A Review of Resistance Mechanisms to Bruton's Kinase Inhibitors in Chronic Lymphocytic Leukemia.

Bruton's Tyrosine Kinase (BTK) inhibitors have become one of the most vital drugs in the therapy of chronic lymphocytic leukemia (CLL). Inactivation of BTK disrupts the B-cell antigen receptor (BCR) signaling pathway, which leads to the inhibition of the proliferation and survival of CLL cells. BTK inhibitors (BTKi) are established as leading drugs in the treatment of both treatment-naïve (TN) and relapsed or refractory (R/R) CLL. Furthermore, BTKi demonstrate outstanding efficacy in high-risk CLL, including patients with chromosome 17p deletion, TP53 mutations, and unmutated status of the immunoglobulin heavy-chain variable region (IGHV) gene. Ibrutinib is the first-in-class BTKi which has changed the treatment landscape of CLL. Over the last few years, novel, covalent (acalabrutinib, zanubrutinib), and non-covalent (pirtobrutinib) BTKi have been approved for the treatment of CLL. Unfortunately, continuous therapy with BTKi contributes to the acquisition of secondary resistance leading to clinical relapse. In recent years, it has been demonstrated that the predominant mechanisms of resistance to BTKi are mutations in BTK or phospholipase Cγ2 (PLCG2). Some differences in the mechanisms of resistance to covalent BTKi have been identified despite their similar mechanism of action. Moreover, novel mutations resulting in resistance to non-covalent BTKi have been recently suggested. This article summarizes the clinical efficacy and the latest data regarding resistance to all of the registered BTKi.

Intervention of exogenous VEGF protect brain microvascular endothelial cells from hypoxia-induced injury by regulating PLCγ/RAS/ERK and PI3K/AKT pathways.

Ischemic stroke rapidly increases the expression level of vascular endothelial growth factor (VEGF), which promotes neovascularization during hypoxia. However, the effect and mechanism of VEGF intervention on cerebrovascular formation remain unclear. Therefore, our research discussed the protective effect of exogenous VEGF on cells in hypoxia environment in cerebral microvascular endothelial cells, simulating ischemic stroke in hypoxic environment. Firstly, we detected the proliferation and apoptosis of cerebral microvascular endothelial cells under hypoxia environment, as well the expression levels of VEGF-E, vascular endothelial growth factor re-ceptor-2 (VEGFR-2), BCL2, PRKCE and PINK1. Moreover, immunofluorescence and western blotting were used to verify the regulation of exogenous VEGF-E on VEGFR-2 expression in hypoxic or normal oxygen environment. Lastly, we manipulated the concentration of VEGF-E in the culture medium to investigate its impact on phospholipase Cγ1 (PLCγ1)/extracellular signaling regulatory protein kinase (ERK) -1/2 and protein kinase B (AKT) pathways. Additionally, we employed a PLCγ1 inhibitor (U73122) to investigate its impact on proliferation and PLCγ1/ERK pathways. The results show that hypoxia inhibited the proliferation of cerebral microvascular endothelial cells, promoted cell apoptosis, significantly up-regulated the expression of VEGF-E, VEGFR-2, PRKCE and PINK1, but down-regulated the expression of BCL2. Interference from exogenous VEGF-E activated PLCγ1/ERK-1/2 and AKT pathways, promoting cell proliferation and inhibiting apoptosis of hypoxic brain microvascular endothelial cells. In summary, exogenous VEGF-E prevents hypoxia-induced damage to cerebral microvascular endothelial cells by activating the PLCγ1/ERK and AKT pathways. This action inhibits the apoptosis pathway in hypoxic cerebral microvascular endothelial cells, thereby safeguarding the blood-brain barrier and the nervous system.

A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes.

The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes.

ERBB1 alleviates secondary brain injury induced by experimental intracerebral hemorrhage in rats by modulating neuronal death via PLC-γ/PKC pathway.

