Endosomal ClC-3 and Nox1

Despite tremendous advances in our knowledge of the role of oxidative species in vascular disease, much debate remains regarding the mechanism by which NADPH oxidases (Nox) impart specific effects in cells. Over the past 2 decades, the term oxidative stress has often been used to describe the effects of Nox-derived reactive oxygen species (ROS). Much credence was initially given to relatively large quantities of ROS as mediators of myriad and varied cellular responses, ranging from connective tissue expression to differentiation, hypertrophy, proliferation, and apoptosis.¹ With the discovery of multiple Nox isoforms and emergence of loss-of-function and gain-of-function technologies manipulating the levels of individual isoforms, a large body of literature emerged suggesting that small, localized ROS derived from individual Nox proteins can impart unique cellular responses. The lack of sophisticated detection methods for localizing perhaps subtle changes in ROS continues to stymie our understanding of how one Nox may confer activation of a specific signaling cascade and render distinct cell changes. The conundrum of why similar whole cell or tissue increases or decreases in ROS confer disparate pheno-types continues to confound leaders in the field.

One such phenotypic change with broad implication for human disease is vascular smooth muscle cell (SMC) hyperplasia and neointimal growth. Central to neointimal formation is the proliferation and migration of SMCs, processes in which ROS and matrix metalloproteinase activation are established factors.^2–5 It is now broadly accepted that (1) inflammation plays an important role in atherosclerosis and SMC activation, which leads to neointimal formation,² and (2) cytokines, such as tumor necrosis factor-α (TNF-α), are important mediators in this response^6,7. Indeed, inflammatory cytokines emerge both as products of SMCs in neointimal formation and propagators of the observed proliferation and migration.^8,9

In this issue, Chu et al¹⁰ shed important new light on this phenomenon, with groundbreaking results that have the potential to change the way we think about redox signaling. To date, prevailing wisdom has suggested that individual Nox isoforms, by virtue of their subcellular localization, relay distinct downstream signaling in each cell or tissue by oxidizing targets in the vicinity of the compartment in which they reside.^11,12 The current findings, in conjunction with recent other findings by this group, are paradigm-shifting in that they suggest that a single Nox isoform, namely Nox1, can have multiple signaling effects by belonging to distinct signaling pathways and by occupying multiple docking sites within the cell. Even though the ultimate effect in this study is the same, one can readily infer that Nox1 would effect distinct signaling changes depending on its localization. Although the concept of a charge-neutralizing antiporter being involved in Nox modulation is not novel,¹³ the pivotal role of ClC-3 in directed and distinct Nox1-mediated signaling is. In their report, “A Critical Role for ClC-3 in Smooth Muscle Cell Activation and Neointima Formation,”¹⁰ the authors intriguingly show that the chloride-proton antiporter ClC-3 is key to (1) TNF-α-mediated but not thrombin-mediated SMC proliferation and (2) development of neointimal hyperplasia in response to flow cessation. The authors use cultures of aortic SMCs from ClC-3 knockout mice and their wild-type controls to investigate the role of TNF-α and thrombin in SMC proliferation, matrix metalloproteinase-9 activity, and protein expression, as well as extracellular signal-regulated kinase 1/2 (Erk1/2) activation. These parameters were all impaired in TNF-α but not in thrombin-treated ClC-3 null cells, consistent with the authors’ recent findings showing distinct localization of TNF-α-induced Nox1-derived ROS to endosomes.¹⁴ In the current study, the intriguing dependence of these effects on ClC-3 were demonstrated in gene-transfer experiments whereby the authors reintroduced the ClC-3 gene into SMC from ClC-3-null mice and showed restoration of the responses to TNF-α. The authors also show that ClC-3 is important for neointimal hyperplasia following carotid artery injury using the flow cessation model: ClC-3-null mice exhibited a reduction in neointimal area 28 days after injury compared with wild-type controls. Furthermore, the authors demonstrate by polymerase chain reaction (PCR) that ClC-3 expression is upregulated both under in vitro TNF-α treatment of SMCs and in vivo after carotid injury. What the authors do not mention is that by virtue of its localization in endosomes, whose importance in signal transduction is becoming increasingly apparent,¹⁵ Nox1 has the potential to relay a signal to varying targets as it moves across the cell.

