Src Family Kinase Inhibitor PP2 Research Articles

Cyclic stretch-induced TGF-β1 and fibronectin expression is mediated by β1-integrin through c-Src- and STAT3-dependent pathways in renal epithelial cells.

Extracellular matrix (ECM) proteins, including fibronectin, may contribute to the early development and progression of renal interstitial fibrosis associated with chronic renal disease. Recent studies showed that β1-integrin is associated with the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). However, the molecular events responsible for β1-integrin-mediated signaling, following UUO, have yet to be determined. In this study, we investigated the mechanism by which mechanical stretch, an in vitro model for chronic obstructive nephropathy, regulates fibronectin and transforming growth factor-β1 (TGF-β1) expression in cultured human proximal tubular epithelium (HK-2) cells. Mechanical stretch upregulated fibronectin and TGF-β1 expression and activated signal transducer and transcription factor 3 (STAT3) in a time-dependent manner. Stretch-induced fibronectin and TGF-β1 were suppressed by a STAT3 inhibitor, S3I-201, and by small interfering RNA (siRNA) targeting human STAT3 (STAT3 siRNA). Similarly, fibronectin and TGF-β1 expression and STAT3 activation induced by mechanical stretch were suppressed by the Src family kinase inhibitor PP2 and by transfection of HK-2 cells with a dominant-negative mutant of c-Src (DN-Src), whereas PP3, an inactive analog of PP2, had no significant effect. Furthermore, mechanical stretch resulted in increased β1-integrin mRNA and protein levels in HK-2 cells. Furthermore, neutralizing antibody against β1-integrin and silencing of β1-integrin expression with siRNAs resulted in decreased c-Src and STAT3 activation and TGF-β1 and fibronectin expression evoked by mechanical stretch. This work demonstrates, for the first time, a role for β1-integrin in stretch-induced renal fibrosis through the activation of c-Src and STAT3 signaling pathways.

Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors

Caffeine, a methylated derivative of xanthine and widely consumed psychoactive substance, acts in several targets in the nervous system. We investigated its role in retinal explants of chick embryo analyzing the role of purinergic receptors in [3H]-GABA release induced by d-aspartate (d-asp). d-Asp increases GABA-release 4.5-fold when compared to basal levels from 13-day-old chick embryo retinal explants. Caffeine 500μM elevated d-asp-induced GABA release in 60%. The release was inhibited in the presence of NNC-711, a GABA transporter-1 (GAT-1) blocker or by MK-801, an N-methyl-d-aspartate receptor (NMDAR) antagonist. Caffeine did not modify [3H]-GABA uptake carried out for 5, 10, 30 and 60min and did not increase the release of d-asp or glutamate at basal or stimulated conditions. The caffeine effect was mimicked by the adenosine A1 receptor antagonist DPCPX and by the adenylyl cyclase (AC) activator forskolin. It was also blocked by the protein kinase A (PKA) inhibitor H-89, tyrosine kinase inhibitor genistein or by the src family kinase (SFK) inhibitor PP1. Forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels were reduced in the presence of the A1 receptor agonist CHA. Western blot analysis revealed that 500μM caffeine increased phosphoGluN2B expression levels in approximately 60% when compared to total GluN2B levels in embryonic E13 retina. The GluN2B subunit-containing NMDAR antagonist ifenprodil inhibited the caffeine effect. Our results suggest that caffeine potentiates d-asp-induced GABA release, which is mediated by GAT-1, via inhibition of adenosine A1 receptor and activation of the PKA pathway. Regulation of NMDAR by phosphorylation of GluN2B subunit by a SFK may also be involved in the effect promoted by caffeine.

Phosphorylation of flotillin-1 by mitochondrial c-Src is required to prevent the production of reactive oxygen species.

