bluuebunny/crossref_metadata_2025_split huggingface dataset:
PLCε Selectively Transduces Thrombin Versus LPA Signals to Astrocyte Proliferation Through Rap1 and Rho
Authors: Citro Simona , Kelley Grant G. , Smrcka Alan , Brown Joan Heller 
Published: June 2021

Phospholipase C epsilon (PLCε) is a recently discovered PLC that, in contrast to other PLCs, responds directly to inputs from Ras and Rho family small G‐proteins. Using primary astrocytes isolated from a PLCε‐deficient mouse strain we show that endogenous G‐protein coupled receptors for LPA, S1P and thrombin utilize predominantly PLCε for inositol phosphate (InsP) and DAG formation. LPA‐ and S1P‐mediated InsP production was sensitive to Pertussis toxin (65±8% and 74±9% inhibition respectively) whereas the thrombin response was reduced by C3 toxin (68±13% inhibition), suggesting that these ligands activate PLCε through different mechanisms, a Gi pathway for LPA and S1P and a Rho mediated pathway for thrombin. Thrombin stimulated [3H]thymidine incorporation mediated by was markedly reduced in KO astrocytes (66±2%) and further studies revealed that PLCε is required for thrombin‐induced sustained ERK phosphorylation. Activation of a Ras family protein, Rap1, was required for ERK phosphorylation and DNA synthesis and the activation of this small G‐protein was abolished in KO cells. Notably LPA‐induced ERK and mitogenic effects were not Rap1 or PLCε dependent. Thus PLCε is selectively utilized by thrombin vs. LPA to transduce mitogenic signals through mechanism distinct from its role in generation of PLC‐derived second messengers.National Institute of Health Grant GM 36927 and GM 53536.
Suppression of sphingomyelin synthase 1 (SMS1) by small interference RNA (siRNA) is associated with enhanced ceramide production and apoptosis after photodamage
Authors: Separovic Duska , Semaan Louie , Villani Maristella , Bielawski Jacek , Luberto Chiara 
Published: June 2021

Rationale:We have shown that overexpression of SMS1, an enzyme that converts de novo‐generated ceramide into sphingomyelin, is accompanied by attenuated ceramide response and apoptotic resistance after photodamage with the photosensitizer Pc 4 (PDT). To test whether SMS1 overexpression‐related effects after PDT can be reversed, in this study SMS1 was downregulated using siRNA for SMS1.Methods:Jurkat T lymphoma/leukemia cells were transfected with SMS 1 siRNA or control (scrambled)‐siRNA. Seventy two‐hours later, cells were treated with PDT, and following 2 h incubation, they were collected and processed for the following analyses: SMS activity was measured using a chemifluorescence assay with subsequent signal detection and quantitation using STORM 860/ImageQuant; sphingolipid mass was determined using high‐performance liquid chromatography‐tandem mass spectrometry; caspase‐3‐like activity (DEVDase) was measured using a spectrofluorimetric assay.Results:Compared to control siRNA‐transfectants, in SMS1 siRNA‐transfected cells the activity of SMS at rest was downregulated with concomitant decrease in sphingomyelin mass. In SMS1 siRNA‐transfected cells increases in ceramides were higher than in control siRNA‐transfectants after PDT. Similar findings were obtained for dihydroceramides suggesting the involvement of de novo ceramide pathway. DEVDase activation was enhanced in SMS1 siRNA‐transfected cells compared to their control counterparts.Conclusions:The data support the notion that downregulation of SMS1 promotes both de novo ceramide metabolism and apoptosis after photodamage. These findings suggest that SMS1 is a potential novel molecular target that can augment the therapeutic efficacy of PDT.
Role of Triacylglycerol Hydrolase and Apolipoprotein E in Hepatic Lipid Droplet Metabolism
Authors: Wang Huajin , Lehner Richard 
Published: June 2021

