Isorhamnetin Downregulates MMP2 and MMP9 to Inhibit Development of Rheumatoid Arthritis through SRC/ERK/CREB Pathway.

OBJECTIVE: To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis (RA). METHODS: Tumor necrosis factor (TNF)- α -induced fibroblast-like synoviocytes (FLS) was exposed to additional isorhamnetin (10, 20 and 40 µ mol/L). Overexpression vectors for matrix metalloproteinase-2 (MMP2) or MMP9 or SRC were transfected to explore their roles in isorhamnetin-mediated RA-FLS function. RA-FLS viability, migration, and invasion were evaluated. Moreover, a collagen-induced arthritis (CIA) rat model was established. Rats were randomly divided to sham, CIA, low-, medium-, and high-dosage groups using a random number table (n=5 in each group) and administed with normal saline or additional isorhamnetin [2, 10, and 20 mg/(kg·day)] for 4 weeks, respectively. Arthritis index was calculated and synovial tissue inflammation was determined in CIA rats. The levels of MMP2, MMP9, TNF-α, interleukin-6 (IL-6), and IL-1 ß, as well as the phosphorylation levels of SRC, extracellular regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding (CREB), were detected in RA-FLS and synovial tissue. Molecular docking was also used to analyze the binding of isorhamnetin to SRC. RESULTS: In in vitro studies, isorhamnetin inhibited RA-FLS viability, migration and invasion (P<0.05). Isorhamnetin downregulated the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß in RA-FLS (P<0.05). The overexpression of either MMP2 or MMP9 reversed isorhamnetin-inhibited RA-FLS migration and invasion, as well as the levels of TNF-α, IL-6, and IL-1 ß (P<0.05). Furthermore, isorhamnetin bound to SRC and reduced the phosphorylation of SRC, ERK, and CREB (P<0.05). SRC overexpression reversed the inhibitory effect of isorhamnetin on RA-FLS viability, migration and invasion, as well as the negative regulation of MMP2 and MMP9 (P<0.05). In in vivo studies, isorhamnetin decreased arthritis index scores (P<0.05) and alleviated synovial inflammation. Isorhamnetin reduced the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß, as well as the phosphorylation of SRC, ERK, and CREB in synovial tissue (P<0.05). Notably, the inhibitory effect of isorhamnetin was more pronounced at higher concentrations (P<0.05). CONCLUSION: Isorhamnetin exhibited anti-RA effects through modulating SRC/ERK/CREB and MMP2/MMP9 signaling pathways, suggesting that isorhamnetin may be a potential therapeutic agent for RA.

Anti-metastatic Potential of Natural Triterpenoid Cucurbitacin B Against Cholangiocarcinoma Cells by Targeting Src Protein.

Owing to the crucial role of Src in cancer metastasis, interruption of Src and its signaling has been considered a promising strategy for cancer metastasis treatment. Cucurbitacin B, a dietary triterpenoid, has been shown to possess anti-proliferative and apoptosis-inducing activities in cholangiocarcinoma (CCA) cells via suppressing the activation of FAK which is a main downstream Src effector. We hypothesized that cucurbitacin B might act as a Src suppressant which conferring anti-metastasis effect against CCA cells. To investigate this, the role of Src in regulating metastasis behavior of CCA cells and the effect of cucurbitacin B on Src-mediated metastatic phenotype of these cells were determined. The results showed that activation of Src significantly enhanced the migratory and invasive abilities of CCA cells. Molecular analysis revealed that Src-facilitated metastasis behavior of CCA cells occurred by modifying expression of a wide range of metastasis-related genes in the cells. Consistent with gene expression results, activation of Src significantly induced the protein expression of 2 important metastasis-associated molecules, MMP-9 and VEGF. Cucurbitacin B markedly suppressed activation of Src and its key effector, FAK. As a consequence, the alteration of expression profiles of metastasis-associated genes induced by Src activator in CCA cells was diminished by cucurbitacin B treatment. The compound also down-regulated Src-induced expression of MMP-9 and VEGF proteins in the cells. Moreover, molecular docking analysis revealed that cucurbitacin B could interact with Src kinase domain and possibly restrain the kinase from being activated by hindering the ATP binding. In conclusion, cucurbitacin B exhibited anti-metastatic property in CCA cells via negatively influencing Src and Src-related oncogenic signaling. This compound may therefore be a potential therapeutic drug for further development as an anti-Src agent for treatment of metastatic CCA.

