Src Kinase Inhibitor PP2 Research Articles

Induction of Neutrophil Chemotaxis by Leptin

Leptin is involved in energy homeostasis, hematopoiesis, inflammation, and immunity. Although hypoleptinemia characterizing malnutrition has been strictly related to increased susceptibility to infection, other hyperleptinemic conditions, such as end-stage renal disease (ESRD), are highly susceptible to bacterial infections. On the other hand, ESRD is characterized by neutrophil functional defects crucial for infectious morbidity, and several uremic toxins capable of depressing neutrophil functions have been identified. In the present study, we investigated leptin's effects on neutrophil function. Our results show that leptin inhibits neutrophil migration in response to classical chemoattractants. Otherwise, leptin is endowed with chemotactic activity toward neutrophils. The two activities, inhibition of the cell response to chemokines and stimulation of neutrophil migration, could be detected at similar concentrations. On the contrary, neutrophils exposed to leptin did not display detectable [Ca2+]i mobilization, oxidant production, or beta2-integrin upregulation. The results demonstrate that leptin is a pure chemoattractant devoid of secretagogue properties but capable of inhibiting neutrophil chemotaxis to classical neutrophilic chemoattractants. This effect is dependent on the activation of intracellular kinases involved in F-actin polymerization and neutrophil locomotion. Indeed, p38 mitogen-activated protein kinase (MAPK) and Src kinase, but not extracellular-regulated kinase (ERK), were activated by short-term incubation with leptin. Moreover, p38 MAPK inhibitor SB203580 and Src kinase inhibitor PP1, but not MEK inhibitor PD98059, blocked neutrophil chemotaxis toward leptin. Serum from patients with ESRD inhibits migration of normal neutrophils in response to N-formyl-methionine-leucyl-phenylalanine (FMLP) with a strict correlation between serum leptin levels and serum ability to suppress neutrophil locomotion. The serum inhibitory activity can be effectively prevented by immune-depletion of leptin. Taking into account the crucial role of neutrophils in host defense, we show that leptin-mediated ability of ERSD serum to inhibit neutrophil chemotaxis appears to be a mechanism contributing to neutrophil dysfunction in ESRD.

A129. Bradykinin cardioprotection is mediated by receptor internalization and mitoKATP opening

Bradykinin (Bk) mimics the cardioprotective effects of ischemic preconditioning (IPC) by a complex series of cellular signaling events that include activation of protein kinase G (PKG), opening of mitochondrial ATP-sensitive K+-channels (mitoKATP), and increased production of reactive oxygen species (ROS). In this study, we investigate the hypothesis that the bradykinin B2 receptor is internalized within a signaling platform that mediates the opening of mitoKATP and cardioprotection. We describe a procedure in which hearts are perfused with Bk in a Langendorff apparatus. Following perfusion, mitochondria are isolated and subjected to purification in a Percoll density gradient. This yields a low-density fraction (henceforth termed the light-layer) and a purified mitochondrial fraction. As determined by western blot analysis, the light-layer contains caveolin-1 and -3, the bradykinin B2 receptor, and PKG as well as other proteins. We find that addition of the light-layer to untreated mitochondria opens mitoKATP similar to KATP openers such as diazoxide, as measured by changes in matrix volume. MitoKATP opening by the light-layer does not require the addition of cyclic-GMP but is sensitive to the mitoKATP blocker 5-hydroxydecanoate, the PKCε peptide antagonist εV1–2, the serine/threonine phosphatase PP2A and the PKG-specific blocker KT5823, indicating that PKG was activated by Bk treatment. Inhibition of the open state was not observed upon addition of the Src kinase inhibitor PP2, the tyrosine phosphatase PTP2B, MEK inhibitor I or the ERK inhibitor 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, HCl. From this data, we conclude that the light-layer from Bk perfused hearts contains a cardioprotective signaling platform that migrates to mitochondria and opens mitoKATP, resulting in a protected state.

