Role Of Src Tyrosine Kinase Research Articles

Src kinase as a potential therapeutic target in non‐alcoholic and alcoholic steatohepatitis

AbstractNon‐alcoholic steatohepatitis (NASH) and alcohol‐associated steatohepatitis (ASH) are the major cause of liver‐related mortality with limited therapeutic options available. In this study, we investigated the role of Src tyrosine kinase in the pathogenesis of (N)ASH. We examined Src kinase expression in livers from patients with NASH, ASH, cirrhosis and biliary atresia, and from preclinical mouse models: methionine choline deficient (MCD)‐diet‐induced NASH, Lieber–DeCarli ethanol (EtOH)‐diet‐induced ASH, carbon tetrachloride (CCl4)‐ and bile duct ligation (BDL)‐induced liver fibrosis. Functional studies, using Src kinase inhibitor KX2‐391, were performed in NASH and ASH mouse models, and in relevant in vitro models. Transcriptomic analysis revealed increased Src kinase expression in human and mouse diseased livers that positively correlated with disease progression. Src kinase inhibition ameliorated lipid biosynthesis (steatosis); monocytes/macrophages infiltration and inflammatory cytokines expression (inflammation) and hepatic stellate cells (HSCs) activation and collagen expression (fibrosis) in MCD‐diet and EtOH‐diet induced liver disease mouse models. In vitro Src inhibition attenuated FFA‐induced hepatocytic lipid accumulation, LPS/IFNγ‐induced nitric oxide release and expression of several inflammatory signatures in macrophages and TGFβ‐induced HSCs activation, contractility and collagen expression. Moreover, Src inhibition diminished gene expression of inflammatory markers in LPS/IFNγ‐stimulated BMDMs and LPS‐stimulated PCLS, and reduced FA accumulation, macrophage activation and collagen deposition in 3D human liver spheroids. Mechanistic studies further revealed that Src kinase mediated the effects through FAK/PI3K/AKT pathway. Our results demonstrate a multicellular and functional role of Src kinase highlighting Src kinase as a promising therapeutic target in (non)alcoholic steatohepatitis.

ErbB1-dependent signalling and vesicular trafficking in primary afferent nociceptors associated with hypersensitivity in neuropathic pain

Effective analgesic treatment for neuropathic pain remains an unmet need, so previous evidence that epidermal growth factor receptor inhibitors (EGFRIs) provide unexpected rapid pain relief in a clinical setting points to a novel therapeutic opportunity. The present study utilises rodent models to address the cellular and molecular basis for the findings, focusing on primary sensory neurons because clinical pain relief is provided not only by small molecule EGFRIs, but also by the anti-EGFR antibodies cetuximab and panitumumab, which are unlikely to access the central nervous system in therapeutic concentrations. We report robust, rapid and dose-dependent analgesic effects of EGFRIs in two neuropathic pain models, matched by evidence with highly selective antibodies that expression of the EGFR (ErbB1 protein) is limited to small nociceptive afferent neurons. As other ErbB family members can heterodimerise with ErbB1, we investigated their distribution, showing consistent co-expression of ErbB2 but not ErbB3 or ErbB4, with ErbB1 in cell bodies of nociceptors, as well as providing evidence for direct molecular interaction of ErbB1 with ErbB2 in situ. Co-administration of selective ErbB1 and ErbB2 inhibitors produced clear evidence of greater-than-additive, synergistic analgesia; highlighting the prospect of a unique new combination therapy in which enhanced efficacy could be accompanied by minimisation of side-effects. Peripheral (intraplantar) administration of EGF elicited hypersensitivity only following nerve injury and this was reversed by local co-administration of selective inhibitors of either ErbB1 or ErbB2. Investigating how ErbB1 is activated in neuropathic pain, we found evidence for a role of Src tyrosine kinase, which can be activated by signals from inflammatory mediators, chemokines and cytokines during neuroinflammation. Considering downstream consequences of ErbB1 activation in neuropathic pain, we found direct recruitment to ErbB1 of an adapter for PI 3-kinase and Akt signalling together with clear Akt activation and robust analgesia from selective Akt inhibitors. The known Akt target and regulator of vesicular trafficking, AS160 was strongly phosphorylated at a perinuclear location during neuropathic pain in an ErbB1-, ErbB2- and Akt-dependent manner, corresponding to clustering and translocation of an AS160-partner, the vesicular chaperone, LRP1. Exploring whether neuronal ion channels that could contribute to hyperexcitability might be transported by this vesicular trafficking pathway we were able to identify Nav1.9, (Nav1.8) and Cav1.2 moving towards the plasma membrane or into proximal axonal locations – a process prevented by ErbB1 or Akt inhibitors. Overall these findings newly reveal both upstream and downstream signals to explain how ErbB1 can act as a signalling hub in neuropathic pain models and identify the trafficking of key ion channels to neuronal subcellular locations likely to contribute to hyperexcitability. The new concept of combined treatment with ErbB1 plus ErbB2 blockers is mechanistically validated as a promising strategy for the relief of neuropathic pain.

