ABSTRACT
Rheumatoid arthritis synovial fibroblasts (RA-SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. New druggable targets in RA-SFs would therefore be of high therapeutic interest. The present study shows that the intermediate-conductance, calcium-activated potassium channel KCa3.1 (KCNN4) is expressed at the mRNA and protein level in RA-SFs, is functionally active, and has a regulatory impact on cell proliferation and secretion of pro-inflammatory and pro-destructive mediators. Whole-cell patch-clamp recordings identified KCa3.1 as the dominant potassium channel in the physiologically relevant membrane voltage range below 0 mV. Stimulation with transforming growth factor ß1 (TGF-ß1) significantly increased transcription, translation, and channel function of KCa3.1. Inhibition of KCa3.1 by the selective, pore-blocking inhibitor TRAM-34, (and, in part, by siRNA) significantly reduced cell proliferation, as well as expression and secretion of pro-inflammatory factors (IL-6, IL-8, and MCP1) and the tissue-destructive protease MMP3. These effects were observed in non-stimulated and/or TGF-ß1-stimulated RA-SFs. Since small molecule-based interference with KCa3.1 is principally well tolerated in clinical settings, further evaluation of channel blockers in models of rheumatoid arthritis may be a promising approach to identify new pharmacological targets and develop new therapeutic strategies for this debilitating disease.
Subject(s)
Arthritis, Rheumatoid/metabolism , Fibroblasts/physiology , Intermediate-Conductance Calcium-Activated Potassium Channels/metabolism , Synovial Membrane/chemistry , Arthritis, Rheumatoid/pathology , Cytokines/genetics , Cytokines/metabolism , Gene Expression Regulation/physiology , Humans , Intermediate-Conductance Calcium-Activated Potassium Channels/genetics , Metalloproteases/genetics , Metalloproteases/metabolism , Pyrazoles/pharmacology , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Synovial Membrane/cytologyABSTRACT
The death ligand TRAIL represents a promising therapeutic strategy for metastatic melanoma, however prevalent and inducible resistance limit its applicability. A new approach is presented here for sensitization to TRAIL. It is based on inhibition of the membrane potassium channel KCa3.1 (IK1), which serves fundamental cellular functions related to membrane potential. The selective inhibitor TRAM-34 did not induce apoptosis by itself but synergistically enhanced TRAIL sensitivity and overrode TRAIL resistance in a large panel of melanoma cell lines. Expression of IK1 was also found in mitochondria, and its inhibition resulted in mitochondrial membrane hyperpolarization and an early activation of Bax. The combination of TRAM-34 and TRAIL resulted in massive release of mitochondrial factors, cytochrome c, AIF and SMAC/DIABLO. Bax knockdown and Bcl-2 overexpression abolished apoptosis. Overexpression of XIAP diminished apoptosis by two-fold, and SMAC knockdown almost completely abolished apoptosis. These data uncover the existence of a rheostat in melanoma cells, consisting of inhibitor of apoptosis proteins and SMAC, which regulates TRAIL sensitivity. Thus, a new strategy is described based on mitochondrial membrane channels, which correspond to Bax activation. As both TRAIL and IK1 inhibitors had shown only minor side effects in clinical trials, a clinical application of this combination is conceivable.
Subject(s)
Apoptosis/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Melanoma/metabolism , Mitochondrial Proteins/metabolism , Potassium Channel Blockers/pharmacology , Pyrazoles/pharmacology , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Apoptosis Regulatory Proteins , BH3 Interacting Domain Death Agonist Protein/metabolism , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Enzyme Activation/drug effects , Humans , Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Up-Regulation , X-Linked Inhibitor of Apoptosis Protein/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity (MIA), secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins α(4)ß(1) and α(5)ß(1), facilitating cell detachment and promoting formation of metastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I (COPI)- and coat protein complex II (COPII)-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca(2+) ions. Interestingly, the Ca(2+)-activated K(+)-channel, subfamily N, member 4 (KCa3.1), known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant-negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.
