Phospholipase Cγ Research Articles

Calcium-sensing receptor protects intestinal integrity and alleviates the inflammatory response via the Rac1/PLCγ1 signaling pathway

This study aimed to test the hypothesis that the calcium-sensing receptor (CaSR) can protect intestinal epithelial barrier integrity and decrease inflammatory response mediated by the Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway. IPEC-J2 monolayers were treated without or with TNF-α in the absence or presence of CaSR antagonist (NPS 2143), CaSR overexpression, and Rac1 silencing, PLCγ1 silencing or spermine. Results showed that spermine increased transepithelial electrical resistance (TER), tight junction protein levels, the protein concentration of Rac1/PLC-γ1 signaling pathway, and decreased paracellular permeability in the presence of TNF-α. NPS2143 inhibited spermine-induced change in above-mentioned parameters. CaSR overexpression increased TER, the levels of tight junction proteins and the protein concentration of CaSR, phosphorylated PLCγ1, Rac1, and IP3, and decreased paracellular permeability and contents of interleukin-8 (IL-8) and TNF-α after TNF-α challenge. Rac1 and PLCγ1 silencing inhibited CaSR-induced increase in barrier function and the protein concentration of phosphorylated PLCγ1, Rac1, and IP3, and decrease in contents of IL-8 and TNF-α after TNF-α challenge. These results suggest that CaSR activation protects intestinal integrity and alleviates the inflammatory response by activating Rac1 and PLCγ1 signaling after TNF-α challenge, and spermine can maintain barrier function via CaSR/Rac1/PLC-γ1 pathway.

Highly Selective Irreversible ITK Inhibitor Cpi-818 Reduces Acute Graft-Versus Host Disease

Highly Selective Irreversible ITK Inhibitor Cpi-818 Reduces Acute Graft-Versus Host Disease

Tryptophan Ameliorates Barrier Integrity and Alleviates the Inflammatory Response to Enterotoxigenic Escherichia coli K88 Through the CaSR/Rac1/PLC-γ1 Signaling Pathway in Porcine Intestinal Epithelial Cells.

BackgroundImpaired intestinal barrier integrity plays a crucial role in the development of many diseases such as obesity, inflammatory bowel disease, and type 2 diabetes. Thus, protecting the intestinal barrier from pathological disruption is of great significance. Tryptophan can increase gut barrier integrity, enhance intestinal absorption, and decrease intestinal inflammation. However, the mechanism of tryptophan in decreasing intestinal barrier damage and inflammatory response remains largely unknown. The objective of this study was to test the hypothesis that tryptophan can enhance intestinal epithelial barrier integrity and decrease inflammatory response mediated by the calcium-sensing receptor (CaSR)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway.MethodsIPEC-J2 cells were treated with or without enterotoxigenic Escherichia coli (ETEC) K88 in the absence or presence of tryptophan, CaSR inhibitor (NPS-2143), wild-type CaSR overexpression (pcDNA3.1-CaSR-WT), Rac1-siRNA, and PLC-γ1-siRNA.ResultsThe results showed that ETEC K88 decreased the protein concentration of occludin, zonula occludens-1 (ZO-1), claudin-1, CaSR, total Rac1, Rho family member 1 of porcine GTP-binding protein (GTP-rac1), phosphorylated phospholipase Cγ1 (p-PLC-γ1), and inositol triphosphate (IP3); suppressed the transepithelial electrical resistance (TEER); and enhanced the permeability of FITC-dextran compared with the control group. Compared with the control group, 0.7 mM tryptophan increased the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; elevated the TEER; and decreased the permeability of FITC-dextran and contents of interleukin-8 (IL-8) and TNF-α. However, 0.7 mM tryptophan+ETEC K88 reversed the effects induced by 0.7 mM tryptophan alone. Rac1-siRNA+tryptophan+ETEC K88 or PLC-γ1-siRNA+tryptophan+ETEC K88 reduced the TEER, increased the permeability of FITC-dextran, and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. NPS2143+tryptophan+ETEC K88 decreased the TEER and the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; increased the permeability of FITC-dextran; and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. pcDNA3.1-CaSR-WT+Rac1-siRNA+ETEC K88 and pcDNA3.1-CaSR-WT+PLC-γ1-siRNA+ETEC K88 decreased the TEER and enhanced the permeability in porcine intestine epithelial cells compared with pcDNA3.1-CaSR-WT+ETEC K88.ConclusionTryptophan can improve intestinal epithelial barrier integrity and decrease inflammatory response through the CaSR/Rac1/PLC-γ1 signaling pathway.

