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Biochemistry -- Faculty Publications Biochemistry, Department of

2009

Copper transport into the secretory pathway isregulated by oxygen in macrophagesCarine WhiteUniversity of Missouri, Columbia

Taiho KambeUniversity of Missouri, Columbia

Yan G. FulcherUniversity of Missouri, Columbia, fulchery@missouri.edu

Sherri W. SachdevUniversity of Missouri, Columbia, sachdevsl@missouri.edu

Ashley I. BushMental Health Research Institute of Victoria, a.bush@mhri.edu.au

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White, Carine; Kambe, Taiho; Fulcher, Yan G.; Sachdev, Sherri W.; Bush, Ashley I.; Fritsche, Kevin; Lee, Jaekwon; Quinn, Thomas P.;and Petris, Michael J., "Copper transport into the secretory pathway is regulated by oxygen in macrophages" (2009). Biochemistry --Faculty Publications. 139.http://digitalcommons.unl.edu/biochemfacpub/139

AuthorsCarine White, Taiho Kambe, Yan G. Fulcher, Sherri W. Sachdev, Ashley I. Bush, Kevin Fritsche, Jaekwon Lee,Thomas P. Quinn, and Michael J. Petris

This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/biochemfacpub/139

1315Research Article

IntroductionCopper is a trace element that is critical for aerobic life. Its ability

to accept and donate electrons has been harnessed by a select group

of enzymes that function in mitochondrial respiration, connective

tissue formation, pigmentation, iron oxidation, neurotransmitter

processing and antioxidant defense (La Fontaine and Mercer, 2007;

Madsen and Gitlin, 2007; Prohaska and Gybina, 2004). However,

this same redox property of copper and its ability to generate reactive

oxygen species, also underscores its potential toxicity. For this

reason, copper-handling proteins have evolved to deliver copper to

specific sites of utilization, thereby preventing the formation of

potentially damaging free ionic copper in the cytoplasm. Copper

uptake in mammalian cells is mediated by CTR1 (SLC31A1), a

ubiquitously expressed homotrimeric transporter (Zhou and

Gitschier, 1997). Once in the cytoplasm, small cytoplasmic proteins

known as copper chaperones deliver copper to distinct target

enzymes by direct protein-protein interactions. The copper

chaperones, CCS and COX17 are involved in copper delivery to

Cu/Zn superoxide dismutase (SOD1) in the cytoplasm, and to

cytochrome c oxidase (CCO) in the mitochondria (Amaravadi et

al., 1997; Casareno et al., 1998). SCO1 and SCO2 are copper

chaperones that are also involved in copper delivery to CCO via a

poorly understood process (Leary et al., 2004). The third target for

copper delivery is the ATP7A protein (or closely related ATP7B

protein), a copper transporter located in the Golgi complex that

receives copper from the ATOX1 copper chaperone in the cytoplasm

(Larin et al., 1999; Petris et al., 1996; Yamaguchi et al., 1996).

ATP7A transports copper into the Golgi lumen to supply copper to

a select group of copper-dependent enzymes, which are either

secreted from cells, or reside within vesicular compartments (Barnes

et al., 2005; El Meskini et al., 2003; Petris et al., 2000; Qin et al.,

2006). An additional function of ATP7A is the export of excess

copper from cells. This export activity is associated with copper-

stimulated trafficking of ATP7A to post-Golgi compartments,

including cytoplasmic vesicles and the plasma membrane (Petris

et al., 1996). The trafficking of ATP7A is triggered when

cytoplasmic copper levels are elevated (Hamza et al., 2003), and

this process requires both copper binding to cytoplasmic regions

of the ATPase as well as its catalytic turnover (Petris et al., 2002;

Strausak et al., 1999). The biological importance of the ATP7A

protein is illustrated by Menkes disease, a lethal disorder of copper

deficiency caused by ATP7A mutations (Kaler, 1998).

An area of copper metabolism of which we have little

understanding is whether specific pathophysiological conditions

lead to adaptive changes in intracellular copper homeostasis.

Hypoxia is a stress that is inherent within the microenvironment of

injured tissues, including dermal wounds and burns (Arnold, 1987),

atherosclerotic plaques (Moreno et al., 2006) and avascular regions

of solid tumors (Bristow and Hill, 2008). Cells of the myeloid

lineage such a macrophages are specifically recruited to these

hypoxic sites and are metabolically adapted to function within this

hostile milieu. For example, it has been known for many years that

neutrophils and macrophages are highly dependent on anaerobic

glycolysis for ATP production, and suppress oxidative

phosphorylation in the presence of hypoxia (Simon et al., 1977).

