Cell proliferation and differentiation, processes that are inversely correlated, are typically regulated during the G1 phase of the cell cycle. Self-renewing stem cells need to proliferate and remain multipotent; indeed, embryonic stem (ES) cells may lack the G1 pathways through which proliferation is regulated. Andäng et al. identified γ-aminobutyric acid type A receptor (GABAAR) subunits and GABA synthetic enzymes in mouse ES cells and peripheral neural crest stem (NCS) cells and combined electrophysiological analysis with use of a voltage-sensitive dye to confirm that the cells contained functional GABAARs. The GABAAR agonist muscimol inhibited proliferation of cultured stem cells, whereas the antagonist bicuculline promoted it, as did knockdown of the GABAAR β3 subunit (GABAAR β3) with RNA interference. Cell cycle distribution analysis indicated that receptor activation promoted the accumulation of ES cells in S phase. Moreover, muscimol stimulated the phosphorylation of histone H2AX (which is involved in the S/G2 DNA damage checkpoint response) by means of the checkpoint kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia, Rad3-related (ATR), even though no overt DNA damage was evident. Furthermore, GABA-dependent inhibition of proliferation depended on H2AX. Muscimol decreased the incorporation of bromodeoxyuridine into blastocysts in vivo, and injection of siRNA directed against GABAAR β3 into zygotes led to the faster development of blastocyst-stage embryos. GABA also regulated proliferation of NCS cells. Thus the proliferation of ES and NCS cells appears to be regulated through autocrine- or paracrine-derived GABA through a pathway that uses components of the DNA damage checkpoint response.
M. Andäng, J. Hjerling-Leffler, A. Moliner, T. K. Lundgren, G. Castelo-Branco, E. Nanou, E. Pozas, V. Bryja, S. Halliez, H. Nishimaru, J. Wilbertz, E. Arenas, M. Koltzenburg, P. Charnay, A. El Manira, C. F. Ibañez, P. Ernfors, Histone H2AX-dependent GABAA receptor regulation of stem cell proliferation. Nature 451, 460-464 (2008). [PubMed]