of AGE formation was dependent on both glyoxal and MG concentration and incubation time. The CD profiles demonstrated that HSA was a‐helical in structure, and low concentrations (2 mM) of glyoxal or methylglyoxal destabilized HSA while high concentrations (40 mM) preserved and stabilized the a‐helical structure of the protein. This was confirmed by thermal melting profiles. Finally, MALDI‐TOF/MS was successfully applied for site‐ and product‐specific relative quantification of AGEs. Supported in part by NCRR/NIH Grant # P20 RR16457.
Protein Aggregation‐Suppression: Molecular Clusters of Arginine are responsible
Authors: Das Utpal , Srinivasan Alagiri
Published: June 2021

Aggregation of unfolded proteins occurs through the exposed hydrophobic surfaces. Any mechanism of aggregation‐supression should explain these hydrophobic interactions. The mechanism of action of arginine as aggregation‐suppressor is not understood. We propose a mechanism based on the hydrophobic interactions of arginine.

Arginine solutions display hydrotropic effect on pyrene. ANS fluorescence studies indicate the presence of hydrophobic environment. Mass spectroscopic analyses and light scattering studies indicate that arginine exists as clusters in the gas phase and in solution. Arginine changes the RPC elution profile and the hydrodynamic volume of Alzheimer's amyloid beta 1–42 (Aβ1–42). Arginine increases the solubility of Aβ1–42 peptide and decreases the aggregation.

Our results show that molecular clusters of arginine in solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces of the proteins and the arginine clusters interact. The masking of hydrophobic surface inhibits protein‐protein aggregation. This study was supported by the Department of Biotechnology, Government of India.
Differentiating TCA and SSRI binding to ionotropic glutamate receptors
Authors: Raabe Richard Carl , Telsey Benjamin Samuel , Yankowski Christie , Stoll Laura , Hall James , Gentile Lisa Nancy
Published: June 2021

Approximately 20.9 million Americans age 18 and older, roughly 9.5 percent of the U.S. adult population, have mood disorders, which range from mild depression to severe bipolar disorder. With 10% of women and 4% of men taking antidepressants, it's imperative to understand their mechanism of action. Clinical antidepressants fall into three main categories: selective serotonin reuptake inhibitors (SSRIs), selective norepinephrine reuptake inhibitors (SNRIs), and tricyclic antidepressants (TCAs). Our research is focused on the binding of antidepressants to ionotropic glutamate receptors (iGluRs). iGluRs are a family (AMPA, kainate, NMDA) of ligand gated ion channels with three transmembrane spanning domains, a re‐entrant loop, an amino terminal domain (ATD), and an extracellular S1S2 domain. A combination of fluorescence quenching studies, Stern‐Volmer analyses, and protease protection assays will be presented that differentiate the binding of each SSRI and TCA to the S1S2 domain of the GluR2 subunit of the AMPA receptor and the NR1‐1b subunit of the NMDA receptor. This data provides further support for a role of the glutamatergic system in antidepressant activity.
Discovering the substrates of β‐1,4‐galactosyltransferase‐1 by use of unnatural UDP‐galactose analogs
Authors: Bond Michelle , Kohler Jennifer
Published: June 2021

The genome encodes seven galactosyltransferases capable of adding galactose in a β1,4 linkage to terminal GlcNAc; however, they are not interchangeable. Despite the fact that in vitro assays of the enzymes do not reveal specificity differences among them, β1,4 galactosyltransferase family members have different biological roles. β1,4‐galactosyltransferase‐1 has been recognized as both an important Golgi‐resident enzyme and as a lectin. Our goal is to identify the cellular substrates of β1,4‐galactosyltransferase‐1 and thereby determine the molecular basis of its biological roles.

To identify the biological substrates of β1,4galactosyltransferase‐1, we are re‐engineering the enzyme to accept an orthogonal and labeled UDP‐galactose analog. We predict that the mutant, but not the wild‐type, enzyme will accept the UDP‐Gal analog. Modified β1,4galactosyltransferase‐1 can then be introduced in place of the wild type enzyme. In this way, we will be able to directly label the substrates of a single β1,4‐galactosyltransferase family member. More generally, we expect that similar methods could be used to decipher the substrates of the over 100 glycosyltransferases identified in the human genome.