Src Inhibition Attenuates Neuroinflammation and Protects Dopaminergic Neurons in Parkinson's Disease Models.

Chronic neuroinflammation is of great importance in the pathogenesis of Parkinson’s disease (PD). During the process of neuroinflammation, overactivated microglia release many proinflammatory factors, which eventually induce neurodegeneration. Inhibition of excessive microglial activation is regarded as a promising strategy for PD treatment. Src is a non-receptor tyrosine kinase that is closely related to tumors. Recently, some reports indicated that Src is a central mediator in multiple signaling pathways including neuroinflammation. The aim of our study was to demonstrate the role of Src in microglial regulation and neuroinflammation. The lipopolysaccharide (LPS)-stimulated BV2 microglia model and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model were applied in this study. The results showed that inhibition of Src could significantly relieve microgliosis and decrease levels of inflammatory factors. Besides, inhibition of Src function reduced the loss of dopaminergic neurons and improved the motor behavior of the MPTP-treated mice. Thus, this study not only verified the critical role of Src tyrosine kinase in neuroinflammation but also further proved that interfering neuroinflammation is beneficial for PD treatment. More importantly, this study shed a light on the hypothesis that Src tyrosine kinase might be a potential therapeutic target for PD and other neuroinflammation-related diseases.

MGluR1‐β‐arrestin 2 Signaling Mediates Induction of Excitatory Synaptic Plasticity

A subset of seven‐transmembrane receptors (7TMRs) couple to both G protein‐ and β‐arrestin (βarr)‐mediated signaling pathways. Our objective is to determine if group I metabotropic glutamate receptors (mGluR1 and mGluR5) engage βarr signaling during the induction of synaptic plasticity in the CNS. In these experiments we tested the role of βarr‐Src‐MAPK signaling in the induction of plasticity at the hippocampal mossy fiber‐CA3 synapse.
 We compared mGluR1‐dependent synaptic plasticity in patch clamp recordings from CA3 pyramidal neurons in hippocampal slices prepared from wildtype, βarr 1 and βarr 2 knockout mice. Potentiation of EPSCs induced by low frequency, paired stimulation of mossy fiber inputs to CA3 pyramidal neurons was absent in mice lacking βarr 2 (91 ± 7% n=9 for βarr 2‐/‐, 125 ± 9% n=18 for wildtype, p=0.01) but intact in mice lacking βarr 1 (128 ± 11% n=8 for βarr 1‐/‐, 122 ± 13% n=17 for wildtype, p=0.74). In contrast, high‐frequency stimulation that induced long‐term potentiation was unaffected by gene targeting of either βarr isoform (190 ± 23% n=9 for βarr 1‐/‐, 153 ± 18% n=6 for wildtype, p=0.22; 163 ± 18% n=7 for βarr 2‐/‐, 167 ± 14% n=6 for wildtype, p=0.89). Pharmacological dissection implicated ERK1/2 of the MAPK pathway (104 ± 8% n=6 for u0126‐treated slices, 139 ± 9% n=15 for vehicle slices, p=0.01), whereas the role of Src tyrosine kinases was equivocal (114 ± 8% n=9 for PP2‐treated slices, 139 ± 9% n=15 for vehicle slices, p=0.06). 
 These data reveal a novel signaling function for βarrs when coupled to mGluR1 and therefore yield insight into new strategies for therapeutic targeting of group I mGluRs, which in recent years has been pursued for a number of neuropathologies.
 Research supported by R21 NS088916‐01 from NINDS to GTS.