SKAP2 is downregulated in the villous tissues of patients with missed abortion and regulates growth and migration in trophoblasts through the WAVE2-ARP2/3 signaling pathway.

INTRODUCTION: Abnormal placental trophoblast function is the main cause of missed abortion (MA). Src kinase-associated phosphoprotein 2 (SKAP2) indirectly affects actin reunion, which is significantly associated with cell migration. METHODS: Twenty women with MA and 20 healthy women who underwent voluntarily induced abortion were included in this study. Immunohistochemistry, qRT-PCR, and western blotting were used to determine SKAP2, WAVE2, and ARP2 expression in the villous tissues. We investigated the effects of SKAP2 and the W336K mutant (blocked SKAP2 Src homology 3 function) on growth and migration in HTR8/SVneo cells using the CCK8 assay, flow cytometry, and transwell assay. The effects of SKAP2 on the WAVE2-ARP2/3 signaling pathway in HTR8/SVneo cells were evaluated by western blotting and immunofluorescence. RESULTS: Compared to the women in the voluntary abortion group, SKAP2 and WAVE2 expression levels were downregulated in the villous of patients with MA. In HTR8/SVneo cells, SKAP2 siRNA silencing regulated the growth and migration, while SKAP2 overexpression promoted growth and migration, and inhibited apoptosis. Additionally, SKAP2 regulated the expression of WAVE2 and ARP2, as well as the colocalization of actin with WAVE2. The SKAP2 W336K mutant could not alter WAVE2 and ARP2 expression, nor HTR8/SVneo cell growth and migration, with or without SKAP2 siRNA transfection. DISCUSSION: SKAP2 could activate the WAVE2-ARP2/3 pathway resulting in an increase of growth and migration in trophoblasts. SKAP2 probably played an important role in MA by affecting the growth and migration of trophoblasts.

BSA-bounded p-cresyl sulfate potentiates the malignancy of bladder carcinoma by triggering cell migration and EMT through the ROS/Src/FAK signaling pathway.

Para-cresyl sulfate (P-CS), a major uremic toxin derived from the metabolites of tyrosine and phenylalanine through liver, existed in the blood of patients with chronic kidney disease (CKD). CKD increases the malignancy in bladder cancers; however, effects of P-CS on bladder cancers are not fully understood. P-CS is conjugated with BSA physiologically, and this study aims to investigate the effects and possible underlying mechanisms of BSA-bounded P-CS on human bladder cancer cells. With P-CS treatment, the intracellular ROS increased in bladder cancer cells. ROS then triggered epithelial-mesenchymal transition (EMT), stress fiber redistribution, and cell migration. With specific inhibitors, the key signals regulating P-CS-treated migration are Src and FAK. This study provided a clinical clue that patients with higher serum P-CS have a higher risk of malignant urothelial carcinomas, and a regulatory pathway of how P-CS regulates bladder cancer migration.

Tat-Src reduced NR2B tyrosine phosphorylation and its interaction with NR2B in levodopa-induced dyskinetic rats model.

Augmented function of N-methyl-d-aspartate receptor subunit 2B (NR2B) and Src protein tyrosine kinase have been demonstrated to get involved in the pathological mechanisms of dyskinesia. In view of functional interactions between NR2B and Src, we investigated the effects of uncoupling NR2B and Src interactions on dyskinesia by using the Src-derived interfering peptide (Tat-Src). In the present study, valid 6-hydroxydopamine-lesioned parkinsonian rats were treated with levodopa intraperitoneally for 22 days to induce dyskinetic rats model. On day 23, dyskinetic rats received either Tat-Src or Tat-sSrc or vehicle with each levodopa dose. The data showed that in dyskinetic rats model intraperitoneal microinjection of Tat-Src improved dyskinetic behaviors and decreased NR2B tyrosine phosphorylation and the interactions of Src with NR2B induced by chronic levodopa treatment. Besides, Tat-Src also attenuated S-nitrosylation (SNO-Src) and the autophosphorylation (p-Src) of Src, which catalyzed NR2B phosphorylation. These findings suggest that targeting NR2B/Src complexes can be one potential treatment for dyskinesia in Parkinson's disease.

