Poldip2, a Novel Regulator of Nox4 and Cytoskeletal Integrity in Vascular Smooth Muscle Cells
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.
Reactive oxygen species (ROS), such as superoxide (O2·−) and hydrogen peroxide (H2O2), are implicated in the development of multiple cardiovascular disease pathologies, including hypertension, atherosclerosis, and restenosis.1 Physiologically, ROS mediate many cellular functions such as proliferation, gene expression, migration, differentiation, and cytoskeletal remodeling.2 One major source of ROS is the NADPH oxidase (Nox) family of enzymes.
The catalytic moieties of NADPH oxidases are homologs of the flavin- and NADPH-binding protein gp91phox (Nox2), termed Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2. Most cell types express multiple Nox enzymes that are differentially regulated and have distinct subcellular localizations, suggesting that these oxidases serve unique roles. For example, Nox1 and Nox4 are the predominant homologs in rodent vascular smooth muscle cells (VSMCs) from large vessels. Whereas Nox1 is primarily found in caveolae, Nox4 is found in the nucleus, in focal adhesions and along stress fibers.3 Nox1 mediates VSMC growth and migration, whereas Nox4 is involved in differentiation.4
Nox enzymes also differ in their mode of regulation. The Nox2-based oxidase consists of 5 subunits. Together, the membrane proteins Nox2 and p22phox comprise the cytochrome b558 membrane complex, which is localized in submembranous vesicles and the plasma membrane. Catalytic activity is initiated by translocation to the membrane of cytosolic subunits p47phox, p67phox, and the small-molecular-weight G protein Rac.4 Nox1 and Nox3 are similarly regulated by the p47phox and p67phox homologs Nox organizer 1 (NoxO1) and Nox activator 1 (NoxA1), respectively, as well as with Rac.4 However, none of the presently known cytosolic regulatory subunits is required for Nox4 activation.5
The mechanism by which Nox4 activity is regulated remains unclear. Some studies suggest that the principal mechanism of Nox4 regulation may be induction at the mRNA level, rather than assembly of an enzyme complex or posttranslational protein modifications.6 However, although it is known that Nox4 requires p22phox, there has been no systematic search for proteins that bind to the Nox4/p22phox complex. In this study, we show that Poldip2 (polymerase [DNA-directed] delta-interacting protein 2)7,8 is a novel Nox4/p22phox-interacting protein and is a potent positive regulator of Nox4 activity in VSMCs. The Nox4/p22phox/Poldip2 complex has profound effects on Rho-dependent cytoskeletal reorganization and cellular migration. These results thus provide mechanistic insight into the unique regulation of Nox4 and the fundamental physiological and pathophysiological cellular functions that Poldip2/Nox4 modulates.
An expanded Materials and Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Rat aortic VSMCs and human aortic smooth muscle cells (passages 6 to 12) were grown in DMEM. HEK293 cells were cultured in DMEM with 10% FBS. Rat VSMCs were used for all VSMC experiments, except where indicated.
Poldip2 goat antibody was custom made by GenScript Corporation (Piscataway, NJ) against the peptide sequence NPAGHGSKEVKGKTC.
Yeast Two-Hybrid Assay
We used the Matchmaker LexA yeast 2-hybrid system (Clontech) and a VSMC cDNA library constructed in pB42AD. The cytosolic tail of rat p22phox (nucleotides 360 to 579) served as bait. Positive colonies were amplified in minimal medium (-His/-Trp/-Ura) and false positives were eliminated by mating with YM4271 yeast bearing the pLexA containing the binding domain alone.
Small Interfering RNA
VSMCs were plated at 40% to 50% confluence on collagen-coated substrate for 4 to 6 hours, washed with OPTI-MEM, and incubated with small interfering (si)RNA plus Oligofectamine complexes for 48 hours. Two stealth siRNAs (Invitrogen) against Poldip2 (siPoldip2) (no. 1 to primer 5′-GCCCACAUAUAUCUCAGAGAUCUCA-3′; no. 2 to primer 5′UGAGAUCUCUGAGAUAUAUGUGGGC3′) or a stealth control siRNA (siCont) were used at 15 nmol/L. Nox4 siRNA (siNox4; 25 nmol/L) was used as described previously,9 with the Allstars Negative Control (Qiagen). Cells were incubated in OPTI-MEM for 2 to 4 days before use.