AIMS: Intracerebral hemorrhage (ICH) is a disease with high rates of disability and mortality. The role of epidermal growth factor receptor 1 (ERBB1) in ICH was elucidated in this study. METHODS: ICH model was constructed by injecting autologous arterial blood into the right basal ganglia. The protein level of ERBB1 was detected by western blot analysis. To up- and downregulation of ERBB1 in rats, intraventricular injection of a lentivirus overexpression vector of ERBB1 and AG1478 (a specific inhibitor of ERBB1) was used. The cell apoptosis, neuronal loss, and pro-inflammatory cytokines were assessed by TUNEL, Nissl staining, and ELISA. Meanwhile, behavioral cognitive impairment of ICH rats was evaluated after ERBB1-targeted interventions. RESULTS: ERBB1 increased significantly in brain tissue of ICH rats. Overexpression of ERBB1 remarkably reduced cell apoptosis and neuronal loss induced by ICH, as well as pro-inflammatory cytokines and oxidative stress. Meanwhile, the behavioral and cognitive impairment of ICH rats were alleviated after upregulation of ERBB1; however, the secondary brain injury (SBI) was aggravated by AG1478 treatment. Furthermore, the upregulation of PLC-γ and PKC in ICH rats was reversed by AG1478 treatment. CONCLUSIONS: ERBB1 can improve SBI and has a neuroprotective effect in experimental ICH rats via PLC-γ/PKC pathway.

Cathelicidin boosts the antifungal activity of neutrophils and improves prognosis during Aspergillus fumigatus keratitis.

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.

Hypericin Alleviates Chronic Kidney Disease-induced Left Ventricular Hypertrophy by Regulation of FGF23-FGFR4 Signaling Pathway.

ABSTRACT: Chronic kidney disease (CKD) is a significant global health threat that imposes a substantial burden on both individuals and societies. CKD frequently correlates with cardiovascular events, particularly left ventricular hypertrophy (LVH), which contributes to the high mortality rate associated with CKD. Fibroblast growth factor 23 (FGF23), a hormone primarily involved in regulating calcium and phosphorus metabolism, has been identified as a major risk factor for LVH in CKD patients. Elevated serum FGF23 levels are known to induce LVH and myocardial fibrosis by activating the fibroblast growth factor receptor 4 (FGFR4) signal pathway. Therefore, targeting FGFR4 and its downstream signaling pathways holds potential as a treatment strategy for cardiac dysfunction in CKD. In our current study, we have discovered that Hypericin, a key component derived from Hypericum perforatum , has the ability to alleviate CKD-related LVH by targeting the FGFR4/phospholipase C gamma 1 (PLCγ1) signaling pathway. Through in vitro experiments using rat cardiac myocyte H9c2 cells, we observed that Hypericin effectively inhibits FGF23-induced hypertrophy and fibrosis by suppressing the FGFR4/PLCγ1/calcineurin/nuclear factor of activated T-cell (NFAT3) signaling pathway. In addition, our in vivo studies using mice on a high-phosphate diet and rat models of 5/6 nephrectomy demonstrated that Hypericin has therapeutic effects against CKD-induced LVH by modulating the FGFR4/PLCγ1/calcineurin/NFAT3 signaling pathway. In conclusion, our research highlights the potential of Hypericin as a candidate for the treatment of CKD-induced cardiomyopathy. By suppressing the FGFR4/PLCγ1 signaling pathway, Hypericin shows promise in attenuating LVH and myocardial fibrosis associated with CKD.

Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

Ginkgetin effectively mitigates collagen and AA-induced platelet activation via PLCγ2 but not cyclic nucleotide-dependent pathway in human.

Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 µM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca2+ ]i) levels and thromboxane A2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoproteinSer157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs.

Nuclear Binding Protein 2/Nesfatin-1 Affects Trophoblast Cell Fusion during Placental Development via the EGFR-PLCG1-CAMK4 Pathway.