We have shown that mitochondrial c-Src regulates reactive oxygen species (ROS) production by phosphorylating the succinate dehydrogenase A of respiratory complex II (CxII). To elucidate the molecular mechanisms underlying ROS production regulated by c-Src in the CxII, we investigated the CxII protein complex derived from cells treated with Src family kinase inhibitor PP2. We identified flotillin-1 as a c-Src target that prevents ROS production from CxII. Phosphorylation-site analysis suggests Tyr56 and Tyr149 on flotillin-1 as sites for phosphorylation by c-Src. A comparison of cells expressing flotillin-1 and its phosphorylation defective mutants confirms the requirement for flotillin-1 phosphorylation for its interaction with CxII and subsequent reduction in ROS production. Our findings suggest a critical role of flotillin-1 in ROS production mediated by c-Src.

Src family kinase inhibitor PP2 enhances differentiation of acute promyelocytic leukemia cell line induced by combination of all-trans-retinoic acid and arsenic trioxide

An all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) combination yields high-quality remission and survival in newly-diagnosed acute promyelocytic leukemia (APL). For subsequent similar data, NCCN guidelines indicate that ATRA plus ATO is one of the recommended regimens for the treatment of patients with APL. We demonstrated SFK (Src family kinase) inhibitor PP2-enhanced APL cell differentiation when combined with either ATRA or ATO with difference in activation of RA-induced genes. In this study, we investigated whether SFK inhibitor PP2 could enhance the differentiation of NB4 APL cells when combined with ATRA and ATO and the changes in the expression of intercellular adhesion molecule-1 (ICAM-1) derived from the retinoic acid receptor (RAR) target gene.Treatment of NB4 cells with 1μM of ATRA, 0.5μM of ATO, or 10μM of PP2 for 72h induced expression of CD11b-positive cells by 13.01%, 11.53% or 13.28%, respectively. However, the combination of ATRA and ATO and the combination of three agents (ATRA, ATO, and PP2) led to a significantly higher expression of CD11b-positive cells (30.96% and 63.17%, respectively). The synergistic effect of the combination of three agents was more significant than the combination of ATRA and ATO. These results were confirmed with NBT staining. These effects were not related to apoptosis. Annexin-V-fluorescein staining revealed that a combination of ATRA and ATO and combination of the three agents did not induce apoptosis in NB4 cells. The expression of ICAM-1 markedly increased in cells treated with the combination of the three agents.These findings suggest that the SFK inhibitor can enhance differentiation of APL cells combined with ATRA and ATO. FDA approved SFK inhibitors, such as dasatinib and bosutinib, may be beneficial for the treatment of APL with a combination of ATRA and ATO.

Protein kinase C and Src family kinases mediate angiotensin II-induced protein kinase D activation and acute aldosterone production

Recent evidence has shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone secretion upon angiotensin II (AngII) stimulation. However, the mechanism by which AngII activates PKD remains unclear. In this study, using both pharmacological and molecular approaches, we demonstrate that AngII-induced PKD activation is mediated by protein kinase C (PKC) and Src family kinases in primary bovine adrenal glomerulosa cells and leads to increased aldosterone production. The pan PKC inhibitor Ro 31-8220 and the Src family kinase inhibitors PP2 and Src-1 inhibited both PKD activation and acute aldosterone production. Additionally, like the dominant-negative serine-738/742-to-alanine PKD mutant that cannot be phosphorylated by PKC, the dominant-negative tyrosine-463-to-phenylalanine PKD mutant, which is not phosphorylatable by the Src/Abl pathway, inhibited acute AngII-induced aldosterone production. Taken together, our results demonstrate that AngII activates PKD via a mechanism involving Src family kinases and PKC, to underlie increased aldosterone production.

Phospholipase Cγ1 Connects the Cell Membrane Pathway to the Nuclear Receptor Pathway in Insect Steroid Hormone Signaling

