Suzanne Jackowski

Cellular Responses to Excess Phospholipid

Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase D-mediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A2.

CELL CYCLE REGULATION OF MEMBRANE PHOSPHOLIPID METABOLISM

This review focuses on the phospholipid metabolism regulated by the cell cycle. Phospholipids are the major cellular constituents required for the assembly of biological membranes, and cells must double their phospholipid mass to form daughter cells. It seems reasonable that this event should coincide with the synthesis of other cellular components such as DNA, stable RNA, etc.; however, the biochemical mechanisms that coordinate macromolecular and bulk membrane phospholipid production are largely unknown. The importance of these regulatory processes to cell physiology is obvious. Discordant regulation of phospholipid accumulation by only a few percent per cell cycle would rapidly result in cells with either a large excess or deficit of membrane surface leading to abnormalities in cell size and/or intracellular lipid accumulation. Thus, stringent control mechanisms must be in place to keep the phospholipid content in tune with the cell cycle. This discussion will explore the state of our knowledge in cultured mammalian cell systems, although cell cycle-regulated phospholipid accumulation occurs in lower eukaryotes such as Saccharomyces cerevisiae (1) and Caulobacter crescentus (2). This review is limited to a discussion of events that are directly tied to the cell cycle. Phospholipid metabolism in response to mitogenic stimulation will not be addressed as these biochemical events are generally associated with the G0 to G1 transition and are ligand-regulated rather than being orchestrated by the cell cycle.

Coordinate Regulation of Phospholipid Biosynthesis and Secretory Pathway Gene Expression in XBP-1(S)-induced Endoplasmic Reticulum Biogenesis

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.

The unfolded protein response transducer IRE1α prevents ER stress-induced hepatic steatosis

The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol‐requiring enzyme 1 α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1α‐mediated signalling in the liver, we generated hepatocyte‐specific Ire1α‐null mice by utilizing an albumin promoter‐controlled Cre recombinase‐mediated deletion. Deletion of Ire1α caused defective induction of genes encoding functions in ER‐to‐Golgi protein transport, oxidative protein folding, and ER‐associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro‐apoptotic UPR trans‐activators. We show that IRE1α is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte‐specific Ire1α deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1α represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer‐binding protein (C/EBP) β, C/EBPδ, peroxisome proliferator‐activated receptor γ (PPARγ), and enzymes involved in triglyceride biosynthesis. IRE1α was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1α in preventing intracellular lipid accumulation, mice with hepatocyte‐specific deletion of Ire1α developed severe hepatic steatosis after treatment with an ER stress‐inducing anti‐cancer drug Bortezomib, upon expression of a misfolding‐prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1α as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries.

Membrane phospholipid synthesis and endoplasmic reticulum function

This review presents an overview of mammalian phospholipid synthesis and the cellular locations of the biochemical activities that produce membrane lipid molecular species. The generalized endoplasmic reticulum compartment is a central site for membrane lipid biogenesis, and examples of the emerging relationships between alterations in lipid composition, regulation of membrane lipid biogenesis, and cellular secretory function are discussed.

Pantothenate Kinase Regulation of the Intracellular Concentration of Coenzyme A

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway in bacteria and is thought to play a similar role in mammalian cells. We examined this hypothesis by identifying and characterizing two murine cDNAs that encoded PanK. The two cDNAs were predicted to arise from alternate splicing of the same gene to yield different mRNAs that encode two isoforms (mPanK1α and mPanK1β) with distinct amino termini. The predicted protein sequence of mPanK1 was not related to bacterial PanK but exhibited significant similarity to Aspergillus nidulansPanK. mPanK1α was most highly expressed in heart and kidney, whereas mPanK1β mRNA was detected primarily in liver and kidney. Pantothenate was the most abundant pathway component (42.8%) in normal cells providing clear evidence that pantothenate phosphorylation was a rate-controlling step in CoA biosynthesis. Enhanced mPanK1β expression eliminated the intracellular pantothenate pool and triggered a 13-fold increase in intracellular CoA content. mPanK1β activityin vitro was stimulated by CoA and strongly inhibited by acetyl-CoA illustrating that differential modulation of mPanK1β activity by pathway end products also contributed to the management of CoA levels. These data support the concept that the expression and/or activity of PanK is a determining factor in the physiological regulation of the intracellular CoA concentration.

