Impairment of adenosine signaling disrupts early embryo development: unveiling the underlying mechanisms

Talita Glaser; Patrícia Martins; Renata Beco; Carolina Adriane Bento; Angelica R Cappellari; Sophia La Banca Oliveira; Christian Albert Merkel; Vanessa Fernandes Arnaud-Sampaio; Claudiana Lameu; Ana Maria Battastini; Henning Ulrich

doi:10.3389/fphar.2023.1328398

Impairment of adenosine signaling disrupts early embryo development: unveiling the underlying mechanisms

Front Pharmacol. 2024 Jan 19:14:1328398. doi: 10.3389/fphar.2023.1328398. eCollection 2023.

Affiliations

¹ Department of Biochemistry, Institute of Chemistry, University of São Paulo, SãoPaulo, Brazil.
² Department of Biochemistry, Federal University of Rio Grande do Sul, Porto Alegre, Brazil.
³ Department of Health (São Paulo-State), Medical School of the University of São Paulo (HCFMUSP), SãoPaulo, Brazil.

^# Contributed equally.

Abstract

Purinergic signaling has been implicated in many biological functions, including development. In this study, we investigate the functions of extracellular adenosine and adenosine receptors using a mouse embryonic stem cell (ESC) line and morula stages isolated from mouse embryos. Feeder-free mouse ESC was investigated in the absence and presence of the leukemia inhibitory factor (LIF), configuring undifferentiated cells and cells undergoing spontaneous differentiation. High alkaline phosphatase (ALPL) and low CD73 levels resulting in low adenosine (eADO) levels were characteristic for pluripotent cells in the presence of the LIF, while LIF deprivation resulted in augmented adenosine levels and reduced pluripotency marker expression, which indicated differentiation. Tracing ESC proliferation by BrdU labeling revealed that the inhibition of ALPL by levamisole resulted in a decrease in proliferation due to less eADO accumulation. Furthermore, caffeine and levamisole treatment, inhibiting adenosine receptor and eADO accumulation, respectively, reduced ESC migration, similar to that observed in the absence of the LIF. Pharmacological approaches of selective adenosine receptor subtype inhibition triggered specific adenosine receptor activities, thus triggering calcium or MAP kinase pathways leading to differentiation. In line with the in vitro data, mouse embryos at the morula stage were sensitive to treatments with A1 and A3 receptor antagonists, leading to the conclusion that A1 receptor and A3 receptor inhibition impairs proliferation and self-renewal and triggers inappropriate differentiation, respectively. The findings herein define the functions of eADO signaling in early development with implications for developmental disorders, in which adenosine receptors or ectonucleotidase dysfunctions are involved, and which could lead to malformations and miscarriages, due to exposure to caffeine.

Keywords: ERK1/2; adenosine; caffeine; calcium signaling; embryonic stem cells; purinergic signaling.

Grants and funding

The authors declare financial support was received for the research, authorship, and/or publication of this article. This work received funding from the São Paulo Research Foundation, FAPESP (Project Nos. 2012/2012/50880-4 and 2018/07366-4 to HU; 2019/17483-0, 2015/19128-2 and 2022/11093-9 to CL; 2013/07937-8 to TG; and 2012/05333-5 to RB), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq Brazil, Project No. 406396/2021 and 308,012/2021–6 to HU), and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Brazil. HU acknowledges financial support and fellowship from the National Institute of Science and Technology in Regenerative Medicine (INCT-REGENERA).