In this study we investigated the impact of hypoxia on copper

Copper is an essential nutrient for a variety of biochemical

processes; however, the redox properties of copper also make

it potentially toxic in the free form. Consequently, the uptake

and intracellular distribution of this metal is strictly regulated.

This raises the issue of whether specific pathophysiological

conditions can promote adaptive changes in intracellular copper

distribution. In this study, we demonstrate that oxygen

limitation promotes a series of striking alterations in copper

homeostasis in RAW264.7 macrophage cells. Hypoxia was

found to stimulate copper uptake and to increase the expression

of the copper importer, CTR1. This resulted in increased

copper delivery to the ATP7A copper transporter and copper-

dependent trafficking of ATP7A to cytoplasmic vesicles.

Significantly, the ATP7A protein was required to deliver copper

into the secretory pathway to ceruloplasmin, a secreted copper-

dependent enzyme, the expression and activity of which were

stimulated by hypoxia. However, the activities of the alternative

targets of intracellular copper delivery, superoxide dismutase

and cytochrome c oxidase, were markedly reduced in response

to hypoxia. Collectively, these findings demonstrate that copper

delivery into the biosynthetic secretory pathway is regulated by

oxygen availability in macrophages by a selective increase in

copper transport involving ATP7A.

Key words: ATP7A, Copper, Hypoxia, Macrophage, Oxygen,

Trafficking

Summary

Copper transport into the secretory pathway isregulated by oxygen in macrophagesCarine White1, Taiho Kambe2, Yan G. Fulcher1, Sherri W. Sachdev2, Ashley I. Bush3, Kevin Fritsche4,Jaekwon Lee5, Thomas P. Quinn2 and Michael J. Petris1,2,*1Department of Nutritional Sciences, University of Missouri, Columbia, MO 65211, USA2Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA3Oxidation Biology Laboratory, Mental Health Research Institute of Victoria, Melbourne, Victoria 3052, Australia4Department of Animal Sciences, University of Missouri, Columbia, MO 65211, USA5The Redox Biology Center, Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA*Author for correspondence (e-mail: petrism@missouri.edu)

Accepted 5 January 2009Journal of Cell Science 122, 1315-1321 Published by The Company of Biologists 2009doi:10.1242/jcs.043216

1316

homeostasis in the murine macrophage cell line, RAW264.7.

Hypoxia resulted in increased copper uptake and enhanced the

expression of the CTR1 transporter. Copper delivery to the ATP7A

protein was also enhanced as evidenced by trafficking from the

Golgi and enhanced copper transport into the secretory pathway to

ceruloplasmin. By contrast, hypoxia triggered a decrease in the

levels of other intracellular copper targets including, CCS, SOD1,

and the copper-binding subunit of CCO, COX1. These findings

suggest that oxygen status can regulate copper allocation to the

secretory pathway for hypoxia-induced cuproenzymes, and reveal

hypoxia as a unique pathophysiological regulator of intracellular

copper hierarchy.

ResultsHypoxia stimulates trafficking of the ATP7A protein inRAW264.7 macrophagesWe began this study by investigating whether reduced oxygen

tension might alter the localization of the ATP7A copper transporter

in the murine macrophage cell line, RAW264.7. The relocalization

of ATP7A from the trans-Golgi network is a key biological indicator

of increased cytoplasmic copper availability and has been

documented in several different cell types (Cobbold et al., 2002;

La Fontaine et al., 1998; Petris et al., 1996). Using

immunofluorescence microscopy, the ATP7A protein was found

within the perinuclear region of RAW264.7 cells exposed to

normoxic conditions (21% O2), consistent with its location in the

trans-Golgi network (Fig. 1A). As expected, treatment of these cells

with copper resulted in the trafficking of ATP7A from the

perinuclear region to cytoplasmic vesicles (Fig. 1A). Significantly,

when these cells were exposed to chronic hypoxia (4% O2 for 96

hours), the ATP7A protein was also dispersed to post-Golgi vesicles

(Fig. 1A). This redistribution of the ATP7A protein required at least

48 hours of hypoxia and was not accelerated by lower levels of

oxygen (data not shown). The return of hypoxic cells to normoxic

conditions restored the location of the ATP7A protein to the

perinuclear region, indicating that the effect of hypoxia on ATP7A

was reversible (Fig. 1A). The intracellular location of the trans-

Golgi marker protein, syntaxin 6 (STX6) and the Golgi matrix

protein, GM130, were not altered by hypoxia in RAW264.7 cells

(Fig. 1B,C), suggesting that the effects on ATP7A were not the result

of a general effect on Golgi structure. The viability of RAW264.7

cells was not altered by these conditions and cells could be

passaged continuously in 4% O2 (data not shown).