Sources of support: NSF Graduate Fellowship, March of Dimes
The presence of an alpha glucomannan polysaccharide (IF‐S) activates NFkB and p38 in hMDDC
Authors: Majano Pedro L , Martin‐Vilchez Samuel , Jimenez Francisca Molina , Alonso‐Lebrero Jose L , Sanz‐Cameno Paloma , Rodriguez Yolanda , Roda‐Navarro Pedro , Trapero Maria , Aragoneses‐Fenoll Laura , Gonzalez Salvador , Lopez‐Cabrera Manuel , Moreno‐Otero Ricardo
Published: June 2021

DC maturation is dependent on NF‐kB activation, which ultimately determines most of the phenotypic and functional parameters associated with this process. We investigated the effects of IF‐S on hMDDC molecular pathway. CD14+ cells were purified from PBMC by positive selection. The CD14+ cells were cultured in presence of GM‐CSF and IL‐4. hMDDC were stimulated with IF‐S. NF‐kB was analyzed by immunofluororescence. Protein levels measurements were done by Western blot using specific antibodies against ERK 1/2, p38, P–ERK 1/2, and P‐p38 and IkBb. TLR activity by luciferase‐based reporter methods using HEK293‐TLR2 and HEK293‐TLR4 cells. The treatment of MDDC with IF‐S induces NF‐kB expression. IF‐S treatment induces a rapid and transient reduction of IkB‐a level, which began to recover after 2 h. In contrast, LPS‐induced downregulation of IkB‐a was not reverted until 24 h post‐treatment. IF‐S induces the phosphorylation of p38 MAPK in a time‐dependent manner and to a similar extent as LPS. Whereas LPS induced a strong phosphorylation of ERK, IF‐S treatment induces only a mild ERK phosphorylation. The results showed that TLR4, but nor TLR2 expressing cells, were capable of activating NF‐kB in response to IF‐S. IF‐S triggeres nuclear translocation of NF‐kB, induces a transient degradation of IkB‐a and phosphorylation of p38 MAPK. In addition, IF‐S promotes NF‐kB activation in a TLR‐4 dependent manner.
Inhibition of Lysozyme Fibrillation by Natural and Synthetic Melanins
Authors: Holmes Earle W. , Thompson Kenneth D
Published: June 2021

Amyloidogenesis, a multistep process in which protein mis‐folding results in the formation of aggregates with cross beta sheet structures, is associated with more than 20 diseases including diabetes, AL amyloidosis, Alzheimer's disease, and prion diseases. Hence, there is an ongoing interest in the development of drugs that inhibit amyloid formation or deposition. In this study, we tested the ability of natural and synthetic polyketides to inhibit lysozyme fibrillation in vitro. Solutions of lysozyme (Hen egg, 20 mg/mL) were incubated at 58°C in 20 mM glycine HCl buffer at 58°C for 8 days in the presence of one of the following compounds (concentration tested): 1. Kraft lignin (125 μg/mL), 2. sulfonated Kraft lignin (190 μg/mL), 3. allomelanin extracted from Echinacea purpurea (210 μg/mL), 4. ES‐EM (166 μg/mL), 5. ES‐CAM (190 μg/mL), 6. C11 (130 μg/mL), or 7. C12 (150 μg/mL). Compounds 4–7 are synthetic melanins obtained From BioActive Polymers.com, (La Grange Park, Illinois, 60526). The incubation mixtures were sampled repeatedly and the kinetics of lysozyme fibrillation were evaluated by Thioflavin T fluorescence, native PAGE, and fluorescence and dark field microscopy. Compounds 6, 4, 3, and 7 inhibited the development of THT fluorescence to levels that were 16%, 42%, 52%, and 61%, respectively of the untreated control mixtures. PAGE analysis of the time points taken during the incubation containing C11 showed that this compound decreased the generation of lysozyme oligomers during the first 48 hours of incubation and delayed the progression of the assembly of lysozyme monomers and oligomers into lysozyme fibers and aggregates. The data suggests that natural and synthetic melanins may be of therapeutic value as fibrillogenesis inhibitors.

