Caveolin-1 influences P2X 7 receptor expression and localization in mouse lung alveolar epithelial cells K. Barth 1, *, K. Weinhold 1, *, A. Guenther 1 , M. T. Young 2 , H. Schnittler 3 and M. Kasper 1 1 Institute of Anatomy, Medical Faculty ‘Carl Gustav Carus’, Dresden University of Technology, Germany 2 Faculty of Life Sciences, University of Manchester, UK 3 Institute of Physiology, Medical Faculty ‘Carl Gustav Carus’, Dresden University of Technology, Germany Extracellular nucleotide signaling in vertebrate cells is mediated by plasma membrane P2 receptors, which may be divided into two categories: G-protein-coupled receptors (P2Y) and ion channels (P2X) [1]. P2X receptors are ATP-gated nonselective cation channels, seven members of which (P2X 1)7 ) have been cloned and characterized in humans, rats and mice [2]. P2X receptors display a unique topology among ion chan- nels. The functional channel is a trimer, with each sub- unit consisting of two transmembrane spans, a large extracellular loop, and cytoplasmic N- and C-termini [2]. The extracellular loop contains 10 conserved cys- teine residues that form five disulfide bonds, some of which are essential for appropriate trafficking of the receptor to the cell surface [3]. Additionally, the extra- cellular loop of P2X receptors contains a number of putative N-glycosylation sites. N-glycosylation of P2X receptors has been shown to be essential for their cor- rect folding, trafficking and function [4–8]. The intracellular C-terminal tail of P2X 7 is consider- ably longer than those of other members of the P2X family, and contains multiple potential protein and lipid interaction motifs [9]. Uniquely for P2X 7 , ion channel activity is coupled through the C-terminus to downstream events, including membrane blebbing, interleukin-1b release and pore formation [10–13]. The presence of two discrete populations of P2X 7 has been demonstrated in the plasma membrane of rat subman- dibular glands [14] and in mouse lymphoma cells [15], leading to the suggestion that P2X 7 may be distributed Keywords alveolar epithelium; Caveolin-1; mouse lung; P2X7 receptor Correspondence M. Kasper, Institute of Anatomy, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Fiedlerstr. 42, D-01307 Dresden, Germany Fax: +49 351 458 6303 Tel: +49 351 458 6080 E-mail: michael.kasper@tu-dresden.de *These authors contributed equally to this work (Received 3 January 2007, revised 11 April 2007, accepted 16 April 2007) doi:10.1111/j.1742-4658.2007.05830.x The P2X 7 receptor has recently been described as a marker for lung alveo- lar epithelial type I cells. Here, we demonstrate both the expression of P2X 7 protein and its partition into lipid rafts in the mouse lung alveolar epithelial cell line E10. A significant degree of colocalization was observed between P2X 7 and the raft marker protein Caveolin-1; also, P2X 7 protein was associated with caveolae. A marked reduction in P2X 7 immunoreacti- vity was observed in lung sections prepared from Caveolin-1-knockout mice, indicating that Caveolin-1 expression was required for full expression of P2X 7 protein. Indeed, suppression of Caveolin-1 protein expression in E10 cells using short hairpin RNAs resulted in a large reduction in P2X 7 protein expression. Our data demonstrate a potential interaction between P2X 7 protein and Caveolin-1 in lipid rafts, and provide a basis for further functional and biochemical studies to probe the physiologic significance of this interaction. Abbreviations b-Cop, b-coatomer protein; Flo-1, flotillin-1; PDI, protein disulfide isomerase; sh, short hairpin; TfR, ransferrin receptor; TRCP1, transient receptor potential channel protein. FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS 3021 among ‘raft’ and ‘nonraft’ domains of the plasma membrane. Lipid rafts are microdomains of the plasma membrane containing a high proportion of sphingo- lipids and cholesterol, assembled in the Golgi apparatus and subsequently delivered to the plasma membrane [16]. Evidence for the involvement of P2X receptors in lipid rafts is emerging; the presence of P2X 1 and P2X 3 receptor subtypes in rafts has also been demonstrated recently [17,18]. Current evidence supports the hypo- thesis that P2X 7 is not firmly embedded in rafts [14], but may interact with an unknown raft component. In this study, the authors imply that P2X 7 receptors in nonraft microdomains of submandibular gland cells are solely involved in ion channel activity, and that re- ceptors present in lipid rafts may regulate the activities of proteins included in signal transduction cascades [14]. More evidence for the association of P2X 7 with lipid rafts may be derived from the proteomic complex described by Kim et al. [19]. In this study, several potential interacting partners of rat P2X 7 were identi- fied, including three chaperone proteins (Hsp70, Hsp71, Hsp90) [19]. In addition, tyrosine phosphoryla- tion of Hsp90 was implicated in the functional regula- tion of ion channel activity [20]. Heat shock proteins identified as part of the P2X 7 complex have