TBK1 at the Crossroads of Inflammation and Energy Homeostasis in Adipose Tissue.

The noncanonical IKK family member TANK-binding kinase 1 (TBK1) is activated by pro-inflammatory cytokines, but its role in controlling metabolism remains unclear. Here, we report that the kinase uniquely controls energy metabolism. Tbk1 expression is increased in adipocytes of HFD-fed mice. Adipocyte-specific TBK1 knockout (ATKO) attenuates HFD-induced obesity by increasing energy expenditure; further studies show that TBK1 directly inhibits AMPK to repress respiration and increase energy storage. Conversely, activation of AMPK under catabolic conditions can increase TBK1 activity through phosphorylation, mediated by AMPK's downstream target ULK1. Surprisingly, ATKO also exaggerates adipose tissue inflammation and insulin resistance. TBK1 suppresses inflammation by phosphorylating and inducing the degradation of the IKK kinase NIK, thus attenuating NF-κB activity. Moreover, TBK1 mediates the negative impact of AMPK activity on NF-κB activation. These data implicate a unique role for TBK1 in mediating bidirectional crosstalk between energy sensing and inflammatory signaling pathways in both over- and undernutrition.

Optogenetics-based localization of talin to the plasma membrane promotes activation of ß3 integrins.

Interaction of talin with the cytoplasmic tails of integrin ß triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In talin KO mice, loss of talin interaction with platelet integrin αIIbß3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell integrin αVß3 affects angiogenesis. In normal cells, talin is autoinhibited and localized in the cytoplasm. Here, we used an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (Arabidopsis cryptochrome 2 fused to the N terminus of talin; N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]) responsive to 450 nm (blue) light was inserted into Chinese hamster ovary cells and endothelial cells also expressing αIIbß3 or αVß3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of Arabidopsis cryptochrome 2-talin to the N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]-decorated plasma membrane. This resulted in ß3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for integrin activation, as membrane-associated Ras-related protein 1 (Rap1)-GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated ß3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of talin in ß3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP.

Optogenetic interrogation of integrin αVß3 function in endothelial cells.

The integrin αVß3 is reported to promote angiogenesis in some model systems but not in others. Here, we used optogenetics to study the effects of αVß3 interaction with the intracellular adapter kindlin-2 (Fermt2) on endothelial cell functions potentially relevant to angiogenesis. Because interaction of kindlin-2 with αVß3 requires the C-terminal three residues of the ß3 cytoplasmic tail (Arg-Gly-Thr; RGT), optogenetic probes LOVpep and ePDZ1 were fused to ß3ΔRGT-GFP and mCherry-kindlin-2, respectively, and expressed in ß3 integrin-null microvascular endothelial cells. Exposure of the cells to 450 nm (blue) light caused rapid and specific interaction of kindlin-2 with αVß3 as assessed by immunofluorescence and total internal reflection fluorescence (TIRF) microscopy, and it led to increased endothelial cell migration, podosome formation and angiogenic sprouting. Analyses of kindlin-2 mutants indicated that interaction of kindlin-2 with other kindlin-2 binding partners, including c-Src, actin, integrin-linked kinase and phosphoinositides, were also likely necessary for these endothelial cell responses. Thus, kindlin-2 promotes αVß3-dependent angiogenic functions of endothelial cells through its simultaneous interactions with ß3 integrin and several other binding partners. Optogenetic approaches should find further use in clarifying spatiotemporal aspects of vascular cell biology.

Interaction of kindlin-2 with integrin ß3 promotes outside-in signaling responses by the αVß3 vitronectin receptor.