Deletion of Adipose Triglyceride Lipase Links Triacylglycerol Accumulation to a More-Aggressive Phenotype in A549 Lung Carcinoma Cells.

Adipose triglyceride lipase (ATGL) catalyzes the rate limiting step in triacylglycerol breakdown in adipocytes but is expressed in most tissues. The enzyme was shown to be lost in many human tumors, and its loss may play a role in early stages of cancer development. Here, we report that loss of ATGL supports a more-aggressive cancer phenotype in a model system in which ATGL was deleted in A549 lung cancer cells by CRISPR/Cas9. We observed that loss of ATGL led to triacylglycerol accumulation in lipid droplets and higher levels of cellular phospholipid and bioactive lipid species (lyso- and ether-phospholipids). Label-free quantitative proteomics revealed elevated expression of the pro-oncogene SRC kinase in ATGL depleted cells, which was also found on mRNA level and confirmed on protein level by Western blot. Consistently, higher expression of phosphorylated (active) SRC (Y416 phospho-SRC) was observed in ATGL-KO cells. Cells depleted of ATGL migrated faster, which was dependent on SRC kinase activity. We propose that loss of ATGL may thus increase cancer aggressiveness by activation of pro-oncogenic signaling via SRC kinase and increased levels of bioactive lipids.

MicroRNA-106a targets autophagy and enhances sensitivity of lung cancer cells to Src inhibitors.

OBJECTIVES: Src tyrosine kinase inhibitors (TKIs) significantly inhibit cell migration and invasion in lung cancer cell lines with minor cytotoxic effects. In clinical trials, however, they show modest activity in combination with chemotherapeutic agents. Possible resistance mechanisms include the induction of cytoprotective autophagy upon Src inhibition. Autophagy is a cellular recycling process that allows cell survival in response to a variety of stress stimuli including responses to various treatments. MATERIAL AND METHODS: We screened autophagic activity in A549, H460, and H1299 NSCLC cell lines treated with two different Src-TKIs (saracatinib, dasatinib) or shRNA targeting SRC. The autophagy response was determined by LC3B-I to -II conversion, increased ULK1 epxression and increased GFP-LC3B dot formation. Autophagy was inhibited by pharmacological (bafilomycin A, chloroquine) or genetic (ULK1 shRNA) means. Expression of miR-106a and miR-20b was analyzed by qPCR, and we used different lentivral vectors for ectopic expression of either miR-106a mimetics, anti-sense miR-106a or different miR-106a-363 cluster constructs. RESULTS: In the current study we found that Src-TKIs induce autophagy in lung adenocarcinoma cell lines and that a combination of autophagy and Src tyrosine kinase inhibition results in cell death. Moreover, Src-TKI induced autophagy depends on the induction of the key autophagy kinase ULK1. This ULK1 upregulation is caused by downregulation of the ULK1-targeting microRNA-106a. An inverse correlation of miR-106a and ULK1 expression was seen in lung adenocarcinoma. Accordingly, ectopic expression of miR-106a in combination with Src-TKI treatment resulted in significant cell death as compared to control transduced cells. CONCLUSIONS: Autophagy protects lung adenocarcinoma cells from Src-TKIs via a newly identified miR-106a-ULK1 signaling pathway. The combined inhibition of Src and ULK1/autophagy might represent a promising treatment option for future clinical trials. Lastly, our data might challenge the term "oncogenic" miR-106a as it can promote sensitivity to Src-TKIs thereby underlining the context-dependent function of miRNAs.

Signaling pathways mediating chemokine induction in keratinocytes by cathelicidin LL-37 and flagellin.

