Calreticulin (CALR) is a 46 kDa endoplasmic reticulum (ER) chaperone responsible for intracellular calcium regulation and protein folding. CALR is also able to perform different functions outside the ER, such as responses to cellular stress. In 2013, several mutations were discovered in exon 9 of CALR gene in Myeloproliferative Neoplasms (MPNs); notably 36 different types of mutations have been reported in 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) patients. These mutations consist of insertions or deletions that induce a frameshift, resulting in a loss of the C-terminal portion domain and the KDEL sequence. In this view, structural alterations of CALR protein might impair its functionality and its cellular localization. Indeed, recent data demonstrated that C-terminal of CALR mutants interacts with the thrombopoietin receptor (MPL), inducing the constitutive activation of JAK-STAT pathway. However, the precise mechanism of action through which CALR mutants contribute to the development of MPNs has only been partially clarified and there is no available information on the function of Wild-Type (WT) CALR during physiological hematopoiesis. In order to elucidate the biological role of WT CALR in the proliferation and differentiation of hematopoietic stem/progenitor cells (HPSCs), we performed gene silencing and overexpression experiments in human CD34+ cells. Our data demonstrated that WT CALR overexpression is able to enhance erythroid and megakaryocyte (MK) differentiation. Consistently, WT CALR silencing induces a marked repression of MK and erythroid lineages. In agreement with these results, gene expression profile (GEP) analysis confirmed that WT CALR is able to affect the expression of genes of erythroid and MK differentiation. Moreover, transcriptome analysis showed the modulation of several genes implicated in oxidative stress, ER stress response, DNA damage and in several pathways already described as able to play a role in MPN development. Next, to investigate the impact of CALR mutations in oxidative and ER stress response, we transduced K562 cells with vectors expressing one of the two commonest CALR mutated variants, either CALRdel52 or CALRins5. We chose to perform experiments in the K562 cell line, devoid of MPL expression, in order to identify additional pathways whose alterations might cooperate with cellular transformation mediated by MPL activation. Firstly, we demonstrated that CALR mutants reduce the capability to respond to ER stress and significantly decrease the activation of the pro-apoptotic unfolded protein response (UPR) pathway. Then, we showed that CALR mutations induce increased sensitivity to oxidative stress, also driving the accumulation of oxidative DNA damage. Finally, we studied the function of the antioxidant gene OXR1, found deregulated in GEP analysis. The downregulation of OXR1 affects the capability of mutant cells to counterbalance reactive oxygen species (ROS) accumulation, suggesting that lack of OXR1 expression might be one of the molecular mechanisms responsible for the impaired oxidative stress response mediated by mutant CALR. Altogether, our results suggest a new role of WT CALR in the regulation of erythroid and MK differentiation, whose deregulation is crucial in MPNs. Moreover, CALR mutants negatively impact on the ability to respond to ER stress, conferring apoptosis resistance to the cells. Finally, the impairment in oxidative stress response due to CALR mutations can lead to genomic instability, thus promoting cell transformation.
Calreticulina (CALR) è uno chaperone da 46 kDa residente nel reticolo endoplasmatico responsabile della regolazione del calcio intracellulare e del folding proteico. CALR è anche in grado di regolare diverse funzioni al di fuori dal reticolo, tra cui la risposta allo stress cellulare. Nel 2013 sono state scoperte diverse mutazioni nell’esone 9 del gene CALR nelle Neoplasie Mieloproliferative (MPNs); in particolare sono state individuate 36 tipi diversi di mutazioni nel 60-70% dei pazienti con Trombocitemia Essenziale (ET) e Mielofibrosi Primaria (PMF) non mutati per JAK2 e MPL. Tali mutazioni consistono in inserzioni o delezioni che portano a frameshift con conseguente perdita del dominio C-terminale e della sequenza KDEL. In quest’ottica, l’alterazione strutturale di CALR comprometterebbe la sua funzionalità e la sua localizzazione cellulare. Infatti è stato dimostrato che il residuo C-terminale di CALR mutato interagisce con il recettore della trombopoietina (MPL), inducendo l’attivazione costitutiva della via JAK-STAT. Tuttavia, il preciso meccanismo d’azione dei mutanti di CALR è stato solo parzialmente chiarito e non ci sono informazioni sulla funzione di CALR Wild-Type (WT) durante l’emopoiesi fisiologica. Per chiarire il ruolo biologico di CALR WT nella proliferazione e nel differenziamento dei progenitori emopoietici (HPSCs), abbiamo eseguito esperimenti di silenziamento e overespressione in cellule CD34+ umane. I nostri dati mostrano che l’overespressione di CALR WT è in grado di promuovere il differenziamento eritroide e megacariocitario (MK). Parallelamente, il silenziamento di CALR WT induce una marcata repressione del lineages eritroide e MK. In accordo con questi risultati, l'analisi del profilo d’espressione genica (GEP) ha confermato che CALR WT è in grado di influenzare l'espressione dei geni del differenziamento eritroide e MK. Inoltre, l’analisi trascrizionale ha mostrato la modulazione di diversi geni coinvolti nella risposta allo stress ossidativo, allo stress del reticolo, al danno al DNA e in pathways biologici alla base dello sviluppo delle MPNs. Successivamente, per