Abstract
Development of the expansive endoplasmic reticulum (ER) present in specialized secretory cell types requires X-box-binding protein-1 (Xbp-1). Enforced expression of XBP-1(S), a transcriptional activator generated by unfolded protein response-mediated splicing of Xbp-1 mRNA, is sufficient to induce proliferation of rough ER. We previously showed that XBP-1(S)-induced ER biogenesis in fibroblasts correlates with increased production of phosphatidylcholine (PtdCho), the primary phospholipid of the ER membrane, and enhanced activities of the choline cytidylyltransferase (CCT) and cholinephosphotransferase enzymes in the cytidine diphosphocholine (CDP-choline) pathway of PtdCho biosynthesis. Here, we report that the level and synthesis of CCT, the rate-limiting enzyme in the CDP-choline pathway, is elevated in fibroblasts overexpressing XBP-1(S). Furthermore, overexpression experiments demonstrated that raising the activity of CCT, but not cholinephosphotransferase, is sufficient to augment PtdCho biosynthesis in fibroblasts, indicating that XBP-1(S) increases the output of the CDP-choline pathway primarily via its effects on CCT. Finally, fibroblasts overexpressing CCT up-regulated PtdCho synthesis to a level similar to that in XBP-1(S)-transduced cells but exhibited only a small increase in rough ER and no induction of secretory pathway genes. The more robust XBP-1(S)-induced ER expansion was accompanied by induction of a wide array of genes encoding proteins that function either in the ER or at other steps in the secretory pathway. We propose that XBP-1(S) regulates ER abundance by coordinately increasing the supply of membrane phospholipids and ER proteins, the key ingredients for ER biogenesis.
Highlights
A key regulator of endoplasmic reticulum (ER) homeostasis is the unfolded protein response (UPR) pathway, a complex signaling system emanating from the ER membrane (8)
We previously showed that fibroblasts overexpressing X-box-binding protein-1 (XBP-1)(S) exhibit enhanced activities of choline cytidylyltransferase (CCT) and CPT1/CEPT1 (10), but the relative role of these alterations in XBP-1(S)-induced PtdCho biosynthesis and ER expansion has not been clarified
Phospholipid Biosynthesis in XBP-1(S)-transduced Fibroblasts—We previously demonstrated that enforced expression of the transcription factor XBP-1(S) is sufficient to up-regulate PtdCho biosynthesis in NIH-3T3 fibroblasts (10)
Summary
PBMNhCPT1-I-GFP encodes full-length human CPT1 (nucleotides 1–1579; GenBank accession #BC050429) and was generated by subcloning a NotI-StuI insert from pCMV-Sport6-hCPT1 (American Type Culture Collection #9271995) into pBMN-IGFP. To assess PtdEtn synthesis, cells were cultured for 2 h at 37 °C in Dulbecco’s modified Eagle’s medium containing 2 Ci/ml [1-3H]ethanolamine hydrochloride (60 Ci/mmol; American Radiolabeled Chemical). CPT activity was assessed as described previously (23) using microsomes prepared from pelleted frozen cells (2 ϫ 107 cells). Two independent stereological sets containing electron micrographs of empty vector-, XBP-1(S)-, and CCT␣-transduced cells at magnifications of 8,000 and 20,000 were analyzed for ER surface area and ER volume as described previously (10). Microarray analysis was performed for each sample using the Affymetrix GeneChip mouse genome arrays MOE430A or MOE430V2 at the Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital. Peaks were identified by comigration with authentic standards, and the amount of each lipid species was calculated using standard curves for each
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