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XBP1: a link between the unfolded protein response, lipid biosynthesis, and biogenesis of the endoplasmic reticulum.

Sriburi R, Jackowski S, Mori K, Brewer JW - J. Cell Biol. (2004)

Bottom Line: When the protein folding capacity of the endoplasmic reticulum (ER) is challenged, the unfolded protein response (UPR) maintains ER homeostasis by regulating protein synthesis and enhancing expression of resident ER proteins that facilitate protein maturation and degradation.Cells overexpressing XBP1(S) exhibit elevated levels of membrane phospholipids, increased surface area and volume of rough ER, and enhanced activity of the cytidine diphosphocholine pathway of phosphatidylcholine biosynthesis.These data suggest that XBP1(S) links the mammalian UPR to phospholipid biosynthesis and ER biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.

ABSTRACT
When the protein folding capacity of the endoplasmic reticulum (ER) is challenged, the unfolded protein response (UPR) maintains ER homeostasis by regulating protein synthesis and enhancing expression of resident ER proteins that facilitate protein maturation and degradation. Here, we report that enforced expression of XBP1(S), the active form of the XBP1 transcription factor generated by UPR-mediated splicing of XBP1 mRNA, is sufficient to induce synthesis of phosphatidylcholine, the primary phospholipid of the ER membrane. Cells overexpressing XBP1(S) exhibit elevated levels of membrane phospholipids, increased surface area and volume of rough ER, and enhanced activity of the cytidine diphosphocholine pathway of phosphatidylcholine biosynthesis. These data suggest that XBP1(S) links the mammalian UPR to phospholipid biosynthesis and ER biogenesis.

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Phospholipid synthesis in NIH-3T3 fibroblasts retrovirally transduced with UPR transcriptional activators. NIH-3T3 cells were transduced with the indicated pBMN-I-GFP retroviral vectors. (A) At 48 h after transduction, cells were harvested and equivalent amounts of cell lysate protein were resolved by standard SDS-PAGE under reducing conditions. Chemiluminescent immunoblotting was performed. A nonspecific band just below the position of pXBP1(S) was detected by the anti-XBP1 antibody. Calnexin served as a loading control. (B) Cells were metabolically labeled with [3H]choline for 2 h, a labeling interval in which [3H]choline is incorporated exclusively into PtdCho. The amount of radiolabel present in cellular lipid extracts was normalized as cpm/105 cells and was plotted as the mean ± SD (n = 3; asterisk denotes P = 0.002 for comparison of XBP1(S) to empty vector). (C) Cells were harvested and total cellular mass of PtdCho, PtdEtn, and cholesterol was determined by flame ionization. Superscript a: average of triplicate determinations from two independent experiments (n = 6) ± SD. Superscript b: control NIH-3T3 values set at 100% for comparison.
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fig1: Phospholipid synthesis in NIH-3T3 fibroblasts retrovirally transduced with UPR transcriptional activators. NIH-3T3 cells were transduced with the indicated pBMN-I-GFP retroviral vectors. (A) At 48 h after transduction, cells were harvested and equivalent amounts of cell lysate protein were resolved by standard SDS-PAGE under reducing conditions. Chemiluminescent immunoblotting was performed. A nonspecific band just below the position of pXBP1(S) was detected by the anti-XBP1 antibody. Calnexin served as a loading control. (B) Cells were metabolically labeled with [3H]choline for 2 h, a labeling interval in which [3H]choline is incorporated exclusively into PtdCho. The amount of radiolabel present in cellular lipid extracts was normalized as cpm/105 cells and was plotted as the mean ± SD (n = 3; asterisk denotes P = 0.002 for comparison of XBP1(S) to empty vector). (C) Cells were harvested and total cellular mass of PtdCho, PtdEtn, and cholesterol was determined by flame ionization. Superscript a: average of triplicate determinations from two independent experiments (n = 6) ± SD. Superscript b: control NIH-3T3 values set at 100% for comparison.

Mentions: To determine whether mammalian UPR transcriptional activators regulate phospholipid biosynthesis, we transduced NIH-3T3 fibroblasts with retroviral vectors encoding either XBP1(U), XBP1(S), or a constitutively active form of ATF6α (ATF6α(373); Yoshida et al., 2000). Flow cytometric assessment of GFP, expressed via the bicistronic mRNA encoded by the retroviral vectors, revealed ≥95% transduction efficiency for all retroviral constructs tested in NIH-3T3 cells (see online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200406136/DC1). As expected, ATF6α(373) induced expression of immunoglobulin heavy chain binding protein (BiP) and the disulfide-isomerase-like protein ERp72, two ER resident chaperones. In addition, ATF6α(373) up-regulated ERdj3 (also known as HEDJ), an ER chaperone cofactor that has been identified as an XBP1 target (Lee et al., 2003) (Fig. 1 A). The XBP1(U) retroviral vector yielded the ∼30-kD pXBP1(U) and no detectable pXBP1(S), whereas the XBP1(S) virus exclusively yielded the ∼54-kD pXBP1(S). Cells transduced with XBP1(S), but not XBP1(U), exhibited increased levels of ERdj3 and BiP (Fig. 1 A). These data demonstrate that ATF6α(373), XBP1(U), and XBP1(S) all functioned as predicted upon enforced expression in NIH-3T3 cells.