Hemagglutinin (HA)-tagged Nox1 (Nox1-HA) and V5-tagged p22phox (V5-p22phox) were prepared as described previously.10 Myc-tagged rat Poldip2 was prepared using the coding region of Poldip2 obtained by PCR.
The AdEasy System was used to prepare adenoviruses with either no insert (green fluorescent protein [AdGFP]), HA-tagged Nox1 (AdNox1HA), antisense Nox1 (AdNox1AS),11 antisense Nox4 (AdNox4AS),9 and N-terminal myc-tagged Poldip2 (AdPoldip2). The LacZ control (AdLacZ), constitutively active RhoA (AdRhoAGV), and dominant negative RhoA adenoviruses (AdRhoTN) were also used. VSMCs were transduced as described previously.9
Glutathione S-Transferase Pull-Down
VSMCs were transfected with AdGFP or AdPoldip2 and labeled with 35S-methionine (20 μCi; 3 hours). Lysates were incubated with glutathione S-transferase (GST) fusion proteins (GST-vector or GST-p22phox) prepared using the TNT T7 Quick coupled transcription/translation system (Promega). Binding partners were detected by autoradiography.
Immunoblotting and Immunoprecipitation
VSMCs were lysed in standard lysis buffer3,10 or in Hunter’s buffer. Whole cell lysates were used for Western blot and immunoprecipitation.9,10 Band intensity was quantified by densitometry using ImageJ software.
RNA Isolation and Quantitative RT-PCR
Total RNA was extracted using the RNeasy kit (Qiagen). Superscipt II (Invitrogen) and random primers were used for reverse transcription. Poldip2 (primer sequences: GTATGAGACGGGACAGCTATTTCTCCA and CTGACATAGTCCAAGCCTGGGATG), nox1, nox4, p22phox, 18S rRNA were measured by amplification of VSMC cDNA using the LightCycler real-time thermocycler and SYBR green dye and normalized to 18S rRNA.9
Immunocytochemistry and Confocal Microscopy
VSMCs were plated, serum deprived for 48 hours, transiently transfected with siRNA for 72 hours, or grown to 50% to 60% confluence before treatment with adenovirus. Immunocytochemistry was carried out as described,3 and images were acquired with a Zeiss LSM 510 META Laser Scanning Confocal Microscope.
Amplex Red Assay
H2O2 was measured using the oxidation of Amplex Red (100 μmol/L) in the presence of horseradish peroxidase.12 H2O2 production was normalized to cellular protein, measured by the Bradford Assay (Bio-Rad).
NADPH oxidase activity in membrane fractions was assessed using 5 μmol/L lucigenin in the presence of 100 μmol/L NADPH and normalized to protein concentration, as described previously.13
Detection of Superoxide
Intracellular O2·− production was evaluated by measuring the conversion of dihydroethidium (DHE) to 2-hydroxyethidium with high-performance liquid chromatography (DHE-HPLC).9 Results are expressed as signal inhibited by polyethylene glycol–conjugated superoxide dismutase.
Tissues from rats were harvested and prepared for immunohistochemistry as described previously.9 All procedures were approved by the Emory University Institutional Animal Care and Use Committee.
Rho Activity Assay
Rho activity was measured by 2 independent methods: Rho pull-down assay and Rho G-LISA.
Migration was measured using Boyden chamber assays, as described previously.14
Results are expressed as means±SEM from at least 3 independent experiments. Statistical significance was assessed using ANOVA, followed by Bonferroni’s multiple comparison post hoc test. A value of P<0.05 was significant.