Oxygen limitation stimulates ATP7A expression and copper-dependent trafficking of ATP7ASince the trafficking of ATP7A from the trans-Golgi network is

known to be triggered by increased copper delivery to this

transporter (Petris et al., 1996), we tested whether a membrane-

permeable copper chelator, tetrathiomolybdate (TTM), could

suppress ATP7A trafficking in response to hypoxia in RAW264.7

cells. As shown in Fig. 2A, TTM inhibited ATP7A relocalization

in response to hypoxia. These findings support the hypothesis that

oxygen limitation increases copper binding to the ATP7A protein,

resulting in its trafficking from the Golgi. We also explored the

possibility that hypoxia may also increase the expression of ATP7A

in RAW264.7 macrophages. Western blot analysis demonstrated that

hypoxia increased ATP7A protein levels above normoxic controls

in a time-dependent manner, beginning between 24 and 48 hours

(Fig. 2B). A similar increase in ATP7A levels was observed in

primary peritoneal macrophages isolated from C57BL mice and

cultured under hypoxic conditions (Fig. 2C). These findings suggest

that hypoxia stimulates copper-dependent trafficking and expression

of the ATP7A protein.

Hypoxia stimulates trafficking of the ATP7A protein in tumor-associated macrophagesWe then sought to confirm whether hypoxia could elicit similar

changes in ATP7A localization in hypoxic macrophages in vivo.

Previous studies have demonstrated that macrophages are recruited

to the hypoxic regions of solid tumors where they play important

roles in promoting angiogenesis (reviewed by Mantovani et al.,

2006). A prostate tumor xenograft model provided an opportunity

to investigate the intracellular distribution of ATP7A in these

hypoxic tumor-associated macrophages. The tumorigenic human

prostate cell line, PC-3, was chosen for these studies since ATP7A

expression was very low in these cells, thus allowing for easy

identification of ATP7A in macrophages recruited to the tumor. PC-

3 tumors were grown in immunocompromised SCID mice for 5

weeks to a size of approximately 1 cm diameter (0.5 g), and then

excised and cryosectioned for immunofluorescence analysis of

ATP7A expression. This revealed abundant ATP7A protein

Journal of Cell Science 122 (9)

Fig. 1. Hypoxia stimulates trafficking of the ATP7A protein.(A) Immunofluorescence analysis of ATP7A protein in RAW264.7 cells grownunder normoxic (21% O2) or hypoxic (4% O2) conditions for 96 hours. Cellswere fixed, permeabilized and probed with antibodies against for ATP7A andanti-rabbit antibodies conjugated to Alexa Fluor 488 (green). Nuclei werelabeled with DAPI (blue). Note the trafficking of ATP7A protein from theperinuclear region in hypoxic cells and copper-treated normoxic cells. ATP7Awas rapidly retrieved to the perinuclear compartment upon the transfer ofhypoxic cells to normoxic media for 30 minutes (HyprNorm). (B,C) Analysisof Golgi marker proteins in hypoxic RAW264.7 cells. Cells were culturedunder hypoxic or normoxic conditions as described in A and probed usingantibodies against the trans-Golgi network marker protein syntaxin 6 (B), orthe Golgi matrix protein, GM130 (C). Nuclei were labeled with DAPI (blue).Scale bars: 7 μm.

1317O2-dependent regulation of copper homeostasis

expression in tumor-associated macrophages that were identified

using the macrophage-specific marker CD-68 (Fig. 3A). As

expected, there was little if any expression of ATP7A in the PC-3

tumor cells (Fig. 3A). Consistent with previous studies, the

macrophages were typically concentrated at the tumor edges, with

occasional infiltration into the tumor body (Biswas et al., 2006;

Lewis and Pollard, 2006; Murdoch et al., 2004). It was noted that

in some macrophages ATP7A was restricted to the perinuclear Golgi

complex, whereas in other macrophages ATP7A was dispersed

throughout the cell in a manner reminiscent of the trafficking seen

earlier in cultured hypoxic RAW264.7 cells (Fig. 3A, lower left

panel). Significantly, this dispersed localization of the ATP7A

protein occurred only in macrophages that coexpressed the HIF1αprotein (Fig. 3B). The HIF1α transcription factor is the master

regulator of gene expression responses to low oxygen, and is

upregulated in macrophages within hypoxic areas of tumors (Burke

et al., 2002; Talks et al., 2000). These findings, together with our

earlier observations in cultured RAW264.7 cells, suggest that

hypoxia triggers copper-dependent trafficking of ATP7A in

macrophages.

