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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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Expression of enzymes that function in the CDP-choline pathway of PtdCho synthesis. Total RNA was prepared from NIH-3T3 fibroblasts harvested at 24 and 48 h after transduction with the indicated retroviral vectors. (A) The relative levels of expression of the CCTα, β2, and β3 isoform cDNAs at the 48-h interval were measured by quantitative real-time PCR. The amount of target RNA was normalized to the endogenous GAPDH reference and related to the amount of target CCTα in NIH-3T3, which was set as the calibrator at 1.0 (superscript a). The mean ± SD (superscript b) of triplicate determinations is shown. (B) Northern blot analysis using 32P-labeled cDNA probes specific for CEPT1 and CPT1, the two genes encoding enzymes possessing CPT activity; ERdj3, a known XBP1 target as a positive control; and γ-actin as a loading control.
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fig4: Expression of enzymes that function in the CDP-choline pathway of PtdCho synthesis. Total RNA was prepared from NIH-3T3 fibroblasts harvested at 24 and 48 h after transduction with the indicated retroviral vectors. (A) The relative levels of expression of the CCTα, β2, and β3 isoform cDNAs at the 48-h interval were measured by quantitative real-time PCR. The amount of target RNA was normalized to the endogenous GAPDH reference and related to the amount of target CCTα in NIH-3T3, which was set as the calibrator at 1.0 (superscript a). The mean ± SD (superscript b) of triplicate determinations is shown. (B) Northern blot analysis using 32P-labeled cDNA probes specific for CEPT1 and CPT1, the two genes encoding enzymes possessing CPT activity; ERdj3, a known XBP1 target as a positive control; and γ-actin as a loading control.

Mentions: There are three isoforms of CCT: α, β2, and β3. CCTα is ubiquitously expressed, whereas the β isoforms exhibit tissue-specific expression that is ∼10-fold lower compared with CCTα (Jackowski et al., 2004). Quantitative real-time RT-PCR revealed no change in transcript levels for any of the CCT isoforms in XBP1(S)-transduced cells (Fig. 4 A). Likewise, Northern blotting revealed no change in the steady-state levels of CPT1 and CEPT1 transcripts (Fig. 4 B). These data suggest that enforced expression of XBP1(S) leads to post-transcriptional and/or post-translational regulation of the CCTα and CPT1/CEPT1 enzymes, thereby augmenting their activities and increasing synthesis of PtdCho (Fig. 5).


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)

Expression of enzymes that function in the CDP-choline pathway of PtdCho synthesis. Total RNA was prepared from NIH-3T3 fibroblasts harvested at 24 and 48 h after transduction with the indicated retroviral vectors. (A) The relative levels of expression of the CCTα, β2, and β3 isoform cDNAs at the 48-h interval were measured by quantitative real-time PCR. The amount of target RNA was normalized to the endogenous GAPDH reference and related to the amount of target CCTα in NIH-3T3, which was set as the calibrator at 1.0 (superscript a). The mean ± SD (superscript b) of triplicate determinations is shown. (B) Northern blot analysis using 32P-labeled cDNA probes specific for CEPT1 and CPT1, the two genes encoding enzymes possessing CPT activity; ERdj3, a known XBP1 target as a positive control; and γ-actin as a loading control.
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fig4: Expression of enzymes that function in the CDP-choline pathway of PtdCho synthesis. Total RNA was prepared from NIH-3T3 fibroblasts harvested at 24 and 48 h after transduction with the indicated retroviral vectors. (A) The relative levels of expression of the CCTα, β2, and β3 isoform cDNAs at the 48-h interval were measured by quantitative real-time PCR. The amount of target RNA was normalized to the endogenous GAPDH reference and related to the amount of target CCTα in NIH-3T3, which was set as the calibrator at 1.0 (superscript a). The mean ± SD (superscript b) of triplicate determinations is shown. (B) Northern blot analysis using 32P-labeled cDNA probes specific for CEPT1 and CPT1, the two genes encoding enzymes possessing CPT activity; ERdj3, a known XBP1 target as a positive control; and γ-actin as a loading control.
Mentions: There are three isoforms of CCT: α, β2, and β3. CCTα is ubiquitously expressed, whereas the β isoforms exhibit tissue-specific expression that is ∼10-fold lower compared with CCTα (Jackowski et al., 2004). Quantitative real-time RT-PCR revealed no change in transcript levels for any of the CCT isoforms in XBP1(S)-transduced cells (Fig. 4 A). Likewise, Northern blotting revealed no change in the steady-state levels of CPT1 and CEPT1 transcripts (Fig. 4 B). These data suggest that enforced expression of XBP1(S) leads to post-transcriptional and/or post-translational regulation of the CCTα and CPT1/CEPT1 enzymes, thereby augmenting their activities and increasing synthesis of PtdCho (Fig. 5).

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