Identification of Poldip2 As a Novel p22phox-Interacting Partner
Using the proline-rich region of the C-terminal tail of p22phox as bait, a likely binding site for regulatory molecules,4 we performed a yeast 2-hybrid screen on a cDNA library from rat VSMCs (cells with high Nox4 expression). After stringent elimination of false positives, we isolated the cDNA of a protein that interacted with p22phox, obtained its full-length sequence using RT-PCR and identified the clone as Poldip2 (GenBank accession no. FJ515740). Poldip2 bears no homology to the classic Nox cytosolic regulatory subunits p47phox or p67phox or their homologs NoxO1 and NoxA1. It consists of 368 amino acids and has a predicted molecular mass of ≈42 kDa, with a potential signal peptide cleavage site after the first N-terminal 48 residues, which would result in a protein of ≈37 kDa. Additional predicted features include an ApaG domain, a PX domain, N-myristoylation sites, phospho-serine/threonine sites, and tyrosine sulfation sites.
Poldip2 Association With Nox Subunits
To verify that Poldip2 associates with p22phox, we initially used a GST pull-down assay. Indeed, 35S-Poldip2 pulls down with GST-p22phox (Figure 1a), confirming the association of these 2 proteins. To further validate the association of Poldip2 and p22phox, we cotransfected HEK293 cells with V5-tagged p22phox (V5-p22phox) and either vector control or Myc-tagged Poldip2 (Myc-Poldip2). Myc-Poldip2 coimmunoprecipitates with V5-p22phox (Figure 1b). To confirm an association between endogenous proteins, we generated and characterized an antibody against Poldip2 (Online Figure I, a and b), which recognizes both the 42- and 37-kDa forms of Poldip2. Endogenous Poldip2 coimmunoprecipitates with p22phox in VSMCs (Figure 1c), as expected from our observations with tagged proteins.
Previously published data show that both Nox1 and Nox4 colocalize with and coimmunoprecipitate with p22phox in rat VSMCs but that other Nox homologs are not expressed.3,15 Therefore, we examined a potential association of Poldip2 with these proteins. VSMCs were transduced with adenovirus to overexpress Myc-Poldip2 (AdPoldip2) (Online Figure I, c) and coimmunoprecipitation revealed an association of Nox4 and Myc-Poldip2 (Figure 1d). To ascertain whether the association with Nox4 is dependent on p22phox, we used a rat VSMC line stably transfected with antisense p22phox (p22AS) in which p22phox expression is ablated (Online Figure I, d).16 Poldip2 coimmunoprecipitates with Nox4 in vector-transfected cells, but not in cells lacking p22phox (Figure 1f), suggesting that Poldip2 requires p22phox to associate with Nox4. As shown in Figure 1e, Poldip2 also coimmunoprecipitates with HA-tagged Nox1 in VSMCs.
Poldip2 Stimulates ROS Production via Nox4
To determine whether Poldip2 regulates oxidase function in VSMCs, we measured NADPH oxidase activity in membrane fractions of VSMCs transduced with AdPoldip2. Overexpressing Poldip2 alone caused a significant increase in basal oxidase activity in a dose-dependent manner when compared to VSMCs transduced with control adenovirus (AdGFP) (Figure 2a). This increase in activity was not caused by an increase in nox1 mRNA (AdGFP: 2.4±1.1×105 versus AdPoldip2: 1.6±0.7×105 copies/μL cDNA; P=NS), nox4 mRNA (AdGFP: 1.3± 0.4×107 versus AdPoldip2: 1.0±0.5×107 copies/μL cDNA; P=NS), or p22phox expression (AdGFP: 4.3±0.2×106 versus AdPoldip2: 3.0±1.1×106 copies/μL cDNA; P=NS), nor by an increase in Nox4 or p22phox protein levels (Online Figure I, f). Because Poldip2 structure does not predict intrinsic oxidase activity, we tested whether this increase in NADPH-dependent O2·− production results from activation of either of the VSMC Nox catalytic subunits. Reduction of Nox4 by expression of antisense Nox4 (AdNox4AS) (Online Figure I, e) before the overexpression of Poldip2 abolishes the Poldip2-mediated increases in NADPH oxidase activity (Figure 2b), suggesting that Nox4 mediates the effects of Poldip2. This dependency on Nox4 is reflected in levels of O2·− and H2O2 in intact cells transduced with both constructs (Figure 2c and 2d). Of interest, Poldip2 was unable to significantly increase H2O2 production in VSMCs lacking p22phox expression (Online Figure III). In contrast, when Nox1 is depleted using antisense Nox1 (AdNox1AS), the increase in H2O2 production caused by Poldip2 overexpression is potentiated rather than inhibited (Figure 2e). Moreover, Poldip2 overexpression significantly increases ROS production by ≈2.5-fold (P<0.05) in both Nox1 wild-type (Nox1y/+; 2.65±0.2-fold increase) and Nox1 knockout (Nox1y/−; 2.56±0.2-fold increase) cells, as measured by electron spin resonance, suggesting that Nox1 is not directly involved in Poldip2-mediated ROS production. The potentiation of ROS production after Nox1 depletion may occur because the absence of Nox1 may increase the amount of p22phox available to stabilize Nox4 or may release a pool of Poldip2 that is then free to interact with Nox4. Taken together, these data strongly indicate that Poldip2 positively regulates Nox4, but not Nox1, activity, leading to subsequent increases in ROS production.
