The UPR ensures the efficient translocation of newly synthesized peptides across the ER membrane and their subsequent folding, maturation, and transport by activating the expression of chaperone genes (16
). Two of the signaling systems that control the UPR are the IRE1/XBP-1 and ATF6 pathways. The relationship between XBP-1 and ATF6, two members of the basic region/leucine zipper class of transcription factors, has been unclear. Here we used DNA microarray analysis to search for genes regulated by XBP-1 and by ATF6α/β. Gene expression in MEFs derived from XBP-1-deficient embryos was compared to that in wt MEFs in the presence or absence of Tm, an agent that evokes the UPR. Similar analyses were carried out in ATF6α- and ATF6β-deficient MEFs generated by RNAi and in MEFs deficient for both XBP-1 and ATF6α. Two major conclusions emerged from this analysis. First, the expression of only a subset of UPR target genes depends on XBP-1. Second, we found that, in contrast to previously published work, neither ATF6α nor β were essential for the expression of a majority of UPR target genes, including BiP.
In the present study, we have identified a series of XBP-1-dependent UPR target genes: ERdj4, p58IPK
, EDEM, RAMP-4, PDI-P5, and HEDJ, all of which appear to act in the ER. ERdj4 (46
), and HEDJ (67
) are localized to the ER and display Hsp40-like ATPase augmenting activity for the Hsp70 family chaperone proteins. EDEM was shown to be critically involved in the ERAD pathway by facilitating the degradation of ERAD substrates (13
). RAMP4 is a recently identified protein implicated in glycosylation and stabilization of membrane proteins in response to stress (42
). PDI-P5 has homology to protein disulfide isomerase, which is thought to be involved in disulfide bond formation (18
). Collectively, these results suggest that the IRE1/XBP-1 pathway is required for efficient protein folding, maturation, and degradation in the ER. Thus, it is not necessarily surprising that XBP-1 controls only a subset of UPR target genes.
Analysis of XBP-1−/−
/iATF6α MEF cells revealed uncompromised induction of some UPR target genes upon ER stress. The PERK/ATF4 pathway is clearly responsible for the activation of some of these genes (10
). This third UPR signaling pathway is activated by the PERK protein kinase. PERK phosphorylates eIF2α, which induces a transient suppression of protein translation, accompanied by induction of transcription factor(s) such as ATF4 (8
). eIF2α is also phosphorylated under various cellular stress conditions by specific kinases, double-strand RNA activated protein kinase PKR, the amino acid control kinase GCN2, and the heme-regulated inhibitor HRI (17
). Since genes that are induced by the PERK pathway are also induced by other stress signals, such as amino acid deprivation, it is likely that PERK-dependent UPR target genes carry out common cellular defense mechanisms, such as cellular homeostasis, apoptosis, and cell cycle (10
). Collectively, we propose that ER stress activates IRE/XBP-1 and PERK/eIF2α pathways to ensure proper maturation and degradation of secretory proteins and to effect common cellular defense mechanisms, respectively.
The reliance of p58IPK
gene expression on XBP-1 is exciting since it connects two of the UPR signaling pathways, IRE1/XBP-1 and PERK. P58IPK
was originally identified as a 58-kDa inhibitor of PKR in influenza virus-infected kidney cells (24
) and described to downregulate the activity of PKR by binding to its kinase domain (15
). It also has a J domain in the C terminus that has been shown to participate in interactions with Hsp70 family proteins (29
). Recently, Katze and coworkers demonstrated that p58IPK
(i) interacts with PERK, which is structurally similar to PKR, (ii) inhibits its eIF2α kinase activity, and (iii) is induced during the UPR by virtue of an ER stress response element in its promoter region (60
). Our data suggest that XBP-1 is the transcription factor that controls p58IPK
expression during the UPR. This has functional consequences since upregulation of p58IPK
upon ER stress may relieve eIF2α phosphorylation and the subsequent protein translation induced by PERK in a negative-feedback manner.
Ohtsuka and Hata reported 23 mouse and human Hsp40/DnaJ homologs which were known genes or novel EST clones found in the DDBJ/GenBank/EMBL DNA database (32
). Potential subcellular localization sites of these Hsp40 members were predicted based on their amino acid sequences and indicated that p58IPK
, ERdj4, and Mtj1 likely have transmembrane domains and may be localized preferentially in the ER. Indeed, these proteins were confirmed to be localized to the ER, where they facilitate the ATPase activity of the Hsp70 chaperones (21
). Recently, two additional ER Hsp40 members, hSec63 (49
) and HEDJ (6
), have been identified. XBP-1-dependent induction of p58IPK
, ERdj4, and HEDJ suggests that an important role of XBP-1 in the UPR is to control the expression of some cochaperones that activate ER resident Hsp70 proteins. Mice that lack XBP-1 die in utero from liver hypoplasia (34
), while mice lacking XBP-1 in the lymphoid system fail to generate plasma cells and hence antibodies (35
). The absolute dependence of these genes, i.e., ERdj4 and p58IPK
, EDEM, Ramp4, PDI-P5, HEDJ, and others still unknown, on XBP-1 for expression suggests that they have an important function in the UPR in plasma cells.
