The Role of Calreticulin in Normal Hematopoiesis and Neoplastic Hematopoiesis of Myeloproliferative Neoplasms
Authors: Shide Kotaro , Kameda Takuro , Kamiunten Ayako , Ozono Yoshinori , Tahira Yuki , Sekine Masaaki , Ono Masaya , Yokomizo Takako , Kubota Sho , Akizuki Keiichi , Kubuki Yoko , Hidaka Tomonori , Sashida Goro , Shimoda Kazuya 
Published: February 2020

Mutations in the Calreticulin (CALR) gene were identified in cases of myeloproriferative neoplasms (MPNs), and various functions of the CALR mutant protein are being elucidated. On the other hand, few data are available on the role of CALR in the hematopoietic system. The knockout (KO) mice of Calr are impaired in expression of transcription factors necessary for cardiac development and are embryonic lethal. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr KO mice.
Mice carrying floxed allele targeted exons 4-7 of Calr (Calrf/+ mice) and Calr heterozygous KO mice (Calr+/− mice) (Tokuhiro et al., Sci Rep 2015) were crossed with Mx1-Cre transgenic mice and obtained mice with three genotypes; Mx1-cre;Calr+/+, Mx1-cre;Calr+/−, and Mx1-cre;Calrf/−. Floxed alleles were then deleted by intraperitoneal injection with polyinosinic: polycytidilic acid.
No differences were found in the peripheral blood (PB) leukocyte count, hemoglobin levels, or platelet count among the three genotypes of mice. The proportions of Mac1+ or Gr1+ myeloid lineage cells, B220+ B cells, and CD3+ T cells among the three groups were comparable. In the bone marrow (BM), cell pellet from Mx1-cre;Calrf/− mice appeared anemic and the proportion of CD71+/Ter119+ erythroid cells and the number of CFU-E were significantly lower in Mx1-cre;Calrf/− BM cells compared to the other two genotypes of BM cells. On the other hand, no difference was found in other mature cells such as myeloid, T, B, or CD41+ megakaryocytes (Mks) in BM. As for HSCs and progenitors, Mx1-cre;Calrf/− mice exhibited a higher proportion of MPP and GMP compared to the other two genotypes of mice. We found no difference in other progenitor compartments including long- and short-term HSCs among the three groups. In contrast to the minor effect on BM and PB cells, The spleen weight in Mx1-cre;Calrf/− mice was about 2-fold heavier than that in Mx1-cre;Calr+/+ mice. In spleens from Mx1-cre;Calrf/− mice, the border between the white and red pulp was obscured. Mks and maturing myeloid cells had markedly infiltrated into the red pulp. In FACS analysis, mature myeloid cells, erythroid cells, and Mks were significantly increased in spleens from Mx1-cre;Calrf/− mice compared to those from the other two genotypes of mice. HSCs and most types of myeloid progenitors were also increased in the spleen. Hematopoiesis in the spleen may compensate for the reduced erythropoiesis in the BM, as no anemia was seen in Mx1-cre;Calrf/− mice. No onset of leukemia or myelofibrosis was observed, and no difference in survival was seen among the three groups following observation for 2 years.
As CALR plays a role as a chaperone in the endoplasmic reticulum (ER) during protein synthesis, we searched for proteins with expression that was reduced by Calr deficiency. The quantitative differences of proteins in Mac1+/Gr1+ BM cells between Mx1-cre;Calrf/− and Mx1-cre;Calr+/+ mice were compared using proteomics analysis by 2DICAL, a shotgun proteomics analysis system (Ono et al. Cellular Proteomics 2006). We found that only a few proteins, including myeloperoxidase and CALR itself, were reduced, and conversely, many proteins were increased by the absence of CALR. List of differentially upregulated proteins higher than those in WT samples were analyzed using Metascape (http://metascape.org) to determine enriched pathways. The most enriched cluster was "protein processing in the ER", and 19 out of 76 upregulated (&gt;1.8-fold) proteins were included in this cluster. In real-time PCR analysis, we confirmed that ER chaperon genes (BiP, Grp94, Hsp40, Pdia3, Pdia4, Pdia6) and genes involved in ER-related degradation (Trap, Bap31, Derl1, p97) were significantly upregulated in Mx1-cre;Calrf/− myeloid cells. These observations suggested that chaperone dysfunction due to Calr deficiency is compensated by upregulation of unfolded protein response (UPR) pathway.
In summary, Calr deficiency induced erythroid hypoplasia in the BM, and induced splenomegaly and extramedullary hematopoiesis. This suggests that the amount of WT CALR expression remaining in CALR mutant cells may modify the phenotype of MPN patients. Absence of myeloperoxidase protein and upregulation of UPR pathway in myeloid cells from Calr KO mice indicates that CALR plays a significant role as a chaperone also in hematopoiesis and that CALR deficient cells are exposed to ER stress.

Disclosures
No relevant conflicts of interest to declare.