studiare l’effetto delle mutazioni di CALR sulla sua funzionalità in risposta allo stress ossidativo e del reticolo, abbiamo infettato le cellule K562 con vettori retrovirali esprimenti una delle due varianti mutate più comuni, CALRdel52 o CALRins5. Abbiamo deciso di utilizzare la linea cellulare K562, priva dell’espressione di MPL, per individuare ulteriori vie di segnalazione alla base della trasformazione MPL-mediata. In primo luogo, i nostri dati mostrano che i mutanti di CALR compromettono la capacità di rispondere allo stress del reticolo e riducono drasticamente l'attivazione dell’apoptosi mediata dalla UPR (Unfolded Protein Response). Inoltre abbiamo dimostrato che le mutazioni di CALR causano una maggiore sensibilità allo stress ossidativo con accumulo di danni ossidativi al DNA. Infine, abbiamo studiato la funzione del gene antiossidante OXR1, deregolato nell’analisi di GEP. La downregolazione di OXR1 altera la capacità delle cellule mutate di controbilanciare l’accumulo di specie reattive dell’ossigeno (ROS), suggerendo il possibile coinvolgimento di OXR1 nell’alterata risposta allo stress ossidativo che si osserva nei mutanti di CALR. Nel complesso, i nostri risultati rivelano un nuovo ruolo di CALR WT nella regolazione del differenziamento eritroide e MK, due lineages coinvolti nelle MPNs. Inoltre, i mutanti di CALR incidono negativamente sulla risposta allo stress del reticolo, causando resistenza all’apoptosi. Infine, l’alterata risposta allo stress ossidativo che si osserva nei mutanti di CALR può causare instabilità genomica, promuovendo così la trasformazione tumorale.
Ruolo di Calreticulina Wild-Type nell’emopoiesi fisiologica ed effetto delle mutazioni di Calreticulina nello sviluppo delle Neoplasie Mieloproliferative / Elena Genovese , 2020 Mar 13. 32. ciclo, Anno Accademico 2018/2019.
Ruolo di Calreticulina Wild-Type nell’emopoiesi fisiologica ed effetto delle mutazioni di Calreticulina nello sviluppo delle Neoplasie Mieloproliferative
GENOVESE, ELENA
2020
Abstract
Calreticulin (CALR) is a 46 kDa endoplasmic reticulum (ER) chaperone responsible for intracellular calcium regulation and protein folding. CALR is also able to perform different functions outside the ER, such as responses to cellular stress. In 2013, several mutations were discovered in exon 9 of CALR gene in Myeloproliferative Neoplasms (MPNs); notably 36 different types of mutations have been reported in 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) patients. These mutations consist of insertions or deletions that induce a frameshift, resulting in a loss of the C-terminal portion domain and the KDEL sequence. In this view, structural alterations of CALR protein might impair its functionality and its cellular localization. Indeed, recent data demonstrated that C-terminal of CALR mutants interacts with the thrombopoietin receptor (MPL), inducing the constitutive activation of JAK-STAT pathway. However, the precise mechanism of action through which CALR mutants contribute to the development of MPNs has only been partially clarified and there is no available information on the function of Wild-Type (WT) CALR during physiological hematopoiesis. In order to elucidate the biological role of WT CALR in the proliferation and differentiation of hematopoietic stem/progenitor cells (HPSCs), we performed gene silencing and overexpression experiments in human CD34+ cells. Our data demonstrated that WT CALR overexpression is able to enhance erythroid and megakaryocyte (MK) differentiation. Consistently, WT CALR silencing induces a marked repression of MK and erythroid lineages. In agreement with these results, gene expression profile (GEP) analysis confirmed that WT CALR is able to affect the expression of genes of erythroid and MK differentiation. Moreover, transcriptome analysis showed the modulation of several genes implicated in oxidative stress, ER stress response, DNA damage and in several pathways already described as able to play a role in MPN development. Next, to investigate the impact of CALR mutations in oxidative and ER stress response, we transduced K562 cells with vectors expressing one of the two commonest CALR mutated variants, either CALRdel52 or CALRins5. We chose to perform experiments in the K562 cell line, devoid of MPL expression, in order to identify additional pathways whose alterations might cooperate with cellular transformation mediated by MPL activation. Firstly, we demonstrated that CALR mutants reduce the capability to respond to ER stress and significantly decrease the activation of the pro-apoptotic unfolded protein response (UPR) pathway. Then, we showed that CALR mutations induce increased sensitivity to oxidative stress, also driving the accumulation of oxidative DNA damage. Finally, we studied the function of the antioxidant gene OXR1, found deregulated in GEP analysis. The downregulation of OXR1 affects the capability of mutant cells to counterbalance reactive oxygen species (ROS) accumulation, suggesting that lack of OXR1 expression might be one of the molecular mechanisms responsible for the impaired oxidative stress response mediated by mutant CALR. Altogether, our results suggest a new role of WT CALR in the regulation of erythroid and MK differentiation, whose deregulation is crucial in MPNs. Moreover, CALR mutants negatively impact on the ability to respond to ER stress, conferring apoptosis resistance to the cells. Finally, the impairment in oxidative stress response due to CALR mutations can lead to genomic instability, thus promoting cell transformation.File | Dimensione | Formato | |
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