Investigation of IRS1 and IRS2 function in hematopoiesis using murine knockout models

Author(s)

Bruna Alves Fenerich

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-03-13

Examining board members

Fabíola Traina; José Barreto Campello Carvalheira; Carla Roberta de Oliveira Carvalho; Fernanda Maris Peria

• Advisor: Fabíola Traina; João Agostinho Machado Neto

Abstract

Insulin receptor substrates 1 and 2 (IRS1/2) are proteins that act on signal transduction, regulating processes such as cell growth, energy metabolism, cell survival and proliferation. IRS1/2 are differentially expressed in hematological malignancies and the pharmacological inhibition of these targets results in antineoplastic effects in preclinical models. Previous studies conducted by our research group indicates that IRS2 is recruited during erythroid, megakaryocytic and granulocytic differentiation. However, the role of IRS1 and IRS2 in signal transduction and their participation in hematopoiesis remains poorly explored. The aim of this study was to investigate the effects of Irs1 and Irs2 knockout on hematopoiesis using murine models. B6.129S2-Irs1S57X/J and B6.129-Irs2tm1Mfw mice were used in C57BL/6 background. For Irs1 mice, hematological parameters of peripheral blood (PB) were evaluated monthly by automated blood count, assessment of fetal development were performed at embryonic days E+9.5, E+12.5, E+15.5 and E+18.5, and investigation of fetal hematopoiesis were performed by flow cytometry. Irs2 knockout mice were evaluated for PB hematological parameters by blood count; constitution of the hematopoietic populations of PB, bone marrow (BM) and spleen by flow cytometry, and short and long-term hematopoietic reconstitution post bone marrow transplantation (BMT). In addition, evaluation of gene expression was performed using PCR array. After 28 intercrosses events, a total of Irs1 101 heterozygotes (HET), 46 wild type (WT) and 1 homozygous mice were obtained, with an average of 4.9 (± 1.6) born alive per female. There was no significant difference in body weight, PB hematological parameters and fetal liver hematopoietic stem cells (HSC) frequency between WT and Irs1 KO animals (all p> 0.05). Analysis of embryonic development indicated that Irs1 KO animals developed up to the gestational age E+18.5, however, Irs1 KO mice were identified as stillborn or died immediately after delivery. Irs2 KO animals showed a reduction in red blood cells (RBC) and hemoglobin (p=0.005 and p=0.003, respectively). Irs2 KO group showed an increase in hematopoietic stem cells (HSCs), including LT-HSC (Irs2 KO: 0.087 % vs. WT: 0.027% vs. HET: 0.022%; all p<0.002) and ST-HSC (Irs2 KO: 0.14 % vs. WT:0.04% vs. HET: 0.03%; all p<0.02), in addition to a significant increase in erythroblasts (Ter119 + cells [Irs2 KO: 69.5% vs. WT: 57.1%; p=0.003]). Analysis of hematopoiesis reconstitution, 4 and 8 weeks post BMT, indicated that the group transplanted with hematopoietic cells from Irs2 KO animals showed a reduction in RBC, hematocrit and hemoglobin (all p<0.05). Platelet count decreased after 8 weeks post-transplantation (Irs2 KO: 288x103/&micro;L vs. WT: 538 x103/&micro;L vs. HET: 530 x103/&micro;L; p0.003 and p<0.004, respectively). Long-term evaluation (12 months post BMT) indicated that chimerism did not differ between groups, with a median of 96.1% and 93.6% for WT and Irs2 KO recipients, respectively. Long-term Irs2 KO recipients exhibited reduction in leukocytes (WT: 13.7 x103/&micro;L vs. Irs2 KO: 8.7 x103/&micro;L) and platelets (WT: 1226 x103/&micro;L vs. Irs2 KO: 266 x103/&micro;L); all p<0.05. There was no difference in RBC, hemoglobin and hematocrit between groups (all p>0.05). Mice transplanted with Irs2 KO cells showed an increase in the frequency of LT-HSC (Irs2 KO: 0.4% vs. WT: 0.08%); ST-HSC (Irs2 KO: 0.14% vs. WT:0.05%); myeloid progenitors (Irs2 KO: 2.80% vs. WT:1.78%) and intermediately mature erythroblasts (Irs2 KO: 22.1% vs. WT:15.9%), all p<0.05. Gene expression profile from Irs2 KO hematopoietic cells indicated modulation of pathways involved in hematopoiesis, including deregulation of Wnt pathway and genes that encode cytokines, growth factors, thyroid hormones and regulators of erythropoiesis and megakaryopoiesis. (AU)

• FAPESP's process: 17/25068-8 - Investigation of IRS1 and IRS2 function in normal hematopoiesis and myelodysplastic syndrome using murine models and human hematopoietic stem cells
• Grantee: Bruna Alves Fenerich
• Support Opportunities: Scholarships in Brazil - Doctorate