[Supported in part by a gift from the Charles and Lee Finkl Amyloidosis Foundation]
Evaluating Anti‐Androgen Resistance by In‐Vitro Selection
Authors: Bourreza Aly
Published: June 2021

Anti‐androgens, such as flutamide and bicalutamide (Casodex®), used alone or in conjunction with chemical castration, have been used in the treatment of prostate cancer (PCa) for decades. However, as many as 30%–40% of patients treated with anti‐androgens acquire a therapy‐resistant phenotype after one to three years of treatment. Furthermore, some patients experience clinical improvement upon cessation of anti‐androgens, a condition known as anti‐androgen withdrawal syndrome. Anti‐androgen withdrawal syndrome has been associated with mutations to the AR that cause an agonist response to anti‐androgens and is one of the most difficult forms of PCa to treat. The goal of this project is to redesign anti‐androgens to evade molecular mechanisms that lead to anti‐androgen withdrawal syndrome. To accomplish this, novel analogs, PLM1 and PLM6, were developed that remain antagonists towards three AR mutants associated with anti‐androgen withdrawal syndrome. In this study, long‐term growth analyses of the human LNCaP cell line in the presence of PLM1, PLM6, or bicalutamide have been performed to identify potential AR mutants that would represent an anti‐androgen withdrawal phenotype. After approximately 4–6 weeks, resistant colonies developed and were selected. DNA sequence data of the resistant colonies showed various AR mutations. PLM6 proved to be superior to PLM1 as fewer resistant colonies were observed compared to bicalutamide and PLM1. This suggests that PLM6 may be effective against this form of resistant PCa. This work is supported by the Howard Hughes Medical Institute Undergraduate Science Education program and the National Institutes of Health, NIDDK; 3‐R01‐DK054257‐09.
Granule polarization is the preferred target for inhibition by NK cell inhibitory receptors.
Authors: Das Asmita , Long Eric
Published: June 2021

NK cells kill target cells by polarized release of cytotoxic granules. Granule polarization and degranulation are controlled by different signals in NK cells. The integrin LFA‐1 signals for polarization but not degranulation, whereas the Fc receptor CD16 induces degranulation but not polarization. Lysis of target cells by NK cells is controlled by inhibitory receptors specific for MHC class I. To test the sensitivity to inhibition of polarization and degranulation separately, NK cells were mixed with insect cells expressing ICAM‐1 (ligand of LFA‐1) and MHC class I in the presence or absence of rabbit IgG (ligand of CD16) bound to the insect cells. Engagement of inhibitory receptors by their MHC ligands completely blocked polarization but not degranulation. Similar results were obtained during natural cytotoxicity (independent of CD16) against human K562 cells transfected with MHC class I. Thus, either degranulation is not sensitive to inhibitory signals or inhibition of degranulation requires additional receptor – ligand interactions. Inhibition of degranulation occurred with the human target cell 221 transfected with MHC class I. These data demonstrate that (1) granule polarization is a preferred target of inhibitory signals, and (2) as yet unknown receptor – ligand interactions contribute to inhibition of degranulation.
Epithelial γδ T cell‐specific costimulation mediated by JAML‐CAR interactions
Authors: Witherden Deborah A , Verdino Petra , Rieder Stephanie E , Haynes Matthew R , Mills Robyn E , Teyton Luc , Fischer Wolfgang , Wilson Ian A , Havran Wendy L
Published: June 2021