been shown to be localized to lipid rafts [21,22]. It is known that subsets of lipid rafts form cell surface invaginations termed caveolae. Caveolae are formed by polymerization of caveolins, hairpin-like palmitoylated integral membrane proteins from lipid rafts that tightly bind cholesterol. An important feature of caveolae is their function during signal transduction [16,23]. However, they are not a prerequisite for signal transduction, as several cell types lacking Caveolin-1, such as lymphocytes and neurons, may transduce sig- nals through rafts. Caveolin-1, the main component of caveolae, negatively regulates several proteins, resulting in inactivation of signaling molecules and modulation of downstream signal transduction [23]. In this study, we analyzed the expression of P2X 7 receptors in the following lung epithelial cell lines: A549, E10, R3⁄ 1, and L2. All lung cell lines were character- ized by a high number of caveolae and strong expres- sion of Caveolin-1 [24–26]. After demonstrating robust levels of P2X 7 protein expression in the lung epithelial cell line E10, we studied the localization of P2X 7 as well as the association of P2X 7 with lipid rafts, and we consider the hypothesis that lipid rafts might contri- bute to specific subcellular targeting and functional activation of the P2X 7 receptor. In addition, we have demonstrated that the levels of P2X 7 protein expres- sion are dependent upon Caveolin-1 expression in two ways. First, we show that P2X 7 protein expression is significantly reduced in Caveolin-1-knockout mice as compared to wild-type mice. Second, we show that short hairpin RNA (shRNA)-mediated downregulation of Caveolin-1 expression leads to significantly reduced levels of P2X 7 protein expression in E10 cells. These data imply that Caveolin-1 regulates the expression level of the P2X 7 receptor. Results Presence of the P2X 7 receptor and its modification in alveolar epithelial cell lines First, we tested the expression of the P2X 7 receptor in permanent alveolar epithelial cell lines with properties more similar to those of AT I cells (R3 ⁄ 1, L2, E10) or AT II cells (A549) using western blot analysis. Ini- tially, we were only able to detect P2X 7 protein in the alveolar epithelial cell line E10 (Fig. 1A, lane 2). Sub- sequently, P2X 7 expression was also detected in R3⁄ 1 cells (Fig. 1B, lane 2), but only after immunoprecipita- tion with the same antibody to concentrate protein from approximately 2.5 · 10 6 cells (5· confluent T25 flasks). This result implies that the expression levels of P2X 7 in R3 ⁄ 1 cells are very low. Therefore, subsequent experiments were performed using the alveolar epithe- lial cell line E10 only. We detected two major bands of 80 kDa and 60 kDa by western blot analysis using antibodies to P2X 7 direc- ted against the extreme C-terminus (amino acids 576– 595) in E10 cell membrane extracts (Fig. 1A). The 80 kDa band was consistent with that previously des- cribed for mouse P2X 7 [8,27], and N-glycosidase F treatment resulted in a decrease in the molecular mass of the 80 kDa band to approximately 68 kDa, consis- tent with the calculated molecular mass of nonglycosyl- ated P2X 7 (Fig. 1C). The 60 kDa band was unchanged by N-glycosidase F treatment (Fig. 1C), and its mole- cular mass was too low to represent nonglycosylated full-length P2X 7 , raising the possibility that it might be a nonspecific band. However, immunoreactivity to both the 80 kDa and 60 kDa bands was abolished following preincubation with the antibody control peptide (Fig. 1D), implying that the 60 kDa band shared at least a portion of the epitope recognized by the C-ter- minal P2X 7 antibody. It is possible that the 60 kDa band represents an alternatively spliced or N-terminally truncated P2X 7 protein. However, because N-glycosyla- tion is required for cell surface expression of functional P2X receptors [6,7], it follows that the 60 kDa band, if specific, represents a truncated, nonfunctional, intra- cellular P2X 7 protein. We therefore assumed that the 80 kDa band represents mature, functional P2X 7 Caveolin-1 and P2X 7 expresssion in lung cells K. Barth et al. 3022 FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS protein, and consequently focused upon this band for the remainder of the study. We also assumed that, in immunohistochemical studies, plasma membrane stain- ing reflects the presence of the 80 kDa band alone. Isolation of lipid rafts and localization of Caveolin-1 and P2X 7 Insolubility in the detergent Triton X-100 is a well- recognized biochemical characteristic of proteins locali- zed in lipid rafts. We therefore analyzed the Triton solubility of P2X 7 protein prepared from E10 cell mem- brane fractions, along with the raft markers Caveolin-1 and flotillin-1 (Flo-1), and the nonraft marker T1a (an integral membrane protein expressed strongly within the apical plasma membrane of both type I alveolar epithe- lial cells and lymphatic endothelial cells in the lung [28,29]). As expected, Caveolin-1 and Flo-1 were pre- dominantly found in the detergent-insoluble