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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Characterization of ire1α–/– HSCs. (A) Phenotype of ire1α–/– HSCs from f...
Characterization of ire1α–/– HSCs. (A) Phenotype of ire1α–/– HSCs from fetal liver and AGM. FACS analysis of CD34 and c-kit in cells from ire1α–/– and ire1α+/+ fetal liver and AGM at E11. The ratio of the number of AGM cells to that of fetal liver cells used for FACS analysis was 2:1. Representative data from 1 of at least 3 separate experiments are shown. (B) BrdU labeling of ire1α–/– and ire1α+/+ fetal livers at E11. The pregnant mouse was injected with 10 mg BrdU at 2 hours before isolation of embryos. Paraffin-embedded sections (5 μM) were stained with anti-BrdU antibody and visualized at ×100 magnification. The proliferating cells in the fetal livers that incorporated BrdU can be identified by dark staining. (C) Proliferation rates of ire1α–/– and ire1α+/– HSCs. Fetal liver cells from WT embryos at E14.5 were first cultured for 2 days to create a hepatic stromal layer. Similar amounts of ire1α–/– and ire1α+/– CD34 and c-kit double-positive HSCs from embryos at E11 were overlaid on the hepatic stromal layer and were cultured for 10 days. The proliferation rates of ire1α–/– and ire1α+/– HSCs were determined by quantification of neomycin gene copies in the genomic DNA isolated from the proliferated ire1α–/– and ire1α+/– cells. The proliferation rate of the ire1α–/– cells was defined as 1. The proliferation rates of other cells were compared with that of the ire1α–/– cells. Experiments were performed at least 3 times, and the SD is indicated.