Functional Study of Na, K-ATPase β3 Subunit Involving in T Lymphocyte Regulation

Abstract

Na, K-ATPase plays crucial functions in the regulation of Na and K ions of cells and important in controlling membrane potential and various cellular functions. The Na, K-ATPase composes of two subunits, α and β subunits. These two subunits were demonstrated to associate together in order to function as Na, K pump. The α subunit plays an enzymatic role and as the binding site of Na and K ions, whereas the β subunit was suggested to play a role as stabilizer. However, the exact function of β subunit is still in doubt and need to study in more aspect of function. During the last decades, several monoclonal antibodies (mAbs) have been produced in our laboratory. One of the generated mAb, named P-3E10 which specific reacts to the Na, K-ATPase β3 subunit, showed an interesting effect in inhibiting T cells proliferation. In this study, by using mAb P-3E10, the function of Na, K-ATPase β3 subunit involving T cell regulation was studied in detail. In this study, we found that engagement of β3 subunit by mAb P-3E10 significantly inhibited the T cell proliferation induced by anti-CD3 mAb. The mAb P-3E10 itself, however, had no effect on unstimulated cells. In order to confirm the inhibitory effect of mAb P-3E10, cell cycle analysis was performed. The mAb P-3E10 induced G0/G1 phase arrest of the activated T cells. Accumulation of 70% of the cells in the G0/G1 phase was observed upon mAb P-3E10 treatment. These results suggest that mAb P-3E10 down-regulated T cell proliferation by suppressing the progression of quiescent cells into the cell cycle. The mechanisms of the inhibitory effect of mAb P-3E10 on activated T cells were further evaluated. It was observed that the surface CD25 expression on the activated T cells was down-regulated by mAb P-3E10. Moreover, the reduction of the IL-2 and IFN- producing cells was also observed. The results suggest that the ligation of the Na, K-ATPase β3 subunit with mAb P-3E10 inhibited T cell proliferation by bringing about a decrease in the IL-2 receptor expression and also by altering the cytokine production, thus indicating the role of the Na, K-ATPase β3 subunit in immunoregulation. As β3 subunit is one of the components of the Na, K-ATPase, we raised the question whether the inhibition of T cell activation by mAb P-3E10 is because of the interference of the Na, K pump. To address this question, we investigated whether mAb P-3E10 had any effect on other cellular functions. If mAb P-3E10 interfere the Na, K pump activity, it should alter other cellular functions as well. It was found that mAb P-3E10 did not inhibit the proliferation of various cell lines tested. Furthermore, mAb P-3E10 did not alter the phagocytic activity of granulocytes and monocytes. These findings suggest that the engagement of the Na, K-ATPase β3 subunit with mAb P-3E10 specifically regulated T cell activation via another pathway and not by interfering with the Na, K-ATPase. To confirm that mAb P-3E10 is not really involved in cellular Na, K-ATPase activity, the effect of mAb P-3E10 on the Na, K-ATPase activity of several cell types was assessed. The results reveal that mAb P-3E10 did not alter the activity of Na, K-ATPase in all the tested cells indicating that mAb P-3E10 has a specific effect on the Na, K-ATPase β3 subunit, without interfering with the Na, K- ATPase activity. As mAb P-3E10 inhibits T cell activation without interfering with the Na, K-ATPase activity, we asked the question whether the β3 subunit is expressed in a non-associated form. To elucidate this question, a sequential immunoprecipitation was carried out. The results showed that the β3 subunit could be precipitated out from the α subunit depleted lysates. This finding indicates that the β3 subunit might express freely from α subunit and play a role different from that of the Na, K-ATPase. Taken together, in this study, we provide the evidence that the β3 subunit of Na, K-ATPase is individually expressed without association with the α subunit. The free form of β3 subunit plays a role in T cell regulation. The knowledge about these β3 subunits that are able to regulate T cell activation may lead to a new approach to immunotherapy in the future.