Ano1 as a regulator of proliferation

Abstract

Ano1 is a Ca2+ activated Cl− channel expressed in several proliferating cell types including various epithelia, gastrointestinal stromal tumors, interstitial cells of Cajal (ICC) and CFPAC‐1 cells. The aim of this study was to determine if Ano1 regulates proliferation. ICC from Ano1(−/−) and Ano1(+/+) mice were cultured with Cl− channel inhibitors or in low Cl− media. CFPAC‐1 cells grown in low Cl− media were pulsed with EdU for 1 h. Incorporation was measured by flow cytometry. For immunoblots on mouse jejunum, antibodies to pRb, phospho(Ser773)‐pRb and GAPDH were used.Fewer proliferating ICC were detected in Ano1(−/−) cultures (15 ± 3.5% Ki67+ ICC vs 25 ± 4% in Ano1(+/+), mean ± SEM, n = 4, p < 0.05). Cl− channel inhibitors DIDS (10 μM), niflumate (10 μM) and tamoxifen (1 μM) decreased ICC proliferation by 53, 53 and 59%, respectively (p < 0.05). This effect was reduced in cultures from Ano1(−/−) mice (29% and 19 % decrease, 22% increase, p > 0.05). Fewer proliferating ICC and CFPAC‐1 cells were detected in low Cl− media (% EdU+ ICC: 120 mM; 19.8 ± 5.3, 12 mM; 8.5 ± 3.2, n = 4, and % EdU+ CFPAC‐1: 120 mM; 29.5, 12 mM; 14.3 p < 0.05). Proportionally less Ser773‐phosphorylated pRb was detected in jejunum from Ano1(−/−) mice (Ano1(+/+); 5.98 ± 0.78, Ano1(−/−); 3.60 ± 0.49, mean ± SEM, n = 7, p < 0.05). In conclusion, Ano1 regulates proliferation possibly by promoting the G1/S transition of the cell cycle. Support: DK57061 and DK68055.