Previous studies have shown that nuclear binding protein 2 (NUCB2) is expressed in the human placenta and increases with an increase in the syncytialization of trophoblast cells. This study aimed to investigate the role of NUCB2 in the differentiation and fusion of trophectoderm cells. In this study, the expression levels of NUCB2 and E-cadherin in the placentas of rats at different gestation stages were investigated. The results showed that there was an opposite trend between the expression of placental NUCB2 and E-cadherin in rat placentas in different trimesters. When primary human trophoblast (PHT) and BeWo cells were treated with high concentrations of Nesfatin-1, the trophoblast cell syncytialization was significantly inhibited. The effects of NUCB2 knockdown in BeWo cells and Forskolin-induced syncytialization were investigated. These cells showed a significantly decreased cell fusion rate. The mechanism underlying NUCB2-regulated trophoblast cell syncytialization was explored using RNA-Seq and the results indicated that the epidermal growth factor receptor (EGFR)-phospholipase C gamma 1 (PLCG1)-calmodulin-dependent protein kinase IV (CAMK4) pathway might be involved. The results suggested that the placental expression of NUCB2 plays an important role in the fusion of trophoblasts during differentiation via the EGFR-PLCG1-CAMK4 pathway.

8-Br-cGMP suppresses tumor progression through EGFR/PLC γ1 pathway in epithelial ovarian cancer.

BACKGROUND: Cyclic guanosine monophosphate (cGMP)-dependent protein kinase I (PKG-I), a serine/threonine kinase, is important in tumor development. The present study determines that the cGMP/PKG I pathway is essential for promoting cell proliferation and survival in human ovarian cancer cells, whereas cGMP analog has been shown to lead to growth inhibition and apoptosis of various cancer cells. The role of cGMP/PKG I pathway in epithelial ovarian cancer (EOC), therefore, remains controversial. We investigated the effect of cGMP/PKG I pathway and the underlying mechanism in EOC. METHODS AND RESULTS: The results showed that exogenous 8-Bromoguanosine-3', 5'-cyclic monophosphate (8-Br-cGMP) (cGMP analog) could antagonize the effects by EGF, including suppressing proliferation, invasion and migration of EOC cells. In vivo, 8-Br-cGMP hampered the growth of the xenograft tumor. Additionally, the expressions of epidermal growth factor receptor (EGFR), matrix metallopeptidase 9 (MMP9), proliferating cell nuclear antigen and Ki67 in xenograft tumor were decreased after 8-Br-cGMP intervention. Further research demonstrated that 8-Br-cGMP decreased the phosphorylation of EGFR (Y992) and downstream proteins phospholipase Cγ1 (PLC γ1) (Y783), calmodulin kinase II (T286) and inhibited cytoplasmic Ca2+ release as well as PKC transferring to cell membrane. It's worth noting that the inhibition was 8-Br-cGMP dose-dependent and 8-Br-cGMP showed similar inhibitory effect on EOC cells compared with U-73122, a specific inhibitor of PLC γ1. CONCLUSIONS: The activation of endogenous PKG I by addition of exogenous 8-Br-cGMP could inhibit EOC development probably via EGFR/PLCγ1 signaling pathway. 8-Br-cGMP/PKG I provide a new insight and strategy for EOC treatment.

Auricular Vagal Nerve Stimulation Inhibited Central Nerve Growth Factor/Tropomyosin Receptor Kinase A/Phospholipase C-Gamma Signaling Pathway in Functional Dyspepsia Model Rats With Gastric Hypersensitivity.