The bidirectional signaling and hemostatic functions of platelet αIIbß3 are regulated by kindlin-3 through interactions with the ß3 cytoplasmic tail. Little is known about kindlin regulation of the related "vitronectin receptor," αVß3. These relationships were investigated in endothelial cells, which express αVß3 and kindlin-2 endogenously. "ß3ΔRGT" knock-in mice lack the 3 C-terminal ß3 tail residues, whereas in "ß3/ß1(EGK)" mice, RGT is replaced by the corresponding residues of ß1. The wild-type ß3 tail pulled down kindlin-2 and c-Src in vitro, whereas ß3ΔRGT bound neither protein and ß3/ß1(EGK) bound kindlin-2, but not c-Src. ß3ΔRGT endothelial cells, but not ß3/ß1(EGK) endothelial cells, exhibited migration and spreading defects on vitronectin and reduced sprouting in 3-dimensional fibrin. Short hairpin RNA silencing of kindlin-2, but not c-Src, blocked sprouting by ß3 wild-type endothelial cells. Moreover, defective sprouting by ß3ΔRGT endothelial cells could be rescued by conditional, forced interaction of αVß3ΔRGT with kindlin-2. Stimulation of ß3ΔRGT endothelial cells led to normal extracellular ligand binding to αVß3, pin-pointing their defect to one of outside-in αVß3 signaling. ß3ΔRGT mice, but not ß3/ß1(EGK) mice, exhibited defects in both developmental and tumor angiogenesis, responses that require endothelial cell function. Thus, the ß3/kindlin-2 interaction promotes outside-in αVß3 signaling selectively, with biological consequences in vivo.

Endothelial Cells Require CD98 for Efficient Angiogenesis-Brief Report.

OBJECTIVE: CD98 regulates integrin signaling and is critical for tumor cell proliferation. It is also expressed on endothelial cells (EC), but its role in angiogenesis is unclear. APPROACH AND RESULTS: We used specific genetic targeting and antibody blockade approaches to examine the function of CD98 in EC proliferation, blood vessel growth, and tumor angiogenesis. It is upregulated on angiogenic ECs, and EC-specific deletion of CD98 in mice inhibited tumor growth, retinal angiogenesis, and EC proliferation. Reconstitution with CD98 mutants showed that integrin and CD98 interaction is necessary for EC survival and growth. Moreover, anti-CD98 treatment inhibited vessel formation and reversed EC-assisted tumor growth. CONCLUSIONS: Our findings demonstrate a requirement for CD98 in EC growth and suggest that CD98-specific reagents could have a dual anticancer effect: directly by inhibiting tumor cell proliferation and indirectly by preventing tumor angiogenesis.

The TBK1/IKKε inhibitor amlexanox improves dyslipidemia and prevents atherosclerosis.

Cardiovascular diseases, especially atherosclerosis and its complications, are a leading cause of death. Inhibition of the noncanonical IκB kinases TANK-binding kinase 1 and IKKε with amlexanox restores insulin sensitivity and glucose homeostasis in diabetic mice and human patients. Here we report that amlexanox improves diet-induced hypertriglyceridemia and hypercholesterolemia in Western diet-fed (WD-fed) Ldlr-/- mice and protects against atherogenesis. Amlexanox ameliorated dyslipidemia, inflammation, and vascular dysfunction through synergistic actions that involve upregulation of bile acid synthesis to increase cholesterol excretion. Transcriptomic profiling demonstrated an elevated expression of key bile acid synthesis genes. Furthermore, we found that amlexanox attenuated monocytosis, eosinophilia, and vascular dysfunction during WD-induced atherosclerosis. These findings demonstrate the potential of amlexanox as a therapy for hypercholesterolemia and atherosclerosis.

An AMPK-caspase-6 axis controls liver damage in nonalcoholic steatohepatitis.

Liver cell death has an essential role in nonalcoholic steatohepatitis (NASH). The activity of the energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) is repressed in NASH. Liver-specific AMPK knockout aggravated liver damage in mouse NASH models. AMPK phosphorylated proapoptotic caspase-6 protein to inhibit its activation, keeping hepatocyte apoptosis in check. Suppression of AMPK activity relieved this inhibition, rendering caspase-6 activated in human and mouse NASH. AMPK activation or caspase-6 inhibition, even after the onset of NASH, improved liver damage and fibrosis. Once phosphorylation was decreased, caspase-6 was activated by caspase-3 or -7. Active caspase-6 cleaved Bid to induce cytochrome c release, generating a feedforward loop that leads to hepatocyte death. Thus, the AMPK-caspase-6 axis regulates liver damage in NASH, implicating AMPK and caspase-6 as therapeutic targets.

