TRPV6 channel modulates proliferation of insulin secreting INS-1E beta cell line

Abstract

Transient receptor potential channel vanilloid type 6 (TRPV6) is a non-selective cation channel with high permeability for Ca²⁺ ions. So far, the role of TRPV6 in pancreatic beta cells is unknown. In the present study, we characterized the role of TRPV6 in controlling calcium signaling, cell proliferation as well as insulin expression, and secretion in experimental INS-1E beta cell model. TRPV6 protein production was downregulated using siRNA by approx. 70%, as detected by Western blot. Intracellular free Ca²⁺ ([Ca²⁺]i) was measured by fluorescence Ca²⁺ imaging using fura-2. Calcineurin/NFAT signaling was analyzed using a NFAT reporter assay as well as a calcineurin activity assay. TRPV6 downregulation resulted in impaired cellular calcium influx. Its downregulation also reduced cell proliferation and decreased insulin mRNA expression. These changes were companied by the inhibition of the calcineurin/NFAT signaling. In contrast, insulin exocytosis was not affected by TRPV6 downregulation. In conclusion, this study demonstrates for the first time the expression of TRPV6 in INS-1E cells and rat pancreatic beta cells and describes its role in modulating calcium signaling, beta cell proliferation and insulin mRNA expression. In contrast, TRPV6 fails to influence insulin secretion.

Role of TRPV channels in regulating various pancreatic β-cell functions: Lessons from <i>in vitro</i> studies

“…A promitogenic role of TRPVs in insulin producing cells is also supported by our recent data. We found that suppression of TRPV6 protein production by siRNA technique resulted in reduced cell proliferation and viability in INS-1E cells (61). Likewise previous studies, these effects were detected in the presence of glucose and serum.…”

“…Recently, however, we reported that TRPV6 mRNA is expressed in rat INS-1E cells and rat pancreatic islets (61). Using double immunofluorescence staining we detected that TRPV6 is expressed in rat β-cells.…”

TRPV6 modulates proliferation of human pancreatic neuroendocrine BON-1 tumour cells

“…The consequences of TRPV6 down-regulation in BON-1 cells on NFAT activity were assessed using NFAT reporter assay (Qiagen) 48 h after TRPV6 siRNA transfection, as previously described in our earlier study [15]. …”

“…The consequences of TRPV6 down-regulation in BON-1 cells on cell cycle were determined using propidium iodide (PI) staining 48 h after siRNA transfection, as described [15]. …”

“…TRPV6 is present in pancreatic adenocarcinoma and insulinoma cells [14,15]. In insulin-producing INS-1E cells, TRPV6 down-regulation impairs cell growth and inhibits insulin mRNA expression [15]. However, studies addressing the expression and function of TRPV6 in pancreatic neuroendocrine tumour cells (NETs) are not yet available.…”