ATF6α induces XBP1-independent expansion of the endoplasmic reticulum

A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6α (ATF6α), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6α drives ER expansion and can do so in the absence of XBP1(S). Overexpression of active ATF6α induces PtdCho biosynthesis and modulates the CDP-choline pathway differently than does enforced expression of XBP1(S). These data indicate that ATF6α and XBP1(S) have the ability to regulate lipid biosynthesis and ER expansion by mechanisms that are at least partially distinct. These studies reveal further complexity in the potential relationships between UPR pathways, lipid production and ER biogenesis.

Distribution of CTP:phosphocholine cytidylyltransferase (CCT) isoforms. Identification of a new CCTbeta splice variant.

CTP:phosphocholine cytidylyltransferase is a major regulator of phosphatidylcholine biosynthesis. A single isoform, CCTα, has been studied extensively and a second isoform, CCTβ, was recently identified. We identify and characterize a third cDNA, CCTβ2, that differs from CCTβ1 at the carboxyl-terminal end and is predicted to arise as a splice variant of the CCTβ gene. Like CCTα, CCTβ2 is heavily phosphorylated in vivo, in contrast to CCTβ1. CCTβ1 and CCTβ2 mRNAs were differentially expressed by the human tissues examined, whereas CCTα was more uniformly represented. Using isoform-specific antibodies, both CCTβ1 and CCTβ2 localized to the endoplasmic reticulum of cells, in contrast to CCTα which resided in the nucleus in addition to associating with the endoplasmic reticulum. CCTβ2 protein has enzymatic activity in vitro and was able to complement the temperature-sensitive cytidylyltransferase defect in CHO58 cells, just as CCTα and CCTβ1 supporting proliferation at the nonpermissive conditions. Overexpression experiments did not reveal discrete physiological functions for the three isoforms that catalyze the same biochemical reaction; however, the differential cellular localization and tissue-specific distribution suggest that CCTβ1 and CCTβ2 may play a role that is distinct from ubiquitously expressed CCTα.

Apoptosis triggered by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine is prevented by increased expression of CTP:phosphocholine cytidylyltransferase.

A HeLa cell line was constructed for the regulation of CTP:phosphocholine cytidylyltransferase (CCT) expression via a tetracycline-responsive promoter to test the role of CCT in apoptosis triggered by exposure of cells to the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). Basal CCT expression in the engineered HeLa cell line was the same as in control HeLa cells lines, and CCT activity and protein were elevated 25-fold following 48 h of induction with doxycycline. Increased CCT expression prevented ET-18-OCH3-induced apoptosis. Acylation of exogenous lysophosphatidylcholine circumvented the requirement for CCT activity by providing an alternate route to phosphatidylcholine, and heightened CCT expression and lysophosphatidylcholine supplementation were equally effective in reversing the cytotoxic effect of ET-18-OCH3. Neither CCT overexpression nor lysophosphatidylcholine supplementation allowed the HeLa cells to proliferate in the presence of ET-18-OCH3, indicating that the cytostatic property of ET-18-OCH3 was independent of its effect on membrane phospholipid synthesis. These data provide compelling genetic evidence to support the conclusion that the interruption of phosphatidylcholine synthesis at the CCT step by ET-18-OCH3 is the primary physiological imbalance that accounts for the cytotoxic action of the drug.

Cloning and characterization of a second human CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CCT) is a key regulator of phosphatidylcholine biosynthesis, and only a single isoform of this enzyme, CCTα, is known. We identified and sequenced a human cDNA that encoded a distinct CCT isoform, called CCTβ, that is derived from a gene different from that encoding CCTα. CCTβ transcripts were detected in human adult and fetal tissues, and very high transcript levels were found in placenta and testis. CCTβ and CCTα proteins share highly related, but not identical, catalytic domains followed by three amphipathic helical repeats. Like CCTα, CCTβ required the presence of lipid regulators for maximum catalytic activity. The amino terminus of CCTβ bears no resemblance to the amino terminus of CCTα, and CCTβ protein was localized to the cytoplasm as detected by indirect immunofluorescent microscopy. Whereas CCTα activity is regulated by reversible phosphorylation, CCTβ lacks most of the corresponding carboxyl-terminal domain and contained only 3 potential phosphorylation sites of the 16 identified in CCTα. Transfection of COS-7 cells with a CCTβ expression construct led to the overexpression of CCT activity, the accumulation of cellular CDP-choline, and enhanced radiolabeling of phosphatidylcholine. CCTβ protein was posttranslationally modified in COS-7 cells, resulting in slower migration during polyacrylamide gel electrophoresis. Expression of CCTβ/CCTα chimeric proteins showed that the amino-terminal portion of CCTβ was required for posttranslational modification. These data demonstrate that a second, distinct CCT enzyme is expressed in human tissues and provides another mechanism by which cells regulate phosphatidylcholine production.