γδ T cells in murine skin (DETC) represent a prototypical γδ intraepithelial lymphocyte population. They express an invariant γδ T cell receptor, Vγ3Vδ1, and play important roles as effectors and regulators of homeostasis through recognition of antigen expressed on damaged or diseased keratinocytes. DETC do not express many coreceptors important for αβ T cell activation. To date, no γδ‐specific accessory molecules have been described. Our recent focus has been to identify molecules on γδ T cells that play a key role in their activation and function. We have identified the first γδ T cell‐specific costimulatory molecule, Junctional Adhesion Molecule‐Like protein (JAML) and identify Coxsackie and Adenovirus Receptor (CAR) as its ligand. Only costimulation of epithelial γδ T cells occurs through JAML, αβ‐and lymphoid γδ T cells are unresponsive. JAML costimulation leads to cell proliferation and cytokine production through activation of MAP kinase pathways. Our results demonstrate a novel receptor‐ligand pair specific for epithelial γδ T cell function. In addition, the crystal structure of JAML reveals a unique Ig domain prototype that undergoes conformational changes upon ligand binding, suggesting a mechanism for initiating signaling through JAML. Experiments are underway to establish the role of JAML‐CAR interactions in epithelial γδ T cell responses during wound repair and other epithelial challenges.

This work was supported by NIH grants AI32751 and AI52257 (W.L.H.), AI042266 and CA58896 (I.A.W.), an Erwin‐Schrödinger Fellowship of the Austrian Science Fund (P.V.) and The Leukemia and Lymphoma Society (S.E.R.).
IFNbeta induces mature DC apoptosis through the caspase 11/caspase 3 pathway
Authors: Yen Jui‐Hung , Ganea Doina
Published: June 2021

IFNβ has been used in the treatment of multiple sclerosis for more than a decade, although the molecular mechanisms are still not clear. In this study, we report for the first time that IFNβ induces apoptosis in dendritic cells (DC) matured with a cytokine cocktail (TNFα+IL‐1+IL‐6+PGE2). In addition, LPS induces IFNβ during DC maturation and leads to DC apoptosis. LPS induced DC apoptosis is abolished in the presence of neutralizing IFNβ antibodies, indicating the role of IFNβ in the apoptosis of mature DC. IFNβ‐induced apoptosis is mediated through induction of caspase‐11 and subsequent activation of caspase 3. DC matured with LPS or cytokine cocktail + IFNβ express caspase 11 mRNA and are positive for activated caspase 3. STAT‐1 signaling is essential for IFNβ induced apoptosis, since DC from STAT‐1 deficient mice are resistant to IFNβ‐induced apoptosis, and do not express caspase 11 or activated caspase 3. Caspase 11 promoter contains STAT‐1 and NFκB binding sites which are essential for caspase 11 expression. We report that treatment with the cytokine cocktail+IFNβ or with LPS induces increased levels of NFkB p65 and STAT‐1 phosphorylation. We propose that signaling through both NFκB and STAT‐1 leads to caspase 11 expression and caspase 3 activation in mature DC, resulting in apoptosis and subsequent downregulation of the inflammatory process.

This work is support by NIH 2RO1AI052306.
Feedback inhibition of indoleamine 2,3‐dioxygenase by BLIMP‐1 in response to tryptophan depletion
Authors: Barnes Nicholas , Stephenson Sophie , Tooze Reuben , Doody Gina
Published: June 2021

BLIMP‐1 protein expression is up‐regulated in the myeloid cell lineage during differentiation and, as previously shown by us, as part of the unfolded protein response downstream of endoplasmic reticulum stress and PERK activation. We hypothesized that BLIMP‐1 would also be implicated in the signaling pathways activated in response to alternative cellular stresses. Indoleamine 2,3‐dioxygenase (IDO) is an immunoregulatory enzyme that depletes tryptophan from the surrounding tissue microenvironment, thereby inducing amino acid starvation and activation of the stress kinase GCN2. Using transfection studies and tryptophan‐depleted media we found that PRDM1, the gene encoding BLIMP‐1 protein, was induced, and that a pharmacologic activator of the GCN2 response caused an up‐regulation of BLIMP‐1 protein in the monocytic U937 cell line. Furthermore, BLIMP‐1 is a transcriptional repressor and has two potential consensus target sites in the IDO promoter. Analysis of BLIMP‐1 binding to these regions of the IDO promoter indicates that BLIMP‐1 is not only capable of binding here, but that BLIMP‐1 also inhibits IFNγ‐induced up‐regulation of the IDO gene. We propose that BLIMP‐1 may function in a negative feedback role to regulate IDO levels in cell types that express these two proteins, thereby maintaining the expression of IDO at a level that successfully balances the outcome of tolerance versus inflammation.
A2A adenosine receptor stimulation enhances arginase I expression in macrophages resulting in a phenotypically unique macrophage
Authors: Black Steven G , Wilson Jeffrey M , Ernst Peter B , Smith Michael F
Published: June 2021

