Induced Complex Formation Research Articles

Fibulin-3 is a novel TGF-β pathway inhibitor in the breast cancer microenvironment.

TGF-β is an important regulator of breast cancer progression. However, how the breast cancer microenvironment regulates TGF-β signaling during breast cancer progression remains largely unknown. Here, we identified fibulin-3 as a secreted protein in the breast cancer microenvironment, which efficiently inhibits TGF-β signaling in both breast cancer cells and endothelial cells. Mechanistically, fibulin-3 interacts with the type I TGF-β receptor (TβRI) to block TGF-β induced complex formation of TβRI with the type II TGF-β receptor (TβRII) and subsequent downstream TGF-β signaling. Fibulin-3 expression decreases during breast cancer progression, with low fibulin-3 levels correlating with a poorer prognosis. Functionally, high fibulin-3 levels inhibited TGF-β-induced EMT, migration, invasion and endothelial permeability, while loss of fibulin-3 expression/function promoted these TGF-β-mediated effects. Further, restoring fibulin-3 expression in breast cancer cells inhibited TGF-β signaling, breast cancer cell EMT, invasion and metastasis in vivo. These studies provide a novel mechanism for how TGF-β signaling is regulated by the tumor microenvironment, and provide insight into targeting the TGF-β signaling pathway in human breast cancer patients.

Surfactant induced complex formation and their effects on the interfacial properties of seawater

The effect of a cationic surfactant, hexadecyltrimethylammonium bromide (CTAB), on the interfacial properties of seawater has been studied by dynamic and equilibrium surface tension and by dilational rheology essays. Important modifications of the surface tension and dilational rheology response have been observed already at the very low CTAB concentrations, where the effects due to the high ionic strength are negligible. The comparison with the effects of CTAB in different seawater models, or in natural seawater fractions, points out the establishment of strong interactions between the surfactant molecules and the lipophilic fraction of organic material dispersed/dissolved in seawater, affecting the interfacial activity of the molecules.Considering the biochemical richness of seawater, these results can be explained assuming interaction mechanisms and adsorption schemes similar to those speculated for protein and other macromolecules in the presence of surfactants, which in fact show similar features. Thus already at the low concentrations the surfactant molecules form highly surface-active complexes with part of the organic fraction of seawater. At the larger surfactant concentrations these complexes compete for adsorption with an excess of free CTAB molecules which, according to the thermodynamic conditions, are most favoured to occupy the liquid interface.The results of this study underline the important role of the sea organic content in enhancing the surface-activity of surfactants, which is relevant for a deeper understand of the direct and indirect effects of these types of pollutants on the physico-chemical environment in the sea coastal areas and develop mitigation strategies.

Having excess levels of PCSK9 is not sufficient to induce complex formation between PCSK9 and the LDL receptor

Proprotein convertase subtilisin/kexin-9 (PCSK9) acts mainly by forming complexes with the LDL receptor at the cell surface, which are then degraded in the lysosome. Studies were performed to determine whether excess levels of PCSK9 was sufficient to induce PCSK9/LDL receptor complex formation in human hepatocyte-like C3A cells. It was demonstrated using ELISA that instead of considering the overall levels of PCSK9 protein that is produced in response to certain treatment, what is critical is how much PCSK9 is actually capable of forming complexes. Despite the high levels, most of the PCSK9 produced as a result of incubating cells with a medium supplemented with BD™ MITO+ serum extender (MITO+ medium) appeared to be inhibited by a secreted factor. Having lower levels of PCSK9/LDL receptor complexes did not prevent an increase in the degradation rate of LDL receptors in MITO+ medium as compared to fetal bovine serum (FBS) containing medium (Regular medium), an effect that did not correlate with an increase in protein levels of the inducible degrader of LDL receptors (IDOL), as demonstrated using Western blotting analysis. Additional studies are required to determine the exact mechanism(s) for the degradation of the LDL receptor and/or to identify the secreted inhibitor of PCSK9.

