Based upon the presented data, we propose that ILK signalling is dysregulated early during the development of human colon cancer, and that selective inhibition of this molecule alone or in combination with the standard therapeutic modality might be a more effective means of treating colon cancer.The putative oncogene, integrin-linked kinase (ILK) is a protein serine/threonine kinase that has been reported to regulate a number of biological properties including anchorage-independent cell cycle progression, tumour cell invasion and apoptosis. Overexpression of ILK has been documented in a wide variety of human malignancies including Ewing's sarcoma (ES), primitive neural ectodermal tumours (PNETs) and prostate tumours (PT). We recently reported that ILK signalling was also dysregulated in patients with the genetic condition familial adenomatous polyposis (FAP), a precursor to colon cancer. In this study, we extended our previous work by investigating the ILK-signalling pathway in sporadic human colon cancer and representative lymph node metastases. The data indicate that the ILK protein is significantly hyperexpressed in malignant acini in relation to normal crypts. Moreover, overexpression of ILK not only coincided with increased MBP phosphotransferase activity but as well with effects on downstream targets like GSK3 Stabilisation of the latter is believed to result in its translocation to the nucleus where it binds to the Tcf-4 (T-cell factor) family of transcription factors resulting in the expression of a number of different genes that have been implicated in oncogenesis. These include cyclin D1, c-myc and the matrix metalloproteinase (MMP)-7 , duplicating the events associated with the mutation of APC. Translocation of β-catenin accompanied the activation of Tcf-4-dependent gene transcription (Mutation of the adenomatous polyposis coli (APC) gene is an integral event in the genesis of colorectal cancer . Monoclonal anti-ILK (IP) and MBP were obtained from Upstate Biotechnology Inc. . Anti-phosphospecific-PKB Ser473 and GSK3β Ser-9 were obtained from New England Biolabs Inc. . Horse-radish peroxidase-conjugated secondary antibodies were obtained through Calbiochem . cAMP-dependent protein kinase inhibitor peptide, EGTA, EDTA, MOPS, PMSF, sodium orthovanadate, leupeptin, aprotinin, benzamidine, dithiothreitol and β-glycerolphosphate were purchased from Sigma, Sigma-Aldrich, Oakville, Ontario.Rabbit polyclonal antibodies for ILK (IB/IHC), PKB and GSK3We obtained a total of 38 cases of human colon cancer through Dr D Owen from the Division of Anatomical Pathology at Vancouver Hospital and Health Sciences Centre (VH&HSC). The 38-paired cases were used for biochemical analysis. In all, 16 of the cases, which were utilised in these studies, were selected for immunohistochemical analysis on the basis that lymph node metastases were present. Ethical consent for these studies was obtained from each of the patients and by boards governing research at the University of British Columbia and VH&HSC.μm in diameter) using a cryostat, and approximately 20 slices were placed in 1 ml of homogenisation buffer containing 20 mM MOPS, 50 mMβ-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP40, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM phenylmethanesulphonylfluoride and 10 μg ml−1 leupeptin as previously described .A total of 400 μl of the kinase reaction buffer , 2 mM NaF, 5 μg of MBP per tube and 0.5 μg of ATP  at 30°C for 20 min. The reaction was terminated with the addition of 10 μl of 4 × sample buffer. The tubes were microfuged at maximum for 1 min and the proteins were resolved on a 14% SDS–PAGE gel. The resulting gel was stained with Coomassie Blue as outlined above and phosphorylation of the substrate was visualised by autoradiography.The beads were pelleted and the reaction was started by the addition of 25 M citrate buffer for 10 min at high power using a 925-W microwave oven. The slides were then incubated with the appropriate primary antibody at a pre-determined concentration overnight at room temperature. Following incubation, the sections were rinsed three consecutive times with PBS and then incubated with the appropriate biotinylated secondary antibody for 1 h followed by incubation with peroxidase-labelled streptavidin. AEC substrate was used as the chromagen and the sections were counterstained with haematoxylin. Three observers independently examined all the stained immunoreactive positivity was assessed by uniform red staining.Formalin-fixed paraffin-embedded tissue sections were obtained through the Division of Anatomical Pathology at VH&HSC. Immunohistochemical staining for ILK (1 : 100) and phospho-Ser473 PKB (1 : 100) was carried out using a standard strepta-vidin–biotin technique. Antigen retrieval was performed by pressure-cooking the sections in a 0.01 The staining intensity  was scored by three independent examiners , and the results are expressed as the mean±s.d.P<0.05 being considered significant using the Student's t-test .The relative amounts of protein were measured by scanning the film using the BioRad gel-doc apparatus into a TIFF format file. The numerical densitometric values were assigned arbitary values on a scale of 1–3 for immunohistochemistry. The band densities and the results are expressed as mean±s.d., with A recent report from our laboratory indicated that the expression and activity of ILK is perturbed in polypoid lesions resected from patients diagnosed with the autosomal dominant condition, FAP . There was approximately a three-fold increase in the expression of ILK in the cancerous acini (2.22) when compared to the normal adjacent tissue  was shown to inhibit MMP-9 promoter activity as well as lead to a reduction in the invasive potential of intestinal and mammary epithelial cells (P<0.00001). There was no significant difference in the expression of ILK between the primary cancer and the metastatic deposit within regional lymph nodes (P=0.1). Based upon these results, it appears that changes in the expression of ILK occur prior to changes in the metastatic potential of colon cancer cells.To characterise the protein expression of ILK in metastatic lesions, 16 cases were selected on the basis that metastatic deposits were present in the regional lymph nodes. The representative results from these studies are presented in Figure 2In our previous study, we demonstrated that overexpression of ILK in colonic polyps coincided with an increase in the immunoprecipitated ILK MBP phosphotransferase activity  has been reported to coincide with a decrease in Ser473 phosphorylation as well as modulate the activity of the respective kinase. Transient and stable overexpression of the ILK protein is known to correlate with an increase in not only Ser473 phosphorylation but also the biochemical activity of PKB.There are a number of reports, which indicate that Ser473 of PKB is phosphorylated by an array of protein kinases including Mapkapk-2, PDK1/PRK2 and PKC. ILK has also been described as the putative PDK2 that phosphorylates this site on PKB. In this regard, ILK has been demonstrated to phosphorylate Ser473 β. However, only modest differences in the Ser473 phosphorylation status of PKB were observed in this animal model malignant cells retain the ability to modulate signalling pathways which ultimately regulate the subcellular distribution of β-catenin and that this regulation is probably a consequence of the ‘second hit’ in the wild-type APC allele  or even quite possibly because of differences in the proposed etiological pathways, that is, MIN β-catenin were shown to occur at the leading edge of the tumour (Since ILK has been reported to enhance tumour cell invasion (e tumour , whereasvs MIN lesions. As well, we are attempting to unravel the mechanism by which ILK is dysregulated in colon cancer. Undoubtedly, this information could provide further insight into the underlying mechanisms involved in the initiation of carcinogenesis within the colon, as well as enhance our understanding of the ‘just right’ model for carcinogenesis.In summary, we suggest that dysregulation of ILK signalling is an important early event in the genesis of human colon cancer. Furthermore, with the recent surge in the development of specific inhibitors to protein kinases, we propose that an inhibitor to ILK should be explored as a possible novel strategy for either the treatment and/or prevention of this disease. An inhibitor to this protein kinase could prove to be as efficacious as the tyrosine kinase inhibitor STI-571, which is used in the treatment of chronic myeloid leukaemia (


Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity. Cell signal transduction is a summation of positive stimuli and homeostatic negative feedback. Positive regulation of protein kinase cascades, such as those activated during mitogenic signaling, differentiation, and pathogenic processes such as inflammation and cancer, is particularly well studied. Most commonly, activation of a kinase cascade results from sequential phosphorylation of protein kinases by other kinases positioned upstream. In contrast, other than the action of phosphatases that remove the activating phosphorylation, inhibitory or negative feedback mechanisms for damping or silencing protein kinase cascades are less well understood. In fact, mainly due to the utility of anti-phosphoepitope antibodies in identifying phosphate on specific proteins, it has become common in the literature for the presence of an activating phosphorylation on a signaling kinase to be interpreted as an ‘active’ kinase pathway. This unfortunate simplification ignores more subtle means of signal regulation.Many signaling events, including UV irradiation, inflammatory cytokines, and mitogen stimulation, are accompanied by generation of reactive oxygen species Compared to the study of protein phosphorylation, study of specific oxidation on proteins is difficult. Antibodies against S-glutathionylation have been described, and protein sulfhydryls have been labeled with fluorescent maleimides or iodoacetamides. We have developed an unbiased method of quantifying reversible cysteine oxidation in proteins using an affinity capture technique we call Purification of Reversibly Oxidized Proteins (PROP). We have applied this technique to study the regulatory oxidation of the p38 MAPK signaling kinase. We have found that p38 is oxidized following exposure of cells to exogenous hydrogen peroxide or prostaglandin J2, and that oxidation results in kinase inactivation despite continued phosphorylation on the activating residues detected by immunoblot. Prostaglandin J2 (PGJ2) is an inflammatory mediator that has been characterized for its role in inducing oxidative signals 2O2, or both. As expected, we found that both osmotic shock and hydrogen peroxide, or both, induced phosphorylation of p38 detected with anti-phospho-p38 antibodies (2O2 treatment did not induce kinase activity despite strongly inducing p38 phosphorylation (lane 2). Additionally, H2O2 treatment of osmotically-shocked cells completely extinguished the p38 kinase activity, despite having no effect on p38 phosphorylation (lane 4). These results suggests that H2O2 blocks p38 kinase activity through a mechanism that does not decrease activating phosphorylation, probably cysteine oxidation.To test the effect of oxidation on p38 kinase activity, we treated cells with hyperosmotic sorbitol to activate p38, with 10 mM Htibodies , top. Wetibodies , bottom.To test this