Background. Macrophages traditionally are grouped into two distinct categories, classically (exposed to LPS or Th1 cytokines) or alternatively (exposed to Th2 cytokines) activated macrophages. Adenosine may also modulate macrophage function through G‐protein‐coupled receptors A1, A2A, A2B, and A3. Methods. To understand the effects of adenosine on macrophage function, LPS stimulated macrophages were treated with an agonist for the A2A receptor (ATL 313). Real time PCR, enzymatic assays, and ELISAs were performed. Results. ATL 313 treatment of LPS stimulated macrophages resulted in a dramatic increase in arginase I expression and enzymatic activity, hallmarks of an alternatively activated macrophage. Expression of other markers of alternatively activated macrophages (Fizz‐1 and YM) were absent in ATL 313 treated cells. In addition, LPS induced expression and activity of iNOS in macrophages were not affected by ATL 313. Conclusion. We propose stimulation of the A2A receptor increases arginase activity, thus altering arginine metabolism resulting in increased production of polyamines and proline. This stimulation results in a unique macrophage phenotype that may promote cellular proliferation and wound healing and inhibit T lymphocyte responses. Supported by NIH AI070491.
Use of Mesenchymal Stem Cells in the Regulation of Inflammation: Novel Concept for Future Therapies in the Lung
Authors: Rojas Mauricio , Xu Jianguo , Iyer Smita , Neujahr David , Brigham Kenneth , Mora Ana L
Published: June 2021

Bone marrow‐derived mesenchymal stem cells (BMDMSCs) appear to be important in the prevention of lung damage after acute and chronic inflammatory process; however the mechanisms in which these cells protect are unknown. Using an endotoxin‐induced Acute Lung Injury (ALI) model and a heterotopic trachea transplant model we demonstrate the ability of BMDMSC to regulate the immune response during acute and chronic inflammatory lung diseases respectively. First, we administrated BMDMSC in endotoxin induced ALI model in mice and pigs. We observed that BMDMSC suppressed the upregulation of pro‐inflammatory cytokines preventing inflammation and edema in the lung. The clinical relevance of these studies is shown by the observed association between the ability to induce mobilization of bone marrow derived cells and the survival of patients with ALI. Second, we studied the effect of BMDSC administration during heterotopic trachea transplant as a model of chronic transplant rejection. Lung transplantation is a viable treatment option for end‐stage pulmonary diseases, but development of obliterative bronchiolitis (OB), reduces survival, accounting for 30% of deaths after the third year. In this model of chronic inflammation, the systemic administration of BMDMSC prevents the development of OB. We were able to demonstrate the total inhibition of inflammation and fibrosis when the mice received a single dose of BMDMSC independent of the strain of mice from where the cells were obtained. To demonstrate the mechanism used by BMDMSC we realize co‐culture experiments and affimetrix analysis. These studies reveled that BMDMSC have the capacity to induce T‐regulatory cells and to produce soluble factors like TGFβ and sIL1‐R that mediate the suppression of the inflammatory immune response.
Mast Cell Deficiency Attenuates Atherosclerosis in Apolipoprotein E‐deficient mice
Authors: Smith Donald D. , Tan Xiaoyu , Tawfik Ossama , Stechschulte Daniel J. , Dileepan Kottarappat N.
Published: June 2021