In addition to the classical nuclear receptor pathway, there is a nongenomic pathway in the cell membrane that regulates gene expression in animal steroid hormone signaling; however, this mechanism is unclear. Here, we report that the insect steroid hormone 20-hydroxyecdysone (20E) regulates calcium influx via phospholipase Cγ1 (PLCG1) to modulate the protein kinase C phosphorylation of the transcription factor ultraspiracle (USP1) in the lepidopteran insect Helicoverpa armigera. The PLCG1 mRNA levels are increased during the molting and metamorphic stages. The depletion of PLCG1 by RNA interference can block 20E-enhanced pupation, cause larvae death and pupation defects, and repress 20E-induced gene expression. 20E may induce the tyrosine phosphorylation of PLCG1 at the cytosolic tyrosine kinase (Src) homology 2 domains and then determine the migration of PLCG1 toward the plasma membrane. The G-protein-coupled receptor (GPCR) inhibitor suramin, Src family kinase inhibitor PP2, and the depletions of ecdysone-responsible GPCR (ErGPCR) and Gαq restrain the 20E-induced tyrosine phosphorylation of PLCG1. PLCG1 participates in the 20E-induced Ca(2+) influx. The inhibition of GPCR, PLC, inositol 1,4,5-trisphosphate receptor, and calcium channels represses the 20E-induced Ca(2+) influx. Through calcium signaling, PLCG1 mediates the transcriptional activation driven by the ecdysone-response element. Through PLCG1 and calcium signaling, 20E regulates PKC phosphorylation of USP1 at Ser-21 to determine its ecdysone-response element binding activity. These results suggest that 20E activates PLCG1 via the ErGPCR and Src family kinases to regulate Ca(2+) influx and PKC phosphorylation of USP1 to subsequently modulate gene transcription for metamorphosis.

Phosphorylation of c-Cbl and p85 PI3K driven by all-trans retinoic acid and CD38 depends on Lyn kinase activity

The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48h followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38+ transfectants. In contrast, cells cultured for 48h following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48h of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation.

Abstract 18201: Src-Dependent Caveolar Trafficking Defects in Hereditary Pulmonary Arterial Hypertension

Patients with hereditary pulmonary arterial hypertension (HPAH) inherit heterozygous germ line mutations in the BMP type 2-receptor, BMPR2, but it is not known how these mutations cause the disease. Bmpr2 mutant mice have endothelial dysfunction and increased vascular permeability, and are more susceptible to the development of pulmonary hypertension. In these studies we explore the mechanisms mediating endothelial barrier dysfunction in Bmpr2+/- mice. Conditionally immortalized pulmonary endothelial cells (ciPECs) from Bmpr2+/- mice have increased permeability to 70kD dextran and albumin, but not to 4kD dextran, suggesting that Bmpr2+/- ciPECs have intact cell-cell junctions but increased transcellular (vesicle mediated) permeability. Caveolae are the predominant vesicle involved in transcellular permeability in endothelium. Transmission EM shows an increase in Caveolar structures in ciPECs and intact endothelium of Bmpr2+/- lungs. Quantitative analysis indicates that Caveolin-1 (Cav1) and Cavin have altered localization from the plasma membrane (PM) in wild type ciPECs to an intracellular localization in Bmpr2+/- ciPECs. In addition, inhibition of Caveolar endocytosis with the Dynamin-2 inhibitor, Dynasore, restores Cav1 localization to the PM in Bmpr2+/- PECs, suggesting there is increased Dynamin-dependent Caveolar endocytosis and trafficking in Bmpr2+/- ciPECs. Bmpr2+/- ciPECs also have increased levels of phosphorylated (pY14) Cav1 associated with increased expression of active Src-kinase (pY416), the kinase responsible for phosphorylating Cav1 and initiating Caveolar endocytosis. The selective Src-family kinase inhibitor PP2, reduces pY14-Cav1 and rescues Cav1 and Cavin localization to the PM in Bmpr2+/- ciPECs. Furthermore, PP2 restores endothelial barrier function in Bmpr2+/- ciPECs demonstrated by a reduction in albumin and 70kD dextran permeability. These data demonstrate a defect in Caveolar trafficking mediated by constitutive activation of Src kinase in Bmpr2+/- ciPECs, and suggest Src kinase inhibitors may ameliorate endothelial barrier dysfunction in HPAH patients.

