Esposito, Federica Maria (2025) Interplay between PIEZO1 and mTOR pathway in the regulation of hepatic iron metabolism. [Tesi di dottorato]

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Abstract

PIEZO1, a mechanosensitive cation channel, is the primary causative gene for Dehydrated Hereditary Stomatocytosis (DHS), an autosomal dominant pleiotropic syndrome characterized by anemia, macrocytosis, and iron overload. PIEZO1 Gain-of-Function (GoF) mutations result in delayed channel inactivation, altered intracellular ion homeostasis, and dysregulated iron metabolism. Recent studies have shown that PIEZO1 is a regulator of hepatic iron metabolism. In mice, expression of a Piezo1-GoF allele downregulates hepcidin, leading to iron accumulation. In human hepatoma cells (Hep3B PIEZO1-KI), PIEZO1-GoF mutations enhance ERK1/2 phosphorylation, disrupting R-SMAD-mediated activation of hepcidin. Through in silico transcriptomic analysis of PIEZO1-GoF human hepatoma cells, we identified dysregulation of the PI3K/AKT and mTOR pathways as key contributors to impaired hepcidin expression. Interesting, the Ragulator complex component LAMTOR4 was significantly downregulated, while phosphorylation of mTOR and its downstream target TFEB was increased. These findings were further confirmed in primary murine hepatocytes from C57BL/6 mice silenced for Lamtor4 and in hepatocytes from Piezo1-GoFconstitutive mice. Our results suggest that mTOR plays a pivotal role in the crosstalk between PIEZO1 signaling and hepcidin regulation in iron homeostasis, mediated by LAMTOR4. To identify potential therapeutic compounds that could restore hepcidin expression and mitigate iron overload, we conducted a high-throughput drug screening of 401 clinically validated drugs targeting the PI3K/AKT/mTOR pathway. Statistical analysis identified compounds that successfully restored HAMP expression to WT levels in Hep3B PIEZO1-KI cells and primary hepatocytes from Piezo1-GoFconstitutive mice. Additional analysis of BMP/SMAD signaling (Id1), inflammation (Saa1), and lipid metabolism (Creb3l3) pathways provided insights into the mechanisms underlying hepcidin restoration. These findings suggest that targeting mTOR signaling could offer a therapeutic strategy for the management of iron overload in PIEZO1-related diseases. Moreover, given the high expression of Piezo1 in liver endothelial cells (LSECs), which are crucial for BMPs-related Hamp transcription, we demonstrate a reduced Bmps expression following increased Ca2+ levels in a Piezo1-dependent manner. This suggests a role for PIEZO1 in LSECs in releasing BMPs to regulate hepcidin expression through crosstalk between LSECs, and hepatocytes.

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