Phosphorylation Of PLCγ2 Research Articles

Kaempferol Suppresses the Activation of Mast Cells by Modulating the Expression of FcεRI and SHIP1.

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of FcεRI on BMMCs, but the mRNA levels of FcεRIα, β, and γ-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface FcεRI on BMMCs was still observed when protein synthesis or protein transporter was inhibited. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NF-E2-related factor 2 (NRF2)-a master transcription factor of antioxidant stress-in BMMCs, the inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we found that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. The kaempferol-induced upregulation of SHIP1 was also observed in peritoneal MCs. The knockdown of SHIP1 by siRNA significantly enhanced IgE-induced degranulation of BMMCs. A Western blotting analysis showed that IgE-induced phosphorylation of PLCγ was suppressed in kaempferol-treated BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating FcεRI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.

Hypothalamic TrkB.FL overexpression improves metabolic outcomes in the BTBR mouse model of autism.

BTBR T+ Itpr3tf/J (BTBR) mice are used as a model of autism spectrum disorder (ASD), displaying similar behavioral and physiological deficits observed in patients with ASD. Our recent study found that implementation of an enriched environment (EE) in BTBR mice improved metabolic and behavioral outcomes. Brain-derived neurotrophic factor (Bdnf) and its receptor tropomyosin kinase receptor B (Ntrk2) were upregulated in the hypothalamus, hippocampus, and amygdala by implementing EE in BTBR mice, suggesting that BDNF-TrkB signaling plays a role in the EE-BTBR phenotype. Here, we used an adeno-associated virus (AAV) vector to overexpress the TrkB full-length (TrkB.FL) BDNF receptor in the BTBR mouse hypothalamus in order to assess whether hypothalamic BDNF-TrkB signaling is responsible for the improved metabolic and behavioral phenotypes associated with EE. Normal chow diet (NCD)-fed and high fat diet (HFD)-fed BTBR mice were randomized to receive either bilateral injections of AAV-TrkB.FL or AAV-YFP as control, and were subjected to metabolic and behavioral assessments up to 24 weeks post-injection. Both NCD and HFD TrkB.FL overexpressing mice displayed improved metabolic outcomes, characterized as reduced percent weight gain and increased energy expenditure. NCD TrkB.FL mice showed improved glycemic control, reduced adiposity, and increased lean mass. In NCD mice, TrkB.FL overexpression altered the ratio of TrkB.FL/TrkB.T1 protein expression and increased phosphorylation of PLCγ in the hypothalamus. TrkB.FL overexpression also upregulated expression of hypothalamic genes involved in energy regulation and altered expression of genes involved in thermogenesis, lipolysis, and energy expenditure in white adipose tissue and brown adipose tissue. In HFD mice, TrkB.FL overexpression increased phosphorylation of PLCγ. TrkB.FL overexpression in the hypothalamus did not improve behavioral deficits in either NCD or HFD mice. Together, these results suggest that enhancing hypothalamic TrkB.FL signaling improves metabolic health in BTBR mice.

NWASP Inhibition Increases Wound Healing via TrKb/PLCγ Signalling.

(1) Background: Chronic wounds represent a major burden to patients and healthcare systems and identifying new therapeutic targets to encourage wound healing is a significant challenge. This study evaluated nWASP as a new therapeutic target in human wound healing and determined how this can be regulated. (2) Methods: Clinical cohorts from patients with chronic wounds were tested for the expression of nWASP and cell models were employed to evaluate the influence of nWASP on cellular functions that are key to the healing process following knockdown and/or the use of nWASP-specific inhibitors. (3) Results: nWASP was significantly elevated at transcript levels in human non-healing chronic wounds versus healing tissues. nWASP inhibitors, wiskostatin and 187-1, along with the knockdown of nWASP, modified both HaCaT and HECV cell behaviour. We then identified two signalling pathways affected by nWASP inhibition: TrkB signalling and downstream PLCγ1 phosphorylation were impaired by nWASP inhibition in HaCaT cells. The healing of wounds in a diabetic murine model was significantly improved with an nWASP inhibitor treatment. (4) Conclusions: This study showed that nWASP activity was related to the non-healing behaviour of chronic wounds and together with the findings in the in vivo models, it strongly suggested nWASP as a therapeutic target in non-healing wounds that are regulated via TrkB and PLCγ1 signalling.

