Maho Niwa

Identification of an Ire1alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma.

Activation of the adaptive Ire1-XBP1 pathway has been identified in many solid tumors and hematologic malignancies, including multiple myeloma (MM). Here, we report the identification of STF-083010, a novel small-molecule inhibitor of Ire1. STF-083010 inhibited Ire1 endonuclease activity, without affecting its kinase activity, after endoplasmic reticulum stress both in vitro and in vivo. Treatment with STF-083010 showed significant antimyeloma activity in model human MM xenografts. Similarly, STF-083010 was preferentially toxic to freshly isolated human CD138(+) MM cells compared with other similarly isolated cell populations. The identification of this novel Ire1 inhibitor supports the hypothesis that the Ire1-XBP1 axis is a promising target for anticancer therapy, especially in the context of MM.

ER stress activates NF-κB by integrating functions of basal IKK activity, IRE1 and PERK.

NF-κB, a transcription factor, becomes activated during the Unfolded Protein Response (UPR), an endoplasmic reticulum (ER) stress response pathway. NF-κB is normally held inactive by its inhibitor, IκBα. Multiple cellular pathways activate IKK (IκBα Kinase) which phosphorylate IκBα leading to its degradation and NF-κB activation. Here, we find that IKK is required for maximum activation of NF-κB in response to ER stress. However, unlike canonical NFκB activation, IKK activity does not increase during ER stress, but rather the level of basal IKK activity is critical for determining the extent of NF-κB activation. Furthermore, a key UPR initiator, IRE1, acts to maintain IKK basal activity through IRE1s kinase, but not RNase, activity. Inputs from IRE1 and IKK, in combination with translation repression by PERK, another UPR initiator, lead to maximal NF-κB activation during the UPR. These interdependencies have a significant impact in cancer cells with elevated IKK/NF-κB activity such as renal cell carcinoma cells (786-0). Inhibition of IKK by an IKK inhibitor, which significantly decreases NF-κB activity, is overridden by UPR induction, arguing for the importance of considering UPR activation in cancer treatment.

ORMDL3 is an inducible lung epithelial gene regulating metalloproteases, chemokines, OAS, and ATF6

Orosomucoid like 3 (ORMDL3) has been strongly linked with asthma in genetic association studies, but its function in asthma is unknown. We demonstrate that in mice ORMDL3 is an allergen and cytokine (IL-4 or IL-13) inducible endoplasmic reticulum (ER) gene expressed predominantly in airway epithelial cells. Allergen challenge induces a 127-fold increase in ORMDL3 mRNA in bronchial epithelium in WT mice, with lesser 15-fold increases in ORMDL-2 and no changes in ORMDL-1. Studies of STAT-6–deficient mice demonstrated that ORMDL3 mRNA induction highly depends on STAT-6. Transfection of ORMDL3 in human bronchial epithelial cells in vitro induced expression of metalloproteases (MMP-9, ADAM-8), CC chemokines (CCL-20), CXC chemokines (IL-8, CXCL-10, CXCL-11), oligoadenylate synthetases (OAS) genes, and selectively activated activating transcription factor 6 (ATF6), an unfolded protein response (UPR) pathway transcription factor. siRNA knockdown of ATF-6α in lung epithelial cells inhibited expression of SERCA2b, which has been implicated in airway remodeling in asthma. In addition, transfection of ORMDL3 in lung epithelial cells activated ATF6α and induced SERCA2b. These studies provide evidence of the inducible nature of ORMDL3 ER expression in particular in bronchial epithelial cells and suggest an ER UPR pathway through which ORMDL3 may be linked to asthma.

Phosphorylation of Eukaryotic Translation Initiation Factor 2α Coordinates rRNA Transcription and Translation Inhibition during Endoplasmic Reticulum Stress

ABSTRACT The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2α plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2α phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting ∼90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2α, suggesting that this pathway may be broadly utilized by stresses that activate eIF2α kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.

Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase

Mutations that impair activity of the ER stress response kinase Ire1 inhibit resolution of the unfolded protein response (see also a related paper by Rubio et al. in this issue).