OBJECTIVE: Functional dyspepsia (FD), which has a complicated pathophysiologic process, is a common functional gastrointestinal disease. Gastric hypersensitivity is the key pathophysiological factor in patients with FD with chronic visceral pain. Auricular vagal nerve stimulation (AVNS) has the therapeutic effect of reducing gastric hypersensitivity by regulating the activity of the vagus nerve. However, the potential molecular mechanism is still unclear. Therefore, we investigated the effects of AVNS on the brain-gut axis through the central nerve growth factor (NGF)/ tropomyosin receptor kinase A (TrkA)/phospholipase C-gamma (PLC-γ) signaling pathway in FD model rats with gastric hypersensitivity. MATERIALS AND METHODS: We established the FD model rats with gastric hypersensitivity by means of colon administration of trinitrobenzenesulfonic acid on ten-day-old rat pups, whereas the control rats were given normal saline. AVNS, sham AVNS, K252a (an inhibitor of TrkA, intraperitoneally), and K252a + AVNS were performed on eight-week-old model rats for five consecutive days. The therapeutic effect of AVNS on gastric hypersensitivity was determined by the measurement of abdominal withdrawal reflex response to gastric distention. NGF in gastric fundus and NGF, TrkA, PLC-γ, and transient receptor potential vanilloid 1 (TRPV1) in the nucleus tractus solitaries (NTS) were detected separately by polymerase chain reaction, Western blot, and immunofluorescence tests. RESULTS: It was found that a high level of NGF in gastric fundus and an upregulation of the NGF/TrkA/PLC-γ signaling pathway in NTS were manifested in model rats. Meanwhile, both AVNS treatment and the administration of K252a not only decreased NGF messenger ribonucleic acid (mRNA) and protein expressions in gastric fundus but also reduced the mRNA expressions of NGF, TrkA, PLC-γ, and TRPV1 and inhibited the protein levels and hyperactive phosphorylation of TrkA/PLC-γ in NTS. In addition, the expressions of NGF and TrkA proteins in NTS were decreased significantly after the immunofluorescence assay. The K252a + AVNS treatment exerted a more sensitive effect on regulating the molecular expressions of the signal pathway than did the K252a treatment. CONCLUSION: AVNS can regulate the brain-gut axis effectively through the central NGF/TrkA/PLC-γ signaling pathway in the NTS, which suggests a potential molecular mechanism of AVNS in ameliorating visceral hypersensitivity in FD model rats.

Human PLCG2 haploinsufficiency results in a novel natural killer cell immunodeficiency.

BACKGROUND: Although most individuals effectively control herpesvirus infections, some suffer from severe and/or recurrent infections. A subset of these patients possess defects in natural killer (NK) cells, lymphocytes that recognize and lyse herpesvirus-infected cells; however, the genetic etiology is rarely diagnosed. PLCG2 encodes a signaling protein in NK-cell and B-cell signaling. Dominant-negative or gain-of-function variants in PLCG2 cause cold urticaria, antibody deficiency, and autoinflammation. However, loss-of-function variants and haploinsufficiency have not been reported to date. OBJECTIVES: The investigators aimed to identify the genetic cause of NK-cell immunodeficiency in 2 families and herein describe the functional consequences of 2 novel loss-of-function variants in PLCG2. METHODS: The investigators employed whole-exome sequencing in conjunction with mass cytometry, microscopy, functional assays, and a mouse model of PLCG2 haploinsufficiency to investigate 2 families with NK-cell immunodeficiency. RESULTS: The investigators identified novel heterozygous variants in PLCG2 in 2 families with severe and/or recurrent herpesvirus infections. In vitro studies demonstrated that these variants were loss of function due to haploinsufficiency with impaired NK-cell calcium flux and cytotoxicity. In contrast to previous PLCG2 variants, B-cell function remained intact. Plcg2+/- mice also displayed impaired NK-cell function with preserved B-cell function, phenocopying human disease. CONCLUSIONS: PLCG2 haploinsufficiency represents a distinct syndrome from previous variants characterized by NK-cell immunodeficiency with herpesvirus susceptibility, expanding the spectrum of PLCG2-related disease.

The hypermorphic PLCγ2 S707Y variant dysregulates microglial cell function - Insight into PLCγ2 activation in brain health and disease, and opportunities for therapeutic modulation.