Myricetin alleviated immunologic contact urticaria and mast cell degranulation via the PI3K/Akt/NF-κB pathway.

Immunologic contact urticaria (ICU) is characterized by the wheal and flare reaction from direct contact with a chemical or protein agent, which involves a type I hypersensitivity mediated by allergen-specific immunoglobulin E (sIgE). Myricetin (Myr), a bioactive flavonoid, exhibits antiinflammatory activities. Our results showed that treatment with Myr could alleviate ICU symptoms, including a decrease in the number of wheals and scratching, and inhibit ear swelling in the IgE/DNFB-induced mice. The serum level of IgE, histamine, interleukin (IL)-4, TNF-α, and MCP-1 were reduced in Myr-treated mice. Myr also attenuated mast cells (MCs) degranulation and H-PGDS, TSLP, IL-33, PI3K, Akt, and NF-κB mRNA levels in ICU model. The IgE-mediated anaphylaxis mouse models demonstrated anti-allergic effects of Myr. In vitro analysis showed that Myr reduced IgE-induced calcium (Ca2+ ) influx, suppressed degranulation, and chemokine release in LAD2 cells (human primary mast cells). Myr can significantly inhibited PLCγ1, Akt, NF-κB, and p38 phosphorylation. In conclusion, the study demonstrated that Myr alleviate ICU symptoms and inhibit mast cell activation via PI3K/Akt/NF-κB signal pathway.

Combination CTLA4Ig and Anti-CD40 Ligand Treatment Modifies T and B Cell Metabolic Profiles and Promotes B Cell Receptor Remodeling in a Mouse Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus is a complex autoimmune disease with significant morbidity that demands further examination of tolerance-inducing treatments. Short-term treatment of lupus-prone NZB/WF1 mice with combination CTLA4Ig and anti-CD40 ligand, but not single treatment alone, suppresses disease for >6 mo via modulation of B and T cell function while maintaining immune responses to exogenous Ags. Three months after a 2-wk course of combination costimulatory blockade, we found a modest decrease in the number of activated T and B cells in both combination and single-treatment cohorts compared with untreated controls. However, only combination treatment mice showed a 50% decrease in spare respiratory capacity of splenic B and T cells. RNA sequencing and gene set enrichment analysis of germinal center (GC) B cells confirmed a reduction in the oxidative phosphorylation signature in the combination treatment cohort. This cohort also manifested increased expression of BCR-associated signaling molecules and increased phosphorylation of PLCγ in GC B cells after stimulation with anti-IgG and anti-CD40. GC B cells from combination treatment mice also displayed a signature involving remodeling of GPI-linked surface proteins. Accordingly, we found a decrease in cell surface expression of the inhibitory molecule CD24 on class-switched memory B cells from aged NZB/W mice that corrected in the combination treatment cohort. Because both a profound decrease in BCR signaling and remodeled immune cell metabolism enhance loss of tolerance in lupus-prone mice, our findings help to explain the restoration of tolerance observed after short-term combination costimulatory blockade.

Chemical composition-based characterization of the anti-allergic effect of Guominkang Formula on IgE-mediated mast cells activation and passive cutaneous anaphylaxis.

