The role of p44/42 activation in tributyltin-induced inhibition of human natural killer cells: effects of MEK inhibitors

Abstract

Destruction of tumor cells is a key function of natural killer (NK) cells. Previous studies have shown that tributyltin (TBT) can significantly reduce the lytic function of the human NK cells with accompanying increases in the phosphorylation (activation) states of the mitogen activated protein kinases (MAPKs), p44/42. The current studies examine the role of p44/42 activation in the TBT-induced reduction of NK-lytic function, by using MAPK kinase (MEK) inhibitors, PD98059 and U0126. A 1 h treatment with PD98059 or U0126 or both decreased the ability of NK cells to lyse K562 tumor cells. PD98059, U0126 or a combination of both inhibitors were able to completely block TBT-induced activation of p44/42. However, when p44/42 activation was blocked by the presence of PD98059, U0126 or the combination, subsequent exposure to TBT was still able to decrease the lytic function of NK cells. These results indicate that TBT-induced activation of p44/42 occurs via the activation of its upstream activator, MEK, and not by a TBT-induced inhibition of p44/42 phosphatase activity. Additionally, as lytic function was never completely blocked by MEK inhibitors, the results indicate that activation of p44/42 pathway is not solely responsible for the activation of lytic function of freshly isolated human NK cells. Finally, the results showed that TBT-induced activation of p44/42 is not solely responsible for the loss of lytic function.

Figure 1. Effect of PD98059 or U0126 or both on K562 lysis of tumor cells

Starting from the left Bar, NK cells were treated with the indicated concentrations of PD98059 (P) or U0126 (U) or both for 1 h. Lysis of the target cells was measured by using a ⁵¹Cr release assay. X-axis represents the indicated concentrations of MEK inhibitors. Y-axis represents the % control lysis activity. * indicates significant difference as compared to control, p<0.05.

Figure 2. Effect of MEK inhibition on TBT-induced inhibition of NK lytic function

Starting from the left Bar 1= NK cells were treated with 100 μM PD98059 (P) followed by exposure to control media for 1 h; Bar 2 = NK cells were treated with 100 μM U0126 (U) followed by exposure to control media for 1 h; Bar 3 = NK cells were treated with control media for 1 h followed by exposure to 300 nM TBT for 1 h ; Bar 4 = NK cells were treated with 100 μM PD98059 (P) followed by exposure to 300 nM TBT for 1 h; and Bar 5 = NK cells were treated with 100 μM U0126 (U) followed by exposure to 300 nM TBT for 1 h. The entire treatment time was 2 h, with the initial treatment remaining during the second treatment. * indicates significant difference as compared to control, p<0.05.

Figure 3. Effect of MEK 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 100 μM PD98059 (P) followed by control media for 1 h, Bar 2 = NK cells were treated with 100 μM U0126 (U) followed by control media for 1 h, Bar 3 = NK cells were treated with control media for 1 h followed by exposure to 200 nM TBT for 1 h, Bar 4 = NK cells were treated with control media for 1 h followed by exposure to 100 nM TBT for 1 h, Bar 5 = NK cells were treated with 100 μM PD98059 (P) for 1 h followed by exposure to 200 nM TBT for 1 h, Bar 6 = NK cells were treated with 100 μM PD98059 (P) for 1 h followed by exposure to 100 nM TBT for 1 h, and Bar 7 = NK cells were treated with 100 μM U0126 (U) for 1 h followed by exposure to 200 nM TBT for 1 h, Bar 8 = NK cells were treated with 100 μM U0126 (U) for 1 h followed by exposure to 100 nM TBT for 1 h. The entire treatment time was 2 h, with the initial treatment remaining during the second treatment. Following the treatments the cells were washed twice with gel media and were incubated for 24 hrs in compound-free media prior to assaying for lytic funciton. * indicates significant difference as compared to control, p<0.05.