Neuropilin 1 (NRP1) hypomorphism combined with defective VEGF-A binding reveals novel roles for NRP1 in developmental and pathological angiogenesis

Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1Y297A/Y297A mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1Y297A/Y297A mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth.

Tyrosine Phosphorylation of CD13 Regulates Inflammatory Cell–Cell Adhesion and Monocyte Trafficking

CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein α-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions.

Obesity-Associated Autoantibody Production Requires AIM to Retain the Immunoglobulin M Immune Complex on Follicular Dendritic Cells

Natural immunoglobulin M (IgM) is reactive to autoantigens and is believed to be important for autoimmunity. Blood pentameric IgM loaded with antigens forms a large immune complex (IC) that contains various elements, including apoptosis inhibitor of macrophage (AIM). Here we demonstrate that this IgM-AIM association contributes to autoantibody production under obese conditions. In mice fed a high-fat diet, natural IgM increased through B cell TLR4 stimulation. AIM associated with IgM and protected AIM from renal excretion, increasing blood AIM levels along with the obesity-induced IgM augmentation. Meanwhile, the AIM association inhibited IgM binding to the Fcα/μ receptor on splenic follicular dendritic cells, thereby protecting the IgM IC from Fcα/μ receptor-mediated internalization. This supported IgM-dependent autoantigen presentation to B cells, stimulating IgG autoantibody production. Accordingly, in obese AIM-deficient (AIM(-/-)) mice, the increase of multiple IgG autoantibodies observed in obese wild-type mice was abrogated. Thus, the AIM-IgM association plays a critical role in the obesity-associated autoimmune process.

Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2–3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

Regulation of error-prone translesion synthesis by Spartan/C1orf124

Translesion synthesis (TLS) employs low fidelity polymerases to replicate past damaged DNA in a potentially error-prone process. Regulatory mechanisms that prevent TLS-associated mutagenesis are unknown; however, our recent studies suggest that the PCNA-binding protein Spartan plays a role in suppression of damage-induced mutagenesis. Here, we show that Spartan negatively regulates error-prone TLS that is dependent on POLD3, the accessory subunit of the replicative DNA polymerase Pol δ. We demonstrate that the putative zinc metalloprotease domain SprT in Spartan directly interacts with POLD3 and contributes to suppression of damage-induced mutagenesis. Depletion of Spartan induces complex formation of POLD3 with Rev1 and the error-prone TLS polymerase Pol ζ, and elevates mutagenesis that relies on POLD3, Rev1 and Pol ζ. These results suggest that Spartan negatively regulates POLD3 function in Rev1/Pol ζ-dependent TLS, revealing a previously unrecognized regulatory step in error-prone TLS.

PKA and cAMP stimulate proliferation of mouse embryonic stem cells by elevating GLUT1 expression mediated by the NF-κB and CREB/CBP signaling pathways

BackgroundRegulation of glucose transporter (GLUT) expression and activity plays a vital role in the supply of glucose to embryonic stem (ES) cells. MethodsTo observe the effect of 6-phenyl cyclic monophosphate (cAMP) on glucose uptake and cell proliferation, 2-deoxyglucose (2-DG) uptake, immunohistochemistry, Western blotting, and immunoprecipitation were carried out. ResultsAmong GLUT isoforms in mouse ES cells, GLUT1 was predominantly expressed and 6-phenyl cAMP increased GLUT mRNA levels. Among cAMP agonists, 6-phenyl cAMP increased 2-DG uptake more than that of 8-p-chlorophenylthio-2′-O-methyl-cAMP. 6-Phenyl cAMP increased GLUT1 expression and translocation from the cytosol to the plasma membrane. 6-Phenyl cAMP increased 2-DG uptake in a time- and concentration-dependent manner due to an increase in Vmax but not Km. 6-Phenyl cAMP increased phosphorylation of nuclear factor-κB (NF-κB) and cAMP response element binding (CREB) and expression of the CREB protein (CBP) and transducer of regulated CREB activity 2 (TORC2) in sequence. 6-Phenyl cAMP induced complex formation of NF-κB/CREB/CBP/TORC2, which are involved in the increase of gluconeogenic enzyme expression. 6-Phenyl cAMP also increased cell cycle regulatory protein expression levels, the proportion of S-phase cells, and proto-oncogene expression via protein kinase A (PKA)-dependent NF-κB signaling. Finally, GLUT1 siRNA blocked the 6-phenyl cAMP-induced increase in ES cell proliferation. We conclude that PKA stimulated the complex formation of CREB/CBP/TORC2 via NF-κB, which induced effective coordination of glucose uptake as well as proliferation in ES cells. General significance6-Phenyl cAMP-induced PKA activation modified the proliferation, which may be beneficial for expanding ES cell use to cell therapy.

