c-Src Kinase Activity Research Articles

Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.

Pleiotrophin-induced endothelial cell migration is regulated by xanthine oxidase-mediated generation of reactive oxygen species

Pleiotrophin (PTN) is a heparin-binding growth factor that induces cell migration through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and integrin alpha v beta 3 (ανβ3). In the present work, we studied the effect of PTN on the generation of reactive oxygen species (ROS) in human endothelial cells and the involvement of ROS in PTN-induced cell migration. Exogenous PTN significantly increased ROS levels in a concentration and time-dependent manner in both human endothelial and prostate cancer cells, while knockdown of endogenous PTN expression in prostate cancer cells significantly down-regulated ROS production. Suppression of RPTPβ/ζ through genetic and pharmacological approaches, or inhibition of c-src kinase activity abolished PTN-induced ROS generation. A synthetic peptide that blocks PTN–ανβ3 interaction abolished PTN-induced ROS generation, suggesting that ανβ3 is also involved. The latter was confirmed in CHO cells that do not express β3 or over-express wild-type β3 or mutant β3Y773F/Y785F. PTN increased ROS generation in cells expressing wild-type β3 but not in cells not expressing or expressing mutant β3. Phosphoinositide 3-kinase (PI3K) or Erk1/2 inhibition suppressed PTN-induced ROS production, suggesting that ROS production lays down-stream of PI3K or Erk1/2 activation by PTN. Finally, ROS scavenging and xanthine oxidase inhibition completely abolished both PTN-induced ROS generation and cell migration, while NADPH oxidase inhibition had no effect. Collectively, these data suggest that xanthine oxidase-mediated ROS production is required for PTN-induced cell migration through the cell membrane functional complex of ανβ3 and RPTPβ/ζ and activation of c-src, PI3K and ERK1/2 kinases.

Integrin αIIbβ3-Mediated c-Src Activation: Differential Binding to Inactive and Active c-Src

Activation of the tyrosine kinase c-Src is a fundamental element of integrin αIIbβ3-mediated outside-in signaling in platelets. Presently, it is thought that c-Src is bound via its SH3 domain to the C-terminal Arg-Gly-Thr (RGT) sequence of the β3 cytoplasmic tail (CT). Although a recent NMR study supports this contention, it is likely that such binding would be precluded in the inactive conformation of c-Src because the linker connecting the SH2 and kinase domains of c-Src physically occludes the putative β3 CT binding site in the SH3 domain. Accordingly, we have re-examined the interaction between c-Src and β3 by immunoprecipitating β3 bound c-Src from resting and agonist-stimulated platelets and then applied the biophysical techniques of surface plasmon resonance (SPR) and NMR spectroscopy to directly characterize the interaction of the SH3 domain with the β3 CT. In unstimulated platelets, there was little or no association between c-Src and β3. However, following platelet stimulation with 1 unit/ml thrombin, c-Src binding to β3 is detectable within 10 sec of stimulation and binding persists for at least the next 90 sec. Phosphorylation of c-Src residue Tyr419 located in its activation loop is an indicator of c-Src activation. We detected the phosphorylation of β3-bound c-Src on Tyr419 at 20 sec following thrombin stimulation. Tyr419 phosphorylation then persisted for the next 40 sec after which it rapidly declined. Thus, platelet stimulation induces the rapid interaction of c-Src with the β3 subunit of αIIbβ3 and this interaction is accompanied by the induction of c-Src kinase activity. Since it has been reported that c-Src binds to β3 via its SH3 domain, we used SPR spectroscopy to measure the strength of this interaction. In initial experiments, we measured binding of the C-terminal β3 heptapeptide NITYRGT to the immobilized SH3 domain and detected NITYRGT binding with a dissociation constant (Kd) of approximately 700 mM. We then repeated the measurements by flowing the SH3 domain over the entire 43 residue β3 CT appended to the SPR chip. Under these conditions, we detected a Kd for SH3 domain binding to the β3 CT of 7.21 ± 2.42 µM. Previously, we reported similar behavior for talin-1 FERM domain binding to the β3 CT. Thus, these experiments suggest that like binding of the talin-1 FERM domain to the β3 CT, the interaction of c-Src with β3 is a two-dimensional ternary interaction in which the membrane surface makes an important contribution. Next, to identify the sites in the SH3 domain that interact with β3, we used NMR spectroscopy. In these experiments, we measured the interaction of the β3 peptide NITYRGT with the 15N-labeled SH3 domain. Because NITYRGT binding to the SH3 domain is weak, we used chemical shift perturbations (CSP) to identify the SH3 domain residues interacting with NITYRGT. We found that NITYRGT interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 CT (NITYEGK) induced only small CSP on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the β3 peptide to the RT-loop. Under these conditions, the β3 peptide induced CSP similar to the negative control. Thus, these studies indicate that the primary interaction of c-Src with the β3 CT occurs in its activated state at a site that overlaps with the polyproline binding site in its SH3 domain and suggest that protein-membrane interactions make an important contribution to the strength of binding. By contrast, interactions of inactive c-Src with β3 are weak and insensitive to β3 CT mutations. DisclosuresNo relevant conflicts of interest to declare.