Guominkang (GMK), a Chinese medicine formula, has been used to treat allergic diseases in clinical settings for many years. To evaluate the antiallergic effect and molecular mechanism of action of GMK extract, RBL-2H3 cell models and passive cutaneous anaphylaxis (PCA) mouse models were established. High performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analyses were performed to characterize the chemical composition of GMK. A total of 94 compounds were identified or tentatively identified from GMK. Three of them, emodin, ursolic acid, and hamaudol, were identified for the first time as potential active compounds in GMK, since they inhibited the degranulation of mast cells. The anti-allergic effect of hamaudol was the first to be discovered. GMK could markedly mitigate the shade of Evans Blue extravasation and ear incrassation in PCA mouse models. Additionally, GMK significantly inhibited the degranulation of mast cells, suppressed mast cell degranulation by reducing Ca2+ influx and the levels of TNF-α, IL-4, and histamine, and markedly inhibited the phosphorylation of Lyn, Syk, PLCγ1, IκBα, and NF-κB p65. Molecular docking results indicated that hamaudol and emodin had strong interaction with FcɛRI and NF-κB related proteins, while ursolic acid only interacted with NF-κB associated proteins. These results suggest GMK suppresses the activation of MCs both in vivo and in vitro. The underlying mechanism of its anti-allergic activity is associated with the inhibition of FcɛRI and NF-κB activation.

Presenilin1 FAD mutants and γ‐secretase inhibitors decrease VEGFR2 signaling and block angiogenic functions of brain endothelial cells

AbstractBackgroundAlzheimer’s disease (AD) is a multifactorial and progressive neurodegenerative disease where cerebrovascular abnormalities are commonly observed. Various factors, including decreased sprouting angiogenesis, can cause a reduction in capillary density observed in AD. In addition, brain vascular alterations occur before the appearance of neuropathology in animal AD models. Brain angiogenesis is a reparative function in response to toxic insults and is regulated by endothelial cells (ECs) and angiogenic factors such as the VEGF; impairment in this function would render the brain vulnerable to insults. In present study, we examine the effects of Presenilin 1 (PS1)‐Familial AD (FAD) mutants and γ‐secretase in the VEGF‐induced angiogenic signaling and functions of brain ECs.MethodPrimary cortical ECs (pCECs) from brains of wild‐type (WT) and knock‐in (KI) mice expressing either PS1 M146V or PS1 I213T FAD mutant in heterozygous state were isolated and cultured. These mice constitute a “humanized” FAD model having the same genotype as human FAD patients. We performed the in vitro angiogenesis assays using pCEC cultures. Processing of VEGFR2 was detected in HEK293 cells overexpressing VEGFR2 in the presence or absence of VEGF‐A and γ‐secretase inhibitor (RO4929097). The same processing was also analyzed by western blotting in PS1 M146V and I213T mutant pCECs treated with VEGF‐A. VEGF‐induced phosphorylation of signaling molecules downstream of VEGFR2 such as p38 kinase and PLCγ1 was also examined in WT and PS1 M146V mutant pCECs by western blotting.ResultWe found that VEGFR2 is processed by γ‐secretase and that this processing together with VEGF‐induced sprouting, tube formation, migration and angiogenic complexes between VE‐cadherin and Rok‐α kinase are decreased in the presence of γ‐secretase inhibitor or by PS1 FAD mutants. Furthermore, the VEGF‐induced phosphorylation of p38 and PLCγ1 is decreased in PS1 FAD mutant pCECs.ConclusionOur findings suggest that VEGFR2 is processed by γ‐secretase and that γ‐secretase promotes VEGF‐induced angiogenic functions of brain ECs through activation of VEGF‐VEGFR2 downstream signaling. Our data also show that a PS1 FAD mutant decreases both the VEGFR2 processing by γ‐secretase and the VEGF‐induced signaling and angiogenic functions of pCECs, providing a mechanism via which FAD mutants affect brain angiogenesis.

Ibrutinib suppresses the activation of neutrophils and macrophages and exerts therapeutic effect on acute peritonitis induced by zymosan.