Poldip2 Colocalizes With Nox4 and p22phox
Because Poldip2 functionally regulates Nox4 activity, we used immunocytochemistry to determine whether Poldip2 colocalizes with Nox4 and p22phox in VSMCs. As shown in Figure 3, Poldip2 and p22phox colocalize in focal adhesions (Figure 3a, upper), along stress fibers (Figure 3a, middle), and in the nucleus of VSMCs, which are the patterns of p22phox distribution reported previously.3 Similar results were obtained using a Myc-tagged Poldip2 construct (Figure 3a, lower). In VSMCs, Nox4 has been detected in comparable locations.3,9 Indeed, colocalization experiments showed a clear overlap between Myc-Poldip2 and Nox4 staining in all 3 major subcellular sites (Figure 3b). Together, these data suggest that Poldip2 associates with p22phox and Nox4 in specific subcellular compartments.
The association between Nox4 and Poldip2 prompted us to examine the expression of Poldip2 in tissues rich in Nox4, such as aorta, lung, and kidney.4 Analysis of tissue distribution using immunohistochemistry, Western blot, and quantitative RT-PCR indicates that Poldip2 is highly expressed in all 3 tissues (Figure 4). In contrast, Poldip2 is barely detectable in spleen and thymus, which are rich in Nox2.
siPoldip2 Decreases ROS Production by Nox4 and Alters VSMC Phenotype
Overexpression of Poldip2 establishes its ability to regulate Nox4 but does not indicate whether Poldip2 is required for Nox4 activation. We have previously shown that basal ROS production in VSMCs is attributable to Nox4 activity9; therefore, we tested the effect of Poldip2 knockdown on basal O2·− and H2O2 production in VSMCs transfected with siRNA to deplete Poldip2 (siPoldip2). As shown in Figure 5a and 5b, siPoldip2 significantly decreases Poldip2 mRNA and protein levels, as well as O2·− and H2O2 production (Figure 5c and 5d), compared to control siRNA (siCont). Interestingly, we observed distinct changes in VSMC morphology after siPoldip2 treatment; cells became elongated, spindly, and seemed to have fewer points of contact with the dish, reminiscent of the phenotype observed in siNox4 treated cells (Figure 5f).
Poldip2 Regulates Proper Nox4 and p22phox Localization, Focal Adhesion Integrity, and Stress Fiber Formation
The localization of Poldip2, Nox4, and p22phox in focal adhesions and the apparent loss of these structures in siPoldip2-treated cells raise the possibility that Nox4 and p22phox localization is impaired when Poldip2 is depleted. Both Nox4 and p22phox protein levels seem slightly reduced in siPoldip2 treated VSMCs; however, the most striking effect is the loss of Nox4 and p22phox localization to focal adhesions (Figure 6a). The profound cytoskeletal phenotype observed in cells treated with siPoldip2 or siNox4 suggests that this enzyme complex may regulate focal adhesion integrity and/or stress fiber formation. As shown in Figure 6a and 6b, siPoldip2 or siNox4 treatment results in wavy, disorganized stress fibers, as detected by phalloidin staining, and complete loss of focal adhesion-like structures, as detected by staining for the resident focal adhesion proteins vinculin and paxillin. In contrast, overexpressing Poldip2 results in an increase in both focal adhesion and stress fiber formation (Figure 6c). These two phenotypic changes are maintained in Nox1y/− cells (Online Figure II). Thus, the Poldip2/Nox4 complex, but not Nox1, appears to be a potent regulator of these important cytoskeletal structures.