Oxygen limitation stimulates the expression of CTR1 andcopper uptake in macrophagesBased on our finding that the trafficking of ATP7A in response to

hypoxia was dependent on copper, we investigated whether this

might occur through increased copper uptake. Radioactive 64Cu

uptake experiments were carried out using RAW264.7 macrophages

that had been pre-exposed to hypoxic or normoxic conditions. A

significant increase in copper uptake was found for RAW264.7 cells

pre-exposed to hypoxia relative to normoxia (Fig. 4A). Interestingly,

this increased copper uptake was associated with a time-dependent

increase in the levels of the CTR1 copper importer (Fig. 4B). A

similar increase in CTR1 expression was observed in murine

primary peritoneal macrophages cultured under hypoxic conditions

(Fig. 4C). Taken together with our earlier results, these findings

suggest that the copper-dependent trafficking the ATP7A protein

is associated with an increase in CTR1 expression and copper

uptake.

Hypoxia stimulates copper transport to ceruloplasmin viaATP7AThe major function of ATP7A is to pump copper into the secretory

pathway to supply copper to secreted cuproenzymes. We

hypothesized that a potential target for this copper delivery in

response to hypoxia might be ceruloplasmin, a cuproenzyme that

requires copper delivery into the secretory pathway. Ceruloplasmin

is a ferroxidase secreted from macrophages and hepatocytes, the

expression and activity of which are stimulated by hypoxia (Martin

et al., 2005; Mukhopadhyay et al., 2000). Hypoxia was found to

Fig. 2. Hypoxia stimulates both copper-dependent trafficking and increasedexpression of ATP7A. (A) RAW264.7 cells were cultured under hypoxic ornormoxic conditions as described in Fig. 1 in the presence or absence of thecopper chelator, tetrathiomolybdate (TTM; 5 nM). ATP7A protein wasdetected using immunofluorescence as described in Fig. 1. Scale bars: 10 μm.(B,C) Immunoblot analysis of ATP7A protein in RAW264.7 cells (B) andprimary peritoneal macrophages (C) cultured under normoxic (N; 21% O2) orhypoxic (H; 4% O2) conditions for the indicated times. Tubulin was detectedas a loading control. Relative ATP7A band intensities at each time pointnormalized against tubulin are shown for each normoxic and hypoxic pair.

Fig. 3. ATP7A trafficking in HIF1α-positive tumor-associated macrophages.(A) ATP7A is strongly expressed in tumor-associated macrophages. Humanprostate cell PC-3 xenograft tumors from SCID mice were cryosectioned andprobed with antibodies against ATP7A (green) or antibodies against themacrophage marker CD68 (red). Nuclei were stained using DAPI (blue).Upper panels show both the tumor mass and the tumor edge, with strongATP7A expression in CD68-positive macrophages associated with the tumoredge Scale bar: 60 μm. The lower panels are higher magnifications of thetumor periphery and reveal extensive coexpression of ATP7A in CD68-positive macrophages, as indicated by yellow signal in the merged image.Scale bar: 30 μm. The arrow and arrowhead (lower left panel) indicateheterogeneous localization of the ATP7A in macrophages, in either aperinuclear or dispersed distribution. (B) The dispersed distribution of theATP7A occurs in HIF1α-positive macrophages. Three different PC-3 tumorcryosections were immunostained for ATP7A (green) and HIF1α staining(red). ATP7A was restricted to the perinuclear region of macrophages negativefor HIF1α expression (arrows), whereas a dispersed distribution of ATP7Aoccurred in HIF1α-positive macrophages (arrowheads) (originalmagnification, �600). Scale bar: 10 μm.

1318

increase both the abundance and the activity of ceruloplasmin

secreted into the culture medium of RAW264.7 cells relative to

normoxia (Fig. 5A,B). To examine whether the increase in

ceruloplasmin activity was dependent on ATP7A copper transport

activity, we depleted ATP7A expression in RAW264.7 cells using

RNAi-mediated gene silencing (ATP7A/RNAi cells; Fig. 5C).

Control cells were transfected with a construct expressing an

irrelevant RNAi against GFP (Fig. 5C). Compared with control cells,

ceruloplasmin activity in ATP7A/RNAi cells was markedly reduced

under hypoxic conditions, suggesting that ATP7A copper transport

activity was required for copper delivery to ceruloplasmin (Fig. 5D).

