Gene symbol: Xbp1
Alternate Names: X box binding protein 1, Tax-responsive element binding protein 5 (Tax5)
1. General Information
X-box binding protein 1 (XBP1) is member of the cAMP response element binding/ Activating transcription factor (CREB/ATF) family of basic region leucine zipper (bZIP) transcription factors (4), and it was initially discovered through analysis of proteins that bound to an X-box motif in the major histocompatibility complex (MHC) class II, downregulated in adenoma (DRA) gene in humans and the MHC class II antigen A, alpha (Aa) gene in mice (20). Xbp1 is the mammalian homologue to the yeast gene Hac1, an important mediator of the unfolded protein response (UPR).
XBP1 transcriptional activity is regulated by inositol-requiring enzyme 1 (IRE1), the transducer of one of three signaling cascades activated by the unfolded protein response.
Under non-stressed conditions, IRE1 is associated with glucose responsive protein 78 (GRP78/BiP) in an inactive state. When unfolded proteins accumulate in the ER lumen they can sequester BiP, or bind directly with IRE1 monomers (7) resulting in its oligomerization and trans-autophosphorylation of its kinase domain. Activation of IRE1 results in endoribonuclease (RNase) activity (25, 31) that splices a 26 base pair fragment out of the Xbp1 mRNA from base pairs 531 to 556 (32). While the unspliced Xbp1 (Xbp1u) generates a protein that is 261 amino acids in size (3, 26, 32), the splicing of Xbp1u mRNA causes a frameshift and generates a 376 amino acid XBP1s that juxtaposes the bZIP DNA binding domain with a potent transactivation domain in the shifted and extended C-terminus. This alteration confers stability and transcriptional activation ability to XBP1s (32). Both Xbp1u and Xbp1s are translated, but XBP1u is believed to be highly labile and is rapidly degraded. Interestingly, recent evidence suggests XBP1u can repress the transcriptional activity of the UPR (28, 33, 34).
The transcriptional targets of XBP1 vary based on tissue type (1, 16). Studies in mouse embryonic fibroblasts (MEFs) lacking Xbp1 revealed that two XBP1 targets are UPR regulators, the ER chaperones DnaJ (Hsp40) homolog, subfamily B, member 9 (DnaJB9; ERdj4; MDG1) and protein kinase inhibitor of 58 kDa (p58IPK; DnaJC3) (17). ChIP-on-chip analysis aimed at determining transcriptional networks governed by XBP1 revealed that XBP1 constitutively binds to genes involved in ER homeostasis, including PRKR-like endoplasmic reticulum kinase (PERK; Eif2ak3) and GRP78/BiP (1).
XBP1 is required for plasma cell differentiation and consequent Ig production (23) and is a transcriptional activator of interleukin-6 (Il6) (12), indicating that XBP1 is crucial for adaptive immunity. XBP1 has also been implicated in adipocyte differentiation and hepatic lipid metabolism, since XBP1 regulates expression of the lipogenic genes acetyl Co-A carboxylase (ACC2), stearoyl Co-A reductase (SCD1) and diacylglycerol acetyltransferase 2 (DGAT2) by binding to their promoter regions (18). Recently, XBP1 has been implicated in bone morphogenetic-2-induced osteoblast differentiation through its transcriptional regulation of osterix, a gene required for bone development (29). XBP1 is also required for the proper differentiation of zymogenic chief cells. Zymogenic chief cells are similar to pancreatic acinar cells in that they are highly secretory and require expression of Mist1 for proper differentiation (10).
Complete loss of Xbp1 through targeted ablation in mice (Xbp1-/-) results in embryonic lethality by embryonic day (E) 14.5. Embryonic lethality was attributed to the development of hypoplastic fetal livers resulting in reduced hematopoiesis and anemia (22), and was rescued by maintaining XBP1 expression in the liver (Xbp1-/-; LiverXbp1) (16). Most Xbp1-/-; LiverXbp1 exhibited perinatal lethality, with symptoms of poor nutritional status, growth retardation and hypoglycemia. Interestingly, Xbp1-/-; LiverXbp1mice display a severe exocrine pancreas phenotype (16) that implicates XBP1 in acinar cell development and function.