Mast cells (MC) are important cells of the immune system and are recognized as participants in the progression of atherosclerosis. To test MC's role in atherosclerosis in vivo, apolipoprotein E‐deficient (ApoE−/−) mice were crossed with MC‐deficient KitW‐sh/W–sh mice to obtain an apoE−/−/KitW‐sh/W–sh double knockout mouse model. To induce atherosclerosis, ApoE−/−, KitW‐sh/W–sh, and ApoE−/−/KitW‐sh/W–sh mice were maintained on a Western Diet for 80 to 90 days. The mice were then killed, blood was collected, and the aorta was perfused in situ first with PBS and then with buffered 10% formalin and processed for cross‐sectional analyses of plaque size in the arch or thoracic region, or processed for en face analyses for plaques after Sudan III staining. H and E slides were digitally scanned on ScanScope XT system and images were evaluated for plaques thickness. Plaque thickness in aortic cross sections were significantly different between ApoE−/− (203μm, n=6) and ApoE−/−/KitW‐sh/W–sh mouse (120μm, n=7) with no lesions in the KitW‐sh/W–sh mouse. The en face analyses of aortas also showed reduced atherosclerotic area in ApoE−/−/KitW‐sh/W–sh mice with no aortic plaque formation in KitW‐sh/W–sh mice. MCs with indications of degranulation were seen only in the aortic walls of ApoE−/− mice. In comparison to ApoE−/− mice, the cholesterol levels were 50% lower in apoE−/−/KitW‐sh/W–sh mice while no appreciable difference in triglycerides were noted. Results demonstrate the involvement of MC in the progression of atherosclerosis. The mechanisms by which MC enhance atherosclerotic plaque development remain to be elucidated. (Supported by NIH R01‐HL070101 and Carey Arthritis Fund)
Dendritic Epidermal T Cells Promote Wound Healing by Production of Vascular Endothelial Growth Factor Mediated by HIF‐1α Signaling
Authors: Kim Chang‐Hoon , Witherden Deborah A , Havran Wendy L
Published: June 2021

Local hypoxia is general consequence of an acute skin wound due to vascular disruption and high oxygen consumption by cells at the edge of the wound. The resident γδ T cells in the skin, Dendritic Epidermal T Cells (DETC), are known to support local tissue homeostasis and wound repair by local production of growth factors, such as keratinocyte growth factors (KGFs) and insulin‐like growth factor (IGF)‐1. We have analyzed the functional role of DETC in response to local tissue hypoxia generated by an acute skin wound. The hypoxic status of the skin wound induced HIF‐1α expression in DETC. HIF‐1α signalling during hypoxia induced expression of vascular endothelial growth factor (VEGF) by DETC. Wound healing was impaired after HIF‐1α gene‐silencing in DETC and this impairment was rescued by VEGF administration. Together this data demonstrates that acute skin wound induces HIF‐1α signaling in DETC, which promote VEGF production with the result of enhancing skin wound healing.

Grant: NIH‐AI‐36964
Adenosine A2B receptors in regulation of dendritic cell differentiation and function
Authors: Ryzhov Sergey , Novitskiy Sergey V. , Zaynagetdinov Rinat , Goldstein Anna E. , Biaggioni Italo , Carbone David P. , Feoktistov Igor , Dikov Mikhail M.
Published: June 2021

Differentiation of functional dendritic cell (DC) crucially depends on the microenvironment. DCs differentiate in hypoxic tumor sites, and inflamed or damaged tissue. Because local concentrations of adenosine reach high physiologically relevant levels in these conditions, we assessed the expression of adenosine receptors and their activation on differentiation of human monocytes and mouse hematopoietic progenitor cells (HPCs) into myeloid DCs. Stimulation of adenosine receptors skews differentiation of DCs toward a distinct cell population characterized by expression of both DC and monocyte/macrophage cell surface markers. Pharmacological analysis and experiments with HPCs from A2B adenosine receptor knockout mice identified A2B receptor as the mediator of adenosine actions on DC differentiation. Unlike myeloid DCs, adenosine‐differentiated DCs have impaired allostimulatory activity and produce high levels of angiogenic, pro‐inflammatory, and immune suppressor/tolerogenic factors. Adenosine‐differentiated DCs promoted tumor growth if injected into Lewis lung carcinoma tumors implanted in mice. We conclude that adenosine signaling through A2B receptor is an important factor of aberrant DC differentiation into tolerogenic, angiogenic and pro‐inflammatory cells. Supported by NIH grants.
Human follicular dendritic cells interact with T cells via expression and regulation of cyclooxygenases and prostaglandin E and I synthases
Authors: Choe Jongseon , Lee In Yong , Cho Whajung , Kangwon Jini Kim.
Published: June 2021