Collision Induced Complex Formation following Electron Capture of SO2–H2O Complex Interacting with Argon Atoms

Electron capture dynamics of SO(2)-H(2)O(Ar)(n) complexes (n = 0-2) have been investigated by means of direct ab initio molecular dynamics (MD) method in order to elucidate the effects of solvent argon on the reaction dynamics of SO(2)-H(2)O. The neutral complex of SO(2)-H(2)O has a C(s) symmetry, and the sulfur of SO(2) interacts with the oxygen of H(2)O with an eclipsed form. In the SO(2)-H(2)O(Ar)(n) complexes, the dipole of H(2)O interacts with the argon atoms in the most stable structure. Following the electron capture of the complex SO(2)-H(2)O, the complex anion SO(2)(-)(H(2)O) is dissociated directly into SO(2)(-) + H(2)O. On the other hand, the electron capture of SO(2)(H(2)O)(Ar)(n) argon complex (n = 1-2) leads to the anion-water complex SO(2)(-)(H(2)O) because the collision of H(2)O with the Ar atom causes a rebound of H(2)O from Ar atom to the SO(2)(-) anion. The argon solvent enhanced the SO(2)(-)(H(2)O) complex formation. The reaction mechanism of SO(2)(H(2)O) in the participation of argon atoms was discussed on the basis of the present results.

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit beta3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, alphavbeta3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser(910) upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from alphavbeta3 integrin. The alphavbeta3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/alphavbeta3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.

TM4SF5 accelerates G1/S phase progression via cytosolic p27 Kip1 expression and RhoA activity

Transmembrane 4 L six family member 5 (TM4SF5) causes epithelial–mesenchymal transition (EMT) for aberrant cell proliferation. However, the effects of TM4SF5 expression on cell cycle are unknown so far. In this study, using hepatocytes that either ectopically or endogenously express TM4SF5 and human hepatocarcinoma tissues, the role of TM4SF5 in G1/S phase progression was examined. We found that TM4SF5 expression accelerated G1/S phase progression with facilitated cyclin D1 and E expression and Rb phosphorylation. Furthermore, TM4SF5 enhanced trafficking of CDK4 and cyclin D1 into the nucleus and induced complex formation between them. However, TM4SF5-facilitated G1/S phase progression was blocked by silencing of p27 Kip1 using siRNA or by infection of active RhoA. Pharmacological inhibition of ROCK accelerated the G1/S phase progression of control TM4SF5-unexpressing cells. Altogether, these observations suggest that TM4SF5 accelerates G1/S phase progression with facilitated CDK4/cyclin D1 entry into the nucleus, which might be supported by TM4SF5-mediated actin reorganization through cytosolic p27 Kip1 expression and Rho GTPase activity.

Feedback Activation of Factor XI by Thrombin Is Essential for Hemostasis In Vivo.