Timely treatment of acute inflammatory reactions induced by fungi or bacteria is essential to prevent infectious damage. Ibrutinib is a Bruton's tyrosine kinase (BTK) inhibitor which is used to treat various lymphoid cancers. It is also known that BTK plays important roles in innate immunity and inflammatory response. In the present study, we investigated the regulatory effects of Ibrutinib on the activation of neutrophils and macrophages and its therapeutic effects on acute peritonitis. In addition, we also studied its anti-inflammatory mechanisms. The results showed that Ibrutinib inhibited the expression and secretion of inflammatory factors in macrophages induced by multiple Toll-like receptor (TLR) agonists. In the study of neutrophils, Ibrutinib selectively suppressed the activation, superoxide release, and calcium influx of neutrophils stimulated by zymosan. Furthermore, in zymosan-induced mice acute peritonitis, Ibrutinib significantly reduced the infiltration of neutrophils into peritoneal cavity, the release of myeloperoxidase (MPO) and β-glucuronidase as well as the production of inflammatory factors in peritoneal cavity. In mechanism study, Ibrutinib selectively inhibited the phosphorylation of PLCγ2, PKCδ, and ERK1/2 in neutrophils induced by zymosan. Collectively, Ibrutinib can significantly inhibit the activation of neutrophils and macrophages by inhibiting BTK-PLCγ2-PKC signaling pathway, and has great potential to be developed into therapeutic drug for acute inflammatory diseases.

Critical role of mitogen-inducible gene 6 in restraining endothelial cell permeability to maintain vascular homeostasis

Although mitogen-inducible gene 6 (MIG6) is highly expressed in vascular endothelial cells, it remains unknown whether MIG6 affects vascular permeability. Here, we show for the first time a critical role of MIG6 in limiting vascular permeability. We unveil that genetic deletion of Mig6 in mice markedly increased VEGFA-induced vascular permeability, and MIG6 knockdown impaired endothelial barrier function. Mechanistically, we reveal that MIG6 inhibits VEGFR2 phosphorylation by binding to the VEGFR2 kinase domain 2, and MIG6 knockdown increases the downstream signaling of VEGFR2 by enhancing phosphorylation of PLCγ1 and eNOS. Moreover, MIG6 knockdown disrupted the balance between RAC1 and RHOA GTPase activation, leading to endothelial cell barrier breakdown and the elevation of vascular permeability. Our findings demonstrate an essential role of MIG6 in maintaining endothelial cell barrier integrity and point to potential therapeutic implications of MIG6 in the treatment of diseases involving vascular permeability.Graphical abstractXing et al. (2022) investigated the critical role of MIG6 in vascular permeability. MIG6 deficiency promotes VEGFA-induced vascular permeability via activation of PLCγ1-Ca2+-eNOS signaling and perturbation of the balance in RAC1/RHOA activation, resulting in endothelial barrier disruption.

Kefir peptides ameliorate osteoporosis in AKR1A1 knockout mice with vitamin C deficiency by promoting osteoblastogenesis and inhibiting osteoclastogenesis

