PLCß2 Promotes VEGF-Induced Vascular Permeability.

BACKGROUND: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need. METHODS: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) ß2. RESULTS: Global knock-out of PLCß2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCß2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCß2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCß2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls. CONCLUSIONS: The results implicate PLCß2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCß2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.

Bitter taste receptor T2R14 detects quorum sensing molecules from cariogenic Streptococcus mutans and mediates innate immune responses in gingival epithelial cells.

Host-pathogen interactions play an important role in defining the outcome of a disease. Recent studies have shown that the bacterial quorum sensing molecules (QSM) can interact with host cell membrane proteins, mainly G protein-coupled receptors (GPCRs), and induce innate immune responses. However, few studies have examined QSM-GPCR interactions and their influence on oral innate immune responses. In this study, we examined the role of bitter taste receptor T2R14 in sensing competence stimulating peptides (CSPs) secreted by cariogenic bacterium Streptococcus mutans and in mediating innate immune responses in gingival epithelial cells (GECs). Transcriptomic and western blot analyses identify T2R14 to be highly expressed in GECs. Our data show that only CSP-1 from S. mutans induces robust intracellular calcium mobilization compared to CSP-2 and CSP-3. By using CRISPR-Cas9, we demonstrate that CSP-1 induced calcium signaling and secretion of cytokines CXCL-8/IL-8, TNF-α, and IL-6 is mediated through T2R14 in GECs. Interestingly, the NF-kB signaling activated by CSP-1 in GECs was independent of T2R14. CSP-1-primed GECs attracted differentiated HL-60 immune cells (dHL-60) and this effect was abolished in T2R14 knock down GECs and also in cells primed with T2R14 antagonist 6-Methoxyflavone (6-MF). Our findings identify S. mutans CSP-1 as a peptide ligand for the T2R family. Our study establishes a novel host-pathogen interaction between cariogenic S. mutans CSP-1 and T2R14 in GECs leading to an innate immune response. Collectively, these findings suggest T2Rs as potential therapeutic targets to modulate innate immune responses upon oral bacterial infections.

Functional characterization of uveal melanoma oncogenes.

Uveal melanoma (UM) is a currently untreatable form of melanoma with a 50% mortality rate. Characterization of the essential signaling pathways driving this cancer is critical to develop target therapies. Activating mutations in the Gαq signaling pathway at the level of GNAQ, GNA11, or rarely CYSLTR2 or PLCß4 are considered alterations driving proliferation in UM and several other neoplastic disorders. Here, we systematically examined the oncogenic signaling output of various mutations recurrently identified in human tumors. We demonstrate that CYSLTR2 → GNAQ/11 → PLCß act in a linear signaling cascade that, via protein kinase C (PKC), activates in parallel the MAP-kinase and FAK/Yes-associated protein pathways. Using genetic ablation and pharmacological inhibition, we show that the PKC/RasGRP3/MAPK signaling branch is the essential component that drives the proliferation of UM. Only inhibition of the MAPK branch but not the FAK branch synergizes with inhibition of the proximal cascade, providing a blueprint for combination therapy. All oncogenic signaling could be extinguished by the novel GNAQ/11 inhibitor YM-254890, in all UM cells with driver mutation in the Gαq subunit or the upstream receptor. Our findings highlight the GNAQ/11 → PLCß â†’ PKC → MAPK pathway as the central signaling axis to be suppressed pharmacologically to treat for neoplastic disorders with Gαq pathway mutations.

Glycogen Synthase Kinase-3 and phospholipase C-beta signalling: Roles and possible interactions in myelodysplastic syndromes and acute myeloid leukemia.

GSK-3 and PLCbeta enzymes are responsible for the regulation of several signalling pathways related to many cellular functions. In hematopoietic cells, GSK-3 deficiency is correlated with an MDS-like phenotype and with leukemogenesis, showing a prognostic potential in AML cells. GSK-3 interacts with Wnt or MAPK signalling, but it is also linked to PI3K/Akt/mTOR pathways to regulate cell proliferation and apoptosis of hematopoietic stem cell progenitors. PLCbeta enzymes are involved in cell cycle progression of hematopoietic, MDS/AML and immune cells, through activation of PKC or calcium signalling. Of note, a PLCbeta1/PKCalpha pathway is modulated during MDS pathogenesis, with a specific involvement of the inositides localized in the nucleus. Here we focus on GSK-3 and PLCbeta signalling, describing the many evidences that underline the pivotal role of both GSK-3 and PLCbeta-dependent pathways in MDS/AML, their association with therapy and their possible interactions.