Abstract 2127Poster Board II-102Background: The revised model of coagulation proposes that factor XI (FXI) can be activated by thrombin, which is generated upon activation of the tissue factor (TF) pathway. This concept, however, has not been tested in vivo. A recent study questioned the existence of this feedback loop and suggested that factor XII (FXII) is the sole activator of FXI. Here, we analyze the feedback activation of FXI in plasma and in genetically altered mice. Methods and results: Fluorescence-based assays indicated that particle-bound thrombin caused thrombin generation in plasma both in the absence of TF and in the presence of active site inhibited factor VIIa. Thrombin failed to activate FXII and thrombin generation was almost completely abolished by an anti-FXIa antibody and in FXI-deficient plasma. Surface bound thrombin induced complex formation of FXI, with its major inhibitor C1 inhibitor, even in FXII-deficient plasma in a time and dose dependent manner. To determine if thrombin-driven FXI activation is important for hemostasis in vivo we used TF deficient mice (low TF), which have severely reduced thrombin formation. Low TF mice were crossed with mice deficient in one of the intrinsic pathway proteases FXII, FXI, or FIX. Double deficiency in TF and either FIX or FXI resulted in the intrauterine death of embryos due to hemorrhage. In contrast low TF/FXII-null mice were viable and the bleeding phenotype was unchanged from low TF animals. Conclusions: Surface-bound thrombin, a model for fibrin clot-protected thrombin, generates thrombin in a FXI dependent manner, independently from FXII. In addition to corroborating an amplifying role of FXI in thrombin generation, we provide the first evidence that at low TF levels FXI is essential in generating a sufficient ambient level of thrombin to permit embryonic development. Disclosures:No relevant conflicts of interest to declare.

Nicotine Stimulates PPARβ/δ Expression in Human Lung Carcinoma Cells through Activation of PI3K/mTOR and Suppression of AP-2α

We previously showed that nicotine stimulates non-small cell lung carcinoma (NSCLC) cell proliferation through nicotinic acetylcholine receptor (nAChR)-mediated signals. Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) has also been shown to induce NSCLC cell growth. Here, we explore the potential link between nicotine and PPARbeta/delta and report that nicotine increases the expression of PPARbeta/delta protein; this effect was blocked by an alpha7 nAChR antagonist (alpha-bungarotoxin), by alpha7 nAChR short interfering RNA, and by inhibitors of phosphatidylinositol 3-kinase (PI3K; wortmannin and LY294002) and mammalian target of rapamycin (mTOR; rapamycin). In contrast, this effect was enhanced by PUN282987, an alpha7 nAChR agonist. Silencing of PPARbeta/delta attenuated the stimulatory effect of nicotine on cell growth, which was overcome by transfection of an exogenous PPARbeta/delta expression vector. Of note, nicotine induced complex formation between alpha7 nAChR and PPARbeta/delta protein and increased PPARbeta/delta gene promoter activity through inhibition of AP-2alpha as shown by reduced AP-2alpha binding using electrophoretic gel mobility shift and chromatin immunoprecipitation assays. In addition, silencing of Sp1 attenuated the effect of nicotine on PPARbeta/delta. Collectively, our results show that nicotine increases PPARbeta/delta gene expression through alpha7 nAChR-mediated activation of PI3K/mTOR signals that inhibit AP-2alpha protein expression and DNA binding activity to the PPARbeta/delta gene promoter. Sp1 seems to modulate this process. This study unveils a novel mechanism by which nicotine promotes human lung carcinoma cell growth.

Heterotrimeric G-protein signaling and light response

Almost all creatures have invented sophisticated mechanisms to adjust their developmental and metabolic processes to the changing light intensities in day and night. Recent findings suggest that one such mechanism is signaling via heterotrimeric G-proteins.1 The Trichoderma reesei (anamorph of Hypocrea jecorina) G-alpha subunit gene gna3 was found to be responsive to light and influenced by the light regulatory protein ENVOY.2 GNA3 significantly impacts regulation of cellulase gene expression only in light.1 While the exact mechanism of this regulation remains to be determined, first hints point to a regulation at the transcriptional level, since we observed light induced complex formation within the gna3 promotor. At least some of the components of this putatively regulatory protein complex also bind to the env1- promotor. These data indicate that the signal related by GNA3 is of light-dependent significance for H. jecorina and that the pathway of heterotrimeric G-protein signaling may be a target of the light perception machinery in fungi.