raft frac- tion (Fig. 2A, second and third panels), and T1a was present only within the nonraft fraction of the mem- brane (Fig. 2A, bottom panel). Interestingly, although the majority of P2X 7 protein was Triton X-100-soluble, a significant proportion was found within the Triton X-100-insoluble fraction (Fig. 2A, top panel), suggest- ing partial localization with lipid rafts. Another main characteristic of lipid rafts is low buoyant density in sucrose gradient centrifugation. We isolated raft-like membranes using three distinct meth- ods; Triton X-100 treatment at 4 °C (Fig. 2B), a deter- gent-free method (Fig. 3A), and Brij35 treatment at 4 °C (Fig. 3B). When rafts were prepared using Tri- ton X-100 treatment followed by sucrose density gradi- ent centrifugation, P2X 7 protein was not detected in the Caveolin-1- and Flo-1-containing low-density raft fractions (Fig. 2B, lanes 3–5), and was only faintly detected in the nonraft fractions (Fig. 2B, lanes 9–13). In contrast, significant amounts of P2X 7 protein were detected in low-density raft fractions when rafts were isolated using either a detergent-free raft isolation pro- cedure (consisting of fine disruption of the membrane by sonication followed by sucrose density gradient cen- trifugation) (Fig. 3A, lanes 2–4) or Brij35 isolation (Fig. 3B, lanes 3–4). P2X 7 protein was also detected in the high-density fractions from both detergent-free (Fig. 3A, lanes 7–9) and Brij35 isolation methods (Fig. 3B, lanes 9–14), along with markers for Flo-1, the Golgi apparatus and endoplasmic reticulum [b-coa- tomer protein (b-Cop) and protein disulfide isomerase (PDI)]. In addition, isolated lipid rafts prepared using either method were devoid of intracellular membrane markers for the Golgi apparatus and the endoplasmic A549 E10 R3/1 L2 80 kDa 60 kDa P2X 7 R -tubulin -0.5h1h24h Deglycosylation 60 kDa epitope blocking P2X 7 Ag + P2X 7 80 kD a 60 kD a 68 kDa 80 kDa 100 kDa 80 kDa A C B D Glycosylated Nonglycosylated Fig. 1. (A) Analysis of P2X 7 receptor expression in alveolar epithelial lung cell lines. P2X 7 receptor expression was measured in cell lysates of A549, E10, R3 ⁄ 1 and L2 cells by SDS ⁄ PAGE and western blot analysis. Fifty micrograms of protein from each sample was loaded on the gel. Western blot analysis was performed using rabbit anti-P2X 7 (1 : 375) and anti-c-tubulin (1 : 1000). c-Tubulin served as a loading control. Representative data from three separate experiments are shown. (B) Immunoprecipitation of P2X 7 from R3 ⁄ 1 cells. The figure represents a western blot for P2X 7 (1 : 1000) from samples immunoprecipitated with the same antibody (0.3 lg) to concentrate P2X 7 protein. Lane 1: 10 lg of rat P2X 7 from transiently transfected HEK cells. Note the appearance of a nonspecific 90 kDa band. Lane 2: total protein from 2.5 · 10 6 R3 ⁄ 1 cells shows a band of the same molecular mass as the positive control (lane 1; 80 kDa). (C) P2X 7 receptors are glycosylated in E10 cells. Membranes were incubated in the presence of N-glycosidase F for 0.5 h, 1 h and 24 h, and compared with nontreated mem- branes (–). Full deglycosylation of the 80 kDa band was indicated by a reduction in molecular mass of approximately 12 kDa to 68 kDa. The mass of the 60 kDa band was unaffected by deglycosylation. Representative data from three separate experiments are shown. (D) Fifty micrograms of E10 cell lysates were run on an SDS gel and blotted for P2X 7 protein with (right side) or without (left side) preincubation with the antigenic blocking peptide. K. Barth et al. Caveolin-1 and P2X 7 expresssion in lung cells FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS 3023 reticulum (Figs 2B and 3A,B). Transferrin receptor (TfR), a marker of the nonraft fraction of the plasma membrane [30,31], was mainly found in high-density fractions (Fig. 3A,B), although very small amounts (compared to Caveolin-1 and P2X 7 ) were also present in the low-density fractions. Immunogold labeling electron microscopy of P2X 7 in alveolar epithelial E10 cells Immunogold labeling of P2X 7 receptors expressed in E10 cells was performed to analyze the subcellular localization of the receptor in detail (Fig. 4, upper panel). Gold particles were mainly associated with caveolae at this resolution, further supporting a locali- zation for P2X 7 in raft-like membranes. Very few gold particles were associated with P2X 7 at the plasma membrane in this study (not shown); this was probably due to poor preservation of the plasma membrane under our experimental conditions rather than any specific lack of receptor expression. When parallel immunostaining of a similar area of E10 cells in a second ultrathin section was performed, the majority of caveolae were also positive with the polyclonal anti- body to Caveolin-1–3 (Fig. 4, lower panel). Double-label immunofluorescence of Caveolin-1 and P2X 7 in alveolar epithelial E10 cells To examine the possible colocalization of the puriner- gic P2X 7 receptor with Caveolin-1 in alveolar epithelial E10 cells, we analyzed the immunocytochemical locali- zation