2. XBP1 and the Exocrine Pancreas
Pancreatic acinar cells are highly secretory in nature and have a high rate of protein production. Since most proteins produced in acinar cells are secreted, there is a high demand for protein folding in the ER when secretion is stimulated, requiring proper management of protein synthesis and folding machinery. This requirement suggests that the pancreatic acinar cell relies on the UPR (and thus the IRE1/XBP1 pathway) for efficient function. Generation of XBP1-reporter transgenic mouse lines has confirmed that XBP1 is constitutively active in the pancreas (13, 27).
Xbp1 expression is first detected in the mouse pancreas at E12.5, coinciding with the second wave of differentiation. Its expression increases to a maximum by E14.5, only to decrease to lower levels by E18.5 (5), suggesting a developmental role for XBP1.
The Glimcher group at Harvard generated knockout mouse lines to examine the role of XBP1 in various tissues (16). A global XBP1 knockout (Xbp1-/-) was embryonic lethal where no viable Xbp1-/- embryos were obtained after E14.5. Embryonic lethality was rescued in the Xbp1-/- mouse line when XBP1 was re-expressed in the liver (Xbp1-/-; LiverXbp1). Gross morphological analysis of Xbp1-/-;LivXbp1 pancreata revealed a 90% decrease in pancreas size when compared to wild type (WT) littermates, where pancreatic tissue consisted of sparsely distributed acini in a loose mesenchymal structure. Transmission electron microscopy (TEM) revealed proper organization of Xbp1-/-;LivXbp1 acinar cells around a lumen, but with significantly fewer and smaller zymogen granules compared to WT litter mates. Additionally, the endoplasmic reticulum in mutant cells was poorly developed with few cisternae. Pancreatic tissue from these mice also showed drastic reductions in amylase and trypsin accumulation and significant acinar cell apoptosis by E18.5 due to dysregulated ER stress. The importance of the IRE1/XBP1 pathway in pancreatic acinar cells is highlighted by the discovery that loss of the PERK pathway in pancreatic cells does not result in ER stress or apoptosis (11). Interestingly, development of the endocrine pancreas was unaffected in Xbp1-/-;LivXbp1 mice (16). Thus, this work established a role for XBP1 specifically in development of the exocrine pancreas.
To more thoroughly determine the role of XBP1 in the pancreatic acinar cell Stephen Konieczny’s group crossed Xbp1flox (18) mice with Mist1-CreER/T2 mice to generate a line that is null for Xbp1 only in acinar cells (Xbp1ΔEx2) (8). Analysis of Xbp1ΔEx2 mice 4 weeks after loss of Xbp1 revealed a 60% decrease in expression of amylase and elastase, and increased activity of the UPR. Structurally, Xbp1ΔEx2 acinar cells exhibited decreased accumulation of zymogen granules and decreased amounts of cytoplasm. Further analysis revealed poorly developed and distended ER with disorganized cisternae, and ribosomes were not longer associated with the ER. Additionally, these cells showed increases in ER stress indicators and eventually succumbed to apoptotic cell death, indicating that XBP1 is essential for maintaining both the UPR and acinar cell homeostasis (8).
Transcriptional targets for XBP1 in the pancreas include Sec61α, ER degradation enhancer, mannosidase alpha-like (EDEM), protein disulfide isomerase (PDI) and the pancreas-specific isoform PDIp. Sec61α is required for translocation of newly synthesized proteins across the ER membrane (21), whereas EDEM plays a role in the degradation of misfolded proteins in the ER (9). As the name implies, the PDIs are required for disulfide bond formation and isomerization of newly synthesized proteins (24) within the ER. PDIp is exclusively expressed in the exocrine pancreas (6) and is able to bind zymogens, suggesting an important role in zymogen folding (30). XBP1 also directly regulates expression of Mist1, an exocrine pancreas developmental gene (1). XBP1 binds to the Mist1 promoter in the C2C12 myoblast cell line, plasma cells and in MIN6 insulinoma cells, and ectopic XBP1s expression induces MIST1 expression (1). While these studies were not carried out in an acinar cell environment, they indicate that XBP1 can regulate expression of Mist1, and that the loss of this interaction in Xbp1-/-;LivXbp1 mice may account for the observed exocrine pancreas phenotype.