Src Family Kinase Inhibitor PP2 Enhances Differentiation Of Acute Promyelocytic Leukemia Cell Line Induced By Combination Of ATRA and Arsenic Trioxide

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia. For subsequent similar data, NCCN guidelines indicate that ATRA plus ATO is an alternative for patients who cannot tolerate anthracycline therapy. We demonstrated that SFK (Src Family Kinase) inhibitor PP2 enhanced acute promyelocytic leukemia (APL) cell differentiation when combined with either ATRA or ATO with difference in activation of RA-induced genes. In this study, we investigated SFK inhibitor PP2 could enhances the differentiation of NB4 APL cells when combined with ATRA and ATO and the changes in the expression of intercellular adhesion molecule-1 (ICAM-1) derived from the retinoic acid receptor (RAR) target gene.Treatment of NB4 cells with 1 nM of ATRA, 0.5 uM of ATO, or 10 uM of PP2 for 72 hours induced expression of CD11b-positive cells by 13.01%, 11.53% or 13.28%, respectively. However, the combination of ATRA and ATO and the combination of three agents (ATRA, ATO, and PP2) led to a significant higher expression of CD11b-positive cells (30.96% and 63.17%, respectively). The synergistic effect of the combination of three agents was more significant than the combination of ATRA and ATO. These results were confirmed by NBT staining. These effects were not related with apoptosis. Annexin-V-fluorescene staining revealed that combination of ATRA and ATO and combination of three agents did not induced apoptosis in NB4 cells. The expression of ICAM-1 was markedly increased in cells treated with the combination of three agents.These findings suggest that the SFK inhibitor can enhances differentiation of APL cells combined with ATRA and ATO. FDA approved SFK inhibitors, such as dasatinib and bosutinib, may be beneficial for the treatment of APL in combination with ATRA and ATO. Disclosures:No relevant conflicts of interest to declare.

Activating Individual αIIbβ3 Molecules Using An Anti-αIIb-Transmembrane Domain Peptide Causes αIIbβ3 Oligomerization and αIIbβ3-Mediated Outside-In Signaling