Upregulation of EphA2 during in vivo and in vitro renal ischemia-reperfusion injury: role of Src kinases

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury in both native kidneys and renal allografts. Disruption of the actin cytoskeleton is a key event with multiple repercussions on cell adhesion and function during IRI. However, receptors involved in regulating cytoskeletal repair following injury have not been identified. In an in vivo model of renal IRI, we used multiprobe RNase protection assay to examine the expression of Eph receptor tyrosine kinases, key regulators of actin dynamics in embryonic development. We found that one receptor in particular, EphA2, was strongly upregulated in the kidney following IRI. Ephrins, the cell-bound ligands of Eph receptors, were also strongly expressed. In cultured renal tubular cells, diverse injurious stimuli mimicking IRI also resulted in upregulation of EphA2 protein expression. Upregulation of EphA2 was inhibited by the Src kinase inhibitor PP2. Conversely, overexpression of Src kinases strongly enhanced the expression of endogenous EphA2 as well as the activity of a human EphA2 promoter construct. Activation of the Erk pathway was necessary, but not sufficient for full induction of EphA2 upreglation by Src kinases. Stimulation of renal tubular epithelial cells with the EphA2 ligand ephrin-A1 caused tyrosine phosphorylation of endogenous EphA2, paxillin, and an unidentified approximately 65-kDa protein and resulted in increased cortical F-actin staining. In summary, under in vitro conditions mimicking IRI, EphA2 expression is strongly upregulated through a Src kinase-dependent pathway. Interactions between upregulated EphA2 and its ephrin ligands may provide critical cell contact-dependent, bidirectional cues for cytoskeletal repair in renal IRI.

Key Role of Src Kinase in S100B-induced Activation of the Receptor for Advanced Glycation End Products in Vascular Smooth Muscle Cells

The receptor for advanced glycation end products (RAGE) and its ligands have been implicated in the activation of oxidant stress and inflammatory pathways in vascular smooth muscle cells (VSMCs) leading to the initiation and augmentation of atherosclerosis. Here we report that non-receptor Src tyrosine kinase and the membrane protein caveolin-1 (Cav-1) play a key role in the activation of RAGE by S100B in VSMCs. S100B increased the activation of Src kinase and tyrosine phosphorylation of caveolin-1 in VSMCs. A RAGE-specific antibody blocked both these effects. An inhibitor of Src kinase, PP2, significantly blocked S100B-induced activation of Src kinase, mitogen-activated protein kinases, transcription factors NF-kappaB and STAT3, superoxide production, tyrosine phosphorylation of Cav-1, VSMC migration, and expression of the pro-inflammatory genes monocyte chemotactic protein-1 and interleukin-6. Cholesterol depletion also inhibited S100B-induced effects indicating the requirement for intact caveolae in RAGE-specific signaling. Nucleofection of either a Src dominant negative mutant, or a Cav-1 mutant lacking the scaffolding domain, or Cav-1 short hairpin RNA significantly reduced S100B-induced inflammatory gene expression in VSMCs. Furthermore, VSMCs derived from insulin-resistant and diabetic db/db mice displayed increased RAGE expression, Src activation, and migration compared with those from control db/+ mice. The RAGE antibody blocked enhanced migration in db/db cells. These studies demonstrate for the first time that, in VSMCs, Src kinase and Cav-1 play important roles in RAGE-mediated inflammatory gene expression and migration, key events associated with diabetic vascular complications.

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

We previously reported that human growth hormone (hGH) increases cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and proliferation in pancreatic beta-cells (Sjöholm A, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033-21040, 2000) and that the hGH-induced rise in [Ca(2+)](i) involves Ca(2+)-induced Ca(2+) release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658-1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca(2+)](i) and insulin release in BRIN-BD11 beta-cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca(2+)](i) and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K(+) concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca(2+)](i) elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K(+). Ovine prolactin increased [Ca(2+)](i) and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca(2+)](i) but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca(2+)](i). Our study indicates that hGH stimulates rise in [Ca(2+)](i) and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells.