Phospholipase Cγ2 Is Essential for Experimental Models of Epidermolysis Bullosa Acquisita

Phospholipase Cγ2 (PLCγ2) mediates tyrosine kinase‒coupled receptor signaling in various hematopoietic lineages. Although PLCγ2 has been implicated in certain human and mouse inflammatory disorders, its contribution to autoimmune and inflammatory skin diseases is poorly understood. In this study, we tested the role of PLCγ2 in a mouse model of epidermolysis bullosa acquisita triggered by antibodies against type VII collagen (C7), a component of the dermo-epidermal junction. PLCγ2-deficient (Plcg2-/-) mice and bone marrow chimeras with a Plcg2-/- hematopoietic system were completely protected from signs of anti-C7-induced skin disease, including skin erosions, dermal‒epidermal separation, and inflammation, despite normal circulating levels and skin deposition of anti-C7 antibodies. PLCγ2 was required for the tissue infiltration of neutrophils, eosinophils, and monocytes/macrophages as well as for the accumulation of proinflammatory mediators (including IL-1β, MIP-2, and LTB4) and reactive oxygen species. Mechanistic experiments revealed a role for PLCγ2 in the release of proinflammatory mediators and reactive oxygen species but not in the intrinsic migratory capacity of leukocytes. The phospholipase C inhibitor U73122 inhibited dermal-epidermal separation of human skin sections incubated with human neutrophils in the presence of anti-C7 antibodies. Taken together, our results suggest a critical role for PLCγ2 in the pathogenesis of the inflammatory form of epidermolysis bullosa acquisita.

Phospholipase Cγ2 regulates endocannabinoid and eicosanoid networks in innate immune cells

Human genetic studies have pointed to a prominent role for innate immunity and lipid pathways in immunological and neurodegenerative disorders. Our understanding of the composition and function of immunomodulatory lipid networks in innate immune cells, however, remains incomplete. Here, we show that phospholipase Cγ2 (PLCγ2 or PLCG2)-mutations in which are associated with autoinflammatory disorders and Alzheimer's disease-serves as a principal source of diacylglycerol (DAG) pools that are converted into a cascade of bioactive endocannabinoid and eicosanoid lipids by DAG lipase (DAGL) and monoacylglycerol lipase (MGLL) enzymes in innate immune cells. We show that this lipid network is tonically stimulated by disease-relevant human mutations in PLCγ2, as well as Fc receptor activation in primary human and mouse macrophages. Genetic disruption of PLCγ2 in mouse microglia suppressed DAGL/MGLL-mediated endocannabinoid-eicosanoid cross-talk and also caused widespread transcriptional and proteomic changes, including the reorganization of immune-relevant lipid pathways reflected in reductions in DAGLB and elevations in PLA2G4A. Despite these changes, Plcg2-/- mice showed generally normal proinflammatory cytokine and chemokine responses to lipopolysaccharide treatment, instead displaying a more restricted deficit in microglial activation that included impairments in prostaglandin production and CD68 expression. Our findings enhance the understanding of PLCγ2 function in innate immune cells, delineating a role in cross-talk with endocannabinoid/eicosanoid pathways and modulation of subsets of cellular responses to inflammatory stimuli.

