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

Abstract

Highly Ca(2+) permeable receptor potential channel vanilloid type 6 (TRPV6) modulates a variety of biological functions including calcium-dependent cell growth and apoptosis. So far, the role of TRPV6 in controlling growth of pancreatic neuroendocrine tumour (NET) cells is unknown. In the present study, we characterize the expression of TRPV6 in pancreatic BON-1 and QGP-1 NET cells. Furthermore, we evaluate the impact of TRPV6 on intracellular calcium, the activity of nuclear factor of activated T-cells (NFAT) and proliferation of BON-1 cells. TRPV6 expression was assessed by real-time PCR and Western blot. TRPV6 mRNA expression and protein production were down-regulated by siRNA. Changes in intracellular calcium levels were detected by fluorescence calcium imaging (fura-2/AM). NFAT activity was studied by NFAT reporter assay; cell proliferation by bromodeoxyuridine (BrdU), MTT and propidium iodine staining. TRPV6 mRNA and protein are present in BON-1 and QGP-1 NET-cells. Down-regulation of TRPV6 attenuates BON-1 cell proliferation. TRPV6 down-regulation is associated with decreased Ca(2+) response pattern and reduced NFAT activity. In conclusion, TRPV6 is expressed in pancreatic NETs and modulates cell proliferation via Ca(2+)-dependent mechanism, which is accompanied by NFAT activation.

Keywords: BON-1; QGP-1; TRPV6; calcium; neuroendocrine tumour; proliferation.

Figure 1. TRPV6 mRNA expression and protein production in NET cells

(A) Real time PCR detection of TRPV6 mRNA expression in QGP-1, BON-1 and LCC-18 cells. (B) Western blot detection of TRPV6 protein in BON-1, QGP-1 and LCC-18 cells. (C) Suppression of TRPV6 mRNA expression in BON-1 cells transfected with siRNA for 48 h in comparison with BON-1 cells transfected with non-targeting construct (nt). (D) Suppression of TRPV6 protein production in BON-1 cells 48 h after siRNA transfection in comparison with nt BON-1 cells. Results are the mean ± S.E.M., obtained from at least n=3.

Figure 2. Effect of TRPV6 down-regulation on Ca²⁺ regulation in BON-1 cells

(A) Measurement of intracellular Ca²⁺ concentration in cells transfected either with TRPV6 or nt siRNA. The basal Ca²⁺ level was measured during the first 200 s followed by Ca²⁺ reduction (left arrow) under Ca²⁺-free condition and a Ca²⁺ increase after 1.5 mM Ca²⁺ exposure at 380 s. (right arrow). Intracellular Ca²⁺ increases above the base line (dashed line) in nt siRNA-transfected cells could be detected after re-addition of extracellular Ca²⁺ (open circles) (n=13). In contrast, this effect could be clearly suppressed in TRPV6 siRNA-transfected BON-1 cells (filled circles) (n=19). Changes in cytosolic free Ca²⁺ are depicted as the ratio of the fluorescence induced by excitation wavelength of 340 and 380 nm by the equation of Grynkiewicz et al. [19]. (B) Summary of the experiments with nt and TRPV6 siRNA-transfected BON-1 cells. Statistical evaluation of [Ca²⁺]_i was performed after 300 s (light grey bars) and 600 s (dark grey bars).

Figure 3. Effects of TRPV6 down-regulation on BON-1 cells proliferation and cell cycle

(A) BON-1 cell proliferation assed 24, 48 and 72 h after transfection with siRNA. (B) The number of viable BON-1 cells studied 48 h after transfection with siRNA using MTT assay. (C) BON-1 cell cycle analysis 48 h after siRNA transfection. (D–F) Expression of CCND1, CCND2 and CDK4 in cells transfected with siRNA for 48 h. Results are the mean ± S.E.M., obtained from at least n=4.

Figure 4. Effects of NFAT suppression on BON-1 cells proliferation

(A) Expression of NFATs in BON-1 and LCC-18 cells. (B) NFAT activity in BON-1 cells treated with 10 μM FK506 or 10 μM CsA for 24 h. BON-1 cell proliferation treated with FK506 (C) or CsA (D) for 24 h. The number of viable BON-1 cells assed after 24 incubation in the presence of FK506 (E) or CsA (F). Results are the mean ± S.E.M., obtained from at least n=4.

Figure 5. NFAT activity and expression in BON-1 cells with down-regulated TRPV6

NFAT activity (A) and expression (B) evaluated in cells transfected either with nt (white bars) or TRPV6 siRNA (grey bars) for 48 h. (C) Effects of 24 h treatment with FK506 on BON-1 cells transfected with nt (white bars) or TRPV6 siRNA (grey bars) for 48 h. Results are the mean ± S.E.M. (n=4).