Cathelicidin LL-37 is a multifunctional immunomodulatory and antimicrobial host defense peptide that has an important role in the immune defenses of the skin and other epithelial barriers. We have previously demonstrated that at physiological concentrations LL-37 synergistically augments the production of immune mediators in response to microbial compounds in human primary keratinocytes. Here we define the signaling mechanisms responsible for this activity. We demonstrate that inhibition of Src family kinases (SFKs) strongly inhibited the synergistic chemokine production in response to LL-37 and flagellin in keratinocytes. SFK activation was induced by LL-37 stimulation and was required for the downstream activation of Akt (protein kinase B) and the transcription factors CREB and ATF1. In cells stimulated with LL-37 and flagellin together, Akt activation was primarily induced by LL-37, while both flagellin and LL-37 contributed to the activation of CREB and ATF1 and consequently chemokine induction. The purinergic receptor P2X7 was identified as the receptor upstream of SFK activation in LL-37-stimulated keratinocytes. Overall, these findings established the P2X7-SFK-Akt-CREB/ATF1 signaling pathway activated by LL-37 in primary keratinocytes. These signaling mechanisms mediated the synergistic effects of LL-37 on chemokine production in flagellin-stimulated keratinocytes, and thus might have a role in the immune defenses of the skin and possibly other epithelial barriers.

Src tyrosine kinase inhibition prevents pulmonary ischemia-reperfusion-induced acute lung injury.

PURPOSE: Pulmonary ischemia-reperfusion is a pathological process seen in several clinical conditions, including lung transplantation, cardiopulmonary bypass, resuscitation for circulatory arrest, atherosclerosis, and pulmonary embolism. A better understanding of its molecular mechanisms is very important. METHODS: Rat left lung underwent in situ ischemia for 60 min, followed by 2 h of reperfusion. The gene expression profiles and Src protein tyrosine kinase (PTK) phosphorylation were studied over time, and PP2, an Src PTK inhibitor, was intravenously administered 10 min before lung ischemia to determine the role of Src PTK in lung injury. RESULTS: Reperfusion following ischemia significantly changed the expression of 169 genes, with Mmp8, Mmp9, S100a9, and S100a8 being the most upregulated genes. Ischemia alone only affected expression of 9 genes in the lung. However, Src PTK phosphorylation (activation) was increased in the ischemic lung, mainly on the alveolar wall. Src PTK inhibitor pretreatment decreased phosphorylation of Src PTKs, total protein tyrosine phosphorylation, and STAT3 phosphorylation. It increased phosphorylation of the p85α subunit of PI3 kinase, a signal pathway that can inhibit coagulation and inflammation. PP2 reduced leukocyte infiltration in the lung, apoptotic cell death, fibrin deposition, and severity of acute lung injury after reperfusion. Src inhibition also significantly reduced CXCL1 (GRO/KI) and CCL2 (MCP-1) chemokine levels in the serum. CONCLUSION: During pulmonary ischemia, Src PTK activation, rather than alteration in gene expression, may play a critical role in reperfusion-induced lung injury. Src PTK inhibition presents a new prophylactic treatment for pulmonary ischemia-reperfusion-induced acute lung injury.

Postexposure treatment with a Src-PTK inhibitor in combination with N-l-acetyl cysteine to reduce noise-induced hearing loss.

Both the antioxidant, N-l-acetyl cysteine (NAC), and the Src inhibitor, KX1-004, have been used to protect the cochlea from hazardous noise. In order to extend our previous work on KX1-004 with noise exposure, the current studies were undertaken with two goals: (1) to test the effectiveness of NAC and KX1-004 in combination with one another when given in a protection paradigm, and (2) to test the NAC+KX1-004 combination in a postexposure rescue paradigm. The noise exposure for the first experiment consisted of a 4-kHz octave band of noise at 107 dB SPL for 2 hours. The combination of NAC and KX1-004 were administered either prior to the noise exposure or post exposure (rescue). The second experiment was undertaken to extend the findings of the first experiment's rescue paradigm. The 4 kHz octave band noise was delivered at 112 dB SPL for 1 hour, with the experimental drugs delivered only in a rescue paradigm. In Experiment 1, animals treated before the 2-hour noise exposure with the combination of NAC and KX1-004 had from 12 to 17 dB less permanent threshold shift when compared to control saline treated animals. Treatment in the rescue paradigm did not produce any reductions in threshold shift from the 2-hour exposure. In the second experiment, with the 1-hour noise, rescue with KX1-004 or KX1-004 plus NAC yielded small, but significant, reductions in threshold shift. There was no additional benefit from the combination of NAC and KX1-004 over KX1-004 by itself.

Src family tyrosine kinases-driven colon cancer cell invasion is induced by Csk membrane delocalization.

