Knockdown Of XBP1 Research Articles

497 ER Stress Transcription Factor XBP1 Regulates NfκB Activation in Intestinal Epithelial Cells

Background: The unfolded protein response (UPR) is a mechanism that allows cells to cope with endoplasmic reticulum (ER) stress due to the accumulation of misfolded proteins. Among the three proximal effectors of the UPR, the IRE1/XBP1 pathway represents the most conserved pathway. We have recently reported that conditional deletion of Xbp1 specifically in the intestinal epithelium ofmice results in spontaneous small intestinal enteritis reminiscent of human inflammatory bowel disease (IBD), and that polymorphisms in the XBP1 gene are associated with both, Crohn's disease (CD) and ulcerative colitis (UC). We have shown that Xbp1-/epithelia deplete Paneth cells due to apoptosis, and exhibit a pro-inflammatory phenotype as evidenced by JNK phosphorylation. We hypothesized that XBP1 knock-down might increase NFκB signaling as a consequence of IRE1 overactivation. Methods: XBP1 expression was silenced in the intestinal epithelial cell (IEC) line MODE-K via a retroviral shRNA vector, and cells stimulated with TLR ligands or TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed by phospho-specific antibodies. DNA binding activity of nuclear extracts to the NFκB consensus sequence was assessed, and expression of a classical target gene of the canonical NFκB pathway, IκBα, measured by qPCR. Results: TNFα stimulation resulted in a marked increase in the extent and duration of IKK phosphorylation in XBP1-silenced MODE-Ks. IkBα phosphorylation was increased after TNFα stimulation at early time-points in Xbp1-silenced relative to control cells, as was nuclear NFκB phosphorylation. NFκB protein was retained in nuclei for a prolonged period of time after TNFα stimulation of Xbp1-silenced cells compared to control-silenced MODE-Ks. NFκB p65 DNA binding activity in nuclear extracts of XBP1-silenced MODE-Ks was also increased after TNFα stimulation. Expression of IκBαmRNA, a downstream target of NFκBwas substantially increased in TNFα and TLR3 ligand-stimulated MODE-K cells. Discussion: We show that unresolved ER stress in IECs as modeled by XBP1 knock-down results in marked overactivation of the NFκB pathway. Similar to increased JNK phosphorylation as we have previously reported, this is most likely due to marked overactivation of IRE1α in XBP1-deficient IECs since phosphorylated IRE1α has previously been shown to physically interact with the adapter proteins TRAF2 and IKK2 under conditions of ER stress. These data show that the pro-inflammatory consequences of hypomorphic XBP1 function includes dysregulation of a key pro-inflammatory signal transduction pathway, NFκB. RSB and AK share senior authorship

Preventing oxidative stress: a new role for XBP1

Antioxidant molecules reduce oxidative stress and protect cells from reactive oxygen species (ROS)-mediated cellular damage and probably the development of cancer. We have investigated the contribution of X-box-binding protein (XBP1), a major endoplasmic reticulum stress-linked transcriptional factor, to cellular resistance to oxidative stress. After exposure to hydrogen peroxide (H(2)O(2)) or a strong ROS inducer parthenolide, loss of mitochondrial membrane potential (MMP) and subsequent cell death occurred more extensively in XBP1-deficient cells than wild-type mouse embryonic fibroblast cells, whereas two other anticancer agents induced death similarly in both cells. In XBP1-deficient cells, H(2)O(2) exposure induced more extensive ROS generation and prolonged p38 phosphorylation, and expression of several antioxidant molecules including catalase was lower. Knockdown of XBP1 decreased catalase expression, enhanced ROS generation and MMP loss after H(2)O(2) exposure, but extrinsic catalase supply rescued them. Overexpression of XBP1 recovered catalase expression in XBP1-deficient cells and diminished ROS generation after H(2)O(2) exposure. Mutation analysis of the catalase promoter region suggests a pivotal role of CCAAT boxes, NF-Y-binding sites, for the XBP1-mediated enhancing effect. Taken together, these results indicate a protective role of XBP1 against oxidative stress, and its positive regulation of catalase expression may at least in part account for this function.

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3'-kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3-Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

The Kinase Inhibitor Sorafenib Induces Cell Death through a Process Involving Induction of Endoplasmic Reticulum Stress

Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1alpha markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1alpha or XBP1, disruption of PERK activity, or inhibition of eIF2alpha phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.