Figure 1.
Effect of intracellular zinc on enhancement of Akt and Erk phosphorylation.
(A) Enhancement of Akt and Erk phosphorylation required intracellular zinc. Serum-starved cells were treated with (+) or without (−) 10 µM TPEN for 1 h, before treatment with 0.5 mg/mL anti-IgM antibody (lane 2 and 3), and then treated with 10 and 20 µM ZnPy (lane 4 and 5) or 10 µM CaI (lane 6). (B) The treatment with ZnPy activated the phosphorylation of Akt and Erk. The abbreviation, “unt.” was defined the untreated sample. Serum-starved cells were treated with 0.5 mg/mL anti-IgM antibody (lane 2), 5 and 10 µM ZnCl2 (lanes 3 and 4), 5 and 10 µM ZnPy (lanes 5 and 6), and 20 µM pyrithione (lane 7) for 10 min. (C) Akt and Erk phosphorylation by ZnPy was enhanced in a time-dependent manner. The abbreviation, “unt.” was defined the untreated sample. Serum-starved cells were treated with 10 µM ZnPy for 5 min (lane 2), 10 min (lane 3), 15 min (lane 4), 30 min (lane 5), and 60 min (lane 6). (D) The inhibitors of PI3K and MEK1/2 inhibited the phosphorylation of Akt and Erk. Serum-starved cells were pretreated with (+) or without (−) LY294002 or U0126, before treatments with 0.5 mg/mL anti-IgM antibody (lanes 1–4) and 10 µM ZnPy (lanes 5–8). All data are representative of three independent experiments.
Figure 2.
Akt and Erk phosphorylation in zinc-transporter-knockout DT40 cells.
(A) Suppression of Akt and Erk phosphorylation in cZip9KO cells. Western blot analysis was performed using exponentially growing WT (lane 1), TKO (lane 2), and cZip9KO (lane 3) cells. (B) Analysis of total PTPase activity. WT (column 1), TKO (column 2), and cZip9KO (column 3) cells were subjected to PTPase assay. Values are expressed as the mean ± standard deviations. Significant difference at the level of *P<0.01 against the activity of WT cells (column 1). (C) ZnPy failed to induce Akt and Erk phosphorylation in cZip9KO cells. Serum-starved WT (lanes 1 and 2), TKO (lanes 3 and 4), and cZip9KO (lanes 5 and 6) cells were treated with (+) or without (−) 10 µM ZnPy for 10 min. (D) Analysis of PTPase activity in serum-starved DT40 cells. After treatment of serum-starved WT (columns 1 and 2), TKO (columns 3 and 4), and cZip9KO (columns 4 and 5) cells treated with (columns 2, 4 and 6) or without (columns 1, 3 and 5) 10 µM ZnPy for 10 min, and subjected to PTPase assay. Values are expressed as the mean ± standard deviations. Asterisk represents significant difference at the level of *P<0.01 for the columns linked by a line. All data are representative of three independent experiments.
Figure 3.
Effect of overexpression of human Zip9 on phosphorylation levels of Akt and Erk in response to zinc treatment and anti-IgM antibody stimulation.
(A) Overexpression of hZip9 restored the phosphorylation of Akt and Erk. Western blot analysis was performed using exponentially growing WT (lane 1), cZip9KO (lane 2), and cZip9KO+hZip9HA (lane 3) cells. (B) Overexpression of hZip9 in cZip9KO cells by ZyPy treatment stimulated the phosphorylation of both proteins. Serum-starved WT (lanes 1–3), cZip9KO (lanes 4–6), and cZip9KO+hZip9HA (lanes 7–9) cells were treated with 10 µM ZnPy for 10 min (lanes 2, 5 and 8) and 30 min (lanes 3, 6 and 9). The abbreviation, “unt.” was defined the untreated sample. (C) Analysis of total PTPase activity. Serum-starved WT (lanes 1–3), cZip9KO (lanes 4–6), and cZip9KO+hZip9HA (lanes 7–9) cells were treated with 10 µM ZnPy for 10 min (lanes 2, 5 and 8) and 30 min (lanes 3, 6 and 9). Values are expressed as the mean ± standard deviations. Significant difference at the level of *P<0.01 for the columns linked by a line. (D) Overexpression of hZip9 restored the response to anti-IgM antibody-stimulated BCR activation. Serum-starved WT (lanes 1 and 2), cZip9KO (lanes 3 and 4), and hZip9-HA-overexpressing cZip9KO (lanes 5 and 6) cells were treated with 0.5 mg/mL anti-IgM antibody for 10 min. All data are representative of three independent experiments.
Figure 4.
ZIP9 is an essential factor for regulating the intracellular zinc level in DT40 cells.
(A) The intracellular zinc release depends on the expression of ZIP9. Serum-starved WT (panels a, b, g and h), cZip9KO (panels c, d, i and j), and hZip9-HA-overexpressing cZip9KO (panels e, f, k and l) DT40 cells were pretreated with 5 µM Newport Green PDX (magnification; ×40), FluoZin-3 (magnification; ×60) and BODIPY TR-ceramide for 30 min before treatment with 10 µM ZnPy (WT: panels b and h, cZip9KO: panels d and j, hZip9-HA-overexpressing cZip9KO: panels f and l) for 10 min. (B) Serum-starved WT (panels a and d), cZip9KO (panels b and e), and hZip9-HA-overexpressing cZip9KO (panels c and f) DT40 cells were pretreated with 5 µM Newport Green PDX (magnification; ×40) and BODIPY TR-ceramide for 30 min before treatment with 0.5 mg/mL anti-IgM antibody (WT: panel d, cZip9KO: panel e, hZip9-HA-overexpressing cZip9KO: panel f) for 10 min. The abbreviation, “unt.” was defined the untreated sample, and white bars were defined as 10 µm length.
Figure 5.
Proposed action sites of intracellular zinc release by ZIP9 in DT40 cells for activation of B cell receptor signaling.
It is the proposed mechanism of Zn-induced PTPase inhibition by ZIP9, which leads to the activation of B cell receptor signaling in DT40 cells. Intracellular zinc is incorporated into the Golgi by ZnT5/6/7. Zinc is released as induced by ZIP9 into the cytosol from the Golgi, which in turn inhibits PTPase activity and induces the phosphorylation of Akt and ERK probably indirectly by regulating upstream components of the signal transduction.