The objective of the study was to address the roles of triacylglycerol hydrolase (TGH) and apolipoprotein E (apoE) in the mobilization of hepatic triacylglycerol (TG) stores for very‐low density lipoprotein (VLDL) assembly.TGH and apoE associate with the same pool of hepatic luminal lipid droplets (LLD) and potentially interact with each other. Lack of TGH or apoE expression alters hepatic TG storage in both the cytosolic lipid droplets (CLD) and in the luminal lipid droplets (LLD), suggesting a crosstalk between the two lipid storage pools. Metabolic labeling studies revealed that ectopic expression of TGH channels the cytosolic rather than the luminal pool of TG for VLDL secretion. Accordingly, it is also observed by confocal imaging that the absence of TGH or apoE alters CLD morphology and distribution in hepatocytes. Apo E deficiency results in redistribution of LLD‐associated proteins, suggesting a role for apoE in altering LLD protein composition. ApoE is also found to translocate to CLD in TGH deficient hepatocytes, indicating correlated roles of TGH and apoE. It is concluded that TGH, in concert with apoE, channels mainly CLD‐TG for VLDL secretion and plays a critical role in regulating lipid droplet formation, morphology and protein composition.Supported by the a grant from the Canadian Institutes of Health Research
Intersectin (ITSN) regulation of Rab family GTPases
Authors: Wong Katy Ann , Chen Yun‐Ju , O’Bryan John P. 
Published: June 2021

ITSN is a multi‐domain scaffolding protein that regulates endocytosis and signal transduction. In addition, ITSN enhances receptor tyrosine kinase (RTK) trafficking in part through regulation of RTK ubiquitylation. We now report that ITSN regulates Rab‐mediated trafficking of RTKs. ITSN and Rab5 co‐localize on early endosomes. To examine the relationship of ITSN with various Rab family GTPases, we used bimolecular fluorescence complementation (BiFC) in which a YFP variant, Venus, is split into amino (VN)‐ and carboxyl (VC)‐terminal fragments, neither of which is fluorescent. When these fragments are fused to proteins that interact, fluorescence is reconstituted thus allowing for temporal and spatial monitoring of protein:protein interactions in vivo. Using BiFC, we shown that ITSN complexes with Rab5 and Rab7, but not with the dominant negative forms of these GTPases indicating that ITSN preferentially associates with active Rab5 and Rab7. In addition, we have identified a new component of the Rab pathway that forms a complex with ITSN and Rab5/Rab7 suggesting a role for this protein in Rab‐mediated trafficking. The localization of ITSN to vesicles as well as ITSN's interaction with the Rab pathway supports the model that ITSN regulates intracellular trafficking.K.A.W. supported in part by NIH PSTP Training Grant (5T32GM070388)
The Role of Neuregulin/erbB Signaling in Endothelial Progenitor Cell Biology
Authors: Safa Radwan Nabeel , Lamparter Mathias , Pentasugglia Laura , Peng Xu Yang , Hatzopoulos Antonis , Sawyer Doug 
Published: June 2021

Neuregulin (NRG) is a ligand for the erbB3 and erbB4 members of the EGF family receptors, and is known to activate signaling pathways that mediate proliferation, survival, migration and differentiation in various cell types. Stimuli such as exercise and ischemia‐reperfusion, which induce NRG shedding also induce endothelial progenitor cell (EPC) mobilization and recruitment. We hypothesized that NRG signaling may a play role in EPC mobilization and homing.Using western blot and immunostaining, we found that a murine embryonic endothelial progenitor cell line expresses NRG ligand and the ErbB‐2, 3 and 4 receptors. Treatment with recombinant NRG led to ErbB2/B3 phosphorylation as well as downstream Akt and endothelial nitric oxide synthase phosphorylation. NRG activated signaling was blocked by the ErbB receptor inhibitors AG1478 and Lapatanib. To examine the role of NRG/ErbB signaling in EPC biology we performed a matrigel angiogenesis assay. In contrast to mature rat cardiac microvascular endothelial cells (CMEC), EPC's in 3 dimensional monoculture do not form microvessel networks. However when EPC's are co‐cultured with CMEC's in matrigel, EPC's incorporate into the developing microvessel network within 24 hrs. This effect was completely inhibited by the erbB receptor inhibitor AG1478.These results support a role for Neuregulin/ErbB signaling in EPC recruitment. A clearer understanding of what role NRG signaling may play in EPC biology has important implications for cardiovascular biology, as well as the pathophysiology of cardiac dysfunction seen in the setting of erbB2 targeted cancer therapeutics.
Determining Role of O‐GlcNAcylation During T‐cell Activation
Authors: Shimoji Shino , Hart Gerald W 
Published: June 2021