Neutralization of Oxidized Phospholipids Ameliorates Non-alcoholic Steatohepatitis.

Oxidized phospholipids (OxPLs), which arise due to oxidative stress, are proinflammatory and proatherogenic, but their roles in non-alcoholic steatohepatitis (NASH) are unknown. Here, we show that OxPLs accumulate in human and mouse NASH. Using a transgenic mouse that expresses a functional single-chain variable fragment of E06, a natural antibody that neutralizes OxPLs, we demonstrate the causal role of OxPLs in NASH. Targeting OxPLs in hyperlipidemic Ldlr-/- mice improved multiple aspects of NASH, including steatosis, inflammation, fibrosis, hepatocyte death, and progression to hepatocellular carcinoma. Mechanistically, we found that OxPLs promote ROS accumulation to induce mitochondrial dysfunction in hepatocytes. Neutralizing OxPLs in AMLN-diet-fed Ldlr-/- mice reduced oxidative stress, improved hepatic and adipose-tissue mitochondrial function, and fatty-acid oxidation. These results suggest targeting OxPLs may be an effective therapeutic strategy for NASH.

uPAR isoform 2 forms a dimer and induces severe kidney disease in mice.

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding ß3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin ß3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for ß3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.

P2 receptors in health and disease.

In the past ten years since the discovery and cloning of members of the P2 receptor family, rapid progress has been made in the field regarding the function and pharmacology of different P2 receptor subtypes. This research resulted in identifying these receptors as important players in the pathology of atherosclerosis, cystic fibrosis, neurodegenerative and autoimmune diseases, among other disorders. The signalling mechanisms whereby P2 receptors mediate pathogenesis are not clear in most cases. Future studies in this field will focus on the integration of signalling pathways coupled to P2 receptors and the generation of specific agonists/antagonists for each receptor subtype to provide strategies for the treatment of a variety of diseases.

P2X7 receptors stimulate AKT phosphorylation in astrocytes.

1. Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X(7) subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2. P2Y and P2X receptor agonists ATP, uridine 5'-triphosphate (UTP) and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X(7) receptor. Activation was maximal at 5 - 10 min and was sustained for 60 min; the EC(50) for BzATP was approximately 50 microM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X(7) receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5. In conclusion, our data indicate that stimulation of astrocytic P2X(7) receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.

The P2Y2 Receptor Interacts with VE-Cadherin and VEGF Receptor-2 to Regulate Rac1 Activity in Endothelial Cells.

Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the Gq protein-coupled P2Y2 nucleotide receptor (P2Y2R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y2R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1.

The primacy of ß1 integrin activation in the metastatic cascade.

After neoplastic cells leave the primary tumor and circulate, they may extravasate from the vasculature and colonize tissues to form metastases. ß1 integrins play diverse roles in tumorigenesis and tumor progression, including extravasation. In blood cells, activation of ß1 integrins can be regulated by "inside-out" signals leading to extravasation from the circulation into tissues. However, a role for inside-out ß1 activation in tumor cell metastasis is uncertain. Here we show that ß1 integrin activation promotes tumor metastasis and that activated ß1 integrin may serve as a biomarker of metastatic human melanoma. To determine whether ß1 integrin activation can influence tumor cell metastasis, the ß1 integrin subunit in melanoma and breast cancer cell lines was stably knocked down with shRNA and replaced with wild-type or constitutively-active ß1. When tumor cells expressing constitutively-active ß1 integrins were injected intravenously into chick embryos or mice, they demonstrated increased colonization of the liver when compared to cells expressing wild-type ß1 integrins. Rescue expression with mutant ß1 integrins revealed that tumor cell extravasation and hepatic colonization required extracellular ligand binding to ß1 as well as ß1 interaction with talin, an intracellular mediator of integrin activation by the Rap1 GTPase. Furthermore, shRNA-mediated knock down of talin reduced hepatic colonization by tumor cells expressing wild-type ß1, but not constitutively-active ß1. Overexpression in tumor cells of the tumor suppressor, Rap1GAP, inhibited Rap1 and ß1 integrin activation as well as hepatic colonization. Using an antibody that detects activated ß1 integrin, we found higher levels of activated ß1 integrins in human metastatic melanomas compared to primary melanomas, suggesting that activated ß1 integrin may serve as a biomarker of invasive tumor cells. Altogether, these studies establish that inside-out activation of ß1 integrins promotes tumor cell extravasation and colonization, suggesting diagnostic and therapeutic approaches for targeting of ß1 integrin signaling in neoplasia.

