NADPH Oxidase 4
Walking the Walk With Poldip2
See related article, pages 249–259
Over the last decade, the NADPH oxidases have been recognized as an important source of reactive oxygen species (ROS) involved in both normal physiological redox-dependent processes, and in the generation of oxidative stress, with a role in the development of cardiovascular disease. As a multisubunit enzyme complex, the NADPH oxidases have the unusual composition of multiple interchangeable homologs of the catalytic, or NADPH oxidase (Nox), subunit that directly interacts with membrane-bound p22phox. It is at the Nox subunit that NADPH binding leads to electron transfer via heme groups to oxygen resulting in the generation of superoxide anion. Recent investigations have identified 7 distinct Nox subunits, some possessing multiple splice variants, but the real challenge has been to characterize the specific cellular functions and the regulation of the various Nox enzymes. The difficulty in doing so is partly attributable to the presence of multiple Nox isoforms within cells, the low and transient levels of ROS produced with Nox activation, limitations in the ability to detect protein levels of specific Nox enzymes, and the potential for one Nox protein to compensate for the change in expression or activity of a different Nox protein. Although significant progress has been made, it can be argued that our knowledge of the regulation of Nox activity has only partially extended beyond what could be inferred from the model of the phagocyte oxidase (ie, gp91phox or Nox2), which has been characterized over the past twenty to thirty years.
One of the most abundant and widely expressed Nox isoforms is Nox4. Originally identified in the kidney,1 Nox4 is usually described as being constitutively active, suggesting a role in resting cellular homeostasis. Even so, activation of Nox4 has been observed under certain experimental conditions.2 Expression levels of Nox4 are reported to be as much as 100-fold greater than other Nox isoforms in vascular smooth muscle cells (SMCs), and Nox4 appears to localize at focal adhesions, within the nucleus, or in the endoplasmic reticulum.
Despite its ubiquitous expression and activity, the primary function of Nox4-derived ROS is not clear. It has been proposed to have a role in oxygen sensing, cell migration, growth, senescence, and SMC differentiation. Surprisingly, despite its first report nearly ten years ago, a Nox4-deficient mouse has yet to be described in the literature.
In this issue of Circulation Research, using a yeast 2-hybrid system with the cytosolic tail of p22phox as bait, Lyle et al significantly advance our understanding of NADPH oxidase regulation by identifying polymerase delta interacting protein 2 (Poldip2) as an unexpected and novel partner of Nox4.3 Very little is known regarding Poldip2, which was originally described to bind to DNA polymerase delta and proliferating cell nuclear antigen.4 Lyle et al3 show that Poldip2 colocalizes with p22phox at sites of Nox4 in SMCs and, when present, increases Nox4 activity by three-fold. In addition, Nox4-Poldip2–derived ROS activate RhoA to strengthen focal adhesions and increase stress fiber formation. Interestingly, either the overexpression or the depletion of Poldip2 inhibits migration of cultured SMCs, suggesting a central role for Nox4-Poldip2 in synchronizing the process of directed cell migration.
Previously, the activity of Nox4 (as determined by the generation of ROS) has been considered to be exclusively dependent on the protein levels of Nox4.5 Lyle et al3 are the first study to identify a protein, other than p22phox, with the capacity to directly regulate the activity of Nox4. Unfortunately, the study does not identify the mechanism by which Poldip2 regulates Nox4 activity. It could be hypothesized that Poldip2 stabilizes the Nox4-p22phox complex or increases available binding sites for Nox4 on focal adhesions and, thereby, increases the cellular levels of functional Nox4. However, there does not appear to be an associated change in Nox4 protein level with changes in Poldip2 expression. Remembering that Nox4 does not require the traditional NADPH oxidase cytosolic subunits (p67phox, p47phox, or their homologs) for activation, Poldip2 may function to emulate the cytosolic subunits in activating Nox4. Structure-based studies will be necessary to identify the mechanism by which Poldip2 facilitates Nox4-derived ROS.
The association of Poldip2 with Nox4 is via p22phox; therefore, it may not be surprising that Lyle et al3 also found colocalization of Poldip2 with Nox1. However, in contrast to findings with depletion of Nox4, the depletion of Nox1 actually augments the increase in ROS associated with expression of Poldip2. This observation is difficult to explain; nonetheless, a distinct difference between Nox1 and Nox4 is that Nox1 is dependent on an activation process involving recruitment of cytosolic subunits to the Nox1-p22phox complex. It is not known whether Poldip2 would modify the agonist-mediated activation of Nox1. It is also unknown whether the association of different Nox isoforms with Poldip2 is specific or, instead, generally applicable to any Nox enzyme that requires p22phox. For example, it will be interesting to determine whether Nox2 associates with Poldip2.