The nonreceptor tyrosine kinases of the Src family (SFK) are frequently deregulated in human colorectal cancer (CRC), and they have been implicated in tumour growth and metastasis. How SFK are activated in this cancer has not been clearly established. Here, we show that the SFK-dependent invasion is induced by inactivation of the negative regulator C-terminal Src kinase, Csk. While the level of Csk was inconsistent with SFK activity in colon cancer cells, its membrane translocation, needed for efficient regulation of membrane-localized SFK activity, was impaired. Accordingly, Csk downregulation did not affect SFK oncogenic activity in these cells, whereas expression of a membrane-localized form of this kinase affected their invasive activity. Downregulation of the transmembrane and rafts-localized Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomain (PAG), was instrumental for the cytoplasmic accumulation of Csk. Re-expression of PAG in cells from late-stage CRC inhibited SFK invasive activity in a Csk-dependent manner. Conversely, inactivation of its residual expression in early-stage CRC cells promoted SFK invasive activity. Finally, this mechanism was specific to CRC as Csk coupling to SFK was readily detected in breast cancer cells. Therefore, Csk mis-localization defines a novel mechanism for SFK oncogenic activation in CRC cells.

Regulation of miRNA expression by Src and contact normalization: effects on nonanchored cell growth and migration.

Transformation by the Src tyrosine kinase (Src) promotes nonanchored cell growth and migration. However, nontransformed cells can force Src-transformed cells to assume a normal morphology and phenotype by a process called 'contact normalization'. It has become clear that microRNA (miRNA) can affect tumorigenesis by targeting gene products that direct cell growth and migration. However, the roles of miRNA in Src transformation or contact normalization have not yet been reported. We examined the expression of 95 miRNAs and found 9 of them significantly affected by Src. In this study, we report that miR-218 and miR-224 were most significantly induced by Src, but not affected by contact normalization. In contrast, miR-126 was most significantly suppressed by Src and was induced by contact normalization in transformed cells. Mir-126 targets Crk, a component of the focal adhesion network that participates in events required for tumor cell migration. Accordingly, we show that miR-126 expression correlates inversely with Crk levels, motility and the invasive potential of human mammary carcinoma cells. Moreover, we show that miR-224 expression promotes nonanchored growth of nontransformed cells. These data reveal novel insights into how Src regulates miRNA expression to promote hallmarks of tumor cell growth and invasion, and how nontransformed cells can affect miRNA expression in adjacent tumor cells to inhibit this process.

Src-family protein tyrosine kinase negatively regulates cerebellar long-term depression.

Protein phosphorylation is a major mechanism for the regulation of synaptic transmission. Previous studies have shown that several serine/threonine kinases are involved in the induction of long-term depression (LTD) at excitatory synapses on a Purkinje neuron (PN) in the cerebellum. Here, we show that Src-family protein tyrosine kinases (SFKs) are involved in the regulation of the LTD induction. Intracellular application of c-Src suppressed LTD. We also show that application of a SFK-selective inhibitor PP2 recovered LTD from the suppression caused by the inhibition of mGluR1 activity. These results indicate that SFKs negatively regulate the LTD induction at excitatory synapses on a cerebellar PN.

An acidic extracellular pH induces Src kinase-dependent loss of beta-catenin from the adherens junction.

Little attention has been paid to the role of adherens junctions (AJs) in acidic extracellular pH (pHe)-induced cell invasion. Incubation of HepG2 cells in acidic medium (pH 6.6) induced cell dispersion from tight cell clusters, and this change was accompanied by downregulation of beta-catenin at cell junctions and a rapid activation of c-Src. Pretreatment with PP2 prevented the acidic pH-induced downregulation of beta-catenin at AJ and in the membrane fractions. The acidic pHe-induced c-Src activation increased tyrosine phosphorylation of beta-catenin and decreased the amount of beta-catenin-associated E-cadherin. The depletion of membrane-bound beta-catenin coincided with enhanced cell migration and invasion, and this acidic pHe-increased cell migration and invasion was prevented by PP2. In conclusion, this study characterizes a novel signaling pathway responsible for acidic microenvironment-promoted migration and invasive behaviors of cancer cells.

Synthesis and evaluation of 3-phenylpyrazolo[3,4-d]pyrimidine-peptide conjugates as Src kinase inhibitors.