Evidence that a lipolytic enzyme--hematopoietic-specific phospholipase C-ß2--promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes.

Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4ß1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-ß2 (PLC-ß2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-ß2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs.

Association of activated Gαq to the tumor suppressor Fhit is enhanced by phospholipase Cß.

BACKGROUND: G proteins are known to modulate various growth signals and are implicated in the regulation of tumorigenesis. The tumor suppressor Fhit is a newly identified interaction partner of Gq proteins that typically stimulate the phospholipase C pathway. Activated Gαq subunits have been shown to interact directly with Fhit, up-regulate Fhit expression and enhance its suppressive effect on cell growth and migration. Other signaling molecules may be involved in modulating Gαq/Fhit interaction. METHODS: To test the relationship of PLCß with the interaction between Gαq and Fhit, co-immunoprecipication assay was performed on HEK293 cells co-transfected with different combinations of Flag-Fhit, Gα16, Gα16QL, pcDNA3 vector, and PLCß isoforms. Possible associations of Fhit with other effectors of Gαq were also demonstrated by co-immunoprecipitation. The regions of Gαq for Fhit interaction and PLCß stimulation were further evaluated by inositol phosphates accumulation assay using a series of Gα16/z chimeras with discrete regions of Gα16 replaced by those of Gαz. RESULTS: PLCß1, 2 and 3 interacted with Fhit regardless of the expression of Gαq. Expression of PLCß increased the affinities of Fhit for both wild-type and activated Gαq. Swapping of the Fhit-interacting α2-ß4 region of Gαq with Gαi eliminated the association of Gαq with Fhit without affecting the ability of the mutant to stimulate PLCß. Other effectors of Gαq including RGS2 and p63RhoGEF were unable to interact with Fhit. CONCLUSIONS: PLCß may participate in the regulation of Fhit by Gq in a unique way. PLCß interacts with Fhit and increases the interaction between Gαq and Fhit. The Gαq/PLCß/Fhit complex formation points to a novel signaling pathway that may negatively regulate tumor cell growth.

Estrogen regulates Hippo signaling via GPER in breast cancer.

The G protein-coupled estrogen receptor (GPER) mediates both the genomic and nongenomic effects of estrogen and has been implicated in breast cancer development. Here, we compared GPER expression in cancerous tissue and adjacent normal tissue in patients with invasive ductal carcinoma (IDC) of the breast and determined that GPER is highly upregulated in cancerous cells. Additionally, our studies revealed that GPER stimulation activates yes-associated protein 1 (YAP) and transcriptional coactivator with a PDZ-binding domain (TAZ), 2 homologous transcription coactivators and key effectors of the Hippo tumor suppressor pathway, via the Gαq-11, PLCß/PKC, and Rho/ROCK signaling pathways. TAZ was required for GPER-induced gene transcription, breast cancer cell proliferation and migration, and tumor growth. Moreover, TAZ expression positively correlated with GPER expression in human IDC specimens. Together, our results suggest that the Hippo/YAP/TAZ pathway is a key downstream signaling branch of GPER and plays a critical role in breast tumorigenesis.

Nuclear PI-PLC ß1 and Myelodysplastic syndromes: from bench to clinics.

Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older adult patients, show ineffective hematopoiesis in one or more of the lineages of the bone marrow. A number of MDS progresses to acute myeloid leukemia (AML) with the involvement of genetic and epigenetic mechanisms affecting PI-PLC ß1. The molecular mechanisms underlying the MDS evolution to AML are still unclear, even though it is now clear that the nuclear signaling elicited by PI-PLC ß1, Cyclin D3, and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Moreover, a correlation between other PI-PLCs, such as PI-PLC ß3, kinases and phosphatases has been postulated in MDS pathogenesis. Here, we review the findings hinting at the role of nuclear lipid signaling pathways in MDS, which could become promising therapeutic targets.

Short Stat5-interacting peptide derived from phospholipase C-ß3 inhibits hematopoietic cell proliferation and myeloid differentiation.

Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN). Our recent study found that phospholipase C (PLC)-ß3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-ß3-dependent manner. PLC-ß3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-ß3 (PLC-ß3-CT) accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-ß3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-ß3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-ß3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.

Physiology and pathology of nuclear phospholipase C ß1.

