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The zinc transporter ZIP12 regulates the pulmonary vascular response to chronic hypoxia

Nature volume 524, pages 356–360 (2015)Cite this article

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Abstract

The typical response of the adult mammalian pulmonary circulation to a low oxygen environment is vasoconstriction and structural remodelling of pulmonary arterioles, leading to chronic elevation of pulmonary artery pressure (pulmonary hypertension) and right ventricular hypertrophy. Some mammals, however, exhibit genetic resistance to hypoxia-induced pulmonary hypertension1,2,3. We used a congenic breeding program and comparative genomics to exploit this variation in the rat and identified the gene Slc39a12 as a major regulator of hypoxia-induced pulmonary vascular remodelling. Slc39a12 encodes the zinc transporter ZIP12. Here we report that ZIP12 expression is increased in many cell types, including endothelial, smooth muscle and interstitial cells, in the remodelled pulmonary arterioles of rats, cows and humans susceptible to hypoxia-induced pulmonary hypertension. We show that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia dependent and that targeted inhibition of ZIP12 inhibits the rise in intracellular labile zinc in hypoxia-exposed pulmonary vascular smooth muscle cells and their proliferation in culture. We demonstrate that genetic disruption of ZIP12 expression attenuates the development of pulmonary hypertension in rats housed in a hypoxic atmosphere. This new and unexpected insight into the fundamental role of a zinc transporter in mammalian pulmonary vascular homeostasis suggests a new drug target for the pharmacological management of pulmonary hypertension.

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Figure 1: The pulmonary vascular response to hypoxia in the F344 rat is influenced by a region of chromosome 17 containing Slc39a12.
Figure 2: Slc39a12 encodes a zinc transporter, ZIP12, which is upregulated in pulmonary vascular tissue from mammals exposed to chronic hypoxia.
Figure 3: ZIP12 knockdown inhibits hypoxia-induced increase in intracellular labile zinc concentration and proliferation of HPASMCs.
Figure 4: Genetic disruption of ZIP12 in WKY rat attenuates hypoxia-induced pulmonary hypertension.

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Acknowledgements

This research was supported by successive grants from British Heart Foundation to M.R.W. and L.Z. (PG/95170, PG/98018, PG/2000137, PG/04/035/16912, PG/12/61/29818, PG/10/59/28478 and RG/10/16/28575). G.A.R. was supported by a Wellcome Trust Senior Investigator Award (WT098424AIA), MRC Programme Grant (MR/J0003042/1) and a Royal Society Research Merit Award. T.A. acknowledges support from European Research Council Advanced Grant ERC-2010-AdG, number 268880. We thank A. I. Garcia-Diaz for advice on genotyping techniques and R. Edwards for advice on antibody production. We thank C. Haley for discussions on the rat genetic map.

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Author notes

  1. Timothy J. Aitman

    Present address: †Present address: Centre for Genomic and Experimental Medicine, Institute of Genetic and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK,

  2. Lan Zhao and Eduardo Oliver: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Experimental Medicine, Centre for Pharmacology and Therapeutics, Imperial College London, Hammersmith Hospital, London, W12 0NN, UK

    Lan Zhao, Eduardo Oliver, Olivier D. Dubois, Emanuele Cotroneo, Chien-Nien Chen, Lei Wang, Cristina Arce & Martin R. Wilkins

  2. Division of Diabetes, Section of Cell Biology and Functional Genomics, Endocrinology and Metabolism, Imperial College London, Hammersmith Hospital, London, W12 0NN, UK

    Guy A. Rutter

  3. Department of Medicine, Section of Epigenomics and Disease, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London, W12 0NN, UK

    Joan Ponsa-Cobas & Jorge Ferrer

  4. Physiological Genomics and Medicine Group, Medical Research Council Clinical Sciences Centre, Hammersmith Hospital, London, W12 0NN, UK

    Klio Maratou, Santosh S. Atanur, Pauline L. Chabosseau & Timothy J. Aitman

  5. Transgenics and Embryonic Stem Cell Laboratory, Medical Research Council Clinical Sciences Centre, Hammersmith Hospital, London, W12 0NN, UK

    Benjamin Moyon & Zoe Webster

  6. Institute of Molecular Biology and Medicine, 3 Togolok Moldo Street, Bishkek, 720040, Kyrgyzstan

    Almaz Aldashev

  7. Department of Pediatrics and Medicine, Division of Critical Care Medicine and Cardiovascular Pulmonary Research Laboratories, University of Colorado Denver, Denver, 80045, Colorado, USA

    Maria G. Frid & Kurt R. Stenmark

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  1. Lan Zhao
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Contributions