possibility, we devised a method to quantify oxidation of specific proteins. We developed and optimized a procedure for Purification of Reversibly Oxidized Proteins (PROP) described in detail in 2O2 induced oxidation of one or more cysteine residues on p38. We used the PROP procedure to measure reversible oxidation on p38. HeLa cells treated as above with hyperosmotic sorbitol and/or H2O2 were subjected to the PROP procedure, and the recovered proteins analyzed for p38 , an aliquot of the input sample was analyzed adjacent to equivalent fractions of serial precipitations using the activated thiol sepharose beads. Essentially no p38 was precipitated from the unoxidized cell extract . Oxidized p38 was recovered in the first pull down from the oxidize cells (lane 6) but not from the repeatedly precipitated extracts (lanes 7 and 8). Thus, the precipitation of oxidized proteins does not recover unoxidized proteins, and a single precipitation is essentially complete.Extracts containing p38 oxidized by Hull down . Using eNext, the efficiency of blocking of unoxidized proteins using NEM in the PROP procedure was tested. In the absence of blocking NEM ; thus NEM blockade of free thiols is efficient. As predicted labeling with biotin-maleimide could be increased in the oxidized cell sample using DTT (lane 8), and less so in unoxidized cells (lane 7). Following precipitation of these cell extracts using activated thiol beads (lanes 9 and 10) essentially no proteins were labeled with biotin-maleimide. Thus, again thiol bead precipitation using activated thiol beads efficiently removed the initially-oxidized proteins from cell extracts.in vitro assays, we sought to confirm that this reflected the activity of p38 in intact cells. We repeated the experiment shown in 2O2. Again PROP revealed oxidation of p38 by H2O2 treatment (bottom two panels).While oxidation of cells reduced the activity of p38 measured in 2O2 is a super-physiological oxidative stimulus, we tested the natural prostaglandin, PGJ2, for oxidation on p38. PGJ2 is an arachidonic acid metabolite associated with inflammation that has been implicated in regulatory oxidation of other proteins, for example p53 Since 10 mM H2O2, and PGJ2, all induced p38 phosphorylation  and PGJ2 .Hyperosmotic sorbitol, H2O2 and PGJ2 induced p38 oxidation, as revealed by PROP .The induction of p38 oxidation by PGJ2 was dose responsive , with di2O2 or PGJ2 was the result of reversible oxidation of p38, as detected using the PROP procedure. To test this hypothesis, cells were treated with combinations of sorbitol, H2O2, or PGJ2 as before. Aliquots of these samples were immunoblotted to assess p38 expression and phosphorylation , and had only minor effects on the mildly activated p38 from cells treated with PGJ2 alone (sample 2). However, DTT treatment of p38 from cells treated with both sorbitol and PGJ2 (sample 3) strongly (2.5 fold) activated p38 compared to the p38 that was not treated with DTT. In fact, DTT restored 104% of the p38 activity from the PGJ2/sorbitol sample (sample 3) compared to DTT-treated p38 from cells treated with sorbitol but no oxidants (sample 6). Thus, in vitro exposure to DTT completely restores the activity of p38 inhibited by PGJ2.2O2 alone (sample 4) or the combination of H2O2 and sorbitol (sample 5), the amount of activity recovered was reduced (66%) compared to the activity recovered from cells treated with sorbitol alone or sorbitol with PGJ2. Thus, this concentration of H2O2 may induce partially non-reversible inhibitory oxidation of p38.DTT treatment also increased the activity of p38 69% from cells treated with sorbitol alone (sample 6), suggesting that a fraction of phosphorylated p38 remains inactive as a kinase owing to oxidation in cells not exposed to deliberate oxidants, and perhaps that oxidation is a normal consequence of kinase activation. If so, this is not detected in the PROP assay , lane 2.The p38 protein kinase has 4 cysteine residues, none of which are predicted to be disulfide bonded. We constructed mutants of p38 containing cysteine-to-serine substitution of all 4 cysteine residues (C4S), four mutants in which each individual cysteine is mutated to serine, as well as four mutants in which single cysteines were added back to the 4CS mutant individually. We tested proteins encoded by each of these mutants for reversible oxidation induced by PGJ2, using PROP. Each of the mutant p38 proteins with individual cysteine to serine substitutions was still oxidized after exposure of cells to PGJ2 as measured by PROP, suggesting that oxidation occurred on more than one cysteine residue (data not shown). Analysis of the mutant p38 proteins in which individual cysteines were added back to the 4CS mutant allowed us to assess the oxidation at each cysteine residue after PGJ2 treatment. Summary results averaging three experiments are shown in http://www.pdb.org/pdb/explore/explore.do?structureId=1WFC). This structure confirms that p38 does not have disulfide bonds. It also shows  but this reagent is suitable for use with the activated thiol agarose beads.2H) or sulfonic (R-SO3H) acids. Additionally, nitroso-thiols can result from endogenous or exogenous nitric oxide. Disulfides, nitroso-thiols, and sulfenic acid are non-enzymatically converted to thiols in the presence of reducing agents such as glutathione or DTT, and are certainly detected using the PROP procedure in which modified proteins are subjected DTT. These modifications are likely also reversible in vivo and therefore represent potential regulatory events. Higher order oxidations (sulfinic and sulfonic) are unlikely to be reversed by DTT treatment during PROP. Based on our observations, it is possible that H2O2 induces some higher-order modifications of p38, given the lack of recovery of full activity by DTT treatment in vitro  or higher-order sulfinic . This observation leads to the prediction that these conserved cysteine residues may serve to control other MAP kinase family members much as they do p38.On the other hand, kinases that are related to one another likely share functional regulatory mechanisms, including the inhibitory redox switch. We identified the surface cysteines C119 and C162 of p38 as major sites of oxidation using mutant studies. On the linear subdomain map of p38 aligned with other protein kinases The ease by which specific cysteines and not others are oxidized may reflect the relative degree ionization of the target cysteine. Some oxidizable cysteines are also targets of electrophilic attack by drugs or signal-regulatory compounds originating outside the cell. For example the oxidized cysteine found in MEKK1 The biological relevance of cysteine modification by electrophilic compounds such as oxidized lipidsWe this inactive kinase retains its phosphorylation and may incorrectly be interpreted as an active kinase when using anti-phospho antibody immunoblot as a surrogate measure of “activity”. Signal transduction investigators are cautioned that the simplicity of an anti-phospho-immunoblot may obscure more subtle means by which signal transduction is regulated. We recommend that for a specific signaling pathway to be deemed “activated”, a direct measurement of the catalytic activity in vitro is indicated, together with an evaluation of natural substrates of the kinase within cells.Our results suggest that oxidation may serve as a redox switch that provides a feedback mechanism for p38 and other signaling kinases. Specifically, initial activation of p38 results from phosphorylation within the “activation loop”, that is detectable using anti-phospho p38 antibodies. Subsequently, activated p38 may become oxidized at one or more cysteine residues, resulting in the loss of kinase activity. Importantly, 2 environment. For experiments, cells were seeded in 6-well plates. Cells were transfected using Lipofectamine 2000  per manufacturer's instructions. Prostaglandin J2  was prepared in DMSO and stored in aliquots at −20°C. Antibodies used for immunoblotting were as follows: anti-p38 , anti-phospho-p38 and anti-phospho-ATF2 , HRP conjugated goat anti-rabbit secondary antibody . Blots were developed using enhanced chemiluminescence with images captured on a Fluor-Chem HD2  in 16 bit mode without image saturation. TIFF files were quantified using ImageJ (http://rsbweb.nih.gov/ij/).Cell culture, reagents and transfections. HeLa cells  were grown in Dulbecco's Minimal Eagle's Medium supplemented with 10% calf serum and penicillin/streptomycin, in 5% COp38 expression vectors. p38 was expressed from pcDNA3.1 containing a synthetic gene  encoding murine p38alpha, isoform 1 (GI:10092590), tagged at the C terminus with a chitin binding domain. For the cysteine mutants, a second synthetic gene was designed that was identical to the first except that all of the cysteine codons were mutated to serine codons (4CS). To prepare the single cysteine add back mutants, restriction sites engineered between each of the cysteine codons were used to swap each individual cysteine codon into the 4CS mutant.PROP procedure. Cells were rinsed quickly with HEPES-buffered saline and fixed in 10% TCA for 10 minutes to prevent post-lytic oxidation. Fixed cells on 35 mm wells were rinsed three times with methanol containing 10 mM N-ethylmaleimide (NEM), then air dried. Dry monolayers were dissolved in 0.25 ml of MLB/G  containing 20 mM NEM. During a 2 hour total incubation period at room temperature, cells were scraped into microfuge tubes, and subjected to sonication (five ½ second pulses with a microtip probe) to shear DNA. Twenty percent of each sample was retained as a ‘Total’ fraction, and precipitated in 4 volumes of methanol. After the two hour incubation, DTT was added to 50 mM to reduce oxidized thiols, and incubated at 50°C for 30 minutes. Proteins were precipitated by addition of 4 volumes of methanol, with incubation for 2 hours or overnight at −20°C, followed by centrifugation at 2300×g for 5 minutes. Pellets were washed 5 times for 5 minutes each by rotational agitation in 100% methanol and recentrifugation, to remove all reducing agents. Drained pellets were redissolved in MLB/G containing activated thiopropyl agarose beads  that had previously been washed rapidly in cold water per manufacturer's instructions, allowing 10 mg of dried beads per 35 mm well. Pellets were dislodged in this solution, and allowed to dissolve and react 2 hours or overnight with rotational agitation at RT, during which proteins with newly revealed thiols formed disulfide bonds with thiols on the beads. Beads were washed twice in MLB/G and three times in MLB . Drained beads, and ‘Total’ fractions, were resuspended in Laemmli sample buffer  and subjected to SDS-PAGE and immunoblotting. Gels were transferred to PVDF membranes and immunoblotted with anti-p38 antibodies. The protein purified on the thiol beads in the PROP procedure represents the oxidized protein present in the cells at the time of lysis, quantified as described above.In vitro kinase assay. Cells were lysed in MLB and immunoprecipitated using immobilized anti-phospho-p38 conjugated beads . Beads were washed 3 times with MLB and once with 50 mM Tris (pH 7.4). p38 activity was assayed using 1 µg ATF2 substrate  in a 20 µL reaction containing 25 mM Tris (pH 7.4), 10 mM magnesium chloride, and 7.5 µM ATP. Samples were incubated for 30 minutes at room temperature, and then stopped by addition of 2X Laemmli sample buffer. Samples were separated by SDS-PAGE and phosphorylated ATF2 detected by immunoblotting.