The existence and function of inositide signaling in the nucleus is well documented and we know that the existence of the inositide cycle inside the nucleus has a biological role. An autonomous lipid-dependent signaling system, independently regulated from its plasma membrane counterpart, acts in the nucleus and modulates cell cycle progression and differentiation.We and others focused on PLCß1, which is the most extensively investigated PLC isoform in the nuclear compartment. PLCß1 is a key player in the regulation of nuclear inositol lipid signaling, and, as discussed above, its function could also be involved in nuclear structure because it hydrolyses PtdIns(4,5)P2, a well accepted regulator of chromatin remodelling. The evidence, in a number of patients with myelodysplastic syndromes, that the mono-allelic deletion of PLCß1 is associated with an increased risk of developing acute myeloid leukemia paves the way for an entirely new field of investigation. Indeed the genetic defect evidenced, in addition to being a useful prognostic tool, also suggests that altered expression of this enzyme could have a role in the pathogenesis of this disease, by causing an imbalance between proliferation and apoptosis. The epigenetics of PLCß1 expression in MDS has been reviewed as well.

Lyn- and PLC-beta3-dependent regulation of SHP-1 phosphorylation controls Stat5 activity and myelomonocytic leukemia-like disease.

Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-ß3-dependent manner. Thus, PLC-ß3-deficient mice develop myeloproliferative neoplasm, like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-ß3 doubly deficient lyn(-/-);PLC-ß3(-/-) mice develop a Stat5-dependent, fatal myelodysplastic/myeloproliferative neoplasm, similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-ß3(-/-) mice that cause the CMML-like disease, phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore, we identify a novel Lyn/PLC-ß3-mediated regulatory mechanism of SHP-1 and Stat5 activities.

Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2.

Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca(2+) responses to Galphai-linked receptors, including the C5aR, but reduced responses to Galphaq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca(2+) responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCbeta isoform-deficient BMDM, we found that ToxB inhibited Ca(2+) signaling through PLCbeta4 but enhanced signaling through PLCbeta3. Effects of ToxB on GPCR Ca(2+) responses correlated with GPCR use of PLCbeta3 versus PLCbeta4. ToxB inhibited UDP Ca(2+) signaling without reducing InsP3 production or the sensitivity of cellular Ca(2+) stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca(2+)coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca(2+) signaling by C5a was prevented by inhibition of PLA(2) or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca(2+) signaling by different GPCR-linked PLCbeta isoforms in macrophages.

Selective activation of the "b" splice variant of phospholipase Cbeta1 in chronically dilated human and mouse atria.

Atrial fibrillation (AF) is commonly associated with chronic dilatation of the left atrium, both in human disease and animal models. The immediate signaling enzyme phospholipase C (PLC) is activated by mechanical stretch to generate the Ca2+-releasing messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and sn-1,2-diacylglycerol (DAG), an activator of protein kinase C subtypes. There is also evidence that heightened activity of PLC, caused by the receptor coupling protein Gq, can contribute to atrial remodelling. We examined PLC activation in right and left atrial appendage from patients with mitral valve disease (VHD) and in a mouse model of dilated cardiomyopathy caused by transgenic overexpression of the stress-activated protein kinase, mammalian sterile 20 like kinase 1 (Mst1) (Mst1-TG). PLC activation was heightened 6- to 10-fold in atria from VHD patients compared with right atrial tissue from patients undergoing coronary artery bypass surgery (CABG) and was also heightened in the dilated atria from Mst1-TG. PLC activation in human left atrial appendage and in mouse left atria correlated with left atrial size, implying a relationship between PLC activation and chronic dilatation. Dilated atria from human and mouse showed heightened expression of PLCbeta1b, but not of other PLC subtypes. PLCbeta1b, but not PLCbeta1a, caused apoptosis when overexpressed in neonatal rat cardiomyocytes, suggesting that PLCbeta1b may contribute to chamber dilatation. The activation of PLCbeta1b is a possible therapeutic target to limit atrial remodelling in VHD patients.

The SPS affair: a complex tale of illicit proliferation.

In this issue of Cancer Cell, Xiao et al. report that PLC-beta3 mutant mice develop myeloprolfierative neoplasms and show that tumor suppressor activity does not require PLC-beta3 catalytic activity. Instead, PLC-beta3 forms a complex with SHP-1 and Stat5 that facilitates Stat5 dephosphorylation. A similar mechanism may be operative in some human leukemias.