Rat parotid gland cell differentiation in three-dimensional culture.

The use of polarized salivary gland cell monolayers has contributed to our understanding of salivary gland physiology. However, these cell models are not representative of glandular epithelium in vivo, and, therefore, are not ideal for investigating salivary epithelial functions. The current study has developed a three-dimensional (3D) cell culture model for rat Par-C10 parotid gland cells that forms differentiated acinar-like spheres on Matrigel. These 3D Par-C10 acinar-like spheres display characteristics similar to differentiated acini in salivary glands, including cell polarization, tight junction (TJ) formation required to maintain transepithelial potential difference, basolateral expression of aquaporin-3 and Na+/K+/2Cl- cotransporter-1, and responsiveness to the muscarinic receptor agonist carbachol that is decreased by the anion channel blocker diphenylamine-2-carboxylic acid or chloride replacement with gluconate. Incubation of the spheres in the hypertonic medium increased the expression level of the water channel aquaporin-5. Further, the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma induced alterations in TJ integrity in the acinar-like spheres without affecting individual cell viability, suggesting that cytokines may affect salivary gland function by altering TJ integrity. Thus, 3D Par-C10 acinar-like spheres represent a novel in vitro model to study physiological and pathophysiological functions of differentiated acini.

The P2Y2 nucleotide receptor requires interaction with alpha v integrins to access and activate G12.

The P2Y2 nucleotide receptor (P2Y2R) interacts with alpha v integrins to activate G(o) and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with alpha v integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit G(q) or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative G alpha12 construct. G12 activation by the P2Y2R also was inhibited by anti-alpha v beta5 integrin antibodies and alpha v integrin antisense oligonucleotides, suggesting that alpha v integrin activity and expression are required for the P2Y2R to activate G12. Co-immunoprecipitation experiments confirmed that G alpha12 protein associates with the wild-type P2Y2R and with alpha v integrins but not with the RGE mutant P2Y2R or with alpha3 integrins. Collectively, these results suggest that alpha v integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis.

P2 receptors: intracellular signaling.

P2 receptors for extracellular nucleotides are divided into two categories: the ion channel receptors (P2X) and the G-protein-coupled receptors (P2Y). For the P2X receptors, signal transduction appears to be relatively simple. Upon activation by extracellular ATP, a channel comprised of P2X receptor subunits opens and allows cations to move across the plasma membrane, resulting in changes in the electrical potential of the cell that, in turn, propagates a signal. This regulated flux of ions across the plasma membrane has important signaling functions, especially in impulse propagation in the nervous system and in muscle contractility. In addition, P2X receptor activation causes the accumulation of calcium ions in the cytoplasm, which is responsible for activating numerous signaling molecules. For the P2Y receptors, signal transduction is more complex. Intracellular signaling cascades are the main routes of communication between G-protein-coupled receptors and regulatory targets within the cell. These signaling cascades operate mainly by the sequential activation or deactivation of heterotrimeric and monomeric G proteins, phospholipases, protein kinases, adenylyl and guanylyl cyclases, and phosphodiesterases that regulate many cellular processes, including proliferation, differentiation, apoptosis, metabolism, secretion, and cell migration. In addition, there are numerous ion channels, cell adhesion molecules and receptor tyrosine kinases that are modulated by P2Y receptors and operate to transmit an extracellular signal to an intracellular response. These intracellular signaling pathways and their regulation by P2 receptors are discussed in this review.