O‐GlcNAcylation is a dynamic post‐translational modification of serine/threonine with &#946;‐linked N‐acetylglucosamine. This cyto‐nuclear modification is found in all multi‐cellular organisms. Regulatory factors NF&#954;B, NFATc1, and Elf‐1 have been identified to be O‐GlcNAc modified upon activation. O‐GlcNAcylation of Elf‐1 is necessary for translocation into nucleus. OGT expression has been positively linked to IL‐2 production. These data identify O‐GlcNAcylation as an integral part of signal transduction during T‐cell activation. We examined O‐GlcNAcylation changes upon PMA/ionomycin activation. Preliminary study showed cyto‐nuclear O‐GlcNAcylation levels increased through the first 2 hrs of the activation. The changes appeared to be protein specific. SILAC was used to identify the proteins with altered modifications. 30 proteins with increased modifications and 18 proteins with decreased modifications were identified.(Supported by NIH grants CA42486 and HD13563. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHU.)
FAK Interacts With MEF2 and Drives the Stretch‐Induced Activation of an Intronic Enhancer of Phospholamban Gene in Cardiomyocytes
Authors: Cardoso Alisson C , Judice Carla C , Deckmann Ana C , Clemente Carolina F.M.Z. , Pereira Ana HM , Franchini Kleber G 
Published: June 2021

Aim: FAK re‐locates at the nucleus and mediates the hypertrophy of cardiomyocytes in response to mechanical stress. Herein, we investigated possible regulatory role of FAK in gene expression in cardiomyocytes.Methods: We identified an intronic sequence of phospholamban gene (Pln‐i1), containing a MEF2 consensus site, by a chromatin immunoprecipitation (ChIP) approach with anti‐FAK antibody. We then used a combination of electrophoretic mobility shift (EMSA), pulldown and reporter genes assays to examine possible FAK/MEF2 interaction and FAK/MEF2 regulation of Pln‐i1 expression.Results: Fractional centrifugation and confocal microscopy confirmed the presence of FAK in the nuclei of stretched cardiomyocytes. EMSA indicated an interaction between MEF2 and Pln‐i1, but not with the fragments or the full‐length FAK. Pulldown assays performed with recombinant fragments of FAK (GST‐FERM and GST‐C‐TERM domains), nuclear extracts from nonstretched and stretched cardiomyocytes and anti‐MEF2 antibody indicated an association between FAK and MEF2. Further assays performed with recombinant GST‐MEF2 and full‐length FAK confirmed the interaction of MEF2 with phosphoFAK but not with non‐phosphorylated FAK. Transfection studies with Pln‐i1 and mut‐Pln‐i1 reporter constructs indicated that the MEF2 site is sufficient to direct the baseline and the stretch induced expression of luciferase. FAK silencing was demonstrated to markedly reduce the stretch‐induced expression of Pln‐i1 reporter construct.Conclusion: These data identify the interaction of C‐terminal FAK with MEF2 and indicate that active FAK may modulate the MEF2‐dependent transcriptional regulation of phospholamban gene.
Bradykinin B2 Receptor Interacts with Integrin α5β1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells
Authors: Kramarenko Inga I , Bunni Marlene , Raymond John R , Garnovskaya Maria N 
Published: June 2021

The vasoactive peptide bradykinin (BK) stimulates proliferation of the inner medullary collecting duct (mIMCD‐3) cells via transactivation of epidermal growth factor receptor (EGFR) by an unusual mechanism that involves collagenases‐2 and ‐3. Since collagenases lack an integral membrane domain, we hypothesized that integrins may target collagenases to the membrane to form a functional signaling complex.We supported a role of integrins in BK‐induced signaling by showing that RGD peptides and neutralizing antibody against α5β1 integrin partially (∼50%) decreased BK‐induced phosphorylation of extracellular signal‐regulated protein kinase (ERK) in mIMCD‐3 cells. Silencing of α5 and β1 expression by transfecting cells with siRNA also caused a significant (∼40%) decrease in BK‐induced ERK activation and EGFR phosphorylation. The effect was more pronounced (∼70% inhibition) in cells that were co‐transfected with siRNAs directed against collagenases. Using immunoprecipitation/Western blotting, we demonstrated association of BK B2 receptor with α5β1 integrin upon BK treatment. Furthermore, we showed that BK induced complex formation between α5β1, collagenases and EGFR.These data provide the first evidence that integrins are involved in BK‐induced proliferation of kidney cells, and might ultimately lead to development of new strategies for treatment of renal tubulointerstitial fibrosis.
5‐HT induces threonine (T375) phosphorylation of ADAM17/TACE cytoplasmic tail
Authors: Gooz Monika , Rocha Claudia , Bell Hannah , Gooz Pal , Luttrell Louis M , Raymond John R 
Published: June 2021