Figure 4. Effect of the MEK inhibitor, PD98059, on the TBT-induced phosphorylation of p44/42 and total p44/42

A.) levels of phospho-p44/42 and total p44/42 compared to control in pure NK cells exposed to 200 or 100 nM TBT for 10 min +/− a 1 h pre-incubation with 100 μM PD98059 (P). The band densities for both control and treated cells were first divided by corresponding β-actin density to correct for very small loading differences. The treatments were then normalized to control. The same procedure was followed for all the experiments. Values are mean ± S.D. from at least three separate experiments using cells from different donors. * indicates significant difference as compared to control, p<0.05. B.) Representative experiment (1) NK cells exposed to media for 1 h followed by exposure to media for 10 min (control) , (2) NK cells exposed to 100 μM PD98059 for 1h followed by exposure to media for 10 min, (3) NK cells exposed to media for 1 h followed by exposure to 200 nM TBT for 10 min (4) NK cells exposed to media for 1h followed by exposure to 100 nM TBT for 10 min, (5) NK cells exposed to 100 μM PD98059 for 1h followed by exposure to 200 nM TBT for 10 min, (6) NK cells exposed to 100 μM PD98059 for 1h followed by exposure to 100 nM TBT for 10 min. The entire treatment time was 1 h and 10 min, with the initial treatment remaining during the second treatment.

Figure 5. Effect of the MEK inhibitor, U0126, on the TBT-induced phosphorylation of p44/42 total p44/42

A.) levels of phospho-p44/42 and total p44/42 compared to control in pure NK cells exposed to 200 or 100 nM TBT for 10 min +/− a 1 h pre-incubation with 100 μM U0126 (U). The band densities for both control and treated cells were first divided by corresponding β-actin density to correct for very small loading differences. The treatments were then normalized to control. The same procedure was followed for all the experiments. Values are mean ± S.D. from at least three separate experiments using cells from different donors. * indicates significant difference as compared to control, p<0.05. B.) Representative experiment (1) NK cells exposed to media for 1 h followed by exposure to media for 10 min (control) , (2) NK cells exposed to 100 μM U0126 for 1h followed by exposure to media for 10 min, (3) NK cells exposed to media for 1 h followed by exposure to 200 nM TBT for 10 min (4) NK cells exposed to media for 1h followed by exposure to 100 nM TBT for 10 min, (5) NK cells exposed to 100 μM U0126 for 1h followed by exposure to 200 nM TBT for 10 min, (6) NK cells exposed to 100 μM U0126 for 1h followed by exposure to 100 nM TBT for 10 min. The entire treatment time was 1 h and 10 min, with the initial treatment remaining during the second treatment.

Figure 6. Effect of MEK inhibitors, PD98059 and U0126, on the TBT-induced phosphorylation of p44/42 and total p44/42

A.) levels of phospho-p44/42 and total p44/42 compared to control in pure NK cells exposed to 200 or 100 nM TBT for 10 min +/− a 1 h pre-incubation with 100 μM PD98059 (P) and 100 μM U0126 (U). The band densities for both control and treated cells were first divided by corresponding β-actin density to correct for very small loading differences. The treatments were then normalized to control. The same procedure was followed for all the experiments. Values are mean ± S.D. from at least three separate experiments using cells from different donors. * indicates significant difference as compared to control, p<0.05. B.) Representative experiment (1) NK cells exposed to media for 1 hr followed by exposure to media for 10 min (control) , (2) NK cells exposed to 100 μM P and 100 μM U for 1h followed by exposure to media for 10 min, (3) NK cells exposed to media for 1 h followed by exposure to 200 nM TBT for 10 min (4) NK cells exposed to media for 1h followed by exposure to 100 nM TBT for 10 min, (5) NK cells exposed to 100 μM P and 100 μM U for 1h followed by exposure to 200 nM TBT for 10 min, (6) NK cells exposed to 100 μM P and 100 μM U for 1h followed by exposure to 100 nM TBT for 10 min. The entire treatment time was 1 h and 10 min, with the initial treatment remaining during the second treatment.