Prostaglandin E2 (PGE2) inhibits mature T cell proliferation and protects T cells from activation‐induced cell death (AICD). We have previously demonstrated that human follicular dendritic cells (FDC) strongly express prostaglandin I synthase (PGIS). In this study, the hypothesis that FDC have regulatory roles on germinal center (GC) T cells by controlling production of PGE2 and PGI2 was tested. Confocal microscopic analyses of human tonsil tissues revealed that FDC indeed expressed PGE synthase (PGES) in addition to PGIS. To confirm these results, we studied the regulation mechanism of prostaglandin production in FDC, utilizing an established human FDC‐like cell line, HK. Specifically in response to TNF‐α, TGF‐β, and LPS, protein expression of cyclooxygenase (COX)‐2 and downstream PGES was up‐regulated with coordinate kinetics while COX‐1 and PGIS were constitutively expressed. The increase of these enzymes was reflected in actual production of PGE2 and PGI2. Furthermore, the up‐regulation of PGE2 and PGI2 production was markedly down‐regulated by indomethacin and a selective COX‐2 inhibitor. PGI2 analog and PGE2 inhibited proliferation and AICD of T cells in dose‐ and time‐dependent manners. Finally, co‐culture experiments revealed that HK cells really inhibit proliferation and AICD of T cells. Put together, these results show an unrecognized pathway of FDC and T cell interactions and differential mechanisms for PGE2 and PGI2 production, suggesting an important implication for development and use of anti‐inflammatory drugs.

This work was supported by the Korea Research Foundation Grant (MOEHRD, Basic Research Promotion Fund) (KRF‐2005‐041‐E00123) and the Vascular System Research Center grant from the Korea Science and Engineering Foundation.
A novel polymorphism in human CD40 enhances B cell activation
Authors: Peters Anna Louisa , Gaffney Patrick M. , Bishop Gail A.
Published: June 2021

CD40 is a member of the TNF‐receptor superfamily expressed on immune cells including B cells. A novel missense SNP in the cytoplasmic domain of human CD40 (hCD40P227A) was identified, and resides on a conserved ancestral haplotype highly enriched in indigenous populations from Mexico and South America. Functional studies indicated that signaling via hCD40P227A stably expressed in mouse B cell lines led to significantly increased phosphorylation of c‐Jun, increased secretion of the pro‐inflammatory cytokines IL‐6 and TNF‐α, and increased Ig production, compared to wild‐type hCD40 and to endogenous mouse CD40. Cooperation between hCD40P227A signaling and BCR‐ or TLR9‐mediated signaling was also enhanced, resulting in elevated synergistic production of IL‐6 and Ig. Binding and degradation of TRAFs by hCD40P227A was not detectably altered. Thus, hCD40P227A is a novel genetic variant with a gain‐of‐function immune phenotype and unique population stratification. Future directions include examination of effects of hCD40P227A in myeloid cells, and production of an hCD40P227A transgenic mouse to study in vivo effects. ALP is supported by an NIH training grant; PMG is supported by grants from the NIH and the Lupus Foundation of Minnesota; GAB is supported by grants from the NIH and a Career Award from the VA.
Defining CD21‐ligand interactions using ligand specific inhibitory peptides
Authors: Kovacs James M , Hannan Jonathan P , Holers V Michael
Published: June 2021