and distribution of both proteins with a laser scanning confocal microscope, using two independent channels (Fig. 5). Caveolin-1 and P2X 7 receptor are endogenously expressed in E10 cells, and their localiza- tion in subconfluent cells was determined. Figure 5A shows a Caveolin-1 staining pattern at the cell surface, confirming the known caveolar location of Caveolin-1. In addition, intracellularly distinguishable punctate patterns of Caveolin-1 staining were found (Fig. 5A). P2X 7 staining appeared as small punctate spots at the A Cav-1 T1 12 3 4 56 7 8910111213 PDI P2X 7 R -Cop Flotillin-1 B Triton X-100 soluble insoluble P2X 7 R Cav-1 Flotillin-1 T1 TfR Fig. 2. (A) Solubility of Caveolin-1, Flo-1 and P2X 7 receptor in Triton X-100. E10 cells were lysed in a buffer containing 1% Triton X-100 to obtain soluble and insoluble fractions. These fractions were adjusted to equal volumes, and an aliquot of each (100 lL) was analyzed by western blot analysis using Caveolin-1-, Flo-1-, T1a- or P2X 7 receptor-specific antibodies. Caveolin-1 and Flo-1 were used as markers of the lipid lipid rafts, and T1a as a marker of the nonraft fraction. Representative data from three separate experiments are shown. (B) Characteri- zation of membrane fractions prepared by Triton X-100 from E10 cells. E10 cells were homogenized in a buffer containing 1% Triton X-100 and subjected to sucrose density gradient centrifugation. Thirteen fractions were collected (fraction 1, top of the gradient; fraction 13, bot- tom of the gradient), and an aliquot of each fraction (20 lL) was resolved by SDS ⁄ PAGE and subjected to western blot analysis with anti- bodies against Caveolin-1, P2X 7 , PDI, TfR, b-Cop and Flo-1. As expected, Caveolin-1 and Flo-1 were enriched in fractions 3–5, representing caveolae-enriched membrane fractions. Representative data from three separate experiments are shown. Caveolin-1 and P2X 7 expresssion in lung cells K. Barth et al. 3024 FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS plasma membrane in a similar pattern as observed for Caveolin-1 staining (Fig. 5B). Merged immunofluores- cence pictures of Caveolin-1 and P2X 7 indicate partial colocalization of both proteins (Fig. 5C). This partial colocation implies that a portion of P2X 7 molecules is located in or associated with lipid rafts of alveolar epi- thelial E10 cells. Caveolin-1-knockout animals show reduced P2X 7 expression levels Immunohistochemical assessment of lungs from wild- type and Caveolin-1-deficient mice revealed significant differences in P2X 7 immunoreactivity. Decreased levels of P2X 7 receptor expression were detected in Caveolin- 1-deficient mice as compared to wild-type mice (Fig. 6A). Wild-type mice exhibited P2X 7 receptor immunoreactivity in both type I cells and pulmonary endothelial cells (Fig. 6A). P2X 7 expression in type I cells of Caveolin-1-deficient mice was strongly decre- ased, and reduced immunoreactivity was also found in other pulmonary cells. shRNA-mediated downregulation and altered localization of Caveolin-1 in E10 cells To reduce the expression of Caveolin-1 protein in E10 cells, we designed two different shRNAs targeted to the mouse Caveolin-1 mRNA. Figure 6B shows Cav-1 12345678 91011121314 P2X 7 R Flotillin-1 -Cop PDI TfR 1 2 3 4 56789 Cav-1 P2X 7 R Flotillin-1 -Cop PDI TfR A B Fig. 3. (A) Characterization of the membrane fractions prepared by sonication from E10 cells. Raft and nonraft membranes were prepared by sonication followed by centrifugation in a discontinuous sucrose gradient. Nine fractions were collected (fraction 1, top of the gradient; fraction 9, bottom of the gradient), and an aliquot of each fraction (20 lL) was resolved by SDS ⁄ PAGE and subjected to western blot analy- sis with antibodies against Caveolin-1, P2X 7 , PDI, TfR, b-Cop and Flo-1. Representative data from three separate experiments are shown. (B) Characterization of membrane fractions prepared by Brij35 from E10 cells. E10 cells were homogenized in a buffer containing 1% Brij35, and subjected to sucrose density gradient centrifugation. Fourteen fractions were collected (fraction 1, top of the gradient; fraction 14, bot- tom of the gradient), and an aliquot of each fraction (20 lL) was resolved by SDS ⁄ PAGE and subjected to western blot analysis with anti- bodies against Caveolin-1, P2X 7 , PDI, TfR, b-Cop and Flo-1. As expected, Caveolin-1 and Flo-1 were enriched in fractions 3 and 4, representing caveolae-enriched membrane fractions. Representative data from three separate experiments are shown. Fig. 4. Immunogold detection of P2X 7 (upper panel) and Caveolin- 1–3 (lower panel) on an ultrathin cryosection of E10 cells. Note the labeling of caveolae (black arrows). The thick arrow indicates a caveosome. Bar ¼ 60 nm. K. Barth et al. Caveolin-1 and P2X 7 expresssion in lung cells FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS 3025 western blots probed for Caveolin-1 and P2X 7 in E10 cells 72 h after transfection with Caveolin-1- specific shRNA. Caveolin-1 expression was efficiently reduced by transfection with