Consistent with this postulate, the addition of copper to the medium

of these cells bypassed the requirement for ATP7A and restored

ceruloplasmin activity, indicating that the effect of ATP7A gene

silencing was due to a blockage of copper delivery to ceruloplasmin

(Fig. 5D). Control experiments indicated that ATP7A silencing did

not alter ceruloplasmin protein levels in the medium relative to

control cells in either hypoxic or normoxic conditions (data not

shown). Taken together with our earlier results, these findings

suggest that hypoxia stimulates an increase in copper delivery to

ceruloplasmin by increasing CTR1-mediated copper uptake as well

as ATP7A-dependent copper delivery into secretory compartments.

Hypoxia differentially affects intracellular copper pathwaysHaving established that hypoxia increases the delivery of copper

to ceruloplasmin via the ATP7A copper transporter, we explored

the effect of hypoxia on two additional targets of intracellular copper

pathways. These include Cu/Zn-superoxide dismutase (SOD1) in

the cytoplasm and CCO in the mitochondria. Hypoxia resulted in

a time-dependent decrease of SOD1 activity in RAW264.7

macrophages (Fig. 6A). Interestingly, this reduction in SOD1

activity was associated with a marked reduction in the level of CCS

protein, which is the copper chaperone required for copper delivery

to SOD1 (Fig. 6B). The activity of the cuproenzyme, CCO, was

also markedly reduced in mitochondrial preparations isolated from

hypoxic RAW264.7 macrophages (Fig. 6C), and this was

accompanied by reduced levels of COX1 protein, a copper

containing subunit of CCO (Fig. 6D). Hypoxia did not result in

detectable changes in other copper chaperones COX17, SCO1 or

SCO2, which are required for copper delivery to CCO (data not

shown). Taken together, these findings suggest that hypoxia

differentially impacts intracellular copper handling pathways by

decreasing copper delivery to SOD1 and CCO, while up-regulating

copper delivery to the secretory pathway via ATP7A.

DiscussionHypoxia is a stress that is commonly encountered by macrophages

as they migrate away from the vasculature. Accordingly, this cell

type is uniquely adapted to function within this hostile

microenvironment. In this study we demonstrate that reduced

oxygen levels promote adaptive changes in intracellular copper

homeostasis in macrophages by specifically enhancing copper

delivery to the biosynthetic pathway via the ATP7A protein

(illustrated in Fig. 7). Copper-dependent trafficking of the ATP7A

protein from the trans-Golgi network is one of the clearest biological

indicators of increased cytoplasmic copper concentrations. Previous

studies have shown that copper-stimulated trafficking is dependent

Journal of Cell Science 122 (9)

Fig. 4. Hypoxia stimulates copper uptake and CTR1 expression in RAW264.7macrophages. (A) Copper uptake activity. RAW264.7 cells were pre-exposedto normoxia (21% O2) or hypoxia (4% O2) for 72 hours and 64Cu uptake wasmeasured over 5 minutes. Values were normalized against total proteinconcentrations (mean + s.d.; n=3; *P<0.05). (B,C) The effect of hypoxia onCTR1 protein levels in RAW264.7 cells (B) and primary peritonealmacrophages (C) cultured under normoxia (N; 21% O2) or hypoxia (H; 4%O2) for the indicated times. Immunoblot analysis was used to detect CTR1protein in lysates using anti-CTR1 antibodies. Tubulin was detected as aloading control. Relative CTR1 band intensities at each time point, normalizedagainst tubulin, are shown for each normoxic and hypoxic pair.

Fig. 5. ATP7A-dependent copper transport is required for hypoxia-stimulatedceruloplasmin activity. (A) Immunoblot analysis of ceruloplasmin (Cp)secreted from RAW264.7 cells grown under normoxic (N; 21% O2) or hypoxic(H; 4% O2) conditions for the indicated times. Conditioned medium wasconcentrated and subjected to non-denaturing SDS-PAGE and immunoblotanalysis with anti-Cp antibodies. Tubulin levels from corresponding celllysates are also shown. Lane 1 is a negative control of concentrated growthmedium alone (M). (B) Hypoxia stimulates ceruloplasmin activity.Ceruloplasmin activity (p-phenylenediamine oxidase activity) was measuredin the concentrated conditioned medium from RAW264.7 cells followingexposure to normoxia (N; 21% O2) or hypoxia (H; 4% O2) for 72 hours.Activity was normalized against total protein content of the corresponding celllysates (mean + s.d.; n=3). (C) RNAi-mediated silencing of the ATP7Aprotein. Immunoblot analysis of ATP7A protein levels in RAW264.7 cellsstably transfected with either ATP7A-RNAi or control-RNAi.(D) Ceruloplasmin activity was measured in conditioned medium from theATP7A-RNAi or control-RNAi cells exposed to hypoxia (N; 21% O2) orhypoxia (H; 4% O2) for 72 hours (mean + s.d.; n=3). Note the failure toactivate ceruloplasmin in ATP7A-depleted cells, and the restoration byaddition of copper to the growth medium (+Cu).