Complement receptor 2 (CR2, CD21) is a cell membrane protein, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B cell immune responses. The most distally located SCRs (SCR1‐2) mediate the interaction of CD21 with its four known ligands (C3d, EBV gp350, IFN‐alpha, CD23). Anti‐CD21 inhibitory mAbs block binding of all ligands equivalently. To develop ligand‐specific inhibitors that would also assist in identifying residues unique to each receptor‐ligand interaction, phage were selected by panning with SCR1‐2 from constrained and un‐constrained libraries, followed by ligand‐driven elution. Derived peptides were tested by competition ELISA. Peptides identified against C3d and gp350 showed low and mid micromolar ligand‐selective inhibition, respectively. The peptide blocking C3d, as well as C3d, were titrated into N‐15 labeled SCR1‐2, which revealed specific chemical shift changes indicative of specific CD21 interactions. These peptides represent the first method of selectively inhibiting single ligands of CD21 and have the potential to determine the effect of inhibiting a single ligand‐CD21 interaction during a complex immune response. R01 CA053615.
COX‐1 inhibitors block MHC‐restricted exogenous antigen presentation in dendritic cells
Authors: Chang Pan‐young , Kim Hyunjin , Jung Ki‐Sung , Lee Young‐Hee , Lee Young‐Ran , Im Sun‐A , Lee Chong‐Kil
Published: June 2021

T lymphocytes recognize antigens in the form of short peptides presented by MHC molecules. Although MHC‐restricted antigen presentation is an essential process for the initiation T cell responses, it has received little attention as a target of therapeutic immunomodulation. In an effort to search for and characterize MHC‐restricted antigen presentation modulators, we screened several thousands of compounds for their ability to inhibit or enhance exogenous antigen presentation in dendritic cells (DCs). We found that COX‐1 inhibitors, aspirin and ibuprofen, efficiently block MHC‐restricted presentation of an exogenous antigen, ovalbumin (OVA), when added to cultures of DC2.4 cells and BM‐DCs for 18 h or longer. The antigen presentation inhibitory activity of ibuprofen was much more potent than aspirin. The COX‐1 inhibitors, however, did not affect phagocytic activity, the expression level of total MHC molecules and co‐stimulatory molecules in DCs. DCs differentiated from BM cells in the presence of COX‐1 inhibitors were also suppressed in the antigen presentation capability, and this suppressive activity was more prominent when developing DCs were exposed with the COX‐1 inhibitors at the early stage of maturation. These results suggest that prolonged administration of COX‐1 inhibitors may impair antigen presentation capability of DCs.
ERAAP is essential for presentation of Toxoplasma gondii GRA6 protein to CD8 T cells
Authors: Blanchard Nicolas , Gonzales Federico , Shastri Anjali , Schaeffer Marie , Robey Ellen A , Shastri Nilabh
Published: June 2021

Toxoplasma gondii is an intracellular parasite that causes severe disease in immunocompromised individuals. Toxoplasma‐specific CD8 T cells confer immunity but their peptide‐MHC I ligands and the antigen processing mechanisms for Toxoplasma antigens are unknown. We generated lacZ‐inducible, Toxoplasma‐specific CD8 T cell hybridomas and used them as a probe to screen a Toxoplasma tachyzoite cDNA library. We identified GRA6, a Toxoplasma protein expressed in dense granules, as the source of a naturally processed, immunodominant, decapeptide presented by the mouse Ld MHC I. Peptide/Ld presentation requires active infection by tachyzoites, proteasomal activity and TAP transport. Moreover presentation by infected macrophages or dendritic cells critically depends upon expression of the ER aminopeptidase associated with antigen processing (ERAAP). Impaired presentation by ERAAP−/− antigen‐presenting cells leads to inefficient priming of GRA6‐specific CD8 T cells during infection and ERAAP‐deficient mice are susceptible to Toxoplasma infection. Thus, GRA6 is the first natural Toxoplasma antigen that elicits an immunodominant and protective CD8 T cell response. Funding by the NIH and the Human Frontier Science Program Organization