The unfolded protein response sensor IRE1α is required at 2 distinct steps in B cell lymphopoiesis
Kezhong Zhang, Hetty N. Wong, Benbo Song, Corey N. Miller, Donalyn Scheuner, Randal J. Kaufman
Kezhong Zhang, Hetty N. Wong, Benbo Song, Corey N. Miller, Donalyn Scheuner, Randal J. Kaufman
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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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IRE1α kinase and RNase activities regulate the production of Ig through ...
IRE1α kinase and RNase activities regulate the production of Ig through spliced XBP1. (A) Depiction of IRE1α trans-dominant-negative vectors. IRE1α K599A, IRE1α kinase mutant; IRE1α K907A, IRE1α RNase mutant. (B) FACS analysis of IgD and IgM in splenocytes from reconstituted rag2–/– mice. (C) Levels of serum IgM and IgG1 in the reconstituted rag2–/– mice. Blood samples were collected from the reconstituted rag2–/– mice at 1 month after transplantation. Levels of serum IgM and IgG1 were determined by ELISA and normalized to the number of GFP-positive B cells. (D) XBP1 splicing and induction of IL-6, secretory IgM, IgG2b, and IgG3 transcripts during plasma cell differentiation in the absence of the IRE1α kinase, RNase, or cytoplasmic domain. GFP and B220 double-positive splenic B cells were sorted from the reconstituted rag2–/– mice and subsequently stimulated with LPS (20 μg/ml) for 48 hours in vitro. Total RNA was isolated from the LPS-treated B cells and subjected to semiquantitative RT-PCR analysis. IgMs, secretory IgM transcript; u-Xbp1, unspliced xbp1 transcript; s-Xbp1, spliced xbp1 transcript. (E) Western blot analysis of expression of IRE1α and XBP1 protein variants in primary B cells after secondary viral transduction. (F) Spliced XBP1 restores Ig production in the IRE1α kinase or RNase mutant B cells, but not in the IRE1α cytoplasmic domain–deleted B cells. Primary B cells after secondary viral transduction were treated with LPS (20 μg/ml) for 48 hours. Levels of secreted IgM and IgG1 in cultured supernatants of the indicated stimulated B cells were determined by ELISA. For B–F, experiments were performed at least 3 times and representative data and SD are shown.