Genomic targets of the IRE1-XBP1s pathway in mediating metabolic adaptation in epithelial plasticity.

Epithelial mesenchymal plasticity (EMP) is a complex cellular reprogramming event that plays a major role in tissue homeostasis. Recently we observed the unfolded protein response (UPR) triggers EMP through the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 spliced (XBP1s) axis, enhancing glucose shunting to protein N glycosylation. To better understand the genomic targets of XBP1s, we identified its genomic targets using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of a FLAG-epitope tagged XBP1s in RSV infection. CUT&RUN identified 7086 binding sites in chromatin that were enriched in AP-1 motifs and GC-sequences. Of these binding sites, XBP1s peaks mapped to 4827 genes controlling Rho-GTPase signaling, N-linked glycosylation and ER-Golgi transport. Strikingly, XBP1s peaks were within 1 kb of transcription start sites of 2119 promoters. In addition to binding core mesenchymal transcription factors SNAI1 and ZEB1, we observed that hexosamine biosynthetic pathway (HBP) enzymes were induced and contained proximal XBP1s peaks. We demonstrate that IRE1α -XBP1s signaling is necessary and sufficient to activate core enzymes by recruiting elongation-competent phospho-Ser2 CTD modified RNA Pol II. We conclude that the IRE1α-XBP1s pathway coordinately regulates mesenchymal transcription factors and hexosamine biosynthesis in EMP by a mechanism involving recruitment of activated pSer2-Pol II to GC-rich promoters.

The IRE1α-XBP1s Arm of the Unfolded Protein Response Activates N-Glycosylation to Remodel the Subepithelial Basement Membrane in Paramyxovirus Infection.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections (LRTI) associated with decreased pulmonary function, asthma, and allergy. Recently, we demonstrated that RSV induces the hexosamine biosynthetic pathway via the unfolded protein response (UPR), which is a pathway controlling protein glycosylation and secretion of the extracellular matrix (ECM). Because the presence of matrix metalloproteinases and matricellular growth factors (TGF) is associated with severe LRTI, we studied the effect of RSV on ECM remodeling and found that RSV enhances the deposition of fibronectin-rich ECM by small airway epithelial cells in a manner highly dependent on the inositol requiring kinase (IRE1α)-XBP1 arm of the UPR. To understand this effect comprehensively, we applied pharmacoproteomics to understand the effect of the UPR on N-glycosylation and ECM secretion in RSV infection. We observe that RSV induces N-glycosylation and the secretion of proteins related to ECM organization, secretion, or proteins integral to plasma membranes, such as integrins, laminins, collagens, and ECM-modifying enzymes, in an IRE1α-XBP1 dependent manner. Using a murine paramyxovirus model that activates the UPR in vivo, we validate the IRE1α-XBP1-dependent secretion of ECM to alveolar space. This study extends understanding of the IRE1α-XBP1 pathway in regulating N-glycosylation coupled to structural remodeling of the epithelial basement membrane in RSV infection.

Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.

Cooperative interaction of interferon regulatory factor -1 and bromodomain-containing protein 4 on RNA polymerase activation for intrinsic innate immunity.

Introduction: The human orthopneumovirus, Respiratory Syncytial Virus (RSV), is the causative agent of severe lower respiratory tract infections (LRTI) and exacerbations of chronic lung diseases. In immune competent hosts, RSV productively infects highly differentiated epithelial cells, where it elicits robust anti-viral, cytokine and remodeling programs. By contrast, basal cells are relatively resistant to RSV infection, in part, because of constitutive expression of an intrinsic innate immune response (IIR) consisting of a subgroup of interferon (IFN) responsive genes. The mechanisms controlling the intrinsic IIR are not known. Methods: Here, we use human small airway epithelial cell hSAECs as a multipotent airway stem cell model to examine regulatory control of an intrinsic IIR pathway. Results: We find hSAECs express patterns of intrinsic IIRs, highly conserved with pluri- and multi-potent stem cells. We demonstrate a core intrinsic IIR network consisting of Bone Marrow Stromal Cell Antigen 2 (Bst2), Interferon Induced Transmembrane Protein 1 (IFITM1) and Toll-like receptor (TLR3) expression are directly under IRF1 control. Moreover, expression of this intrinsic core is rate-limited by ambient IRF1• phospho-Ser 2 CTD RNA Polymerase II (pSer2 Pol II) complexes binding to their proximal promoters. In response to RSV infection, the abundance of IRF1 and pSer2 Pol II binding is dramatically increased, with IRF1 complexing to the BRD4 chromatin remodeling complex (CRC). Using chromatin immunoprecipitation in IRF1 KD cells, we find that the binding of BRD4 is IRF1 independent. Using a small molecule inhibitor of the BRD4 acetyl lysine binding bromodomain (BRD4i), we further find that BRD4 bromodomain interactions are required for stable BRD4 promoter binding to the intrinsic IIR core promoters, as well as for RSV-inducible pSer2 Pol II recruitment. Surprisingly, BRD4i does not disrupt IRF1-BRD4 interactions, but disrupts both RSV-induced BRD4 and IRF1 interactions with pSer2 Pol II. Conclusions: We conclude that the IRF1 functions in two modes- in absence of infection, ambient IRF1 mediates constitutive expression of the intrinsic IIR, whereas in response to RSV infection, the BRD4 CRC independently activates pSer2 Pol II to mediates robust expression of the intrinsic IIR. These data provide insight into molecular control of anti-viral defenses of airway basal cells.

