The unfolded protein response gene Ire1α is required for tissue renewal and normal differentiation in the mouse tongue and esophagus.

The IRE1α-XBP1s signaling branch of the unfolded protein response is a well-characterized survival pathway that allows cells to adapt to and resolve endoplasmic reticulum stress. Recent data has broadened our understanding of IRE1α-XBP1s signaling beyond a stress response and revealed a physiological mechanism required for the differentiation and maturation of a wide variety of cell types. Here we provide evidence that the IRE1α-XBP1s signaling pathway is required for the proliferation and maturation of basal keratinocytes in the mouse tongue and esophageal epithelium. Mice with conditional targeted deletion of either Ire1α or Xbp1 in keratin 14 expressing basal keratinocytes displayed severe thinning of the lingual and esophageal mucosa that rendered them unable to eat. In IRE1α null epithelium harvested at an earlier timepoint, genes regulating cell proliferation, cell-cell adhesion, and keratinization were significantly downregulated; indirect immunofluorescence revealed fewer proliferating basal keratinocytes, downregulation of E-cadherin, and thinning of the loricrin-positive granular and cornified layers. The number of Tp63-positive basal keratinocytes was reduced in the absence of IRE1α, and expression of the Wnt pathway transcription factor LEF1, which is required for the proliferation of lingual transit amplifying cells, was also significantly downregulated at the transcript and protein level. Together these results reveal an essential role for IRE1α-XBP1s in the maintenance of the stratified squamous epithelial tissue of the tongue and esophagus.

ER stress and distinct outputs of the IRE1α RNase control proliferation and senescence in response to oncogenic Ras.

Oncogenic Ras causes proliferation followed by premature senescence in primary cells, an initial barrier to tumor development. The role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in regulating these two cellular outcomes is poorly understood. During ER stress, the inositol requiring enzyme 1α (IRE1α) endoribonuclease (RNase), a key mediator of the UPR, cleaves Xbp1 mRNA to generate a potent transcription factor adaptive toward ER stress. However, IRE1α also promotes cleavage and degradation of ER-localized mRNAs essential for cell death. Here, we show that oncogenic HRas induces ER stress and activation of IRE1α. Reduction of ER stress or Xbp1 splicing using pharmacological, genetic, and RNAi approaches demonstrates that this adaptive response is critical for HRas-induced proliferation. Paradoxically, reduced ER stress or Xbp1 splicing promotes growth arrest and premature senescence through hyperactivation of the IRE1α RNase. Microarray analysis of IRE1α- and XBP1-depleted cells, validation using RNA cleavage assays, and 5' RACE identified the prooncogenic basic helix-loop-helix transcription factor ID1 as an IRE1α RNase target. Further, we demonstrate that Id1 degradation by IRE1α is essential for HRas-induced premature senescence. Together, our studies point to IRE1α as an important node for posttranscriptional regulation of the early Ras phenotype that is dependent on both oncogenic signaling as well as stress signals imparted by the tumor microenvironment and could be an important mechanism driving escape from Ras-induced senescence.

The multiple roles of the unfolded protein response regulator IRE1α in cancer.

Cancer is associated with a number of conditions such as hypoxia, nutrient deprivation, cellular redox, and pH changes that result in accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) and trigger a stress response known as the unfolded protein response (UPR). The UPR is a conserved cellular survival mechanism mediated by the ER transmembrane proteins activating transcription factor 6, protein kinase-like endoplasmic reticulum kinase, and inositol-requiring enzyme 1α (IRE1α) that act to resolve ER stress and promote cell survival. IRE1α is a kinase/endoribonuclease (RNase) with multiple activities including unconventional splicing of the messenger RNA (mRNA) for the transcription factor X-Box Binding Protein 1 (XBP1), degradation of other mRNAs in a process called regulated IRE1α-dependent decay (RIDD) and activation of a pathway leading to c-Jun N-terminal kinase phosphorylation. Each of these outputs plays a role in the adaptive and cell death responses to ER stress. Many studies indicate an important role for XBP1 and RIDD functions in cancer and new studies suggest that these two functions of the IRE1α RNase can have opposing functions in the early and later stages of cancer pathogenesis. Finally, as more is learned about the context-dependent role of IRE1α in cancer development, specific small molecule inhibitors and activators of IRE1α could play an important role in counteracting the protective shield provided by ER stress signaling in cancer cells.

IRE1α regulates ROS and immune responses after UVB irradiation.

