Low concentrations of nitric oxide delay the differentiation of embryonic stem cells and promote their survival

Abstract

Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to embryonic stem cell (ESC) biology has not been characterized. Exposure of ESCs to low concentrations (2–20 μM) of the NO donor diethylenetriamine NO adduct confers protection from apoptosis elicited by leukaemia inhibitory factor (LIF) withdrawal. NO blocked caspase 3 activation, PARP degradation, downregulation of the pro-apoptotic genes Casp7, Casp9, Bax and Bak1 and upregulation of the anti-apoptotic genes Bcl-2 111, Bcl-2 and Birc6. These effects were also observed in cells overexpressing eNOS. Exposure of LIF-deprived mESCs to low NO prevented the loss of expression of self-renewal genes (Oct4, Nanog and Sox2) and the SSEA marker. Moreover, NO blocked the differentiation process promoted by the absence of LIF and bFGF in mouse and human ESCs. NO treatment decreased the expression of differentiation markers, such as Brachyury, Gata6 and Gata4. Constitutive overexpression of eNOS in cells exposed to LIF deprivation maintained the expression of self-renewal markers, whereas the differentiation genes were repressed. These effects were reversed by addition of the NOS inhibitor L-NMMA. Altogether, the data suggest that low NO has a role in the regulation of ESC differentiation by delaying the entry into differentiation, arresting the loss of self-renewal markers and promoting cell survival by inhibiting apoptosis.

Potential for pharmacological manipulation of human embryonic stem cells

“…Sinomenine (or cocculine) is a morphine derivative with anti-rheumatic effects thought to be primarily mediated via the release of histamine (Yamasaki, 1976); but other effects such as inhibition of prostaglandin, leukotriene and NO synthesis may also be involved (Liu et al, 1994). An unrelated study has shown that exposure of ESCs to low concentrations of diethylenetriamine NO (DETA-NO) adduct maintains hESC pluripotency to a similar extent as bFGF (Tejedo et al, 2010), although no definitive mechanism was provided. Gatifloxacin is an antibiotic of the fourth-generation fluoroquinolone family (Burka et al, 2005), while flurbiprofen is a member of the phenylalkanoic acid derivative family of non-steroidal antiinflammatory drugs (NSAIDs) used to treat the inflammation and pain of arthritis.…”

“…Antimycin A Mitochondrial respiratory chain (Varum et al, 2009) BIO GSK3b (Bone et al, 2009;James et al, 2005;Sato et al, 2004) Butyryl CoA Energy release/storage (Ware et al, 2009) CHIR99021 GSK3b (Tsutsui et al, 2011) DETA-NO NO donor (Tejedo et al, 2010) Dorsomorphin ALK2, 3 and 6 (Gonzalez et al, 2011) EHNA ? (Burton et al, 2010a,b) FBP ?…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…Murine bone marrow stromal (OP9) cell apoptosis was reduced in cells treated with the NO donor S-nitroso-N-acetylpenicillamine or YC-1 (300). In embryonic stem cells, treatment with low concentrations of an NO donor delayed the process of differentiation and maintained expression of the key self-renewal transcription factors: octamer-binding transcription factor 4 (Oct4), Nanog, and sex determining region Y-box 2 (Sox2) (270). Hematopoietic stem cell sensitivity to the self-renewal effects of NO may decrease with cell age (99).…”

Regulation of pancreatic β-cell survival by nitric oxide

“…Thus, treatment of mouse and human ES cells with 2 μM DETA-NO protects them from apoptosis induced by cell differentiation by upregulating the expression of anti-apoptotic genes, such as Bcl2, Bcl2-l and Birc6; by downregulating pro-apoptotic genes, such as Casp7, Casp9, Bax and Bak1; and by inhibiting caspase-3 activation by cleavage. 114 Several mechanisms have been described for the antiapoptotic actions of NO, such as protein activation by modification of prosthetic groups 102,115 and cysteine modification by S-nitrosylation. 29,111,112,[116][117][118] Both mechanisms can regulate signaling pathways 17,101,102,104 and gene expression.…”