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
View: Text | PDF
Article Development

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

×

Figure 5

Options: View larger image (or click on image) Download as PowerPoint
The IRE1α cytoplasmic domain is required for BCR formation and plasma ce...
The IRE1α cytoplasmic domain is required for BCR formation and plasma cell differentiation. (A) Depiction of viral transduction and bone marrow cell transplantation. Lentivirus (IRE1α), lentivirus expressing IRE1α protein variants; Retrovirus (XBP1), retrovirus expressing spliced XBP1 protein. (B) FACS analysis of B220 and IgM in splenocytes from the reconstituted rag2–/– mice. (C) Western blot analysis of expression of the IRE1α transgenes in B220+ splenic B cells sorted from the reconstituted rag–/– mice as described in B. (D) 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. (E) Western blot analysis of expression of IRE1α and XBP1 protein variants in primary splenic B cells after secondary viral transduction. (F) Levels of secreted IgM and IgG1 in LPS-stimulated B cells. The samples are labeled the same as described in E. 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 medium were determined by ELISA. V/C, control virus–transduced; IRE1α °C, IRE1α cytoplasmic domain deletion; IRE1α °C + IRE1α, IRE1α °C plus WT IRE1α protein, RV, control retrovirus; XPB1s, retrovirus expressing spliced XBP1 protein. For B–F, experiments were performed at least 3 times and representative data and SD are shown.