Src Tyrosine Kinase Pathway Research Articles

Signal Transduction in Hepatocellular Carcinoma: Insights from Zebrafish

The zebrafish has been an increasingly popular animal model for human diseases as it offers the combined advantages compared to various animal models and cell based assays; in particular, the feasibility of high throughput studies as an economical vertebrate model. In this past decade, we and several other laboratories have developed various hepatocellular carcinoma (HCC) models using the zebrafish and demonstrated the conservation of HCC between zebrafish and human at both histopathological and molecular levels. In this review, we focus on the conservation of signal transductions during hepatocarcinogenesis between zebrafish and human. Based on existing zebrafish HCC models, indeed many alterations of signal pathways that cause human liver cancers can also result in HCC in zebrafish, such as Ras pathway, EGFR pathway, Wng/β-catenin pathway, TGF-β pathway, PI3K/AKT pathway, JAK/STAT pathway, Hippo pathway, src tyrosine kinase pathway, etc. In future, zebrafish may be used for better quantification of signaling molecules and thus to aid development of more effective therapeutic methods.

Activation of the Calcium Sensing Receptor potentially inhibits Kv1.5 channel activity

The Calcium sensing receptor (CaSR) is class of G protein coupled receptors (GPCR) that can be activated by extracellular Ca2+ and Mg2+. Activation of CaSR can stimulate the phospholipase C (PLC) signaling pathway that leads to synthesis of inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG). Voltage‐gated potassium (Kv) channels activity is essential for establishing resting membrane potential in a variety of cell types including pulmonary artery smooth muscle cells (PASMC). Indeed, stimulation or inhibition of K+ channels can easily determine cell fate, as K+ channel activity is associated with cell proliferation, survival and apoptosis. It has been reported that some K+ channel classes are regulated by GPCRs. For example, Kv1.2, and members of the two‐pore domain K+ channel family (TASK‐1 and TASK‐3) were shown to be inhibited by GPCR activation. Interestingly, Kv1.5 channel activity can also be suppressed by stimulation of GPCR via the Src tyrosine kinase pathway. However there are no reports to date that have shown any functional interaction between CaSR and Kv1.5. In this present study, we examined the effects of CaSR activation on Kv1.5 channel activity by transfection of KCNA5, which encodes for the Kv1.5 pore‐forming α subunit, alone or cotransfection of KCNA5/CaSR in HEK293 cells. Overexpression of KCNA5 and CaSR/KCNA5 in HEK293 cells were confirmed by Western blot and Immunofluorescence. Kv1.5 current amplitude (IKv1.5) was significantly attenuated by 1.8 mM external Ca2+ in cells transfected with both CaSR/KCNA5 but not in cells transfected with KCNA5 alone. To determine whether this Ca2+ induced decrease of IKv1.5 was due to activation of PLC‐DAG‐IP3 signaling cascade we used the PLC inhibitor U‐73122. Preliminary data showed that 10uM U‐73122 was capable of antagonizing the external Ca2+ induced decrease in IKv1.5. This would suggests that activation of CaSR by way of external Ca2+ is capable of inhibiting Kv1.5 channel activity via PLC signaling pathwaySupport or Funding InformationResearch supported by NIH Grants (HL115014, HL066012 and HL098053).

High-dose IL-2 induces rapid albumin uptake by endothelial cells through Src-dependent caveolae-mediated endocytosis.

High-dose interleukin-2 (HDIL2) treatment of patients with metastatic melanoma and renal cell carcinoma is associated with durable responses, but therapy is accompanied by significant toxicity related to vascular leak syndrome (VLS). Currently, the cause of VLS is not well defined; however, based on the role of endothelial cell (EC) permeability in VLS and the commonly observed hypoalbuminemia in patients receiving HDIL2 therapy, we established an in vitro approach utilizing primary human pulmonary microvascular ECs to monitor the effect of HDIL2 therapy on albumin uptake. We found that HDIL2 treatment of ECs results in albumin colocalization with caveolin-1 leading to albumin uptake by ECs. This albumin uptake occurs through caveolae-mediated but not clathrin-mediated endocytosis and is abrogated with inhibition of the Src tyrosine kinase pathway. These findings provide insight into how IL-2 induces VLS and may help identify potential targets for prevention of toxicity without affecting the therapeutic activity of HDIL2.

Serotonin contracts the rat mesenteric artery by inhibiting 4-aminopyridine-sensitive Kv channels via the 5-HT2A receptor and Src tyrosine kinase

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, α-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.

Liver-Specific Expressions of HBx and src in the p53 Mutant Trigger Hepatocarcinogenesis in Zebrafish

Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.

