Regulation of Fas/Fas Ligand-Mediated Apoptosis in Megakaryocytes by Nuclear Factor of Activated T Cells.

Abstract 4023

Poster Board III-959

Bone marrow megakaryocytes are the precursors of peripheral blood platelets and therefore constitute an integral part of primary haemostasis, thrombosis and wound healing. Platelets have also been implicated in the regulation of inflammation and other immune responses, but the role of megakaryocytes in this context is less clear. The calcineurin-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) is a master regulator of cytokine production in T lymphocytes and therefore central for T cell-dependent immune reactions. We have previously shown that NFAT is strongly expressed in megakaryocytes and is required for the transcription of specific megakaryocytic genes. The biological role of NFAT in megakaryocytes, however, is unknown.

Here we show that activation of the calcineurin/NFAT pathway in either primary megakaryocytes or CMK megakaryocytic cells forces the cells to go into apoptosis. Cell death in megakaryocytes is induced by treating the cells with the calcium ionophore ionomycin and at least partially suppressed by either the pan-caspase inhibitor zVAD or the calcineurin inhibitor cyclosporin A (CsA). Ionomycin stimulation of megakaryocytes leads to the expression of Fas Ligand (FasL), a pro-apoptotic member of the tumor necrosis factor superfamily. Expression of FasL was detectable as early as six hours after stimulation on the membrane of ionomycin-treated megakaryocytes, was augmented in cells stably overexpressing NFATc2, and was suppressed in cells either pretreated with CsA or expressing the specific peptide inhibitor of NFAT, VIVIT. To investigate the physiological relevance of FasL expression on megakaryocytes, we performed cocultures of megakaryocytes with Fas-expressing T lymphocytes, in which CMK cells were either left unstimulated or prestimulated with ionomycin and then added to Jurkat cells. The presence of ionomycin-stimulated CMK cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells. Overexpression of NFATc2 in CMK cells enhanced their potency to induce apoptosis in Jurkat cells, while cells expressing VIVIT were less effective. Apoptosis induction of Jurkat cells by stimulated CMK cells was partially blocked by the presence of either a neutralizing antibody against FasL or an antagonistic antibody to Fas during the coculture period, indicating involvement of the FasL/Fas apoptosis pathway.

These results have several implications. First, they represent the first clear evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, namely the regulation of Fas/FasL-dependent apoptosis. Second, they underline that the biological role of megakaryocytes is not restricted to the production of proteins and other cellular structures for platelet assembly, but that this population of cells fulfills an independent regulatory function in the context of the surrounding tissue. Our results suggest that this regulatory function may include the induction of Fas/FasL-dependent apoptosis in bystander cells under certain conditions, for example the elimination of activated T cells in inflammatory situations.