Src tyrosine kinase mediates platelet-derived growth factor BB-induced and redox-dependent migration in metanephric mesenchymal cells

The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild-type and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-l-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2',7'-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4.

Inhibition of Src tyrosine kinase activity by squamosamide derivative FLZ attenuates neuroinflammation in both in vivo and in vitro Parkinson's disease models

The participation of neuroinflammation in the pathogenesis of Parkinson's disease (PD) has long been validated. Excessive activated microglia release a large number of pro-inflammatory factors, damage surrounding neurons and eventually induce neurodegeneration. Inhibition of microglial over-activation might be a promising strategy for PD treatment. FLZ (formulated as: N-(2-(4-hydroxy-phenyl)-ethyl)-2-(2, 5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide, the code name: FLZ), a natural squamosamide derivative from a Chinese herb, has been shown to inhibit over-activated microglia and protect dopaminergic neurons in previous studies, but the mechanism remains unclear. In the present study, we further investigated the mechanism in lipopolysaccharide (LPS)-induced in vivo and in vitro PD models. FLZ treatment significantly improved the motor dysfunction of PD model rats induced by intra-nigral injection of LPS and this beneficial effect of FLZ attributed to the inhibition of microglial over-activation and the protection on dopaminergic neurons in the substantia nigra (SN). In vitro mechanistic study revealed that the inhibitive effect of FLZ on microglia was mediated by suppressing Src kinase related inflammatory signaling pathway activation and subsequent NF-κBp65 nuclear translocation, inhibiting nitric oxide (NO) and reactive oxygen species (ROS) production, decreasing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. In conclusion, the present study supports that FLZ exerts neuroprotection against LPS-induced dopaminergic neurodegeneration through its anti-inflammatory effect, which is mediated by suppressing Src tyrosine kinase and the downstream inflammatory signaling pathway. Furthermore, this study defines a critical role of Src tyrosine kinase in neuroinflammation, and suggests that particular tyrosine kinase inhibition may be a potential anti-inflammatory approach for PD treatment.

Role of Src Tyrosine Kinases in Experimental Pulmonary Hypertension

Pulmonary arterial hypertension is a progressive pulmonary vascular disorder with high morbidity and mortality. Compelling evidence suggests that receptor tyrosine kinases, such as platelet-derived growth factor (PDGF) are closely involved in the pathogenesis of pulmonary arterial hypertension. We investigated the effects of 2 novel PDGF inhibitors, nilotinib/AMN107 (Abl kinases/PDGF receptor inhibitor) and dasatinib/BMS-354825 (Abl kinases/PDGF receptor/Src inhibitor), on the proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) and on the hemodynamics and pulmonary vascular remodeling in experimental pulmonary hypertension, and determined the expression and regulation of Src family kinases. Human PASMCs were stimulated by PDGF alone or multiple growth factors to induce proliferation and migration in vitro. Dasatinib (0.03 μmol/L), nilotinib (0.3 μmol/L), and imatinib (1 μmol/L) potently inhibited PDGF-induced signal transducer and activator of transcription 3 and Akt phosphorylation. All 3 inhibitors decreased PDGF-induced proliferation, cell cycle gene regulation, and migration. In contrast, only dasatinib inhibited multiple growth factor-induced PASMC proliferation, and this was associated with the inhibition of Src phosphorylation. Combination of specific Src inhibitors (phosphoprotein phosphatase 1, phosphoprotein phosphatase 2) with either imatinib or nilotinib reduced multiple growth factor-induced proliferation to a similar extent as dasatinib. Importantly, Src phosphorylation increased in pulmonary arterial hypertension PASMCs compared with control PASMCs. Finally, in vivo dasatinib (15 mg/kg per body weight) treatment caused a complete reversal of pulmonary vascular remodeling and achieved similar effectiveness as imatinib (100 mg/kg per body weight) in both monocrotaline- and hypoxia-induced pulmonary hypertension models. We suggest that dual inhibition of PDGF receptor and Src kinases potently inhibits mitogenic and motogenic responses to growth factors in PASMCs and pulmonary vascular remodeling in vivo so that dual inhibition may represent an alternative therapeutic approach for pulmonary arterial hypertension.

