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A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation.

Prakasam HS, Gallo LI, Li H, Ruiz WG, Hallows KR, Apodaca G - Mol. Biol. Cell (2014)

Bottom Line: Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved.Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17(S811A) mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage.Furthermore, expression of ADAM17(S811A) in bladder tissues impairs A1AR-induced apical exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.

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A1AR-stimulated exocytosis is dependent on ADAM17. (A) Uroepithelial lysates obtained from rat tissues transduced in situ with scrambled shRNA or ADAM17 shRNA were resolved by SDS–PAGE and Western blots probed with rabbit-anti ADAM17 antibody (top) or mouse anti–β-actin (bottom). (B) Quantification of ADAM17 expression in rat tissue treated with scrambled shRNA or ADAM17 shRNA. Data are mean ± SEM (n ≥ 4). The means of the two treatment groups are significantly different (p < 0.05). (C) Immunolocalization of ADAM17 expression (red) in rat tissue treated with scrambled or ADAM17-specific shRNAs. Nuclei (blue) are labeled with TOPRO-3. Scale bar, 13 μm. The cell junctions are marked with arrows and the umbrella cells with an asterisk. (D) CCPA (500 nM) was added to rat tissue treated with scrambled or ADAM17 shRNA and total EGFR or receptor phosphorylated at Y1173 was detected by Western blot. (E) Quantification of effects of scrambled or ADAM17-specific shRNA treatment on EGFR activation. Data are mean ± SEM (n = 4). The two treatment groups were significantly different (p < 0.05). (F) WGA-FITC (50 μg/ml) was added to the mucosal hemichamber of untreated rat tissue (control) or that treated with 500 nM CCPA. After 120 min, the tissue was incubated at 4°C with N-acetylglucosamine to remove surface lectin, fixed, and then processed for immunofluorescence. In these whole-mounted tissues, internalized WGA-FITC is shown in green, phalloidin-labeled actin in red, and TOPRO-3-labeled nuclei in blue. Bar, 15 μm. Quantification of the fluorescence intensity of WGA-FITC below the apical membrane. Data are mean ± SEM (n ≥ 8). The two treatment groups were significantly different (p < 0.05). (G) Rat tissues were transduced with hGH alone (control) or transduced with hGH and either scrambled or ADAM17 shRNAs. The excised bladder tissue was mounted in an Ussing stretch chamber and left untreated (control), treated with CCPA (500 nM), or stretched by filling the mucosal hemichamber. The mucosal fluid was collected and concentrated, the tissues were lysed, and hGH was detected in the secreted fraction (1/20 of total) or tissue lysates (1/13 of total) using Western blot. (H) Quantification of effects of ADAM17-specific shRNAs on hGH secretion. Data are mean ± SEM (n ≥ 3). Statistically significant effects (p < 0.05) are indicated with an asterisk.
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Figure 4: A1AR-stimulated exocytosis is dependent on ADAM17. (A) Uroepithelial lysates obtained from rat tissues transduced in situ with scrambled shRNA or ADAM17 shRNA were resolved by SDS–PAGE and Western blots probed with rabbit-anti ADAM17 antibody (top) or mouse anti–β-actin (bottom). (B) Quantification of ADAM17 expression in rat tissue treated with scrambled shRNA or ADAM17 shRNA. Data are mean ± SEM (n ≥ 4). The means of the two treatment groups are significantly different (p < 0.05). (C) Immunolocalization of ADAM17 expression (red) in rat tissue treated with scrambled or ADAM17-specific shRNAs. Nuclei (blue) are labeled with TOPRO-3. Scale bar, 13 μm. The cell junctions are marked with arrows and the umbrella cells with an asterisk. (D) CCPA (500 nM) was added to rat tissue treated with scrambled or ADAM17 shRNA and total EGFR or receptor phosphorylated at Y1173 was detected by Western blot. (E) Quantification of effects of scrambled or ADAM17-specific shRNA treatment on EGFR activation. Data are mean ± SEM (n = 4). The two treatment groups were significantly different (p < 0.05). (F) WGA-FITC (50 μg/ml) was added to the mucosal hemichamber of untreated rat tissue (control) or that treated with 500 nM CCPA. After 120 min, the tissue was incubated at 4°C with N-acetylglucosamine to remove surface lectin, fixed, and then processed for immunofluorescence. In these whole-mounted tissues, internalized WGA-FITC is shown in green, phalloidin-labeled actin in red, and TOPRO-3-labeled nuclei in blue. Bar, 15 μm. Quantification of the fluorescence intensity of WGA-FITC below the apical membrane. Data are mean ± SEM (n ≥ 8). The two treatment groups were significantly different (p < 0.05). (G) Rat tissues were transduced with hGH alone (control) or transduced with hGH and either scrambled or ADAM17 shRNAs. The excised bladder tissue was mounted in an Ussing stretch chamber and left untreated (control), treated with CCPA (500 nM), or stretched by filling the mucosal hemichamber. The mucosal fluid was collected and concentrated, the tissues were lysed, and hGH was detected in the secreted fraction (1/20 of total) or tissue lysates (1/13 of total) using Western blot. (H) Quantification of effects of ADAM17-specific shRNAs on hGH secretion. Data are mean ± SEM (n ≥ 3). Statistically significant effects (p < 0.05) are indicated with an asterisk.

