Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells

Abstract

Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

Figure 1

Effects of exposures of human NK cells to TBT for 10 minutes on the phosphorylation state of PKC. A) Fold increase in phospho-PKC as compared to control cells following 10 min exposures to 300 nM to 50 nM TBT. Values are mean ±S.D. from at least three separate experiments using different donors (triplicate determinations for each experiment, n ≥ 9). An asterisk indicates a significant increase as compared to control (p<0.05). B) Representative western blot of the effect of 10 min exposures of NK cells to 25 nM to 300 nM TBT on the phosphorylation state of PKC. Upper bands: (1) control cells, (2) NK cells exposed to 300 nM TBT, (3) NK cells exposed to 200 nM TBT, (4) NK cells exposed to 100 nM TBT, (5) NK cells exposed to 50 nM TBT, (6) NK cells exposed to 25 nM TBT. lower bands: β-actin for the corresponding lanes.

Figure 2

Effect of Bisindolylmaleimide I on the lytic function of NK cells. Starting from the left Bar, NK cells were treated with the indicated concentrations of Bisindolylmaleimide I (Bis I) for 1 h. Lysis of the target cells was measured by using a ⁵¹Cr release assay. X-axis represents the indicated concentrations of PKC inhibitor. Y-axis represents the % control lysis activity. *indicates significant difference compared to the control (p<0.05)

Figure 3

Effect of PKC inhibition on TBT-induced decreases in NK lytic function in 24 h period following a 1 h exposure TBT. Starting from the left Bar 1 = NK cells were treated with 25 μM Bisindolylmaleimide I (Bis I) followed by control media for 1 h, Bar 2 = NK cells were treated with control media for 1 h followed by exposure to 200 nM TBT for 1 h, Bar 3 = NK cells were treated with control media for 1 h followed by exposure to 100 nM TBT for 1 h, Bar 4 = NK cells were treated with 25 μM Bisindolylmaleimide I (Bis I) for 1 h followed by exposure to 200 nM TBT for 1 h and Bar 5 = NK cells were treated with 25 μM Bisindolylmaleimide I (Bis I)for 1 h followed by exposure to 100 nM TBT for 1 h., washed twice with gel media and were incubated for 24 hrs. The lysis of the target cells were measured the same way as fig. 6. X-axis represents treatment conditions. Y-axis represents the % control lysis. *indicates significant difference compared to the control (p<0.05)

Figure 4

Effect of the PKC inhibitor, Bisindolylmaleimide I, on the TBT-induced phosphorylation of p44/42. A.) Fold increase in the levels of phospho-p44/42 compared to control. Values are mean ± S.D. from at least three separate experiments using cells from different donors *indicates significant difference compared to the control (p<0.05). B.) Representative experiment of Phospho-p44/42 (Upper bands), total p44/42 (Middle bands) and β-actin (Lower Bands): (1) control cells treated with TBT free media for 1h, (2) NK cells exposed to 25 μM bisindolylmaleimide I for 1 h followed by 10 min exposure to control media (3) NK cells exposed to control for 1 h followed by 200 μM TBT for 10 min (4) NK cells exposed to control for 1 h followed by 100 μM TBT for 10 min, (5) NK cells exposed to 25 μM bisindolylmaleimide I for 1 h followed by 200 μM TBT for 10 min, (6) NK cells exposed to 25 μM bisindolylmaleimide I for 1 h followed by 100 μM TBT for 10 min

Figure 5

Effect of the PKC inhibitor, Bisindolylmaleimide I, on the TBT-induced phosphorylation of p38. A.) Fold increase in the levels of phospho-p38 compared to control. Values are mean ± S.D. from at least three separate experiments using cells from different donors *indicates significant difference compared to the control (p<0.05). B.) Representative experiment of Phospho-p38 (Upper bands), total p38 (Middle bands) and β-actin (Lower Bands): Lanes are defined as in the legend for Figure 4.

Figure 6

Effect of the PKC inhibitor, Bisindolylmaleimide I, on the TBT-induced phosphorylation of JNK. Representative experiment of Phospho-JNK (Upper bands), total JNK (Middle bands) and β-actin (Lower Bands): (1) control cells treated with TBT free media for 1h, (2) NK cells exposed to 25 μM bisindolylmaleimide I for 1 h followed by 10 min exposure to control media (3) NK cells exposed to control for 1 h followed by 200 μM TBT for 10 min (4) NK cells exposed to control for 1 h followed by 100 μM TBT for 10 min, (5) NK cells exposed to 25 μM bisindolylmaleimide I for 1 h followed by 200 μM TBT for 10 min, (6) NK cells exposed to 25 μM bisindolylmaleimide I for 1 h followed by 100 μM TBT for 10 min. lower bands: β-actin for the corresponding lanes.