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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.044123

Journal of Cell Science 122, 1238-1247 (2009)
Published by The Company of Biologists 2009
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Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells

Sylvia Luxen1, Deborah Noack1, Monika Frausto1, Suzel Davanture1, Bruce E. Torbett2 and Ulla G. Knaus1,*

1 Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA
2 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA


Figure 1
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  		Fig. 1. ER exit of Duox1-DuoxA1 (343aa) and Duox2-DuoxA2. Analysis of NCI-H661 cells transiently expressing Duox1-DuoxA1 (343aa) or Duox2-DuoxA2. (A) Duox-dependent H2O2 production was determined with and without addition of ionomycin or thapsigargin. n=3 each; *P<0.05; **P<0.01; ***P<0.001 versus unstimulated. Graphs show mean ± s.e.m. Immunoblots were probed for Duox ({alpha}-Duox1,2), DuoxA ({alpha}-Myc) and {alpha}-tubulin. (B) Colocalization of Duox1-DuoxA1-Myc and Duox2-DuoxA2-Myc. Inserts depict particular locations of Duox1-DuoxA1 and Duox2-DuoxA2 at higher magnification. Colocalization is shown in white in the merged images in the right column. Calnexin served as ER marker. Scale bar, 10 µm.

 

Figure 2
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  		Fig. 2. Heme-dependent heterodimer formation of Duox-DuoxA. (A) Coimmunoprecipitation of DuoxA ({alpha}-Myc IP) with the respective Duox isoforms in transiently transfected H661 cells. Control IP (ctrl) was done with {alpha}-GST. Immunoblotting (IB) of IPs and total cell lysates (TCL) was performed with {alpha}-Duox1,2 or {alpha}-Myc antibodies. (B,C) H661 cells stably expressing Duox1-DuoxA1 (343aa) or Duox2-DuoxA2 were cultured for 4 days in the presence or absence of 10 µg/ml SA prior to (B) {alpha}-DuoxA (Myc IP) or {alpha}-GST (ctrl IP) immunoprecipitation, followed by immunoblotting (IB) as indicated, or (C) H2O2 production determined with or without addition of ionomycin. n=3 each; ***P<0.001 versus untreated. Graphs show mean ± s.e.m.

 

Figure 3
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  		Fig. 3. Differential expression and localization of DuoxA1 splice variants. (A) Schematic representation of DuoxA1-(1-3). Variants share the putative five transmembrane (TM) structure. DuoxA1-1 (298aa) lacks two of three putative N-glycosylation sites. DuoxA1-3 contains an extended C terminus. Green, extracellular regions; yellow, TM domains; blue, intracellular regions; *, N-glycosylation sites. (B) RT-PCR using undifferentiated (2D) und ALI-differentiated SAEC and NHBE cells (14 days; 3D). (C-E) Analysis of NCI-H661 cells transiently transfected with Duox1 and EGFP-fused DuoxA1-1, DuoxA1-2 and DuoxA1-3. (C) H2O2 production was measured after stimulation with ionomycin or thapsigargin. **P<0.01; ***P<0.001 versus unstimulated. Data are presented as mean values ± s.e.m.; n=3 each. Immunoblot detection of Duox1 ({alpha}-Duox1,2), DuoxA1 ({alpha}-GFP) and {alpha}-tubulin in TCL. (D) Localization of Duox1 ({alpha}-Duox1,2) and DuoxA1 (GFP) variants by immunofluorescence. Colocalization is shown in white. Scale bar, 10 µm. (E) Immunoblotting (IB) for Duox1 ({alpha}-Duox1,2) and DuoxA1 ({alpha}-GFP) was performed after immunoprecipitation (IP {alpha}-GFP). Control IP (ctrl) was done with {alpha}-GST. TCL was probed for Duox1 and DuoxA1 variant expression as indicated.

 

Figure 4
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  		Fig. 4. DuoxA1-2 can substitute for DuoxA2 in functional Duox2-DuoxA heterodimerization. Transient expression of Duox1, Duox2, DuoxA1-2 and DuoxA2 in NCI-H661 cells. (A) H2O2 production was measured in response to stimulation with ionomycin. **P<0.01; ***P<0.001 versus unstimulated. Shown are mean values ± s.e.m.; n=3 each. Expression of Duox1, Duox2, DuoxA1-2 ({alpha}-Myc), DuoxA2 ({alpha}-Myc) and {alpha}-tubulin was determined by immunoblotting. (B) Localization of Duox1-DuoxA1-2-Myc, Duox1-DuoxA2-Myc, Duox2-DuoxA1-2-Myc and Duox2-DuoxA2-Myc was determined by immunofluorescence in NCI-H661 cells. Colocalization of Duox ({alpha}-Duox) and DuoxA ({alpha}-Myc) is shown in white. Scale bar, 10 µm. (C) Detection of Duox2 or Duox1 after {alpha}-Myc IP of DuoxA1-2 and DuoxA2, respectively. {alpha}-GST IP (ctrl) served as control. IPs and TCL of NCI-H661 cells were probed for Duox ({alpha}-Duox) and DuoxA ({alpha}-Myc) expression.