Mentions: To provide further evidence that ADAM17 is required for EGFR transactivation, we exploited our previously described in situ viral transduction approach (Khandelwal et al., 2008, 2010), in this case to express ADAM17-specific short hairpin RNAs (shRNAs) or scrambled shRNAs in the rat bladder uroepithelium. We used rat bladders in these studies because the volume capacity of the bladder, and therefore the number of virus particles needed, was relatively small (∼500 μl) compared with the volumes required to fill the rabbit bladder (∼60–100 ml). This technique targets the umbrella cell layer and achieves transduction efficiencies of 70–95% (Khandelwal et al., 2008, 2010). Indeed, we were able to achieve >90% knockdown of ADAM17 expression (Figure 4, A and B). On examining the expression and distribution of ADAM17 in cross sections of uroepithelium, we observed that knockdown of ADAM17 was largely confined to the umbrella cells, and expression of ADAM17 in the underlying cell layers was largely undisturbed (Figure 4C).


A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation.

Prakasam HS, Gallo LI, Li H, Ruiz WG, Hallows KR, Apodaca G - Mol. Biol. Cell (2014)

A1AR-stimulated exocytosis is dependent on ADAM17. (A) Uroepithelial lysates obtained from rat tissues transduced in situ with scrambled shRNA or ADAM17 shRNA were resolved by SDS–PAGE and Western blots probed with rabbit-anti ADAM17 antibody (top) or mouse anti–β-actin (bottom). (B) Quantification of ADAM17 expression in rat tissue treated with scrambled shRNA or ADAM17 shRNA. Data are mean ± SEM (n ≥ 4). The means of the two treatment groups are significantly different (p < 0.05). (C) Immunolocalization of ADAM17 expression (red) in rat tissue treated with scrambled or ADAM17-specific shRNAs. Nuclei (blue) are labeled with TOPRO-3. Scale bar, 13 μm. The cell junctions are marked with arrows and the umbrella cells with an asterisk. (D) CCPA (500 nM) was added to rat tissue treated with scrambled or ADAM17 shRNA and total EGFR or receptor phosphorylated at Y1173 was detected by Western blot. (E) Quantification of effects of scrambled or ADAM17-specific shRNA treatment on EGFR activation. Data are mean ± SEM (n = 4). The two treatment groups were significantly different (p < 0.05). (F) WGA-FITC (50 μg/ml) was added to the mucosal hemichamber of untreated rat tissue (control) or that treated with 500 nM CCPA. After 120 min, the tissue was incubated at 4°C with N-acetylglucosamine to remove surface lectin, fixed, and then processed for immunofluorescence. In these whole-mounted tissues, internalized WGA-FITC is shown in green, phalloidin-labeled actin in red, and TOPRO-3-labeled nuclei in blue. Bar, 15 μm. Quantification of the fluorescence intensity of WGA-FITC below the apical membrane. Data are mean ± SEM (n ≥ 8). The two treatment groups were significantly different (p < 0.05). (G) Rat tissues were transduced with hGH alone (control) or transduced with hGH and either scrambled or ADAM17 shRNAs. The excised bladder tissue was mounted in an Ussing stretch chamber and left untreated (control), treated with CCPA (500 nM), or stretched by filling the mucosal hemichamber. The mucosal fluid was collected and concentrated, the tissues were lysed, and hGH was detected in the secreted fraction (1/20 of total) or tissue lysates (1/13 of total) using Western blot. (H) Quantification of effects of ADAM17-specific shRNAs on hGH secretion. Data are mean ± SEM (n ≥ 3). Statistically significant effects (p < 0.05) are indicated with an asterisk.