Caveolin-1-specific shRNA (Fig. 6B, left side), whereas transfection with scrambled shRNAs (control 1–3) did not affect the expression of Caveolin-1 (Fig. 6B, right side). Levels of P2X 7 protein were significantly reduced in E10 cells treated with two different shRNA constructs (Fig. 6B), implying that Caveolin-1 was able to influence the expression of P2X 7 protein. The results showed that the potent inhibition of P2X 7 expression could reach 59% (shRNA1) and 52% (shRNA2), and was specific to the Caveolin-1-derived shRNAs but not to the scrambled shRNAs. Under all experimental conditi- ons, levels of c-tubulin protein in cell lysates remained constant. Double immunofluorescence labeling for Caveolin-1 and P2X 7 of Caveolin-1 shRNA-treated E10 cells revealed a partial loss of colocalization of P2X 7 , with a remaining low amount of Caveolin-1 protein and an altered intracellular distribution of P2X 7 (Fig. 7). Discussion The P2X 7 receptor was introduced as a novel marker for alveolar epithelial type I cells [32], which cover more than 95% of the alveolar surface in lungs. In our study, we tested four alveolar epithelial cell lines with characteristics of type I cells [33], and among them only the E10 cell line expressed a robust amount of the P2X 7 receptor. We determined a molecular mass of 80 kDa of the P2X 7 receptor in E10 cells after immunoblotting, consistent with the detected size of A Cav-1 B P2X 7 R C Cav-1/ P2X 7 R Cav-1/ P2X 7 R D Fig. 5. (A–C) Immunofluorescence demonstration of Caveolin-1 (A; fluorescein isothiocyanate) and P2X 7 (B; Texas Red) in E10 cells. (D) Higher magnification of the labeled part in (C) (merged picture). Caveolin-1 and P2X 7 expresssion in lung cells K. Barth et al. 3026 FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS the P2X 7 receptor after immunoprecipitation in R3 ⁄ 1 cells. Treatment of the E10 cells with N-glycosidase F resulted in a decrease in the apparent molecular mass of the 80 kDa product to about 68 kDa, and indicates that the P2X 7 receptor of the E10 cells is glycosylated. Hu et al. [34] have published a similar result for P2X 4 in cardiac myocytes. They demonstrated that the gly- cosylation of the P2X 4 receptor is required for its localization to the cytoplasmic membrane. Glycosyla- tion of the P2X 2 receptor is also required for their functional surface expression [6,7]. In the present study, we further show that two populations of the purinergic receptor P2X 7 are detect- able in the plasma membrane of E10 cells. A discrete population of P2X 7 was associated with lipid rafts, as determined by cofractionation with the raft markers Caveolin-1 and Flo-1 after preparation of lipid rafts in the presence of Brij35 and detergent-free membrane preparation. These fractions did not contain Golgi apparatus and endoplasmic reticulum, as shown by detection of the organelle-specific protein markers b-Cop and PDI, respectively. A second population of P2X 7 was present in higher-density membranes. Interestingly, the majority of the P2X 7 receptor was extracted by treatment with 1% ice-cold Triton X-100, contrary to what would be expected for raft-embedded proteins. In addition, raft-like membranes prepared using Triton X-100 did not contain any P2X 7 receptor protein. It is possible that detergent may disrupt inter- actions of proteins with components of the rafts, and that this effect could be particularly relevant for pro- teins that may interact weakly with rafts, such as P2X 7 . Nevertheless, our studies provide evidence to support the possibility of an association of a pro- portion of P2X 7 receptor molecules with lipid rafts. Immunoelectron microscopy revealed preferential A B wild-type mice (+/+) knockout mice (-/-) control shRNA1 shRNA2 Cav-1 -tubulin P2X 7 R (80 kDa) control shRNA shRNA shRNA control1 control2 control3 Cav-1 P2X 7 R (80 kDa) -tubulin control shRNA1 shRNA2 0 20 40 60 80 100 120 percent (%) P2X 7 R(80kDa) Fig. 6. (A) Immunoperoxidase demonstration of P2X 7 in alveolar epithelial cells of wild-type (+ ⁄ +) and Caveolin-1-knockout (- ⁄ -) lungs. Wild- type: Arrow indicates a negative alveolar epithelial type II cell and a neighboring positive type I cell. Note the additional strong immunoreac- tivity of endothelial cells in a larger blood vessel (asterisk) and of capillary endothelial cells. Alveolar macrophages (arrowheads) were in most cases negative. Knockout: Note the reduced immunoreactivity for P2X 7 in the entire lung parenchyma. Arrows indicate the loss of P2X 7 from the alveolar lining layer. Bar ¼ 10 lm. (B) The effect of Caveolin-1 downregulation in E10 cells. After transfection of E10 cells with two dif- ferent shRNA constructs of Caveolin-1 (shRNA1 and shRNA2) and three different scrambled shRNAs (control 1–3), cells were harvested, and the expression levels of Caveolin-1 and of P2X 7 receptor were analyzed by western blot analysis with mouse monoclonal anti-Caveolin-1 or rabbit polyclonal anti-P2X 7 receptor. c-Tubulin served as a loading control. Representative data from three separate experiments are shown. K. Barth