A Disintegrin and Metalloenzyme (ADAM) 17 Activation Is Regulated by α5β1 Integrin in Kidney Mesangial Cells

The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation.We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT.Our data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases

Curcumin Alleviates Matrix Metalloproteinase-3 and -9 Activities during Eradication of Helicobacter pylori Infection in Cultured Cells and Mice

Current therapy-regimens against Helicobacter pylori (Hp) infections have considerable failure rates and adverse side effects that urge the quest for an effective alternative therapy. We have shown that curcumin is capable of eradicating Hp-infection in mice. Here we examine the mechanism by which curcumin protects Hp infection in cultured cells and mice. Since, MMP-3 and -9 are inflammatory molecules associated to the pathogenesis of Hp-infection, we investigated the role of curcumin on inflammatory MMPs as well as proinflammatory molecules. Curcumin dose dependently suppressed MMP-3 and -9 expression in Hp infected human gastric epithelial (AGS) cells. Consistently, Hp-eradication by curcumin-therapy involved significant downregulation of MMP-3 and -9 activities and expression in both cytotoxic associated gene (cag)+ve and cag-ve Hp-infected mouse gastric tissues. Moreover, we demonstrate that the conventional triple therapy (TT) alleviated MMP-3 and -9 activities less efficiently than curcumin and curcumin's action on MMPs was linked to decreased pro-inflammatory molecules and activator protein-1 activation in Hp-infected gastric tissues. Although both curcumin and TT were associated with MMP-3 and -9 downregulation during Hp-eradication, but unlike TT, curcumin enhanced peroxisome proliferator-activated receptor-γ and inhibitor of kappa B-α. These data indicate that curcumin-mediated healing of Hp-infection involves regulation of MMP-3 and -9 activities

Cell free assays show ADAM17 binding to α5β1 integrin and changes in ADAM17 activity.

<p>(A) Recombinant ADAM17 binds purified α5β1 integrin and recombinant β1 integrin in a cell free binding assay. Plates pre-coated with antibodies against α5β1 integrin or β1 integrin were incubated with purified α5β1 integrin or with β1 recombinant integrin, individually. Recombinant ADAM17 was then added at the indicated concentrations and ADAM17 binding was measured using a colorimetric assay at 450 nm as described in Methods. Data are expressed as mean±S.D. *p<0.05, **p<0.01 <i>vs</i> control; data from 4 experiments with 3 parallels/each condition are shown. (B) Purified α5β1 integrin and recombinant β1 integrin decrease ADAM17 enzymatic activity. Recombinant ADAM17 (10 ng/ml) was incubated alone or together with either purified α5β1 integrin (25 ng/ml or 250 ng/ml) or with β1 recombinant integrin (12 ng/ml or 120 ng/ml), in OG buffer in the presence of a quenched fluorogenic ADAM17 substrate. Enzyme activity was expressed as the rate of change of relative fluorescence units (ΔRFUs⁻¹); *p<0.05, **p<0.01 <i>vs</i> control; data from four experiments with eight parallels/each condition are shown.</p

Manganase²⁺ stimulation decreases association of ADAM17 to β1 integrin and leads to increased ADAM17 activity.

<p>(A) Control and manganese stimulated (1 mM MnCl₂ in PIPES buffer for 2 min) mesangial cell lysates were immunoprecipitated with ADAM17 antibody. Samples were resolved on a 3–8% Tris-acetate gel and probed for β1 integrin and α5 integrin by Western blotting. Immunoblotting for ADAM17 served as loading control. Arrows point to specific and non-specific (NS) bands. One representative blot out of three is shown. (B) Mesangial cells were transfected with AP-HB-EGF construct and stimulated with 1 mM MnCl₂ in PIPES buffer. HB-EGF shedding was expressed as mean±S.D. of fold increase in the rate of change of relative fluorescence units (RFU); **p<0.01 <i>vs</i> control; n = three experiments, 6 parallels/each condition.</p

Co-localization of ADAM17 and α5β1 integrin in rat mesangial cells.

<p>Control (C) and 1 µM 5-HT -stimulated mesangial cell were fixed, permeabilized, and (A) co-immunostained using ADAM17 antibody (green) and β1 integrin antibody (red) as indicated in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033350#s4" target="_blank">Material and Methods</a>”. Arrows indicate co-localization of ADAM17 and α5β1 integrin immunopositive areas (yellow). For the negative controls we omitted the primary antibodies and used PBS followed by secondary antibodies. (B) Parallel samples were incubated with oligonucleotide-labeled PLA probes after incubation with primary antibodies. PLA signals as fluorescence dots were imaged and quantified. As negative control we used either ADAM17 or α5β1 integrin antibody alone followed by the oligonucleotide-labeled PLA probes. Cartoon explains binding of the fluorescence detection reagent only to antibodies in close proximity; **p<0.01. Representative examples out of three experiments are shown.</p

ADAM17 co-precipitates with α5β1 integrin and dissociates from the integrin during GPCR stimulation.