Activating αIIbβ3 in platelets is followed by αIIbβ3 clustering and αIIbβ3-mediated outside-in signaling. However, the molecular bases for the latter two processes are not well understood. It has been found that activating αIIbβ3 in platelets is followed by trans-autoactivation of the tyrosine kinase c-Src constitutively associated with the β3 cytosolic tail. In turn, activated c-Src initiates a signaling cascade that culminates in the re-organization of the platelet cytoskeleton, platelet spreading, and fibrin clot retraction. Previously, we observed that replacing Gly708 in the β3 transmembrane helix with Asn causes constitutive αIIbβ3 activation, as well as ligand binding-independent αIIbβ3 clustering when the β3 mutant is expressed in CHO cells. Here, we have asked whether activating αIIbβ3 in platelets in the absence of ligand binding is sufficient to cause c-Src activation. Exogenous peptides that interact with the membrane-embedded segments of αIIbβ3, such as the CHAMP (Computed Helical Anti-Membrane Protein) peptide anti-αIIb TM, cause agonist-independent activation of single αIIbβ3 molecules. To test whether anti-αIIb TM causes c-Src activation in the absence of ligand binding to αIIbβ3, we suspended gel-filtered human platelets in buffer containing an inhibitory cocktail of EDTA, apyrase, and eptifibatide that completely prevents anti-αIIb TM-stimulated platelet aggregation as well as FITC-labeled fibrinogen binding to αIIbβ3 and extracted aliquots of the stimulated platelets with 1% NP-40 in a buffer containing a cocktail of protease and phosphatase inhibitors. c-Src was immunoprecipitated from the extracts and immunoblotted for phosphorylation of c-Src tyrosine residue 416, an indicator of c-Src activation. We found that stimulating platelets with anti-αIIb TM caused Tyr416 phosphorylation. This effect required the presence of αIIbβ3 because it did not occur when β3-knockout mouse platelets were incubated with the peptide. Further, Tyr416 phosphorylation was prevented by the Src-family kinase inhibitor PP2, but not by the inactive analogue PP3, implying that it resulted from the trans-autophosphorylation of β3-associated c-Src. Tyr416 phosphorylation was not dependent on an anti-αIIb TM-induced rearrangement of the platelet cytoskeleton because it was not inhibited by the actin polymerization inhibitors cytochalasin D and latrunculin A. Similar results were seen when immunoprecipitated β3 was immunoblotted for phosphorylated Tyr416. The tyrosine kinase Syk is also associated with the β3 cytosolic tail. Like c-Src, we found that incubating platelets with anti-αIIb TM caused Syk phosphorylation that was independent of ligand binding to αIIbβ3, was catalyzed by activated c-Src because it was inhibited by PP2 but not PP3, and like c-Src trans-autophosphorylation, was unaffected by cytochalasin D and latrunculin A. By contrast, we found that anti-αIIb TM-induced αIIbβ3 activation was not sufficient to cause phosphorylation of FAK when ligand binding to αIIbβ3 was prevented by EDTA, apyrase, and eptifibatide. Anti-αIIb TM-induced c-Src activation in the absence of ligand binding to αIIbβ3 implies that the peptide causes ligand-binding-independent αIIbβ3 clustering. To demonstrate that this is the case, we incubated suspended CHO cells expressing wild-type αIIbβ3 with anti-αIIb TM, fixed the cells with formalin, and stained the cells with the FITC-labeled αIIbβ3-specific monoclonal antibody A2A9. Like CHO cells expressing β3 Gly708Asn, the αIIbβ3 of CHO cells incubated with anti-αIIb TM was present in patches distributed over the cell surface, consistent with the formation of αIIbβ3 clusters. By contrast, in the absence of anti-αIIb TM, αIIbβ3 was present in a homogenous ring at the CHO cell periphery. These studies demonstrate that the CHAMP peptide anti-αIIb TM can activate β3-associated c-Src and Syk under conditions to preclude ligand binding to αIIbβ3. This indicates that activation of individual αIIbβ3 molecules can initiate αIIbβ3 oligomerization in the absence of ligand binding and cytoskeletal reorganization. Thus, these studies suggest that not only is resting αIIbβ3 poised to undergo a conformational change that exposes its ligand binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well. Disclosures:No relevant conflicts of interest to declare.

Hypoxia Induces Metalloproteinase-9 Activation and Human Vascular Smooth Muscle Cell Migration Through Low-Density Lipoprotein Receptor–Related Protein 1–Mediated Pyk2 Phosphorylation

Hypoxia disturbs vascular function by promoting extracellular matrix remodeling. Extracellular matrix integrity and composition are modulated by metalloproteinases (MMPs). Our aim was to investigate the role of low-density lipoprotein receptor-related protein 1 (LRP1) in regulating MMP-9/MMP-2 activation and vascular smooth muscle cells (VSMCs) migration in response to hypoxia, and to elucidate the LRP1-signaling pathways involved in this process. Western blot analysis showed that hypoxia induced a sustained phosphorylation of proline-rich tyrosine kinase 2 concomitantly with LRP1 overexpression in human VSMCs (hVSMCs). Deletion of LRP1 using small-interfering RNA technology or treatment of hVSMCs with the Src family kinase inhibitor PP2 impaired hypoxia-induced phosphorylation of proline-rich tyrosine kinase 2 levels. Coimmunoprecipitation experiments showed that the higher amounts of phosphorylation of proline-rich tyrosine kinase 2/LRP1β immunoprecipitates in hypoxic hVSMCs were abolished in PP2-treated hVSMCs. Both LRP1 silencing and PP2 treatment were highly effective in the prevention of hypoxia-induced MMP-9 activation and hVSMC migration. Cellular subfractionation experiments revealed that PP2 effects may be caused by impairment of hypoxia-induced nuclear factor-κβ translocation to the nucleus. ELISA measurements showed that LRP1 silencing but not PP2 treatment increased interleukin-1β, interleukin-6, and monocyte chemoattractant protein-1 secretion by hypoxic hVSMCs. Our findings determine a crucial role of LRP1-mediated Pyk2 phosphorylation on hypoxia-induced MMP-9 activation and hVSMC migration and therefore in hypoxia-induced vascular remodeling. Both LRP1 silencing and PP2 treatments also influence hypoxia-induced proinflammatory effects in hVSMCs. Therefore, further studies are required to establish therapeutical strategies that efficiently modulate vascular remodeling and inflammation associated with hypoxia-vascular diseases.