Src inhibition reduces NR2B surface expression and synaptic plasticity in the amygdala

The Src protein tyrosine kinase plays a central role in the regulation of N-methyl-d-aspartate receptor (NMDAR) activity by regulating NMDAR subunit 2B (NR2B) surface expression. In the amygdala, NMDA-dependent synaptic plasticity resulting from convergent somatosensory and auditory inputs contributes to emotional memory; however, the role of Src tyrosine kinase has not been investigated. We have synthesized a Src-derived peptide, Tat-Src (40-58), that crosses the blood-brain barrier following injection and accumulates intracellularly. Tat-Src (40-58) blocks the interaction of Src with NMDA receptors. Following injection, mice demonstrate impaired amygdala-dependent cued fear conditioning, as well as impairments in an amygdala-dependent nonassociative social recognition task. The Src inhibitor decreased NR2B phosphorylation in amygdala tissue and reduced NR2B surface expression in cultured amygdala neurons with a concomitant reduction in NMDA multimer-containing dendritic puncta. In addition, preincubation of this inhibitory peptide blocked amygdalar long-term potentiation in the lateral to basolateral pathway in vitro. These results indicate that Src is a key regulator of NMDAR trafficking in the amygdala. Furthermore, Src-dependent phosphorylation of NR2B supports amygdala plasticity and amygdalar-dependent learning.

CAMP-GEF cytoprotection by Src tyrosine kinase activation of phosphoinositide-3-kinase p110 β/α in rat hepatocytes

Cyclic AMP protects against hepatocyte apoptosis by a protein kinase A-independent cAMP-GEF/phosphoinositide-3-kinase (PI3K)/Akt signaling pathway. However, the signaling pathway coupling cAMP-GEF with PI3K is unknown. The aim of this study was to investigate the role of Src tyrosine kinases (Src-TYK) and PI3K-p110 isoforms in this pathway. Studies were done in rat hepatocytes using the hydrophobic bile acid glycochenodeoxycholic acid (GCDC) to induce apoptosis. cAMP-binding guanine nucleotide exchange factors (cAMP-GEFs) were selectively activated by using 4-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (CPT-2-Me-cAMP), which sequentially phosphorylated Src-TYK (within 1 min) followed by Akt (within 5 min). The Src inhibitors PP2 and SU6656 inhibited basal and CPT-2-Me-cAMP-mediated Src and Akt phosphorylation. These inhibitors had no effect on CPT-2-Me-cAMP-mediated activation of Rap GTPases. CPT-2-Me-cAMP induced transient Src dependent autophosphorylation of the epidermal growth factor receptor (EGFR). Inhibition of the EGFR with AG 1478 partially inhibited the ability of CPT-2-Me to phosphorylate Akt. Whereas PP2 completely abolished the protective effect of CPT-2-Me-cAMP in GCDC induced apoptosis, AG 1478 partially inhibited the cytoprotective effect. CPT-2-Me-cAMP treatment resulted in Src-dependent activation of the p110 beta and alpha subunits of PI3K, but only the latter was sensitive to inhibition with AG 1478. In conclusion, activation of cAMP-GEFs results in phosphorylation of Src-TYK and Akt and activation of the p110 beta/alpha subunits of PI3K. Maximal cAMP-GEF-mediated Akt phosphorylation as well as protection from bile acid-induced apoptosis requires activation of Src-TYK and the EGFR. These studies support the existence of two pathways: cAMP-GEF/Rap/Src/PI3Kbeta/Akt and cAMP-GEF/Rap/Src/EGFR/PI3Kalpha/Akt, both of which are necessary for maximal cytoprotective effect of cAMP-GEFs in hepatocytes.

