Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells

Abstract

Rationale: NADPH oxidases (Noxes) regulate vascular physiology and contribute to the pathogenesis of vascular disease. In vascular smooth muscle cells (VSMCs), the interactions of individual Nox homologs with regulatory proteins are poorly defined.

Objective: The objective of this study was to identify novel NADPH oxidase regulatory proteins.

Methods and results: Using a yeast 2-hybrid screen, we identified a novel p22phox binding partner, Poldip2, and demonstrated that it associates with p22phox, NADPH oxidase (Nox)1, and Nox4 and colocalizes with p22phox at sites of Nox4 localization. Poldip2 increases Nox4 enzymatic activity by 3-fold and positively regulates basal reactive oxygen species production in VSMCs (O2(.-): 86.3+/-15.6% increase; H2O2: 40.7+/-4.5% increase). Overexpression of Poldip2 activates Rho (180.2+/-24.8% increase), strengthens focal adhesions, and increases stress fiber formation. These phenotypic changes are blocked by dominant negative Rho. In contrast, depletion of either Poldip2 or Nox4 results in a loss of these structures, which is rescued by adding back active Rho. Cell migration, which requires dynamic cytoskeletal remodeling, is impaired by either excess (70.1+/-14.7% decrease) or insufficient Poldip2 (63.5+/-5.9% decrease).

Conclusions: These results suggest that Poldip2 associates with p22phox to activate Nox4, leading to regulation of focal adhesion turnover and VSMC migration, thus linking reactive oxygen species production and cytoskeletal remodeling. Poldip2 may be a novel therapeutic target for vascular pathologies with a significant VSMC migratory component, such as restenosis and atherosclerosis.

Figure 1. Poldip2 associates with Nox1 and Nox4 in a p22phox-dependent manner

a. VSMCs were transfected with vector control (−) or Poldip2 (+), labeled with S-Methionine, and used in a GST-pulldown assay. Binding partners were detected by autoradiography. Positive control: in vitro translated Poldip2 (lane 1). b. HEK 293 cells were co-transfected with V5-tagged p22phox (V5-p22phox) and either empty vector (−) or vector expressing Myc-tagged Poldip2 (+; Myc-Poldip2). Cells were immunoprecipitated (IP) with V5 antibody and immunoblotted (IB) with a Myc (upper) or V5 (lower) antibody. c. Human VSMC lysates were immunoprecipitated with rabbit IgGs (IgG) or p22phox antibody and immunoblotted with a Poldip2 (upper) or p22phox (lower) antibody. Lysis buffer (LB) is a negative control in all IP experiments. Poldip2 is detected as a 37 kDa band; IgGs interfere with resolution of the 42 kDa band. d. VSMCs were transduced with no adenovirus (No Ad), 125 µL/dish of control adenovirus (AdGFP) or Myc-tagged Poldip2 adenovirus (AdPoldip2). Lysates were immunoprecipitated with a Myc antibody and immunoblotted with a Nox4 (upper) or Myc (lower) antibody. e. VSMCs transduced with AdGFP or HA-tagged Nox1 (Nox1HA) adenovirus were immunoprecipitated with goat IgGs or a Nox1 antibody and immunoblotted with a Poldip2 (upper) or HA (lower) antibody. f. VSMCs stably transfected with empty vector (Vector) or antisense p22phox (p22AS) vector were immunoprecipitated with rabbit IgGs or a Nox4 antibody and immunoblotted with a Poldip2 (upper) or Nox4 (lower) antibody.