Regulation of TrkB receptor translocation to lipid rafts by adenosine A(2A) receptors and its functional implications for BDNF-induced regulation of synaptic plasticity.

Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A(2A) receptor activation, we hypothesized that activation of A(2A) receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A(2A) receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansyl cadaverine (100 μM) did not modify the effects of the A(2A) receptor agonists, but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A(2A) receptor activation in TrkB localization was mimicked by 5 μM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors RpcAMPs and PKI-(14-22) and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF upon hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts, induced by the activation of adenosine A(2A) receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.

Non-receptor-tyrosine Kinases Integrate Fast Glucocorticoid Signaling in Hippocampal Neurons

Despite numerous descriptions of rapid effects of corticosterone on neuronal function, the intracellular mechanisms responsible for these changes remain elusive. The present comprehensive analysis reveals that signaling from a membrane-located G protein-coupled receptor activates PKC, Akt/PKB, and PKA, which subsequently trigger the phosphorylation of the tyrosine kinases Pyk2, Src, and Abl. These changes induce rapid cytoskeletal rearrangements (increased PSD-95 co-clustering) within the post-synaptic density; these events are accompanied by increased surface NMDA receptor expression, reflecting corticosterone-induced inhibition of NMDA receptor endocytosis. Notably, none of these signaling mechanisms require de novo protein synthesis. The observed up-regulation of ERK1/2 (downstream of NMDA receptor signaling) together with the fact that c-Abl integrates cytoplasmic and nuclear functions introduces a potential mechanism through which rapid signaling initiated at the plasma membrane may eventually determine the long term integrated response to corticosterone by impacting on the transcriptional machinery that is regulated by classical, nuclear mineralocorticoid, and glucocorticoid receptors.

Src Family Kinase Inhibitor PP2 Has Different Effects on All-Trans-Retinoic Acid or Arsenic Trioxide-Induced Differentiation of an Acute Promyelocytic Leukemia Cell Line

PurposeLeukemic promyelocytes have the unique ability to undergo differentiation after exposure to retinoic acid and both differentiation and apoptosis after exposure to arsenic trioxide (ATO). Recent studies have shown that inhibition of Src family kinases (SFKs) resulted in enhancement of retinoic acid-induced myeloid differentiation.Materials and MethodsIn this study, we investigated the question of whether the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATO as well as when combined with all-trans-retinoic acid (ATRA). In addition, we attempted to determine the difference in retinoic acid-induced gene expression between cells treated with PP2 in combination with ATRA and in combination with ATO.ResultsSFK inhibitor PP2 induced significant enhancement of ATRA- or ATO-induced differentiation of NB4 cells. A significantly stronger synergistic effect was observed when PP2 was combined with ATRA than when combined with ATO. Flow cytometric analysis demonstrated a significant increase in CD11b-positive granulocytes up to 60.73% and 31.58%, respectively. These results were confirmed by nitroblue tetrazolium staining. These effects were not related to apoptosis. Results of Annexin-V-fluorescein staining revealed that PP2 combined with ATRA or PP2 combined with ATO did not induce apoptosis in NB4 cells. Retinoic acid-induced gene expression was different in both groups. Intercellular adhesion molecule-1 expression showed a significant increase in cells treated with PP2 in combination with ATRA, whereas cathepsin D expression showed a significant increase in cells treated with PP2 in combination with ATO.ConclusionOur data showed that SFK inhibitor PP2 enhanced acute promyelocytic leukemia cell differentiation when combined with either ATRA or ATO with difference in activation of retinoic acid-induced genes.

