Pdx1 is transcriptionally regulated by EGR-1 during nitric oxide-induced endoderm differentiation of mouse embryonic stem cells

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.C.S.-A. was a doctoral PFIS fellow of ISCIII (FI11/00301). This study was supported by grants from Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI105/2010, TCMR06/009, and PI-0022) to F.J.B. and F.M.; from Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (CTS-7127/2011) to F.J.B.; from ISCIII cofunded by Fondos FEDER (RED-TERCEL RD 16-011-0034, along with PICI PICI21/00016 and GVA-COVID19/2021/047 to B.S.); from Servicio Andaluz de Salud (SAS 11245), and from Ministerio de Economía y Competitividad- Secretaría de Estado de Investigación Desarrollo e Innovación (IPT-2011-1615-900000). Along with this, there was support to PAIDI group CTS576, and by the European Regional Development Fund (FEDER) and the Consejería de Economía, Conocimiento, Empresas y Universidades de la Junta de Andalucía, within the framework of the operational program FEDER Andalucía 2014–2020. Specific Objective 1.2.3 “Promotion and generation of frontier knowledge and knowledge oriented to the challenges of society, development of emerging technologies” led the reference research project (UPO-1381598) of J.R.T.Peer reviewe

Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages

AICAR Stimulates the Pluripotency Transcriptional Complex in Embryonic Stem Cells Mediated by PI3K, GSK3β, and β-Catenin

Pluripotent stem cells maintain the property of self-renewal and differentiate into all cell types under clear environments. Though the gene regulatory mechanism for pluripotency has been investigated in recent years, it is still not completely understood. Here, we show several signaling pathways involved in the maintenance of pluripotency. To investigate whether AMPK is involved in maintaining the pluripotency in mouse embryonic stem cells (mESCs) and elucidating the possible molecular mechanisms, implicated D3 and R1/E mESC lines were used in this study. Cells were cultured in the absence or presence of LIF and treated with 1 mM and 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), 2 mM metformin, compound C, and the PI3K inhibitor LY294002 for 24, 72, and 120 h. The levels of Nanog, Oct3/4, and REX1 and Brachyury, Notch2, and Gata4 mRNAs and Nanog or OCT3/4 protein levels were analyzed. Alkaline phosphatase and the cellular cycle were determined. The pGSK3β, GSK3β, p-β-catenin, and β-catenin protein levels were also investigated. We found that AMPK activators such as AICAR and metformin increase mRNA expression of pluripotency markers and decrease mRNA expression of differentiation markers in R1/E and D3 ES cells. AICAR increases phosphatase activity and arrests the cellular cycle in the G1 phase in these cells. We describe that AICAR effects were mediated by AMPK activation using a chemical inhibitor or by silencing this gene. AICAR effects were also mediated by PI3K, GSK3β, and β-catenin in R1/E ES cells. According to our findings, we provide a mechanism by which AICAR increases and maintains a pluripotency state through enhanced Nanog expression, involving AMPK/PI3K and p-GSK3β Ser21/9 pathways backing up the AICAR function as a potential target for this drug controlling pluripotency. The highlights of this study are that AICAR (5-aminoimidazole-4-carboxamied-1-b-riboside), an AMP protein kinase (AMPK) activator, blocks the ESC differentiation and AMPK is a key enzyme for pluripotency and shows valuable data to clarify the molecular pluripotency mechanism.Ministerio de Economía y Competitividad- Secretaria de Estado de Investigacion Desarrollo e Innovacio ́ n- Bernat Soria ́ −Innpacto Proyect, No. IPT-2011-1615-900000; Instituto de Salud Carlos III, Gobierno de España - Bernat Soria, No. TERCEL RD06/0010/0025; Consejeri ́ a de Salud Junta de Andalucía - Francisco Javier Bedoya Bergua, No. PI-0105- 2010; Consejeri ́ a de Economi ́ a Innovacion Ciencia y Empleo - ́ Junta de Andalucia - Francisco Javier Bedoya, No. CTS-7127/ 2011; Servicio Andaluz de Salud - JR Tejedo, No SAS 11245; Ministerio de Economi ́ a y Competitividad- Secretari ́ a de Estado de Investigacion Desarrollo e Innovacio n- JR Tejedo, ́ Innpacto Proyect, NoPT-2011-1615-900000.Peer reviewe

AICAR Stimulates the Pluripotency Transcriptional Complex in Embryonic Stem Cells Mediated by PI3K, GSK3β, and β-Catenin

Pluripotent stem cells maintain the property of self-renewal and differentiate into all cell types under clear environments. Though the gene regulatory mechanism for pluripotency has been investigated in recent years, it is still not completely understood. Here, we show several signaling pathways involved in the maintenance of pluripotency. To investigate whether AMPK is involved in maintaining the pluripotency in mouse embryonic stem cells (mESCs) and elucidating the possible molecular mechanisms, implicated D3 and R1/E mESC lines were used in this study. Cells were cultured in the absence or presence of LIF and treated with 1 mM and 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), 2 mM metformin, compound C, and the PI3K inhibitor LY294002 for 24, 72, and 120 h. The levels of Nanog, Oct3/4, and REX1 and Brachyury, Notch2, and Gata4 mRNAs and Nanog or OCT3/4 protein levels were analyzed. Alkaline phosphatase and the cellular cycle were determined. The pGSK3β, GSK3β, p-β-catenin, and β-catenin protein levels were also investigated. We found that AMPK activators such as AICAR and metformin increase mRNA expression of pluripotency markers and decrease mRNA expression of differentiation markers in R1/E and D3 ES cells. AICAR increases phosphatase activity and arrests the cellular cycle in the G1 phase in these cells. We describe that AICAR effects were mediated by AMPK activation using a chemical inhibitor or by silencing this gene. AICAR effects were also mediated by PI3K, GSK3β, and β-catenin in R1/E ES cells. According to our findings, we provide a mechanism by which AICAR increases and maintains a pluripotency state through enhanced Nanog expression, involving AMPK/PI3K and p-GSK3β Ser21/9 pathways backing up the AICAR function as a potential target for this drug controlling pluripotency. The highlights of this study are that AICAR (5-aminoimidazole-4-carboxamied-1-b-riboside), an AMP protein kinase (AMPK) activator, blocks the ESC differentiation and AMPK is a key enzyme for pluripotency and shows valuable data to clarify the molecular pluripotency mechanism.España Ministerio de Economía y Competitividad Proyect, No. IPT-2011-1615-900000España Instituto de Salud Carlos III, No. TERCEL RD06/0010/0025España Consejería de Salud Junta de Andalucía No. PI-0105- 2010España Consejería de Economía Innovación Ciencia y Empleo - Junta de Andalucia No. CTS-7127/ 201