The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.3 using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration.We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-PThese data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes. Angiogenesis, the formation of new vessels from existing vasculature, is required for different physiological processes such as development, reproduction, wound healing, and tissue regeneration Binding of VEGFs and FGFs to their respective receptors leads to receptor phosphorylation and subsequent activation of signalling proteins including the Ras pathway, the Src family tyrosine kinases, phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1.PI3Ks are a conserved family of lipid kinases that catalyze the phosphorylation of the D3 position of the inositol ring of phosphoinositides at the plasma membrane or in specific cellular membrane compartments. Interaction between the newly generated 3-phosphorylated phosphoinositides and distinct structural motifs, such as pleckstrin homology (PH) domain, can regulate the activity of different proteins through their membrane targeting or direct modulation of their enzymatic activity 3) PLCγ1 hydrolyzes phosphatidylinositol-4,5-bisphosphate to generate the second messengers inositol-1,4,5-trisphosphate and diacylglycerol which in turn can activate several downstream effectors including specific protein kinase C (PKC) isoforms leading to many cellular responses in vitro). Moreover our data demonstrate that a PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt through PKCα activation, unveiling a novel role for PLCγ1 as a mediator of the PI3K-dependent Akt activation and as a key regulator of different Akt-dependent processes.Here we show that FGF-2 activates PLCγ1 in human umbilical vein endothelial cells (HUVECs) and that this enzyme is critical for the FGF-2-dependent cell migration and capillary tube formation  and incubated with 25 µg DNA in a cuvette for 10 min at room temperature. Cells were electroporated  by using a Bio-Rad Gene Pulser instrument (time constant ∼26 sec) and then left in the cuvette for a further 10 min at room temperature before plating.HUVEC were purchased from TCS CellWorks and grown in EGM kit  supplemented with 10% FBS. For expression of GFP-PH PLCγ1, HUVEC were electroporated as described For siRNA-based experiments, confluent HUVEC plated in 6 well plates were transfected by incubating in OPTIMEM containing oligofectAMINE (Invitrogen) and 200 nM siRNA. After 24 h cells were serum starved overnight in M199+0.5% FBS before being used for experiments. Alternatively, number of cells was determined 24 h or 48 h after transfection by manual counting.3H]myo inositol (PerkinElmer) in inositol free medium M199 supplemented with 2% FBS. After 24 h, cells were incubated in M199 containing 10 mM Hepes and 20 mM LiCl for 15 min before stimulation, then lysed in 0.1 M HCOOH. Total inositol phosphates were separated by anion exchange chromatography by using AG 1-X8 resin  and the total amount assessed by liquid radioactivity counting.Cells were labelled with [3 production was assessed by thin layer chromatography (TLC) analysis as described 32P]phosphoric acid (PerkinElmer) in phosphate free medium M3786 (Sigma) for 3 h before stimulation as indicated. Phospholipids were extracted, dried under nitrogen, resuspended in 100 µl chloroform∶methanol (3∶2) and spotted onto silica plates pre-soaked in water∶methanol (3∶2) containing 1.2% (w/v) oxalic acid and oven baked for 15 min at 110°C. TLC plates were developed in chloroform: acetone: methanol: acetic acid: water (90∶36∶30∶27∶18).PtdIns-3,4,5-PAssays of cell migration were performed by using Transwell chambers  as described Microscopy was performed by using a Zeiss laser confocal microscope system (LSM 510) connected to an Axiovert 100M (Zeiss) and a Zeiss 63X objective. Images were acquired and loaded by using a TCS NT program (version 1.6.587). No further processing of the images was done except for changes in brightness/contrast to better visualize the data. Images were collected at 3.5 µm from the bottom and all conditions were imaged at the same distance from the glass.2, 4 µM Fluo-4 for 45 min at 37°C. Cells were then washed twice with HBSS containing 0.5% BSA, 2 mM CaCl2 and incubated at 37°C in the same solution for 30 min to allow de-esterification of Fluo-4. Where indicated, 10 µM LY29400 was added after the first 15 minutes of the de-esterification passage for 15 min. Samples were analyzed using the Zeiss confocal microscope LSM 510 equipped with a chamber for live imaging at 37°C supplied with 5% CO2 using a 10X objective. After recording basal fluorescent, cells were stimulated with 100 ng/ml FGF-2 in the presence or in the absence of 10 µM LY294002 and fluorescence was measured for 10 min. At the end of each experiment, cells were stimulated with 1 mM ATP. ZEN Carl Zeiss software was used for acquisition and analysis of raw data. Fluorescence values of 40 cells per chamber were analyzed and expressed as mean of fluorescence.Changes in intracellular calcium were determined using the fluorescent calcium indicator Fluo-4 AM (Invitrogen). Cells were seeded on Lab-Tek chambered #1.0 borosilicate coverglass (LABTEK) for confocal live microscopy and serum starved overnight. Each well was then incubated with 200 µl of HBSS containing 0.5% BSA, 2 mM CaCl3H]-labelled HUVEC  and our data indicate that the role of PLCγ1 in tubulogenesis is associated with its role in cell migration.Here we show that PLCγ1 is activated upon FGF-2 stimulation in HUVEC and that upstream activation of PI3K is required for full activation of the enzyme. PLCγ1 activation is necessary for FGF-2-induced capillary tube formation 3, we demonstrate here that FGF-2 activates PI3K in HUVECs and that this enzyme is involved in PLCγ1 activation. Although FGF-induced PLCγ1 phosphorylation and activation were previously observed and characterised in fibroblasts and in cell lines overexpressing FGFR1 The mechanisms of FGF-dependent activation of PI3K and PLCγ1 have been controversial. Early studies showed that p85, the regulatory subunit of PI3K, did not coimmunoprecipitate with FGFR1 and no PI3K activity was detected in anti FGFR1 immunoprecipitates 3 and its role in membrane targeting of the enzyme Our study indicates that PI3K activation is required for PLCγ1 activation, consistent with our previous work describing the interaction of PLCγ1 PH domain with the PI3K product PtdIns-3,4,5-Pin vitro. Recent data have suggested that PI3K and PLCγ1 have opposite effects on capillary tube formation in vitro with PI3K promoting tubule formation and PLCγ1 regulating tubule regression 3 binding and growth factor-induced membrane localization of PLCγ1 We further show that the PI3K-dependent PLCγ1 activation is required for FGF-2-dependent migration of HUVEC. Previous data indicated that PLCγ1 was not required for migration downstream of FGFR1 activation 2-modified low density lipoprotein In an effort to define the mechanism of PLCγ1-dependent regulation of EC tubulogenesis we found that activation of a PLCγ1/PKCα pathway is necessary for FGF-2-induced Akt phosphorylation. Akt is involved in physiological and pathological angiogenesis through effects in both EC and cells producing endothelial signals such as tumour cells EC lining the lumen of all blood vessels play essential roles in maintaining normal vascular function and in vascular homeostasis Figure S1Effects of downregulation of PLCγ1 and PLCγ2 on HUVEC. (A) Expression levels of PLCγ1 or PLCγ2 in HUVEC transfected with the indicated siRNAs. Equal loading was confirmed using anti Akt or anti ERK antibodies. (B) HUVEC were transfected with scrambled siRNA or siRNAs targeting PLCγ1 or PLCγ2. After 24 h, cells were starved overnight in M199+0.5% FBS and then the number of cells was determined by manual counting. Data are expressed as percentage of cells transfected with scrambled siRNA and are means±SEM of values obtained from 5 independent experiments. *p<0.05, **p<0.01 vs HUVEC expressing siRNA PLCγ1. In these experiments siRNA targeting PLCγ2 was from Qiagen.(1.56 MB TIF)Click here for additional data file.Figure S2FGF-2 activates PLCγ1 in a mechanism involving PI3K activation. HUVEC were labelled with [3H]myo inositol for 24 h and then pre-treated with 100 nM wortmannin for 15 min before stimulation with 100 ng/ml FGF-2. Data indicate the total amount of inositol phosphates generated at the indicated times and are expressed as percentages of the basal inositol phosphates. Data are means±SEM of values obtained from 1–2 independent experiments.(3.73 MB TIF)Click here for additional data file.Figure S3PI3K regulates FGF-2-induced PLCγ1 recruitment to the plasma membrane. HUVEC were transfected with a GFP-tagged PLCγ1 PH domain and plated on glass coverslips. After 24 h cells were serum deprived overnight in M199+0.5% FBS and then left untreated or pre-treated with 100 nM wortmannin for 15 min before stimulation with 100 ng/ml FGF-2 for 10 min. Coverslips were fixed and analyzed by confocal microscopy.(6.24 MB TIF)Click here for additional data file.Figure S4VEGF-mediated activation of PLCγ1 is not dependent on PI3K activation. HUVEC were labelled with [3H]myo inositol for 24 h and then pre-treated with 10 µM LY294002 (A) or 100 nM wortmannin (B) for 15 min before stimulation with 10 ng/ml VEGF. Data indicate the total amount of inositol phosphates generated at the indicated times and are expressed as percentages of the basal inositol phosphates. Data are means±SEM of values obtained from 1–3 (A) or 1–4 independent experiments (B).(6.07 MB TIF)Click here for additional data file.