Phospholipase C-gamma 2 (PLCγ2) is highly expressed in hematopoietic and immune cells, where it is a key signalling node enabling diverse cellular functions. Within the periphery, gain-of-function (GOF) PLCγ2 variants, such as the strongly hypermorphic S707Y, cause severe immune dysregulation. The milder hypermorphic mutation PLCγ2 P522R increases longevity and confers protection in central nervous system (CNS) neurodegenerative disorders, implicating PLCγ2 as a novel therapeutic target for treating these CNS indications. Currently, nothing is known about what consequences strong PLCγ2 GOF has on CNS functionality, and more precisely on the specific biological functions of microglia. Using the PLCγ2 S707Y variant as a model of chronic activation we investigated the functional consequences of strong PLCγ2 GOF on human microglia. PLCγ2 S707Y expressing human inducible pluripotent stem cells (hiPSC)-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, compared to PLCγ2 wild type. Despite the increase in PLCγ2 enzymatic activity, the PLCγ2 S707Y hiPSC-derived microglia display diminished functionality for key microglial processes including phagocytosis and cytokine secretion upon inflammatory challenge. RNA sequencing revealed a downregulation of genes related to innate immunity and response, providing molecular support for the phenotype observed. Our data suggests that chronic activation of PLCγ2 elicits a detrimental phenotype that is contributing to unfavourable CNS functions, and informs on the therapeutic window for targeting PLCγ2 in the CNS. Drug candidates targeting PLCγ2 will need to precisely mimic the effects of the PLCγ2 P522R variant on microglial function, but not those of the PLCγ2 S707Y variant.

Bruton's Tyrosine Kinase Inhibitors with Distinct Binding Modes Reveal Differential Functional Impact on B-Cell Receptor Signaling.

Small molecule inhibitors of Bruton's tyrosine kinase (BTK) have been approved for the treatment of multiple B-cell malignancies and are being evaluated for autoimmune and inflammatory diseases. Various BTK inhibitors (BTKi) have distinct potencies, selectivity profiles, and binding modes within the ATP-binding site. On the basis of the latter feature, BTKis can be classified into those that occupy the back-pocket, H3 pocket, and the hinge region only. Hypothesizing that differing binding modes may have differential impact on the B-cell receptor (BCR) signaling pathway, we evaluated the activities of multiple BTKis in B-cell lymphoma models in vitro and in vivo. We demonstrated that, although all three types of BTKis potently inhibited BTK-Y223 autophosphorylation and phospholipase C gamma 2 (PLCγ2)-Y1217 transphosphorylation, hinge-only binders were defective in inhibiting BTK-mediated calcium mobilization upon BCR activation. In addition, PLCγ2 activation was effectively blocked by back-pocket and H3 pocket binders but not by hinge-only binders. Further investigation using TMD8 cells deficient in Rac family small GTPase 2 (RAC2) revealed that RAC2 functioned as a bypass mechanism, allowing for residual BCR signaling and PLCγ2 activation when BTK kinase activity was fully inhibited by the hinge-only binders. These data reveal a kinase activity-independent function of BTK, involving RAC2 in transducing BCR signaling events, and provide mechanistic rationale for the selection of clinical candidates for B-cell lymphoma indications.

PLCG2-associated immune dysregulation (PLAID) comprises broad and distinct clinical presentations related to functional classes of genetic variants.