1319O2-dependent regulation of copper homeostasis

on ATP7A copper transport activity as well as copper binding to

cysteines within its amino-terminal region (Strausak et al., 1999;

Petris et al., 2002). Our finding that hypoxia stimulated the

relocalization of ATP7A to post-Golgi vesicles in a copper-

dependent manner is consistent with increased copper binding to

this protein, as well as an increase in its copper transport activity

into the secretory pathway. This was supported by the finding that

the increased activity of ceruloplasmin, an enzyme that acquires

copper within secretory compartments, was dependent on ATP7A

expression in hypoxic cells.

In contrast to ceruloplasmin, the abundance and/or activity of

CCS, SOD1 and CCO were reduced by hypoxia. These findings

provide the first evidence that the pathways of intracellular copper

distribution can be differentially regulated in response to an

environmental stress. By reducing the flow of copper from CCS to

SOD1, this may provide a mechanism to redirect copper to the

ATP7A protein. Like SOD1, the activity of CCO was also

diminished by hypoxia in RAW264.7 cells and this was associated

with a decrease in levels of COX1 protein, the subunit of CCO

containing the CuB site. Although a decrease in CCO activity has

been previously reported in hypoxic macrophages to facilitate the

metabolic shift from oxidative phosphorylation to glycolysis

(Murphy et al., 1984; Simon et al., 1977), our findings highlight

the possibility that COX1 depletion serves an additional purpose

of diverting precious copper stores to secretory compartments via

ATP7A.

The finding that ceruloplasmin was a recipient of increased

copper delivery to the secretory pathway is in agreement with the

function of this protein in iron homeostasis. Ceruloplasmin is a

ferroxidase required for cellular iron export, which is a critical step

in the loading of iron onto transferrin in the blood (Nittis and Gitlin,

2002). This process is an adaptive response to hypoxia to meet the

increased iron demand of hematopoiesis (Sarkar et al., 2003). Thus,

the prioritization of copper delivery to ceruloplasmin via ATP7A

may ultimately function to regulate iron homeostasis in response

to hypoxia. The finding that ATP7A trafficking in hypoxic cells

occurred concurrently with ceruloplasmin activation raises the

possibility that under hypoxic conditions, copper delivery to

ceruloplasmin may occur in post-Golgi vesicles rather than in the

trans-Golgi network where copper loading normally takes place.

Thus, ATP7A trafficking may not simply reflect an increased flux

Fig. 6. Hypoxia downregulates alternative copper pathways in RAW264.7macrophages. (A) Effect of hypoxia on SOD1 protein and activity. RAW264.7cells were grown under normoxic (N; 21% O2) or hypoxic (H; 4% O2)conditions for the indicated times. Cell lysates were subjected to non-denaturing SDS-PAGE for the in-gel SOD1 activity assay (top panel).Immunoblots from the same lysates were probed with anti-SOD1 antibodies todetect SOD1 protein. Tubulin was detected as a loading control. RelativeSOD1 band intensities at each time point, normalized against tubulin, areshown for each normoxic and hypoxic pair. (B) Effect of hypoxia on theabundance of CCS, the copper chaperone for SOD1. The same lysates as in Awere subjected to SDS-PAGE and immunoblot analysis with anti-CCSantibodies and relative band intensities at each time point normalized againsttubulin are shown for each normoxic and hypoxic pair. (C) Analysis of CCOactivity in hypoxic (shaded bars) and normoxic (solid bars) conditions.Activity was measured in mitochondrial preparations isolated from RAW264.7cells cultured as described in A. Values were normalized against totalmitochondrial protein (mean + s.d.; n=3; *P<0.05). (D) Analysis of COX1protein levels in RAW264.7 cells cultured under normoxic (N; 21% O2) orhypoxic (H; 4% O2) conditions for the indicated times. Mitochondrialpreparations from B were subjected to SDS-PAGE and probed with antibodiesagainst the copper-binding subunit COX1 of the CCO complex. Immunoblotswere probed with an antibody against porin to indicate protein loading, andrelative COX1 band intensities at each time point, normalized against porin,are shown for each normoxic and hypoxic pair.