H-1-benzopyran-3-carboxylate and 3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate. Both drugs were able to inhibit cell invasion markedly in a Boyden chamber assay, the bromo derivative being more potent than the reference matrix metalloprotease (MMP) inhibitor GI 129471. In vivo, tumour growth was reduced when nude mice grafted with HT1080 or MDA-MB231 cells were treated i.p. 3 days week−1 with the bromo coumarin derivative. These effects were not associated with the inhibition of urokinase, plasmin, MMP-2 or MMP-9. The mechanism of action of the drugs remains to be elucidated. However, these two coumarin derivatives may serve as new lead compounds of an original class of antitumour agents.In search for new anticancer agents, we have evaluated the antiinvasive and antimigrative properties of recently developed synthetic coumarin derivatives among which two compounds revealed important activity: 3-chlorophenyl 6-acetoxymethyl-2-oxo-2 Some of them were potent inhibitors of serine proteases such as α-chymotrypsin (α-CT), human leucocyte elastase (HLE) and thrombin (THR) (H-1-benzopyran-3-carboxylate (compound 4) and 3-bromophenyl 6-acetoxy-methyl-2-oxo-2H-1-benzopyran-3-carboxylate (compound 7)  instrument in dimethyl-d6 sulphoxide with hexamethyl disilazane as an internal standard; chemical shifts are reported in δ values (p.p.m.) relative to internal HMDS. The abbreviations s (=singlet), d (=doublet) and m (=multiplet) are used throughout. Elemental analyses  were realised on a Carlo-Erba EA 1108-elemental analyser and were within ±0.4% of the theoretical values. All reactions were routinely checked by thin layer chromatography on silica gel Merck 60F 254.Melting points were determined on a Büchi-Tottoli capillary apparatus and are uncorrected. Infrared spectra were recorded as KBr pellets on a Perkin-Elmer 1750 FT spectrophotometer. The 6, 7 and 8 are reported in Table 1The coumarin derivatives were obtained according to previously described synthetic procedures . The phy−1) fetal calf serum (FCS), penicillin–streptomycin , 2 mM glutamine and 10 mM HEPES buffer at 37°C in a humid atmosphere (5% CO2 and 95% O2).Human fibrosarcoma HT1080 cells and human breast MDA-MB 231 adenocarcinoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v vμm pore size) of Transwell cell culture chamber inserts  were coated with 100 μl of type IV collagen purified from human placenta diluted at 200 μg ml−1 in phosphate-buffered saline (PBS). Coats were left to air-dry overnight in a laminar flow hood under UV light. Uniformity of the coating was assessed by Coomassie blue staining. Before use, coated plates and inserts were washed twice with water and incubated for at least 1 h in serum-free DMEM at 37°C (Plates (24-well)  and 6.5 mm polycarbonate filters  were collected by trypsin–ethylenediaminetetraacetic acid treatment, washed with serum-containing medium and allowed to recover from trypsinisation for at least 30 min at 37°C. Cells were then washed twice in serum-free DMEM and diluted in the same medium supplemented with 0.1% bovine serum albumin  to a cell density of 6 × 10μM) on HT1080 cell invasion and migration was assayed using Transwell cell culture inserts (Costar) and compared to the reference compound GI 129471 (1 μM), an MMP inhibitor  or GI 129471 . Cells (6 × 104) suspended in DMEM supplemented with 0.1% BSA  were seeded in the upper wells. Chambers were subsequently incubated for 48 and 4 h for chemoinvasion and chemotaxis assays, respectively, in a humid atmosphere at 37°C . After incubation, filters were removed from the chambers, fixed in 4% paraformaldehyde for 15 min (4°C), permeabilised for 10 min in methanol (−20°C) and stained with Giemsa (4%) for 15 min. The upper surface of the filters was scrapped with a cotton swab and cells having reached the lower surface of the filters were visually counted using a light microscope  at 20-fold magnification. Each assay was performed in triplicate. Results were expressed as percentages (±s.d.) of the migration of control cells.For chemoinvasion assays, type IV collagen-coated membranes (see above) were used, whereas uncoated membranes were employed for chemotaxis assays. Lower wells of chambers were filled with DMEM supplemented with 20% FCS and 1% BSA  as chemoattractant, in the presence or absence of coumarin derivative  on cell metabolism, HT1080 cell suspension (6 × 105 cells ml−1) was plated on type IV collagen-coated 96-well plates, 200 μl well−1 (1.2 × 105 cells), in the same culture conditions as described above and incubated for 48 h in the presence of different concentrations of the coumarin derivative . Media were subsequently harvested and replaced by 100 μl of DMEM supplemented with 10% WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate, Boehringer Mannheim, Germany) and plates were incubated for 2 h at 37°C. Cellular metabolism was determined by measuring the absorbance of the medium between 420 and 480 nm (with a reference filter of 595 nm) on a microtitre plate reader . At the concentrations tested, the synthetic coumarin derivatives did not significantly affect cellular metabolism (data not shown).In order to assess the potential influence of coumarin derivatives  were plated on plastic or type IV collagen or matrigel in 24-well plates . At each time points , the medium was removed, and the cells were washed with PBS and frozen at −20°C. The DNA content was determined by spectrofluorimetry (Cells (6.8 × 10−1). The suspension (1 × 106 cells injection site−1) was injected subcutaneously (s.c.) on both body flanks of 6–8-week-old female athymic nude mice . The coumarin derivatives in PBS were injected intraperitoneally (30 mg kg−1) three times per week during the whole assay.Matrigel (basement membrane proteins) was extracted from the mouse Engelbreth–Holm–Swarm (EHS) tumour as previously described  and smaller (b) diameters of the apparent tumour were measured and served for tumour volume calculation according to b2 × 0.4 . Injecteμl onto 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulphate (SDS), 2 mg ml−1 casein and 0.16 μM human plasminogen. Following electrophoresis, gels were incubated for 1 h in 2.5% (v v−1) Triton X-100 for removal of SDS and then for 1 h at 37°C in 50 mM Tris-HCl, 0.1 M NaCl, pH 7.6. Plasminogen activator (PA) activity was visualised as clear bands of lysis upon staining with 0.1% Coomassie blue R250. Purified human high-molecular-weight two-chain urokinase plasminogen activator (HMW-uPA) and low-molecular-weight urokinase plasminogen activator (LMW-uPA) were run in each gel as positive controls. An analogous protocol was followed to detect enzymatic activities towards MMP-2 and MMP-9 except that the 7% SDS–PAGE contained 1 mg ml−1 bovine gelatin. The gels were incubated at 37°C for about 18 h in 5 mM Tris, pH 7.6 containing 0.2 M NaCl, 5 mM CaCl2 and 0.02 (w v−1) Brij35. Protein staining was performed during 1 h with a 0.25% solution of Coomassie brillant blue R250 in 50% methanol and 7% acetic acid and destaining in a solution of 20% methanol and 50% acetic acid. Purified pro-MMP-2, pro-MMP-9 and MMP-2 were used as positive controls. MMP-2 was freshly prepared by incubating pro-MMP-2 with 100 μMp-aminophenylmercuric acetate  for 30 min at 37°C. Some experiments were carried out with the culture medium after activation by 100 μM APMA (30 min at 37°C). The effects of compounds 4 and 7 (1–100 μM) incubated in the culture medium were checked for both types of zymography.The culture medium of human fibrosarcoma HT1080 cells (DMEM) was removed and concentrated 20-fold in a Vivaspin 6 ml concentrator. It was incubated for 30 min at ambient temperature in nonreducing sample buffer and run under Laemmli conditions  by loadiα-CT and LMW-uPA (MW=33 000 Da) were purchased from Sigma, HMW-uPA (MW=54 000 Da) from Choay, trypsin (TRY) and THR from Boehringer-Indelheim (Germany), and MMP-2 and MMP-9 from Calbiochem. Human leucocyte elastase and PM were from Elastase Products Co. and Biogenic, respectively. Except for THR, HMW-uPA, MMP-2 and MMP-9, enzyme concentrations were determined by active-site titrations using the appropriate titrant (p-nitroanilide substrates: succininyl-Ala2-Pro-Phe-p-nitroanilide (Suc-AAPF-p-NA), benzoyl-Arg-p-nitroanilide (Bz-R-p-NA) and methoxysuccinyl-Ala2-Pro-Val-p-nitroanilide (MeO-Suc-AAPV-p-NA) for α-CT, TRY and HLE, respectively (obtained from Sigma); or S-2238 (H-D-Ile-Pro-Arg-p-nitroanilide), S-2444 (<Glu-Gly-Arg-p-nitroanilide) and S-2251  for THR, HMW- and LMW-uPA, and PM, respectively (from Biogenic). Matrix metalloprotease-2 previously activated by treatment with APMA were assayed at 412 nm with the thiopeptide Ac-Pro-Leu-Gly-Sleu-Leu-Gly-Oet in the presence of 5,5′-dithiobis(2-nitrobenzenic) acid (DTNB). The amidolytic activities of the enzymes were followed in 0.025 M sodium phosphate, 0.05 KCl, pH 7.5 for α-CT; 0.1 M HEPES, 0.5 M NaCl, 0.1% (v v−1) Tween 80, pH 8.0 for HLE; 0.01 M HEPES, 0.01 M Tris, 0.1 M NaCl, 0.1% (v v−1) PEG6000, pH 7.5 for THR; 0.1 M Tris, 0.01 M CaCl2, pH 7.5 for TRY; 0.025 M NaH2PO4, 0.1 M NaCl, 0.05% (v v−1) Tween 80, pH 7.5 for HMW-and LMW-uPA; 0.1 M NaH2PO4, 25% (v v−1) glycerol, pH 7.5 for PM; and 0.1 M Tris, 0.15 M NaCl, 0.01 M CaCl2, 0.005% (w w−1) NaN3, 0.03% (w w−1) Brij35, pH 7.5 for MMP-2 and MMP-9. All assays contained 10% (v v−1) DMSO and were run at 25°C in a Perkin-Elmer Lambda 5 or Kontron Uvikon 941 spectrophotometer equipped with a thermostated cell holder.Bovine  titrant . SeveralThe kinetic parameters for the inhibition were determined either by the preincubation method for LMW-uPA and PM , or by the progress curve method (HLE) when the inhibition was too fast to be measured accurately using the preincubation method. The minimum kinetic scheme for the inactivation process is described as follows:KI the apparent enzyme-inhibitor dissociation constant and ki the inactivation rate constant at infinite inhibitor concentration. Using the preincubation method, the pseudo-first-order constants for inactivation kobs were obtained from the least-squares analysis of semilogarithmic plots of remaining activity [E]/[E]0 against time , LMW-uPA and HMW-uPA  and PM .The products resulting from the spontaneous hydrolysis of compounds μM concentration as reference compound. In such a chemoinvasion assay, this reference compound led to a 60% inhibition of HT1080 cell invasion.To determine whether coumarin derivatives could reduce the invasive behaviour of tumour cells, we measured the ability of cells treated or not with the coumarin derivative to pass through type IV collagen-coated Transwell cell culture inserts (chemoinvasion assay). Cell invasion in the absence of coumarin derivative was considered as 100%  reduced the invasion by 50% at 1 μM and 65% at 10 μM. The 6-methyl 2 (Y=CH3) and the 6-acetoxymethyl 4 (Y=CH2OCOCH3) coumarin affected the invasion in a similar manner. At 1 μM, both compounds led to a 50% inhibition, and at 10 μM, a 70 and 90% inhibition were observed for the methyl 2 and the acetoxymethyl 4 derivatives, respectively. In contrast, cell invasiveness was not markedly affected by addition of the 6-chloromethyl-substituted derivative 3 (Y=CH2Cl) (only 30% invasion inhibition at 10 μM). Thus, these data indicate that an acetoxymethyl group in the 6-position gave rise to better inhibition of cell invasion (Coumarin derivatives 1–4  reduced invasion .5) and the 3-halogenophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate derivatives 6–8. The different halogen atoms  present on the 3′-position gave rise to different effects on cell invasion (5 (X′=H) has a lower inhibitory effect on cell chemoinvasion than that observed with GI 129471. Indeed, when this derivative was used at 1 μM, a 30% inhibition of cell invasion was observed. At 10 μM, it gave rise to a similar inhibition (50–60%) as that observed by using the reference compound at 1 μM. Substitution by a halogen atom improved the inhibitory capacity of coumarin derivatives as compared to the nonsubstituted