A Cell-Autonomous Myeloproliferative Phenotype Caused by Loss of Latexin in Stem and Progenitor Cells. (2012)

Abstract Abstract 2314 We have previously shown a novel role for Latexin (Lxn), a carboxypeptidase inhibitor, in influencing the size of murine hematopoietic stem cell (HSC) population. Lxn negatively regulates HSC population size by enhancing apoptosis while repressing proliferation and self-renewal. However, the major mechanisms underlying this phenotype remain unknown. To further study the functionality of latexin in hematopoiesis, we generated a knock-out mouse model (Lxn−/− mouse) with a truncation of Lxn exon 2 to exon 5. Blood cell counts showed that lymphocytes, neutrophils, eosinophils, and monocytes were significantly increased from 50% to 100%, indicating an overall myeloid expansion in the periphery. The Lxn−/− mice also showed hypercellular bone marrow (BM), splenomegaly, and increased colony formation in methylcellulose. We found that the frequencies and absolute numbers of Lineage-/Sca-1+/c-Kit+ (LSK) and multi-potent progenitor (MPP) cells were significantly increased, while short-term and long-term HSCs remained unchanged in Lxn−/− mice. We next evaluated both lymphoid and myeloid progenitor subpopulations within the MPP compartment. Absolute numbers of granulocyte-monocyte progenitor (GMP) cells as well as common lymphoid progenitor (CLP) cells were significantly increased by 60–80% in Lxn−/− mice compared with wild type animals. We then performed cobblestone-area-forming-cell (CAFC) assay, an in vitro functional assay to evaluate the numbers of functional progenitors and stem cells. As a result, cobblestone area forming cells were significantly up-regulated at days 7, 14, 28 and 35, indicating that numbers of both mature progenitors and functional stem cells in BM were increased. To further investigate the observed progenitor and stem cell expansion in Lxn−/− mice, we studied the apoptotic status of BM cells, using the apoptotic marker Annexin V. The apoptosis frequencies were reduced approximately 50% in Lxn deficient LSK cells and MPP cells, suggesting that Lxn positively regulates apoptosis apoptosis in HSCs, as reported previously. Moreover, we used the competitive repopulation assay to determine functional capacities of Lxn−/− HSCs. In this assay, equal numbers (1×106) of Lxn−/− (or wild type) whole BM cells were transplanted along with competitors into lethally irradiated recipient animals. We analyzed the peripheral blood and BM of recipients at 15 weeks post transplantation. In both BM and PB, Lxn−/− cells showed a 50% increase in engraftment. We noted that there was significant up-regulation in granulocytes and monocytes (50%), as well as B cell lymphocytes (50%), but not in T cells, which is consistent with what we observed in Lxn−/− mice. These findings reveal that the myeloproliferation in Lxn−/− mice is transplantable and thus is intrinsic. To gain insights into the molecular mechanisms underlying the phenotypes created by loss of Lxn expression, we carried out gene profiling between Lxn and wild type MPP cells. Microarray experiments were performed on BM cells using Affymetrix Gene Chip. To study the pathway changes in response to loss of Lxn expression, we performed the gene set enrichment analysis (GSEA). Five enriched pathways have been identified as altered in Lxn−/− MPP cells that include: cell communication, ECM receptor interaction, proteasome, cell cycle and neuroactive ligand receptor interaction. In conclusion, these data showed that loss of Lxn leads to proliferative hematopoiesis with clonal expansion at the stem and progenitor cell levels, and that this myeloproliferative phenotype is cell autonomous and transplantable. This work was supported by NIH HD-060915 and the Edward P. Evans Foundation Disclosures: No relevant conflicts of interest to declare.