Endothelial cell cortactin phosphorylation by Src contributes to leukocyte transendothelial migration in vitro

The underlying mechanisms that regulate leukocyte transendothelial migration (TEM) remain incompletely understood. Cortactin is a substrate of Src tyrosine kinases and a regulator of cytoskeletal dynamics. Previous studies demonstrated a role for Src phosphorylation of cortactin in clustering of E-selectin and ICAM-1 around adherent leukocytes. Here, an in vitro flow model was used to investigate the role of Src-induced cortactin phosphorylation in TNF-α-activated human umbilical vein endothelium (HUVEC) during polymorphonuclear leukocyte (PMN) TEM of HUVEC. Pretreatment of HUVEC with Src kinase inhibitors PP2 or SU6656 reduced PMN transmigration (45± 8% & 36± 6%, respectively). Live cell imaging of GFP-tagged cortactin in HUVEC revealed re-distribution of cortactin to transmigrating PMN. Reduction of cortactin protein in HUVEC by small interfering RNA (siRNA) also impaired TEM to a similar degree. Interestingly, Src inhibitors or cortactin siRNA reduced PMN TEM at cell-cell borders, but did not alter adhesion. The reduced TEM by cortactin siRNA was rescued by reconstitution with wild-type murine cortactin. Tyrosine phosphorylation of cortactin was important for TEM, because expression of a cortactin mutant, in which the tyrosine phosphorylation sites were mutated to phenylalanine (cortacin3F), failed to rescue TEM. Moreover, expression of cortactin3F alone partially blocked PMN transmigration. These data suggest a model whereby cortactin regulates PMN transmigration through its tyrosine phosphorylation by Src family kinases. Supported by NIH grants HL53393 and HL 36028.

Src family kinases phosphorylate TLR2 in airway cells in response to bacterial ligands

TLR2 is apically localized in airway epithelial cells where it can initiate proinflammatory signaling. In response to bacterial stimulation, TLR2 undergoes tyrosine phosphorylation that is required for PI3K recruitment and the activation of distal signaling cascades. In human airway cells, activation of TLR2 induces the release of Ca2+ from internal stores. We hypothesize that this response is also dependent upon TLR2 phosphorylation and sought to identify the kinases responsible. Stimulation of human airway epithelial cells with theTLR2 agonist P3C induces TLR2 tyrosine phosphorylation. Since TLR2 was shown to undergo tyrosine phosphorylation on residues 616 and 761, we generated Y616A/Y761A mutants and tested their response to TLR2 activation. Stable transfection of 1HAEo- cells with the mutant or a TLR2 dominant negative construct, blocked the expected Ca2+ flux, as detected by confocal imaging using fluo-3/AM loaded cells, as opposed to cells transfected with a vector control. Similarly, cells expressing mutant forms of TLR2 generated significantly less NF-kB activation than cells expressing wild type TLR2. To determine which tyrosine kinases are associated with TLR2, co-immunoprecipitation experiments were done, indicating an association between TLR2 and c-Src. The involvement of c-Src in signaling was documented by experiments showing that the Src kinase inhibitors PP1 and PP2 blocked P3C induced TLR2 tyrosine phosphorylation, as well as the P3C induced Ca2+ flux. A dose-dependent decrease in P3C induced IL-8 expression was also demonstrated in cells treated with the PP1 and PP2. These experiments suggest that response of TLR2 to bacterial ligands, the recruitment of PI3K, PLCγ and Ca2+ release require TLR2 tyrosine phosphorylation by Src kinases. Supported by NIH HL73989.

Oxidative signaling in renal epithelium: Critical role of cytosolic phospholipase A 2 and p38 SAPK

