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Cadmium favors F-actin depolymerization in rat renal mesangial cells by site-specific, disulfide-based dimerization of the CAP1 protein

  • Inorganic Compounds
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Abstract

Cadmium is a toxic metal that produces oxidative stress and has been shown to disrupt the actin cytoskeleton in rat renal mesangial cells (RMC). In a survey of proteins that might undergo Cd2+-dependent disulfide crosslinking, we identified the adenylyl cyclase-associated protein, CAP1, as undergoing a dimerization in response to Cd2+ (5–40 µM) that was sensitive to disulfide reducing agents, was reproduced by the disulfide crosslinking agent diamide, and was shown by site-directed mutagenesis to involve the Cys29 residue of the protein. Reactive oxygen species are not involved in the thiol oxidation, and glutathione modulates background levels of dimer. CAP1 is known to enhance cofilin’s F-actin severing activity through binding to F-actin and cofilin. F-actin sedimentation and GST-cofilin pulldown studies of CAP1 demonstrated enrichment of the CAP1 dimer’s association with cofilin, and in the cofilin–F-actin pellet, suggesting that Cd2+-induced dimer increases the formation of a CAP1–cofilin–F-actin complex. Both siRNA-based silencing of CAP1 and overexpression of a CAP1 mutant lacking Cys29 (and therefore, incapable of dimerization in response to Cd2+) increased RMC viability and provided some protection of F-actin structures against Cd2+. It is concluded that Cd2+ brings about disruption of the RMC cytoskeleton in part through formation of a CAP1 dimer that increases recruitment of cofilin to F-actin filaments.

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Acknowledgements

This work was funded by Grant #418208 to DMT from the Natural Sciences and Engineering Research Council of Canada.

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Authors and Affiliations

  1. Laboratory Medicine and Pathobiology 1 King’s College Circle, University of Toronto, Toronto, ON, M5S 1A8, Canada

    Ying Liu, Weiqun Xiao & Douglas M. Templeton

  2. Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Manasi Shinde & Jeffrey Field

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  1. Ying Liu
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Correspondence to Douglas M. Templeton.

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204_2017_2142_MOESM1_ESM.tiff

Supplementary Fig. 1 Viability studies of cells exposed to various metal ions. Cells were exposed to various concentrations of Fe, Cu, or Cd salts, [M], under conditions reported in Fig. 6 of the main text. Viability was measured with an MTT assay (dimethylthiazolyl diphenyltetrazolium bromide, Sigma-Aldrich (St. Louis, MO)) as described previously (Liu and Templeton 2008). A significant decrease in viability was seen with (A) FeCl3 at 400 µM and above, and (B) FeSO4 and (C) CuSO4 only by 800 µM. Viability was decreased with (D) 20 µM CdCl2 and above. All values are from 4 measurements in a single experiment, with significance vs. no added metal at p<0.05 (*) or p<0.01 (**). (TIFF 838 KB)

204_2017_2142_MOESM2_ESM.tiff

Supplementary Fig. 2 Comparison of CAP1 dimer response to Cd 2+ in human and rodent cell lines. Different cell lines were held in serum free medium for 6 h without the addition of Cd2+, or supplemented with CdCl2 at the indicated concentrations up to 40 µM, or treated with diamide. Images are of Western blots of CAP1 protein in cell lysates from typical experiments. The positions of CAP1 monomer and dimer are shown to the right of panels C) and E). A) Human embryonic kidney cells. B) Human epithelial HeLa cells, with rodent-derived RMC shown in the right-most lane as a positive control for dimer. C) Mouse fibroblast NIH3T3 cell line. D) HeLa and E) NIH3T3 cells were treated for 45 min with increasing concentrations of diamide (compare Fig. 3, main text). (TIFF 1972 KB)

204_2017_2142_MOESM3_ESM.tiff

Supplementary Fig. 3 Time dependence of dimer at 40 µM CdCl 2 in HEK cells. HEK cells were transfected with either wt or C29A CAP1 and extracts were subjected to Western blotting for dimer expression. A) Cells treated for 6 h in serum-free conditions with or without 40 µM CdCl2. B) Time course from 0-24 h of wt or C29A CAP1-transfected cells exposed to 40 µM CdCl2 in 10 % FBS. (TIFF 1277 KB)

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Liu, Y., Xiao, W., Shinde, M. et al. Cadmium favors F-actin depolymerization in rat renal mesangial cells by site-specific, disulfide-based dimerization of the CAP1 protein. Arch Toxicol 92, 1049–1064 (2018). https://doi.org/10.1007/s00204-017-2142-3

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