Glypican-1 stimulates Skp2 autoinduction loop and G1/S transition in endothelial cells.

The heparan sulfate proteoglycan glypican-1 (GPC1) is involved in tumorigenesis and angiogenesis and is overexpressed frequently in tumor and endothelial cells (ECs) in human gliomas. We demonstrated previously that in brain EC, GPC1 regulates mitotic cyclins and securin as well as mitosis and that GPC1 is required for progression through the cell cycle. To characterize the molecular mechanism underlying cell cycle regulation by GPC1, we systematically investigated its effects on key G(1)/S checkpoint regulators and on major signaling pathways reportedly activated by Dally (Division abnormally delayed) the Drosophila GPC1 homologue. We found that elevated GPC1 affected a wide range of G(1)/S checkpoint regulators, leading to inactivation of the G(1)/S checkpoint and increased S phase entry, apparently by activating the mitogen-independent Skp2 autoinduction loop. Specifically, GPC1 suppressed CDK inhibitors (CKIs), including p21, p27, p16, and p19, and the D cyclins, and induced CDK2 and Skp2. GPC1 may trigger the Skp2 autoinduction loop at least partially by suppressing p21 transcription as knockdown of p21 by RNAi can mimic the effect of GPC1 on the cell cycle regulators related to the loop. Moreover, multiple mitogenic signaling pathways, including ERK MAPK, Wnt and BMP signaling, were significantly stimulated by GPC1 as has been reported for Dally in Drosophila. Notably, the c-Myc oncoprotein, which is frequently up-regulated by both ERK and Wnt signaling and functions as a potent transcription repressor for CKIs as well as D cyclins, was also significantly induced by GPC1. These findings provide mechanistic insights into how GPC1 regulates the cell cycle and proliferation.

Angiogenesis induced by signal transducer and activator of transcription 5A (STAT5A) is dependent on autocrine activity of proliferin.

Multiple secreted factors induce the formation of new blood vessels (angiogenesis). The signal transduction events that orchestrate the numerous cellular activities required for angiogenesis remain incompletely understood. We have shown previously that STAT5 plays a pivotal role in angiogenesis induced by FGF2 and FGF8b. To delineate the signaling pathway downstream of STAT5, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in mouse brain endothelial cells (EC). We found that the conditioned medium from CA-STAT5A but not from dominant-negative STAT5A overexpressing EC is sufficient to induce EC invasion and tube formation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. Conversely, CA-STAT5A-induced conditioned medium had no effect on EC proliferation. Using a comparative genome-wide transcription array screen, we identified the prolactin family member proliferin (PLF1 and PLF4) as a candidate autocrine factor. The CA-STAT5A-dependent transcription and secretion of PLF by EC was confirmed by quantitative RT-PCR and Western blotting, respectively. CA-STAT5A binds to the PLF1 promoter region, suggesting a direct transcriptional regulation. Knockdown of PLF expression by shRNA or by blocking of PLF activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. Similarly, the ability of concentrated conditioned medium from CA-STAT5A transfected EC to induce angiogenesis in Matrigel plugs in vivo was abolished when PLF was depleted from the medium. These observations demonstrate a FGF/STAT5/PLF signaling cascade in EC and implicate PLF as autocrine regulator of EC invasion and tube formation.

RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response.

Introduction: The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. Methods: To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor. Results: We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. Discussion: We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.