L.Z. and M.R.W. were principal investigators on grants from the British Heart Foundation, developed concepts and supervised the project. L.Z., M.R.W. and E.O. designed and implemented the experiments. T.A. gave conceptual advice on the congenic program and whole-genome sequencing. L.Z., M.R.W., T.A., K.M., E.O. and O.D.D. conducted the congenic breeding program. S.S.A. analysed whole-genome sequence data and performed the Polyphen analysis. L.Z., E.O. and O.D.D., with the support of B.M. and Z.W., generated the ZIP12 transgenic rat. L.Z., E.C. and L.W. performed immunohistochemistry and immunofluorescence. E.O. conducted the in vitro cell culture experiment. C.A. and E.O. performed the angiogenesis assay. G.R. supervised and E.O. and P.L.C. conducted the intracellular labile zinc measurement experiments. C.-N.C. and E.O. performed ChIP–PCR. J.P.-C. and E.O. cloned the HRE construct and performed luciferase reporter assays. M.G.F., K.R.S. and A.A. provided cattle and human lung sections. E.O. performed statistical analysis. L.Z., M.R.W. and E.O. interpreted the data and wrote the manuscript. T.A., G.R., J.F., S.S.A. and K.D. edited the manuscript.

Corresponding author

Correspondence to Lan Zhao.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation of congenic and sub-congenic strains.

Congenic rat lines were produced by introgression of the F344 chromosome 17 QTL segment onto the WKY genetic background by repeated backcrossing. Congenic rat strain R47A (WKY.F344-D17Got91/D17Rat51) contains 15 Mbp from the F344 donor region that maps to the distal end of the QTL on a WKY background. Three sub-congenic strains, SubA (WKY.F344-D17Got91/D17Rat47), SubB (WKY.F344-D17Rat47/D17Rat51) and SubC (WKY.F344-D17Rat131/D17Rat51), were produced containing separate fragments of the R47A donor region by backcrossing (R47A × WKY) F1 with WKY parental rats. Three recombination events within the R47A congenic interval divided the congenic interval into three smaller and overlapping sub-congenic intervals (Fig. 1 and main text).

Extended Data Figure 2 Dissection of QTL and cardiovascular phenotype of rat strains.

a, An illustrative genetic map showing the relationship of the congenic strains (R42, R47A), subcongenic strains (SubA, SubB, SubC) and Slc39a12 to the original QTL (defined by a LOD score >3 (ref. 2)) on a physical map of chromosome 17 (using Rat Genome Assembly version 5.0). b, The hypoxia-resistant F344 phenotype tracks with the congenic R47A line. Rats were kept in 10% O2 for 2 weeks and right ventricular hypertrophy was significantly attenuated in the congenic R47A strain (0.32 ± 0.03, n = 13, **P < 0.01) compared with WKY rats (0.37 ± 0.03, n = 15), whereas congenic R42 rats (0.36 ± 0.03, n = 17) were similar (NS) to WKY rats. In normoxia, WKY, F344, R47A, SubA, SubB and SubC rats show no significant differences in (c) mPAP, (d) right ventricular hypertrophy and (e) vascular muscularization (n = 8 each group); f, systemic blood pressure (SBP) is similar in all strains in both normoxia and hypoxic conditions. g, F344, R47A and SubB rats exhibit attenuated pulmonary vascular remodelling after 2 weeks exposure to a 10% O2 atmosphere compared with WKY, SubA and SubC rats (n = 6 each group). Values are expressed as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 compared with WKY (percentage of fully muscularized and partly muscularized vessels); ##P < 0.01, ###P < 0.001 compared with WKY (percentage of non-muscularized vessels) after one-way ANOVA followed by Bonferroni correction for multiple testing.

Extended Data Figure 3 Hypoxia-induced pulmonary vascular remodelling in parental strains.

a, Upper panel sequence shows the WKY protein sequence (688aa); lower panel shows the truncated F344 protein sequence (553aa). Stars (*) mark the mutated amino acids compared with WKY protein. Dotted line indicates the C-terminal truncated region in F344. The grey square highlights the metalloprotease motif. b, Prominent ZIP12 immunostaining is seen in remodelled pulmonary arterioles in the chronically hypoxic WKY rat alongside vessels with a double elastic lamina (stained with elastic Van Gieson) but not F344 lungs exposed to hypoxia. Red arrow, vessel with double elastic lamina; blue arrow, vessel with single elastic lamina.

Extended Data Figure 4 ZIP12 upregulation in response to hypoxia exposure and measurements of intracellular labile zinc concentration and proliferation of HPASMCs in normoxic conditions.

a, Upregulation of ZIP12 in HPASMCs exposed to hypoxia, in contrast to other zinc transporters (n = 6). b, Representative western blots demonstrating increased HIF-1α and HIF-2α expression in HPASMCs after exposure to hypoxia for 24 h. c, Confocal laser scanning images of HPASMCs transfected with eCALWY-4 probe. Intracellular free zinc was not affected by transfection with ZIP12 siRNA in normoxia. d, Representative traces showing the changes in fluorescence ratio using the eCALWY-4 probe. e, Quantification of intracellular zinc levels (n = 10). f, ZIP12 siRNA did not affect proliferation of HPASMCs in normoxic conditions (n = 5).