et al. Caveolin-1 and P2X 7 expresssion in lung cells FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS 3027 localization of the P2X 7 receptor in caveolae. Double- label immunofluorescence of Caveolin-1 and P2X 7 receptor in alveolar epithelial E10 cells confirmed the biochemical data. The results are consistent with the findings of Garcia-Marcos et al. [14] and Bannas et al. [15]. They observed, in rat submandibular glands and mouse lymphoma cells, that the P2X 7 receptor mole- cules were distributed among ‘raft’ and ‘nonraft’ domains in the plasma membrane. One conclusion that may be drawn from our data is that the P2X 7 receptor is not an integral component of the rafts, but rather is weakly associated with them, thus supporting the hypothesis that some of the P2X 7 receptor molecules interact weakly with an unknown component of the rafts. In Caveolin-1-knockout mice, both the formation of caveolae and the expression of the P2X 7 receptor were strongly reduced. This indicates that the Caveolin-1 protein itself or the formation of the caveolae may directly affect the expression of the P2X 7 receptor. The putative physiologic role of caveolae and Caveolin-1 could be to centralize, concentrate and colocalize part- ner proteins, signaling proteins and effectors within lipid rafts. For some proteins, Caveolin-1 may aid localization of proteins to the plasma membrane through a physical interaction. Brazer et al. [35] showed that the transient receptor potential channel protein (TRPC1) (amino acids 271–349) contains a Caveolin-1-binding motif between amino acids 322 and 349. Deletion of this binding domain altered localiza- tion of TRPC1 to the plasma membrane. Ion channels are important components of many signal transduction pathways; therefore, this localization may ensure that the channels are located in proximity to the signaling molecules that modulate them. Many signaling mole- cules have been shown to be preferentially associated with rafts [36]. To examine the possibility of a direct interaction between Caveolin-1 and P2X 7 , we attempted pull-down experiments using glutathione S-transferase fusion con- structs of Caveolin-1. The results of these experiments gave no hints of any direct interaction (data not shown). Given that our biochemical data reduce the likelihood of a strong interaction between Caveolin-1 and the P2X 7 receptor, as only a proportion of P2X 7 receptor molecules is associated with Caveolin-1- containing lipid rafts, the lack of a direct interaction between Caveolin-1 and P2X 7 is unsurprising. Cav-1 Cav-1 P2X 7 R P2X 7 R merge merge E10/ control E10/ cav-1 shRNA Fig. 7. Immunofluorescence demonstration of Caveolin-1 and P2X 7 distribution in control and in Caveolin-1 shRNA-transfected E10 cells. Note the loss of colocalization of Caveolin-1 and P2X 7 in Cav-1 shRNA treated cells. Caveolin-1 and P2X 7 expresssion in lung cells K. Barth et al. 3028 FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS The results presented in this study support the evi- dence of active alveolar ion transport in alveolar epithelial cells by adding P2X 7 , a potentially Caveo- lin-1-regulated nonselective cation channel, to the list of alveolar epithelial type I cell-specific channels [37]. The exact role of the P2X 7 receptor in alveolar liquid homeostasis remains open. However, the P2X 7 recep- tor not only gates the opening of cationic channels, but also couples to various downstream signaling events in the cell [10–14]. The P2X 7 receptor is also thought to be able to mediate cell death by apoptosis [38]; prolonged P2X 7 stimulation with extracellular ATP leads to the opening of nonselective transmem- brane pores and the initiation of proapoptotic signals. Interestingly, mechanical ventilation of rats modifies purine rgic r eceptor m RNA l evels and, in paral- lel, Fas and Fas ligand m RNA l evels in the lung [39]. Taken together, these data suggest a complex func- tion of P2X 7 receptors in the lung. The study of P2X 7 receptor expression in alveolar epithelial cells will pro- vide further insights into the characterization of the P2X 7 receptor, as well as enabling investigation of its dual biological role in terms of Caveolin-1-dependent signal transduction and ⁄ or ion channel function in type I cells of the lung. Experimental procedures Cell lines and reagents The mouse E10 lung cell line was kindly provided by M. Williams (Pulmonary Center, Boston University School of Medicine, Boston, MA, USA). The source of A549, L2 and R3 ⁄ 1 cells has been previously described [25,40]. DMEM was purchased from PAA Laboratories GmbH (Co ¨ lbe, Germany). Fetal bovine serum was obtained from HyClone Perblo Science Deutschland GmbH (Bonn, Germany). l-Glutamine and trypsin ⁄ EDTA were purchased from BIOCHROM AG Seromed (Berlin, Germany). Dimethylsulfoxide and loading buffer X were obtained from AppliChem GmbH (Darmstadt, Germany). Eppend- orf MasterMix was purchased from Eppendorf (Wesseling- Berzdorf, Germany). Cell culture E10 cells were cultured in DMEM ⁄ Ham’s F12 medium (1 : 1) supplemented