EGFR tyrosine kinase regulates human small-conductance Ca2+-activated K+ (hSKCa1) channels expressed in HEK-293 cells

SKCa (small-conductance Ca(2+)-activated K(+)) channels are widely distributed in different tissues, including the brain, pancreatic islets and myocardium and play an important role in controlling electrical activity and cellular functions. However, intracellular signal modulation of SKCa channels is not fully understood. The present study was designed to investigate the potential regulation of hSKCa1 (human SKCa1) channels by PTKs (protein tyrosine kinases) in HEK (human embryonic kidney)-293 cells expressing the hSKCa1 (KCNN1) gene using approaches of whole-cell patch voltage-clamp, immunoprecipitation, Western blotting and mutagenesis. We found that the hSKCa1 current was inhibited by the broad-spectrum PTK inhibitor genistein, the selective EGFR (epidermal growth factor receptor) kinase inhibitors T25 (tyrphostin 25) and AG556 (tyrphostin AG 556), but not by the Src-family kinases inhibitor PP2. The inhibitory effect of these PTK inhibitors was significantly antagonized by the PTP (protein tyrosine phosphatase) inhibitor orthovanadate. The tyrosine phosphorylation level of hSKCa1 channels was reduced by genistein, T25 or AG556. The reduced tyrosine phosphorylation was countered by orthovanadate. Interestingly, the Y109F mutant hSKCa1 channel lost the inhibitory response to T25 or AG556, and showed a dramatic reduction in tyrosine phosphorylation levels and a reduced current density. These results demonstrate the novel information that hSKCa1 channels are inhibited by genistein, T25 and AG556 via EGFR tyrosine kinase inhibition, which is related to the phosphorylation of Tyr(109) in the N-terminus. This effect may affect electrical activity and cellular functions in brain, pancreatic islets and myocardium.

Abstract 4283: Src phosphorylates splicing factor 45 (SPF45) and regulates SPF45 nucleocytoplasmic shuttling, cell migration and invasion.

Abstract Src phosphorylates splicing factor 45 (SPF45) and regulates SPF45 nucleocytoplasmic shuttling, cell migration and invasion Yuying Liu1, LaShardai Conaway1, Jennifer Rutherford Bethard1, Amber Thompson Bradley1 and Scott T. Eblen1,2 From Department of Cell and Molecular Pharmacology and Experimental Therapeutics1 and the Hollings Cancer Center2 Medical University of South Carolina, Charleston, SC, 29425 SPF45 is a bifunctional protein involved in splicing and DNA repair and is overexpressed in several forms of cancer. Previously, we have shown that SPF45 overexpression regulates ovarian cancer cell proliferation and adhesion in a MAP kinase phosphorylation-dependent manner. In this study, we investigated novel roles of SPF45 and their regulation by the tyrosine kinase Src. We show by cell fractionation that endogenous SPF45 localizes in both the nucleus and cytoplasm and that exposure of cells to UV irradiation or osmotic shock increased SPF45 cytoplasmic accumulation. Consistent with this result, heterokaryon assays using SKOV-3 cells stably expressing Myc-SPF45 showed that SPF45 shuttles between the nucleus and cytoplasm and that pretreatment of the cells with the Src family kinase inhibitor PP2 inhibited nucleocytoplasmic shuttling. Src strongly induced SPF45 tyrosine phosphorylation and co-immunoprecipitated with SPF45 after co-transfection into COS-1 cells. Src phosphorylated SPF45 in vitro on five tyrosine residues (Y100, Y108, Y194, Y214 and Y246), as determined by mass spectrometry. Sequential mutation of these sites to phenylalanine inhibited SPF45 tyrosine phosphorylation in cells co-transfected with Src, with mutation of all 5 sites (Myc-SPF45-Y5F) completely inhibiting SPF45 tyrosine phosphorylation. Myc-SPF45-Y5F stably expressed in SKOV-3 cells showed reduced nucleocytoplasmic shuttling compared to wild type Myc-SPF45. SKOV-3-Myc-SPF45 cells demonstrated enhanced migration and invasion compared to vector control cells, while SKOV-3-Myc-SPF45-Y5F cells migrated significantly less than SKOV-3-Myc-SPF45 cells. These data are the first to show that SPF45 is a shuttling protein, a Src substrate, and that Src phosphorylation of SPF45 regulates nucleocytoplasmic shuttling and SPF45-induced cell migration and invasion. Citation Format: Yuying Liu, Lashardai Neniara Conaway, Jennifer Rutherford Bethard, Amber Thompson Bradley, Scott Eblen. Src phosphorylates splicing factor 45 (SPF45) and regulates SPF45 nucleocytoplasmic shuttling, cell migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4283. doi:10.1158/1538-7445.AM2013-4283