Role of Src Tyrosine Kinase in the Atherogenic Effects of the 12/15-Lipoxygenase Pathway in Vascular Smooth Muscle Cells

The 12/15-Lipoxygenase (12/15-LO) and its metabolite 12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] mediate proatherogenic responses in vascular smooth muscle cells (VSMCs). We examined the role of the nonreceptor tyrosine kinase Src in the signaling and epigenetic chromatin mechanisms involved in these processes. Rat VSMCs (RVSMCs) were stimulated with 12(S)-HETE (0.1 micromol/L) in the presence or absence of the Src inhibitor PP2 (10 micromol/L). Src activation and downstream signaling events including inflammatory gene expression and chromatin histone H3-Lys-9/14 acetylation were examined by immunoblotting, RT-PCR, and chromatin immunoprecipitation assays, respectively. 12(S)-HETE significantly activated Src, focal adhesion kinase, Akt, p38MAPK, and CREB. Expression of monocyte chemoattractant protein-1 and interleukin-6 genes and histone H3-Lys-9/14 acetylation on their promoters were also increased by 12(S)-HETE. PP2 inhibited these responses as well as 12(S)-HETE-induced VSMC migration. Furthermore, dominant negative mutants of Src, CREB, and a histone acetyltransferase p300 significantly blocked 12(S)-HETE-induced inflammatory gene expression. In addition, growth factor induced Src signaling and downstream events including H3-Lys-9/14 acetylation and migration were significantly attenuated in VSMCs derived from 12/15-LO(-/-) mice relative to WT. Src kinase signaling plays a central role in the proatherogenic responses mediated by 12/15-LO and its oxidized lipid metabolite 12(S)-HETE in VSMCs.

The Src tyrosine kinase pathway regulates thecal CYP17 expression and androstenedione secretion

In order to evaluate the role of Src tyrosine kinase in thecal cell steroidogenesis, a pharmacological approach was utilized by treating enriched populations of mouse ovarian theca-interstitial cells in vitro with a direct Src kinase inhibitor, PP2. Inhibition of Src with PP2 increased both basal and forskolin-stimulated androstenedione secretion, and increased cytochrome P450 17-alpha hydroxylase-lyase (CYP17) promoter activity and steady state mRNA. PP2 did not change thecal levels of StAR mRNA. Inhibition of mitogen-activated protein kinase kinase, a downstream regulator of Src activity, using PD98059 also increased forskolin-stimulated secretion of androstenedione above forskolin alone, but had no effect on basal secretion of androstenedione. Src inhibition increased mitogen-activated protein kinase phosphatase-1 protein and decreased phosphorylation of SF-1, which correlated with increased CYP17 promoter activity and mRNA levels. These results implicate Src tyrosine kinase in the regulation of CYP17 and thecal androgen secretion.