CD24 activates the NLRP3 inflammasome through c-Src kinase activity in a model of the lining epithelium of inflamed periodontal tissues.

Chronic periodontitis is characterized by perturbation of the epithelial attachment to the tooth with subsequent migration of the lining epithelium and formation of a cleft or pocket. This non-keratinized lining epithelium provides initial responses to bacterial products by signalling through receptors of innate immunity to activate inflammasome pathways. These comprise an intracellular network of regulatory and effector molecules leading to synthesis and activation of pro-inflammatory cytokines. Conversely, CD24 is characteristically strongly expressed by the pocket epithelium and is reported to function as an important negative regulator for danger signals, protecting tissues from excessive leukocyte activity. The objective of the study was to determine the impact of ligation of CD24 on expression of inflammasome components. An epithelial mimic of pocket epithelium was used to evaluate activation of the NLRP3 inflammasome pathway. Surprisingly, antibody ligation of CD24 enhanced expression of NLRP3 together with co-activators ASC and caspase-1 resulting in burst release of activated interleukin (IL)-18. Potent product inhibition was detected with IL-18 suppressing expression of NLRP3, ASC, and caspase-1. Scant distribution of these products within pocket epithelium compared with healthy gingival attachment provided indication of potential cycling of NLRP3 inflammasome expression. As subjects with mild chronic periodontitis have increased titres of serum antibodies auto-reactive with CD24 compared with those of subjects with severe periodontitis, a molecular mechanism for regulated expression of the NLRP3 inflammasome mediated by c-Src kinase activity, is proposed. This pathway could be regionally disrupted by products of pathogenic bacteria with profound downregulation in the dysbiosis associated with severe disease.

A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition.

Serum starvation regulates E-cadherin upregulation via activation of c-Src in non-small-cell lung cancer A549 cells.

E-cadherin is essential for the integrity of adherens junctions between lung epithelial cells, and the loss of E-cadherin allows cell motility and is thought to promote lung cancer metastasis. While the downregulation of E-cadherin expression has been well characterized and is seen with transforming growth factor-β1 (TGF-β1) exposure, few studies have focused on E-cadherin upregulation. Here, we show that serum starvation causes increased E-cadherin expression via the activation of c-Src kinase in non-small-cell lung cancer A549 cells. Serum starvation increased E-cadherin protein levels in a time- and dose-dependent manner. E-cadherin mRNA transcripts were unchanged with starvation, while protein translation inhibition with cycloheximide attenuated E-cadherin protein induction by starvation, suggesting that E-cadherin is regulated at the translational level by serum starvation. c-Src is a nonreceptor tyrosine kinase known to regulate protein translation machinery; serum starvation caused early and sustained activation of c-Src in A549 cells followed by E-cadherin upregulation. Furthermore, overexpression of a dominant negative c-Src attenuated the induction of E-cadherin by serum deprivation. Finally, we observed that TGF-β1 treatment attenuated the serum activation of c-Src as well as E-cadherin expression when cells were deprived of serum. In conclusion, our data demonstrate that the c-Src kinase is activated by serum starvation to increase E-cadherin expression in A549 cells, and these phenomena are antagonized by TGF-β1. These novel observations implicate the c-Src kinase as an upstream inducer of E-cadherin protein translation with serum starvation and TGF-β1 diametrically regulating c-Src kinase activity and thus E-cadherin abundance in A549 cells.