XBP1: a link between the unfolded protein response, lipid biosynthesis, and biogenesis of the endoplasmic reticulum.

Sriburi R, Jackowski S, Mori K, Brewer JW - J. Cell Biol. (2004)

Phospholipid synthesis in NIH-3T3 fibroblasts retrovirally transduced with UPR transcriptional activators. NIH-3T3 cells were transduced with the indicated pBMN-I-GFP retroviral vectors. (A) At 48 h after transduction, cells were harvested and equivalent amounts of cell lysate protein were resolved by standard SDS-PAGE under reducing conditions. Chemiluminescent immunoblotting was performed. A nonspecific band just below the position of pXBP1(S) was detected by the anti-XBP1 antibody. Calnexin served as a loading control. (B) Cells were metabolically labeled with [3H]choline for 2 h, a labeling interval in which [3H]choline is incorporated exclusively into PtdCho. The amount of radiolabel present in cellular lipid extracts was normalized as cpm/105 cells and was plotted as the mean ± SD (n = 3; asterisk denotes P = 0.002 for comparison of XBP1(S) to empty vector). (C) Cells were harvested and total cellular mass of PtdCho, PtdEtn, and cholesterol was determined by flame ionization. Superscript a: average of triplicate determinations from two independent experiments (n = 6) ± SD. Superscript b: control NIH-3T3 values set at 100% for comparison.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172532&req=5

fig1: Phospholipid synthesis in NIH-3T3 fibroblasts retrovirally transduced with UPR transcriptional activators. NIH-3T3 cells were transduced with the indicated pBMN-I-GFP retroviral vectors. (A) At 48 h after transduction, cells were harvested and equivalent amounts of cell lysate protein were resolved by standard SDS-PAGE under reducing conditions. Chemiluminescent immunoblotting was performed. A nonspecific band just below the position of pXBP1(S) was detected by the anti-XBP1 antibody. Calnexin served as a loading control. (B) Cells were metabolically labeled with [3H]choline for 2 h, a labeling interval in which [3H]choline is incorporated exclusively into PtdCho. The amount of radiolabel present in cellular lipid extracts was normalized as cpm/105 cells and was plotted as the mean ± SD (n = 3; asterisk denotes P = 0.002 for comparison of XBP1(S) to empty vector). (C) Cells were harvested and total cellular mass of PtdCho, PtdEtn, and cholesterol was determined by flame ionization. Superscript a: average of triplicate determinations from two independent experiments (n = 6) ± SD. Superscript b: control NIH-3T3 values set at 100% for comparison.
Mentions: To determine whether mammalian UPR transcriptional activators regulate phospholipid biosynthesis, we transduced NIH-3T3 fibroblasts with retroviral vectors encoding either XBP1(U), XBP1(S), or a constitutively active form of ATF6α (ATF6α(373); Yoshida et al., 2000). Flow cytometric assessment of GFP, expressed via the bicistronic mRNA encoded by the retroviral vectors, revealed ≥95% transduction efficiency for all retroviral constructs tested in NIH-3T3 cells (see online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200406136/DC1). As expected, ATF6α(373) induced expression of immunoglobulin heavy chain binding protein (BiP) and the disulfide-isomerase-like protein ERp72, two ER resident chaperones. In addition, ATF6α(373) up-regulated ERdj3 (also known as HEDJ), an ER chaperone cofactor that has been identified as an XBP1 target (Lee et al., 2003) (Fig. 1 A). The XBP1(U) retroviral vector yielded the ∼30-kD pXBP1(U) and no detectable pXBP1(S), whereas the XBP1(S) virus exclusively yielded the ∼54-kD pXBP1(S). Cells transduced with XBP1(S), but not XBP1(U), exhibited increased levels of ERdj3 and BiP (Fig. 1 A). These data demonstrate that ATF6α(373), XBP1(U), and XBP1(S) all functioned as predicted upon enforced expression in NIH-3T3 cells.

Bottom Line: When the protein folding capacity of the endoplasmic reticulum (ER) is challenged, the unfolded protein response (UPR) maintains ER homeostasis by regulating protein synthesis and enhancing expression of resident ER proteins that facilitate protein maturation and degradation.Cells overexpressing XBP1(S) exhibit elevated levels of membrane phospholipids, increased surface area and volume of rough ER, and enhanced activity of the cytidine diphosphocholine pathway of phosphatidylcholine biosynthesis.These data suggest that XBP1(S) links the mammalian UPR to phospholipid biosynthesis and ER biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.

ABSTRACT
When the protein folding capacity of the endoplasmic reticulum (ER) is challenged, the unfolded protein response (UPR) maintains ER homeostasis by regulating protein synthesis and enhancing expression of resident ER proteins that facilitate protein maturation and degradation. Here, we report that enforced expression of XBP1(S), the active form of the XBP1 transcription factor generated by UPR-mediated splicing of XBP1 mRNA, is sufficient to induce synthesis of phosphatidylcholine, the primary phospholipid of the ER membrane. Cells overexpressing XBP1(S) exhibit elevated levels of membrane phospholipids, increased surface area and volume of rough ER, and enhanced activity of the cytidine diphosphocholine pathway of phosphatidylcholine biosynthesis. These data suggest that XBP1(S) links the mammalian UPR to phospholipid biosynthesis and ER biogenesis.

Show MeSH
Related in: MedlinePlus