GAIN-OF-FUNCTION MUTATIONS IN PIEZO1 DIRECTLY IMPAIR HEPATIC IRON METABOLISM BY ALTERATIONS OF BMP/SMADS PATHWAY
Author(s): ,
Immacolata Andolfo
Affiliations:
Dip. Medicina Molecolare e Biotecnologie Mediche,University of Naples, Federico II, Napoli, Italy,Napoli,Italy;Ceinge biotecnologie avanzate,Napoli,Italy
,
Barbara Eleni Rosato
Affiliations:
Dip. Medicina Molecolare e Biotecnologie Mediche,University of Naples, Federico II, Napoli, Italy,Napoli,Italy;Ceinge biotecnologie avanzate,Napoli,Italy
,
Francesco Manna
Affiliations:
Dip. Medicina Molecolare e Biotecnologie Mediche,University of Naples, Federico II, Napoli, Italy,Napoli,Italy;Ceinge biotecnologie avanzate,Napoli,Italy
,
Gianluca De Rosa
Affiliations:
Dip. Medicina Molecolare e Biotecnologie Mediche,University of Naples, Federico II, Napoli, Italy,Napoli,Italy;Ceinge biotecnologie avanzate,Napoli,Italy
,
Roberta Marra
Affiliations:
Dip. Medicina Molecolare e Biotecnologie Mediche,University of Naples, Federico II, Napoli, Italy,Napoli,Italy;Ceinge biotecnologie avanzate,Napoli,Italy
,
Domenico Girelli
Affiliations:
Department of Medicine,University of Verona,Verona,Italy
,
Roberta Russo
Affiliations:
Dip. Medicina Molecolare e Biotecnologie Mediche,University of Naples, Federico II, Napoli, Italy,Napoli,Italy;Ceinge biotecnologie avanzate,Napoli,Italy
Achille Iolascon
Affiliations:
Dip. Medicina Molecolare e Biotecnologie Mediche,University of Naples, Federico II, Napoli, Italy,Napoli,Italy;Ceinge biotecnologie avanzate,Napoli,Italy
EHA Library. Andolfo I. Jun 15, 2019; 267486; S903
Dr. Immacolata Andolfo
Dr. Immacolata Andolfo
Contributions
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Abstract

Abstract: S903

Type: Oral Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:00 - 17:15

Location: Hall G102

Background

Dehydrated hereditary stomatocytosis (DHS), or xerocytosis, is an autosomal dominant hemolytic anemia characterized by cation leak resulting in altered intracellular cation content and cell volume alterations. Two causative genes have been identified until now: PIEZO1 (DHS1), a selective cation channel activated by mechanical force, and KCNN4 (DHS2), the Gardos channel, a Ca2+-dependent K+ channel. In DHS1 erythrocytes several electrophysiological studies have demonstrated that most of PIEZO1 missense mutations are gain-of-function. Indeed, these mutations cause the dehydration of the erythrocytes due to excessive potassium efflux and calcium influx. DHS1 patients present as major complication severe hepatic iron overload with several cases of hepatosiderosis. We hypothesized a specific implication of PIEZO1 gain-of-function mutations in the pathogenesis of the iron overload in DHS1 because: i) KCNN4 mutated patients showed a milder degree of iron overload compared to PIEZO1 mutated ones; ii) the iron overload is independent of the transfusion rate in PIEZO1 patients; iii) the iron overload is independent of the degree of the anemia in DHS1 patients.

Aims
To dissect the pathogenetic mechanism of iron overload in DHS1.

Methods

Hepcidin and ERFE dosage in plasma samples of DHS patients; cells culture, transfection assays and drug treatment; qRT-PCR; immunoblotting; calcium assay.

Results

We first analyzed the expression of the hepatic hormone, hepcidin, demonstrating its decreased levels in the plasma of DHS1 patients compared to healthy controls. Moreover, we measured in the same plasma samples the expression of ERFE, the negative erythroid regulator of hepcidin, demonstrating no impairment of its expression in DHS1. Thus, we assumed that an ERFE-independent regulated mechanism controls the hepcidin expression.

By means of two hepatic cellular models, HepG2 and HuH7 cells, expressing PIEZO1-WT and two PIEZO1 gain-of-function mutants, we highlighted the altered expression of several genes/proteins involved in iron metabolism as Hamp, Ferritin, TFR1, TMPRSS6, and HJV in the mutants.

Then, we assessed the intracellular calcium concentration of our cells demonstrating a higher content of calcium in the mutants compared to the WT. Thus, we hypothesized that the link between the PIEZO1 mutations and the iron overload could be the increased intracellular calcium that in turn triggers the ERK signaling. We assayed the phosphorylation of ERK1/2, and we found increased p-ERK1/2 in the PIEZO1-mutants in our systems. Of note, the activation of ERK1/2 kinases inhibits the BMP-SMADs pathway, the major regulator of Hamp transcription. Indeed, we found reduced phosphorylation of SMAD1/5/8 in the mutants, converging on the suppression of transcription of Hamp.

To demonstrate the direct link between PIEZO1 mutations and iron overload, we selectively inhibited PIEZO1 by GSMTX-4. We showed the rescue of the phenotype observed in the PIEZO1-mutants in both the hepatic cell systems. 

Conclusion

The pathomechanism of iron overload in DHS1 provides the interplay between calcium and iron pathways that converge by negative regulating hepcidin expression. This is the first demonstration of a link between a cation channel alteration and iron metabolism that could shed light on new therapeutic strategies of iron overload in DHS1 and also in other iron loading anemia characterized by possible alterations of PIEZO1, as sickle cell disease and thalassemia.

Session topic: 29. Iron metabolism, deficiency and overload

Keyword(s): ERK, Hepcidin, Iron overload

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