An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance

The HER2-targeted therapy trastuzumab is widely used for the treatment of patients with metastatic breast tumors overexpressing HER2. However, an objective response is observed in only 12% to 24% of patients treated with trastuzumab as a single agent and initial responders regress in <6 months (1-3). The reason for the clinical failure of trastuzumab in this setting remains unclear. Here we show that local lymph node-positive disease progression in 89% of breast cancer patients with HER2-positive tumors involves the HER2 oncogenic variant HER2Delta16. We further show that ectopic expression of HER2Delta16, but not wild-type HER2, promotes receptor dimerization, cell invasion, and trastuzumab resistance of NIH3T3 and MCF-7 tumor cell lines. The potentiated metastatic and oncogenic properties of HER2Delta16 were mediated through direct coupling of HER2Delta16 to Src kinase. Cotargeting of HER2Delta16 and Src kinase with the single-agent tyrosine kinase inhibitor dasatinib resulted in Src inactivation, destabilization of HER2Delta16, and suppressed tumorigenicity. Activated Src kinase was also observed in 44% of HER2Delta16-expressing breast carcinomas underscoring the potential clinical implications of coupled HER2Delta16 and Src signaling. Our results suggest that HER2Delta16 expression is an important genetic event driving trastuzumab-refractory breast cancer. We propose that successful targeted therapeutics for intervention of aggressive HER2-positive breast cancers will require a strategy to suppress HER2Delta16 oncogenic signaling. One possibility involves a therapeutic strategy employing single-agent tyrosine kinase inhibitors to disengage the functionally coupled oncogenic HER2Delta16 and Src tyrosine kinase pathways.

Airway smooth muscle: telling the eosinophil how to behave

Airway smooth muscle: telling the eosinophil how to behave

Kir2.x inward rectifier potassium channels are differentially regulated by adrenergic α 1A receptors

Inhibition of I K1 currents by adrenergic α 1 receptors has been observed in cardiomyocytes and has been linked to arrhythmogenesis in an animal model. Both PKC-dependent and PKC-independent pathways have been implied in this regulation. The underlying molecular mechanisms, however, have not been elucidated to date. The molecular basis of native I K1 current is mainly formed by Kir2.1 (KCNJ2), Kir2.2 (KCNJ12) and Kir2.3 (KCNJ4) channels that are differentially regulated by protein kinases. We therefore sought to investigate the role of those different Kir2.x channel subunits in this regulation and to identify the major signalling pathways involved.Adrenergic α 1A receptors (the predominant cardiac isoform) were co-expressed with cloned Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes and electrophysiological experiments were performed using two-microelectrode voltage clamp. Native I K1 currents were measured with the whole-cell patch clamp technique in isolated rat ventricular cardiomyocytes.Activation of co-expressed adrenergic α 1A receptors by phenylephrine induced differential effects in Kir2.x channels. No effect was noticed in Kir2.1 channels. However, a marked inhibitory effect was observed in Kir2.2 channels. This regulation was not attenuated by inhibitors of PKC, CamKII and PKA (chelerythrine, KN-93, KT-5720), and mutated Kir2.2 channels lacking functional phosphorylation sites for PKC and PKA exhibited the same effect as Kir2.2 wild-type channels. By contrast, the regulation could be suppressed by the general tyrosine kinase inhibitor genistein and by the src tyrosine kinase inhibitor PP2 indicating an essential role of src kinases. This finding was validated in rat ventricular cardiomyocytes where co-application of PP2 strongly attenuated the inhibitory regulation of I K1 current by adrenergic α 1 receptors. The inactive analogue PP3 was tested as negative control for PP2 and did not reproduce the effects of PP2. In Kir2.3 channels, a marked inhibitory effect of α 1A receptor activation was observed. This regulation could be attenuated by inhibition of PKC with chelerythrine or with Ro-32-0432, but not by tyrosine kinase inhibition with genistein.In summary, on the molecular level the inhibitory regulation of I K1 currents by adrenergic α 1A receptors is probably based on effects on Kir2.2 and Kir2.3 channels. Kir2.2 is regulated via src tyrosine kinase pathways independent of protein kinase C, whereas Kir2.3 is inhibited by protein kinase C-dependent pathways. Src tyrosine kinase pathways are essential for the inhibition of native I K1 current by adrenergic α 1 receptors. This regulation may contribute to arrhythmogenesis under adrenergic stimulation.

Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling

Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can be induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin's poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin-rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.