225 The role of PCγ2 in the in vitro activation of neutrophils by type VII collagen immune complexes

Phospholipase Cγ2 (PLCγ2) is a protein expressed predominantly in hematopoietic cells and has a great importance in immunoreceptor signaling. Our group previously found that PLCγ2 deficient mice were protected from autoantibody-mediated skin blistering model induced by the systemic injection of antibodies against type VII collagen (C7), which is located in the basement membrane. The inflammation mediated mainly by neutrophils was completely abrogated in PLCγ2 deficient mice, but the mechanism is presently unknown. We aimed to examine the expression of PLCγ2 in the main circulating leukocyte populations and investigate the in vitro activation of PLCγ2 deficient neutrophils by C7-anti-C7 immune complexes. Expression of PLCγ2 in circulating leukocyte populations was measured by flow cytometry after intracellular labelling. We used bone marrow derived neutrophils to examine in vitro cell activation plated onto immobilized C7-anti-C7 immune complex surfaces. Type VII collagen or anti-C7 served as control surfaces. Cell spreading was evaluated using phase-contrast microscopy. Production of reactive oxygen species (ROS) was measured in a cytochrome-C based reduction assay. PLCγ2 was present in neutrophils, eosinophils, monocytes and B cells but not in T cells of the wild type mice. While examining the effector functions of neutrophils activated by C7-anti-C7 immune complexes, we found that nearly a third of the cells of the wild type mice spread and a great amount of ROS was produced. On the contrary, both neutrophil spreading and ROS production were completely absent in immune complex-treated PLCγ2 deficient cells. Our results show that the PLCγ2 is expressed in the major myeloid cell populations-expression of the circulating immune cells and plays a crucial role in the spreading and ROS-production of neutrophils activated by C7-anti-C7 immune complexes in vitro, which can explain why PLCγ2 deficient mice are protected in autoantibody mediated skin blistering.

194 Phospholipase Cγ2 is Essential in Autoantibody-induced Skin Blistering

Phospholipase Cγ2 (PLCγ2) is a signaling molecule of tyrosine kinase-coupled receptors in cells of hematopoietic origin. Gain-of-function mutations of PLCγ2 have been reported to cause a complex syndrome involving inflammation and bullous skin eruptions in both mice and humans. Previously we showed that PLCγ2 deficient mice remained protected in an autoimmune skin blistering model induced by the passive transfer of autoantibodies against collagen type VII (C7), which is a key anchoring fibril in the dermal-epidermal junction.

Overcoming Acquired Epigenetic Resistance to BTK Inhibitors.

In diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought.See related commentary by Pasqualucci, p. 555. This article is highlighted in the In This Issue feature, p. 549.

Phospholipase Cγ1 represses colorectal cancer growth by inhibiting the Wnt/β-catenin signaling axis

As essential phospholipid signaling regulators, phospholipase C (PLC)s are activated by various extracellular ligands and mediate intracellular signal transduction. PLCγ1 is involved in regulating various cancer cell functions. However, the precise in vivo link between PLCγ1 and cancer behavior remains undefined. To investigate the role of PLCγ1 in colorectal carcinogenesis, we generated an intestinal tissue-specific Plcg1 knock out (KO) in adenomatous polyposis coli (Apc) Min/+ mice. Plcg1 deficiency in ApcMin/+ mice showed earlier death, with a higher colorectal tumor incidence in both number and size than in wild-type mice. Mechanistically, inhibition of PLCγ1 increased the levels of its substrate phosphoinositol 4,5-bisphosphate (PIP2) at the plasma membrane and promoted the activation of Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) to enhance β-catenin signaling. Enhanced cell proliferation and Wnt/β-catenin signaling were observed in colon tumors from Plcg1 KO mice. Furthermore, low PLCγ1 expression was associated with a poor prognosis of colon cancer patients. Collectively, we demonstrated the role of PLCγ1 in vivo as a tumor suppressor relationship between the regulation of the PIP2 level and Wnt/β-catenin-dependent intestinal tumor formation.