The H⁺/Cl⁻ antiporter channel, ClC-3, one of 9 members of the ClC family of Cl⁻ channels or Cl⁻/H⁺ exchangers, localizes in membranes of endosomes and lysosomes and is expressed in a broad variety of cells, including SMCs.¹⁶ Historically, ClC-3 was more often described in the regulation of cell volume. Later, a correlative link to cell proliferation was made.¹⁷ Almost at the same time, ClC-3 upregulation was associated with the hypertrophy of pulmonary artery SMCs in monocrotaline-induced pulmonary hypertension,¹⁸ supporting a role for this channel in vascular remodeling. Moreover, it has been reported that short interfering RNA against ClC-3 results in attenuation of SMC proliferation in a pathway that involved cell cycle arrest.¹⁹

The mitogen-activated protein kinases are important signaling mediators in numerous pathways and are invoked in virtually all discussions of redox signaling. Furthermore, Erk1/2 activation has long been shown to be involved in neointima formation.^20,21 In addition, Erk1/2 was implicated in TNF-α-induced matrix metalloproteinase-9 expression in human aortic SMC, the latter of which renders the SMC capable of migration to the intima across matrix-rich vessels.⁷ Moreover, thrombin has been shown to activate AP-1 by way of Erk1/2, in a Nox-dependent mechanism.²² Taken together, the above evidence is consistent with a role of inflammatory cytokines, ClC-3, Erk1/2, and matrix metalloproteinase-9 in the development of vascular remodeling and neointimal formation.

The current work expands on the authors’ previous findings demonstrating a requirement of ClC-3 in endosome-dependent Nox1 activity in SMC²³ and ties those findings to a more recent discovery by the same group connecting TNF-α to activation of Nox1-derived endosomal ROS.¹⁴ Admittedly, the authors state that one limitation of the study is that flow cessation may not adequately reflect mechanical models of neointimal hyperplasia, such as those evoked by angioplasty. What the authors do not discuss is that the in vivo model used also does not reflect the inflammatory implications in neointimal hyperplasia arising during atherosclerosis. Perhaps more importantly, a review of the literature suggests that neointimal formation arising from flow cessation involves a nonuniform thrombus formation and thus thrombin-mediated effects.^24,25 Therefore, the results of the study need to be interpreted with some caution in that, depending on the region of the blood vessel studied, a thrombin-mediated hyperplasia could prevail. In fact, the results of ClC-3 deletion may have been more pronounced in tissues or regions selected expressly for the lack of a thrombus.

Enthusiasm for these findings might be tempered by a narrow view of the data. That is, some might argue that ultimately TNF-α and thrombin mediate the same cellular and tissue end effect via ERK 1/2. This, however, should in no way be taken to imply that blocking TNF-α- and thrombin-mediated Nox1 activation will have the same effect. Clearly, the pathways diverge at ClC-3. This is likely to have other unique signaling effects concomitant with Nox1 as the endosome travels to other parts of the cell. Moreover, independently of Nox1, ClC-3 is likely to elicit effects on signaling via charge changes within the endosome or heretofore undescribed interactions with other signaling molecules. It will be interesting to test for the possibility of unique kinase activation in populations of cells that exhibit disparate distribution of endosomes. Clearly, myriad opportunities abound in deconstructing the expectedly complex and sophisticated signaling effects of this novel discovery.

Further to the above, the current findings with respect to a role of ClC-3 in neointimal formation by themselves are highly significant in advancing our understanding of this complex pathological process. They introduce a potentially novel target in SMC proliferation in the context of Nox1 and ROS and thus open the door to new combinatorial therapies for the prevention of neointimal formation. Furthermore, studies suggesting exquisite regulation of ClC-3 channel activity by phosphorylation hint at other opportunities for regulating Nox1 and hyperplasia.²⁶ The current findings by Chu et al are undoubtedly trailblazing. The only remaining uncertainty is which trail to take first.