3-Phenylpyrazolo[3,4-d]pyrimidine (PhPP) derivatives substituted with an alkyl or aryl carboxylic acid at the N1-endocyclic amine, such as PhPP-CH(2)COOH (IC(50)=250 microM), and peptides Ac-CIYKYY (IC(50)=400 microM) and Ac-YIYGSFK (IC(50)=570 microM) were weak inhibitors of polyE(4)Y phosphorylation by active c-Src. A series of PhPP-peptide conjugates were synthesized using PhPP as an ATP mimic and CIYKYY or YIYGSFK as a peptide substrate to improve the inhibitory potency against active c-Src kinase. PhPP derivatives were attached to the N terminus or the side chain of amino acids in the peptide template. Two N-terminal substituted conjugates, PhPP-CH(2)CO-CIYKYY (IC(50)=0.38 microM) and PhPP-CH(2)CO-YIYGSFK (IC(50)=2.7 microM), inhibited the polyE(4)Y phosphorylation by active c-Src significantly higher than that of the parent compounds. The conjugation of PhPP with the peptides produced a synergistic inhibition effect possibly through creation of favorable interactions between the conjugate and the kinase domain as shown by molecular modeling studies.

Inhibition of SRC tyrosine kinases suppresses activation of nuclear factor-kappaB, and serine and tyrosine phosphorylation of IkappaB-alpha in lipopolysaccharide-stimulated raw 264.7 macrophages.

Involvement of protein tyrosine kinases (PTK) in lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-kappaB) activation has been demonstrated. Studies investigated the role of PTK and the underlying mechanisms by which PTK play a role in LPS induction of pathways leading to NF-kappaB activation in macrophages. Inhibitors of PTK-genistein, herbimycin A, or AG126-blocked LPS-induced NF-kappaB activation. Genistein also blocked pervanadate-induced NF-kappaB activation. Furthermore, Src TK selective inhibitors-damnacanthal or PP1-blocked LPS-induced NF-kappaB activation over a range of nanomolar concentrations. Genistein, damnacanthal, or PP1 blocked the LPS-induced serine phosphorylation, the degradation of IkappaB-alpha, and the consequent translocation of the p65 subunit of NF-kappaB to the nucleus. In addition to serine phosphorylation of IkappaB-alpha, LPS-induced NF-kappaB activation also required tyrosine phosphorylation of IkappaB-alpha. These TK inhibitors blocked substantially LPS induction of tyrosine phosphorylation of IkappaB-alpha. Furthermore, cSrc and Lck were physically associated with IkappaB-alpha. These results suggest that the LPS-induced NF-kappaB pathways are dependent on both serine and tyrosine phosphorylation of IkappaB-alpha, and that Src TK, such as cSrc and Lck, are key components of the LPS signaling pathway through at least two different mechanisms associated with NF-kappaB activation.

SH2 domain-mediated activation of an SRC family kinase is not required to initiate Ca2+ release at fertilization in mouse eggs.

SRC family kinases (SFKs) function in initiating Ca2+ release at fertilization in several species in the vertebrate evolutionary line, but whether they play a similar role in mammalian fertilization has been uncertain. We investigated this question by first determining which SFK proteins are expressed in mouse eggs, and then measuring Ca2+ release at fertilization in the presence of dominant negative inhibitors. FYN and YES proteins were found in mouse eggs, but other SFKs were not detected; based on this, we injected mouse eggs with a mixture of FYN and YES Src homology 2 (SH2) domains. These SH2 domains were effective inhibitors of Ca2+ release at fertilization in starfish eggs, but did not inhibit Ca2+ release at fertilization in mouse eggs. Thus the mechanism by which sperm initiate Ca2+ release in mouse eggs does not depend on SH2 domain-mediated activation of an SFK. We also tested the small molecule SFK inhibitor SU6656, and found that it became compartmentalized in the egg cytoplasm, thus suggesting caution in the use of this inhibitor. Our findings indicate that although the initiation of Ca2+ release at fertilization of mammalian eggs occurs by a pathway that has many similarities to that in evolutionarily earlier animal groups, the requirement for SH2 domain-mediated activation of an SFK is not conserved.

The influence of SRC-family tyrosine kinases on Na,K-ATPase activity in lens epithelium.