one. Addition of chloro  and iodo  derivatives at 1 μM led to an inhibition quite similar to that observed with the reference compound. Interestingly, fluoro  and bromo  derivatives appeared more potent, inducing an 80% inhibition at similar concentration. Moreover, chloro (compound 4) and bromo (compound 7) derivatives used at 10 μM dramatically decreased the cell invasiveness (±90% invasion inhibition).We next synthesised the 3-phenyl (invasion . Additio4 and 7) appeared to be very potent inhibitors of cell invasion, their anti-invasion and anticancer capacities were further studied in vitro and in vivo. We compared the ability of cells treated or not with the two selected coumarin derivatives to invade through type IV collagen-coated filters (chemoinvasion) and through uncoated filters (chemotaxis). Chemoinvasion corresponds to the capacity to invade through an extracellular matrix, while chemotaxis relies on the ability to migrate in response to a chemotactic factor. Addition of the chloro derivative 4 caused a potent and dose-dependent decrease of HT1080 cell chemoinvasion confirming the results reported in μM, 50% inhibition at 1 μM and 80% at 10 μM). In contrast, cell migration through uncoated filters was not markedly affected by the reference compound at 1 μM (20–30% chemotaxis inhibition vs 60% inhibition of chemoinvasion)  reduced cell invasion and migration in a dose-dependent manner, this compound was a more potent inhibitor of chemoinvasion than chemotaxis .4 and 7), we have evaluated their activity against serine proteases and MMPs released by HT1080 cells. We first performed plasminogen and gelatin zymographic analysis on HT1080 cell conditioned media. Plasminogen activator activity was detected in all samples. Densitometric analysis of zymograms revealed greater amounts of HMW-uPA than LMW-uPA. Gelatin zymography demonstrated the presence of pro-MMP-2 (66 kDa), pro-MMP-9 (92 kDa), two MMP-2 activated forms (62 and 59 kDa) and a 120 kDa gelatinolytic species  acted as a suicide substrate leading to enzyme irreversible inhibition of α-CT, whereas HLE was transiently inactivated through the formation of a stable acyl-enzyme  demonstrating that the observed irreversible process is not because of the formation of a stable acyl-enzyme. Hydroxylamine is known to cleave acyl-enzymes , which markedly decreased the cell invasiveness at 10 μM , were unable to act as uPA, PM or MMPs inhibitors.Thereafter, we compared the inhibitory potencies of the two selected compounds to that of other coumarin derivatives against purified enzymes  on the development of tumours induced by the injection into nude mice of human breast adenocarcinoma and fibrosarcoma cells. Treatment of mice with bromo derivative (compound 7) was found to reduce both the incidence  and growth of tumours induced by sc injection of HT1080 cells . In sharp contrast, the chloro derivative did not significantly affect tumour growth.We next investigated the influence of the two selected compounds rivative , since tP<0.05).The effects of both compounds were also evaluated on the tumorigenicity of mammary MDA-MB231 cells. The MDA-MB231 cell inoculation led to 100% tumours within 2–3 weeks. While the bromo derivative did not affect tumour incidence, it again reduced significantly tumour growth   . MoreoveH-1-benzopyran-3-carboxylate led to a better anti-invasive effect than that observed in the absence of any substituent. In the same way, an acetoxymethyl or a methyl group in the 6-position conferred an improved effect.Using noncytotoxic concentrations of compounds, our studies illustrated that some coumarin derivatives markedly inhibited the HT1080 cell invasiveness. The inhibition capacity varied according to the substituent present in the 6-position of the coumarin, and according to the nature of the halogen atom in the 3-position of the phenyl ring. In general, substitution by a halogen atom  in the ‘meta’ position of the phenyl ring relative to the ester oxygen atom of 2-oxo-2H-1-benzopyran-3-carboxylate (compound 4) has almost the same potent inhibitory activity on cellular invasion (chemoinvasion) and on cell migration (chemotaxis). In sharp contrast, the corresponding 3-bromo derivative (compound 7) led to a low inhibitory effect on chemotaxis, similar to that observed with GI 129471 used as reference compound (30% inhibition at 1 μM). Interestingly, the bromo derivative was more potent in the chemoinvasion assay than the reference compound and the chloro-substituted analogue. These data emphasise the interest of the bromo derivative as an anti-invasive inhibitor. On the contrary, the chloro derivative which affected both chemoinvasion and chemotaxis is likely to modulate cell mobility in a manner independent of a protease inhibition. In accordance with these in vitro observations, our in vivo study in two experimental models demonstrates that only the bromo derivative markedly affected tumour growth. It reduced both the incidence and the growth of tumour induced by HT1080 cell injection. Furthermore, it affected the growth of more aggressive breast adenocarcinoma MDA-MB231 derived tumours.It is worth noting that 3-chlorophenyl 6-acetoxymethyl-2-oxo-24 and 7 were not found to act as an uPA, PM, MMP-2 or MMP-9 inhibitor. Moreover, the coumarin derivative 3, which was found to be a strong inactivator of α-CT and THR and which possessed an inhibitory potency towards uPA, did not markedly decrease cell invasiveness. Thus, the observed activity of the coumarin derivatives on HT1080 cell invasion could not be ascribed to their inhibitory activity against the enzymes tested. Both compounds were found to be unable to inhibit the angiogenesis evaluated in the aortic ring assay (data not shown) according to the procedure previously described (in vivo and in vitro results, the bromo coumarin derivative appears to be very promising as potential antitumoral agent. To the best of our knowledge, this study provides for the first time evidence that a coumarin derivative displays marked anti-invasive properties in vitro and antitumour activities in vivo.The two selected coumarin derivatives in vitro and in vivo. Although coumarin derivatives might constitute an alternative to MMP inhibitors as anticancer agents, further biological investigations are required before any clinical trial.The disappointment of MMP inhibitors in clinical trials as a result of the expression of a high toxicity (


Modulation of SIRT1 activity or protein levels using nicotinamide or RNAi did not modify JNK1 activity as measured by its ability to phosphorylate cJUN. In contrast, human SIRT1 was phosphorylated by JNK1 on three sites: Ser27, Ser47, and Thr530 and this phosphorylation of SIRT1 increased its nuclear localization and enzymatic activity. Surprisingly, JNK1 phosphorylation of SIRT1 showed substrate specificity resulting in deacetylation of histone H3, but not p53. These findings identify a mechanism for regulation of SIRT1 enzymatic activity in response to oxidative stress and shed new light on its role in the stress protection pathway.SIRT1 is a NAD-dependent deacetylase that regulates a variety of pathways including the stress protection pathway. SIRT1 deacetylates a number of protein substrates, including histones, FOXOs, PGC-1α, and p53, leading to cellular protection. We identified a functional interaction between cJUN N-terminal kinase (JNK1) and SIRT1 by coimmunoprecipitation of endogenous proteins. The interaction between JNK1 and SIRT1 was identified under conditions of oxidative stress and required activation of JNK1  Given the roles of JNK1 and SIRT1 in stress protection pathways, we tested for functional interactions between these proteins. In the present study, we report post-translational modification of SIRT1 as a result of JNK1 activation. JNK1 directly phosphorylates SIRT1 on three specific residues, concentrating it in the nucleus and resulting in selective activation of SIRT1, as measured by deacetylation of histone H3 (but not p53).2O2  were immunoprecipitated and associated proteins were identified by Western blotting. An interaction between SIRT1 and JNK1 was observed only when cells were treated with the known JNK1 activators anisomycin or H2O2 . Anisomyted JNK1 . A similnd pJNK1 .2O2 in the presence or absence of JNKi, a cell permeable inhibitor of JNK1. Immunohistochemistry demonstrated SIRT1 was localized both in cytoplasmic and nuclear compartments in control C2C12 cells  or chronically (knockdown by siRNA). Acute inhibition of SIRT1 activity did not significantly change the phosphorylation or activity of JNK1 by anisomycin treatment . In addiin vitro kinase assays and autoradiography with JNK1, or a non-specific kinase, namely glycogen synthase kinase-3β (GSK3β). Human SIRT1 contains two previously identified phosphorylation sites (Ser27 and Ser47) of unknown function. Recombinant purified hSIRT1 (FL hSIRT1) protein was phosphorylated by JNK1 but not GSK3β . In contrast to hSIRT1, mSIRT1 does not contain the Ser27 and Thr530 phosphorylation sites . An N-tein vitro, phosphorylation of Mt-SIRT1 by JNK1 was dramatically reduced  levels even in cells over-expressing SIRT1 (either WT- or Mt-SIRT1)  protein was generated by site-directed mutagenesis of Ser27, Ser47 and Thr530 to alanine residues. Whereas recombinant FL hSIRT1 was effectively phosphorylated by JNK1  reduced . To asse reduced . Cells tced  . Similar2O2. Second, we showed that SIRT1 localization changes after treatment with H2O2, an effect blocked by treatment of cells with a JNK1 inhibitor. Third, we confirmed JNK1 as a kinase involved in phosphorylating SIRT1 at previously identified sites (Ser27 and Ser47), and identified a novel JNK1 phosphorylation site (Thr530) on SIRT1. Fourth, we demonstrated a functional role of JNK1 phosphorylation of SIRT1, selective deacetylation of histone H3 but not another SIRT1 substrate, namely p53. Mutations of these sites into alanines abolished JNK1-mediated phosphorylation of SIRT1 and abrogated the functional changes seen with respect to histone H3. Finally, we determined that the SIRT1:JNK1 interaction did not lead to a change in JNK1 activity, as measured by levels of phosphorylated cJun.In this paper we describe a novel mechanism whereby oxidative stress, via JNK1, regulates SIRT1 activity by altering its subcellular localization and activity. First, we demonstrated that SIRT1 and JNK1 functionally interact, but only when JNK1 is activated by anisomycin or HThe findings presented here provide a molecular mechanism for activation of SIRT1 in response to oxidative stress. This is the first report identifying JNK1 as a kinase that functionally modifies SIRT1, increasing SIRT1 activity and altering SIRT1 localization. Specifically, phosphorylation of SIRT1 increases deacetylation of histone H3. By modifying chromatin SIRT1 induction might modulate a set of genes that will confer protection from oxidative stress. In support of this hypothesis, Berthiaume and colleagues 2O2 mediated phosphorylation of SIRT1. In addition, cyclin dependent kinases (Cyclin B/Cdk1) phosphorylate SIRT1, increasing deacetylase activity and regulating cell cycle progression Recently, it has been shown that JNK2 can phopshorylate SIRT1 at Ser27 under basal conditions, increasing SIRT1 protein stability 2O2-induced apoptosis was attenuated by SIRT1 overexpression 2O2-induction of p53 acetylation was decreased in mesangial cells overexpressing SIRT1, similar to data we obtained in a different cell line (HEK293T). In addition, neither overexpression nor knockdown of SIRT1 affected H2O2-mediated phosphorylation of MAP kinases . This is in agreement with our finding that SIRT1 has no effect on JNK1 activity. Tanno et al SIRT1 is a cell survival factor that may increase resistance to oxidative stress in mammalian cells. In normal human fibroblast IMR-90 cells, H2O2 for 1 h, resulting in minimal cell death, whereas the JNK1:PARP-1 association required longer (∼6 h) cellular H2O2 exposure. Perhaps during prolonged H2O2 exposure, DNA