Src Inhibitors in Metastatic Bone Disease

Src tyrosine kinase was the first gene product shown to have an essential function in bone using recombinant DNA technology after its expression was knocked out in mice approximately 15 years ago. Since then, our understanding of the regulation of bone catabolism has advanced significantly with the identification of other key enzymes that regulate osteoclast formation, activation, and survival after their knockout in mice or recognition of mutations in them in humans. This led to the discovery or development of specific inhibitors of some of these key enzymes, including Src, as proof-of-concept lead compounds or potential clinical candidates for the prevention of diseases associated with increased bone resorption, such as osteoporosis and metastatic bone disease. Although bisphosphonates have been prescribed with proven and improving efficacy for the prevention of bone loss for >30 years, adverse effects, such as upper gastrointestinal tract symptoms, and the requirement to take them at least 2 hours before food have limited patient compliance. Thus, with growing knowledge of the pathways regulating osteoclast function and the appreciation that some of these are active also in tumor cells, drug companies have made efforts to identify small-molecular lead compounds for development into new therapeutic agents for the prevention of bone loss with efficacy that matches or supersedes that of bisphosphonates. In this article, we review our current understanding of the signaling pathways that regulate osteoclast formation, activation, and survival with specific reference to the role of Src tyrosine kinase and downstream signaling and highlight in a variety of models of increased bone resorption the effects of Src kinase inhibitors that have been targeted to bone to limit potential adverse effects on other cells.

Rac1 Modulates Sphingosine 1-Phosphate-mediated Activation of Phosphoinositide 3-Kinase/Akt Signaling Pathways in Vascular Endothelial Cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that activates G protein-coupled S1P receptors and initiates a broad range of responses in vascular endothelial cells. The small GTPase Rac1 is implicated in diverse S1P-modulated cellular responses in endothelial cells, yet the molecular mechanisms involved in S1P-mediated Rac1 activation are incompletely understood. We studied the pathways involved in S1P-mediated Rac1 activation in bovine aortic endothelial cells (BAEC) and found that S1P-induced Rac1 activation is impaired following chelation of G protein betagamma subunits by transfection of betaARKct. Treatment with the Src tyrosine kinase inhibitor PP2 completely attenuated S1P-mediated Rac1 activation; however, pretreatment of BAEC with wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, had no effect on Rac1 activation while completely blocking S1P-induced Akt phosphorylation. We used Rac1-specific small interfering RNA (siRNA) duplexes to "knock down" endogenous Rac1 expression and found that siRNA-mediated Rac1 knockdown significantly impaired basal as well as S1P-induced phosphorylation of protein kinase Akt, as well as several downstream targets of Akt including endothelial nitric-oxide synthase and glycogen synthase kinase 3beta. By contrast, S1P-induced phosphorylation of the mitogen-activated protein kinases ERK1/2 was unperturbed by siRNA-mediated Rac1 knockdown. We found that overexpression of the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 markedly enhanced Rac1 activity, whereas a dominant negative Tiam1 mutant significantly attenuated S1P-mediated Rac1 activation. Taken together, these studies identify G protein betagamma subunits, Src kinase and the GEF Tiam1 as upstream modulators of S1P-mediated Rac1 activation, and establish a central role for Rac1 in S1P-mediated activation of PI 3-kinase/Akt/endothelial nitric-oxide synthase signaling in vascular endothelial cells.

Src tyrosine kinase-dependent migratory effects of antithrombin in leukocytes

Tyrosine kinases are known to play a critical role in the regulation of leukocyte function. Antithrombin mediates its effects via syndecan-4 which is known to be linked to the Src tyrosine kinases. In this study, we investigated the role of Src tyrosine kinases in antithrombin-regulated leukocyte migration and Src tyrosine kinase phosphorylation in response to stimulation with antithrombin. Neutrophils and monocytes obtained from forearm venous blood were pre-treated by various Src-family selective tyrosine kinase inhibitors with or without antithrombin followed by washing and assessment of their migratory response toward antithrombin, interleukin-8, or RANTES using Boyden microchemotaxis chambers. Activation status of the two major Src tyrosine kinase phosphorylation sides Tyr416 and Tyr527 was tested using Western blot analysis. Dose-dependent reversal of the antithrombin-mediated effects on neutrophil and monocyte migration was induced by the selective Src kinase inhibitors PP1 and PP2. In Western blot analyses, antithrombin increased Tyr416 and decreased Tyr527 phosphorylation of Src tyrosine kinases in a time- and dose-dependent manner. Moreover, co-incubation with antithrombin lowered the level of RANTES-induced Tyr416 phosphorylation. Therefore, Src tyrosine kinases linked to signaling of antithrombin-binding sites on leukocytes may play an important role in modulating effects on cells function.