<p>(A) Control (−) and 1 µM 5-HT stimulated (+) mesangial cell lysates were immunoprecipitated (IP) with either ADAM17 antibody or Ig control (Ig C), resolved on 3–8% Tris-acetate gel and probed for the presence of β1 integrin and α5 integrin by Western blotting. (B) ADAM17 blot shows that equal amount of ADAM17 were precipitated from each sample. Arrows point to specific and non-specific (NS) bands. One representative example out of four experiments is shown.</p

Integrin expression regulates ADAM17 sheddase activity.

<p>(A) Time-dependent release of alkaline phosphatase (AP)-tagged HB-EGF by unstimulated (C) and 5-HT stimulated cells. Cells were transfected with AP-HB-EGF expressing plasmid and 2 days after transfection they were stimulated with 5-HT for the indicated time. AP activity of cell supernatants was determined using Attophos substrate. Data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units; *p<0.05, **p<0.01 <i>vs</i> control at same time point, n = eight experiments, three parallels/each condition. (B) β1 integrin silencing promotes 5-HT-induced AP-HB-EGF shedding. Cells were transfected with AP-HB-EGF expression plasmid together with β1 integrin siRNA (β1-siRNA) or a non-targeting (nt−) siRNA. Successful silencing of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. (C) β1 integrin overexpression inhibits 5-HT induced AP-HB-EGF release in mesangial cells. Cells were transfected with AP-HB-EGF and with β1 integrin expressing plasmid (β1-plasmid) or control DNA (Co-DNA). Successful overexpression of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. Activity data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units (RFU); *p<0.05, **p<0.01 <i>vs</i> unstimulated control nt-siRNA or control DNA-transfected cells; ^# p<0.05 and ^## p<0.01 <i>vs</i> 5-HT stimulated nt-siRNA or DNA-transfected cells; n = five experiments, three parallels/each condition.</p

TRPP2 and TRPV4 Form an EGF-Activated Calcium Permeable Channel at the Apical Membrane of Renal Collecting Duct Cells

<div><p>Objective</p><p>Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.</p> <p>Methods and Results</p><p>We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium.</p> <p>Conclusion</p><p>We conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.</p> </div

Effect of gene silencing on the 23-pS single-channel currents.

<p>(<b>a</b>–<b>b</b>) Western blots showing knockdown of TRPP2 (left) and TRPV4 (right) by TRPP2- and TRPV4-specific shRNAs. (<b>c</b>–<b>f</b>) Summary of the data obtained from western blots (<b>c & d</b>; n = 5) and quantitative real-time PCR analysis (<b>e & f</b>; n = 3). Ctrl, EV, sh1, sh2 and sh3 represent non-tranfected (control), empty vector, shRNA1, shRNA2 and shRNA3 transfected cells, respectively. P2 and V4 indicate that the shRNAs were specific to TRPP2 and TRPV4, respectively. * indicates significant difference from control and empty vector transfected group. (<b>g</b> & <b>h</b>) Sample single-channel traces were recorded from cilia (-) cells expressing shRNA3s specific to TRPP2 and TRPV4, using excised inside-out patch configuration. (<b>i</b> & <b>j</b>) Summary of i-V plots constructed from the single-channel recordings as shown (<b>g</b>) and (<b>h</b>) demonstrating altered biophysical properties of the channels. Knockdown TRPP2 and TRPV4 resulted in the appearance of TRPV4-like channels (<b>i</b>) with a single-channel conductance of 115.7 ± 1.98 pS for outward and 51.8 ± 0.91 pS for inward currents, respectively and TRPP2-like currents with a single-channel conductance of 86.8 ± 1.78 pS (n = 5-9 for different data points).</p

EGF-induced activation of TRPP2\TRPV4 channels mediates hyperproliferation of cilia (-) cells.

<p>(<b>a</b>) Plot shows the rate of cell proliferation. Under control conditions, cell proliferation rate was significantly higher in cilia (-) cells (the red dashed line) than in cilia (+) cells (the red solid line). EGF significantly enhanced the rate of cell proliferation in cilia (-) cells but not in cilia (+) cells. The effect of EGF on cell proliferation was abolished by AG1478, PD98059 and BAPTA in cilia (-) cells (n = 5 for each condition). (<b>b</b> & <b>c</b>) Western blots confirm the effectiveness of knockdown of both TRPP2 and TRPV4, where Ctrl, EV, sh3-P2\sh3-V4 represent non-tranfected (control), cells co-transfectd with empty vector, sh3-P2\sh3-V4 specific to TRPP2 and TRPV4, respectively. (<b>d</b> & <b>e</b>) Summary of the results obtained from western blots analysis; both TRPP2 and TRPV4 protein expression levels were reduced by ~75-80%. (<b>f</b>) Cell proliferation assays were performed at 96 hour time point after seeding the cells in the presence or in the absence of EGF. Plot shows that the rate of EGF-induced proliferation in cilia (-) cells was significantly reduced by knockdown TRPP2\TRPV4 channels. KO indicates knockdown, * indicates that under control condition the rate of cell proliferation was significantly higher in the presence of EGF. # indicates a significant difference compared to control.</p