Nox4 and Poldip2 Stabilize Focal Adhesions and Promote Stress Fiber Formation Through RhoA Activation
Because both focal adhesion turnover and stress fiber formation are mediated through RhoA activation,17 we hypothesized that Poldip2/Nox4 may exert their effects by activating RhoA. Previous work has shown that RhoA is activated by ROS, suggesting that RhoA is a potential target of this complex.18 Overexpression of Poldip2 results in a substantial increase in RhoA activation (180.2%±24.8 increase versus AdGFP, P<0.05), and this increase is significantly blocked by treatment with the potent antioxidant N-acetyl cysteine (Figure 6d).
To evaluate the role of RhoA in mediating the effects of Poldip2/Nox4, we transduced siPoldip2-treated VSMCs with constitutively active RhoA (AdRhoGV) to determine whether we could rescue the loss of focal adhesions and stress fibers. Indeed, as shown in Figure 7a, RhoGV countered the phenotypic loss of stress fibers and focal adhesions caused by Poldip2 depletion. Additionally, dominant negative RhoA (AdRhoTN) blocked the phenotypic increase in stress fiber and focal adhesion formation in VSMCs overexpressing Poldip2 (Figure 7b). Taken together, these data strongly indicate that Poldip2 functionally regulates focal adhesion and stress fiber formation through a RhoA-dependent pathway.
Nox4 and Poldip2 Regulate VSMC Migration
VSMCs participate in the development of vascular lesions through their ability to proliferate and migrate.19 Migration, in particular, is dependent on dynamic focal adhesion turnover and cytoskeletal reorganization. Because either upregulation or downregulation of Poldip2/Nox4 negatively impacts focal adhesion turnover (Figure 6a and 6b), we hypothesized that either overexpression or knockdown of these proteins would impair cell migration. As shown in Figure 8a, overexpression of Poldip2, which strengthens focal adhesions, blocks migration in response to platelet-derived growth factor (10 nmol/L, 4 hours), perhaps because focal adhesions cannot release from the trailing edge of the cell to allow forward movement. Knockdown of Poldip2 or Nox4, which induces a loss of focal adhesions, also inhibits migration (Figure 8b and 8c), in this case, presumably, because new focal adhesions cannot form or mature at the leading edge of the cell.
In this study, we identify a novel role for Poldip2 as a unique positive regulator of Nox4 via its association with p22phox. Poldip2, together with Nox4, has profound effects on Rho-dependent cytoskeletal remodeling. Very little is known about the physiological role of Poldip2, except as a regulator of cell division.20 On the other hand, Nox4 is functionally linked to senescence, proinflammatory responses, oxygen sensing, migration,21 differentiation,9 proliferation, and apoptosis.4 The fact that Nox4 regulates such diverse physiological and pathophysiological responses suggests that it regulates a fundamental cellular process. Our data showing a functional association of Nox4 with Poldip2 and subsequent modulation of cytoskeletal integrity provide a possible mechanism to explain the known functions of both proteins.