BACKGROUND: Pathogenic variants of phospholipase C gamma 2 (PLCG2) cause 2 related forms of autosomal-dominant immune dysregulation (ID), PLCγ2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammatory PLAID (APLAID). Since describing these conditions, many PLCG2 variants of uncertain significance have been identified by clinical sequencing of patients with diverse features of ID. OBJECTIVE: We sought to functionally classify PLCG2 variants and explore known and novel genotype-function-phenotype relationships. METHODS: Clinical data from patients with PLCG2 variants were obtained via standardized questionnaire. PLCG2 variants were generated by mutagenesis of enhanced green fluorescent protein (EGFP)-PLCG2 plasmid, which was overexpressed in Plcg2-deficient DT-40 B cells. B-cell receptor-induced calcium flux and extracellular signal-regulated kinase phosphorylation were assayed by flow cytometry. In some cases, stimulation-induced calcium flux was also measured in primary patient cells. RESULTS: Three-fourths of PLCG2 variants produced functional alteration of B-cell activation, in vitro. Thirteen variants led to gain of function (GOF); however, most functional variants defined a new class of PLCG2 mutation, monoallelic loss of function (LOF). Susceptibility to infection and autoinflammation were common with both GOF and LOF variants, whereas a new phenotypic cluster consisting of humoral immune deficiency, autoinflammation, susceptibility to herpesvirus infection, and natural killer cell dysfunction was observed in association with multiple heterozygous LOF variants detected in both familial and sporadic cases. In some cases, PLCG2 variants produced greater effects in natural killer cells than in B cells. CONCLUSIONS: This work expands the genotypic and phenotypic associations with functional variation in PLCG2, including a novel form of ID in carriers of heterozygous loss of PLCG2 function. It also demonstrates the need for more diverse assays for assessing the impact of PLCG2 variants on human disease.

Signaling pathways regulating the extracellular digestion of lipoprotein aggregates by macrophages.

The interaction between aggregated low-density lipoprotein (agLDL) and macrophages in arteries plays a major role in atherosclerosis. Macrophages digest agLDL and generate free cholesterol in an extracellular, acidic, hydrolytic compartment known as the lysosomal synapse. Macrophages form a tight seal around agLDL through actin polymerization and deliver lysosomal contents into this space in a process termed digestive exophagy. Our laboratory has identified TLR4 activation of MyD88/Syk as critical for digestive exophagy. Here we use pharmacological agents and siRNA knockdown to characterize signaling pathways downstream of Syk that are involved in digestive exophagy. Syk activates Bruton's tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2). We show that PLCγ2 and to a lesser extent BTK regulate digestive exophagy. PLCγ2 cleaves PI(4,5)P2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Soluble IP3 activates release of Ca2+ from the endoplasmic reticulum (ER). We demonstrate that Ca2+ release from the ER is upregulated by agLDL and plays a key role in digestive exophagy. Both DAG and Ca2+ activate protein kinase Cα (PKCα). We find that PKCα is an important regulator of digestive exophagy. These results expand our understanding of the mechanisms of digestive exophagy, which could be useful in developing therapeutic interventions to slow development of atherosclerosis.

Scaffold protein SH3BP2 signalosome is pivotal for immune activation in nephrotic syndrome.

Despite clinical use of immunosuppressive agents, the immunopathogenesis of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) remains unclear. Src homology 3-binding protein 2 (SH3BP2), a scaffold protein, forms an immune signaling complex (signalosome) with 17 other proteins, including phospholipase Cγ2 (PLCγ2) and Rho-guanine nucleotide exchange factor VAV2 (VAV2). Bioinformatic analysis of human glomerular transcriptome (Nephrotic Syndrome Study Network cohort) revealed upregulated SH3BP2 in MCD and FSGS. The SH3BP2 signalosome score and downstream MyD88, TRIF, and NFATc1 were significantly upregulated in MCD and FSGS. Immune pathway activation scores for Toll-like receptors, cytokine-cytokine receptor, and NOD-like receptors were increased in FSGS. Lower SH3BP2 signalosome score was associated with MCD, higher estimated glomerular filtration rate, and remission. Further work using Sh3bp2KI/KI transgenic mice with a gain-in-function mutation showed ~6-fold and ~25-fold increases in albuminuria at 4 and 12 weeks, respectively. Decreased serum albumin and unchanged serum creatinine were observed at 12 weeks. Sh3bp2KI/KI kidney morphology appeared normal except for increased mesangial cellularity and patchy foot process fusion without electron-dense deposits. SH3BP2 co-immunoprecipitated with PLCγ2 and VAV2 in human podocytes, underscoring the importance of SH3BP2 in immune activation. SH3BP2 and its binding partners may determine the immune activation pathways resulting in podocyte injury leading to loss of the glomerular filtration barrier.

Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.