Influence of Vincristine, Clinically Used in Cancer Therapy and Immune Thrombocytopenia, on the Function of Human Platelets.

Vincristine is a clinically used antimicrotubule drug for treating patients with lymphoma. Due to its property of increasing platelet counts, vincristine is also used to treat patients with immune thrombocytopenia. Moreover, antiplatelet agents were reported to be beneficial in thrombotic thrombocytopenic purpura (TTP). Therefore, we investigated the detailed mechanisms underlying the antiplatelet effect of vincristine. Our results revealed that vincristine inhibited platelet aggregation induced by collagen, but not by thrombin, arachidonic acid, and the thromboxane A2 analog U46619, suggesting that vincristine exerts higher inhibitory effects on collagen-mediated platelet aggregation. Vincristine also reduced collagen-mediated platelet granule release and calcium mobilization. In addition, vincristine inhibited glycoprotein VI (GPVI) signaling, including Syk, phospholipase Cγ2, protein kinase C, Akt, and mitogen-activated protein kinases. In addition, the in vitro PFA-100 assay revealed that vincristine did not prolong the closure time, and the in vivo study tail bleeding assay showed that vincristine did not prolong the tail bleeding time; both findings suggested that vincristine may not affect normal hemostasis. In conclusion, we demonstrated that vincristine exerts antiplatelet effects at least in part through the suppression of GPVI signaling. Moreover, this property of antiplatelet activity of vincristine may provide additional benefits in the treatment of TTP.

Anti-Inflammatory and Anti-Allergic Effects of Saponarin and Its Impact on Signaling Pathways of RAW 264.7, RBL-2H3, and HaCaT Cells.

Saponarin{5-hydroxy-2-(4-hydroxyphenyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]-7-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one}, a flavone found in young green barley leaves, is known to possess antioxidant, antidiabetic, and hepatoprotective effects. In the present study, the anti-inflammatory, anti-allergic, and skin-protective effects of saponarin were investigated to evaluate its usefulness as a functional ingredient in cosmetics. In lipopolysaccharide-induced RAW264.7 (murine macrophage) cells, saponarin (80 μM) significantly inhibited cytokine expression, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, inducible nitric oxide synthase, and cyclooxygenase (COX)-2. Saponarin (80 μM) also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 involved in the mitogen-activated protein kinase signaling pathway in RAW264.7 cells. Saponarin (40 μM) significantly inhibited β-hexosaminidase degranulation as well as the phosphorylation of signaling effectors (Syk, phospholipase Cγ1, ERK, JNK, and p38) and the expression of inflammatory mediators (tumor necrosis factor [TNF]-α, IL-4, IL-5, IL-6, IL-13, COX-2, and FcεRIα/γ) in DNP-IgE- and DNP-BSA-stimulated RBL-2H3 (rat basophilic leukemia) cells. In addition, saponarin (100 μM) significantly inhibited the expression of macrophage-derived chemokine, thymus and activation-regulated chemokine, IL-33, thymic stromal lymphopoietin, and the phosphorylation of signaling molecules (ERK, p38 and signal transducer and activator of transcription 1 [STAT1]) in TNF-α- and interferon (IFN)-γ-stimulated HaCaT (human immortalized keratinocyte) cells. Saponarin (100 μM) also significantly induced the expression of hyaluronan synthase-3, aquaporin 3, and cathelicidin antimicrobial peptide (LL-37) in HaCaT cells, which play an important role as skin barriers. Saponarin remarkably inhibited the essential factors involved in the inflammatory and allergic responses of RAW264.7, RBL-2H3, and HaCaT cells, and induced the expression of factors that function as physical and chemical skin barriers in HaCaT cells. Therefore, saponarin could potentially be used to prevent and relieve immune-related skin diseases, including atopic dermatitis.

The protective effect of allicin on myocardial ischemia-reperfusion by inhibition of Ca2+ overload-induced cardiomyocyte apoptosis via the PI3K/GRK2/PLC-γ/IP3R signaling pathway.