The AKR1A1 protein is a member of the aldo-keto reductase superfamily that catalyzes the transformation of D-glucuronate to L-gulonate in the synthesis of L-ascorbic acid (vitamin C, Vit C). We previously demonstrated that AKR1A1 knockout mice (AKR1A1eGFP/eGFP) with Vit C deficiency exhibited aberrant bone formation and osteoporosis. In this study, we aimed to evaluate the osteoprotective effects of kefir peptides (KPs) in AKR1A1eGFP/eGFP mice and uncover the underlying mechanism of KPs in the modulation of bone remodeling. Six male CD-1 mice and 24 male AKR1A1eGFP/eGFP mice were used in this study, in which the AKR1A1eGFP/eGFP mice were randomly divided into four groups (n = 6). KPs treatment for 12 weeks exerted several effects in AKR1A1eGFP/eGFP mice including the reduction of serum proinflammatory cytokines (IL-1β, IL-6, TNF-α), bone resorption markers (CTX-1, RANKL), and the increase of serum bone formation markers (P1NP, OPG, OC). μ-CT analysis indicated that KPs prevented the bone loss in the femurs of AKR1A1eGFP/eGFP mice by significantly increasing the trabecular parameters of bone mineral density, bone volume and bone number. Nanoindentation analysis demonstrated that KPs enhanced the elasticity and hardness of femoral cortical bones in AKR1A1eGFP/eGFP mice. KPs promoted bone marrow mesenchymal stem cells (BMMSCs)-derived osteoblast differentiation and mineralization by upregulating positive regulators of osteoblastogenesis (Runx2, β-catenin, BMP-2, NFATc1). Conversely, KPs inhibited bone marrow macrophages (BMMs)-derived osteoclast differentiation and bone resorption, which was demonstrated by the facts that KPs suppressed RANKL-induced p38, NF-κB, Akt, PLCγ2 and CREB-1 phosphorylation, decreased the nuclear translocation of NFATc1 and c-Fos. Our findings demonstrate the efficacy of KPs in the prevention of osteoporosis in AKR1A1eGFP/eGFP mice and also unveil the dual effects of KPs in osteogenic promotion and osteoclastic inhibition. This study supports the use of KPs as nutritional supplements for the prevention of osteoporosis.

Versicotide G suppresses osteoclastogenesis and prevents osteolysis

Excessive formation and function of osteoclasts cause various osteolytic bone diseases. Natural products are a potential source for the discovery of new therapeutic candidates to treat bone destruction diseases. In this study, chemical informatics and bioassay guided examination of the marine-derived Aspergillus versicolor F77 fungus chemically resulted in the isolation of seven cyclopeptides, of which versicotides G-J (1–4) are new cyclohexapeptides. Their structures were identified by spectroscopic data in association with Marfey method and single crystal X-ray diffraction data for configurational assignments. Bioassay revealed that versicotide G (1, VG) is the most active among the analogs to suppress the receptor activator of nuclear factor-KB ligand (RANKL)-induced osteoclastogenesis in bone marrow derived monocytes (BMMs) without affecting BMMs viability. VG also suppressed RANKL-induced actin-ring formation and resorbing function of osteoclast dose-dependently. Mechanistically, VG attenuated RANKL-induced intracellular calcium elevation by inhibiting PLCγ1 phosphorylation and blocking the activation of downstream phosphatase calcineurin. In addition, VG abrogated the expression and translocation of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), leading to the downregulation of the expression of osteoclast-specific genes and the abolishment of the osteoclast formation. In the in vivo test, VG suppressed osteoclast formation and bone loss in Ti-induced calvarial osteolytic mouse model.These findings imply that VG is a promising candidate for the remedy of bone destruction-related diseases.

CD4+ T-cell cooperation promoted pathogenic function of activated naïve B cells of patients with SLE

ObjectiveTo explore cooperation between activated naïve (aNAV) B cells and CD4+ T cells in the pathogenesis of SLE through autoantibody production, T-cell differentiation and inflammatory cytokine secretion.MethodsPeripheral blood mononuclear cell...

Disruption of retinal inflammation and the development of diabetic retinopathy in mice by a CD40-derived peptide or mutation of CD40 in Müller cells

