The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins (2). This influx is thought to occur as a consequence of TRPM2 channel activation via ADP-ribose (ADPr) binding to the cytoplasmic, C-terminal domain of the channel. This domain, termed NudT9-H (NudT9 homology), contains an apparent Nudix enzymatic motif and shares significant homology to the mitochondrial NudT9 enzyme (4, 5), a member of the Nudix ADPr hydrolase family. Indeed, electrophysiology experiments have shown that low micromolar levels of ADPr activate the TRPM2 channel (4, 6), and that this activation is mediated through the NudT9-H domain (7, 8), implicating a direct ADPr and NudT9-H interaction.The NudT9-H domain has been reported to have hydrolytic activity toward ADPr, albeit at a ϳ100-fold lower rate than NudT9 (4, 9). Although it was initially thought that the ADPr hydrolase activity of TRPM2 might have a role in regulating TRPM2 gating via hydrolysis of bound ADPr, recent data have demonstrated that mutations in putative catalytic residues of NudT9-H do not affect TRPM2 channel gating, leaving the function of this activity unclear (8). Some studies suggest that hydrogen peroxide directly activates the TRPM2 channel (3, 10); however, it has been reported recently that this oxidative agent acts indirectly by triggering the release of ADPr from an intracellular compartment (8, 11). The mechanism of TRPM2 activation by hydrogen peroxide may prove to be more complex, as hydrogen peroxide and ADPr have also been suggested to act cooperatively in TRPM2 channel activation (12).In this article, we have investigated the ability of TRPM2 to sensitize cells to cellular insults other than those inducing oxidative stress. Here, we demonstrate that puromycin, a well known pleiotropic cell stress agent, selectively targets TRPM2-expressing cells, leading to cell death. Our studies suggest that Sir2 enzymes play an important role in mediating this response. We found that TRPM2 has the capacity to sense OAADPr, the unique metabolite of the Sir2 protein deacetylase reaction, and induce cell death in response to puromycin. Furthermore, we report the first direct evidence that OAADPr binds to the NudT9-H domain and modulates TRPM2 channel activity. Our data have important implications for the potential physiological roles of ADPr-related molecules as secondary messengers. This report links Sir2 enzymes (sirtuins) with the TRPM2 channel through the Sir2 metabolite OAADPr. This newly discovered association provides a plausible mechanism for previously described sirtuin functions, which include control of stress response pathways and metabolic regulation. EXPERIMENTAL PROCEDURESCell Cul...

We have examined the role of histone acetylation in the very earliest steps of differentiation of mouse embryonic stem cells in response to withdrawal of leukemia inhibitory factor (LIF) as a differentiation signal. The cells undergo dramatic changes in morphology and an ordered program of gene expression changes representing differentiation to all three germ layers over the first 3-5 days of LIF withdrawal. We observed a global increase in acetylation on histone H4 and to a lesser extent on histone H3 over this time period. Treatment of the cells with trichostatin A (TSA), a histone deacetylase inhibitor, induced changes in morphology, gene expression, and histone acetylation that mimicked differentiation induced by withdrawal of LIF. We examined localized histone acetylation in the regulatory regions of genes that were transcriptionally either active in undifferentiated cells, induced during differentiation, or inactive under all treatments. There was striking concordance in the histone acetylation patterns of specific genes induced by both TSA and LIF withdrawal. Increased histone acetylation in local regions correlated best with induction of gene expression. Finally, TSA treatment did not support the maintenance or progression of differentiation. Upon removal of TSA, the cells reverted to the undifferentiated phenotype. We concluded that increased histone acetylation at specific genes played a role in their expression, but additional events are required for maintenance of differentiated gene expression and loss of the pluripotent state.

The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERα, ERβ), are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen dependent manner by endogenous ERs or by transiently overexpressed ERα. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation

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