XBP-1 and ATF6α have both been implicated in the function of the UPR. It has been shown by others that ATF6 is involved in the induction of a subset of UPR target genes (61
) and that ATF6α(1-373) was sufficient for the induction of several UPR target genes, including BiP and CHOP (33
). Thus, our failure to uncover UPR target genes regulated by ATF6 was unexpected, especially since we found that the activity of the UPRE and ERSE reporters was completely absent or significantly diminished in ATF6α knockdown cells. It was puzzling that the induction of BiP mRNA was only marginally reduced in the absence of either ATF6α, ATF6β, or both, given that BiP induction was completely abolished in S2P-deficient CHO cells that failed to process ATF6α and presumably ATF6β as well (23
). A trivial explanation is the presence of residual ATF6α in the iATF6α MEFs. However, our failure to detect any processed ATF6α protein, coupled with the inhibition of ATF6α-dependent UPRE and ERSE activity, in this cell line makes this somewhat unlikely. This discrepancy may be due to differences in the cell type studied. One explanation is that XBP-1 may compensate for ATF6α, especially since they share similar DNA-binding specificities (unpublished observations). Alternatively, an intriguing possibility is that there is an additional UPR transcription factor that is activated through proteolysis by S2P similar to ATF6α and ATF6β. It is also certainly possible that there are ATF6α-specific UPR target genes that were not identified in our analysis, a scenario that is suggested by our observation that XBP-1 and ATF6α can synergistically activate the UPRE.
It has been proposed that XBP-1 is situated downstream of ATF6α as an explanation for the observation that ATF6α transactivates but in vitro-translated ATF6α fails to bind the UPRE reporter (64
). Although it has been reported that ATF6α transactivates the XBP-1 promoter, we found no evidence for the regulation of endogenous XBP-1 by ATF6α, since XBP-1 mRNA was normally induced in iATF6α cells. On the contrary, our data suggested that ATF6α was situated downstream of XBP-1 since the induction of mouse ATF6α mRNA upon ER stress was partially compromised in the absence of XBP-1. However, given that the induction of ATF6α by XBP-1 is modest and that ATF6α is primarily regulated by posttranslational mechanisms, we suggest that these two factors are situated largely in parallel pathways.
ATF6β is structurally related to ATF6α, with highest similarity in the b-zip domain (11
). Both ATF6α and ATF6β are proteolytically processed upon ER stress to release the N-terminal transactivator fragment. Interestingly, however, we found no evidence for a requirement of ATF6β for UPRE or ERSE reporter activity or for the induction of UPR target genes. It remains to be determined whether ATF6β is completely redundant with XBP-1 and ATF6α or has a specific, yet-to-be-determined role in UPR target gene expression.
The results obtained with reporter assays versus endogenous gene expression of certain UPR target genes such as BiP deserve comment. ERSE and UPRE motifs are extensively characterized DNA sequences that are responsive to ER stress (37
). UPRE was first identified as an artificial consensus DNA sequence that bound recombinant ATF6α protein (57
) and independently was found to be strikingly similar to the optimal XBP-1 binding sequences (4
). In contrast to the UPRE, whose motif has yet to be identified in the authentic promoter of any endogenous genes, ERSE CCAAT(N)9
CCACG sequences (with N being a GC or GA rich region of 9 bp) is frequently found in the promoter region of well-known UPR target genes such as BiP, grp94, and CHOP (37
). It has been shown that the CCAAT motif is occupied by the constitutive transcription factor NF-Y and that the CCACG region is responsible for the inducible expression observed upon ER stress (37
). Both XBP-1 and ATF6 bind to the CCACG motif only in the presence of NF-Y (64
We have shown that both XBP-1 and ATF6α regulate ERSE and UPRE reporters and that both BiP and CHOP promoters also failed to be induced in XBP-1/ATF6α double deficient cells by Tm (unpublished observations). However, neither XBP-1 nor ATF6 is a significant regulator of endogenous BiP expression. We conclude that there is an additional cis
-acting element in the BiP and CHOP promoters that is responsible for their ER stress-induced expression. In contrast, the reporter gene assays, taken together with endogenous gene expression of certain UPR genes such as Armet and Grp94, suggested that XBP-1 and ATF6 might compensate for each other. The promoter region of the mouse Grp94 and Armet genes has three well-conserved ERSE motifs and one ERSE-II motif, respectively (unpublished observations). ERSE-II is similar to the ERSE motif, which consists of CCAAT and CCACG motifs separated by a single nucleotide in opposite directions (19
). Thus, the ERSE and the ERSE-II motifs may be the critical control elements for Grp94 and Armet but not for BiP and CHOP. Rather, PERK-dependent transcription factors (i.e., ATF4) may control the induction of CHOP and a series of UPR target genes implicated in amino acid metabolism (8
). It was proposed that PERK activates unidentified transcription factor(s) in addition to ATF4, since only a subset of PERK-dependent UPR target genes was affected in ATF4-deficient cells (10
). Deletion of XBP-1, ATF6, and PERK in various combinations will be necessary to establish the roles of these UPR signaling pathways.