Figure 2. Poldip2 stimulates ROS production via Nox4

a. VSMCs were transduced with no adenovirus (No Ad), 37.5 or 225 µL/dish of AdGFP or 37.5 µL/dish to 225 µL/dish of AdPoldip2. Lucigenin enhanced chemiluminescence was used to measure NADPH oxidase activity in membrane fractions. Bars are mean ± S.E.M. of 3–4 independent experiments, †p<0.05 vs. AdGFP. Positive control: VSMCs stimulated with 100 nmol/L Ang II for 4 h. *p<0.05 vs. No Ad - Ang. b. VSMCs were transduced with AdGFP or AdNox4AS for 48 h prior to transduction with AdGFP (−) or AdPoldip2 (+) for 72 h. Lucigenin enhanced chemiluminescence was used to measure NADPH oxidase activity. Bars are mean ± S.E.M. of four independent experiments, *p<0.001 vs. No Ad- AdPoldip2; †p<0.01 vs. AdGFP-AdPoldip2; ‡p<0.001 vs. AdGFP+AdPoldip2. c. VSMCs were transduced with the same adenoviral combinations as panel b prior to using DHE-HPLC to measure O₂^•− production (bars are mean ± S.E.M. of 4 independent experiments, *p<0.001 vs. No Ad-AdPoldip2; †p<0.001 vs. AdGFP-AdPoldip2; ‡p<0.001 vs. AdGFP+AdPoldip2). d. VSMCs transduced with the same adenoviral combinations as in panel b were used for the Amplex red assay to measure H₂O₂ production (bars are mean ± S.E.M. of 4 independent experiments, *p<0.05 vs. No Ad- AdPoldip2; †p<0.01 vs. AdGFP-AdPoldip2; ‡ p<0.001 vs. AdGFP+ AdPoldip2). e. VSMCs were transduced with AdGFP or adenovirus expressing AdNox1AS for 48 h before transducing cells with AdGFP (−) or AdPoldip2 (+) for 72 h. The Amplex Red Assay was used to measure H₂O₂. Bars are mean ± S.E.M. of 4 independent experiments, *p<0.05 vs. No Ad-AdPoldip2; †p<0.05 vs. AdGFP-AdPoldip2; ‡p<0.001 vs. AdGFP+AdPoldip2).

Figure 3. Poldip2 co-localizes with Nox4 and p22phox

a, upper. Confocal images of VSMCs double labeled with anti-Poldip2 (red) and anti-p22phox (green) antibodies. Nuclei are labeled with DAPI (blue). Images acquired at the focal adhesion (upper panel) and stress fiber (lower panel) planes are depicted. Arrows indicate areas of co-localization (yellow) in the merge. a, lower. VSMCs were transduced with AdPoldip2 and double labeled with anti-Myc (pseudo-colored blue) and anti-p22phox (red) antibodies. Arrows indicate areas of co-localization (purple) in the merge. b, upper. VSMCs were transduced with AdPoldip2 and double labeled with anti-Myc (pseudocolored blue) and anti-Nox4 (red) antibodies. Arrows indicate areas of co-localization (purple) in the merge at the focal adhesion (upper panel) and stress fiber (lower panel) planes. b, lower. VSMCs were double labeled with anti-Poldip2 (red) and anti-Nox4 (green) antibodies. Arrows indicate areas of co-localization (yellow) in the merge. Scale bars, 10 µm.

Figure 4. Tissue distribution of Poldip2

a. Quantitative real-time PCR of poldip2 expression in tissues from Sprague Dawley rats; mRNA is normalized to 18S. Bars are mean ± S.E.M. of triplicates from a representative experiment, repeated twice. b. Western blot of Poldip2 protein expression in rat tissue lysates. The 42 kDa Poldip2, containing the predicted signal peptide, and the 37 kDa Poldip2, after peptide cleavage, are shown. c. Immunohistochemical distribution of Poldip2 and Nox4 in rat aorta (L-lumen, M-media, A-adventitia), kidney (G-glomerulus, T-tubule), and lung (B-bronchiole) tissues using an anti-Poldip2 antibody. Poldip2 signal appears brown. Nuclei are counterstained with hematoxylin (blue/purple).

Figure 5. siPoldip2 alters VSMC phenotype and decreases basal ROS production by Nox4

a. Quantitative real-time PCR of poldip2 expression in VSMCs transfected with control siRNA (siCont) or siRNA against Poldip2 (siPoldip2) for the indicated times. Bars are mean ± S.E.M. of three independent experiments, *p<0.001 vs. siCont. b. Western blot analysis of Poldip2 expression in protein extracts from VSMCs transfected for 4 days. CDK4 (lower blot) serves as a loading control. c. DHE-HPLC was used to assess intracellular O₂^•− production in VSMCs were treated as described in b. Bars are mean ± S.E.M. of four independent experiments, *p<0.01 vs. siCont. d. Amplex Red Assay was used to assess H₂O₂ production. Bars are means ± S.E.M. of 4 independent experiments, *p<0.001 vs. siCont. e. VSMCs were transiently transfected for 4 (upper) or 5 (lower) days and visualized by phase contrast microscopy; scale bars are 200 µm.

