B- and T-cell Development Both Involve Activity of the Unfolded Protein Response Pathway

Ryan Brunsing,Sidne A Omori,Frank Weber,Alicia Bicknell,Leslie Friend,Robert Rickert,Maho Niwa

doi:10.1074/jbc.m801395200

Abstract

The unfolded protein response (UPR) signaling pathway regulates the functional capacity of the endoplasmic reticulum for protein folding. Beyond a role for UPR signaling during terminal differentiation of mature B cells to antibody-secreting plasma cells, the status or importance of UPR signaling during hematopoiesis has not been explored, due in part to difficulties in isolating sufficient quantities of cells at developmentally intermediate stages required for biochemical analysis. Following reconstitution of irradiated mice with hematopoietic cells carrying a fluorescent UPR reporter construct, we found that IRE1 nuclease activity for XBP1 splicing is active at early stages of T- and B-lymphocyte differentiation: in bone marrow pro-B cells and in CD4(+)CD8(+) double positive thymic T cells. IRE1 was not active in B cells at later stages. In T cells, IRE activity was not detected in the more mature CD4(+) T-cell population but was active in the CD8(+) cytotoxic T-cell population. Multiple signals are likely to be involved in activating IRE1 during lymphocyte differentiation, including rearrangement of antigen receptor genes. Our results show that reporter-transduced hematopoietic stem cells provide a quick and easy means to identify UPR signaling component activation in physiological settings.

Highlights

The bone marrow of vertebrate adults, undergo dramatic changes in cellular architecture during differentiation to functionally specialized cells
Evidence suggests that the IRE1 kinase activity functions to regulate activation of the IRE1 RNase activity, which cleaves an intron from mRNA coding for a key unfolded protein response (UPR)-specific transcription factor (XBP1 in mammalian cells, HAC1 in yeast) [5, 6, 16, 19]
Following these initial experiments to validate the utility of our XBP1-GFP reporter, we reasoned that our ability to detect IRE1 RNase activity in small cell populations would be of value in examining whether IRE1 is activated during earlier stages of hematopoiesis

Summary

MATERIALS AND METHODS

Cell Culture and UPR Induction—Chinese hamster ovary (CHO) cells were grown in Dulbecco’s modified Eagle’s medium/F12 (Cellgro) medium supplemented with 5% fetal calf serum (Invitrogen), 100 units/ml penicillin, and 100 ␮g/ml streptomycin at 37 °C in 5% CO2. Upon stable transfection of the reporter in CHO cells, GFP expression during tunicamycin treatment was correlated with the extent of splicing of the UPR intron from XBP1/GFP mRNA. PCR primers complementary to the sequence unique to endogenous XBP1 showed that the extent of reporter splicing was similar to that of endogenous XBP1 mRNA (Fig. 1C) These results confirm that the XBP1 fragment of the reporter containing the UPR intron is suffibone marrow was collected as described above. XBP1 splicthymus, and lymph nodes were collected using common surgi- ing was further examined during recovery from UPR activation, cal techniques and homogenized in separate wells of a 6-well following removal and washout of tunicamycin In this case, dish containing 4 ml of PBS. After lysing the red blood cells as described above, cells were Because tunicamycin blocks an early step of protein glycosylaresuspended to 107 cells/ml in PBS for staining

RESULTS

Findings

DISCUSSION