Mercury intoxication disrupts tonic signaling in B cells, and may promote autoimmunity due to abnormal phosphorylation of STIM-1 and other autoimmunity risk associated phosphoproteins involved in BCR signaling

Abstract

Epidemiological studies link exposure to mercury with autoimmune disease. Unfortunately, in spite of considerable effort, no generally accepted mechanistic understanding of how mercury functions with respect to the etiology of autoimmune disease is currently available. Nevertheless, autoimmune disease often arises because of defective B cell signaling. Because B cell signaling is dependent on phosphorylation cascades, in this report, we have focused on how mercury intoxication alters phosphorylation of B cell proteins in antigen-non stimulated (tonic) mouse (BALB/c) splenic B cells. Specifically, we utilized mass spectrometric techniques to conduct a comprehensive unbiased global analysis of the effect of inorganic mercury (Hg²⁺) on the entire B cell phosphoproteome. We found that the effects were pleotropic in the sense that large numbers of pathways were impacted. However, confirming our earlier work, we found that the B cell signaling pathway stood out from the rest, in that phosphoproteins which had sites which were affected by Hg²⁺, exhibited a much higher degree of connectivity, than components of other pathways. Further analysis showed that many of these BCR pathway proteins had been previously linked to autoimmune disease. Finally, dose response analysis of these BCR pathway proteins showed STIM1_S575, and NFAT2_S259 are the two most Hg²⁺ sensitive of these sites. Because STIM1_S575 controls the ability of STIM1 to regulate internal Ca²⁺, we speculate that STIM1 may be the initial point of disruption, where Hg²⁺ interferes with B cell signaling leading to systemic autoimmunity, with the molecular effects pleiotropically propagated throughout the cell by virtue of Ca²⁺ dysregulation.

Keywords: Autoimmunity; B Cell; Immunotoxicology; Inorganic Mercury.

Figure 1:

Effect of Hg²⁺ on median abundance of phosphorylated serine, threonine and tyrosine sites in tonic B cells, as determined by mass spectrometry. Error bars represent the standard error of the mean. (* P < 0.03, ** P < 0.05, *** P < 0.01)

Figure 2:

Volcano plots depicting total phosphosite response to mercury at all concentrations examined. B cells were exposed to various concentrations of Hg²⁺, and then for each exposure condition, phosphosites across the entire phosphoproteome were analyzed by mass spectrometry. For each site, the difference in phosphorylation in Hg²⁺ exposed cells with respect to unexposed control cells was determined. Volcano plots depicting total phosphosite response to each concentration of mercury were then constructed, and plotted above. Black points represent phosphosites whose phosphorylation is not significantly different from controls after exposure to mercury. Red points represent phosphosites whose level of phosphorylation is significantly different from controls, (q < 0.1). Large red dots at the top of each volcano plot indicate the percentage of all identified phosphosites which significantly respond, (either with increased or decreased phosphorylation), to Hg²⁺ exposure.

Figure 3:

The PIANO platform was utilized to perform enrichment analysis of the 2222 established GO pathways in order to determine the percentage of GO pathways which were significantly affected by exposure to Hg²⁺ at different concentrations (FDR < 0.05). (To increase resolution in the figure, the results for the 1 and 2μM exposures are magnified and replicated as an insert).

Figure 4:

Graphical depiction of protein: protein interactions between Hg²⁺ sensitive phosphoproteins in B cells determined by reference to the STRING database. Phosphoproteins belonging to the BCR signaling pathway, and to a lesser extent the actin cytoskeleton, are more highly interconnected than Hg²⁺ sensitive phosphoproteins belonging to other pathways in the B cell. (For clarity, nodes and edges associated with proteins located within the central area of the plot are enlarged, and reproduced in the upper right quadrant of the figure).

Figure 5:

Complete Hg²⁺ dose response profiles of phosphosites belonging to autoimmune linked BCR signaling proteins, which are sensitive to at least 5 μM Hg²⁺, an environmentally relevant exposure (Laiosa et al., 2007). (* p < 0.05, ** P < 0.01, *** P < 0.001)