Upregulated TTYH2 expression is critical for the invasion and migration of U2OS human osteosarcoma cell lines.

Ion channels have recently emerged as stable biomarkers and anticancer targets particularly when the applications of the currently available therapeutic regimens are limited, as in case of osteosarcoma, a malignant bone tumor. Here, we evaluated the expression of TTYH2, a presumably calcium-activated chloride channel, in a human osteosarcoma cell line U2OS. We used small-interfering RNA (siRNA)-mediated gene silencing to demonstrate the downregulation in the expression of TTYH2 that resulted in the decrease in the invasion and migration, but not proliferation, of U2OS cells. The expression levels of Slug and ZEB1, the transcription factors involved in epithelial-mesenchymal transition, significantly reduced after TTYH2 silencing. Based on these results, we suggest that TTYH2 may serve as a novel target for the treatment of osteosarcoma.

PTPN6 regulates the cell-surface expression of TRPM4 channels in HEK293 cells.

Transient receptor-potential, cation channel, subfamily M, member 4 (TRPM4) channels regulate a variety of physiological and pathological processes; however, their roles as functional channels under diverse conditions remain unclear. In this study, cytosolic protein tyrosine phosphatase non-receptor type 6 (PTPN6) interacted with TRPM4 channels. We confirmed their interaction by performing co-immunoprecipitation (Co-IP) assays following heterologous PTPN6 and TRPM4 channel expression in HEK293 cells. Furthermore, biomolecular fluorescence complementation (BiFC) image analysis confirmed TRPM4-PTPN6 binding. In addition, immunoblotting and Co-IP analyses revealed that TRPM4 expression significantly decreased in the membrane fraction of cells after PTPN6 was silenced with a specific short-hairpin RNA (shRNA-PTPN6). In agreement, TRPM4-induced changes in whole-cell currents were not detected in PTPN6-silenced HEK cells, in contrast to cells transfected with a scrambled RNA (scRNA) or in naïve HEK cells. These data suggest that PTPN6 inhibits TRPM4 channel activity by disrupting TRPM4 expression. Furthermore, TRPM4 channels were expressed in the membrane of naïve cells and scRNA transfectants, but not in those of PTPN6-silenced cells. These results indicated that PTPN6 is critically associated with TRPM4 trafficking. This role of PTPN6 in TRPM4 membrane localization was also demonstrated in HeLa cells. TRPM4 overexpression significantly enhanced cell proliferation in untreated HeLa cells, but not in HeLa cells with silenced PTPN6 expression. These findings indicate that PTPN6-dependent TRPM4 expression and trafficking to the plasma membrane is critical for cell proliferation in both HEK293 and HeLa cells. Therefore, PTPN6 is a novel therapeutic target for treating pathologic diseases involving TRPM4.

Acute Hypoxia Activates an ENaC-like Channel in Rat Pheochromocytoma (PC12) Cells.

Cells can resist and even recover from stress induced by acute hypoxia, whereas chronic hypoxia often leads to irreversible damage and eventually death. Although little is known about the response(s) to acute hypoxia in neuronal cells, alterations in ion channel activity could be preferential. This study aimed to elucidate which channel type is involved in the response to acute hypoxia in rat pheochromocytomal (PC12) cells as a neuronal cell model. Using perfusing solution saturated with 95% N(2) and 5% CO(2), induction of cell hypoxia was confirmed based on increased intracellular Ca(2+) with diminished oxygen content in the perfusate. During acute hypoxia, one channel type with a conductance of about 30 pS (2.5 pA at -80 mV) was activated within the first 2~3 min following onset of hypoxia and was long-lived for more than 300 ms with high open probability (P(o), up to 0.8). This channel was permeable to Na(+) ions, but not to K(+), Ca(+), and Cl(-) ions, and was sensitively blocked by amiloride (200 nM). These characteristics and behaviors were quite similar to those of epithelial sodium channel (ENaC). RT-PCR and Western blot analyses confirmed that ENaC channel was endogenously expressed in PC12 cells. Taken together, a 30-pS ENaC-like channel was activated in response to acute hypoxia in PC12 cells. This is the first evidence of an acute hypoxia-activated Na(+) channel that can contribute to depolarization of the cell.