Objective: UV irradiation of the skin induces photo damage and generates cytotoxic intracellular reactive oxygen species (ROS), activating the unfolded protein response (UPR) to adapt or reduce these UVB-mediated damages. This study was designed to understand the role of the UPR mediator IRE1α in the antioxidant response following UVB irradiation of mouse skin and keratinocytes. Methods: We used mice with an epidermal deletion of IRE1α and primary mouse keratinocytes to examine effects of UV on different parameters of the antioxidant response in the presence and absence of functional IRE1α. Results: In the absence of IRE1α, PERK activity and protein levels are significantly compromised following UVB irradiation. Additionally, the loss of IRE1α suppressed phosphorylation of the PERK target, nuclear factor erythroid-2-related factor 2 (NRF2), and NRF2-dependent antioxidant gene expression after UVB irradiation. Interestingly, IRE1α-deficient keratinocytes exhibit elevated basal ROS levels, while a robust ROS induction upon UVB exposure is abolished. Because UVB-induced ROS plays an essential role in regulating skin inflammation, we analyzed recruited immune cell populations and the expression of pro-inflammatory cytokines, Il-6 and Tnfα in mice with epidermally-targeted deletion of Ire1α. Following UVB irradiation, there was significantly less recruitment of neutrophils and leukocytes and reduced expression of pro-inflammatory cytokine genes in the skin of mice lacking IRE1α. Furthermore, keratinocyte proliferation was also significantly reduced after chronic UVB exposure in the skin of these mice. Conclusions: Collectively, our findings indicate that IRE1α is essential for basal and UVB-induced oxidative stress response, UV-induced skin immune responses, and keratinocyte proliferation. Significance: These findings shed new light on the protective function of IRE1α in the response to UV. IRE1α plays an important role in the regulation of ROS, PERK stability, and antioxidant gene expression in response to UVB in mouse keratinocytes and epidermis.

The Endoplasmic Reticulum Stress Sensor IRE1α Regulates the UV DNA Repair Response through the Control of Intracellular Calcium Homeostasis.

The unfolded protein response is activated by UVB irradiation, but the role of a key mediator, IRE1α, is not clear. In this study, we show that mice with an epidermal IRE1α deletion are sensitized to UV with increased apoptosis, rapid loss of UV-induced cyclopyrimidine dimer‒positive keratinocytes, and sloughing of the epidermis. In vitro, Ire1α-deficient keratinocytes have increased UVB sensitivity, reduced cyclopyrimidine dimer repair, and reduced accumulation of γH2AX and phosphorylated ATR, suggesting defective activation of nucleotide excision repair. Knockdown of XBP1 or pharmacologic inhibition of the IRE1α ribonuclease did not phenocopy Ire1α deficiency. The altered UV response was linked to elevated intracellular calcium levels and ROS, and this was due to dysregulation of the endoplasmic reticulum calcium channel InsP3R. Pharmacologic, genetic, and biochemical studies linked the regulation of the Ins3PR, intracellular calcium, and normal UV DNA damage response to CIB1 and the IRE1α‒TRAF2‒ASK1 complex. These results suggest a model where IRE1α activation state drives CIB1 binding either to the InsP3R or ASK1 to regulate endoplasmic reticulum calcium efflux, ROS, and DNA repair responses after UV irradiation.

Jmjd6a regulates GSK3ß RNA splicing in Xenopus laevis eye development.

It has been suggested that Jmjd6 plays an important role in gene regulation through its demethylation or hydroxylation activity on histone and transcription factors. In addition, Jmjd6 has been shown to regulate RNA splicing by interaction with splicing factors. In this study, we demonstrated that Jmjd6a is expressed in developing Xenopus laevis eye during optic vesicle formation and retinal layer differentiation stages. Knockdown of Jmjd6a by an antisense morpholino resulted in eye malformation including a deformed retinal layer and no lens formation. We further found down-regulation of gene expression related to eye development such as Rx1, Otx2, and Pax6 in Jmjd6a morpholino injected embryos. Jmjd6 interacts with splicing factor U2AF25 and GSK3ß RNA in the anterior region of Xenopus embryos. Knockdown of Jmjd6a led to deletion of GSK3ß RNA exon 1 and 2, which resulted in generation of N'-terminal truncated GSK3ß protein. This event further caused decreased phosphorylation of ß-catenin and subsequently increased ß-catenin stability. Therefore, our result may suggest that Jmjd6a plays an important role in Xenopus eye development through regulation of GSK3ß RNA splicing and canonical Wnt/ß-catenin signaling.