RELA∙8-Oxoguanine DNA Glycosylase1 Is an Epigenetic Regulatory Complex Coordinating the Hexosamine Biosynthetic Pathway in RSV Infection.

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a negative-sense RNA virus that is the causative agent of severe lower respiratory tract infections in children and is associated with exacerbations of adult lung disease. The mechanisms how severe and/or repetitive virus infections cause declines in pulmonary capacity are not fully understood. We have recently discovered that viral replication triggers epithelial plasticity and metabolic reprogramming involving the hexosamine biosynthetic pathway (HBP). In this study, we examine the relationship between viral induced innate inflammation and the activation of hexosamine biosynthesis in small airway epithelial cells. We observe that RSV induces ~2-fold accumulation of intracellular UDP-GlcNAc, the end-product of the HBP and the obligate substrate of N glycosylation. Using two different silencing approaches, we observe that RSV replication activates the HBP pathway in a manner dependent on the RELA proto-oncogene (65 kDa subunit). To better understand the effect of RSV on the cellular N glycoproteome, and its RELA dependence, we conduct affinity enriched LC-MS profiling in wild-type and RELA-silenced cells. We find that RSV induces the accumulation of 171 N glycosylated peptides in a RELA-dependent manner; these proteins are functionally enriched in integrins and basal lamina formation. To elaborate this mechanism of HBP expression, we demonstrate that RSV infection coordinately induces the HBP pathway enzymes in a manner requiring RELA; these genes include Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT)-1/2, Glucosamine-Phosphate N-Acetyltransferase (GNPNAT)-1, phosphoglucomutase (PGM)-3 and UDP-N-Acetylglucosamine Pyrophosphorylase (UAP)-1. Using small-molecule inhibitor(s) of 8-oxoguanine DNA glycosylase1 (OGG1), we observe that OGG1 is also required for the expression of HBP pathway. In proximity ligation assays, RSV induces the formation of a nuclear and mitochondrial RELA∙OGG1 complex. In co-immunoprecipitaton (IP) experiments, we discover that RSV induces Ser 536-phosphorylated RELA to complex with OGG1. Chromatin IP experiments demonstrate a major role of OGG1 in supporting the recruitment of RELA and phosphorylated RNA Pol II to the HBP pathway genes. We conclude that the RELA∙OGG1 complex is an epigenetic regulator mediating metabolic reprogramming and N glycoprotein modifications of integrins in response to RSV. These findings have implications for viral-induced adaptive epithelial responses.

The Hexosamine Biosynthetic Pathway Links Innate Inflammation With Epithelial-Mesenchymal Plasticity in Airway Remodeling.

Disruption of the lower airway epithelial barrier plays a major role in the initiation and progression of chronic lung disease. Here, repetitive environmental insults produced by viral and allergens triggers metabolic adaptations, epithelial-mesenchymal plasticity (EMP) and airway remodeling. Epithelial plasticity disrupts epithelial barrier function, stimulates release of fibroblastic growth factors, and remodels the extracellular matrix (ECM). This review will focus on recent work demonstrating how the hexosamine biosynthetic pathway (HBP) links innate inflammation to airway remodeling. The HBP is a core metabolic pathway of the unfolded protein response (UPR) responsible for protein N-glycosylation, relief of proteotoxic stress and secretion of ECM modifiers. We will overview findings that the IκB kinase (IKK)-NFκB pathway directly activates expression of the SNAI-ZEB1 mesenchymal transcription factor module through regulation of the Bromodomain Containing Protein 4 (BRD4) chromatin modifier. BRD4 mediates transcriptional elongation of SNAI1-ZEB as well as enhancing chromatin accessibility and transcription of fibroblast growth factors, ECM and matrix metalloproteinases (MMPs). In addition, recent exciting findings that IKK cross-talks with the UPR by controlling phosphorylation and nuclear translocation of the autoregulatory XBP1s transcription factor are presented. HBP is required for N glycosylation and secretion of ECM components that play an important signaling role in airway remodeling. This interplay between innate inflammation, metabolic reprogramming and lower airway plasticity expands a population of subepithelial myofibroblasts by secreting fibroblastic growth factors, producing changes in ECM tensile strength, and fibroblast stimulation by MMP binding. Through these actions on myofibroblasts, EMP in lower airway cells produces expansion of the lamina reticularis and promotes airway remodeling. In this manner, metabolic reprogramming by the HBP mediates environmental insult-induced inflammation with remodeling in chronic airway diseases.