with 10% fetal bovine serum and 2.5 mml-glutamine. They were grown at 37 °Cina5% CO 2 atmosphere. Cells were seeded at a density of 3 · 10 4 cellsÆmL )1 and passaged continuously. The culture conditions for A549, L2 and R3 ⁄ 1 cells have been described previously [25,40]. Triton X-100 solubility Confluent cells of a T75 flask were washed twice with ice- cold NaCl ⁄ P i (pH 7.2). Five hundred microliters of MBS (25 mm Mes, pH 6.5, 150 mm NaCl) containing 1% Tri- ton X-100 plus protease inhibitors was added to the cells. After 30 min of incubation on ice, the soluble fraction was collected. The remaining Triton X-100-insoluble fraction was dissolved by adding 500 lL of 1% SDS to the T75 flask, and passed through a 26-gauge needle 10 times in order to lower its viscosity. Equal volumes of the Triton X-100-soluble and Triton X-100-insoluble fraction were separated by SDS ⁄ PAGE and subjected to western blot analysis as described above. Western blot analysis Protein concentrations were determined using the bicin- choninic assay (Pierce, Bonn, Germany) according to the manufacturer’s guidelines. Samples were loaded onto 12% SDS polyacrylamide gels and separated according to Laemmli [41]. Total protein concentrations of the lysates were deter- mined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, Ireland). Fifty micrograms of total protein of each sample was redissolved in 6 · SDS sample buffer [300 mm Tris ⁄ HCl, pH 6.8; 30% (w ⁄ v) glycerol; 10% (w ⁄ v) SDS; 0.1% bromophenol blue; 100 mm dithiothrei- tol). After being boiled for 5 min at 95 °C, samples were loaded on a 12% SDS polyacrylamide gel. The separated proteins were transferred to a poly(vinylidene difluo- ride) membrane. After blocking of the membrane in NaCl ⁄ P i -T (137 mm NaCl, 2.7 mm KCl, 6.7 mm Na 2 HPO 4 . 2H 2 O, 1.5 mm KH 2 PO 4 , 0.1% Tween-20; pH 7.2–7.5) con- taining 5% nonfat dry milk for 1 h at room temperature or overnight at 4 °C, it was incubated with monoclonal mouse anti-Caveolin-1 (clone 2297, dilution 1 : 1000 v/v; BD Bio- sciences, Pharmingen, San Jose, CA, USA), polyclonal rab- bit anti-P2X 7 (dilution 1 : 375 v/v; Sigma-Aldrich, Inc., St Louis, MO, USA), monoclonal mouse anti-Flo-1 (clone 18, dilution 1 : 1000 v/v; BD Biosciences), monoclonal mouse anti-(human TfR) (clone H68.4, dilution 1 : 500 v/v; Zymed Laboratories Inc., South San Francisco, CA, USA), poly- clonal rabbit anti-PDI (dilution 1 : 750 v/v; StressGen Bio- technologies Corp., Victoria, Canada), polyclonal rabbit anti-b-Cop (dilution 1 : 750 v/v; Ongogene Research Prod- ucts, Boston, MA, USA) and monoclonal mouse anti- c-tubulin (clone GTU-88, dilution 1 : 1000 v/v; Sigma- Aldrich) for 2 h at room temperature or overnight at 4 °C. Next, the membrane was washed three times for 10 min. Incubation with secondary horseradish peroxidase-conju- gated antibodies [ECL anti-(mouse IgG), dilution 1 : 4000 v/v (Amersham Biosciences, Little Chalfont, UK), or goat anti-(rabbit IgG), dilution 1 : 2000 v/v (Bio-Rad K. Barth et al. Caveolin-1 and P2X 7 expresssion in lung cells FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS 3029 Laboratories, Hercules, CA, USA)] for 1 h at room tem- perature was followed by washing again (three times for 10 min). The chemiluminescent signal was generated by using ECL western blot analysis Detection Reagents (Amersham Biosciences, Uppsala, Sweden) and detected by Image Reader LAS-3000 (Fujifilm Medical Systems USA Inc., Stanford, CT, USA). Immunoprecipitation One hundred micrograms of total protein from HEK cells transiently transfected with rat P2X 7 (positive control), or the total protein from 2.5 · 10 6 R3 ⁄ 1 rat lung alveolar epi- thelial cells, was immunoprecipitated overnight at 4 °Cin IP buffer (10 mm Tris ⁄ HCl, pH 7.4, 150 mm NaCl, 1 mm MgCl 2 ,1mm CaCl 2 , protease inhibitors) using 0.3 lgof anti-P2X 7 bound to protein G–sepharose beads (GE Health- care, Little Chalfont, UK). The beads were washed exten- sively in IP buffer and boiled for 5 min in SDS ⁄ PAGE sample buffer. Protein was separated by SDS ⁄ PAGE and immunoblotted for P2X 7 using a 1 : 1000 v/v dilution of the same antibody used in the immunoprecipitation. Preparation of detergent-insoluble membrane fractions Preparation of detergent-insoluble membrane fractions was carried out as described previously [42], with minor modifi- cations. Confluent cells of six T75 flasks were washed twice with ice-cold NaCl ⁄ P i (pH 7.2) and then scraped in 1 mL of ice-cold MBS containing 1% Triton X-100 plus protease inhibitors. After 30 min, the lysate was centrifuged at 2000 g for 10 min (Allegra TM 64R, Beckman F2402H rotor; Beckman Coulter, Fullerton, CA, USA). The supernatant was removed from the pellet and stored on ice. The pellet was resuspended in ice-cold MBS containing protease inhib- itors, and homogenized by sonication (three times with 30 s bursts). The homogenate was mixed with sucrose to a con- centration of 40%, and 2 mL was placed at the bottom of the centrifuge tube. Sucrose (35%, 1.4 mL) and 0.8 mL of 5% sucrose in MBS containing protease inhibitors