PIEZO1 GAIN-OF-FUNCTION VARIANTS REGULATE HEPCIDIN EXPRESSION BY MODULATING mTOR/LAMTOR4 SIGNALLING

Barbara Eleni ROSATO^1,2, Roberta RUSSO^1,2, Laura SILVESTRI ^1,2, Immacolata ANDOLFO^1,2, Rossana CARLEO³, Vanessa D’ONOFRIO^1,2, Federica Maria ESPOSITO^1,2, Mariateresa PETTINATO³, Alessia PAGANI³, Roberta MARRA ^1,2, Mario CAPASSO^1,2, Achille IOLASCON^1,2

¹Department of Molecular Medicine and Medical Biotechnology, University of Naples 'Federico II', Naples, Italy., Naples, Italy
²CEINGE Advanced Biotechnology Franco Salvatore, Naples, Italy., Naples, Italy
³Regulation of Iron Metabolism Unit, Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy., Milan, Italy
⁴School of Medicine, Vita-Salute San Raffaele University, Milan, Italy., Milan, Italy

PIEZO1 is a mechanoreceptor that plays a crucial role in various biological processes. Gain-of-function (GoF) variants in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS), a pleiotropic syndrome characterized by anemia and transfusion-independent iron overload. Interestingly, the expression of hepcidin, the master regulator of iron metabolism, is reduced in DHS patients and in PIEZO1 mutant cells via an unknown mechanism.

To learn more about the mechanism, we developed a human Hep3B cell line expressing the PIEZO1-R2456H variant and performed transcriptomic and proteomic analyses. Interestingly, we found that the mTOR signaling is one of the pathways most affected by PIEZO1-R2456H. We show that the expression of Late Endosomal/Lysosomal Adaptor, MAPK And mTOR Activator 4 (LAMTOR4) is strongly downregulated in PIEZO1-R2456H cells. The Ragulator complex plays a crucial role in the mTOR pathway by binding mTORC1 to the lysosome. Since mTORC1 is closely linked to the BMP-SMAD pathway, we knocked down Lamtor4 in murine primary hepatocytes and analyzed hepcidin and the BMP-SMAD pathway. We found that autophagy is increased in the absence of Lamtor4. In parallel, hepcidin is downregulated, likely due to inhibition of the BMP-SMAD pathway, being Id1 also downregulated. However, siLamtor4 hepatocytes properly modulate BMP-SMAD signaling in response to activators or inhibitors, suggesting that LAMTOR4 is upstream of BMP receptors. Further studies are underway to better decipher the mechanism.

Exploring the PIEZO1/LAMTOR4/mTOR axis as a novel player in modulating hepcidin expression will be useful to understand the pathogenic mechanism of DHS and identify novel drug targets to alleviate DHS-associated iron overload.