Aims/hypothesisCD40 expressed in Müller cells is a central driver of diabetic retinopathy. CD40 causes phospholipase Cγ1 (PLCγ1)-dependent ATP release in Müller cells followed by purinergic receptor (P2X7)-dependent production of proinflammatory cytokines in myeloid cells. In the diabetic retina, CD40 and P2X7 upregulate a broad range of inflammatory molecules that promote development of diabetic retinopathy. The molecular event downstream of CD40 that activates the PLCγ1–ATP–P2X7–proinflammatory cytokine cascade and promotes development of diabetic retinopathy is unknown. We hypothesise that disruption of the CD40-driven molecular events that trigger this cascade prevents/treats diabetic retinopathy in mice.MethodsB6 and transgenic mice with Müller cell-restricted expression of wild-type (WT) CD40 or CD40 with mutations in TNF receptor-associated factor (TRAF) binding sites were made diabetic using streptozotocin. Leucostasis was assessed using FITC-conjugated concanavalin A. Histopathology was examined in the retinal vasculature. Expression of inflammatory molecules and phospho-Tyr783 PLCγ1 (p-PLCγ1) were assessed using real-time PCR, immunoblot and/or immunohistochemistry. Release of ATP and cytokines were measured by ATP bioluminescence and ELISA, respectively.ResultsHuman Müller cells with CD40 ΔT2,3 (lacks TRAF2,3 binding sites) were unable to phosphorylate PLCγ1 and release ATP in response to CD40 ligation, and could not induce TNF-α/IL-1β secretion in bystander myeloid cells. CD40–TRAF signalling acted via Src to induce PLCγ1 phosphorylation. Diabetic mice in which WT CD40 in Müller cells was replaced by CD40 ΔT2,3 failed to exhibit phosphorylation of PLCγ1 in these cells and upregulate P2X7 and TNF-α in microglia/macrophages. P2x7 (also known as P2rx7), Tnf-α (also known as Tnf), Il-1β (also known as Il1b), Nos2, Icam-1 (also known as Icam1) and Ccl2 mRNA were not increased in these mice and the mice did not develop retinal leucostasis and capillary degeneration. Diabetic B6 mice treated intravitreally with a cell-permeable peptide that disrupts CD40–TRAF2,3 signalling did not exhibit either upregulation of P2X7 and inflammatory molecules in the retina or leucostasis.Conclusions/interpretationCD40–TRAF2,3 signalling activated the CD40–PLCγ1–ATP–P2X7–proinflammatory cytokine pathway. Src functioned as a link between CD40–TRAF2,3 and PLCγ1. Replacing WT CD40 with CD40 ΔT2,3 impaired activation of PLCγ1 in Müller cells, upregulation of P2X7 in microglia/macrophages, upregulation of a broad range of inflammatory molecules in the diabetic retina and the development of diabetic retinopathy. Administration of a peptide that disrupts CD40–TRAF2,3 signalling reduced retinal expression of inflammatory molecules and reduced leucostasis in diabetic mice, supporting the therapeutic potential of pharmacological inhibition of CD40–TRAF2,3 in diabetic retinopathy.Graphical abstract

Yohimbine, an α2-Adrenoceptor Antagonist, Suppresses PDGF-BB-Stimulated Vascular Smooth Muscle Cell Proliferation by Downregulating the PLCγ1 Signaling Pathway.

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.

Unfolded Protein Response Differentially Modulates the Platelet Phenotype.

Unfolded protein response (UPR) is a multifaceted signaling cascade that alleviates protein misfolding. Although well studied in nucleated cells, UPR in absence of transcriptional regulation has not been described. Intricately associated with cardiovascular diseases, platelets, despite being anucleate, respond rapidly to stressors in blood. We investigate the UPR in anucleate platelets and explore its role, if any, on platelet physiology and function. Human and mouse platelets were studied using a combination of ex vivo and in vivo experiments. Platelet lineage-specific knockout mice were generated independently for each of the 3 UPR pathways, PERK (protein kinase RNA [PKR]-like endoplasmic reticulum kinase), XBP1 (X-binding protein), and ATF6 (activating transcription factor 6). Diabetes patients were prospectively recruited, and platelets were evaluated for activation of UPR under chronic pathophysiological disease conditions. Tunicamycin induced the IRE1α (inositol-requiring enzyme-1alpha)-XBP1 pathway in human and mouse platelets, while oxidative stress predominantly activated the PERK pathway. PERK deletion significantly increased platelet aggregation and apoptosis and phosphorylation of PLCγ2, PLCβ3, and p38 MAPK. Deficiency of XBP1 increased platelet aggregation, with higher PLCβ3 and PKCδ activation. ATF6 deletion mediated a relatively modest effect on platelet phenotype with increased PKA (protein kinase A). Platelets from diabetes patients exhibited a positive correlation between disease severity, platelet activation, and protein aggregation, with only IRE1α-XBP1 activation. Moreover, IRE1α inhibition increased platelet aggregation, while clinically approved chemical chaperone, sodium 4-phenylbutyrate reduced the platelet hyperactivation. We show for the first time, that UPR activation occurs in platelets and can be independent of genomic regulation, with selective induction being specific to the source and severity of stress. Each UPR pathway plays a key role and can differentially modulate the platelet activation pathways and phenotype. Targeting the specific arms of UPR may provide a new antiplatelet strategy to mitigate thrombotic risk in diabetes and other cardiovascular diseases.