PURPOSE: Na,K-adenosine triphosphatase (ATPase) is essential for the regulation of cytoplasmic ion concentrations in lens cells. Earlier studies demonstrated that tyrosine phosphorylation by Lyn kinase, a Src-family member, inhibits Na,K-ATPase activity in porcine lens epithelium. In the present study, experiments were conducted to compare the ability of other Src-family kinases (Fyn, Src, and Lck) and Fes, a non-Src-family tyrosine kinase, to alter Na,K-ATPase activity. METHODS: Membranes prepared from porcine lens epithelium were incubated with partially purified tyrosine kinases in buffer containing 1 mM adenosine triphosphate (ATP). ATP hydrolysis in the presence and absence of ouabain was used to measure Na,K-ATPase activity. Western blot analysis was used to examine phosphotyrosine-containing proteins and tyrosine kinase expression. RESULTS: Fyn reduced Na,K-ATPase activity by approximately 30%. In contrast, Src caused a approximately 38% increase of Na,K-ATPase activity. Na,K-ATPase activity in membrane material treated with Lck or Fes was not significantly altered, even though Lck and Fes treatment induced robust tyrosine phosphorylation. Added exogenously, each tyrosine kinase induced a different pattern of membrane protein tyrosine phosphorylation. As judged by immunoprecipitation, Src, Fyn, Lyn, and Lck elicited tyrosine phosphorylation of the Na,K-ATPase alpha1 protein. Src, Fyn, Lyn, Lck, and Fes were each detectable in the epithelium by Western blot. CONCLUSIONS: The results indicate considerable variation in the Na,K-ATPase activity response of lens epithelium to different tyrosine kinases. This could perhaps explain why inhibition of Na,K-ATPase activity is reported to be caused by tyrosine phosphorylation in some tissues, whereas stimulation of Na,K-ATPase activity is observed in other tissues.

A comparison of the protective effects of systemic administration of a pro-glutathione drug and a Src-PTK inhibitor against noise-induced hearing loss.

Both the antioxidant, n-l-acetyl cysteine (L-NAC) and the Src inhibitor, KX1-004, have been used to protect the cochlea from hazardous noise. To date, KX1-004 has only been used locally on the round window. In the current study, the two drugs were administered systemically. LNAC was delivered intraperitoneally at a dose of 325 mg/kg while KX1-004 was administered subcutaneously at a dose of 50 mg/kg. The noise exposure consisted of a 4 kHz octave band of noise at 100 dB SPL for 6 hours/day for 4 days. The drugs were administered once each day, 30 minutes prior to the onset of the noise exposure. The animals' hearing was estimated using the evoked response records from surgically-implanted chronic electrodes in the inferior colliculi. Animals treated with LNAC and KX1-004 had from 10 to 20 dB less temporary threshold shift at day 1 and an average 10 dB less permanent threshold shift by day 21 when compared to control saline treated animals. There were no significant side effects (i.e.: appetite loss, weight loss, lethargy, etc.) related to either of the drug treatments. KX1-004 produced at least as much protection as L-NAC, but at a significantly lower concentration.

[Effect of nocodazole on the activation of transcription factors STAT1 and STAT3 in A431 cells]. / Vliianie nokodazola na aktivatsiiu transkriptsionnykh faktorov STAT1 i STAT3 v kletkakh A431.

The STAT transcription factors (signal transducers and activators of transcription), STAT1 and STAT3, are involved in signal transduction from growth factors and different cytokine receptors. STAT1 and STAT3 activation mechanisms are not sufficiently investigated, but they are known to depend upon both cell type and stimulus for either of them. Recently, we have shown that nocodazole blocked EGF-induced STAT1 transport to the nucleus. Here, we have compared STAT1 and STAT3 activation in response to IFNgamma, IFNalpha and epidermal growth factor (EGF) in A431 cells. We have shown the STAT1 activation by all these agents; unlike, STAT3 was activated by EGF only. STAT1 and STAT3 activation upon EGF is blocked by both nocodazole and Src-kinase family inhibitor. STAT1 activation upon IFNgamma influence is blocked by nocodazole, but does not depend on the activity of Src-family kinases. The increased STAT3 phosphorylation results from a combined action of Src-kinase inhibitor and IFNgamma. IFNalpha-induced activation of STAT1 was not inhibited by either nocodazole or Src-kinase inhibitor. Taken together, the data obtained suggest that the activation of both STAT1 and STAT3 in A431 cells is accomplished by different mechanisms.