The association of Poldip2 and p22phox/Nox4 was first detected in our yeast 2-hybrid screen designed to search for new p22phox-interacting proteins. Further investigation using multiple techniques (Figure 1a through 1c and Figure 2a) confirm this interaction and show a clear functional association between Nox4 and Poldip2. Both Myc-tagged Poldip2 and endogenous Poldip2 associate with Nox4 and p22phox (Figure 1d and 1f). In VSMCs, Nox4 and Poldip2 colocalize specifically in focal adhesions, stress fibers, and the nucleus (Figure 2b). Nox4 and Poldip2 also colocalize in similar cell types and cell structures in tissues from mouse aorta, kidney, and lung (Figure 4c). Interestingly, knockdown of either Poldip2 or Nox4 using siRNA causes the same cytoskeletal and phenotypic changes in VSMCs (Figures 5f and 6⇑a). The ability of Nox4 antisense to block Poldip2-induced increases in NADPH oxidase activity and ROS production (Figure 2a through 2d) indicates that Poldip2/Nox4-mediated ROS generation is responsible for these changes. However, as shown in Figure 1e, Poldip2 can also associate with HA-tagged Nox1, suggesting that Poldip2 may complex with the other Nox enzyme expressed in VSMCs and raising the possibility that an alteration in Nox1 activity could explain the phenotype. This seems unlikely because antisense Nox1 does not inhibit Poldip2-stimulated ROS production (Figure 2e) and Nox1 is not found in focal adhesions or the nucleus.3 Furthermore, overexpression or knockdown of Poldip2 in Nox1y/− VSMCs has the same effect on ROS production and cytoskeletal remodeling as it does in wild-type VSMCs, suggesting that Nox1 is not involved in the cytoskeletal effects of Poldip2. Nonetheless, we cannot rule out the possibility that Poldip2 may also influence Nox1-specific signaling.
There appear to be multiple levels of regulation of Nox4 by Poldip2. First, Poldip2 increases Nox4-dependent ROS production (Figure 2), suggesting Poldip2 may function similarly to other NADPH oxidase regulatory proteins such as p47phox, p67phox, NoxO1, or NoxA1. Second, Poldip2 appears to be involved in targeting Nox4 to specific subcellular compartments in VSMCs. Nox4 and p22phox have been detected in stress fibers, the nucleus, and focal adhesions.9 In the absence of Poldip2, Nox4 and p22phox no longer localize in focal adhesions but are still present in the nucleus (Figure 6 and unpublished observations, 2008). Therefore, binding to Poldip2 may ensure proper Nox4 and p22phox localization to cytoskeletal structures. In cells with low or absent Poldip2 expression, Nox4 may thus have different subcellular distributions. This is supported by the observation that Nox4 is detected in the endoplasmic reticulum in endothelial cells.22,23 A third possibility is that Poldip2 mediates processing of Nox4 and p22phox in the endoplasmic reticulum and Golgi. For other Nox homologs, such as Nox2 and Nox3, heme incorporation and interaction with p22phox during transit through the endoplasmic reticulum are required for complex stabilization and full enzyme activity.24,25 Moreover, Duox1 and -2 require coexpression of a maturation factor (DuoxA1 or DuoxA2) for proper processing.26 These proteins mediate the endoplasmic reticulum-to-Golgi transition of the Duox proteins and participate in their maturation and translocation to the plasma membrane. Because Nox4 is highly expressed in the endoplasmic reticulum in some cells, this potential function requires further investigation.
As noted earlier, the fact that Nox4 regulates such diverse physiological and pathophysiological responses suggests that it modulates a fundamental cellular process that may influence multiple functions such as proliferation, apoptosis, senescence, and migration. The association between Poldip2 and Nox4 predicts that Poldip2 should mediate these functions as well. Our data clearly implicate the cytoskeleton as a target of Poldip2. siPoldip2 causes cells to become elongated and spindly, with few detectable focal adhesions and wavy stress fibers (Figures 5f and 6⇑a). In contrast, overexpression of Poldip2 increases the thickness of stress fibers and causes focal adhesions to elongate and mature (Figure 6c). It is likely that Nox4 mediates these effects, because others have suggested that ROS are potent regulators of cytoskeletal remodeling. Moldovan et al27 showed that actin polymerization in migrating endothelial cells is blocked by the flavin containing oxidase inhibitor diphenylene iodonium. Similar results were found for LDL-induced reorganization of the actin cytoskeleton.28 Wu et al29 identified oxidative modifications of focal adhesion proteins induced by overexpression of a NADPH oxidase component, which raises the interesting possibility that ROS produced in focal adhesions may regulate their integrity by specifically modifying resident proteins.