Fig. 7. Schematic model of hypoxia-induced changes in copper homeostasisresulting in ATP7A-mediated copper transport to the secretory pathway.Effects of hypoxia (H) include: (1) increased expression of the CTR1 copperimporter and increased copper uptake; (2) decreased CCS expression; (3)reduced activity of SOD1; (4) reduced CCO activity and reduced expression ofCOX1; (5) increased expression of ATP7A; (6) copper-dependent traffickingof ATP7A; and (7) ATP7A-dependent copper transport to the secretorypathway of ceruloplasmin.

1320

of copper to this protein, but facilitate copper-loading of

ceruloplasmin in post-Golgi compartments. Indeed, a recent study

demonstrating a subset of ATP7A protein was required in post-Golgi

melanosomes for the copper loading of tyrosinase is consistent with

this model (Setty et al., 2008). A particularly intriguing finding of

our study was the strong expression of ATP7A in tumor-associated

macrophages. Copper has been shown to play an important role in

angiogenesis, and copper chelation via TTM has proved to be an

effective suppressor of tumor growth in animals (Alessandri et al.,

1984; Camphausen et al., 2004; Cox et al., 2003; Cox et al., 2001;

Pan et al., 2003a; Pan et al., 2003b; Pan et al., 2002; Redman et

al., 2003; Teknos et al., 2005). It is therefore tempting to speculate

that the adaptive changes in macrophage copper homeostasis

described in this study may underlie the role of copper in tumor

growth.

A notable finding of our study was that the changes in copper

homeostasis in response to hypoxia appear to be restricted to

macrophages. ATP7A trafficking and/or changes in SOD1 and CCO

activity were not observed in our analysis of cultured cells from a

variety of sources including HeLa (cervical carcinoma), HEK293

(human embryonic kidney), N2a (neuroblastoma), primary human

aortic endothelial cells and primary rat smooth muscle cells (data

not shown). The macrophage-specific effects of hypoxia on copper

homeostasis may reflect inflammatory responses, since hypoxia is

known to specifically activate inflammatory pathways in

macrophages (Rius et al., 2008). Consistent with this postulate, our

unpublished studies demonstrate that pro-inflammatory agents can

stimulate copper-dependent ATP7A trafficking in macrophages

under normoxic conditions. It will be of interest to determine

whether other physiological conditions regulate changes in the

intracellular distribution of copper in other mammalian cell types.

For example, copper transport via ATP7A is required for melanin

production via tyrosinase, norepinephrine synthesis via dopamine

β-hydroxylase, and collagen cross-linking via lysyl oxidase, and

each of these cuproenzymes is stimulated by particular physiological

cues in specific cell types. The challenge of future studies will be

to address whether this involves adaptive changes that promote an

increase in ATP7A-dependent copper transport into the secretory

pathway.

Materials and MethodsReagents and antibodiesAll reagents were from Sigma (St Louis, MO), unless otherwise indicated. The rabbit

polyclonal CTR1 antibody (Nose et al., 2006) was kindly provided by Dennis Thiele

(Duke University, Durham, NC). The rabbit polyclonal antibody against ATP7A was

raised against the C-terminal portion of the protein and was a generous gift from

Elizabeth Eipper (Steveson et al., 2003). Additional affinity purified anti-ATP7A

antibodies were raised in rabbits against the synthetic peptide NH2-

CDKHSLLVGDFREDDDTTL-COOH (Bethyl Laboratories, Mongomery, TX). Anti-

tubulin antibody, and secondary HRP-conjugated antibodies were purchased from

Roche Molecular Biochemicals. Antibodies against COXI, and fluorescent Alexa

Fluor 488- and Alexa Fluor 594-conjugated antibodies were from Invitrogen

(Carlsbad, CA). Antibodies against GM130 and syntaxin 6 were purchased from BD

Transduction Laboratories (San Jose, CA). Antibodies against CD68 and HIF1αwere

purchased from Serotec (Raleigh, NC) and Novus Biologicals (Littleton, CO),

respectively. Antibodies against Cu/Zn-SOD, CCS and ceruloplasmin antibodies were

purchased from Stressgen (Ann Arbor, MI), Santa Cruz Biotechnology (Santa Cruz,

CA), and Abcam (Cambridge, MA), respectively.