The Src-Family Kinase Inhibitor PP2 Rescues Inducible Differentiation Events in Emergent Retinoic Acid-Resistant Myeloblastic Leukemia Cells

Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47phox expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid’s biological effects in WT HL60 cells.

Src and fibroblast growth factor 2 independently regulate signaling and gene expression induced by experimental injury to intact articular cartilage

To investigate whether cartilage injury activates protein tyrosine kinases distinct from fibroblast growth factor (FGF)-related signaling, and whether they contribute to injury-induced gene responses. Phosphokinases and protein tyrosine phosphorylation were assayed by Western blotting of cartilage lysates. Immunoprecipitation and Western blotting with 4G10 antibody and immunoprecipitation kinase assay were carried out. Tyrosine-phosphorylated proteins on silver-stained gels of injured cartilage lysates were identified by mass spectrometry. Messenger RNA induction in cartilage explants was assessed by quantitative reverse transcriptase-polymerase chain reaction. Protein tyrosine phosphorylation occurred within seconds of injury to the surface of intact articular cartilage, as did activation of MAPKs and IKK. Activation did not reoccur upon reinjury of cultured explants. The prominent tyrosine-phosphorylated proteins focal adhesion kinase, paxillin, and cortactin were identified as substrates of Src family kinases. The Src family kinase inhibitor PP2 blocked injury-induced tyrosine phosphorylation. It did not prevent activation of the MAPKs and IKK but differentially inhibited 8 of 10 inflammatory response genes that were induced by injury. In contrast, FGF signaling blockade with PD173074 reduced all MAPK and IKK activation by ∼50% and inhibited a different subset of genes but had no effect on Src-like signaling. Injury to the surface of intact articular cartilage activates Src-like kinases as well as MAPKs and IKK (implying NF-κB activation). FGF-2 contributes to MAPK/IKK activation but not to Src-like signaling, suggesting that the latter is a parallel pathway that also regulates the injury-induced inflammatory gene response.

Trypsin causes platelet activation independently of known protease-activated receptors

To identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, the Gq inhibitor YM-254890 had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was abolished by either p160ROCK inhibitor, Y27632 or H1152. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. Surprisingly, trypsin caused a similar shape change in PAR4-desensitised PAR3 null murine platelets as in PAR4null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, the Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signalling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signalling pathway in platelets that is independent of PARs. This protease-induced novel signalling pathway regulates platelet shape change through SFKs and p160ROCK.

Src family kinase inhibitor PP2 accelerates differentiation in human intestinal epithelial cells

The proto-oncogene Src is an important protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and survival. Here, we investigated the involvement of Src family kinases (SFKs) in human intestinal cell differentiation. We first observed that Src activity peaked in early stages of Caco-2/15 cell differentiation. Inhibition of SFKs with PP2, a selective SFK inhibitor, accelerated the overall differentiation program. Interestingly, all polarization and terminal differentiation markers tested, including sucrase-isomaltase, lactase-phlorizin hydrolase and E and Li-cadherins were found to be significantly up-regulated after only 3days of treatment in the newly differentiating cells. Further investigation of the effects of PP2 revealed a significant up-regulation of the two main intestinal epithelial cell-specific transcription factors Cdx2 and HNF1α and a reduction of polycomb PRC2-related epigenetic repressing activity as measured by a decrease in H3K27me3, two events closely related to the control of cell terminal differentiation in the intestine. Taken together, these data suggest that SFKs play a key role in the control of intestinal epithelial cell terminal differentiation.