damage accumulates and massive PARP-1 activation quickly depletes cellular NAD+ (a SIRT1 co-substrate), leading to decreased SIRT1 activity and necrotic cell death. Conversely, acute (or low-level) exposure to oxidative stress activates SIRT1, promoting the chance for cellular recovery.The cellular balance between SIRT1 and poly(ADP-ribose) polymerase-1 (PARP-1) activity may determine the effects of oxidative stress on cell survival. In contrast to the present study, activation of JNK1 (via oxidative stress) has previously been shown to mediate cell death via PARP-1 activation C. elegans and D. melanogaster has clearly demonstrated the existence of regulatory pathways that control the rate of aging in these organisms, such as the insulin-like pathway, the JNK pathway and the Sir2 deacetylase pathway Drosophila and in C.elegans that activation of the stress responsive JNK pathway has major implications for modulating lifespan. In both organisms it seems that the effect of overexpression of JNK on increased lifespan was mediated by nuclear localization of dFoxo/Daf16 and induction of its target genes During the last two decades progress in the genetics of aging in invertebrate models such as In conclusion, oxidative stress activates JNK1, which directly phosphorylates SIRT1 at Ser27, Ser47, and Thr530, concentrating it into the nucleus. In the nucleus, phosphorylated SIRT1 selectively deacetylates histone H3, but not p53. These findings establish a molecular mechanism linking oxidative stress to SIRT1.5 density were plated onto a 10 cm dish. For overexpression of SIRT1, 10 µg of wild-type and mutant plasmids were used for each dish. The cells were harvested at 72 hours post-transfection. For knockdown (KD) of SIRT1, SiRNAs targeting the SIRT1 and control siRNA were used at 20 nM concentration.Human embryonic kidney (293T) and mouse myoblast (C2C12) cells from ATCC were cultured in Dulbecco's modified Eagle medium (DMEM). The media was supplemented with 10% fetal bovine serum (FBS) and 10 mM HEPES. The day before transfection, 293T cells at 3×10The SIRT1 phospho-mutant construct was subcloned into the pCMV Tag 4A (Flag tag) vector from Stratagene. Ser27Ala, Ser47Ala and Thr530Ala mutations were performed by using site-directed mutagenesis. The Ser27Ala mutagenesis was performed by using the Advantage-GC 2 polymerase kit. The GC-melt concentration was used from 0–1.5 M as recommended. PCR was performed at 94°C for 3 min, then 30 cycles of 94°C for 30 sec and 68°C for 6 min and one additional 68°C incubation for 6 min. A DpnI restriction digest was used to eliminate parental DNA stands and the mixture was transformed using Stratagene XL1-Blue Supercompetent cells. In addition a PstI silent restriction site was incorporated into the mutant primers for candidate screening. The Ser47Ala, Thr530Ala and stop mutation sites were mutated by using the QuikChange II XL Site-Directed Mutagenesis Kit. All mutations were sequence verified and primers are listed in 2O2 for 30 min or with 10 µg per ml (10 mg ml−1 stock in distilled water) anisomycin for 30 min at 37°C. To block the JNK1 kinase activation, cells were treated with 1 µM cell-permeable JNK inhibitor 1,  overnight at 37°C before being exposed to anisomycin for 30 min. Cells were treated with 10 mM nicotinamide (1 M stock solution in distilled water) or 50 µM Sirtinol (50 mM stock in DMSO) overnight before treatment with H2O2 or anisomycin. Controls for the experiments were treated with distilled water and/or DMSO. For detection of acetylated Histone 3 (H3), the cells were incubated overnight with Trichostatin A (TSA) at 400 nM concentration.To activate JNK1 activity, cells were treated with 500 µM (13.3 M stock supplied) HThe rabbit anti-SIR2 polyclonal antibody was purchased from Millipore . Rabbit anti JNK antibody, rabbit anti-phospho JNK pAb were purchased from Cell Signaling Technology . For immunofluorescence, anti-mouse JNK1 mAb was purchased from BD Bioscience . Rabbit anti-Histone H3, Acetylated (1–20), pAb was purchased from EMD Chemicals, Inc. Rabbit anti-Histone H3 pAb was purchased from Cell Signaling Technology.3VO4, 1 mM PMSF, 5 µM pepstatin, 1 mM DTT, 10 U/ml aprotinin, 0.5 mM Na-Pyrophosphate and 1 complete mini protease inhibitor cocktail tablet per 10 ml. The lysate was incubated on ice for 30 min followed by centrifugation at 14,000 rpm for 20 min at 4°C. The protein concentration was determined using the Coomassie plus protein assay reagent. Fifty µg lysates were denatured by adding SDS loading buffer and heated at 99°C for 10 min. The denatured samples were size fractionated by 4–12% SDS-PAGE and transferred onto a 0.4 µ PVDF membrane. Primary antibodies (listed below) at 1∶1000 dilutions were used overnight at 4°C on a platform shaker. After incubation for 1 h with the secondary antibody , the signals were detected by ECL enhanced chemiluminescence reagent using Fujifilm scanner.For Western blot analysis, cells were lysed in RIPA lysis buffer supplemented with 10 mM NaF, 2 mM NaFor co-immunoprecipitation assays, protein A-Sepharose beads were equilibrated with RIPA buffer and pre-coated with BSA (0.5% for 1 hour). Immunoprecipitation assays were then performed using 500 to 1,200 µg of total protein. Lysates were pre-cleared by the addition of 30 µl of 50% slurry of pre-coated protein A-sepharose beads per 500 µg of protein, by rocking at 4°C for 1 h. The supernatant was collected by centrifugation and the SIRT1 protein was immunoprecipitated with rabbit anti-mouse SIR2α polyclonal antibody (5 µl per 1000 µg of protein) from Upstate  by rocking overnight at 4°C. Fifty to 100 µl of 50% slurry (pre-coated protein A Sepharose beads) were added to the mixture and incubated for 2 h at 4°C with rocking. The beads were washed three times with RIPA buffer containing protease and phosphatase inhibitors. The immunoprecipitates were eluted by boiling for 5 min in SDS sample buffer and subjected to SDS-PAGE analysis and Western blotting as described above with SIRT1 and JNK1 antibodies.2O2 and/or JNK inhibitor treatment. After treatment, the cells were washed twice in PBS and fixed in 4% (in distilled water) para-formaldehyde fixative for 30 min; they were then washed and permeabilized three times (10 min each time) with 0.5% Triton-X-100 in PBS. For immunostaining on fixed cells, the dishes were rinsed with PBS containing 0.05% Tween-20 (PBST) and incubated in blocking reagent  for 1 h at room temperature. The next stage involved overnight incubation at 4°C in a 1∶50 dilution of rabbit anti-SIR2 and mouse anti-JNK1 antibodies followed by three washes with PBST before incubation in Alexa-488-conjugated goat anti-rabbit or donkey anti-mouse antibody at a dilution of 1∶2000 for 1 h in dark conditions. The cells were then washed three times in PBST and once in PBS before being exposed to 300 nM DAPI and examined in a Zeiss LSM 510 laser-scanning confocal microscope.C2C12 cells were cultured as described above. The cells were plated on a Mat-Tek 35 mm glass bottom dish for HPathScan phospho-c-Jun (Ser63) and total c-Jun sandwich ELISA kit were used for measuring indirect JNK1 activity. Cell extracts from control and SIRT1 knockdown were used for measuring phosphorylation of c-Jun kinase. The ratio of phospho c-JUN over total c-JUN was used to measure the level of activation.in vitro kinase studies, recombinant full-length human SIRT1 (FL hSIRT1) was purchased from Biomol International, LP . Truncated SIRT1 (amino acids 170–701) was obtained by subcloning poly-histidine, S-tagged SIRT1 into a pNAT40 vector containing a PreScission protease cleavage site after the S-tag. The resulting construct was transformed into BL21DE3 cells, which were grown to an A600 of 0.6–0.8 prior to induction with isopropyl-β-D-thiogalactopyranoside (IPTG) for 6 h. Cells were harvested and stored at −80°C until used. The histidine, S-tagged SIRT1 protein was isolated using affinity, anion exchange, and size exclusion chromatography. Briefly, cells were lysed (using a French pressure cell) in lysis buffer containing 50 mM Tris pH 8, 300 mM NaCl, 2 mM TCEP, and Complete protease inhibitor cocktail tablets . Cell debris was removed by centrifugation . The supernatant was loaded onto a nickel column , washed with lysis buffer, and eluted using an imidazole gradient (0–500 mM). SIRT1 fractions were pooled and subject to PreScission protease  treatment per manufacturer's instructions to remove affinity tag. The cleaved product was dialyzed against lysis buffer containing 50 mM NaCl, loaded onto a mono Q column  and eluted using a NaCl gradient (50–1000 mM). SIRT1 fractions were pooled, loaded onto a Superdex-200 column, and eluted in 50 mM Tris pH 8, 500 mM NaCl, 2 mM TCEP, and 5% glycerol. SIRT1 fractions were pooled at stored at −80°C until used. The mouse GST-SIRT1 fusion proteins were obtained from Department of Diabetes, Mass General Hospital, Massachusetts under MTA contract (Maria Alexander Bridges).For In vitro kinase assay-Equal amounts (1 µg) of the FL SIRT1 and GST-SIRT1 fusion proteins were incubated with the activated kinase (10 U) in kinase-specific reaction buffer containing 10 µCi (0.37 MBq) Easytide γ-33P–adenosine triphosphate (NEG602H). The kinases used for these assays were JNK1 and GSK3β kinase. The kinase reactions were incubated in kinase buffer at 30°C for 30 minutes and then stopped by the addition of Laemmli sample buffer. The reactions were boiled and separated by SDS-PAGE. The gels were dried and protein was detected by autoradiography. For detecting the phosphorylation status of wild-type and mutant SIRT1 proteins, both wild-type and mutant constructs were overexpressed in 293T cells along with their control plasmid. The equal amount of overexpressed proteins was immunoprecipitated using rabbit anti SIR2 and sepharose Protein A beads. The pulled down proteins were washed 3x times with kinase buffer. After washing, the proteins bound to beads, were resuspended with 20 µl kinase buffer and kinased with recombinant JNK1.in vitro studies, were prepared in similar fashion, but with no need to extract peptides from the gel matrix. The samples were digested overnight at 37°C for trypsin and room temperature for chymotrypsin and GluC. Peptides from the in-gel digests were extracted using 50% acetonitrile, 45% water and 5% formic acid with vortexing. After proteolysis peptides were dried down and rehydrated in a small volume for loading to the liquid chromatography mass spectrometry (LC/MS) instrument. Liquid chromatography tandem mass spectrometry (LC/MS/MS) was performed using a nano-flow RP HPLC  flowing at 250 nL/min through a 10 cm×75 µM capillary column  using silica based C18 particles . The gradient was over 55 minutes from 5 to 35% acetonitrile in 0.1% formic acid. Mass spectrometry of eluting peptides was achieved using either a Q-Tof Global  or LTQ FT (Thermo) set to acquire in data dependent mode. Processed data was analyzed using Mascot (Matrix Sciences) to identify the proteins from the gel bands and sites of phosphorylation on detected peptides. The reference databases used were the human/mouse/rat RefSeq protein database as well as a small custom protein database.In a typical experiment, endogenous SIRT1 was immunoprecipitated from 293T cells using the rabbit anti-SIR2 antibody. The complex was run on a SDS denaturing gel. After CBB staining and destaining the gel, gel bands were excised, washed using water/acetonitrile (1/1) with 25 mM ammonium bicarbonate and vortexing for 20 minutes. After dehydrating the bands, the proteins therein were reduced using 10 mM DTT for 45 minutes at 58°C. Next the proteins were alkylated on the cysteines using iodoacetamide at room temperature, in the dark. The gel pieces were then dried and rehydrated using the tryptic, chymotryptic or GluC enzyme solution  in 50 mM ammonium bicarbonate. Solution based samples, such as those from the Table S1The primers used to create the mutant SIRT1 are described in this table.(0.03 MB DOC)Click here for additional data file.


