Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase

TRPM7 is a ubiquitous divalent-selective ion channel with its own kinase domain. Recent studies have shown that suppression of TRPM7 protein expression by RNA interference increases resistance to ischemia-induced neuronal cell death in vivo and in vitro, making the channel a potentially attractive pharmacological target for molecular intervention. Here, we report the identification of the 5-lipoxygenase inhibitors, NDGA, AA861, and MK886, as potent blockers of the TRPM7 channel. Using a cell-based assay, application of these compounds prevented cell rounding caused by overexpression of TRPM7 in HEK-293 cells, whereas inhibitors of 12-lipoxygenase and 15-lipoxygenase did not prevent the change in cell morphology. Application of the 5-lipoxygenase inhibitors blocked heterologously expressed TRPM7 whole-cell currents without affecting the protein's expression level or its cell surface concentration. All three inhibitors were also effective in blocking the native TRPM7 current in HEK-293 cells. However, two other 5-lipoxygenase specific inhibitors, 5,6-dehydro-arachidonic acid and zileuton, were ineffective in suppressing TRPM7 channel activity. Targeted knockdown of 5-lipoxygenase did not reduce TRPM7 whole-cell currents. In addition, application of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), the product of 5-lipoxygenase, or 5-HPETE's downstream metabolites, leukotriene B4 and leukotriene D4, did not stimulate TRPM7 channel activity. These data suggested that NDGA, AA861, and MK886 reduced the TRPM7 channel activity independent of their effect on 5-lipoxygenase activity. Application of AA861 and NDGA reduced cell death for cells overexpressing TRPM7 cultured in low extracellular divalent cations. Moreover, treatment of HEK-293 cells with AA861 increased cell resistance to apoptotic stimuli to a level similar to that obtained for cells in which TRPM7 was knocked down by RNA interference. In conclusion, NDGA, AA861, and MK886 are potent blockers of the TRPM7 channel capable of attenuating TRPM7's function during cell stress, making them effective tools for the biophysical characterization and suppression of TRPM7 channel conductance in vivo

Protein array autoantibody profiles for insights into systemic lupus erythematosus and incomplete lupus syndromes

The objective of this study was to investigate the prevalence and clinical significance of a spectrum of autoantibodies in systemic lupus erythematosus and incomplete lupus syndromes using a proteome microarray bearing 70 autoantigens. Microarrays containing candidate autoantigens or control proteins were printed on 16-section slides. These arrays were used to profile 93 serum samples from patients with systemic lupus erythematosus (SLE (n = 33), incomplete LE (ILE; n = 23), first-degree relatives (FDRs) of SLE patients (n = 20) and non-autoimmune controls (NC; n = 17). Data were analysed using the significance analysis of microarray (SAM) and clustering algorithms. Correlations with disease features were determined. Serum from ILE and SLE patients contained high levels of IgG autoantibodies to 50 autoantigens and IgM autoantibodies to 12 autoantigens. Elevated levels of at least one IgG autoantibody were detected in 26% of SLE and 19% of ILE samples; elevated IgM autoantibodies were present in 13% of SLE and 17% of ILE samples. IgG autoantibodies segregated into seven clusters including two specific for DNA and RNA autoantigens that were correlated with the number of lupus criteria. Three IgG autoantibody clusters specific for collagens, DNA and histones, were correlated with renal involvement. Of the four IgM autoantibody clusters, two were correlated negatively with the number of lupus criteria; none were correlated with renal disease. The IgG : IgM autoantibody ratios generally showed a stepwise increase in the groups following disease burden from NC to SLE. Insights derived from the expanded autoantibody profiling made possible with the antigen array suggest differences in autoreactivity in ILE and SLE. Determining whether the IgM aurotreactivity that predominates in ILE represents an early stage prior to IgG switching or is persistent and relatively protective will require further longitudinal studies

Pharmacodynamics of recombinant human DNase I in serum

Recombinant human deoxyribonuclease I (rhDNase) may be an effective therapeutic for the treatment of systemic lupus erythematosus (SLE). The pharmacodynamics of rhDNase in serum was investigated using two activity assays: one based on hydrolysis of a radiolabelled phage DNA and the other based on hydrolysis of human chromatin. The concentration of endogenous immunoreactive DNase in sera from 16 normal subjects was 3.2 ± 1.4 ng/ml (mean ± s.d.); however, low levels or no nuclease activity were detected in the same sera, suggesting the presence of DNase inhibitors. We assessed the ability of rhDNase to degrade DNA in undiluted serum, since the observed inhibition of endogenous DNase was reversed upon dilution. Addition of rhDNase to undiluted serum at a concentration of 50–100 ng/ml was necessary for degradation of radiolabelled phage DNA. The activity of rhDNase added to serum from normal subjects and SLE patients was similar. rhDNase degraded human chromatin and chromatin/anti-DNA immune complexes in serum with similar potency (EC50 ≈ 100–200 ng/ml). A 500-fold variation in the chromatin/anti-DNA stoichiometry did not significantly affect the digestion of these immune complexes by rhDNase in buffer. These results indicate that a minimum rhDNase concentration of 50–100 ng/ml in serum was required to achieve detectable catalytic activity and that the presence of antibodies to DNA did not inhibit the degradation of DNA/anti-DNA immune complexes

Cross-reaction of anti-DNA autoantibodies with membrane proteins of human glomerular mesangial cells in sera from patients with lupus nephritis

Anti-DNA autoantibodies were thought to play a major role in the pathogenesis of lupus nephritis (LN). A recent study revealed that affinity-purified anti-DNA antibodies had a cross-reaction with human glomerular mesangial cells (HMC). However, whether the cross-reaction was antigen–antibody-mediated was unclear. The aim of the current study was to investigate the binding of anti-DNA antibodies to HMC membrane proteins and to characterize the target antigens. Affinity-purified IgG anti-DNA antibodies were purified by DNA-cellulose chromatography in sera from nine patients with biopsy-proven active lupus nephritis. In vitro cultured primary HMCs were disrupted by sonication and HMC membranes were obtained by differential centrifugation. The membranes of human umbilical vein endothelial cells (HUVEC), human proximal renal tubular epithelial cell line (HK2) and peripheral mononuclear cells (PMC) were obtained as controls. Binding of anti-DNA antibodies to the membrane proteins was investigated by Western blot analysis using soluble membrane proteins as antigens. Both HMC membrane and affinity-purified anti-DNA antibodies were treated with DNase I to exclude DNA bridging. All nine affinity-purified anti-DNA antibodies could blot the HMC membrane proteins, and there were at least three bands at 74 kDa, 63 kDa and 42 kDa that could be blotted. Among the nine IgG preparations, all nine (100%) could blot the 74 kDa band; eight (88·9%) could recognize 63 kDa and 42 kDa protein bands separately. After DNase treatment, the same bands could still be blotted by most affinity-purified anti-DNA antibodies. Affinity-purified anti-DNA antibodies could also blot similar bands on membrane proteins of other cells, but some bands were different. In conclusion, anti-DNA autoantibodies could cross-react directly with cell membrane proteins of human glomerular mesangial cells and might play an important role in the pathogenetic mechanism in lupus nephritis