Purpose: To investigate the protective effect and mechanism of allicin on myocardial ischemia-reperfusion (MI/R) injury.Methods: We investigated the mechanisms by which allicin attenuated the MI/R injury by focusing on phosphoinositide 3-kinase, G protein coupled receptor kinases 2, phospholipase Cγ and cardiomyocyte apoptosis. Sixty male mice were randomly assigned into three groups: repeated MI/R (model), sham-operated (control), and MI/R+ allicin group (allicin). Ultrasound examination was used to examine the cardiac function. Masson staining was used to evaluate the myocardial infarct area. TUNEL assay was performed to examine the anti-apoptotic effect of allicin. Differentially expressed genes (DEGs) and pathways were analyzed by mRNA microarray analysis. Immunofluorescence staining and western blot were carried out to detect the effect of allicin on the PI3K. A pan-PLC activator, m-3M3FBS, was applied to investigate whether allicin induced cardiomyocyte apoptosis was via the GRK2/PLC/IP3R signaling pathway.Results: Masson staining and the TUNEL assay revealed that allicin reduced infarct size and played an anti-apoptotic role in M/IR. Ultrasound examination revealed that allicin improved cardiac function after M/IR injury. Gene ontology analysis indicated that the calcium signaling pathway and PI3KCA(PI3K) were selected. Immunofluorescence staining and western blot exposed that PI3K was activated by allicin during MI/R injury. Fura-2AM staining revealed that the PI3K -mediated GRK2/PLC-γ/IP3R pathway may be involved in the protective effect of allicin on MI/R injury.Conclusions: Allicin has a protective effect on MI/R injury. This effect might be associated with the inhibition of Ca2+ overload-induced apoptosis and the inhibition of the PI3K -mediated GRK2/PLC-γ/IP3R signaling pathway.

Neutrophil Phospholipase Cγ2 Drives Autoantibody-Induced Arthritis Through the Generation of the Inflammatory Microenvironment.

Gain-of-function mutations and genome-wide association studies have linked phospholipase Cγ2 (PLCγ2) to various inflammatory diseases, including arthritis in humans and mice. PLCγ2-deficient (Plcg2-/- ) mice are also protected against experimental arthritis. This study was undertaken to test how PLCγ2 triggers autoantibody-induced arthritis in mice. PLCγ2 was deleted from various mouse cellular lineages. Deletion efficacy and specificity were tested by immunoblotting and intracellular flow cytometry. Autoantibody-induced arthritis was triggered by K/BxN serum transfer. The role of neutrophil PLCγ2 was further investigated by analysis of the inflammatory exudate, competitive in vivo migration assays, and in vitro functional studies. PLCγ2 deficiency in the entire hematopoietic compartment completely blocked autoantibody-induced arthritis. Arthritis development was abrogated by deletion of PLCγ2 from myeloid cells or neutrophils but not from mast cells or platelets. Neutrophil infiltration was reduced in neutrophil-specific PLCγ2-deficient (Plcg2Δ PMN ) mice. However, this was not due to an intrinsic migration defect since Plcg2Δ PMN neutrophils accumulated normally when wild-type cells were also present in mixed bone marrow chimeras. Instead, the Plcg2Δ PMN mutation blocked the accumulation of interleukin-1β, macrophage inflammatory protein 2 (MIP-2), and leukotriene B4 (LTB4 ) in synovial tissues and reduced the secondary infiltration of macrophages. These findings were supported by in vitro studies showing normal chemotactic migration but defective immune complex-induced respiratory burst and MIP-2 or LTB4 release in PLCγ2-deficient neutrophils. Neutrophil PLCγ2 is critical for arthritis development, supposedly through the generation of the inflammatory microenvironment. PLCγ2-expressing neutrophils exert complex indirect effects on other inflammatory cells. PLCγ2-targeted therapies may provide particular benefit in inflammatory diseases with a major neutrophil component.

