Sterol‐induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double‐bond position in the sterol tetracyclic ring structure

Abstract

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7-dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 mm cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 . Enrichment of fibroblasts with 7-dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane-bound CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme, was increased by 47^4% in cholesterol-enriched cells whereas its activity was unchanged in lathosterol-enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down-regulated acetate-incorporation into cholesterol, and upregulated sterol esterification in the sterol-enriched fibroblasts. Using 31 P-NMR to measure the lamellar-to-hexagonal (L a ±H II ) phase transition in multilamellar lipid dispersions, lathosterolcontaining membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and either cholesterol or lathosterol (70 : 30 mol/mol), differential scanning calorimetry also revealed that the L a ±H II -transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the L a ±H II -transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.Keywords: sterol analogues; sterol enrichment; regulation; phosphatidylcholine; synthesis.Cholesterol and phospholipids are essential structural and regulatory components of cellular membranes. While phospholipids build up the membrane bilayer, cholesterol is needed to modulate the physical properties of these phospholipids. The amount of free cholesterol in the cell is strictly regulated, because excess free cholesterol is harmful for the proper function of membranes. Cholesterol synthesis mainly occurs in the endoplasmic reticulum (ER), with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase being the ratelimiting enzyme [1,2]. Cell cholesterol homeostasis is in part regulated by a family of membrane-bound transcription factors called sterol regulatory element binding proteins (SREBPs) which regulate multiple genes involved in cholesterol biosynthesis and uptake (reviewed in [3]). In cells depleted of sterols, a two-step proteolytic process releases the N-terminal segment of the SREBP. This polypeptide then enters the nucleus and activates transcription of genes encoding the LDL receptor and several e...

OSBP-related protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids

“…To be able to study the interaction of ORP2 and cholesterol in solution, cholesterol was complexed with methyl-b-cyclodextrin (mbCD; Sigma-Aldrich) according to (30). Briefly, 1 mg of mbCD was dissolved in 1 ml of binding buffer (10 mM HEPES, pH 7.4, 50 mM KCl, and 0.02% NP-40) and added on the top of the film of cholesterol and [ 3 H]cholesterol (44 Ci/mmol; GE-HealthcareAmersham) in a glass tube.…”

Effects of Enrichment of Fibroblasts with Unesterified Cholesterol on the Efflux of Cellular Lipids to Apolipoprotein A-I

“…Thus, caveolin mRNA levels are upregulated by FC loading of fibroblasts, and the rate of FC efflux is proportional to the caveolin mRNA levels (23). Likewise, FC loading of macrophages and fibroblasts stimulates phosphatidylcholine (PC) biosynthesis and up-regulation of CTP:phosphocholine cytidyltransferase (24,25). Active trafficking of lipid vesicles between the plasma membrane and the cell interior is critical because disruption of Golgi membranes by agents such as brefeldin (26,27) and of calcium-dependent retroendocytosis (28) inhibits apoA-I-mediated FC and PL efflux.…”

“…To better define these contributions, we have examined how FC enrichment of fibroblasts affects FC and PL efflux to apoA-I. Since FC loading of fibroblasts has opposite effects on PC and sphingomyelin (SM) biosynthesis (25,30), the contribution of PL synthesis to ABCA1-mediated release of lipids to apoA-I was investigated. Enrichment of the cells with FC enhances efflux of FC because of increases in both the amount of FC in the plasma membrane and the expression of ABCA1; the latter effect combined with increased PC biosynthesis stimulates release of PC.…”

“…PC biosynthesis in fibroblasts is also up-regulated in response to FC loading (25). To investigate the importance of PL synthesis with regard to the enhanced apoA-I-mediated efflux observed from FC-enriched fibroblasts, we depleted the choline content of the cells and examined the effect on FC efflux.…”

Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent pathway

“…Previous studies of lipid efflux from cells by synthetic peptides were done prior to the discovery of ABCA1 and were done under various cellular conditions in which ABCA1 may or may not have been expressed. Furthermore, inducing endogenous ABCA1 expression in cell lines by various differentiation agents and or cholesterol loading results in numerous changes in gene expression (26), as well as changes in membrane lipid composition (27,28), which could also modulate lipid efflux by the peptides.…”