The unfolded protein response sensor IRE1α is required at 2 distinct steps in B cell lymphopoiesis
Kezhong Zhang, … , Donalyn Scheuner, Randal J. Kaufman
Kezhong Zhang, … , Donalyn Scheuner, Randal J. Kaufman
Published February 1, 2005
Citation Information: J Clin Invest. 2005;115(2):268-281. https://doi.org/10.1172/JCI21848.
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The unfolded protein response sensor IRE1α is required at 2 distinct steps in B cell lymphopoiesis

Abstract

B lymphocyte differentiation is coordinated with the induction of high-level Ig secretion and expansion of the secretory pathway. Upon accumulation of unfolded proteins in the lumen of the ER, cells activate an intracellular signaling pathway termed the unfolded protein response (UPR). Two major proximal sensors of the UPR are inositol-requiring enzyme 1α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, and ER-resident eukaryotic translation initiation factor 2α (eIF2α) kinase (PERK). To elucidate whether the UPR plays an important role in lymphopoiesis, we carried out reconstitution of recombinase-activating gene 2–deficient (rag2–/–) mice with hematopoietic cells defective in either IRE1α- or PERK-mediated signaling. IRE1α-deficient (ire1α–/–) HSCs can proliferate and give rise to pro–B cells that home to bone marrow. However, IRE1α, but not its catalytic activities, is required for Ig gene rearrangement and production of B cell receptors (BCRs). Analysis of rag2–/– mice transplanted with IRE1α trans-dominant-negative bone marrow cells demonstrated an additional requirement for IRE1α in B lymphopoiesis: both the IRE1α kinase and RNase catalytic activities are required to splice the mRNA encoding X-box–binding protein 1 (XBP1) for terminal differentiation of mature B cells into antibody-secreting plasma cells. Furthermore, UPR-mediated translational control through eIF2α phosphorylation is not required for B lymphocyte maturation and/or plasma cell differentiation. These results suggest specific requirements of the IRE1α-mediated UPR subpathway in the early and late stages of B lymphopoiesis.

Authors

Kezhong Zhang, Hetty N. Wong, Benbo Song, Corey N. Miller, Donalyn Scheuner, Randal J. Kaufman

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Development of B cells is blocked at the early stage in the absence of I...
Development of B cells is blocked at the early stage in the absence of IRE1α. (A) FACS analysis of cell surface markers CD43 and B220 in bone marrow cells from rag2–/– mice after 4 weeks of reconstitution with ire1α–/– or ire1α+/+ fetal liver and AGM cells. (B) Genotypes of the CD43+B220– cells sorted from the reconstituted bone marrow. The upper panel shows amplification of segments from the neo gene and the murine ire1α gene. The first 2 lanes show CD43+B220– cells sorted from the ire1α–/– and ire1α+/+ reconstituted bone marrow. The third lane shows CD43+B220– cells sorted from nonirradiated rag2–/– mice. The fourth and fifth lanes show ire1α–/– and ire1α+/+ MEFs, which served as controls for the genotype of ire1α–/– and ire1α+/+ cells, respectively. The lower panel shows amplification of the rag2 gene WT (rag2+/+) and knockout (rag2–/–) alleles. Through calibration of the amplified gene alleles, the ratios of donor cells to recipient cells in both ire1α–/– and ire1α+/+ reconstituted CD43+B220– populations were found to be more than 10:1. (C) RT-PCR analysis of expression of ire1α and neo genes in the CD43+B220– cells sorted from the reconstituted bone marrow. The samples are the same as described in B. (D–F) FACS analysis of cell surface markers BP-1 and HSA (D), B220 and IgM (E), and TER119 and Mac-1 (F) in ire1α–/– and ire1α+/+ reconstituted bone marrow cells. (G) Genotypes of TER119+ cells sorted from the reconstituted bone marrow. The first 2 lanes show TER119+ cells sorted from the ire1α–/– and ire1α+/+ reconstituted bone marrow. The third lane shows TER119+ cells from nonirradiated rag2–/– mice. The fourth and fifth lanes show ire1α–/– and ire1α+/+ MEFs. Through calibration of the amplified gene alleles, the ratios of donor cells to recipient cells in both ire1α–/– and ire1α+/+ reconstituted TER119+ populations were found to be more than 8:1. For A–G, experiments were performed at least 3 times and representative data are shown.