Previous studies from this laboratory have demonstrated a critical role of cytosolic phospholipase A 2 (cPLA 2) and arachidonic acid in angiotensin II (Ang II) AT2 receptor-mediated signal transduction in renal epithelium. In primary proximal tubular epithelial cells exposed to hydrogen peroxide (H 2O 2), both the selective cPLA 2 inhibitors and the cPLA 2 antisense oligonucleotides significantly attenuated H 2O 2-induced arachidonic acid liberation and activation of p38 SAPK, ERK1/2, and Akt1. This H 2O 2-induced kinase activation was significantly attenuated by a Src kinase inhibitor PP2, or by transient transfection of carboxyl-terminal Src kinase (CSK) that maintained Src in the dormant form. Under basal conditions, Src coimmunoprecipitated with epidermal growth factor receptor (EGFR), while H 2O 2 increased EGFR phosphorylation in the complex. We observed that inhibition of EGFR kinase activity with AG1478 significantly attenuated H 2O 2-induced p38 SAPK and ERK1/2 activation, but did not inhibit Akt1 activation. Furthermore, it seems that p38 SAPK is upstream of ERK1/2 and Akt1, since a p38 SAPK inhibitor SB203580 significantly blocked H 2O 2-induced activation of ERK1/2 and Akt1. Interestingly, overexpression of the dominant-negative p38 SAPK isoform α inhibited ERK1/2 but not Akt1 activation. Our observations demonstrate that in these nontransformed cells, activation of cPLA 2 is a converging point for oxidative stress and Ang II, which share common downstream signaling mechanisms including Src and EGFR. In addition, p38 SAPK provides a positive input to both growth and antiapoptotic signaling pathways induced by acute oxidative stress.

Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn2+

Epidemiological studies have implicated zinc (Zn2+) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn2+-induced EGFR activation in HAEC, we treated HAEC with 500 microM ZnSO4 for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn2+ results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn2+-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn2+ treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn2+. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn2+ or V4+ was significantly diminished. Moreover, exposure of HAEC to Zn2+ also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn2+-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn2+ exposure.

Src Kinase Regulates Nitric Oxide-induced Dedifferentiation and Cyclooxygenase-2 Expression in Articular Chondrocytes via p38 Kinase-dependent Pathway

Nitric oxide (NO) in articular chondrocytes regulates dedifferentiation and inflammatory responses by modulating MAP kinases. In this study, we investigated whether the Src kinase in chondrocytes regulates NO-induced dedifferentiation and cyclooxygenase-2 (COX-2) expression. Methods: Primary chondrocytes were treated with various concentrations of SNP for 24 h. The COX-2 and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin E 2 (PGE 2 ) was determined by using a PGE 2 assay kit. Expression and distribution of p-Caveolin and COX-2 in rabbit articular chondrocytes and cartilage explants were determined by immunohistochemical staining and immunocytochemical staining, respectively. Results: SNP treatment stimulated Src kinase activation in a dose-dependent manner in articular chondrocytes. The Src kinase inhibitors PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine], a significantly blocked SNP-induced p38 kinase and caveolin-1 activation in a dose-dependent manner. Therefore, to determine whether Src kinase activation is associated with dedifferentiation and/or COX-2 expression and PGE 2 production. As expected, PP2 potentiated SNP-stimulated dedifferentiation, but completely blocked both COX-2 expression and PGE 2 production. And also, levels of p-Caveolin and COX-2 protein expression were increased in SNP-treated primary chondrocytes and osteoarthritic and rheumatoid arthritic cartilage, suggesting that p-Caveolin may playa role in the inflammatory responses of arthritic cartilage. Conclusion: Our previously studies indicated that NO caused dedifferentiation and COX-2 expression is regulated by p38 kinase through caveolin-1 (1). Therefore, our results collectively suggest that Src kinase regulates NO-induced dedifferentiation and COX-2 expression in chondrocytes via p38 kinase in association with caveolin-1.

Endothelial Cell Cortactin Phosphorylation by Src Contributes to Polymorphonuclear Leukocyte Transmigration In Vitro

The underlying mechanisms that regulate leukocyte transendothelial migration through the vascular endothelium remain unclear. Cortactin is a substrate of Src tyrosine kinases and a regulator of cytoskeletal dynamics. Previous studies demonstrated a role for Src phosphorylation of cortactin in clustering of E-selectin and intercellular cell adhesion molecule-1 around adherent leukocytes. In the current study, we used an in vitro flow model to investigate the role of Src-induced cortactin phosphorylation in endothelium during polymorphonuclear leukocyte (PMN) transmigration through human umbilical vein endothelium (HUVEC) monolayers preactivated with tumor necrosis factor-alpha. Inhibition of Src in HUVEC using Src kinase inhibitors PP2 and SU6656 reduced PMN transmigration by 45+/-8% and 36+/-6%, respectively. Live cell imaging of green fluorescent protein-tagged cortactin in HUVEC revealed redistribution of cortactin in the region surrounding transmigrating PMN. Knockdown of cortactin in HUVEC by small interfering RNA also impaired transmigration to a similar degree, and this phenotype was rescued by reexpression of wild-type cortactin. Analysis of the location of initial arrest and locomotion of PMN adherent to HUVEC demonstrated that inhibition of Src tyrosine kinases or pretreatment with cortactin small interfering RNA reduced PMN transmigration at endothelial cell-to-cell junctions and not adhesion. Tyrosine phosphorylation of cortactin was important for transmigration, because expression of a mutant, in which the tyrosine phosphorylation sites were mutated to phenylalanine (cortactin3F), failed to rescue PMN transmigration. Moreover, expression of cortactin3F alone partially blocked PMN transmigration. These data suggest a model whereby tyrosine phosphorylation of cortactin by Src family kinases regulates PMN transmigration.