ORMDL3 transgenic mice have increased airway remodeling and airway responsiveness characteristic of asthma

Orosomucoid-like (ORMDL)3 has been strongly linked with asthma in genetic association studies. Because allergen challenge induces lung ORMDL3 expression in wild-type mice, we have generated human ORMDL3 zona pellucida 3 Cre (hORMDL3zp3-Cre) mice that overexpress human ORMDL3 universally to investigate the role of ORMDL3 in regulating airway inflammation and remodeling. These hORMDL3zp3-Cre mice have significantly increased levels of airway remodeling, including increased airway smooth muscle, subepithelial fibrosis, and mucus. hORMDL3zp3-Cre mice had spontaneously increased airway responsiveness to methacholine compared to wild-type mice. This increased airway remodeling was associated with selective activation of the unfolded protein response pathway transcription factor ATF6 (but not Ire1 or PERK). The ATF6 target gene SERCA2b, implicated in airway remodeling in asthma, was strongly induced in the lungs of hORMDL3zp3-Cre mice. Additionally, increased levels of expression of genes associated with airway remodeling (TGF-β1, ADAM8) were detected in airway epithelium of these mice. Increased levels of airway remodeling preceded increased levels of airway inflammation in hORMDL3zp3-Cre mice. hORMDL3zp3-Cre mice had increased levels of IgE, with no change in levels of IgG, IgM, and IgA. These studies provide evidence that ORMDL3 plays an important role in vivo in airway remodeling potentially through ATF6 target genes such as SERCA2b and/or through ATF6-independent genes (TGF-β1, ADAM8).

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

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.

Ire1 Has Distinct Catalytic Mechanisms for XBP1/HAC1 Splicing and RIDD

SUMMARY An evolutionarily conserved unfolded protein response (UPR) component, IRE1, cleaves XBP1/HAC1 introns in order to generate spliced mRNAs that are translated into potent transcription factors. IRE1 also cleaves endoplasmic-reticulum-associated RNAs leading to their decay, an activity termed regulated IRE1-dependent decay (RIDD); however, the mechanism by which IRE1 differentiates intron cleavage from RIDD is not well understood. Using in vitro experiments, we found that IRE1 has two different modes of action: XBP1/HAC1 is cleaved by IRE1 subunits acting cooperatively within IRE1 oligomers, whereas a single subunit of IRE1 performs RIDD without cooperativity. Furthermore, these distinct activities can be separated by complementation of catalytically inactive IRE1 RNase and mutations at oligomerization interfaces. Using an IRE1 RNase inhibitor, STF-083010, selective inhibition of XBP1 splicing indicates that XBP1 promotes cell survival, whereas RIDD leads to cell death, revealing modulation of IRE1 activities as a drug-development strategy.

A Surveillance Pathway Monitors the Fitness of the Endoplasmic Reticulum to Control Its Inheritance

The endoplasmic reticulum (ER) plays an essential role in the production of lipids and secretory proteins. Because the ER cannot be generated de novo, it must be faithfully transmitted or divided at each cell division. Little is known of how cells monitor the functionality of the ER during the cell cycle or how this regulates inheritance. We report here that ER stress in S. cerevisiae activates the MAP kinase Slt2 in a new ER stress surveillance (ERSU) pathway, independent of the unfolded protein response. Upon ER stress, ERSU alters the septin complex to delay ER inheritance and cytokinesis. In the absence of Slt2 kinase, the stressed ER is transmitted to the daughter cell, causing the death of both mother and daughter cells. Furthermore, Slt2 is activated via the cell surface receptor Wsc1 by a previously undescribed mechanism. We conclude that the ERSU pathway ensures inheritance of a functional ER.

A novel role in cytokinesis reveals a housekeeping function for the unfolded protein response

The unfolded protein response (UPR) pathway helps cells cope with endoplasmic reticulum (ER) stress by activating genes that increase the ERs functional capabilities. We have identified a novel role for the UPR pathway in facilitating budding yeast cytokinesis. Although other cell cycle events are unaffected by conditions that disrupt ER function, cytokinesis is sensitive to these conditions. Moreover, efficient cytokinesis requires the UPR pathway even during unstressed growth conditions. UPR-deficient cells are defective in cytokinesis, and cytokinesis mutants activate the UPR. The UPR likely achieves its role in cytokinesis by sensing small changes in ER load and making according changes in ER capacity. We propose that cytokinesis is one of many cellular events that require a subtle increase in ER function and that the UPR pathway has a previously uncharacterized housekeeping role in maintaining ER plasticity during normal cell growth.