Tumor suppression by phospholipase C-beta3 via SHP-1-mediated dephosphorylation of Stat5.

Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-beta3-deficient mice develop myeloproliferative disease, lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-beta3 deficiency was suggested in mouse lymphomas in PLC-beta3(-/-) and in Emicro-myc;PLC-beta3(+/-) mice and human Burkitt's lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-beta3 is likely a tumor suppressor.

Ins(1,4,5)P(3) regulates phospholipase Cbeta1 expression in cardiomyocytes.

The functional significance of the Ca2+-releasing second messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3), IP(3)) in the heart has been controversial. Ins(1,4,5)P(3) is generated from the precursor lipid phosphatidylinositol(4,5)bisphosphate (PIP(2)) along with sn-1,2-diacylglycerol, and both of these are important cardiac effectors. Therefore, to evaluate the functional importance of Ins(1,4,5)P(3) in cardiomyocytes (NRVM), we overexpressed IP(3) 5-phosphatase to increase degradation. Overexpression of IP(3) 5-phosphatase reduced Ins(1,4,5)P(3) responses to alpha(1)-adrenergic receptor agonists acutely, but with longer stimulation, caused an overall increase in phospholipase C (PLC) activity, associated with a selective increase in expression of PLCbeta1, that served to normalise Ins(1,4,5)P(3) content. Similar increases in PLC activity and PLCbeta1 expression were observed when Ins(1,4,5)P(3) was sequestered onto the PH domain of PLCdelta1, a high affinity selective Ins(1,4,5)P(3)-binding motif. These findings suggested that the available level of Ins(1,4,5)P(3) selectively regulates the expression of PLCbeta1. Cardiac responses to Ins(1,4,5)P(3) are mediated by type 2 IP(3)-receptors. Hearts from IP(3)-receptor (type 2) knock-out mice showed heightened PLCbeta1 expression. We conclude that Ins(1,4,5)P(3) and IP(3)-receptor (type 2) regulate PLCbeta1 and thereby maintain levels of Ins(1,4,5)P(3). This implies some functional significance for Ins(1,4,5)P(3) in the heart.

The angiotensin receptor type 1-Gq protein-phosphatidyl inositol phospholipase Cbeta-protein kinase C pathway is involved in activation of proximal tubule Na+-ATPase activity by angiotensin(1-7) in pig kidneys.

In a previous study, we observed that angiotensin(1-7) (Ang(1-7)) stimulates proximal tubule Na+-ATPase activity through the angiotensin receptor type 1 (AT1R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1-7) on Na+-ATPase activity through AT1R involves a Gq protein-phosphatidyl inositol-phospholipase Cbeta (PI-PLCbeta) pathway because: (1) the effect was reversed by GDPbetaS, a non-hydrolysable GDP analogue, and by a monoclonal Gq protein antibody; (2) the effect was similar and not additive to that of GTPgammaS, a non-hydrolysable GTP analogue; (3) Ang(1-7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCbeta substrate; and (4) U73122, a specific inhibitor of PI-PLCbeta, abolished Ang(1-7)-induced stimulation of Na+-ATPase activity. Angiotensin(1-7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT1R. The stimulatory effects of Ang(1-7) and PMA on Na+-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na+-ATPase activity. These data show that Ang(1-7) stimulates Na+-ATPase activity through the AT1R-Gq protein-PI-PLCbeta-PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.

Nuclear phospholipase C beta1 and cellular differentiation.

Phosphoinositides (PI) are the most extensively studied lipids involved in cell signaling pathways. The bulk of PI is found in membranes where they are substrates for enzymes, such as kinases, phosphatases and phospholipases, which respond to the activation by cell-surface receptors. The outcome of the majority of signaling pathways involving lipid second messengers results in nuclear responses finally driving the cell into differentiation, proliferation or apoptosis. Some of these pathways are well established, such as that of PI-specific phospholipase C (PI-PLC), which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) into the two second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). Two independent cycles of PI are present inside the cell. One is localized at the plasma membrane, while the most recently discovered PI cycle is found inside the nuclear compartment. The regulation of the nuclear PI pool is totally independent from the plasma membrane counterpart, suggesting that the nucleus constitutes a functionally distinct compartment of inositol lipids metabolism. In this report we will focus on the signal transduction-related metabolism of nuclear PI and review the most convincing evidence that the PI cycle is involved in differentiation programs in several cell systems.