The unfolded protein response (UPR)-activated transcription factor X-box-binding protein 1 (XBP1) induces microRNA-346 expression that targets the human antigen peptide transporter 1 (TAP1) mRNA and governs immune regulatory genes

Abstract

To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3'-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.

FIGURE 1.

miR-346 levels increase during ER stress. A, results of the miRNA profiling arrays. Calu-3 cells were treated with two different ER stressors, TM (5 μg/ml) and proteasome inhibition (ALLN, 100 μg/ml), for 12 h prior to isolation of RNA + miRNA and Affymetrix miRNA array hybridization. The Venn diagram illustrates that the number of miRNAs enhanced was 47 and 39 by TM and ALLN, respectively. Two miRNAs were induced by both mechanisms. B, relative increase in miR-346 and miR-885-3p levels. Two miRNAs, miR-346 and miR-885-3p, were enhanced by both ER stressors. miR-346 was selected for further studies based on the ∼4-fold increase by both ER stressors. C, chromosomal location of the miR-346 gene. The miR-346 gene is localized to human chromosome 10, in intron 2 of the GRID1 gene. The locations of GRID1 and miR-346 are indicated on the model of chromosome 10. D, time course studies on the elevation of sXBP1 mRNA and miR-346 levels following ER stress induction. Calu-3 cells were treated with TM (5 μg/ml) for the time periods indicated, and RNA + miRNA were isolated at the time points specified followed by qRT-PCR quantification of sXBP1 mRNA as reporter of UPR activation (left). miR-346 levels were measured at the time points indicated (right). Results are plotted as -fold increase in sXBP1 mRNA or miR-346 over control, prior to the addition of TM (n = 4).

FIGURE 2.

Overexpression of sXBP1 enhances miR-346 levels in HeLa cells. A, overexpression of sXBP1 and uXBP1 constructs in HeLa cells. HeLa cells were transfected with empty vector (EV), sXBP1, and uXBP1 constructs. To assess transfection efficiency, XBP1 mRNA levels were measured by qRT-PCR (n = 3). B, immunofluorescence of XBP1 protein expression. These experiments were performed to confirm that functional sXBP1 is translated following transfection with the sXBP1 construct. In control cells, XBP1 expression cannot be seen (top left panel, nuclei are stained with DAPI). Induction of ER stress (TM) results in XBP1 mRNA splicing, sXBP1 translation, and nuclear targeting (top right panel, DAPI and anti-XBP1 antibody staining). Transfected cells (EV, sXBP1, or uXBP1) were stained with ER-Tracker (red), and XBP1 protein was detected with an anti-XBP1 antibody that recognizes both the sXBP1 and the uXBP1 proteins. Both sXBP1 and uXBP1 proteins enter the nucleus (green). In cells expressing sXBP1, the ER is expanded (red, middle panel) as expected. C, measurement of miR-346 levels. Using the same samples as in A, miR-346 levels were also determined. Results are plotted as -fold increase over EV-transfected, control samples (n = 4). A significant increase in miR-346 levels was observed in cells transfected with sXBP1.

FIGURE 3.

miR-346 expression is XBP1-dependent. Wild type (Xbp1^+/+) and Xbp1-deficient (Xbp1^−/−) mouse embryonic fibroblasts (MEF) cells were treated with TM (5 μg/ml) for the time periods specified to activate the UPR. A, relative miR-346 levels. miR-346 levels were measured by qRT-PCR in Xbp1^+/+ and Xbp1^−/− MEFs to assess the XBP1 dependence of the miR-346 induction. The largest increase in miR-346 levels occurred 8 h after UPR induction in XBP1^+/+ MEF cells. ER stress did not increase miR-346 levels in XBP1^−/− MEFs. B, Dnajb9 induction is XBP1-dependent. Relative Dnajb9 mRNA levels were also determined from MEF cells. As demonstrated previously, the increased levels of Dnajb9 mRNA were XBP1-dependent. C, CHOP/GADD153 induction is XBP1-independent. Relative CHOP/GADD153 mRNA levels were determined in control and ER-stressed MEFs. In agreement with previous results, the increased levels of CHOP/GADD153 mRNA were independent of XBP1 (n = 4).

FIGURE 4.

ER stress-associated miR-346 induction. Step 1, induction of miR-346 transcription. ER stress activates IRE1α, which splices the XBP1 mRNA, resulting in the translation of sXBP1 protein. sXBP1 enters the nucleus and activates pri-miR-346 transcription. Step 2, target mRNA degradation. Pri-miR-346 enters the miRNA maturation pathway, and in the cytosol, miR-346 is generated. Mir-346 binds to the 3′-UTR of target transcripts and recruits the RNA-induced silencing complex, leading to mRNA decay.