We have shown recently that TACE transactivates epidermal growth factor receptor (EGFR), and induces ERK phosphorylation (ERK‐P) and cellular proliferation upon 5‐HT2A receptor stimulation in renal mesangial cells.In this study our aim was to identify kinases responsible for TACE phosphorylation and potential activation during inter‐receptor cross‐talk. We detected robust threonine phosphorylation of TACE as early as 6 minutes following 5‐HT stimulation using a phospho‐threonine/proline antibody. This suggested phosphorylation of threonine 375, as this is the only threonine next to a proline present in the cytoplasmic tail of TACE. Using chemical inhibitors (naltrindole and PDK1/Akt/Flt dual pathway inhibitor) we showed that ERK phosphorylation is PDK1 dependent. Additionally, a MAPK cascade seems to have a role in TACE phosphorylation in that a MEK inhibitor (10 μM of PD989059) significantly attenuated 1 μM 5‐HT‐induced TACE phosphorylation. To study kinase‐induced activation of the enzyme we used a quenched fluorogenic TACE substrate. We found attenuation of 5‐HT‐induced TACE activity in PD989059‐pretreated cells. No serine phosphorylation of TACE upon 5‐HT stimulation was detected. Our data suggest that (1) either MEK itself or members of the MAPK family can be (directly or indirectly) responsible for TACE phosphorylation and activation during inter‐receptor crosstalk, and (2) multiple kinases contribute to TACE phosphorylation.This work was supported by NIH DK070054‐03 to M.G., T32 DK07752‐08 to C.L.R., NIH NCCR P20 RR016434 to P.G., NIH R01 DK55524 to L.M.L. and REAP from the VA Research Service.
Role of BAFF/APRIL in GALT Development
Authors: Yeramilli Venkata A. , Knight Katherine L. 
Published: June 2021

In rabbits, B cells develop in the bone marrow and migrate to GALT where they proliferate, form organized follicles, and develop the primary antibody repertoire by Ig gene diversification. While the molecular events leading to Ig gene rearrangements in the bone marrow are well understood, little is known about the mechanisms regulating B cell proliferation and homeostasis in GALT. We investigated the role(s) played by BAFF and APRIL in stimulating GALT B cells. By in situ hybridization, we found BAFF transcripts in the appendix to be highly expressed in B cell follicles. In striking contrast to human B cells, we detected BAFF expression on both naïve peripheral and GALT B cells. In vitro, purified rabbit BAFF induced splenocytes to undergo proliferation, but not B cells isolated from GALT. However, addition of BAFF to cultures of GALT B cells transiently prolonged their survival. Following in vivo injection of neonatal rabbits with a soluble decoy receptor (TACI‐Ig), we found a marked reduction in both the size and number of B cell follicles and also in Ki‐67 expression, demonstrating that BAFF/APRIL is required for B cell proliferation in GALT. We are currently investigating whether B cells residing in GALT are immature or mature B cells. Since rabbit B cells express BAFF, we propose that B cells in GALT are stimulated in an autocrine fashion.Supported by NIH AI50260
The Appendix as a Site of Immune Development in Response to Select Bacterial Species
Authors: Severson Kari M. , Mallozzi Michael , Driks Adam , Knight Katherine L. 
Published: June 2021

The human appendix was recently proposed to function as a reservoir for beneficial, commensal bacteria (Randal Bollinger, R. et al. J. Theor. Biol. (2007), doi: 101016/j/ktbo/2007.08.032). The only other mammalian species with an appendix is rabbit, which may provide a model system to study the function of the human appendix. In rabbits, the appendix is a site where B cells proliferate and diversify Ig genes in response to the intestinal microbiota.Our goal is to determine the mechanism by which the intestinal microbiota stimulates B cell proliferation in the appendix. We injected individual bacterial species into germ‐free appendices, and found that the spore‐former, Bacillus anthracis, stimulates B cell proliferation. We hypothesize that B. anthracis stimulates B cells in the appendix via a B cell superantigen. We probed western blots of bacterial lysates with rabbit IgM and identified a 30kD protein in B. anthracis spores that bound IgM. A purified, recombinant form of this protein stimulated calcium flux in appendix B cells, indicating that it can activate B cells. These data suggest that the appendix develops in part, through superantigen‐like stimulation of B cells by the intestinal microbiota. We propose that, in addition to serving as a reservoir for beneficial microbes, the human appendix, like that of rabbits, is a site of immune development in response to select bacterial species.Supported by NIH AI50260
Regulation of CD4 cell memory by PSGL‐1
Authors: Lin Melissa Michelle , Bradley Linda M 
Published: June 2021