Lipid metabolism is a novel and practical source of potential targets for antiviral discovery against porcine parvovirus

How parvovirus manipulates host lipid metabolism to facilitate its propagation, pathogenicity and consequences for disease, is poorly characterized. Here, we addressed this question using porcine parvovirus (PPV) to understand the complex interactions of parvovirus with lipid metabolism networks contributing to the identification of novel and practical antiviral candidates. PPV significantly alters host lipid composition, characteristic of subclasses of phospholipids and sphingolipids, and induces lipid droplets (LDs) formation via regulating calcium-independent PLA2β (iPLA2β), phospholipase Cγ2 (PLCγ2), diacylglycerol kinase α (DKGα), phosphoinositide 3-kinase (PI3K), lysophosphatidic acid acyltransferase θ (LPAATθ), and sphingosine kinases (SphK1 and SphK2). PPV utilizes and exploits these enzymes as well as their metabolites and host factors including MAPKs (p38 and ERK1/2), protein kinase C (PKC) and Ca2+ to induce S phase arrest, apoptosis and incomplete autophagy, all benefit to PPV propagation. PPV also suppresses prostaglandin E2 (PGE2) synthesis via downregulating cyclooxygenase-1 (COX-1), indicating PPV hijacks COX-1-PGE2 axis to evade immune surveillance. Our data support a model where PPV to establishes an optimal environment for its propagation and pathogenicity via co-opting host lipid metabolism, being positioned as a source of potential targets.

The suppressive effect of tamarixetin, isolated from Inula japonica, on degranulation and eicosanoid production in bone marrow-derived mast cells.

The study aimed to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in immunoglobulin E /antigen-induced mouse bone marrow-derived mast cells (BMMCs). Tamarixetin isolated from Inula japonica was infected into BMMCs. The inhibitory effect of tamarixetin were analyzed by quantifying β-hexosaminidase, eicosanoid generation, intracellular calcium measurement, and Western blot analysis. Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of protein kinase B (Akt) and its downstream signal molecules including IκB kinase and nuclear factor-κB. Besides, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 and p38 mitogen-activated protein kinase.Conclusion: In summary, tamarixetin inhibits degranulation and eicosanoid generation through the phospholipase Cγ1 as well as Akt pathways. It could be potential for the prevention of allergic inflammatory diseases in BMMCs.

ANXA7 Regulates Platelet Lipid Metabolism and Ca2+ Release in Arterial Thrombosis.

[Figure: see text].

Phospholipase Cgamma Pathway-Dependent mTOR Signaling Controls IL-7-Mediated Early B Cell Development