Cell cultureAll cell lines were obtained from the American Type Culture Collection and were

maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% fetal

bovine serum and 100 i.u./ml penicillin and streptomycin (Invitrogen) in 5% CO2 at

37°C. Primary macrophages were isolated by peritoneal lavage. C57BL/6J mice were

injected with 2 ml of thioglycolate medium into the peritoneum to elicit macrophage

infiltration. After 72 hours, macrophages were isolated by peritoneal lavage using

ice-cold PBS. Cells were seeded in six-well plates for each experiment as described.

Hypoxic atmospheres were generated by displacement with N2 and CO2 gas using a

trigas hypoxic incubator. RNAi-mediated silencing of ATP7A in RAW264.7 cells

was performed by stable transfection of a pRS vector expressing a 29 nucleotide

short hairpin (sh) RNA against ATP7A (Origene, Rockville, MD) followed by selection

in 25 μg/ml puromycin. Control cells were transfected with the same vector

expressing shRNA against GFP. Lipofectamine 2000 (Invitrogen) was used in all

transfections.

Copper uptakeRadioactive copper (64Cu) was purchased from the Mallinckrodt Institute of Radiology,

Washington University (Saint Louis, MO). Cells were pre-cultured in triplicate for

72 hours in 6-well plates under either normoxic (21% O2) or hypoxic (4% O2)

conditions, and then exposed to 1 μM 64Cu for 5 minutes, washed extensively in ice-

cold PBS and radioactivity measured using a gamma counter. Counts were normalized

against total protein.

Immunocytochemistry and PC-3 tumor xenograftsImmunofluorescence microscopy and western blot analysis were performed as

described previously (Mao et al., 2007). PC-3 prostate carcinoma cells (5�106) were

injected subcutaneously into one flank of anesthetized 4-week-old ICRSC-M SCID

mice obtained from Taconic (Germantown, NY). Mice were maintained in an approved

pathogen-free institutional housing and studies were conducted as outlined in the

NIH Guidelines for the Care and Use of Laboratory Animals and the Policy and

Procedures for Animal Research of the Harry S. Truman Veterans Memorial Hospital.

After a period of 4 weeks solid tumors of appropriately 1 cm diameter were excised

from anesthetized mice and flash frozen in isopentane. Frozen tumors were

cryosectioned, fixed in acetone for 10 minutes, washed in phosphate-buffered saline

(PBS) and blocked overnight in 1% casein in PBS. Immunostaining was performed

using antibodies against ATP7A, CD68 or HIF1α, followed by staining with Alexa

Fluor 488 anti-rabbit and Alexa Fluor 594 anti-mouse antibodies, as indicated in the

figure legends. Nuclei were stained with 4�,6-diamidino-2-phenylindole (DAPI).

Enzyme assaysCCO activity assays were performed using mitochondrial preparations from

RAW264.7 cells obtained using the Cell Mitochondria Isolation Kit from Sigma.

CCO activity was measured using a CCO assay kit (Sigma) according to the

manufacturer’s instructions and activity was normalized against mitochondrial protein

content.

Superoxide dismutase assays were performed as described previously (Flohe and

Otting, 1984). Briefly, RAW264.7 cell lysates were fractionated using nondenaturing

12% polyacrylamide gel electrophoresis and superoxide dismutase activity was

detected by incubation of gels in nitro blue tetrazolium at room temperature.

Ceruloplasmin activity in concentrated conditioned media was determined by its p-

phenylenediamine oxidase activity as previously described (Sunderman and Nomoto,

1970). RAW264.7 cells were grown in 6-well plates and the conditioned medium

was collected and concentrated using Amicon Ultra-4 filter tubes (Millipore). A low

level of endogenous ceruloplasmin activity in concentrated medium alone was

subtracted from that of conditioned medium for each experiment. Ceruloplasmin

activity was normalized against total protein content in the cell pellets. Ceruloplasmin

protein levels were detected in concentrated medium using immunoblot analysis.

We thank Dennis Thiele (Duke University, Durham, NC) for anti-CTR1 antiserum and Betty Eipper (University of Connecticut HealthCenter, Farmington, CT) for anti-ATP7A antiserum. We thank membersof the Petris lab for their support. This work was supported by grantsfrom the National Institutes of Health DK66333 and DK59893 to M.J.P.and DK79209 to J.L. The production of 64Cu at Washington UniversitySchool of Medicine is supported by a grant from the NIH-NCI R24CA86307. T.K. was supported, in part, by a fellowship from the UeharaMemorial Foundation and the Naito Foundation. Deposited in PMCfor release after 12 months.

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