Paeonol suppresses chondrosarcoma metastasis through up-regulation of miR-141 by modulating PKCδ and c-Src signaling pathway.

Chondrosarcoma, a primary malignant bone cancer, has potential for local invasion and distant metastasis, especially to the lungs. Patients diagnosed with it show poor prognosis. Paeonol (2'-hydroxy-4'-methoxyacetophenone), the main active compound of traditional Chinese remedy Paeonia lactiflora Pallas, exhibits anti-inflammatory and anti-tumor activity; whether paeonol regulates metastatic chondrosarcoma is largely unknown. Here, we find paeonol do not increase apoptosis. By contrast, at non-cytotoxic concentrations, paeonol suppresses migration and invasion of chondrosarcoma cells. We also demonstrate paeonol enhancing miR-141 expression and miR-141 inhibitor reversing paeonol-inhibited cell motility; paeonol also reduces protein kinase C (PKC)δ and c-Src kinase activity. Since paeonol inhibits migration and invasion of human chondrosarcoma via up-regulation of miR-141 via PKCδ and c-Src pathways, it thus might be a novel anti-metastasis agent for treatment of metastatic chondrosarcoma.

6-Shogaol exerts anti-proliferative and pro-apoptotic effects through the modulation of STAT3 and MAPKs signaling pathways.

6-shogaol (6SG), one of active ingredients in ginger (Zingiber officinale), is known to exhibit anti-proliferative, anti-metastatic, and pro-apoptotic activities through a mechanism that is not fully elucidated. Because the aberrant activation of STAT3 and MAPKs have been associated with regulation of proliferation, invasion, and metastasis of tumors, we hypothesized that 6SG modulates the activation of STAT3 and MAPKs activation in tumor cells. We found that 6SG strongly inhibited constitutive phosphorylation of STAT3 through inhibition of the activation of upstream JAK2 and c-Src kinases and nuclear translocation of STAT3 on both MDA-MB231 and DU145 cells. Also, 6SG caused the activation of JNK, p38 MAPK, and ERK. Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented 6SG-induced apoptosis. 6SG induced apoptosis as characterized by cleavage of PARP, accumulation of cells in subG1 phase, positive Annexin V binding, down-regulation of STAT3-regulated proteins, and activation of caspase-8, -9, -3 in both MDA-MB231 cells. Compared with other analogues of 6SG, such as 6-gingerol (6G), 8-gingerol (8G), and 10-gingerol (10G), 6SG was found to be the most potent blocker of STAT3 activation. We observed that the administration of 6SG alone significantly suppressed the growth of the tumor. As compared to the vehicle control, 6SG also suppressed the expression of STAT3-regulated gene products such as Bcl-2, Bcl-xL, and Survivin in tumor tissues. Overall, these findings suggest that 6SG can interfere with multiple signaling cascades involved in tumorigenesis and can be used as a potential therapeutic candidate for both the prevention and treatment of cancer.

Endoplasmic Reticulum Stress Induces Epithelial-Mesenchymal Transition through Autophagy via Activation of c-Src Kinase

Background: Endoplasmic reticulum (ER) stress has been implicated in inducing epithelial-mesenchymal transition (EMT). ER stress is also known to induce autophagy. However, it is unclear whether ER stress-induced autophagy contributes to EMT. We hypothesized that ER stress might induce EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. Method: All experiments were performed using HK-2 cells. Protein expression was measured by Western blot analysis. Immunofluorescence and small interfering RNA (siRNA) experiments were performed. Results: Chemical ER stress inducers such as tunicamycin (TM, 0.2 μ<smlcap>M</smlcap>) and thapsigargin (TG, 0.2 μ<smlcap>M</smlcap>) induced EMT, as shown by upregulation of α-smooth muscle actin and downregulation of E-cadherin. ER stress inhibitors such as 4-PBA and salubrinal suppressed both TM- and TG-induced EMT. TM and TG also induced autophagy, as evidenced by upregulation of LC3-II and beclin-1, which were abolished by pretreatment with ER stress inhibitors. Transfection with siRNA targeting ER stress protein (IRE-1) blocked the TM- or TG-induced EMT and autophagy. Autophagy inhibitors such as 3-methyladenine and bafilomycin inhibited the TM- or TG-induced EMT. Transfection with siRNA targeting autophagy protein (beclin-1) also blocked the TM- or TG-induced EMT. Both TM and TG induced activation of c-Src kinase. Inhibitor of c-Src kinase (PP2) suppressed the TM- or TG-induced autophagy and EMT. Conclusion: ER stress by TM or TG induced EMT through autophagy via activation of c-Src kinase in tubular epithelial cells.

Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism.

The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 h resulted in a dramatic modulation of tight junction protein distribution from a linear organization into wavy structure. Continuation of cyclic stretch for 6 h led to redistribution of tight junction proteins from the intercellular junctions into the intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src, and myosin light-chain kinase (MLCK). Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions, and actin cytoskeleton. Paracellular permeability measured by a novel method demonstrated that cyclic stretch increases paracellular permeability by a JNK, Src kinase, and MLCK-dependent mechanism. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin, and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC colocalizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src colocalizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src, and MLCK-dependent mechanism.

A Novel Mechanism by Which Tissue Transglutaminase Activates Signaling Events That Promote Cell Survival

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

Biochemical evidence for gap junctions and Cx43 expression in immortalized human chondrocyte cell line: a potential model in the study of cell communication in human chondrocytes

The development of chondrocytic cell lines has enabled the investigation of the role of cellular phenotype and mechanisms in articular cartilage biology and physiopathology of several rheumatic diseases. Among them, the T/C-28a2 cell line has become a common tool in cartilage research. Recent results from our group have revealed that primary human chondrocytes in tissue and in monolayer culture contain high levels of connexin 43 (Cx43) and are able to directly communicate through gap junction (GJ) channels. These results challenge the existing thesis of cartilage physiology, that chondrocytes do not have the capacity to physically communicate with each other. Established cell lines offer the advantage of convenience and uniformity; however, the establishment process may cause a disruption of GJ. This study was performed to investigate if T/C-28a2 cells contain Cx43 protein and form functional channels. Cx43 was characterized by RT-qPCR, Western blotting, and immunohistochemistry (IHC). Electrophysiology experiments, Lucifer Yellow (LY) uptake, electroporation in situ and scrape loading assay were performed to test the functionality of GJs. T/C-28a2 cells express Cx43. Electrophysiology experiments and LY uptake confirmed the capacity of these cells to communicate through GJ channels, although these cells contain significant levels of active c-Src kinase, presumably due to their immortalization with the Simian Virus 40 large T antigen. The results were validated using primary chondrocytes (PC). These results reveal that the T/C-28a2 line may provide a useful invitro model for the study of Cx43 function and cell communication to understand the physiology of chondrocytes and cartilage.

Simulation of the Conformational Transition Pathway for the Activation of Full-Length C-Src Kinase using the String Method

The Src family kinases are important cellular signaling proteins that share a common catalytic domain and two peptide-binding regulatory domains called SH2 and SH3. X-ray crystal structures of c-Src kinase are available for the inactive assembled state and an active-like re-assembled state, but the transition pathway and its dependence on the configuration of the SH2 and SH3 regulatory modules is still largely unsolved. Using molecular dynamics simulations and the string method with swarms-of-trajectories we have determined the transition pathway in both the assembled and re-assembled active-like conformations of the SH2/SH3 domains. Comparison of the active-like conformation of the catalytic domain in the two SH2/SH3 states shows that the re-assembled SH2/SH3 promotes a further opening of the catalytic site. The mechanism of the A-loop opening is also influenced by the SH2/SH3 domains. The calculated pathway provides novel insight into the influence of the regulatory models on kinase activation.

