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| Cell cycle: GABA keeps embryonic stem cells in check
γ-aminobutyric acid (GABA) receptor-mediated signaling causes S-phase cell cycle arrest in embryonic stem (ES) cells and neural crest stem (NCS) cells by promoting phosphorylation of histone H2AX. Cell cycle progression in differentiated cells is controlled by a series of checkpoints that regulate proliferation by halting cell division at pre-defined points in the cell cycle. Checkpoints in differentiated cells are necessary to avoid transmission of DNA mutations and development of genomic instability. However, mouse ES cells may lack a G1/S checkpoint, and it is not yet understood how the cell cycle is regulated in these multipotent cells. In Nature, Ernfors and colleagues now report that GABA-mediated signaling triggers a novel post-replicative checkpoint that is a critical regulator of cell cycle progression in ES and NCS cells.
ES and NCS cells were found to contain both functional GABAA receptors (GABAAR) and the vesicular GABA transporter VGAT. Differentiating ES cells lost expression of VGAT, suggesting a potential role for GABA signaling in ES cells. Indeed, the selective GABAAR agonist muscimol inhibited cell division in cultured ES and NCS cells. Conversely, inhibiting endogenous GABA signaling with small interfering RNA (siRNA) against the GABAAR β3 subunit or with bicuculline, a competitive antagonist of GABAAR, increased cellular division and proliferation. These data indicate that activation of GABAAR blocks cell cycle progression. Muscimol treatment caused stem cells to accumulate in S phase with a concomitant increase in phosphorylated histone H2AX (γ-H2AX). Although accumulation of γ-H2AX is a hallmark of DNA damage, the stem cells exhibited no evidence of DNA damage. In adult cells, the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ataxia telangiectasia mutated (ATM), ataxia telangiectasia Rad3-related (ATR) and DNA protein kinase (DNA-PK) phosphorylate H2AX and activate a post-replicative checkpoint in response to DNA damage. Surprisingly, all three kinases were also found to regulate stem cell proliferation. Genetic depletion of ATR overcame the cell cycle block induced by muscimol, and the levels of phosphorylated ATM increased with muscimol treatment and decreased in the presence of siRNA against GABAAR β3. Pharmacologic inhibition of ATM and DNA-PK also blocked the muscimol-mediated accumulation of γ-H2AX while increasing BrdU incorporation and decreasing the overall percentage of cells in S phase. Therefore, GABA signaling induces a post-replicative checkpoint in ES and NCS cells by promoting PIKK-mediated phosphorylation of H2AX, even in the absence of DNA damage. Similar to the results observed in vitro, cellular proliferation in whole blastocysts in vivo also decreased following muscimol treatment. Furthermore, blastocysts derived from zygotes expressing siRNA against GABAAR β3 developed faster and contained smaller cells than untreated zygotes. Ernfors and colleagues have defined a novel GABA signaling pathway in stem cells that determines cell cycle progression in vivo. This pathway defines a new mechanism for blocking cell cycle progression in S phase that is independent of DNA damage, and illustrates a previously unappreciated means of regulating ES cell proliferation at the S/G2 transition. An important future direction of this work will be to elucidate the biological mechanism that links GABA signaling to PIKK activation. Emily J. Chenette Reference | |
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