Alterations in Reproductive Function in Src Tyrosine Kinase Knockout Mice

The role of Src tyrosine kinase in regulating reproductive processes in female mice was investigated using Src wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice. Ovarian Src kinase activity transiently increased in Src +/+ mice following eCG administration. Src knockout mice were infertile. Estrous cycles and vaginal opening in Src knockouts were variable and altered compared with Src +/+ and +/- mice. Follicle development was compromised in Src knockout mice as evidenced by reduced numbers of large pre-antral and antral follicles compared to Src +/+ mice. Corpora lutea were not observed in the ovaries of Src knockout mice; however, administration of eCG and hCG did result in ovulation. Serum LH and FSH on d 40 and 52 of age did not differ between Src wild-type and knockout females. Results from these studies reveal that female Src knockout mice are infertile due to reduced follicle development and anovulation.

Activation of SRC Tyrosine Kinases in Response to ICAM-1 Ligation in Pulmonary Microvascular Endothelial Cells

Previous studies demonstrated that ICAM-1 ligation on human pulmonary microvascular endothelial cells (ECs) sequentially induces activation of xanthine oxidase and p38 MAPK. Inhibition of these signaling events reduces neutrophil migration to the EC borders. This study examined the role of SRC tyrosine kinases in ICAM-1-initiated signaling within these ECs. Cross-linking ICAM-1 on tumor necrosis factor-alpha-pretreated ECs induced an increase in the activity of SRC tyrosine kinases. This increase was inhibited by allopurinol (a xanthine oxidase inhibitor), Me2SO (a hydroxyl radical scavenger), or deferoxamine (an iron chelator). Phenylarsine oxide, a tyrosine phosphatase inhibitor, reduced the base-line activity of SRC as well as the increase in SRC activity induced by ICAM-1 cross-linking. Specific inhibition of the protein expression of the SRC homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) by an antisense oligonucleotide prevented the induced SRC activation but had no effect on the basal SRC activity. Activation of SRC tyrosine kinases was accompanied by tyrosine phosphorylation of ezrin at Tyr-146, which was inhibited by PP2, an SRC tyrosine kinase inhibitor. Moreover, PP2 completely inhibited p38 activation, suggesting a role for SRC tyrosine kinases in p38 activation. These data demonstrate that ICAM-1 ligation activates SRC tyrosine kinases and that this activation requires SHP-2 as well as production of reactive oxygen species generated from xanthine oxidase. Activation of SRC tyrosine kinases in turn leads to tyrosine phosphorylation of ezrin, as well as activation of p38, a kinase previously identified to be required for cytoskeletal changes induced by ICAM-1 ligation and for neutrophil migration along the EC surface.

Inhibition of Src tyrosine kinase stimulates adrenal androgen production.

A unique characteristic of the primate adrenal is the ability to produce 19-carbon steroids, often called the adrenal androgens. Although it is clear that the major human adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), are produced almost solely in the adrenal reticularis, the mechanisms regulating production are poorly understood. Herein, we tested the hypothesis that the Src family of tyrosine kinases are involved in the regulation of adrenal androgen production. The NCI-H295R human adrenal cell line and primary human adrenal cells in culture were used to study adrenal androgen production and expression of enzymes involved in steroidogenesis. To examine the role of Src tyrosine kinase, cells were treated with PP2, a specific Src inhibitor. Alternatively, adrenal cells were transfected with an expression vector containing a dominant-negative form of Src. PP2 treatment inhibited basal cortisol production while significantly increasing the production of DHEA and DHEA-S (together referred to as DHEA(S)) in both adrenal cell models. The effect of PP2 on steroidogenesis occurred along with a rapid induction of steroidogenic acute regulatory (StAR) protein synthesis as revealed by Western analysis. Treatment with PP2 also increased mRNA levels for StAR, and cholesterol side-chain cleavage (CYP11A) and 17alpha-hydroxylase/17,20-lyase (CYP17) enzymes. Treatment of adrenal cells with the cAMP agonist dibutyryladenosine cyclic monophosphate (dbcAMP), stimulated the production of cortisol and DHEA(S). However, treatment of adrenal cells with a combination of PP2 and dbcAMP enhanced the production of DHEA(S) while inhibiting cortisol production. During dbcAMP treatment PP2 was able to augment the expression of CYP17 and to inhibit the induction of 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) levels. Increasing the CYP17 to HSD3B2 ratio is likely to promote the use of steroid precursors for the production of DHEA(S) and not for cortisol. Taken together these data suggest that the inhibition of Src tyrosine kinases causes adrenal cells to adopt a reticularis phenotype both by the production of DHEA(S) and by the steroidogenic enzymes expressed.

