Calreticulin binds to gentamicin and reduces drug-induced ototoxicity

Abstract

Aminoglycosides like gentamicin are among the most commonly used antibiotics in clinical practice and are essential for treating life-threatening tuberculosis and Gram-negative bacterial infections. However, aminoglycosides are also nephrotoxic and ototoxic. Although a number of mechanisms have been proposed, it is still unclear how aminoglycosides induce cell death in auditory sensory epithelia and subsequent deafness. Aminoglycosides bind to various intracellular molecules, such as RNA and phosphoinositides. We hypothesized that aminoglycosides, based on their tissue-specific susceptibility, also bind to intracellular proteins that play a role in drug-induced ototoxicity. By conjugating an aminoglycoside, gentamicin, to agarose beads and conducting a gentamicin-agarose pull-down assay, we have isolated gentamicin-binding proteins (GBPs) from immortalized cells of mouse organ of Corti, HEI-OC1. Mass spectrometry identified calreticulin (CRT) as a GBP. Immunofluorescence revealed that CRT expression is concentrated in strial marginal cells and hair cell stereocilia, primary locations of drug uptake and cytotoxicity in the cochlea. In HEI-OC1 cells treated with gentamicin, reduction of CRT expression using small interfering RNA (siRNA) reduced intracellular drug levels. CRT-deficient mouse embryonic fibroblast (MEF) cells as well as CRT siRNA-transfected wild-type MEFs also had reduced cell viability after gentamicin treatment. A pull-down assay using deletion mutants of CRT determined that the carboxyl C-domain of CRT binds to gentamicin. HeLa cells transfected with CRT C-domain deletion mutant construct were more susceptible to gentamicin-induced cytotoxicity compared with cells transfected with full-length CRT or other deletion mutants. Therefore, we conclude that CRT binding to gentamicin is protective against gentamicin-induced cytotoxicity.

FIG. 1.

(A) CRT and GRP94 are GBPs in HEI-OC1 cells. GBPs were pulled down from total cell lysate of HEI-OC1 cells, resolved by SDS-gel electrophoresis, and stained by Coomassie blue. Most of the protein bands were similar between nonpermissive cells at 39°C and permissive cells at 33°C, but two bands were more intense in nonpermissive cells at 39°C. Mass spectrometric analysis on these bands identified CRT and GRP94. (B) Western blot analysis on total cell lysate showed more protein expression of CRT and GRP94 in cells at 39°C compared with 33°C.

FIG. 2.

CRT expression in the cochlea by immunofluorescence. In the organ of Corti, CRT expression was localized in sensory hair cell bundles (A and C). In the stria vascularis, CRT expression was concentrated near the lumenal membranes of marginal cells (D–H, arrowheads). CRT expression is in green and actin in red in (C, F, and H) in the online version. CRT expression was also present in kidney proximal (p) and distal (d) tubules (I). GRP94 expression was weak and unremarkable in the organ of Corti (J) and stria vascularis (K).

FIG. 3.

CRT expression knockdown decreases intracellular gentamicin levels. CRT siRNA or scrambled control siRNA-transfected HEI-OC1 cells at 39°C were treated with gentamicin for 6 h. (A) Immunofluorescence using CRT and gentamicin antibodies (CRT Ab and GT Ab) showed that CRT siRNA reduced cellular CRT and gentamicin immunofluorescence. (B) Intensity of gentamicin immunofluorescence was significantly lower in CRT siRNA-transfected cells compared with control siRNA (**p < 0.01).

FIG. 4.

GRP94 expression knockdown has no effect on intracellular gentamicin levels. GRP94 siRNA or scrambled control siRNA-transfected HEI-OC1 cells at 39°C were treated with gentamicin for 6 h. (A) Immunofluorescence using GRP94 and gentamicin antibodies (GRP94 Ab and GT Ab) showed that GRP4 siRNA did not significantly alter cellular gentamicin immunofluorescence, despite knocking down GRP94 expression. (B) There was no significant difference in gentamicin immunofluorescence between GRP94 siRNA and control siRNA cells.

FIG. 5.

CRT-deficient cells are more susceptible to gentamicin treatment. (A) K41 (crt^+/+) and K42 (crt^−/−) cells were treated with gentamicin for 6 h at 37°C. Gentamicin immunofluorescence in K42 cells were lower compared with most K41 cells. (B) Gentamicin fluorescence intensities, on average, were significantly lower in K42 cells compared with K41 cells (**p < 0.01). (C) Cell viability measured by MTT assay revealed that K42 cells were more susceptible to gentamicin compared with K41 cells after 3 days of treatment (**p < 0.01). Cells cultured for 3 days with no gentamicin treatment had no significant difference in viability. (D) Gentamicin (10mM) treatment for 3 days increased CRT protein expression levels in K41 cells. There was no CRT expression detected in K42 cells as these cells are CRT deficient.

FIG. 6.

CRT knockdown increases susceptibility to gentamicin cytotoxicity. (A) K41 cells transfected with CRT siRNA showed reduced intracellular gentamicin levels compared with cells transfected with control siRNA, after 6 h of treatment at 37°C. (B) Quantification of fluorescence intensities showed that intracellular gentamicin levels in CRT siRNA-transfected K41 cells were significantly lower than control siRNA cells (**p < 0.01). (C) Western blotting confirmed that the two different siRNA for CRT reduced the protein expression efficiently in K41 cells, unlike the two scrambled control siRNA. (D) MTT assay on cells treated with gentamicin for 3 days showed that CRT siRNA#2-transfected K41 cells had lower viability compared with control siRNA#2.

FIG. 7.

CRT binds to gentamicin through the C-domain, and it contributes to cellular resistance to gentamicin cytotoxicity. (A) HeLa cells were transfected with GFP fusions of full-length CRT (CRT-GFP) or deletion mutants of the N-, P-, and C-domains (ΔN-GFP, ΔP-GFP, and ΔC-GFP) that retained the ER localization signal. Confocal microcopy confirmed that all the GFP fusion proteins had similar intracellular distributions. (B) Gentamicin-agarose pull-down assay was performed using 293T cells transfected with the GFP fusions of full-length CRT or deletion mutants. Western blotting revealed that the mutant lacking the C-domain was not pulled down by gentamicin-agarose. (C) HeLa cells were transfected with the GFP fusions, and cells were treated with gentamicin after 24 h. Cell viability was measured after 3 days of gentamicin treatment (10mM). The bars represent the ratio to cells of the respective transfectants not treated with gentamicin (the number is set as 1 for cells with no gentamicin treatment). The C-domain deletion mutant showed significantly lower viability compared with full-length CRT-GFP (**p < 0.01).

FIG. 8.

Schematic diagram of CRT structure. CRT consists of a globular N-domain, a proline-rich P-domain, and an acidic C-domain. The N-domain includes polypeptide and carbohydrate-binding sites, and the P-domain includes the binding site for ERp57, an oxidoreductase that forms complexes with CRT for the chaperone function (Oliver et al., 1997). The C-domain contains a large number of negatively charged amino acids that bind to Ca²⁺. Our pull-down assay shows that gentamicin binds to the C-domain.