Simultaneous Expression of Caveolin-1 and E-Cadherin in Ovarian Carcinoma Cells Stabilizes Adherens Junctions through Inhibition of src-Related Kinases

Cadherin-mediated adhesion plays an important role in maintaining cell-cell contacts and reducing tumor metastasis. However, neo-expression of E-cadherin in ovarian carcinoma does not prevent the release and spread of cells from the primary tumor. Because caveolin-1 is down-regulated concomitantly with E-cad expression, we investigated whether the stability of adherens junctions in ovarian carcinoma was affected by caveolin-1 expression. We used IGROV1 cells transfected with caveolin-1 (IGtC3), mock-transfected control cells (IGtM87), and SKOV3 cells that endogenously express caveolin-1. Simultaneous expression of caveolin-1 and E-cadherin favored membrane distribution of E-cadherin and its associated catenin (p120ctn), even when caveolin-1 was only focally associated with adherens junctions. Silencing of caveolin-1 induced intracellular E-cadherin redistribution in IGtC3 and SKOV3 cells. Treatment with the specific src kinase inhibitor PP1 increased E-cadherin expression in IGtM87 and SKOV3 cells and enhanced membrane localization of both E-cadherin and p120ctn. However, PP1 could not completely reverse the detrimental effects on cell-cell adhesion induced by Ca2+ depletion in IGtM87 cells. Together, our data suggest that caveolin-1 expression indirectly promotes cell-cell adhesion in ovarian carcinoma cells by a mechanism involving inhibition of src-related kinases. Thus, down-regulation or loss of caveolin-1 might contribute significantly to the spread of tumor cells from the primary tumor.

Mitochondrial reactive oxygen species and c-Src play a critical role in hypoxic response in vascular smooth muscle cells

Thickened atherosclerotic plaques are prone to be hypoxic because of poor perfusion. In this study, we tested (a) whether reactive oxygen species (ROS) and c-Src play roles in hypoxic induction of HIF-1alpha protein and PAI-1 gene expression in the rabbit aortic smooth muscle cell line C2/2 cells and primary cultures of rat aortic smooth muscle cells, and (b) how mitochondria act on the hypoxia-induced signaling mechanism. Hypoxic exposure of C2/2 cells increased H2O2 generation, c-Src phosphorylation, HIF-1alpha protein expression, and PAI-1 gene expression. Catalase, a scavenger of H2O2, inhibited the hypoxia-induced ROS generation and PAI-1 gene expression. Src kinase inhibitors PP1 and PP2 inhibited hypoxia-induced HIF-1alpha protein and PAI-1 gene expression. Ablation of mitochondrial respiration by rotenone abolished hypoxia-induced ROS generation, c-Src phosphorylation, HIF-1alpha protein expression, and PAI-1 gene expression. Induction of HIF-1alpha protein and PAI-1 gene expression in response to hypoxia was regulated by ROS production and c-Src activation in vascular smooth muscle cells. Mitochondria linked the hypoxic signal to c-Src, which in turn led to HIF-1alpha protein and PAI-1 gene expression. These results provide evidence that hypoxia induces the ROS-mediated and c-Src-dependent signaling cascades which are closely associated with angiogenesis and thrombosis in atherosclerotic vasculature.