Studies have also aimed at determining XBP1’s role during pancreatic injury. Experimental induction of pancreatitis using L-arginine in rats or CCK analogs in isolated rat pancreatic acini induced splicing of Xbp1 (14, 15). Furthermore, studies from our lab and others have shown that prolonged exposure to ethanol initiated splicing of Xbp1 in pancreatic tissue (2, 19). While long-term ethanol feeding in mice does not lead to pancreatic deficiency, long-term ethanol feeding of Xbp1 heterozygote (Xbp1+/-) mice, which exhibit a 30% decrease in the level of pancreatic XBP1, resulted in areas of acinar cell necrosis, stroma deposition and the presence of tubular complexes. There was also a 25% decrease in the number of zymogen granules per cell and a 30% reduction in amylase expression in Xbp1+/- ethanol-fed mice. Other hallmarks of injury in Xbp1+/- mice included increased tissue inflammation, increased vacuolization of acinar cells and increased autophagy (19). To date, transcriptional targets of XBP1 during pancreas injury have not been determined. Elucidation of these targets is essential for the complete understanding of the role of XBP1 and its impact in exocrine pancreas pathology.
3. Reagents available for the study of XBP1
Multiple XBP1 antibodies are commercially produced. Antibodies that are commonly used in published studies of XBP1 include:
- Rabbit anti-XBP1 (M-186) from Santa Cruz (cat. # sc-7160). This antibody has been successfully used for Western blotting, and ChIP in various tissues and for immunofluorescence in cultured cells overexpressing XBP1.
- Rabbit anti-XBP1 from Biolegend (cat. # 619502). This antibody has successfully been used for Western blotting and ChIP in various tissues (18).
b. cDNA clones
Addgene distributes a clone for both unspliced (pFLAG.XBP1u.CMV2) and spliced Xbp1 (pFLAG.XBP1s.CMV2) conjugated with a FLAG tag (3).
c. siRNA, shRNA
Xbp1-specific siRNA is commercially available through Santa Cruz Biotechnology (sc-38628). Santa Cruz Biotechnology also carries an Xbp1 shRNA plasmid (sc-38628-SH) and lentiviral particle (sc-38628-V).
d. Genetically modified mice
Mice generated with global loss of Xbp1 (Xbp1-/-) exhibit embryonic lethality at approximately E12.5 (23). Reintroduction of Xbp1 in the liver (Xbp1-/-;LivXbp1) rescued embryonic lethality, generating a viable mouse line in which to study the role of Xbp1 (16). A mouse line harbouring loxP sites in the first and second intron of the Xbp1 gene (Xbp1flox) have also been generated (18), and crossing with Mist1-CreER/T mice produced an acinar cell specific Xbp1 null mouse line (Xbp1ΔEx2) (8).
