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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 21, 2005 as doi:10.1096/fj.05-4564fje. |
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Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA
1 Correspondence: Division of Cardiology, Emory University School of Medicine, 101 Woodruff circle, WMB 319, Atlanta, GA 30322, USA. Email: tfukai{at}emory.edu
SPECIFIC AIMS
Extracellular superoxide dismutase (SOD3), a secretory copper enzyme, plays an important role in atherosclerosis and hypertension by modulating the levels of extracellular superoxide anion (O2•–) in the vasculature. Little is known about the mechanisms by which SOD3 obtains its catalytic copper cofactor. The Menkes ATPase (MNK) has been shown to transport cytosolic copper to the secretory pathway in nonvascular cells. We performed the present study to determine whether MNK is required for the activation of SOD3 in the vasculature.
PRINCIPAL FINDINGS
1. MNK is abundantly expressed in vascular smooth muscle cells, vascular endothelial cells, fibroblasts, and aorta
2. Vascular MNK is required for full activation of SOD3, not SOD1, by transporting copper to SOD3
To determine whether MNK is required for the full activity of SOD3, we used cultured MNK mutant mouse fibroblasts. The specific activity of SOD3 was markedly decreased in MNK mutant fibroblasts, which was partially restored by addition of copper in the culture medium (Fig. 1
). In contrast, the activity, protein, and specific activity of SOD1 were not changed in MNK mutant fibroblasts. Consistent with the results obtained with cultured fibroblasts, the specific activity of SOD3, but not SOD1, was robustly decreased in aortas from MNK mutant mice. These findings suggest that MNK is required for copper loading to SOD3 for its full activity in vascular tissue as well as in cultured fibroblasts.
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3. SOD3 colocalizes with MNK in the trans-Golgi network
Since we found that specific activity of SOD3 is dependent on MNK, we next examined where MNK delivers copper to SOD3 in the secretory pathway. Confocal immunofluorescence analysis showed that MNK and SOD3 partially colocalized with 
-adaptin, a trans-Golgi network resident protein, in the peri-nuclear region. Subcellular fractionation analysis demonstrated that MNK and SOD3 partially cofractionated with adaptin, but not GM130, a cis-Golgi resident protein. These results suggest that SOD3 colocalizes with MNK in the trans-Golgi network of the secretory pathway.
4. SOD3 directly interacts with MNK in a copper dependent manner
To determine how SOD3 obtains copper through MNK, we performed coimmunoprecipitation of SOD3 and MNK in mouse fibroblasts. As shown in Fig. 2
A, SOD3 was coimmunoprecipitated with MNK, which was enhanced by addition of copper in a dose-dependent manner. In contrast, their association was inhibited by the copper chelator BCS. In vitro pull-down assays demonstrated that interaction of SOD3 and MNK is direct and specific, because recombinant SOD3 bound to MNK protein but not to unrelated protein Na, K, ATPase (Fig. 2B
). These data suggest that MNK directly interacts with SOD3 in a copper-dependent manner.
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5. MNK plays an important role in modulating vascular O2•– levels by modulating vascular SOD3 activity
In aortas from the MNKblo mutant mice, activity of SOD3, but not SOD1, was markedly decreased. To determine the functional significance of MNK in modulating SOD3 activity in vivo, we measured O2•– production in mouse aortas from control and MNKblo mutant mice using lucigenin-enhanced chemiluminescence. We found that vascular O2•– was markedly increased in aortas from MNK mutant mice compared with those from control littermates. To estimate O2•– production in mouse aortas in situ, we also used the dihydroethidium (DHE) fluorescence method. DHE staining clearly demonstrated that O2•– production was markedly increased in all layers of aortas from MNK mutant mice. The fluorescence signal was markedly decreased by addition of SOD, suggesting that DHE signal mainly reflects an increase in O2•–. Taken together, these findings suggest that MNK plays an important role in regulating vascular O2•– levels at least in part by modulating vascular SOD3 activity.
6. MNK is highly expressed in the intimal lesions of atherosclerotic vessels
To gain further insight into the role of MNK in modulating SOD3 activity in atherosclerosis, we performed immunohistochemical analysis of MNK protein in aortas from control and ApoE–/– mice. Aortas from control mouse showed a staining of MNK and SOD3 in medial layer whereas those from ApoE–/– mice demonstrated their intense staining in the intimal lesion of atherosclerosis. These results suggest that both MNK and SOD3 are highly expressed in the intimal lesion of atherosclerotic vessels.