Mechanisms of Protease-activated Receptor-4 Actions in Cardiomyocytes: ROLE OF Src TYROSINE KINASE

Protease-activated receptor (PAR)-4 is a low affinity thrombin receptor with slow activation and desensitization kinetics relative to PAR-1. This study provides novel evidence that cardiomyocytes express functional PAR-4 whose signaling phenotype is distinct from PAR-1 in cardiomyocytes. AYPGKF, a modified PAR-4 agonist with increased potency at PAR-4, activates p38-mitogen-activated protein kinase but is a weak activator of phospholipase C, extracellular signal-regulated kinase, and cardiomyocyte hypertrophy; AYPGKF and thrombin, but not the PAR-1 agonist SFLLRN, activate Src. The observation that AYPGKF and thrombin activate Src in cardiomyocytes cultured from PAR-1(-/-) mice establishes that Src activation is via PAR-4 (and not PAR-1) in cardiomyocytes. Further studies implicate Src and epidermal growth factor receptor (EGFR) kinase activity in the PAR-4-dependent p38-mitogen-activated protein kinase signaling pathway. Thrombin phosphorylates EGFRs and ErbB2 via a PP1-sensitive pathway in PAR-1(-/-) cells that stably overexpress PAR-4; the Src-mediated pathway for EGFR/ErbB2 transactivation underlies the protracted phases of thrombin-dependent extracellular signal-regulated kinase activation in PAR-1(-/-) cells that overexpress PAR-4 and in cardiomyocytes. These studies identify a unique signaling phenotype for PAR-4 (relative to other cardiomyocyte G protein-coupled receptors) that is predicted to contribute to cardiac remodeling and influence the functional outcome at sites of cardiac inflammation.

Association of Lyn tyrosine kinase to the GM-CSF and IL-3 receptor common betac subunit and role of Src tyrosine kinases in DNA synthesis and anti-apoptosis.

After GM-CSF or IL-3 stimulation, the activation of JAK2 tyrosine kinase and members of the Src family of tyrosine kinases takes place, followed by phosphorylation of betac tyrosine residues and the recruitment of SH2 containing molecules to the receptor complex. The exact role of Src kinases such as Lyn in this and other downstream signal transduction events remains unclear. We investigated the association of Lyn kinase with betac using synthetic peptides derived from the eight betac tyrosine residues and the Box 1 motif. We found that Lyn kinase GST fusion proteins bind to peptides corresponding to the membrane proximal region of betac and to peptides containing specific betac derived phosphorylated tyrosine residues. We also determined that betac tyrosine residues Y1,2 as well as Y7 and Y8 can act as substrates of Lyn. We further analysed the role of the Src kinases in DNA synthesis and anti-apoptosis downstream of GM-CSF by using the Src kinase inhibitor PP1 in murine BA/F3 cells stably expressing a series of mutant betac receptors. Lyn binds to betac derived peptides through multiple interactions, and may play an important role in betac phosphorylation. Src family kinases also play an essential role in GM-CSF mediated DNA synthesis, as well as an important role in anti-apoptosis in response to GM-CSF.