An intriguing and yet unexplained observation is the localization of Nox4 within the nucleus.6 It is difficult to understand how a membrane-associated protein can be found in a membrane-free space, such as the interior of the nucleus, and what function an ROS-generating system would serve there. Lyle et al3 demonstrate that, in addition to its association with the cytosolic region of Nox4, Poldip2 also associates with Nox4 in the nucleus. The interaction of Poldip2 with DNA polymerase delta and proliferating cell nuclear antigen suggests a role in DNA modification and/or synthesis. In this context, Nox4 may regulate the redox environment within the nucleus, resulting in redox modification of DNA or associated proteins. The authors also propose a potential role for Nox4 in regulation of DNA repair. If true, this could have important implications not only in cancer biology, but also in the use of antioxidants, and particularly in therapies directed at inhibiting Nox4, in the treatment of cardiovascular disease in patients.
The migration of SMCs from the media to the intima is central to the development of cardiovascular disease. For a cell to migrate, it must be polarized, which indicates that the molecular processes at the front and the back of a moving cell are different. Migrating cells continuously form and disassemble adhesions such that strengthening of focal complexes at the front of the cell provide traction over which the cell body moves, whereas the release of adhesions in the rear of the cell allows forward displacement. Lyle et al3 show that the Nox4-Poldip2 complex is necessary for formation of focal adhesions and migration of SMCs (Figure). In this model, the dynamic regulation of focal adhesions by Nox4-Poldip2 locally is important for efficient directional cell migration. It has also recently been shown that Nox1 activation is necessary for SMC migration.7,8 Essentially nothing is known regarding how these processes are integrated temporally and spatially across the migrating cell.
In summary, the exciting new finding by Lyle et al3 identifies Poldip2 as a novel positive regulator of Nox4 activity in SMCs. Through the redox-dependent activation of RhoA, Nox4-Poldip2 affects the cytoskeletal rearrangement necessary for cell migration. Although studies were performed in SMCs, these findings may have far reaching implications as it is expected that Poldip2 will regulate Nox4 in other cell types as well. This is supported by the observation that expression levels of Poldip2 in various tissues is related to the expression of Nox4.3 Future studies determining the mechanism by which Poldip2 increases Nox4 activity, its regulatory role with other Nox isoforms, and the function of Poldip2-Nox4 in the nucleus will greatly extend our understanding of NADPH oxidases in diverse areas of redox biology and their potential as therapeutic targets in cardiovascular disease.
Sources of Funding
Supported by NIH grant HL081750 and by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. The opinions expressed in this editorial do not represent the views of the Department of Veterans Affairs or the United States Government.
Geiszt M, Kopp JB, Varnai P, Leto TL. Identification of Renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci U S A. 2000; 97: 8010–8014.
Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007; 87: 245–313.
Lyle AN, Deshpande NN, Taniyama Y, Seidel-Rogol B, Pounkova L, Du P, Papaharalambus C, Lassegue B, Griendling KK. Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells. Circ Res. 2009; 105: 249–259.
Liu L, Rodriguez-Belmonte E, Mazloum N, Xie B, Lee M. Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase delta and proliferating cell nuclear antigen. J Biol Chem. 2003; 278: 10041–10047.
Serrander L, Cartier L, Bedard K, Banfi B, Lardy B, Plastre O, Sienkiewicz A, Forro L, Schlegel W, Krause KH. NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation. Biochem J. 2007; 406: 105–114.
Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK. Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24: 677–683.
Lee MY, Martin AS, Mehta PK, Dikalova AE, Garrido AM, Lyons E, Krause KH, Banfi B, Lambeth JD, Lassegue B, Griendling KK. Mechanisms of vascular smooth muscle NADPH Oxidase 1 (Nox1) contribution to injury-induced neointimal formation. Arterioscler Thromb Vasc Biol. 2009; 29: 480–487.
Schroder K, Helmcke I, Palfi K, Krause KH, Busse R, Brandes RP. Nox1 mediates basic fibroblast growth factor-induced migration of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2007; 27: 1736.