Rapid and transient activation of the ERK MAPK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on JAB1/CSN5 and Src kinase activity

Macrophage migration inhibitory factor (MIF) is a 12.5 kD polypeptide that serves as a critical regulator of cell functions such as gene expression, proliferation or apoptosis. However, the signal transduction pathways through which MIF takes part in cellular regulation are only incompletely understood. MIF leads to CD74-dependent “sustained” activation of ERK1/2 MAPK, but MIF's role in “transient” ERK activation and the involved upstream pathways are unknown. Here we report that the transient ERK pathway was markedly activated by MIF. This effect involved the phosphorylation and activation of Raf-1, MEK, ERK, and Elk-1. Of note, rapid and transient ERK phosphorylation by MIF was measurable in MIF-deficient cells, suggesting that MIF acted in a non-autocrine fashion. Applying the inhibitor genistein, a tyrosine kinase (TPK) activity was identified as a critical upstream signalling event in MIF-induced transient ERK signalling. Experiments using the Src kinase inhibitor PP2 indicated that the involved TPK was a Src-type tyrosine kinase. A role for an upstream Src kinase was proven by applying Src-deficient cells which did not exhibit transient ERK activation upon treatment with MIF, but in which MIF-induced ERK signalling could be restored by re-expressing Src. Intriguingly, JAB1/CSN5, a signalosome component, cellular binding protein of MIF and regulator of cell proliferation and survival, had a marked, yet dual, effect on MIF-induced ERK signalling. JAB1 overexpression inhibited sustained, but not transient, ERK phosphorylation. By contrast, JAB1-knock-down by siRNA revealed that minimum JAB1 levels were necessary for transient activation of ERK by MIF. In conclusion, MIF rapidly and transiently activates the ERK pathway, an effect that has not been recognized previously. This signalling pathway involves the upstream activation of a Src-type kinase and is co-regulated by the cellular MIF binding protein JAB1/CSN5. Our study thus has unravelled a novel MIF-driven signalling pathway and an intricate regulatory system involving extra- and possibly intracellular MIF, and which likely critically participates in controlling cell proliferation and survival.

MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent on activation of different signaling cascades

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understood. In the current investigation, we demonstrate activation of p42/44 ERK1/2 and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44 ERK1/2 MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44 ERK1/2 MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44 ERK1/2, but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity.

The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

We recently demonstrated that the chemokine CXCL16 is expressed in aortic smooth muscle cells (ASMC) and induces ASMC adhesion and proliferation (Chandrasekar, B., Bysani, S., and Mummidi, S. (2004) J. Biol. Chem. 279, 3188-3196). Here we reort that interleukin (IL)-18 positively regulates CXCL16 transcription in rat ASMC. We characterized the cis-regulatory region of CXCL16 and identified a functional activator protein-1 (AP-1) binding motif. Deletion or mutation of this site attenuated IL-18-mediated CXCL16 promoter activity. Gel shift, supershift, and chromatin immunoprecipitation assays confirmed AP-1-dependent CXCL16 expression. CXCL16 promoter-reporter activity was increased by constitutively active c-Fos and c-Jun and decreased by dominant negative or antisense c-Fos and c-Jun. Src kinase inhibitors PP1 and PP2, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, Akt inhibitor, the c-Jun N-terminal kinase (JNK) inhibitor SP600125, antisense JNK and dominant negative MyD88, interleukin-1 receptor-associated kinase (IRAK)-1, IRAK4, and phosphatidylinositol 3-kinase expression all attenuated IL-18-mediated AP-1 binding and reporter activity, CXCL16 promoter-reporter activity, and CXCL16 expression. Thus IL-18 induced CXCL16 expression via a MyD88 --> IRAK1-IRAK4-TRAF6 (tumor necrosis factor receptor-associated factor 6) --> c-Src--> PI3K --> Akt --> JNK --> AP-1 pathway. Importantly, IL-18 stimulated ASMC proliferation in a CXCL16-dependent manner. These data provide for the first time a mechanism of IL-18-mediated CXCL16 gene transcription and CXCL16-dependent ASMC proliferation and suggest a role for IL-18-CXCL16 cross-talk in atherogenesis and restenosis following angioplasty.