FIGURE 5.

Confirmation of predicted miR-346 targets by qRT-PCR. A–D, mRNA levels of HLA-B (A), mRNA levels of HLA-C (B), mRNA levels of HLA-F (C), and mRNA levels of IFI6 (D). Relative mRNA levels of four selected mRNAs from supplemental Fig. 3 were tested by qRT-PCR to confirm the effects of miR-346 on the post-transcriptional regulation of these genes. Three different conditions were applied. 1) Cells were transfected with a control miRNA (C67); 2) ER stress was induced with TM (C67 TM); 3) cells were transfected with miR-346 mimic (mim 346). As suggested by the mRNA profiling following miR-346 mimic expression, both ER stress and miR-346 expression caused a significant decrease in all four mRNAs. Results are plotted as relative mRNA levels as compared with mRNA levels in cells transfected with the control miRNA, C67 (n = 4).

FIGURE 6.

The human TAP1 mRNA is a direct target of miR-346. A, the predicted seeding of miR-346 to the 3′-UTR of the human TAP1 mRNA. The human TAP1 mRNA 3′-UTR and the predicted binding site of miR-346 are shown with the 6-mer canonical seeding site highlighted in gray. AU elements in the vicinity of the predicted miR-346 binding are indicated with a rectangle. B, ER stress reduces TAP1 mRNA levels in Calu-3 and HeLa cells. Cells were treated with different ER stressors, TM, proteasome inhibition (PI), thapsigargin (TG), and brefeldin A (BFA), for 12 h, and relative TAP1 mRNA levels were measured. Results are plotted relative to untreated control TAP1 mRNA level (n = 4). C, ER stress reduces TAP1 protein levels. The effects of ER stress (TM) were tested on human TAP1 protein levels in HeLa cells. Cells were treated with TM (5 μg/ml) for the time periods specified, and 30 μg of total protein lysate was analyzed by SDS-PAGE and Western blot analysis for TAP1 expression. Actin was detected as a loading control, and densitometry was performed using ImageJ (National Institutes of Health). The percentage values represent the percentage of untreated control (100%). D, ER stress has no effect on mouse TAP1 mRNA levels. Mouse embryonic fibroblasts (Xbp1^+/+ and Xbp1^−/−) were treated with TM (5 μg/ml) for 18 h to induce ER stress. The relative mouse TAP1 mRNA levels were determined by qRT-PCR using mouse TAP1-specific primer sets. Results are plotted relative to untreated control (n = 3).

FIGURE 7.

The miR-346 antagomir protects TAP1 from ER stress or miR-346 expression. A, ER stress and miR-346 mimic reduce human TAP1 mRNA levels. A control miRNA (C67), miR-346 mimic (mim), and miR-346 antagomir (inh) were expressed in control untreated and TM-treated (5 μg/ml for 20 h) HeLa cells. The relative human TAP1 mRNA levels were measured by qRT-PCR. Results are plotted as -fold change relative to untreated, C67-transfected TAP1 mRNA levels (n = 4). B, miR-346 mimic expression reduces human TAP1 protein levels. Western blots were performed following transfection of HeLa cells with C67 (C), Mir-346 mimic (mim), or antagomir (inh). Actin levels were measured as a loading control. miR-346 mimic expression reduced TAP1 protein levels to 27% of untreated controls. Densitometry was performed using ImageJ (National Institutes of Health). Three individual experiments were performed that gave similar results. C, expression of miR-346 antagomir protects TAP1 from ER stress-associated reduction. Cells were untreated (control) or treated with TM, and then miR-346 mimic (TM mim) or antagomir (TM inh) was expressed in TM-treated cells to study whether the miR-346 antagomir can reduce the effect of ER stress on TAP1 protein levels. TAP1 protein levels were assessed by densitometry (ImageJ) following Western blotting (control represents 100%). β-Actin was used as a loading control. D, TAP1 protein levels decrease following miR-346 mimic expression without significant changes in ER structure. ER structure was visualized by anti-KDEL polyclonal (rabbit) antibody (red). TAP1 was detected with anti-TAP1 monoclonal (mouse) antibody (green), and nuclei were visualized by staining with DAPI (blue). A reduction in TAP1 (green) is apparent following miR-346 mimic expression. Merged images represent co-localization of TAP1 and KDEL sequences (yellow). Bar: 5 μm.

FIGURE 8.

The biological effects of miR-346 during ER stress. The ER stress-induced sXBP1 transcription factor activates miR-346 expression. TAP1 is a direct target of miR-346. Reduced TAP1 mRNA and protein levels lead to inhibition of peptide influx into the ER. Reduced ER peptide influx contributes to decreased MHC class I-associated antigen presentation.