- Acosta-Alvear D, Zhou Y, Blais A, Tsikitis M, Lents NH, Arias C, Lennon CJ, Kluger Y and Dynlacht BD. XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks. Mol Cell 27: 53-66, 2007. PMID: 17612490
- Alahari S, Mehmood R, Johnson CL and Pin CL. The Absence of MIST1 Leads to Increased Ethanol Sensitivity and Decreased Activity of the Unfolded Protein Response in Mouse Pancreatic Acinar Cells. PLoS One 6: e28863, 2011. PMID: 22216129
- Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG and Ron D. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415: 92-96, 2002. PMID: 11780124
- Clauss IM, Chu M, Zhao JL, and Glimcher LH. The basic domain/leucine zipper protein hXBP-1 preferentially binds to and transactivates CRE-like sequences containing an ACGT core. Nucleic Acids Res 24: 1855-1864, 1996. PMID: 8657566
- Clauss IM, Gravallese EM, Darling JM, Shapiro F, Glimcher MJ and Glimcher LH. In situ hybridization studies suggest a role for the basic region-leucine zipper protein hXBP-1 in exocrine gland and skeletal development during mouse embryogenesis. Dev Dyn 197: 146-156, 1993. PMID: 7693055
- Desilva MG, Lu J, Donadel G, Modi WS, Xie H, Notkins AL and Lan MS. Characterization and chromosomal localization of a new protein disulfide isomerase, PDIp, highly expressed in human pancreas. DNA Cell Biol 15: 9-16, 1996. PMID: 8561901
- Gardner BM and Walter P. Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response. Science 333: 1891-1894, 2011. PMID: 21852455
- Hess DA, Humphrey SE, Ishibashi J, Damsz B, Lee AH, Glimcher LH and Konieczny SF. Extensive pancreas regeneration following acinar-specific disruption of Xbp1 in mice. Gastroenterology 141: 1463-1472, 2011. PMID: 21704586
- Hosokawa N, Wada I, Hasegawa K, Yorihuzi T, Tremblay LO, Herscovics A and Nagata K. A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation. EMBO Rep 2: 415-422, 2001. PMID: 11375934
- Huh WJ, Esen E, Geahlen JH, Bredemeyer AJ, Lee AH, Shi G, Konieczny SF, Glimcher LH, and Mills JC. XBP1 controls maturation of gastric zymogenic cells by induction of MIST1 and expansion of the rough endoplasmic reticulum. Gastroenterology 139: 2038-2049, 2010. PMID: 20816838
- Iida K, Li Y, McGrath BC, Frank A, and Cavener DR. PERK eIF2 alpha kinase is required to regulate the viability of the exocrine pancreas in mice. BMC Cell Biol 8: 38, 2007. PMID: 17727724
- Iwakoshi NN, Lee AH, Vallabhajosyula P, Otipoby KL, Rajewsky K and Glimcher LH. Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1. Nat Immunol 4: 321-329, 2003. PMID: 12612580
- Iwawaki T, Akai R, Kohno K and Miura M. A transgenic mouse model for monitoring endoplasmic reticulum stress. Nat Med 10: 98-102, 2004. PMID: 14702639
- Kubisch CH and Logsdon CD. Secretagogues differentially activate endoplasmic reticulum stress responses in pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol 292: G1804-1812, 2007. PMID: 17431218
- Kubisch CH, Sans MD, Arumugam T, Ernst SA, Williams JA and Logsdon CD. Early activation of endoplasmic reticulum stress is associated with arginine-induced acute pancreatitis. Am J Physiol Gastrointest Liver Physiol 291: G238-245, 2006. PMID: 16574987
- Lee AH, Chu GC, Iwakoshi NN and Glimcher LH. XBP-1 is required for biogenesis of cellular secretory machinery of exocrine glands. EMBO J 24: 4368-4380, 2005. PMID: 16362047
- Lee AH, Iwakoshi NN and Glimcher LH. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 23: 7448-7459, 2003. PMID: 14559994
- Lee AH, Scapa EF, Cohen DE and Glimcher LH. Regulation of hepatic lipogenesis by the transcription factor XBP1. Science 320: 1492-1496, 2008. PMID: 18556558
- Lugea A, Tischler D, Nguyen J, Gong J, Gukovsky I, French SW, Gorelick FS and Pandol SJ. Adaptive unfolded protein response attenuates alcohol-induced pancreatic damage. Gastroenterology 140: 987-997, 2011. PMID: 21111739
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- Reimold AM, Iwakoshi NN, Manis J, Vallabhajosyula P, Szomolanyi-Tsuda E, Gravallese EM, Friend D, Grusby MJ, Alt F and Glimcher LH. Plasma cell differentiation requires the transcription factor XBP-1. Nature 412: 300-307, 2001. PMID: 11460154
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