Capillarisin inhibits constitutive and inducible STAT3 activation through induction of SHP-1 and SHP-2 tyrosine phosphatases

Signal transducers and activators of transcription (STAT)-3 is a latent cytosolic transcription factor that has been closely associated with survival, proliferation, chemoresistance, and metastasis of tumor cells. Whether the anti-proliferative, pro-apoptotic, and anti-metastatic effects of capillarisin (CPS), derived from Artemisia capillaris (Compositae), are linked to its capability to inhibit STAT3 activation was investigated. We found that CPS specifically inhibited both constitutive and inducible STAT3 activation at tyrosine residue 705 but not at serine residue 727 in human multiple myeloma cells. Besides the inhibition of STAT3 phosphorylation, CPS also abrogated STAT3 constitutive activity and nuclear translocation. The suppression of STAT3 was mediated through the inhibition of activation of upstream JAK1, JAK2, and c-Src kinases. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate treatment reversed the CPS-induced down-regulation of JAK1/2 and STAT3, thereby suggesting the involvement of a PTP. Indeed, knockdown of the SHP-1 and SHP-2 genes by small interfering RNA suppressed the ability of CPS to inhibit JAK1 and STAT3 activation, suggesting the critical role of both SHP-1 and SHP-2 in its possible mechanism of action. CPS downregulated the expression of STAT3-regulated antiapoptotic and proliferative gene products; and this correlated with suppression of cell viability, the accumulation of cells in sub-G1 phase of cell cycle and induction of apoptosis. Moreover, CPS potentiated bortezomib-induced apoptotic effects in MM cells, and this correlated with down-regulation of various gene products that mediate cell proliferation (Cyclin D1 and COX-2), cell survival (Bcl-2, Bcl-xl, IAP1, IAP2, and Survivin), invasion (MMP-9), and angiogenesis (VEGF). Thus, overall, our results suggest that CPS is a novel blocker of STAT3 activation and thus may have a potential in negative regulation of growth, metastasis, and chemoresistance of tumor cells.

Maintenance of the stemness in CD44+ HCT-15 and HCT-116 human colon cancer cells requires miR-203 suppression

The purpose of this study was to isolate cancer stem cells (CSCs, also called tumor-initiating cells, TICs) from established human colorectal carcinoma (CRC) cell lines, characterize them extensively and dissect the mechanism for their stemness. Freshly isolated CD44+ and CD44− cells from the HCT-15 human colon cancer cell line were subjected to various analyses. Interestingly, CD44+ cells exhibited higher soft agar colony-forming ability and in vivo tumorigenicity than CD44− cells. In addition, the significant upregulation of the protein Snail and the downregulation of miR-203, a stemness inhibitor, in CD44+ cells suggested that this population possessed higher invasion/metastasis and differentiation potential than CD44− cells. By manipulating the expression of CD44 in HCT-15 and HCT-116 cells, we found that the levels of several EMT activators and miR-203 were positively and negatively correlated with those of CD44, respectively. Further analyses revealed that miR-203 levels were repressed by Snail, which was shown to bind to specific E-box(es) present in the miR-203 promoter. In agreement, silencing miR-203 expression in wild-type HCT-116 human colon cancer cells also resulted in an increase of their stemness. Finally, we discovered that c-Src kinase activity was required for the downregulation of miR-203 in HCT-15 cells, which was stimulated by the interaction between hyaluronan (HA) and CD44.Taken together, CD44 is a critical molecule for modulating stemness in CSCs. More importantly, we show for the first time that the downregulation of miR-203 by HA/CD44 signaling is the main reason for stemness-maintenance in colon cancer cells.

Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src

c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.

AMP-dependent Kinase Inhibits Oxidative Stress-induced Caveolin-1 Phosphorylation and Endocytosis by Suppressing the Dissociation between c-Abl and Prdx1 Proteins in Endothelial Cells

Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H2O2 exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.

