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.

Targeted deletion of TGFß1 in basal keratinocytes causes profound defects in stratified squamous epithelia and aberrant melanocyte migration.

Transforming Growth Factor Beta 1 (TGFß1) is a multifunctional cytokine that regulates proliferation, apoptosis, and epithelial-mesenchymal transition of epithelial cells. While its role in cancer is well studied, less is known about TGFß1 and regulation of epithelial development. To address this, we deleted TGFß1 in basal keratinocytes of stratified squamous epithelia. Newborn mice with a homozygous TGFß1 deletion had significant defects in proliferation and differentiation of the epidermis and oral mucosa, and died shortly after birth. Hair follicles were sparse in TGFß1 depleted skin and had delayed development. Additionally, the Wnt pathway transcription factor LEF1 was reduced in hair follicle bulbs and nearly absent from the basal epithelial layer. Hemizygous knockout mice survived to adulthood but were runted and had sparse coats. The skin of these mice had irregular hair follicle morphology and aberrant hair cycle progression, as well as abnormally high melanin expression and delayed melanocyte migration. In contrast to newborn TGFß1 null mice, the epidermis was hyperproliferative, acanthotic and inflamed. Expression of p63, a master regulator of stratified epithelial identity, proliferation and differentiation, was reduced in TGFß1 null newborn epidermis but expanded in the postnatal acanthotic epidermis of TGFß1 hemizygous mice. Thus, TGFß1 is both essential and haploinsufficient with context dependent roles in stratified squamous epithelial development and homeostasis.

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.

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.

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response.

Background: The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR).  This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis.  The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK) or activating transcription factor 6 (ATF6).  Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity.  The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1), as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR.  Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1.