 

Figure 5
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  		Fig. 5. Expression analysis of endogenous Duox in differentiated lung epithelial cells. (A) Upper panel: validation of {alpha}-Duox1, {alpha}-Duox2 and {alpha}-DuoxA1 antibodies. TCL of cells expressing Duox1-DuoxA1-2-Myc or Duox2-DuoxA2-Myc were probed with isoform-specific antibodies as indicated. {alpha}-tubulin served as loading control. {alpha}-Duox1,2 and {alpha}-Myc blotting served as expression control. Lower panel: cells expressing Duox1-DuoxA1-2 or Duox2-DuoxA2 were stained with DAPI and preimmune sera derived from rabbits used for Duox1, Duox2 or DuoxA1 antibody generation as indicated. Images in both channels are depicted in white. Scale bar, 10 µm. (B) Real-time PCR of DUOX1 and DUOX2 in SAEC, either undifferentiated (2D; 0 days) or differentiated in ALI-culture for 7, 14 and 21 days (3D). Columns show the means ± s.e.m. and represent three different samples, which were individually normalized to actin. **P<0.01; ***P<0.001 versus transcript levels in 2D samples; n=3. (C) Upregulation of Duox1 protein during differentiation. Immunoblot analysis of endogenous Duox1 in undifferentiated (2D) and differentiated [14 and 23 days (d); 3D] SAEC. Lysates of NCI-H661 cells transfected with Duox1-DuoxA1-2 were used as control (+).

 

Figure 6
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  		Fig. 6. Detection of endogenous Duox1 and DuoxA1 protein in primary human lung epithelial cells (2D). Lentivirally transduced SAEC expressing the indicated combinations were used for localization studies. SAEC transduced with vector (EV) or not transduced (N.T.) served as control. Comparison of {alpha}-Duox1,2 antibody staining with {alpha}-Duox1 or with {alpha}-Duox2 antibodies (all green); and of {alpha}-Myc (red) staining with {alpha}-DuoxA1 (green) antibody. Endogenous coexpression of DuoxA1 and Duox1 was visualized by co-staining of DuoxA1 (green) with Cy3-labeled Duox1 antibody (red). Colocalization in merged pictures is shown in white. Separate staining of endogenous Duox1 and DuoxA1 in right panels 4 and 5 is depicted in white with nuclei in blue (DAPI). Scale bars, 10 µm.

 

Figure 7
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  		Fig. 7. Reintroduction of functional Duox1 or Duox2 augments cellular migration. NCI-H661 cells stably expressing empty vector (EV), DuoxA1-2, Duox1-DuoxA1-2, DuoxA2 or Duox2-DuoxA2. (A) Cells were seeded on top of Transwell inserts and migration was initiated by addition of FCS to the bottom of the chamber. Cells underneath the filter were counted after 5.5 hours. *P<0.05; **P<0.01; ***P<0.001 versus EV. Data are shown as mean values ± s.e.m.; n=3 each. (B) Confluent monolayers of cells expressing EV, Duox1-DuoxA1-2 or Duox2-DuoxA2 were wounded and wound closure was determined after 4.5 hours. Data were collected from 12 random fields and are expressed as the percentage of wounds closed. **P<0.01 compared to EV. (C) Cells expressing Duox1-DuoxA1-2 or Duox2-DuoxA2 were grown to confluency and localization of Duox-DuoxA was determined 1 hour after wounding of the monolayer by immunostaining for Duox (Duox1,2; green) and DuoxA (Myc; red). Arrows indicate plasma membrane localization of Duox1 and Duox2, respectively. The images on the far right show single migrating cells. Colocalization is indicated in white. Scale bar, 30 µm.

 

Figure 8
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  		Fig. 8. Distinct localization of endogenous Duox1, DuoxA1 and Duox2 in differentiated airway epithelial cells (3D). ALI-differentiated SAEC (14 days) were stained for mucin (MUC5AC; red, R), cilia (β-tubulin IV; red, R) and Duox1, Duox2 or DuoxA1 (all green, G) using isoform-specific antibodies. Nuclear staining was with DAPI (blue, B). Per insert, several Z-sections at 0.3 µm intervals were taken throughout the entire depth of multiple cell layers. Orthogonal views correspond to the crosslines in the rectangular overview and show two color overlays of green and blue (GB), red and blue (RB) and three color merge (red, green, blue; RGB), with colocalization indicated in yellow. Scale bar, 10 µm.

 

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© The Company of Biologists Ltd 2009