Early B cell lymphopoiesis is intricately regulated by signals from various receptors including the pre-B cell receptor (pre-BCR) and the interleukin-7 receptor (IL-7R), but the molecular mechanism underlying the regulation is not fully understood. The Phospholipase Cγ (PLCγ) signaling pathway plays a critical role in antigen receptor-mediated activation of mature B cells, but the role of this pathway in cytokine function and early B cell development is completely unknown. Here we generated inducible PLCγ1/PLCγ2 double-deficient mice and found that the mutant mice exhibited a complete blockage of early B cell development at the pre-pro-B cell stage, similar to IL-7R-deficient but not pre-BCR-deficient mice. Of note, PLCγ1/PLCγ2 double-deficient mice had normal numbers of the HSC-containing Lin-c-Kit+Sca1+ (LSK) cells, myeloid progenitor-containing Lin-c-Kit+Sca1- (LK) cells and myeloid lineage cells compared to wild-type mice. PLCγ1/PLCγ2 double-deficient mice displayed a marked reduction in IL-7-induced rearrangement of the distal VHJ558 family gene segments. In addition, PLCγ1/PLCγ2 double deficiency altered the expression of lymphoid-lineage-affiliated genes in common lymphoid progenitors (CLPs) and the expression of B cell lineage-affiliated genes, IL-7 signaling pathway-regulated genes and cell cycle pathway-related genes in B cell progenitors. Further, PLCγ1/PLCγ2 double-deficient Lin- BM progenitors co-cultured with OP-9 stromal cells plus IL-7 failed to give rise to B cells whereas wild-type control BM progenitors generated B cells. PLCγ1/PLCγ2 double-deficient B progenitors exhibited a decrease in the level of IL-7-induced BrdU uptake compared to the corresponding wild-type control cells. Biochemically, the PLCγ pathway was activated upon IL-7 engagement in Rag1-deficient pro-B cells that lack the pre-BCR. PLCγ1/PLCγ2-double deficiency or inhibition of PLCγ pathway by PLC inhibitors impaired IL-7-induced activation of mTORC1 but not Stat5. Consistently, inhibition of the PLCγ pathway impaired IL-7-mediated metabolism of B cell progenitors. It's known that PLCγ activates the DAG/PKC-dependent and IP3/Ca2+-dependent signaling pathways, the two major downstream signaling cascades. We found that inhibition of the PKC-dependent, but not the Ca2+-dependent, pathway impaired IL-7-mediated B cell production and mTORC1 but not Stat5 activation. In addition, the PKC agonist PDBu induced mTORC1 activation in wild-type B cell progenitors, and stimulated 3H-thymidine incorporation in PLCγ1/PLCγ2 double-deficient B cell progenitors to the same extent as it did in wild-type control cells. Moreover, disruption of conventional Akt/TSC/Rheb signaling axis did not affect IL-7-controlled mTORC1 activation or early B cell development. Taken together, our current study for the first time demonstrates that the PLCγ pathway is critical for cytokine-mediated biological function, such as IL-7R-regulated early B lymphopoiesis, and this pathway directs IL-7R-mediated early B cell development through a novel and thitherto-unknown PLCγ/DAG/PKC-dependent but not conventional Akt/Rheb-dependent activation of mTORC1. DisclosuresNo relevant conflicts of interest to declare.

Recruitment of phospholipase Cγ1 to the non-structural membrane protein pK15 of Kaposi Sarcoma-associated herpesvirus promotes its Src-dependent phosphorylation.

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman’s Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells. Here, we show that a phosphorylated Y481EEVL motif in pK15 preferentially binds into the PLCγ1 C-terminal SH2 domain (cSH2), which is involved in conformational changes occurring during the activation of PLCγ1 by receptor tyrosine kinases. We determined the crystal structure of a pK15 12mer peptide containing the phosphorylated pK15 Y481EEVL motif in complex with a shortened PLCγ1 tandem SH2 (tSH2) domain. This structure demonstrates that the pK15 peptide binds to the PLCγ1 cSH2 domain in a position that is normally occupied by the linker region connecting the PLCγ1 cSH2 and SH3 domains. We also show that longer pK15 peptides containing the phosphorylated pK15 Y481EEVL motif can increase the Src-mediated phosphorylation of the PLCγ1 tSH2 region in vitro. This pK15-induced increase in Src-mediated phosphorylation of PLCγ1 can be inhibited with the small pK15-derived peptide which occupies the PLCγ1 cSH2 domain. Our findings thus suggest that pK15 may act as a scaffold protein to promote PLCγ1 activation in a manner similar to the cellular scaffold protein SLP-76, which has been shown to promote PLCγ1 activation in the context of T-cell receptor signaling. Reminiscent of its positional homologue in Epstein-Barr Virus, LMP2A, pK15 may therefore mimic aspects of antigen-receptor signaling. Our findings also suggest that it may be possible to inhibit the recruitment and activation of PLCγ1 pharmacologically.

Resistance Mutations to BTK Inhibitors Originate From the NF-κB but Not From the PI3K-RAS-MAPK Arm of the B Cell Receptor Signaling Pathway.

