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Human Macrophage ATP7A is Localized in the trans-Golgi Apparatus, Controls Intracellular Copper Levels, and Mediates Macrophage Responses to Dermal Wounds

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Abstract

The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.

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Abbreviations

calcein AM:

Calcein acetoxymethyl ester

cPLA2α :

Cytosolic phospholipase A2α

DAPI:

4′,6-Diamidino-2-phenylindole

DIC:

Differential interference contrast

ICP-MS:

Inductively coupled plasma mass spectrometry

PBMC:

Peripheral blood mononuclear cell

PBS:

Phosphate-buffered saline

PDGF:

Platelet-derived growth factor

PMA:

Phorbol-12-myristate-13-acetate

SXRF:

Synchrotron X-ray fluorescence

VEGF:

Vascular endothelial growth factor

VSMC:

Vascular smooth muscle cell

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Acknowledgments

This work was supported by a National Scientist Development Grant (0835268N) from the American Heart Association and grants HL-076684 and HL-62984 from the National Institute of Health. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. We thank Birgit Ehmer and Chet Closson for assisting with the immunofluorescence microscopy and confocal microscopy, Dr. Oyebode Olakanmi for developing the protocol to isolate human PBMC-derived macrophages, and Dr. Dennis McGraw for providing human alveolar macrophages.

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Authors and Affiliations

  1. Division of Cardiovascular Disease, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH, 45267, USA

    Ha Won Kim, Qilin Chan, Scott E. Afton, Neal L. Weintraub & Zhenyu Qin

  2. Department of Chemistry, College of Arts and Sciences, University of Cincinnati, Cincinnati, OH, 45267, USA

    Qilin Chan, Scott E. Afton & Joseph A. Caruso

  3. X-Ray Science Division, Argonne National Laboratory, Argonne, IL, 60439, USA

    Barry Lai

  4. Division of Vascular Surgery, Department of Surgery, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA

    Zhenyu Qin

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  1. Ha Won Kim
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  2. Qilin Chan
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Correspondence to Zhenyu Qin.

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Kim, H.W., Chan, Q., Afton, S.E. et al. Human Macrophage ATP7A is Localized in the trans-Golgi Apparatus, Controls Intracellular Copper Levels, and Mediates Macrophage Responses to Dermal Wounds. Inflammation 35, 167–175 (2012). https://doi.org/10.1007/s10753-011-9302-z

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