Type of PaperY192 within the SH2 Domain of Lck Regulates TCR Signaling Downstream of PLC-γ1 and Thymic Selection.

Signaling via the TCR, which is initiated by the Src-family tyrosine kinase Lck, is crucial for the determination of cell fates in the thymus. Because of its pivotal role, ablation of Lck results in a profound block of T-cell development. Here, we show that, in addition to its well-known function in the initiation of TCR signaling, Lck also acts at a more downstream level. This novel function of Lck is determined by the tyrosine residue (Y192) located in its SH2 domain. Thymocytes from knock-in mice expressing a phosphomimetic Y192E mutant of Lck initiate TCR signaling upon CD3 cross-linking up to the level of PLC-γ1 phosphorylation. However, the activation of downstream pathways including Ca2+ influx and phosphorylation of Erk1/2 are impaired. Accordingly, positive and negative selections are blocked in LckY192E knock-in mice. Collectively, our data indicate that Lck has a novel function downstream of PLCγ-1 in the regulation of thymocyte differentiation and selection.

Allantoin Inhibits Compound 48/80-Induced Pseudoallergic Reactions In Vitro and In Vivo.

Pseudoallergic reactions are hypersensitivity reactions mediated by an IgE-independent mechanism. Since allantoin (AT)-mediated pseudoallergy has not been studied, in this study, our objective is to investigate the anti-pseudoallergy effect of AT and its underlying mechanism. In vitro, β-hexosaminidase (β-Hex) and histamine (HIS) release assays, inflammatory cytokine assays, toluidine blue staining, and F-actin microfilament staining were used to evaluate the inhibitory effect of AT in RBL-2H3 cells stimulated with Compound 48/80 (C48/80). Western blot analysis is further performed to investigate intracellular calcium fluctuation-related signaling pathways. In vivo, Evans Blue extraction, paw swelling, and the diameter of Evans Blue extravasation were evaluated, and skin tissues are examined for histopathological examination in mice with passive cutaneous anaphylaxis (PCA) induced by C48/80. Body temperature is measured, and the levels of cytokines are further determined by ELISA kits in mice with active systemic anaphylaxis (ASA) induced by C48/80. The results show that AT dose-dependently inhibited degranulation in C48/80-stimulated RBL-2H3 cells by inhibiting β-Hex and HIS release, reducing the levels of TNF-α, IL-8, and MCP-1, inhibiting shape changes due to degranulation and disassembling the F-actin cytoskeleton. Furthermore, AT dose-dependently inhibits the phosphorylation of PLCγ and IP3R. In vivo, AT decreased Evans Blue extravasation, paw swelling, and the diameter of Evans Blue extravasation and significantly ameliorate pathological changes and mast cell degranulation in C48/80-induced PCA. Furthermore, AT help the mice recover from the C48/80-induced decrease in body temperature and decreased the levels of cytokines in C48/80-treated ASA mice. Our results indicate that allantoin inhibits compound 48/80-induced pseudoallergic reactions. AT has the potential to be used in IgE-independent anti-allergic and anti-inflammatory therapies.