Extended Data Figure 5 Design of specific Slc39a12 ZFN and confirmation of mutant line.

a, CompoZr Custom Zinc Finger Nucleases (Sigma-Aldrich) for the rat Slc39a12 gene were designed to target exon 8. b–d, Cel-I surveyor assay and gene sequencing confirmed NHEJ-induced mutations in at least one pup (mutant 77). e, The 4-bp (AGTT) deletion followed by 2-bp insertion (TA) into mutant 77 caused a frame-shift in coding, introducing a stop codon leading to a truncated protein. Red star refers to stop codon. c, We subsequently genotyped next generation litters using SwaI (cutting point: 5′-ATTTAAAT-3′), showing 100% digestion for homozygous pups (−/−), 50% for heterozygous (+/−) and no DNA digestion for WT rats (+/+).

Extended Data Figure 6 ZIP12 knockout attenuated hypoxia-induced pulmonary vascular remodelling.

a, Representative lung sections from WT and ZIP12−/− rats 2 weeks after hypoxia exposure. Elastic van Gieson staining showing double elastic lamina (red arrow) in WT but single elastic laminae (blue arrow) in ZIP12−/− rats. b, Genetic disruption of ZIP12 in WKY rat attenuated pulmonary vascular remodelling after exposure for 2 weeks to a 10% O2 atmosphere compared with WT rats (n = 5 each group). *P < 0.01 compared with WT (percentage of fully muscularized vessels); ##P < 0.01, ###P < 0.001 compared with WT (percentage of non-muscularized vessels) after one-way ANOVA followed by Bonferroni’s multiple comparison test. c, Ki67 staining showing reduced proliferation in hypoxic ZIP12−/− rat lungs compared with the WT strain. *P < 0.01 compared with WT. d, Representative sections from hypoxic WT and ZIP12−/− rats lungs showing differences in staining with the proliferation marker, Ki67. e–g, Genetic disruption of ZIP12 in WKY rat did not influence (e) systemic blood pressure (SBP) or (f) cardiac output (CO) but attenuated hypoxia-induced increases in (g) PVR (n = 7 each group). Values are expressed as the mean ± s.e.m. *P < 0.05, **P < 0.01 compared with normoxic rats, #P < 0.05 compared with WT hypoxic rats after one-way ANOVA followed by Bonferroni correction for multiple testing. h, ZIP12-targeted siRNA inhibition attenuates stress fibre formation in HPASMCs in hypoxia (n = 5 each group). **P < 0.01 compared with normoxia control group, #P < 0.05 compared with hypoxia control group. i, Representative pictures of actin stress fibre in HPASMCs. j, Ex vivo angiogenesis studies demonstrated that vascular outgrowth from ZIP12−/− pulmonary vessels in response to hypoxia was attenuated compared with vessels from WT rats (n = 12 each group, 2 rings per rat, 6 ZIP12−/− and 6 WT rats). *P < 0.05 compared with normoxia WT group; #P < 0.05, ##P < 0.01 and ###P < 0.001 compared with hypoxia ZIP12−/− group. k, Representative pictures of pulmonary arteriole ring outgrowth at day 6.

Extended Data Figure 7 Carbonic anhydrase (CAIX) expression.

a, Representative sections demonstrating increased CAIX expression in remodelled pulmonary arterioles in the lungs of rats exposed to alveolar hypoxia (2 weeks), monocrotaline (MCT, 3 weeks) or a chronic iron-deficient diet (4 weeks). b, c, No CAIX staining was detected in pulmonary arteries of low-altitude (normoxia control, CO calf) calves and sea-level humans, but prominent CAIX immunostaining was observed in the remodelled pulmonary arteries of calves with severe pulmonary hypertension (Hx calf), in cattle with naturally occurring pulmonary hypertension (‘Brisket disease’, BD) as well as patients with IPAH.

Extended Data Figure 8 Genetic disruption of ZIP12 in WKY rat attenuated monocrotaline-induced pulmonary hypertension.

a, mPAP, (b) right ventricular hypertrophy and (c) pulmonary arteriole muscularization (n = 5 each group). Values are expressed as the mean ± s.e.m. *P < 0.05, **P < 0.01 compared with WT monocrotaline group after unpaired Student’s t-test. d, Representative lung sections from WT and ZIP12−/− rats 3 weeks after monocrotaline injection. Elastic van Gieson staining showing double elastic lamina (red arrow) in WT but single elastic laminae (blue arrow) in ZIP12−/− rats.

Extended Data Table 1 Frameshift and non-synonymous coding mutations in the refined congenic interval of F344 and the other hypoxia-susceptible strains, WKY, spontaneously hypertensive (SHR) and fawn-hooded (FHH) rat strains
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Extended Data Table 2 Polymorphism markers for congenic strain genotyping
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Zhao, L., Oliver, E., Maratou, K. et al. The zinc transporter ZIP12 regulates the pulmonary vascular response to chronic hypoxia. Nature 524, 356–360 (2015). https://doi.org/10.1038/nature14620

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