CONCLUSIONS AND SIGNIFICANCE
In the current study, we demonstrated that MNK is highly expressed in the vasculature and that MNK is required for its full activation of vascular SOD3 by transporting copper to SOD3 in the trans-Golgi network, thereby regulating vascular O2•– levels. The copper transport to SOD3 by MNK is mediated through direct protein-protein interaction. Atherosclerotic vessels highly express both MNK and SOD3. These studies provide a novel insight into a vascular MNK as a critical modulator of "superoxide" stress, which may contribute to cardiovascular disease.
MNK belongs to a large family of cation-transporting P-type ATPase and plays a key role in transporting copper from the cytosol into the secretory pathways. However, little is known about its role in the vasculature. Here we provide the first evidence that MNK is highly expressed in the vascular tissues and cells, and that MNK is required for full activation of the secretory copper enzyme SOD3 using cultured fibroblasts and aortas from MNK mutant mice. Activation of the cytosolic copper enzyme SOD1 is MNK independent. SOD3-specific activity purified from MNK mutant cells is partially normalized by copper addition. These findings suggest that MNK plays a critical role in delivering copper to SOD3 for its full activation.
SOD3 is a secretory copper enzyme containing a classical signal peptide that will direct SOD3 from the endoplasmic reticulum through the trans-Golgi network to the plasma membrane, thereby being secreted into the extracellular space. Since we found that specific activity of SOD3 is dependent on MNK, we next investigated the mechanisms by which MNK delivers copper to SOD3 in the secretory pathway. Both immunofluorescence and subcellular fractionation analysis revealed that SOD3 partially colocalizes with MNK and adaptin, a marker of the trans-Golgi network, suggesting that MNK transports copper to SOD3 in the trans-Golgi network. Moreover, coimmunoprecipitation and in vitro pull-down assays demonstrated that MNK directly interacts with SOD3 in vivo and in vitro. Thus, MNK delivers copper to SOD3 in the trans-Golgi network through direct binding to SOD3. We also found that interaction of MNK with SOD3 is copper dependent. To our knowledge, this is the first evidence that MNK directly interacts with its target secretory copper enzyme in a copper dependent manner.
Since the level of intracellular free copper is extraordinarily restricted, MNK requires cytosolic copper carrier proteins to deliver copper to SOD3 in the trans-Golgi network. We recently demonstrated that the copper chaperone Atox1 is involved in copper delivery to SOD3. Although several proteins such as MNK, WND, and FKBP52 receive copper from Atox1, but it is likely that SOD3 may obtain copper through Atox1-MNK pathway in the vascular cells (Fig. 3
).
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We have shown that SOD-inhibitable O2•– levels are significantly increased in aortas from MNK mutant mice in which vascular SOD3 is markedly decreased. We also found that MNK protein expression is enhanced in the intimal lesion of atherosclerosis where SOD3 expression is increased. Since bioavailability of NO• can be regulated by extracellular O2•– which is controlled by SOD3, MNK may play an important role in regulating available NO• by modulating SOD3 activity, thereby contributing to vascular function in atherosclerosis.
Some Menkes mutant mice, where SOD3 activity is markedly decreased, show vascular abnormalities, including formation of aortic aneurysm, which is associated with oxidative stress. Thus, it is conceivable that MNK may be involved in aortic aneurysm formation by modulating vascular redox state via regulating SOD3 activity.
In summary, the present study demonstrates that MNK plays an essential role in regulating SOD3 activity through transporting copper to SOD3 in the trans-Golgi network, thereby modulating vascular O2•– levels. In the cardiovascular system, SOD3 is highly expressed in blood vessels and plays a major role to increase nitric oxide bioactivity by scavenging extracellular O2•–. Our study should provide novel insight into how redox state of blood vessel is tightly controlled by copper transport systems.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4564fje;
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P < 0.01 vs. untreated cells. *P < 0.05 vs. untreated cells. B) In vitro pull-down assays using recombinant SOD3 with V5-His tag in mouse fibroblasts. Human recombinant SOD3 with V5-His tag was incubated with mouse fibroblast cell lysates and mixture was immunoprecipitated (IP) with anti-His antibody, followed by immunoblot analysis (IB) with anti-MNK, anti-Na, K-ATPase, or anti-V5 antibody. IgG was included as a negative control for immunoprecipitation. Cell lysates were included as a positive control.