Long‐term immunity against infections depends upon the generation and maintenance of memory T cells. CD4 cells play a pivotal role in the induction of immunity to influenza viruses by regulating the responses of both CD8 cells and B cells. A fundamental feature of CD4 cells is their capacity to generate functionally distinct subsets that confer different protective functions. Recently, we found that the capacity to generate glycosylated, biologically active selectin ligands is acquired by a subset of CD4 effectors after influenza virus infection and that P‐selectin is induced in the lungs. In this study, we used CD4 cells from OT‐II TCR transgenic mice that lack the fucosyl transferases necessary for glycosylation (FucT IV/IV) of selectins to investigate the role of those receptors in the CD4 cell response to influenza. We demonstrate that selectin binding is not required to generate a normal primary response to PR8 influenza virus. However, FucT IV/VII −/− OT‐II cells exhibit elevated production of IFN‐gamma, increased homeostatic turnover, and a failure to undergo secondary expansion as memory cells. Our data support the overall hypothesis that mechanisms regulated by adhesion molecules can be essential for the delivery of signals which control CD4 cell responses and their homeostatic maintenance, thereby sanctioning the development and persistence of memory.This research is supported by NIH Grant P01 AI046530.
Induction of Th17 responses by human skin dendritic cells is dependent on IL‐15 and IL‐6
Authors: Mathers Alicia R , Janelsins Brian M , Rubin Joseph P , Tkacheva Olga A , Larregina Adriana T 
Published: June 2021

Human skin dendritic cells (sDCs) are potent Ag presenting cells that prime and bias naïve CD4+ T cells into Th1 or Th2 effector lymphocytes. Recent data demonstrated that human monocyte derived DCs secreting IL‐12 induced Th1 responses but abrogated a Th17 bias. However, it is unknown if human sDCs that do not secrete IL‐12 and promote Th1 differentiation by secretion of IL‐23 are also capable of initiating proinflammatory IL‐17 responses. Here we analyzed the stimulatory properties of sDCs regarding their ability to promote the differentiation of both Th1 and Th17 responses. DCs purified from cultures of human skin explants, stimulated the proliferation of naïve CD4+ T cells that differentiated into two distinct effector Th cell subsets secreting IFN‐γ and IL‐17 and which expressed the skin homing molecules CCR4 and CCR10. The Th1‐biasing function of sDCs relied on the production of IL‐23. Th17 responses depended on the combined effects of IL‐15 and stabilized IL‐6:IL‐6 Ab complexes that stimulated CD4+ T cells through a molecular mechanism involving IL‐6R trans‐signaling, an effect that was inhibited by specific blockade of the IL‐6R. Additionally, IL‐17 production was independent of IL‐23 and suppressed by TGF‐β. We conclude that human sDCs induce coexistent Th1 and Th17 effector T cells with potential to home to the skin which is relevant for the development of cutaneous inflammation and autoimmunity.Supported by NIH grants R01CA100893 to ATL and T32CA82084 fellowship to ARM.
Effects of IL‐6 on Foxp3+ Treg subsets
Authors: Zheng Song Guo , Wang Juhua , Horwitz David A 
Published: June 2021

TGF‐β promotes either effector or regulatory T cell differentiation depending upon other cytokines in the local environment. While IL‐2 and TGF‐β induce naive T cells to become Foxp3+ regulatory cells (iTregs), the combination of IL‐6 and TGF‐β induce pro‐inflammatory IL‐17‐producing cells (Th17). Others have reported that IL‐6 can utilize TGF‐β produced by thymus‐derived natural Treg cells (nTreg) to convert them to Th17. We report that TGF‐β‐induced Tregs were completely resistant to IL‐6, and Foxp3 expression and suppressive activity in vitro and in vivo was unaffected. We then observed that the combination of IL‐2 and TGF‐β enables nTregs to also become resistant to IL‐6 by down‐regulating IL‐6R. Thus, besides generating iTregs, IL‐2 and TGF‐β stabilizes the suppressive phenotype of nTregs. The different sensitivities of Foxp3+ nTregs and iTregs to IL‐6 may reflect different roles for these two subsets in the adaptive immune response, and emphasizes the central role of the combination of IL‐2 and TGF‐β to control immunologic homeostasis.This work was supported by grants from the Arthritis National Research Foundation, Clinical Research Feasibility Fund, James H. Zumberge Faculty Research Innovation Fund, the Arthritis Foundation, ExCell Therapeutics, Nora Eccles Treadwell Foundation, and BD Biosciences.