Persimmon peel extract attenuates PDGF-BB-induced human aortic smooth muscle cell migration and invasion through inhibition of c-Src activity

The unregulated migration and invasion of human aortic smooth muscle cells (HASMCs) into the intima is a crucial step in the development of atherosclerosis. Recently, the oriental persimmon extract (Diospyros kaki Thunb. cv. Fuyu) has been investigated for its anti-atherogenic properties, but the molecular mechanisms involved remain unclear. We investigated the inhibitory effects of persimmon peel and flesh extract on the platelet-derived growth factor (PDGF) BB-induced MMP-1 expression using Western blot, and abnormal migration and invasion of HASMCs using a modified Boyden chamber assay and a wound healing assay. We also evaluated the inhibitory effects of persimmon peel extract on aortic vessel thickening using a rat aortic sprouting assay. Persimmon peel (PPE), but not flesh extract (PFE), inhibited PDGF-BB-induced MMP-1 expression, cell migration and invasion in HASMCs, while suppressing the rat aortic sprouting. Western blot and in vitro kinase assay data demonstrated that PPE inhibited Src kinase activity and subsequently attenuated PDGF-BB-induced phosphorylation of MAPK and Akt signalling pathways. Taken together, our results indicate that persimmon peel might possess a potential anti-atherogenic effect through attenuation of ASMCs migration and invasion and aortic sprouting by direct inhibition of the c-Src kinase activity.

Kisspeptin activation of TRPC4 channels in female GnRH neurons requires PIP2 depletion and cSrc kinase activation.

Kisspeptin signaling via its Gαq-coupled receptor GPR54 plays a crucial role in modulating GnRH neuronal excitability, which controls pituitary gonadotropins secretion and ultimately reproduction. Kisspeptin potently depolarizes GnRH neurons primarily through the activation of canonical transient receptor potential (TRPC) channels, but the intracellular signaling cascade has not been elucidated. Presently, we have established that kisspeptin activation of TRPC channels requires multiple membrane and intracellular signaling molecules. First, phosphatidylinositol-4,5-bisphosphate (PIP(2)) hydrolysis by phospholipase Cβ is required because whole-cell dialysis of Dioctanoylglycerol-PIP(2) (DiC8-PIP(2)) inhibited the kisspeptin activation of TRPC channels, and the phosphatidylinositol 4-kinase inhibitor wortmannin, which attenuates PIP(2) synthesis, prolonged TRPC channel activation. Using single cell RT-PCR, we identified that the mRNA for the PIP(2)-interacting TRPC channel subunit, TRPC4α, is expressed in GnRH neurons. Depletion of intracellular Ca(2+) stores by thapsigargin and inositol 1,4,5-trisphosphate had no effect, indicating that the TRPC channels are not store-operated. Neither removing extracellular Ca(2+) nor buffering intracellular Ca(2+) with EGTA or BAPTA had any effect on the kisspeptin activation of the TRPC channels. However, the Ca(2+) channel blocker Ni(2+) inhibited the kisspeptin-induced inward current. Moreover, inhibition of protein kinase C by bisindolylmaleimide-I or calphostin C had no effect, but activation of protein kinase C by phorbol 12,13-dibutyrate occluded the kisspeptin-activated current. Finally, inhibition of the cytoplasmic tyrosine kinase cSrc by genistein or the pyrazolo-pyrimidine PP2 blocked the activation of TRPC channels by kisspeptin. Therefore, TRPC channels in GnRH neurons are receptor-operated, and kisspeptin activates TRPC channels through PIP(2) depletion and cSrc tyrosine kinase activation, which is a novel signaling pathway for peptidergic excitation of GnRH neurons.

D-pinitol Inhibits Prostate Cancer Metastasis through Inhibition of αVβ3 Integrin by Modulating FAK, c-Src and NF-κB Pathways

Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. D-pinitol, a 3-methoxy analogue of d-chiro-inositol, was identified as an active principle in soy foods and legumes, and it has been proven to induce tumor apoptosis and metastasis of cancer cells. In this study, we investigated the anti-metastasis effects of D-pinitol in human prostate cancer cells. We found that D-pinitol reduced the migration and the invasion of prostate cancer cells (PC3 and DU145) at noncytotoxic concentrations. Integrins are the major adhesive molecules in mammalian cells and have been associated with the metastasis of cancer cells. Treatment of prostate cancer cells with D-pinitol reduced mRNA and cell surface expression of αvβ3 integrin. In addition, D-pinitol exerted its inhibitory effects by reducing focal adhesion kinase (FAK) phosphorylation, c-Src kinase activity and NF-κB activation. Thus, D-pinitol may be a novel anti-metastasis agent for the treatment of prostate cancer metastasis.