Crosstalk between integrins and FGF receptors has been implicated in FGF signaling, but the specifics of the crosstalk are unclear. We recently discovered that 1) FGF1 directly binds to integrin αvβ3, 2) the integrin-binding site and FGF receptor (FGFR) binding site are distinct, and 3) the integrin-binding-defective FGF1 mutant (R50E) is defective in inducing FGF signaling although R50E still binds to FGFR and heparin and induces transient ERK1/2 activation.We tested if excess R50E affect DNA synthesis and cell survival induced by WT FGF1 in BaF3 mouse pro-B cells expressing human FGFR1. R50E suppressed DNA synthesis and cell proliferation induced by WT FGF1. We tested if WT FGF1 and R50E generate integrin-FGF1-FGFR ternary complex. WT FGF1 induced ternary complex formation (integrin-FGF-FGFR1) and recruitment of SHP-2 to the complex in NIH 3T3 cells and human umbilical endothelial cells, but R50E was defective in these functions. It has been reported that sustained ERK1/2 activation is integrin-dependent and crucial to cell cycle entry upon FGF stimulation. We thus determined the time-course of ERK1/2 activation induced by WT FGF1 and R50E. We found that WT FGF1 induced sustained activation of ERK1/2, but R50E was defective in this function.Our results suggest that 1) R50E is a dominant-negative mutant, 2) Ternary complex formation is involved in FGF signaling, 3) The defect of R50E to bind to integrin may be directly related to the antagonistic action of R50E. Taken together, these results suggest that R50E has potential as a therapeutic in cancer. Fibroblast growth factors (FGFs) constitute a family of heparin-binding polypeptides involved in the regulation of biological responses, such as growth, differentiation, and angiogenesis Since FGF signaling enhances multiple biological processes that promote tumor progression Integrins are a family of cell adhesion receptors that recognize extracellular matrix (ECM) ligands and cell surface ligands Recently we reported that FGF1 directly bound to integrin αvβ3 and localized the integrin-binding site in FGF1 within or close to the heparin-binding site, but distinct from the FGFR-binding site In the present study, we demonstrate that the R50E mutant of FGF1 suppressed FGF signaling induced by WT FGF1 in a dominant-negative fashion. We studied the mechanism of the antagonistic action of R50E. R50E induced transient ERK1/2 activation, but did not induce sustained ERK1/2 activation, which is integrin-dependent and directly related to cell cycle entry Our previous study shows that integrin-binding-defective R50E is defective in inducing DNA synthesis, chemotaxis, and cell proliferation, while it can bind to FGFR1 and heparin If the integrin αvβ3-binding site in FGF1 is distinct from that of FGFR1 To test if FGF1 induces the FGF1-αvβ3 complex formation in other cell types, we stimulated the serum-starved HUVEC with wt FGF1 and immunoprecipitated αvβ3 from cell lysates using anti-FGFR1 mAb, and analyzed the immunoprecipitates by Western blotting with anti-β3 antibody. We detected integrin β3 in the immune complex only when wt FGF1 was used . As a reIt has been reported that the non-receptor tyrosine kinase Src is recruited to activated fibroblast growth factor receptor (FGFR) complexes through the adapter protein factor receptor substrate 2 (FRS2) It has been reported that IGF1 induces recruitment of the SH2 domain-containing protein phosphatase SHP-2 to the tyrosine phosphorylated integrin β3 cytoplasmic domain The present results suggest that R50E is defective in inducing ternary complex formation because it is defective in direct binding to integrins In the present study we establish that the integrin-binding-defective R50E mutant of FGF1 is a dominant-negative antagonist of FGF1. We demonstrated that excess R50E suppressed DNA synthesis and cell proliferation induced by WT FGF1. Because FGF1 binds to all known FGFR isoforms It has been reported that receptor binding of heparin-binding growth factors (HB-GFs), such as FGF, is regulated by interactions with heparan sulfate proteoglycans We studied the mechanism of the dominant-negative effect of R50E. We presented evidence that WT FGF1 co-precipitation of integrin αvβ3 and FGFR1, and recruitment of SHP-2 to the complex, while R50E was defective in these functions. Since R50E can still bind to FGFR1 and heparin It has been established that the duration of ERK pathway signaling determines the proliferative response of cells WT FGF1, the R50E mutant of FGF1, and the FGFR1 D2D3 fragment were synthesized as described 3VO4.). Protein concentrations in the cell lysates were determined using the BCA protein assay kit (Thermo Scientific). Equal amounts of proteins were analyzed by SDS-PAGE in a 4–12% polyacrylamide gel and standard Western blot analysis protocol. Bound antibodies were detected with HRP-conjugated anti-mouse or anti-rabbit IgG (BioRad), and Super Signal WestPico (Thermo Scientific). Blots were imaged and quantified by Multi Gauge V3.0 software (Fuji film Co.).NIH 3T3 cells or HUVEC were grown to 70–80% confluence, and starved in DMEM supplemented with 0.4% FCS for 16 h. The cells were then treated with wild type (WT) or mutant FGF1 (5 ng/ml) in the presence of 5 µg/ml heparin for 15 min–24 h at 37°C. Then, cells were washed twice with ice-cold PBS, and lysed with the lysis buffer , 1 mM PMSF, 20 mM NaF, and 1 mM Na2, protease inhibitor cocktail (Sigma), 1 mM PMSF, 20 mM NaF, 1 mM Na3VO4 and 1.0% NP-40. After a 30-min incubation on ice, we clarified the lysates by centrifugation at 14,000 rpm for 20 min and adjusted the protein concentration to 2.5 mg in 750 µl lysis buffer, then incubated with 20 µl of protein G-Sepharose (Invitrogen) conjugated with 1.5 µg of anti-FGFR1 (M2F12) mAb (Santa Cruz), 5 µg of rat anti-integrin β3 mAb (MBL), or mouse anti human integrin β3 mAb (AV10) for 24 h at 4°C. After washing with the same lysis buffer except for 0.5% NP-40 three times, we solubilized the immune complex by adding SDS sample buffer to the resin and analyzed by Western blot analysis.Cells cultured in 10-cm dishes were suspended in 300 µl of lysis buffer containing 20 mM Hepes, pH 7.4, 100 mM NaCl, 10% glycerol, 1 mM MgClThe FGFR D2D3 fragment, which contains the FGF-binding site, and the 3xA mutant of FGF1 were prepared as described 4 cells/well), and then incubated with DMEM containing 10% FBS at 37°C in 5% CO2 atmosphere. Twenty µl of MTS reagent was added to each well at the indicated time period. Relative cell number was measured based on increased absorbance at 490 nm.We maintained mouse pro B BaF3 cells that express human FGFR1c  in a medium containing 0.5 ng/ml IL-3 as described Statistical analysis was performed using Prism software (GraphPad software).


Serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) has been implicated as a novel component of the mammalian target of rapamycin (mTOR) signaling pathway. Recently we have demonstrated that mTOR regulates cell motility in part through p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) pathways. Little is known about the role of PP2A in the mTOR-mediated cell motility. Here we show that rapamycin inhibited the basal or insulin-like growth factor 1 (IGF-1)-induced motility of human Ewing sarcoma (Rh1) and rhabdomyosarcoma (Rh30) cells. Treatment of the cells with rapamycin activated PP2A activity, and concurrently inhibited IGF-1 stimulated phosphorylation of Erk1/2. Inhibition of Erk1/2 with PD98059 did not significantly affect the basal mobility of the cells, but dramatically inhibited IGF-1-induced cell motility. Furthermore, inhibition of PP2A with okadaic acid significantly attenuated the inhibitory effect of rapamycin on IGF-1-stimulated phosphorylation of Erk1/2 as well as cell motility. Consistently, expression of dominant negative PP2A conferred resistance to IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. Expression of constitutively active MKK1 also attenuated rapamycin inhibition of IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. The results suggest that rapamycin inhibits cell motility, in part by targeting PP2A-Erk1/2 pathway. The mammalian target of rapamycin (mTOR), a member of the phosphoinositide-3′ kinase-related kinase family, is a central controller of cell proliferation, growth and survival Cas) Clinical trials have demonstrated that rapamycin and its analogs   are promising anticancer drugs. They share same mechanism and specifically block the function of mTOR, inhibiting growth of numerous solid tumors  with only mild side effects PP2A, a serine/threonine (Ser/Thr) protein phosphatase, is a heterotrimeric holoenzyme composed of a catalytic subunit (PP2Ac), an A subunit , and members of the B subunit families, such as B (PR55), B′ (PR61), B″ (PR72), and B′″ (PR93/PR110) 2-adrenergic receptor agonists inhibits motility of keratinocytes PP2A negatively regulates Erk1/2 2. Human embryonic kidney (HEK) 293 , 293TD and 293A cells  were grown in antibiotic-free Dulbecco's modified Eagle medium (DMEM) (Mediatech) supplemented with 10% heat-inactivated FBS and non-essential amino acid (Mediatech) at 37°C and 5% CO2. For experiments where cells were deprived of serum, cell monolayers were washed with phosphate buffered saline (PBS), and incubated in the serum-free DMEM. Rh1 and Rh30 clones Human rhabdomyosarcoma Rh30 and Ewing sarcoma Rh1  were grown in antibiotic-free RPMI 1640 medium  supplemented with 10% fetal bovine serum (FBS)  at 37°C and 5% COThe recombinant adenovirus expressing the green fluorescence protein (GFP) (Ad-GFP) was described previously Lentiviral shRNAs to mTOR and GFP were described previously Western blotting was performed as described previously 2). The phosphatase activity of immunoprecipitated PP2A was assayed with a Ser/Thr Phosphatase Assay kit 1 using KRpTIRR as the substrate peptide (Millipore) following the manufacturer's instructions. Absorbance was measured at 595 nm using a Wallac 1420 Multilabel Counter . Because the optical density (OD) values varied from experiment to experiment, PP2A activity was calculated using the fold change (arbitrary units) in each experiment. Finally all data (from different batches of experiments) were pooled for statistical analysis.Cells were lysed in 50 mM Tris-HCl buffer, pH 7.0, containing 1% Nonidet P-40, 2 mM EDTA, and protease inhibitor cocktail . PP2Ac was immunoprecipitated with antibodies to PP2Ac , and protein A/G-agarose (Santa Cruz Biotechnology). Subsequently, the beads were washed three times with the above lysis buffer, and twice with the phosphatase assay buffer . The data were analyzed by one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's Our previous studies have shown that rapamycin suppresses IGF-1 stimulated motility in