A major target of Nox4/Poldip2 appears to be RhoA. Rho is activated in Poldip2-overexpressing cells, and dominant negative Rho inhibits cytoskeletal changes induced by Poldip2 overexpression (Figures 6d and 7⇑b). Moreover, expression of constitutively active Rho reverses the loss of stress fibers and focal adhesions induced by knockdown of Poldip2 (Figure 7a). RhoA is among the most important factors regulating focal adhesion turnover. Basal Rho activity is required to maintain cell substrate adhesion,30 the maturation of focal adhesions from focal complexes is mediated by Rho-dependent actin–myosin contraction of stress fibers,17 and focal adhesion dissolution requires inhibition of Rho.31 Rho also regulates microtubule stabilization,32 suggesting an additional mechanism to explain the siPoldip2 phenotype. This suggests that other cytoskeleton-mediated events such as cell division, proliferation, and differentiation-associated stress fiber formation, may also be mediated by the Poldip2/Nox4 axis. Moreover, it explains why both Nox1 and Nox4 are required for platelet-derived growth factor–induced VSMC migration. Nox1 is involved in signaling events at the cell membrane, regulating cofilin-mediated actin remodeling,33 whereas Nox4/Poldip2 likely regulates focal adhesion turnover in the trailing edge of the cell.
In this regard, it is of interest to examine what little is presently known about the functions of Poldip2. Poldip2 (also known as PDIP38 and mitogenin I) was originally identified as a proliferating cell nuclear antigen– and DNA polymerase δ–interacting protein, implicating a possible function in the regulation of gene expression, DNA duplication, or DNA repair.7 In the present context, this is of particular importance because we also see Nox4 in the nucleus, but the role of Nox4 in these processes is unknown. Although Poldip2 is reportedly associated with the mitochondria, these studies were performed in HeLa cells transfected with GFP-tagged Poldip2, which may confer different subcellular localizations compared to endogenous Poldip2.8,34 In fact, a more recent study reported localization of Poldip2 in the nucleus, cytoplasm, and plasma membrane in epithelial and endothelial cells.35 Interestingly, these authors found an interaction of Poldip2 with carcinoembryonic antigen-related cell adhesion molecule-1 (CD66a) at the plasma membrane, which induces shuttling of Poldip2 to the nucleus, a process that requires cytoskeletal integrity. Additional reports link Poldip2 to mitotic spindle organization and chromosome segregation, which both require proper microtubule and cytoskeletal dynamics.20 Taken in conjunction with the observations reported here, these data substantiate a role for Poldip2 as a novel modulator of cytoskeletal coordination, a function important in VSMC migration and endothelial barrier function.36
In light of these observations, we propose that the ability of Poldip2 to regulate Nox4 enzymatic activity and to modulate the cytoskeleton via RhoA may underlie many of the described functions of Poldip2. Although Poldip2 likely has additional binding partners, its association with Nox4 seems to be a major determinant of cell phenotype. Conversely, previous knowledge of cell processes affected by Poldip2 suggests new potential functions for Nox4 in the regulation of DNA repair. The identification of Poldip2 as a Nox4 regulatory protein provides an important new mechanism for regulation of basal ROS production. Previous studies have focused on transcriptional control of Nox4 as the principal mechanism of regulation,6 but our data implicate Poldip2 as an additional critical regulator not only of Nox4 activity, but also of its subcellular localization. It is thus conceivable that the interaction of these 2 proteins to coordinate ROS generation and cytoskeletal organization represents a novel mechanism that explains their shared and individual functions.
We thank Dr David Lambeth and Dr Mark Quinn for kindly providing the Nox4 and p22phox antibodies. We thank Dr Karl-Heinz Krause for providing the nox1 knockout animals. We also thank Dr Aviv Hassid for providing the LacZ, Rho-TN, and Rho-GV adenoviruses.
Sources of Funding
This work was supported by NIH grants HL38206 and HL05863 (to K.K.G.), an American Heart Association Predoctoral Grant (to A.N.L.), and Pharmacological Sciences Training Grant T32GM008602.
This manuscript was sent to Mark Taubman, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received January 7, 2009; revision received June 19, 2009; accepted June 24, 2009.
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