BTK-independent regulation of calcium signalling downstream of the B-cell receptor in malignant B-cells

BTK inhibitors (BTKi) have dramatically improved outcomes for patients with chronic lymphocytic leukaemia (CLL) and some forms of B-cell lymphoma. However, new strategies are needed to enhance responses. Here we have performed a detailed analysis of the effects of BTKi on B-cell receptor (BCR)-induced signalling using primary malignant cells from CLL patients and B-lymphoma cell lines. Although BTK is considered as a key activator of PLCγ2, BTKi (ibrutinib and acalabrutinib) failed to fully inhibit calcium responses in CLL samples with strong BCR signalling capacity. This BTKi-resistant calcium signalling was sufficient to engage downstream calcium-dependent transcription and suppress CLL cell apoptosis and was entirely independent of BTK and not just its kinase activity as similar results were obtained using a BTK-degrading PROTAC. BTK-independent calcium signalling was also observed in two B-lymphoma cell lines where BTKi had little effect on the initial phase of the calcium response but did accelerate the subsequent decline in intracellular calcium. In contrast to BTKi, calcium responses were completely blocked by inhibition of SYK in CLL and lymphoma cells. Engagement of BTK-independent calcium responses was associated with BTK-independent phosphorylation of PLCγ2 on Y753 and Y759 in both CLL and lymphoma cells. Moreover, in CLL samples, inhibition of RAC, which can mediate BTK-independent activation of PLCγ2, cooperated with ibrutinib to suppress calcium responses. BTK-independent calcium signalling may limit the effectiveness of BTKi to suppress BCR signalling responses and our results suggest inhibition of SYK or dual inhibition of BTK and RAC as alternative strategies to strengthen pathway blockade.

Evening Primrose Extracts Inhibit PDGF-BB-Induced Vascular Smooth Muscle Cell Proliferation and Migration by Regulating Cell-Cycle-Related Proteins.

The proliferation and migration of vascular smooth muscle cells (VSMCs) are important factors in the occurrence of cardiovascular diseases, such as blood flow abnormalities, stroke and atherosclerosis. Evening primrose, known as Oenothera biennis, is a plant native to Korea that exerts physiological activities, such as antioxidant effects, the inhibition of lipid accumulation and the prevention of muscle atrophy. However, the function of evening primrose stem (EVP) in the regulation of VSMC proliferation and migration and the underlying mechanisms have not been identified. In this study, the effect of EVP on the platelet-derived growth factor (PDGF)-induced proliferation and migration of VSMCs was investigated. The results show that PDGF-BB-induced proliferation of VSMCs was inhibited by EVP at concentrations of 25, 50 or 100 μg/mL in a concentration-dependent manner, and a migration assay showed that EVP inhibited cell migration. Cell cycle analysis was performed to confirm the mechanism by which cell proliferation and migration was inhibited. The results indicate that proteins involved in the cell cycle, such as cyclin, CDK and phosphorylated Rb, were downregulated by EVP at concentrations of 100 μg/mL, thereby increasing the proportion of cells in the G0/G1 phase and inhibiting cell cycle progression. In the PDGF receptor (PDGFR) signaling pathway, phosphorylation of the PDGFR was inhibited by EVP at concentrations of 100 μg/mL, and PLCγ phosphorylation was also decreased. The PDGF-BB-induced effect of EVP on the proliferation of VSMCs involved the inhibition of Akt phosphorylation and the reduction in the phosphorylation of MAPK proteins such as ERK, P38 and JNK. In conclusion, the results demonstrate that EVP inhibited PDGF-BB-induced VSMC proliferation and migration by regulating cell-cycle-related proteins.

A kinetic model of phospholipase C-γ1 linking structure-based insights to dynamics of enzyme autoinhibition and activation

Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain–pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.