Focal Adhesion Kinase Signaling Regulates Cardiogenesis of Embryonic Stem Cells

The signaling steps that induce cardiac differentiation in embryonic stem (ES) cells are incompletely understood. We examined the effect of adhesion signaling including Src and focal adhesion kinase (FAK) on cardiogenesis in mouse ES cells using alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein or luciferase as reporters. Cardiac transcription factors including Nkx2.5 and Tbx5 mRNA were first expressed at day 4 in hanging drop embryoid bodies, and adhesion of embryoid bodies to surfaces at or before that day strongly inhibited differentiation of ES cells to cardiomyocytes. Since adhesion signaling could suppress cardiogenesis through Src kinases, embryoid bodies were exposed to the small molecule PP2, known as a Src family kinase inhibitor. PP2 during embryoid body adhesion dramatically increased cardiomyocyte differentiation and decreased mRNA expression of neuronal cellular adhesion molecule and alpha-fetoprotein, neuroectodermal, and endodermal markers, respectively. Surprisingly, although there was an interaction between Src and FAK in cardiogenesis, the procardiogenic effect of PP2 appeared incompletely explained by Src kinase inhibition, since another Src family kinase inhibitor, SU6656, failed to induce cardiogenesis. Instead, PP2 specifically inhibited adhesion-induced FAK phosphorylation. In ES cells stably expressing FAK-related nonkinase, which functions as a dominant negative FAK, cell migration from embryoid bodies was inhibited, whereas alpha-myosin heavy chain expression and myosin-stained cardiomyocytes were increased, suggesting that reducing cell motility may contribute to cardiogenesis. These data indicate that FAK is a key regulator of cardiogenesis in mouse ES cells and that FAK signaling within embryoid bodies can direct stem cell lineage commitment.

Up-regulation of Vascular Endothelial Growth Factor-A by Active Membrane-type 1 Matrix Metalloproteinase through Activation of Src-Tyrosine Kinases

Membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) are two key molecules involved in pericellular proteolysis and cell proliferation during tumor growth and angiogenesis. Our previous data showed that MT1-MMP overexpression in human breast carcinoma MCF7 cells induced an up-regulation of VEGF expression. This effect was associated in vivo with accelerated tumor growth and angiogenesis. We now provide evidence that MT1-MMP overexpression specifically affected VEGF-A production and failed to influence that of other VEGF family members (VEGF, B, C, D, or PlGF) or their receptors. The up-regulation of VEGF-A by MT1-MMP was related to an increased transcriptional activation rather than to a modification of mRNA stability. It was blocked by synthetic MMP inhibitors, TIMP2, but not TIMP-1 and abolished by a partial deletion of the catalytic domain or the cytoplasmic tail of MT1-MMP. Analysis of the signal transduction mechanisms demonstrated that MT1-MMP acts through a signaling pathway involving Src tyrosine kinases. Thus, our results provide new insight into the mechanisms of action of MT1-MMP during angiogenesis and suggest that the full enzymatic activity of MT1-MMP is required for a specific up-regulation of VEGF-A through an activation of Src tyrosine kinase pathways.

Endothelin-1 Decreases Gap Junctional Intercellular Communication by Inducing Phosphorylation of Connexin 43 in Human Ovarian Carcinoma Cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

Galpha12 regulates epithelial cell junctions through Src tyrosine kinases.

Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that Galpha12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated Galpha12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of Galpha12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active Galpha12 (QLalpha12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QLalpha12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QLalpha12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QLalpha12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QLalpha12-expressing MDCK cells as assessed by Src autophosphorylation, and beta-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that Galpha12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.

The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus

Estrogen replacement therapy in women is associated with improvement of cognitive deficits and reduced incidence of Alzheimer's disease. The present study indicates that estrogen is neuroprotective against N-methyl-d-aspartate (NMDA)- and kainate-mediated neurotoxicity, an effect mediated by tyrosine kinase/mitogen-activated protein kinase (MAPK) pathways. Estrogen also stimulates tyrosine phosphorylation of NMDA receptors via an src tyrosine kinase/MAPK pathway. Finally, estrogen-mediated enhancement of long-term potentiation in hippocampal slices is mediated by activation of an src tyrosine kinase pathway. Thus, estrogen, by activating an src tyrosine kinase and the extracellular signal-related protein kinase/MAPK signaling pathway, both enhances NMDA receptor function and long-term potentiation and retains neuroprotective properties against excitotoxicity. These findings warrant further evaluation of the usefulness of estrogenic compounds for the treatment of Alzheimer's disease and other neurodegenerative diseases.

The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus.

Estrogen replacement therapy in women is associated with improvement of cognitive deficits and reduced incidence of Alzheimer's disease. The present study indicates that estrogen is neuroprotective against N-methyl-d-aspartate (NMDA)- and kainate-mediated neurotoxicity, an effect mediated by tyrosine kinase/mitogen-activated protein kinase (MAPK) pathways. Estrogen also stimulates tyrosine phosphorylation of NMDA receptors via an src tyrosine kinase/MAPK pathway. Finally, estrogen-mediated enhancement of long-term potentiation in hippocampal slices is mediated by activation of an src tyrosine kinase pathway. Thus, estrogen, by activating an src tyrosine kinase and the extracellular signal-related protein kinase/MAPK signaling pathway, both enhances NMDA receptor function and long-term potentiation and retains neuroprotective properties against excitotoxicity. These findings warrant further evaluation of the usefulness of estrogenic compounds for the treatment of Alzheimer's disease and other neurodegenerative diseases.

Acute loss of cell-cell communication caused by G protein-coupled receptors: a critical role for c-Src.

Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.