Contributions
Abstract: S276
Type: Oral Presentation
Session title: Focus on iron metabolism
Background
Dehydrated hereditary stomatocytosis (DHS) is an autosomal dominant condition characterized by alterations in red blood cell membrane permeability to the cations. The most frequently mutated gene in DHS is PIEZO1 that encodes a mechanoreceptor, a cation channel activated by mechanical stimuli. Pathogenic variants in PIEZO1 result in a gain-of-function (GOF) phenotype with delayed inactivation of the cation channel that leads to erythrocyte dehydration. The most harmful complication is the hepatic iron overload (Andolfo et al, AJH 2018; Picard et al Haematologica 2019). We already demonstrated the involvement of PIEZO1 at hepatic level by alterations of ERK1/2 and BMP/SMADs pathways that led to the suppression of hepcidin (Andolfo et al, AJH 2020). This finding was recently confirmed and extended by the study of the mouse model carrying a GOF variant of PIEZO1 that develop age-onset iron overload and specific alterations of the macrophage phagocytic activity (Ma et al, Cell 2021). These recent studies suggest that PIEZO1 is an important genetic factor involved in iron metabolism but the molecular mechanism by which it regulates iron metabolism in the interplay among RBCs, hepatocytes and macrophage is still unknown.
Aims
This study aims to dissect the mechanism that links PIEZO1 GOF variants to the suppression of hepcidin by investigation of new players of intracellular signaling pathways.
Methods
Creation of Hep3b engineered R2456H-PIEZO1 knock-in (KI) cells by CRISPR/Cas9; qRT-PCR and western blotting analysis; calcium and potassium ion channel assay; differential proteomics analysis, and RNA sequencing.
Results
To study the involvement of PIEZO1 in the regulation of iron homeostasis we established a KI Hep3b cell model heterozygous for the most frequent variant of PIEZO1, R2456H, by CRISPR/Cas9 technology (Hep3b-GOF-PIEZO1). Hep3b-GOF-PIEZO1 cells showed (i) no alteration of PIEZO1 gene and protein expression; (ii) increased intracellular calcium concentration compared to Hep3b-WT cells; (iii) hyper activation of the potassium channels activity compared to Hep3b-WT; (iv) reduced HAMP expression, according to the decreased phosphorylation of the BMP/SMADs pathway. To further dissect the alteration of the transcriptome and proteome of Hep3b-GOF-PIEZO1 model, we performed both differential proteomics and RNA-Seq. Differential proteomics identified 225 total proteins differentially regulated in Hep3b-GOF-PIEZO1 cells compared to Hep3b-WT ones (Protein with Log2 Difference ≥ ± 1 and -Log p-value > 1.3 were considered significantly enriched), 147 upregulated and 78 downregulated. RNA-Seq identified 5689 genes differentially regulated in Hep3b-GOF-PIEZO1 cells compared to Hep3b-WT ones (p<0.05, post-hoc correction by FDR), 1695 up-regulated and 1823 down-regulated (-0.5≤fold change≤0.5). Using this multiomics integrated approach, Hep3b-GOF-PIEZO1 cells showed alterations in several proteins belonging to MAPK pathways, and revealed new genes and proteins that links the increased calcium concentration to the activation of the intracellular pathways as TGF-b signaling and R-RAS.
Conclusion
We herein deeply dissected the mechanism by which PIEZO1 GOF mutations cause hepatic iron overload in DHS. The creation of a hepatic engineered cell line for PIEZO1 R2456H variant confirmed the effect on the cation flux and the deregulation of iron status. Proteomic and transcriptomic data give us the opportunity to improve our knowledge identifying new actors in the regulation of the complex landscape of iron metabolism.
Keyword(s): Anemia, Hepcidin, Iron overload, Signal transduction
Abstract: S276
Type: Oral Presentation
Session title: Focus on iron metabolism
Background
Dehydrated hereditary stomatocytosis (DHS) is an autosomal dominant condition characterized by alterations in red blood cell membrane permeability to the cations. The most frequently mutated gene in DHS is PIEZO1 that encodes a mechanoreceptor, a cation channel activated by mechanical stimuli. Pathogenic variants in PIEZO1 result in a gain-of-function (GOF) phenotype with delayed inactivation of the cation channel that leads to erythrocyte dehydration. The most harmful complication is the hepatic iron overload (Andolfo et al, AJH 2018; Picard et al Haematologica 2019). We already demonstrated the involvement of PIEZO1 at hepatic level by alterations of ERK1/2 and BMP/SMADs pathways that led to the suppression of hepcidin (Andolfo et al, AJH 2020). This finding was recently confirmed and extended by the study of the mouse model carrying a GOF variant of PIEZO1 that develop age-onset iron overload and specific alterations of the macrophage phagocytic activity (Ma et al, Cell 2021). These recent studies suggest that PIEZO1 is an important genetic factor involved in iron metabolism but the molecular mechanism by which it regulates iron metabolism in the interplay among RBCs, hepatocytes and macrophage is still unknown.
Aims
This study aims to dissect the mechanism that links PIEZO1 GOF variants to the suppression of hepcidin by investigation of new players of intracellular signaling pathways.
Methods
Creation of Hep3b engineered R2456H-PIEZO1 knock-in (KI) cells by CRISPR/Cas9; qRT-PCR and western blotting analysis; calcium and potassium ion channel assay; differential proteomics analysis, and RNA sequencing.
Results
To study the involvement of PIEZO1 in the regulation of iron homeostasis we established a KI Hep3b cell model heterozygous for the most frequent variant of PIEZO1, R2456H, by CRISPR/Cas9 technology (Hep3b-GOF-PIEZO1). Hep3b-GOF-PIEZO1 cells showed (i) no alteration of PIEZO1 gene and protein expression; (ii) increased intracellular calcium concentration compared to Hep3b-WT cells; (iii) hyper activation of the potassium channels activity compared to Hep3b-WT; (iv) reduced HAMP expression, according to the decreased phosphorylation of the BMP/SMADs pathway. To further dissect the alteration of the transcriptome and proteome of Hep3b-GOF-PIEZO1 model, we performed both differential proteomics and RNA-Seq. Differential proteomics identified 225 total proteins differentially regulated in Hep3b-GOF-PIEZO1 cells compared to Hep3b-WT ones (Protein with Log2 Difference ≥ ± 1 and -Log p-value > 1.3 were considered significantly enriched), 147 upregulated and 78 downregulated. RNA-Seq identified 5689 genes differentially regulated in Hep3b-GOF-PIEZO1 cells compared to Hep3b-WT ones (p<0.05, post-hoc correction by FDR), 1695 up-regulated and 1823 down-regulated (-0.5≤fold change≤0.5). Using this multiomics integrated approach, Hep3b-GOF-PIEZO1 cells showed alterations in several proteins belonging to MAPK pathways, and revealed new genes and proteins that links the increased calcium concentration to the activation of the intracellular pathways as TGF-b signaling and R-RAS.
Conclusion
We herein deeply dissected the mechanism by which PIEZO1 GOF mutations cause hepatic iron overload in DHS. The creation of a hepatic engineered cell line for PIEZO1 R2456H variant confirmed the effect on the cation flux and the deregulation of iron status. Proteomic and transcriptomic data give us the opportunity to improve our knowledge identifying new actors in the regulation of the complex landscape of iron metabolism.
Keyword(s): Anemia, Hepcidin, Iron overload, Signal transduction