084 | Modulation of the autophagic process by S1P under hypoxia: dissecting the contribution of S1P1 and S1P5 receptors

Beatrice Pranzo¹, Marco Greco¹, Luisa Rosato¹, Rita Paroni², Maria Rosaria Coscia³, Paola Italiani⁴, Giacomo Strapazzon⁵, Sara Ottolenghi², Federico Maria Rubino², Alessia Ametrano³, Michele Samaja², Giulia Maria Lionetto⁶, Michele Maffia¹ | ¹Department of Experimental Medicine, University of Salento, Lecce, Italy; ²Department of Health Sciences, University of Milano, Italy; ³Institute of Biochemistry and Cell Biology, National Research Council of Italy, Naples, Italy; ⁴Institute for Biomedical Research and Innovation, National Research Council of Italy, Palermo, Italy; ⁵Institute of Mountain Emergency Medicine, Eurac Research, Bolzano, Italy; ⁶Department of Biological and Environmental Sciences and Technologies, Lecce, Italy.

Abstract

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates cellular fate by integrating proliferation, differentiation, and apoptosis through both intracellular signalling and extracellular activation of five G protein-coupled receptors (S1PR1-5). S1P is a central modulator of cellular adaptation to environmental stress, including hypoxia, by regulating survival, metabolism, and autophagy. Dysregulated hypoxia-induced autophagy is observed in several pathological conditions, including neurodegenerative and cardiovascular diseases, where S1P signalling, especially through S1PR1 and the inhibition of mTOR, supports protective adaptive responses, although the underlying mechanisms remain incompletely understood. This study evaluated the role of S1P in the regulation of autophagic processes during hypoxia, by also focusing on the comprehension of S1PR1- and S1PR5-mediated pathways. Differentiated SH-SY5Y cells were used as a model of study, considering their dopaminergic-like characteristics. The characterization of the S1P receptor expression profile was performed by qPCR. Subsequently, we created a chemical hypoxic model using DMOG (dimethyloxalylglycine), a prolyl hydroxylase inhibitor that stabilizes HIF-1α (Hypoxia Inducible Factor-1α) allowing the subsequent formation of the functional HIF-1 complex. This was confirmed by assessing HIF-1α levels via Western blot and the transcriptional levels of target genes such as BNIP3 and GLUT1/3. Through the analysis of autophagy-related markers p62 and LC3-I/II via Western blot, and by quantifying autophagosomes with a commercial fluorescent probe based on an enhanced version of monodansylcadaverine, we first confirmed that DMOG induces autophagy in SH-SY5Y cells. Furthermore, to assess selective organelle turnover, we evaluated mitophagy and quantified mitochondria/lysosome colocalization fluorometrically. In contrast, treatment with exogenous S1P alone inhibited autophagy, and this inhibitory effect was also observed when S1P was combined with DMOG. To analyse the roles of specific receptors, we used CS2100 and A971432 as selective agonists for S1PR1 and S1PR5, respectively. CS2100 inhibited autophagy, whereas A971432 promoted it. Interestingly, when either agonist was administered together with DMOG, both treatments exerted an overall inhibitory effect on autophagy. Our findings highlight the “vectorial” nature of S1P signalling in balancing adaptive versus maladaptive autophagy, suggesting that S1P acts as a dynamic molecule that improves the cellular response to hypoxic stress. The receptor-specific divergences observed may indicate that S1P signalling plays a role in establishing the threshold between protective autophagy, which supports cellular homeostasis, and its dysregulation, which may instead compromise cellular viability.

Project PRIN 2022 “The double face of hypoxia in health and disease” (JANUS), Cod. 2022E7FZEJ funded by the Italian Ministry of University and Research. Project “POS Salute – Traiettoria 3 SISAGEN_CARDIO “Sistema Integrato SAnitario per le malattie Genetiche CARDIOvascolari” cod. T3-AN-18 e della Convenzione “DiMeS Unisalento – Camera di Commercio di Lecce” – Responsabile della Ricerca prof. Michele Maffia.