The P2Y2 nucleotide receptor interacts with alphav integrins to activate Go and induce cell migration.

Extracellular ATP and UTP induce chemotaxis, or directed cell migration, by stimulating the G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R). Previously, we found that an arginine-glycine-aspartic acid (RGD) integrin binding domain in the P2Y(2)R enables this receptor to interact selectively with alpha(v)beta(3) and alpha(V)beta(5) integrins, an interaction that is prevented by mutation of the RGD sequence to arginine-glycine-glutamic acid (RGE) (Erb, L., Liu, J., Ockerhausen, J., Kong, Q., Garrad, R. C., Griffin, K., Neal, C., Krugh, B., Santiago-Perez, L. I., Gonzalez, F. A., Gresham, H. D., Turner, J. T., and Weisman, G. A. (2001) J. Cell Biol. 153, 491-501). This RGD domain also was found to be necessary for coupling the P2Y(2)R to G(o)- but not G(q)-mediated intracellular calcium mobilization, leading us to investigate the role of P2Y(2)R interaction with integrins in nucleotide-induced chemotaxis. Here we show that mutation of the RGD sequence to RGE in the human P2Y(2)R expressed in 1321N1 astrocytoma cells completely prevented UTP-induced chemotaxis as well as activation of G(o), Rac, and Vav2, a guanine nucleotide exchange factor for Rac. UTP also increased expression of vitronectin, an extracellular matrix protein that is a ligand for alpha(v)beta(3)/beta(5) integrins, in cells expressing the wild-type but not the RGE mutant P2Y(2)R. P2Y(2)R-mediated chemotaxis, Rac and Vav2 activation, and vitronectin up-regulation were inhibited by pretreatment of the cells with anti-alpha(v)beta(5) integrin antibodies, alpha(v) integrin antisense oligonucleotides, or the G(i/o) inhibitor, pertussis toxin. Thus, the RGD-dependent interaction between the P2Y(2)R and alpha(v) integrins is necessary for the P2Y(2)R to activate G(o) and to initiate G(o)-mediated signaling events leading to chemotaxis.

P2X(7) nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons.

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X(7) nucleotide receptor (P2X(7)R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X(7)R agonist BzATP were inhibited by the P2X(7)R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X(7)R antisense oligonucleotide indicating a direct involvement of the P2X(7)R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X(7)R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X(7)Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3.

Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes.

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCalpha and epsilon in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCalpha from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.

Src homology 3 binding sites in the P2Y2 nucleotide receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2, and growth factor receptors.

Many G protein-coupled receptors activate growth factor receptors, although the mechanisms controlling this transactivation are unclear. We have identified two proline-rich, SH3 binding sites (PXXP) in the carboxyl-terminal tail of the human P2Y(2) nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y(2) receptor in 1321N1 astrocytoma cells stimulated with the P2Y(2) receptor agonist UTP. A mutant P2Y(2) receptor lacking the PXXP motifs was found to stimulate calcium mobilization and serine/threonine phosphorylation of the Erk1/2 mitogen-activated protein kinases, like the wild-type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Src-dependent tyrosine phosphorylation of the proline-rich tyrosine kinase 2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor. Dual immunofluorescence labeling of the P2Y(2) receptor and the EGFR indicated that UTP caused an increase in the co-localization of these receptors in the plasma membrane that was prevented by the Src inhibitor PP2. Together, these data suggest that agonist-induced binding of Src to the SH3 binding sites in the P2Y(2) receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y(2) receptor and allows Src to efficiently phosphorylate the EGFR.