various tumor cell lines in part by inhibiting mTORC1-mediated 4E-BP1 and S6K1 pathways To understand whether rapamycin activates PP2A through inhibition of mTOR, Rh1 and Rh30 cells stably expressing a rapamycin-resistant mutant mTOR (S2035I), mTORrr, that prevents binding of FKBP-rapamycin but has intact kinase activity, were used. We found that expression of mTORrr did not significantly reduce the basal PP2A activity, but conferred high resistance to rapamycin inhibition of PP2A activity in Rh30 cells . SimilarSince Erk1/2, negatively regulated by PP2A To unveil whether rapamycin inhibits phosphorylation of Erk1/2 through inhibition of mTOR, Rh1 and Rh30 cells stably expressing mTORrr were used. As shown in It has been described that insulin or IGF-1 stimulates activation of S6K1, which is attributed to mTOR repression of PP2A activity against S6K1 Next, we investigated the role of PP2A in mTOR-mediated cell motility. To this end, a monolayer of Rh1 or Rh30 cells were grown in 6-well plates to 80% confluence, and serum-starved in DMEM for 24 h. Cell motility was assessed by the wound healing assay, as described previously To further determine the role of PP2A in the regulation of Erk1/2 activity and cell motility, Rh1 and Rh30 cells were engineered to stably express HA-tagged dominant negative PP2A . Consistent with previous findings To address whether activation of Erk1/2 by IGF-1 is responsible for increased cell motility, cells were exposed to PD98059  alone, or in combination with rapamycin. We found that PD98059 did not alter the basal level of Erk1/2 phosphorylation (data not shown), but at concentrations of 1–50 µM, significantly inhibited IGF-1-induced activation of Erk1/2 in Rh1 cells . PD98059To further define the role of Erk1/2 in mTOR-mediated cell motility, we generated recombinant adenoviruses Ad-MKK1-R4F and Ad-MKK1-K97M, encoding FLAG-tagged constitutively active and dominant negative MKK1, respectively. Infection of Rh30 cells with Ad-MKK1-R4F and Ad-MKK1-K97M, but not Ad-GFP (control virus), resulted in expression of high levels of FLAG-tagged MKK1 mutants . ExpressRecently we have demonstrated that rapamycin inhibits F-action reorganization and cell motility by inhibition of mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways The phosphatase activity of PP2Ac is modulated by its association with PP2A-A, -B regulatory subunits and α4 protein We found that PD98059 (10 µM) alone did not significantly affect the basal mobility of cells, but dramatically inhibited IGF-1-induced cell motility . HoweverHere we show that rapamycin inhibited IGF-1 stimulated phosphorylation of Erk1/2, which is related to activation of PP2A. This is consistent with recent findings that rapamycin inhibited IGF-1 or EGF stimulated phosphorylation of Erk1/2, not through inhibiting the upstream kinase, as rapamycin did not inhibit growth factor-induced phosphorylation of MKK1 (Ser217/221) It has been described that IGF-1 activates Erk1/2 by dissociating from Shc from PP2Ac In the studies, we also assessed the roles of other PP2A family members, such as PP4, PP6 and PP5, in mTOR-mediated cell motility, since these Ser/Thr protein phosphatses have been reported as novel downstream molecules of mTOR as well In summary, here we have shown that rapamycin inhibits IGF-1 stimulated cell motility by activating PP2A and concurrently inhibiting phosphorylation of Erk1/2. Pharmacological or genetic inhibition of PP2A attenuated the inhibitory effect of rapamycin on IGF-1-stimulated phosphorylation of Erk1/2 as well as cell motility, whereas expression of constitutively active MKK1 results in robust phosphorylation of Erk1/2 and confers resistance to rapamycin inhibition of IGF-1 stimulated cell motility. The results suggest that rapamycin inhibits cell motility, in part by targeting PP2A-Erk1/2 pathway.


The Wnt pathway is a key regulator of development and tumorigenesis. Dpr (Dact/Frodo) influences Wnt signaling in part through the interaction of its PDZ-B domain with Dsh's PDZ domain. Studies have shown that XDpr1a and its close relative, Frodo, are involved in multiple steps of the Wnt pathway in either inhibitory or activating roles. We found that XDpr1a is phosphorylated by casein kinase Iδ/ε (CKIδ/ε), an activator of Wnt signaling, in the presence of XDsh. Abrogating XDpr1a's ability to bind XDsh through mutation of XDpr1a's PDZ-B domain blocks CK1δ/ε's phosphorylation of XDpr1a. Conversely, XDsh possessing a mutation in its PDZ domain that is unable to bind XDpr1a does not promote XDpr1a phosphorylation. Phosphorylation of XDpr1a and XDsh by CKIδ/ε decreases their interaction. Moreover, the phosphorylation of XDpr1a by CKIδ/ε not only abrogates XDpr1a's promotion of β-catenin degradation but blocks β-catenin degradation. Our data suggest that XDpr1a phosphorylation by CKIδ/ε is dependent on the interaction of XDpr1a's PDZ-B domain with XDsh's PDZ domain, and that the phosphorylation state of XDpr1a determines whether it inhibits or activates Wnt signaling. Xenopus development and cell cycle regulators in mammalian cells In canonical Wnt signaling, Wnt activates β-catenin-dependent transcription via a phosphorylation-regulated signal transduction cascade. In the absence of Wnt, the cytoplasmic β-catenin degradation complex, whose core components include β-catenin, adenomatous polyposis coli (APC), axin, and glycogen synthase kinase 3β (GSK3β), is stable and casein kinase Iα (CKIα) primes β-catenin by phosphorylating Ser45, which is required for GSK3β to phosphorylate three upstream Ser/Thr residues PSD, Discs large, and ZO1), and a C-terminal DEP domain  Xenopus Dapper1a (XDpr1a) and the highly related protein Functional regulator of disheveled in ontogenesis (Frodo), are novel mediators of Wnt signaling which were isolated in independent yeast two-hybrid screens for proteins that interact with Xenopus Dsh (XDsh) Dsh has three highly conserved domains: an N-terminal DIX domain , a central PDZ domain  inhibits Xwnt8- and XDsh-induced body axes and reduces Wnt-dependent reporter activity in in vitro, i.e., Dsh, APC, axin, and β-catenin, and CKIε phosphorylates axin and β-catenin in vivoin vitro and in vivo, and that CKIδ and CKIε both act upstream of the B56α regulatory subunit of PP2A in Xenopus body axis formation, which is a Wnt-dependent process Phosphorylation plays a key role in the regulation of Wnt signaling, and CKI family members appear to have several targets in the Wnt pathway. CKIα phosphorylates β-catenin to negatively regulate the pathway The circumstances that determine whether Dpr inhibits or activates Wnt signaling are not known. An appealing hypothesis is that posttranslational modifications of Dpr may regulate its role in Wnt signaling. Since phosphorylation is a common mechanism to alter a protein's activity, we analyzed the phosphorylation of Dpr and found that XDpr1a is phosphorylated by CKIδ/ε in the presence of XDsh. CKIδ's phosphorylation of XDpr1a reduces its interaction with XDsh and transforms XDpr1a from a promoter to an inhibitor of β-catenin degradation. Our data suggest that XDpr1a inhibits Wnt signaling when unphosphorylated and bound to XDsh, but activates Wnt signaling when phosphorylated by CKIδ/ε and associated with other Wnt players.in vitro, GSK3β and CKIδ/ε. We found that CKIδ, but not GSK3β, was able to phosphorylate XDpr1a, as exemplified by a gel shift , and two that do not (XDpr1aΔMTTV and XDpr1aMNTV) NTV, containing a T822N point mutation in its PDZ-B domain, behaved similarly to XDpr1aΔMTTV, and did not exhibit a mobility shift  does not To further show the importance of XDsh in the phosphorylation of XDprIa by CKIδ, we examined whether a mutation in XDsh that reduces its interaction with XDpr1a's PDZ-B domain still promotes the phosphorylation of XDpr1a by CKIδ. The region of XDsh's PDZ domain that binds to XDpr1a's PDZ-B was identified by X-ray crystallography in vitro coimmunoprecipitation assay. We immunoprecipitated Myc-tagged XDpr1a from a reaction containing HA-tagged XDsh in the absence or presence of CKIδ. The presence of CKIδ dramatically decreased the immunoprecipitation of XDsh with XDpr1a  using TNT SP6 Quick Coupled Transcription/Translation System  according to the manufacturer's instructions. Unlabeled proteins were prepared with TNT SP6 Quick Coupled Transcription/Translation System using cold methionine. XDpr1a and XDsh TNT reactions were mixed at a ratio of 1:1 in a reaction also containing 5 mM MgCl2 and 0.5 mM ATP. Rat CKIδ lacking its autoinhibitory C-terminal domain  or GSK3β , was added to a final concentration of 1.1 µM, and the phosphorylation reactions were carried out for 1.5 hours at 30°C. Where specified, [35S]methionine-labeled luciferase was added to the phosphorylation reactions, and bands were quantitated after denaturing SDS-PAGE using a Molecular Dynamics PhosphorImager and ImageQuant software. Where specified, [γ-33P]ATP was added to the CKIδ reactions followed by a 1.5 hour incubation at 30°C.in vivo labeling, HEK293 cells were transfected with Flag:XDpr1a, HA:XDsh, and CKIε, or Flag:XDpr1a with empty vector, using Lipofectamine Plus  and metabolically labeled with [32P]orthophosphoric acid . Cells were homogenized in lysis buffer , followed by anti-Flag immunoprecipitations, SDS-PAGE, and visualization using a Molecular Dynamics PhosphorImager. XDpr1a's molecular weight was determined in the absence or presence of CKIε and XDsh from three experimental trials using GelScape .For 33P]ATP-labeled Myc:XDpr1a was immunoprecipitated in the presence of anti-Myc beads for 2 hours at room temperature. Immunoprecipitates were washed three times with 50 mM Tris-HCl pH 7.4, 137 mM NaCl, followed by SDS-PAGE and visualization by autoradiography. XDpr1a's molecular weight was determined in the absence or presence of CKIδ and XDsh from three experimental trials using GelScape . [35S]methionine-labeled Myc:XDpr1a was immunoprecipitated in the presence of anti-Myc antibodies and protein A agarose at room temperature for 2 hours. Immunoprecipitates were washed three times with 50 mM Tris-HCl pH 7.4, 137 mM NaCl, followed by denaturing SDS-PAGE, visualization by autoradiography, and quantitation using a Molecular Dynamics PhosphorImager and ImageQuant software. The charted coimmunoprecipitation data is the percentage of XDsh in the immunoprecipitate , divided by the percentage of XDpr1a in the immunoprecipitate , normalized to 1.0 in the absence of CKIδ .β-catenin was synthesized using TNT T7 coupled wheat germ extract system . Degradation assays were performed six times, with aliquots removed at 0, 0.5, 1.0, and 2.0 hours. Aliquots were resolved using SDS-PAGE, imaged using a Molecular Dynamics PhosphorImager, and quantitated using ImageQuant software.