Substance P receptor in U373 MG human astrocytoma cells activates mitogen-activated protein kinases ERK1/2 through Src

Substance P (SP) acting through substance P receptor (SPR) increases the proliferation of glioblastoma cells. At the molecular level, stimulation of SPR in human U373 MG glioblastoma cells results in phosphorylation of mitogen-activated protein kinases ERK1/2. Examination of the underlying mechanism reveals that SPR mediates ERK1/2 phosphorylation in a calcium-dependent manner. Surprisingly, blockade of epidermal growth factor receptor (EGFR), which is transactivated by SPR, has a minimal effect on SPR-mediated ERK1/2 phosphorylation. However, SPR-mediated ERK1/2 phosphorylation is significantly reduced by the Src kinase inhibitor PP2. Interestingly, ERK1/2 in U373 MG cells is also activated by several other mitogenic G protein-coupled receptors (GPCRs) including alpha(1B)-adrenergic, M(3)-muscarinic, and H(1)-histaminergic in an Src-dependent manner. We conclude that c-Src is a mediator of SP-stimulated ERK1/2 phosphorylation in human U373 MG glioblastoma cells.

Regulation of vitamin D receptor expression via estrogen-induced activation of the ERK 1/2 signaling pathway in colon and breast cancer cells

We previously demonstrated that 17beta-estradiol (E2) regulates the transcription and expression of the vitamin D receptor (VDR) in rat colonocytes and duodenocytes in vivo. The aim of the present study was to assess whether the extracellular signal-regulated kinase (ERK) induced by E2 is involved in regulating VDR expression. We compared E2-associated signaling activity in HT29 colon cancer cells, a non-classical E2-target, with that in MCF-7 breast cancer cells, the natural targets of the hormone. E2 did not affect proliferation of HT29 cells, but enhanced proliferation of MCF-7 cells. Vitamin D inhibited proliferation of both cell lines and the combined treatment induced potentiation of vitamin D activity. E2 upregulated VDR transcription and protein expression concomitantly with ERK 1/2 phosphorylation in both cell lines. PD98059, a specific mitogen-activated protein kinase (MAPK) inhibitor, prevented E2-mediated activation of ERK 1/2, with concomitant inhibition of VDR expression. ICI182780 inhibited VDR expression in HT29 and MCF-7 cell lines. A conjugate of E2 and bovine serum albumin upregulated phosphorylation of ERK 1/2 and concomitantly enhanced VDR expression in a similar fashion as the nonconjugated hormone. Expression of ERalpha and ERbeta was detected in MCF-7 and HT29 cell lines respectively, which localized to the nuclei, cytosol and caveolar membrane rather than non-caveolar membrane. Disruption of lipid rafts/caveolae by depleting cellular cholesterol with the cholesterol-binding reagent beta-methylcyclodextrin blocked ERK 1/2 phosphorylation concomitantly with VDR upregulation. The tyrosine phosphorylation inhibitor suramin and src kinase inhibitor PP2 inhibited both ERK 1/2 phosphorylation and VDR expression. E2 induced phosphorylation of Raf and Jun in a time-dependent manner. The Ras/Raf dependent inhibitor of transactivation sulindac sulfide also blocked E2 effects. The specific c-Jun phosphorylation inhibitor SP600125 dose dependently inhibited c-Jun phosphorylation and VDR expression. The MAPK/ERK kinase inhibitor PD 98059 downregulated both c-Jun phosphorylation and VDR expression indicating that upstream and downstream events in the signaling cascade are all related to the control of VDR expression. Taken together, our data suggest that E2 binds to receptors compartmentalized to membranal caveolar domains in HT29 and MCF-7 cells, inducing ERK 1/2 activation and transcriptional activity, which finally results in upregulation of expression of the VDR gene.

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery.

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.

Activation of Src kinase Lyn by the Kaposi sarcoma–associated herpesvirus K1 protein: implications for lymphomagenesis

AbstractThe K1 gene of Kaposi sarcoma–associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-κB) inhibitor Bay 11-7085 or an anti–vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-κB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence–deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-κB activation, indicating that ITAM sequences were required for the Lyn kinase–mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-κB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.