Heat Shock Protein 90 Inhibitor Induces Apoptosis and Attenuates Activation of Hepatic Stellate Cells

Activated hepatic stellate cells (HSCs) are major participants in hepatic fibrosis; thus, the induction of HSC apoptosis has been proposed as an antifibrotic treatment strategy. Heat shock protein (Hsp) 90 is a molecular chaperone that stabilizes major signal transduction proteins, and its inhibitors have antitumor activity. In this study, the susceptibility of HSCs to an Hsp90 inhibitor was evaluated. LX-2 cells, an immortalized human HSC line, 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an Hsp90 inhibitor, and monensin, an acidic sphingomyelinase inhibitor, were used in this study. Cellular apoptosis was quantified by 4',6-diamidino-2-phenylindole dihydrochloride staining, and signaling cascades were explored using immunoblotting and immunoprecipitation techniques. Nuclear factor (NF) kappaB activities were evaluated by immunofluorescent microscopy and enzyme-linked immunosorbent assay. Collagen alpha1 and alpha-smooth muscle actin expressions were determined by real-time reverse transcription-polymerase chain reaction and immunoblotting, respectively. It was found that 17AAG induced HSC apoptosis and that caspase 8 cleavage preceded the downstream activation of apoptotic signaling cascades. Furthermore, this caspase 8 activation was dependent on ceramide generation by acidic sphingomyelinase. In addition, 17AAG prevented NFkappaB nuclear translocation and activation, specifically by inducing complex formation between NFkappaB and the glucocorticoid receptor. In accordance, NFkappaB-dependent cellular FLICE-like inhibitory protein expression level was found to be reduced by 17AAG. Finally, 17AAG down-regulated collagen alpha1 and alpha-smooth muscle actin expression levels in HSCs before inducing apoptosis. These results demonstrate that the Hsp90 inhibitor induces HSC apoptosis via a sphingomyelinase- and NFkappaB-dependent mechanism. Because this inhibitor also reduces HSC activation before apoptosis, Hsp90 inhibitor treatment might be therapeutically useful as an antifibrotic strategy in a variety of liver diseases.

Effect of phenazine compounds XR11576 and XR5944 on DNA topoisomerases

Previous in vitro cleavage data showed that XR11576 and XR5944 stabilised topoisomerase I and topoisomerase II complexes on DNA in a dose-dependent fashion. However, some studies indicated a possible topoisomerase-independent mechanism of action for these drugs. Three methods, the TARDIS assay, immunoband depletion and the K(+)/SDS assay have been used to assess topoisomerase complex formation induced by XR11576 or XR5944 in human leukaemic K562 cells. TARDIS and immunoband depletion assays demonstrated that XR11576 and XR5944 induced complex formation for both topoisomerase I and topoisomerase II (alpha and beta) in a dose- and time-dependent manner, following exposure times of 24 and 48 h at concentrations of 1 or 10 microM. The K(+)/SDS assay showed the formation of protein/DNA complexes after a 1 h exposure to 1 or 10 muM XR11576. Our data confirm that XR11576 or XR5944 can form topoisomerase complexes, after long periods of exposure.

Mechanism of dietary salt-mediated increase in intravascular production of TGF-β1

Clinical and preclinical studies have demonstrated an important effect of arterial pathobiology on the progressive loss of renal function that occurs in chronic kidney disease. Chronic kidney disease, in turn, promotes alterations in vascular function. A modulating role for dietary salt has been suggested, with the amount of salt intake regulating endothelial cell production of transforming growth factor-beta1 (TGF-beta1), a fibrogenic growth factor that promotes arteriosclerosis and glomerulosclerosis. The purpose of the present studies was to determine how the interaction between dietary salt intake and vasculature promoted the production of TGF-beta1 in rats. Two different vascular tissues, aortic rings and glomeruli, were chosen for study. Dietary salt induced, in a dose-dependent fashion, activation of proline-rich tyrosine kinase-2 (Pyk2) and further identified c-Src as an important binding partner of Pyk2 in these tissues. Use of pharmacological inhibitors and dominant negative strategies confirmed that dietary salt induced complex formation of Pyk2 and c-Src with downstream activation of p38 and p42/44 mitogen-activated protein kinases and generation of TGF-beta1. The experiments defined the molecular signaling events that promoted the production of TGF-beta1, a key growth factor involved in the vascular response to increased salt intake.