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Figure 4: A1AR-stimulated exocytosis is dependent on ADAM17. (A) Uroepithelial lysates obtained from rat tissues transduced in situ with scrambled shRNA or ADAM17 shRNA were resolved by SDS–PAGE and Western blots probed with rabbit-anti ADAM17 antibody (top) or mouse anti–β-actin (bottom). (B) Quantification of ADAM17 expression in rat tissue treated with scrambled shRNA or ADAM17 shRNA. Data are mean ± SEM (n ≥ 4). The means of the two treatment groups are significantly different (p < 0.05). (C) Immunolocalization of ADAM17 expression (red) in rat tissue treated with scrambled or ADAM17-specific shRNAs. Nuclei (blue) are labeled with TOPRO-3. Scale bar, 13 μm. The cell junctions are marked with arrows and the umbrella cells with an asterisk. (D) CCPA (500 nM) was added to rat tissue treated with scrambled or ADAM17 shRNA and total EGFR or receptor phosphorylated at Y1173 was detected by Western blot. (E) Quantification of effects of scrambled or ADAM17-specific shRNA treatment on EGFR activation. Data are mean ± SEM (n = 4). The two treatment groups were significantly different (p < 0.05). (F) WGA-FITC (50 μg/ml) was added to the mucosal hemichamber of untreated rat tissue (control) or that treated with 500 nM CCPA. After 120 min, the tissue was incubated at 4°C with N-acetylglucosamine to remove surface lectin, fixed, and then processed for immunofluorescence. In these whole-mounted tissues, internalized WGA-FITC is shown in green, phalloidin-labeled actin in red, and TOPRO-3-labeled nuclei in blue. Bar, 15 μm. Quantification of the fluorescence intensity of WGA-FITC below the apical membrane. Data are mean ± SEM (n ≥ 8). The two treatment groups were significantly different (p < 0.05). (G) Rat tissues were transduced with hGH alone (control) or transduced with hGH and either scrambled or ADAM17 shRNAs. The excised bladder tissue was mounted in an Ussing stretch chamber and left untreated (control), treated with CCPA (500 nM), or stretched by filling the mucosal hemichamber. The mucosal fluid was collected and concentrated, the tissues were lysed, and hGH was detected in the secreted fraction (1/20 of total) or tissue lysates (1/13 of total) using Western blot. (H) Quantification of effects of ADAM17-specific shRNAs on hGH secretion. Data are mean ± SEM (n ≥ 3). Statistically significant effects (p < 0.05) are indicated with an asterisk.
Mentions: To provide further evidence that ADAM17 is required for EGFR transactivation, we exploited our previously described in situ viral transduction approach (Khandelwal et al., 2008, 2010), in this case to express ADAM17-specific short hairpin RNAs (shRNAs) or scrambled shRNAs in the rat bladder uroepithelium. We used rat bladders in these studies because the volume capacity of the bladder, and therefore the number of virus particles needed, was relatively small (∼500 μl) compared with the volumes required to fill the rabbit bladder (∼60–100 ml). This technique targets the umbrella cell layer and achieves transduction efficiencies of 70–95% (Khandelwal et al., 2008, 2010). Indeed, we were able to achieve >90% knockdown of ADAM17 expression (Figure 4, A and B). On examining the expression and distribution of ADAM17 in cross sections of uroepithelium, we observed that knockdown of ADAM17 was largely confined to the umbrella cells, and expression of ADAM17 in the underlying cell layers was largely undisturbed (Figure 4C).

Bottom Line: Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved.Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17(S811A) mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage.Furthermore, expression of ADAM17(S811A) in bladder tissues impairs A1AR-induced apical exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.

Show MeSH
Related in: MedlinePlus