Figure 6. Poldip2 regulates proper Nox4 and p22phox localization, focal adhesion integrity and stress fiber formation

a. Confocal images of VSMCs either untreated or transiently transfected with 15 nmol/L of either siCont or siPoldip2. Nox4. single labeled with anti-Nox4 (red) antibody, p22phox. double labeled with anti-p22phox (green) and phalloidin (red) to stain stress fibers, Vinculin. double labeled with anti-vinculin (green) and phalloidin (red), Paxillin. double labeling with anti-paxillin (green) and phalloidin (red). Yellow appears where stress fibers (red) insert into focal adhesions (green). Nuclei are labeled with DAPI (blue). Images in a acquired at the focal adhesion plane. b. Confocal images of VSMCs either untreated or transiently transfected with 25 nmol/L of Allstars negative control siRNA (siCont) or siNox4. Vinculin. double labeled with anti-vinculin (green) and phalloidin (red). Paxillin. double labeled with anti-paxillin (green) and phalloidin (red). images in b acquired at the focal adhesion plane. c. Confocal images of VSMCs transduced with AdGFP or AdPoldip2 (detected as green in GFP panels) before labeling with phalloidin (purple) or with vinculin (red). Scale bars in a, b, and c are 10 µm. d. VMSCs that were non-transduced (No Ad) or transduced for 72 h with AdPoldip2 or AdGFP and that were either untreated or treated with 20 mmol/L N-acetyl cysteine (NAC) for 24 h prior to a Rho activity assay to measure active, GTP-bound RhoA. Western blots of GTP-bound RhoA (upper) and total RhoA (lower) are from a representative experiment. The bar graph quantifies three independent Rho activity assays. Bars are mean ± S.E.M., *p<0.01 vs. AdGFP-NAC; †p<0.05 vs. AdPoldip2-NAC.

Figure 7. Nox4 and Poldip2 regulate focal adhesion and stress fiber maintenance through RhoA activation

a. Confocal images VSMCs either untreated or transiently transfected with 15 nmol/L of either siCont or siPoldip2 prior to transduction with either control adenovirus (AdLacZ) or an HA-tagged constitutively active Rho (AdRhoGV). Cells were triple labeled with anti-HA antibody (HA; red) to detect cells transduced with AdRhoGV, Phalloidin (green), to detect stress fibers, and anti-vinculin (pseudo-colored cyan) to detect focal adhesions. Images acquired at the stress fiber (upper) and focal adhesion (lower) planes. b. VSMCs non-transduced (No Ad) or transduced for 24 h with AdGFP or AdPoldip2 prior to transduction with either AdLacZ or an HA-tagged dominant negative Rho (AdRhoTN). Cells transduced with AdGFP or AdPoldip2 appear green from GFP. Cells were double labeled with anti-HA antibody (HA; red) to detect cells transduced with AdRhoTN, and Phalloidin (pseudo-colored cyan) to detect stress fibers. Nuclei are labeled with DAPI (blue). Images acquired at the stress fiber plane. Scale bars in a and b are 10 µm.

Figure 8. Poldip2 regulates VSMC migration

The Boyden Chamber Assay was used to measure cell migration in response to 10 nmol/L platelet-derived growth factor (PDGF) for 4 h. a. VSMCs were transduced with no adenovirus (No Ad), AdGFP or AdPoldip2 prior to the Boyden Chamber Assay, *p<0.01 vs. AdGFP. b. VSMCs were transfected with no siRNA (Untreated), siCont or siPoldip2 prior to the Boyden Chamber Assay, *p<0.01 vs. siCont. c. VSMCs were transduced with no adenovirus (No Ad), AdGFP or AdNox4AS prior to the Boyden Chamber Assay, *p<0.01 vs. AdGFP.