LIMITATION OF INFARCT SIZE BY ERYTHROPOIETIN IS ASSOCIATED WITH TRANSLOCATION OF Akt TO THE MITOCHONDRIA AFTER REPERFUSION

1. The aim of the present study was to determine the critical timing of Akt activation and its interaction with the mitochondrial permeability transition pore (mPTP) in the mechanism of infarct size limitation by erythropoietin (Epo). 2. In an isolated, buffer-perfused preparation, rabbit hearts were subjected to 30 min ischaemia/2 h reperfusion. Infusion of Epo (1 unit/mL) before ischaemia reduced infarct size from 36.6 +/- 2.6% of the risk area to 15.4 +/- 3.2%, whereas a 10-fold higher dose of Epo infused for 65 min commencing 5 min before reperfusion failed to afford significant cardioprotection. The protection afforded by Epo pretreatment was abolished by coinfusion of 5 micromol/L LY294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor. Infusion of Epo induced phosphorylation of Akt, extracellular signal-regulated kinase, glycogen synthase kinase 3beta and p70s6 kinase before ischaemia and tended to enhance reperfusion-induced phosphorylation of these protein kinases. Erythropoietin increased phospho-Akt in the mitochondria and induced complex formation of Akt with adenine nucleotide translocase (ANT), a major subunit of mPTP, upon reperfusion. 3. In another series of experiments, cardiomyocytes were isolated from rat hearts and loaded with Rhod-2 to determine mitochondrial Ca(2+) levels. Increases in mitochondrial Ca(2+) levels following exposure to 1 mmol/L ouabain for 30 min were similar in untreated and Epo-pretreated cells. However, ouabain-induced hypercontracture was significantly suppressed from 45.1 +/- 1.6 to 39.2 +/- 1.9% by Epo. 4. In conclusion, activation of PI3-K-Akt signalling before ischaemia is crucial for Epo-induced myocardial protection and this protection may be achieved by complex formation of activated Akt with mPTP components upon reperfusion, leading to elevation of the threshold for opening of mPTP.

Bradykinin B2 Receptor Interacts with Integrin α5β1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

The vasoactive peptide bradykinin (BK) stimulates proliferation of the inner medullary collecting duct (mIMCD‐3) cells via transactivation of epidermal growth factor receptor (EGFR) by an unusual mechanism that involves collagenases‐2 and ‐3. Since collagenases lack an integral membrane domain, we hypothesized that integrins may target collagenases to the membrane to form a functional signaling complex.We supported a role of integrins in BK‐induced signaling by showing that RGD peptides and neutralizing antibody against α5β1 integrin partially (∼50%) decreased BK‐induced phosphorylation of extracellular signal‐regulated protein kinase (ERK) in mIMCD‐3 cells. Silencing of α5 and β1 expression by transfecting cells with siRNA also caused a significant (∼40%) decrease in BK‐induced ERK activation and EGFR phosphorylation. The effect was more pronounced (∼70% inhibition) in cells that were co‐transfected with siRNAs directed against collagenases. Using immunoprecipitation/Western blotting, we demonstrated association of BK B2 receptor with α5β1 integrin upon BK treatment. Furthermore, we showed that BK induced complex formation between α5β1, collagenases and EGFR.These data provide the first evidence that integrins are involved in BK‐induced proliferation of kidney cells, and might ultimately lead to development of new strategies for treatment of renal tubulointerstitial fibrosis.