Abstract
The unfolded protein response (UPR) is a cell-signalling mechanism that regulates endoplasmic reticulum (ER) homeostasis with the help of three ER transmembrane protein sensors: inositol-requiring enzyme 1 (IRE-1α), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Recent studies reported that transcription of UPR-specific genes is significantly upregulated in calreticulin (CALR)-mutated megakaryocyte progenitors. CALR is a major Ca2+ binding protein in the ER. Mutations in exon 9 of CALR are the second most common genetic cause of myeloproliferative neoplasms (MPNs), incurring a characteristic increase of megakaryocytes in the bone marrow. This discovery led to the hypothesis that CALR mutations may affect ER Ca2+ homoeostasis and related signalling processes that might influence aberrant megakaryopoiesis. This thesis aimed to assess the involvement of UPR signalling and Ca2+ homeostasis in normal and CALR-mutant megakaryocyte maturation.
A model system of PMA (phorbol myristate acetate)-induced in vitro maturation of human megakaryocytic cell lines, K-562 and MEG-01, was employed in this thesis to determine the involvement of the UPR during megakaryocyte maturation. RT-qPCR results revealed a significant upregulation in the transcription of IRE1α or ATF6 pathway-specific UPR genes, CHOP, XBP-1t, HERPUD1, and ERDJ4, during in vitro megakaryocyte maturation. Treatment with UPR inhibitors showed that megakaryocyte maturation significantly decreases when the IRE-1α/XBP-1s pathway is blocked, but not PERK or ATF6. However, in vitro differentiated, and undifferentiated megakaryocytes showed similar temporal responses to ER stress activation by dithiothreitol (DTT) and thapsigargin (Tg). Moreover, activation of UPR by thapsigargin (Tg), which induces UPR via depletion of ER calcium stores, showed a dose-dependent decrease in PMA-induced megakaryocyte maturation. Notably, inhibition of ER calcium entry and IRE1α endonuclease activity suppressed the thrombopoietin-induced megakaryocyte differentiation in human peripheral blood-derived CD45+ cells.
Dual-guide CRISPR/Cas9 approach was used to generate the most common CALR mutations (type I, 52-bp deletion) in K-562 cells. Four clonal lines of type I-like CALR mutant cells were generated, exhibiting characteristics similar to those in MPN patients, and the clones were validated using western blotting and mass spectrometry. RT-qPCR revealed no significant difference in the baseline transcriptional activation of UPR genes in the CALR-mutant clones, as compared to WT K-562 cells, suggesting the effects of CALR mutations were independent of UPR signalling. Mag-fluo-4, AM, a fluorescent ER Ca2+ indicator, showed that type I-like CALR mutant cells have higher intraluminal ER Ca2+ than wild-type (WT) cells. Moreover, the cytoplasmic Ca2+ indicator Fura-2, AM, displayed an elevated store depletion of Ca2+ in CALR mutant cells upon ionomycin treatment. Notably, western blotting analysis revealed a significant increase in baseline phosphorylation of ERK1/2 in CALR mutant cells.
Together, the results of this thesis demonstrate that UPR signalling is involved in PMA-induced in vitro megakaryocyte maturation, ER Ca2+ homeostasis together with IRE-1α/XBP-1s pathway play a role in the megakaryocyte maturation process, the effect of CALR mutations on in vitro megakaryocyte maturation is independent of UPR signalling, and CALR-mutant cells have elevated baseline ER Ca2+ levels and MAPK pathway activation, suggesting involvement of ER Ca2+ homeostasis in CALR-mutated MPN pathogenesis, which warrants future investigation.