ENPP1-dependent USP2 ubiquitination governs SQSTM1-mediated autophagy-dependent ferroptosis in trophoblast cells and exacerbates placental dysfunction in gestational diabetes mellitus.

Downregulation of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been implicated in autophagic cell death. However, how ENPP1 regulates the interplay between autophagy and ferroptosis to maintain trophoblast homeostasis in the context of gestational diabetes mellitus (GDM) remains unclear. To determine ENPP1's role in autophagy-dependent ferroptosis and its contribution to GDM-related placental injury, clinical placental tissues, hyperglycemia-treated HTR8/SVneo trophoblasts, and streptozotocin-induced GDM mice were analyzed. Ubiquitination assays, co-immunoprecipitation, functional studies, and therapeutic interventions were conducted. ENPP1 was significantly reduced in GDM placentas and correlated with increased ferroptosis and lipid peroxidation. Mechanistically, ENPP1 recruited USP2 (ubiquitin-specific peptidase 2) to inhibit the ubiquitination and autophagic degradation of SQSTM1 (sequestosome 1), thereby enhancing its stability. ENPP1 loss promoted NCOA4-mediated ferritinophagy, leading to iron overload and ferroptosis. Restoring ENPP1 or inhibiting autophagy alleviated placental thinning and fetal growth restriction in GDM mice. ENPP1 regulates autophagy-dependent ferroptosis via the USP2-SQSTM1 axis, and its deficiency contributes to placental dysfunction.