Na⁺ entry through heteromeric TRPC4/C1 channels mediates (-)Englerin A-induced cytotoxicity in synovial sarcoma cells

The sesquiterpene (-)Englerin A (EA) is an organic compound from the plant Phyllanthus engleri which acts via heteromeric TRPC4/C1 channels to cause cytotoxicity in some types of cancer cell but not normal cells. Here we identified selective cytotoxicity of EA in human synovial sarcoma cells (SW982 cells) and investigated the mechanism. EA induced cation channel current (Icat) in SW982 cells with biophysical characteristics of heteromeric TRPC4/C1 channels. Inhibitors of homomeric TRPC4 channels were weak inhibitors of the Icat and EA-induced cytotoxicity whereas a potent inhibitor of TRPC4/C1 channels (Pico145) strongly inhibited Icat and cytotoxicity. Depletion of TRPC1 converted Icat into a current with biophysical and pharmacological properties of homomeric TRPC4 channels and depletion of TRPC1 or TRPC4 suppressed the cytotoxicity of EA. A Na⁺ /K⁺-ATPase inhibitor (ouabain) potentiated EA-induced cytotoxicity and direct Na⁺ loading by gramicidin-A caused Pico145-resistant cytotoxicity in the absence of EA. We conclude that EA has a potent cytotoxic effect on human synovial sarcoma cells which is mediated by heteromeric TRPC4/C1 channels and Na⁺ loading

Characterization of antibody-defined epitopes on HLA-DRB1*04 molecules

We have previously identified antibody-defined epitopes on the HLA-DRB1*04 molecules that are associated with development of rheumatoid arthritis (RA). The expression of a NFLD.D11-defined epitope (D11⁺0401) on B cell lines (BCL) is dependent on HLA-DM. NFLD.D13 recognizes DRB1*0404, but not DRB1*0401 in DM⁺ BCL (D13⁺0404). However, in DM⁻ BCL, NFLD.D13 recognized an epitope on HLA-DRB1*0401 molecules (D13⁺0401). It was speculated that these epitopes arise either through acquisition of peptides from differentiation antigens of through differential processing and acquisition of a peptide from a common protein. Initially, we show that these epitopes were not detected on the surface of synovial fibroblasts from RA patients, despite the up-regulation of DR, DM molecules and compartments necessary for class II antigen processing and presentation. Further investigation showed that the expression of the D11⁺0401 and D13⁺0404 epitopes was restricted to professional antigen presenting cells, with the exception of the melanoma cell MDA MD 435.0401, which expressed D11⁺0401. To Analyze the intracellular mechanisms by the D11⁺0401 and D13⁺0404 are generated, we treated normal and DM⁻ BCL with various inhibitors, including brefeldin A and protease inhibitors, and employed confocal and immunoelectron microscopy to dissect the compartments within the endocytic pathway where these epitopes form. We found that both the D11⁺0401 and D13⁺0404 epitopes from within CD63⁺CD82⁺ lysosomal compartments in normal BCL. The D11⁺0401 epitope requires cytoplasmic and endosomal cysteine proteases, whereas D13⁺0404 epitope expression is protease independent. Both epitopes appear to require tetraspan protein-enriched microdomains for proper surface expression. Interestingly, the D13⁺0401 epitope also required cysteine proteases for its expression, suggesting that a unique set of peptides are responsible for the formation of these DRB1*04 epitopes