were layered on the top of the lysate. The gradient was centri- fuged at 200 000 g for 20 h in an MLS 50 rotor (Beckman Coulter). Fractions of 13 300 lL were collected, beginning from the top of the tube. Experiments were performed at 4 °C. For the preparation of Brij35-insoluble membranes, cells were washed with ice-cold NaCl ⁄ P i twice on ice, scraped, and pelleted by centrifugation (5 min, 130 g) with a Beck- man F2402H rotor (Allegra TM 64R centrifuge). The pellet was resuspended in 1 mL of 25 mm Hepes (pH 6.5, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethanesulfonyl fluoride, protease inhibitors) + 1% Brij35, treated with ultrasound (30 s, 80%, 0.7 per second per stroke), and then incubated for 30 min at 4 °C. The protein sample was mixed with an equal amount of 80% sucrose and overlaid with 1.4 mL of 35% sucrose followed by a layer of 0.8 mL of 5% sucrose. The gradient was centrifuged at 200 000 g (Optima TM Max Ultrazentrifuge, Beckman Coulter) for 18 h at 4 °C. Four- teen 300 lL fractions were collected, beginning from the top of the tube. Preparation of low-density membrane fractions under detergent-free conditions Detergent-free preparation of low-density fractions was car- ried out as described previously [43], with minor modifica- tions. Confluent cells from six T75 flasks were washed twice with ice-cold NaCl ⁄ P i (pH 7.2), and then scraped in 700 lL of ice-cold MBS containing 500 mm Na 2 CO 3 (pH 11) plus protease inhibitors. The cell suspension was homogenized with a sonicator (three 30 s bursts). The homogenate was mixed with sucrose to a concentration of 40%, and 2 mL was placed at the bottom of the centrifuge tube. Sucrose (35%, 1.4 mL) and 0.8 mL of 5% sucrose in MBS contain- ing 500 mm Na 2 CO 3 (pH 11) and protease inhibitors were layered on the top of the lysate. The gradient was centri- fuged at 200 000 g for 20 h in an MLS 50 rotor (Beckman Coulter). Five 300 lL fractions, followed by four fractions of 600 lL, were collected, beginning from the top of the tube. Experiments were performed at 4 ° C. Deglycosylation Deglycosylation was carried out using the N-glycosidase F Deglycosylation Kit (Roche Diagnostics, Roche Applied Science, Indianapolis, IN, USA). The concentration of the samples was 25 lg of total protein in a volume of 5 l L. We followed the protocol for complete deglycosylation according to the manufacturer’s instructions. Five micro- liters of reduced denaturation buffer was added to each sample, with ensuing incubation at 95 °C for 3 min. The samples were then mixed with 10 lL of reaction buffer and 10 lLofN-glycosidase F or 10 lL of reaction buffer (con- trol). The incubation times were 30 min, 1 h and overnight at 37 °C. For western blot analysis, the samples were mixed with 6· SDS sample buffer. Cryoimmunogold electron microscopy To determine the localization of P2X 7 , mouse lung E10 cells were fixed in 2% (w ⁄ v) paraformaldehyde and 0.5% (w ⁄ v) glutaraldehyde in 0.1 m phosphate buffer (PB) (pH 7.4) for 2 min at 37 °C and 2 h at room temperature. The fixed samples were mixed with 10% gelatine in 0.1 m PB at 37 °C and incubated overnight in cold 2.3 m sucrose in 0.1 m NaCl⁄ Pi at 4 °C. Finally, the probes were frozen in liquid nitrogen. Ultrathin sections (65–70 nm) were cut on an Ultracut S microtome (Leica Microsysteme GmbH, Vienna, Austria) with a cryoattachment (Leica EM-FCS) at ) 110 °C and Caveolin-1 and P2X 7 expresssion in lung cells K. Barth et al. 3030 FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... then were incubated with 0.1 m NaCl ⁄ Pi (pH 7.4) containing 0.1% glycine for 10 min, and washed in NaCl ⁄ Pi for 10 min at room temperature Before incubation (overnight at 4 °C) with either anti -P2X7 or polyclonal rabbit antibodies to caveolin 1–3 (BD Biosciences) (1 : 100), cells were incubated twice for 15 min with PBG (0.1 m NaCl ⁄ Pi with cold water fish skin gelatine and BSA-C; Aurion, Wageningen,... Young MT, Pelegrin P & Surprenant A (2007) Amino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP Mol Pharmacol 71, 92–100 Denlinger LC, Fisette PL, Sommer JA, Watters JJ, Prabhu U, Dubyak GR, Proctor RA & Bertics PJ (2001) Cutting edge: the nucleotide receptor P2X7 contains multiple protein- and lipid interaction motifs including a potential binding site for bacterial... Wageningen, The Netherlands) Washing in PBG was followed by incubation with goat anti-(rabbit IgG), 12 nm gold (dilution 1 : 30 v/v) (Dianova, Hamburg, Germany) After six additional 5 min washings in PBG, and a further six 5 min washings in NaCl ⁄ Pi, incubation with 2.5% (w ⁄ v) glutaraldehyde in NaCl ⁄ Pi, and further brief washes in destilled water, sections were stained by incubation with 2% methylcellulose... 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Caveolin-1 in uences P2X 7 receptor expression and localization in mouse lung alveolar epithelial cells K. Barth 1, *, K. Weinhold 1, *, A of Caveolin-1 and P2X 7 distribution in control and in Caveolin-1 shRNA-transfected E10 cells. Note the loss of colocalization of Caveolin-1 and P2X 7 in