Since the first clinical report in 2013, inhibitors of the intracellular kinase BTK (BTKi) have profoundly altered the treatment paradigm of B cell malignancies, replacing chemotherapy with targeted agents in patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström’s macroglobulinemia. There are over 20 BTKi, both irreversible and reversible, in clinical development. While loss-of-function (LoF) mutations in the BTK gene cause the immunodeficiency X-linked agammaglobulinemia, neither inherited, nor somatic BTK driver mutations are known. Instead, BTKi-sensitive malignancies are addicted to BTK. BTK is activated by upstream surface receptors, especially the B cell receptor (BCR) but also by chemokine receptors, and adhesion molecules regulating B cell homing. Consequently, BTKi therapy abrogates BCR-driven proliferation and the tissue homing capacity of the malignant cells, which are being redistributed into peripheral blood. BTKi resistance can develop over time, especially in MCL and high-risk CLL patients. Frequently, resistance mutations affect the BTKi binding-site, cysteine 481, thereby reducing drug binding. Less common are gain-of-function (GoF) mutations in downstream signaling components, including phospholipase Cγ2 (PLCγ2). In a subset of patients, mechanisms outside of the BCR pathway, related e.g. to resistance to apoptosis were described. BCR signaling depends on many proteins including SYK, BTK, PI3K; still based on the resistance pattern, BTKi therapy only selects GoF alterations in the NF-κB arm, whereas an inhibitor of the p110δ subunit of PI3K instead selects resistance mutations in the RAS-MAP kinase pathway. BTK and PLCγ2 resistance mutations highlight BTK’s non-redundant role in BCR-mediated NF-κB activation. Of note, mutations affecting BTK tend to generate clone sizes larger than alterations in PLCγ2. This infers that BTK signaling may go beyond the PLCγ2-regulated NF-κB and NFAT arms. Collectively, when comparing the primary and acquired mutation spectrum in BTKi-sensitive malignancies with the phenotype of the corresponding germline alterations, we find that certain observations do not readily fit with the existing models of BCR signaling.

Ca2+ signalling is critical for autoantibody-induced blistering of human epidermis in pemphigus.

Pemphigus is a severe bullous autoimmune skin disease. Pemphigus foliaceus (PF) is characterized by antidesmoglein (Dsg) 1 IgG causing epidermal blistering; mucosal pemphigus vulgaris (mPV) by anti-Dsg3 IgG inducing erosions in the mucosa; and mucocutaneous pemphigus vulgaris (PV) by affecting both, with autoantibodies targeting Dsg1 and Dsg3. To characterize the Ca2+ flux pathway and delineate its importance in pemphigus pathogenesis and clinical phenotypes caused by different antibody profiles. Immunoprecipitation, Ca2+ flux analysis, Western blotting, immunofluorescence staining, dissociation assays and a human skin exvivo model were used. PV IgG and PF IgG, but neither Dsg3-specific monoclonal antibody (AK23) nor mPV IgG, caused Ca2+ influx in primary human keratinocytes. Phosphatidylinositol 4-kinase α interacts with Dsg1 but not with Dsg3. Its downstream target - phospholipase-C-γ1 (PLC) - was activated by PV IgG and PF IgG but not AK23 or mPV IgG. PLC releases inositol 1,4,5-trisphosphate (IP3) causing IP3 receptor (IP3R) activation and Ca2+ flux from the endoplasmic reticulum into the cytosol, which stimulates Ca2+ release-activated channels (CRAC)-mediated Ca2+ influx. Inhibitors against PLC, IP3R and CRAC effectively blocked PV IgG and PF IgG-induced Ca2+ influx; ameliorated alterations of Dsg1 and Dsg3 localization, and reorganization of keratin and actin filaments; and inhibited loss of cell adhesion invitro. Finally, inhibiting PLC or IP3R was protective against PV IgG-induced blister formation and redistribution of Dsg1 and Dsg3 in human skin exvivo. Ca2+ -mediated signalling is important for epidermal blistering and dependent on the autoantibody profile, which indicates different roles for signalling complexes organized by Dsg1 and Dsg3. Interfering with PLC and Ca2+ signalling may be a promising approach to treat epidermal manifestations of pemphigus.