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

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

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

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

~23-pS channel permeates divalent cation and mediates EGF-induced Ca²⁺ influx.

<p>Cilia (-) cells were pretreated with 10 ng/mL EGF at the apical membrane prior to recordings. (<b>a</b>–<b>c</b>) The representative traces were recorded using excised inside-out patch mode at V_M = 100 mV from cilia (-) cells and the dashed gray lines indicate closed current level. (<b>a</b>`–<b>c</b>`) The gray dashed lines indicate the fitted single-channel conductance (23.4 ± 0.76 pS) of the currents recorded under control condition (representative single-channel traces are shown in Figure S1; n = 13-21 for different data points). (<b>a</b> & <b>a</b>`) This representative recording contained at least four active channels. While the single-channel conductance of inward currents remained the same as control the single-channel conductance of outward currents was dramatically reduced to 2.79 ± 0.33 pS (n = 9-14 for different data points) by substitution of intracellular Na<sup>+</sup> with equimolar NMDG<sup>+</sup> and the reversal potential shifted to depolarization potentials (the red arrow head). (<b>b</b> & <b>b</b>`) The representative trace was recorded in the presence of equimolar intracellular K⁺ to extracellular Na⁺, which affected neither the single-channel conductance (the blue dashed line) nor the reversal potential (the blue arrow head). (<b>c</b> & <b>c</b>`) The representative trace was recorded in the presence of equimolar intracellular Ba²⁺ to extracellular Na⁺. Replacement of Na⁺ by Ba²⁺ did not alter the single-channel conductance of inward current, but altered the single-channel conductance of outward current (the green dashed line) and the reversal potential of the currents (where the green arrow head indicates the reversal potential that was corrected for the junction potential). (<b>d</b> & <b>e</b>) Manganese quenching assays demonstrate that EGF promotes Ca²⁺ entry in cilia (-) cells, but not in cilia (+) cells (data not shown); while double knockdown of TRPP2\TRPV4 channels or TRPV4, the effect of EGF on Ca²⁺ entry was abolished (n = 5); while knock down of TRPV4, EGF also promotes Ca²⁺ entry, albeit with less extent compared to wild-type cilia (-) cells (n = 5). * indicates significant difference compared to TRPV4 knocked down cilia (-) cells while stimulated with EGF.</p

Cilia (-) cells has a much higher activity of EGFR and p-ERK than cilia (+) cells.

<p>(<b>a</b>) The representative western blots showing the expression of EGFR, ERK, and p-ERK before and after 10 ng/mL EGF treatment from both apical and basolateral sides in both cell types for the indicated time. (<b>b</b>) Summary of EGFR expression levels, all data points were normalized to the value obtained from cilia (-) cells under control condition, i.e. no EGF simulation (n = 5 for each data point). (<b>c</b>) Summary of p-ERK expression levels, all data points were normalized to the value obtained from cilia (-) cells under control condition, i.e. no EGF simulation (n = 6 for each data point). * indicates p-ERK expression level of cilia (-) cells is significantly greater than that of cilia (+) cells. (<b>d</b> & <b>e</b>) The representative western blots showing the expression ERK and p-ERK with and without stimulation by 10 ng/mL EGF from both apical and basolateral sides in both cell types for 10 min, in the presence of variety of inhibitors. Where Crtl indicates control and AG, GF, GO, PD, and SB represent AG1478, GF109203, GO6976, PD98059, and SB202190, respectively. Cells were preincubated with EGF for 60 min, respectively. (<b>f</b> & <b>g</b>) Summary of p-ERK expression levels, all data points were normalized to the value obtained under control condition, i.e. no EGF simulation (n = 5 for each data point). * indicates the values were dramatically less than those of control and EGF stimulation. ^# indicates p-ERK expression level is significantly greater compared to control.</p