Transient Receptor Potential Channel A1 (TRPA1) Regulates Sulfur Mustard-Induced Expression of Heat Shock 70 kDa Protein 6 (HSPA6) In Vitro

Abstract

The chemosensory transient receptor potential ankyrin 1 (TRPA1) ion channel perceives different sensory stimuli. It also interacts with reactive exogenous compounds including the chemical warfare agent sulfur mustard (SM). Activation of TRPA1 by SM results in elevation of intracellular calcium levels but the cellular consequences are not understood so far. In the present study we analyzed SM-induced and TRPA1-mediated effects in human TRPA1-overexpressing HEK cells (HEKA1) and human lung epithelial cells (A549) that endogenously exhibit TRPA1. The specific TRPA1 inhibitor AP18 was used to distinguish between SM-induced and TRPA1-mediated or TRPA1-independent effects. Cells were exposed to 600 µM SM and proteome changes were investigated 24 h afterwards by 2D gel electrophoresis. Protein spots with differential staining levels were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano liquid chromatography electrospray ionization tandem mass spectrometry. Results were verified by RT-qPCR experiments in both HEKA1 or A549 cells. Heat shock 70 kDa protein 6 (HSPA6) was identified as an SM-induced and TRPA1-mediated protein. AP18 pre-treatment diminished the up-regulation. RT-qPCR measurements verified these results and further revealed a time-dependent regulation. Our results demonstrate that SM-mediated activation of TRPA1 influences the protein expression and confirm the important role of TRPA1 ion channels in the molecular toxicology of SM.

Keywords: 2D gel electrophoresis; AP18; HEK293; HSP70; MALDI-TOF MS(/MS); TRPA1; nanoHPLC-ESI MS/MS; proteomics; sulfur mustard.

Figure 1

Representative 2D CBB-stained gel electrophoresis of HEKA1 cells. The proteome of HEKA1 control, 600 µM SM-exposed or 2 µM AP18-pre-incubated and SM-exposed cells was investigated. Isoelectric focusing of cell lysates was performed by 7 cm strips (pH 4–7 and pH 6–11, linear). Proteins were separated by 10% Bis-Tris gels. (A) Overview gel displaying 22 differential protein spots after SM exposure compared to controls (white open circles). White frames indicate three SM-induced and TRPA1-regulated proteins. (B) Zoom of the HSPA6 spot and (C,D) zoom of the two protein spots which proteins are listed in Table 1. Experiments were carried out with n = 3 per group. Molecular weight is indicated on the left and the pI value on top of the gels.

Figure 2

MALDI-TOF MS(/MS) measurements of a protein spot after tryptic cleavage identified HSP6A. (A) Peptide mass fingerprint of HSPA6 identified by MASCOT database matching resulted in a score of 86.6. (B) MS/MS spectrum of the ion at m/z 1487.5 resulted in a sequence tag of 13 amino acids. The complete series of y-ions could be found. (C) A sequence coverage of 35.5% was found for HSPA6 (UniProtKB-P17066). Assigned peptides are indicated with a yellow background. The sequence of the peptide subjected to MS/MS fragmentation depicted in (B) (amino acids 39–51) is highlighted with a red background.

Figure 3

RT-qPCR measurements for potentially SM-affected and TRPA1-regulated genes. mRNA levels of (A) HSPA6 in HEKA1 cells, (B) HSPA6 in A549 cells and (C) CAPRIN1, ELAVL1, FHL1, GPHN, NOSIP, NCL, SFXN1 and STRN4 in HEKA1 cells were analyzed 24 h after 600 µM SM exposure by RT-qPCR. White bars indicate the fold change values after SM exposure while grey bars illustrate the effect of 2 µM AP18 pre-incubation on mRNA levels. The dashed lines represent normalized levels of the control samples and dotted lines indicate ±1.5-fold change ranges. Significant differences (p < 0.05) are displayed by asterisks (*). Error bars represent the 95% confidence intervals. Data are derived from independent biological experiments (n = 3). Gene names correspond to the following proteins: Caprin-1 (CAPRIN1), ELAV like protein 1 (ELAVL1), Four and a half LIM domain protein 1 (FHL1), Gephyrin (GPHN), Nitric oxide synthase interacting protein (NOSIP), Nucleolin (NCL), Sideroflexin 1 (SFXN1) and Striatin 4 (STRN4).

Figure 4

Schematic overview suggesting TRPA1-mediated induction of HSPA6 after SM exposure. SM activates TRPA1 channels with a still unknown extra- or intracellular binding site (bold dotted lines). SM as well as AITC treatment increase intracellular Ca²⁺ ([Ca²⁺]_i) levels. AP18 prevents both SM- and AITC-induced TRPA1 activation. [Ca²⁺]_i affects mitochondrial function thereby producing ROS which triggers HSPA6 induction. SM may also cause ROS formation through disturbance of mitochondrial function or other yet not well-defined mechanisms thereby potentially contributing to HSPA6 induction (dashed line) without involvement of TRPA1. Elevation of [Ca²⁺]_i also results in additional biological effects (open triangles). Text boxes list references describing the illustrated effects. Activation, increase or induction is marked with “+” while impairment of mitochondria is indicated with “−”.