Expression of cholesteryl glucoside by heat shock in human fibroblasts

Abstract

ABSTRACT We investigated the heat-induced alteration of glycolipids in human cultured cells, TIG-3 fibroblasts, to show the expression of steryl glucoside by heat shock. A glycolipid band was detected on a thin-layer chromatography plate in lipid extracts from TIG-3 cells exposed to high temperature (42°C) for 15 and 30 minutes, while it was hardly detectable without heat shock. Both cholesterol and glucose were almost exclusively detected by gas liquid chromatography as degradation products of the lipid. The structure of the lipid molecule was elucidated by electrospray mass spectrometry to be a cholesteryl glucoside. This is the first report to show the occurrence of a steryl glucoside in mammalian cells, and this substance is considered to have a significant role in heat shock responses in mammalian cells.

INTRODUCTION

Many organisms are exposed to various types of stresses, such as heat shock, active oxygen, and heavy metals. In response to these stresses, they produce heat shock proteins (Hsps) to protect themselves. It is well known that the transcriptional induction of heat shock genes is mediated by the heat shock transcription factors (Hsfs) in eukaryotes (Kingston et al 1987; Paker and Topol 1984; Sorger and Pelham 1987; Sorger and Pelham 1988; Sorger et al 1987; Wiederrecht et al 1987; Wu 1985; Wu et al 1987) and that Hsfs are activated by multimerization (Cunniff et al 1991; Rabindran et al 1993; Sorger and Nelson 1989; Clos et al 1990) and phosphorylation (Larson et al 1988). The activated Hsfs bind to the heat shock element (Hse) in promotors of heat shock genes and then stimulate their transcription (Kingston et al 1987; Sorger et al 1987; Zimarino and Wu 1987). But the very early events that cause activation of Hsfs are still unclear.

In a previous report (Murakami-Murofushi et al 1997), we showed that heat stress induces a rapid glycosylation of membrane sterol in the myxoamoebae of a true slime mold, Physarum polycephalum, and an immediate activation of sterol glucosyltransferase was also demonstrated. The purified glycolipid was determined to be a poriferasterol monoglucoside by structural studies (Murakami-Murofushi et al 1997). This was a very rapid reaction after heat shock, and afterward an activation of a Ca²⁺-dependent protein kinase (Maruya et al 1997) and an induction of some stress proteins (Shimada et al 1992) occurred. We suggested, therefore, that steryl glucoside might have an important role in the stress-responsible signal transduction.

In this study, we investigated the change of membrane lipids in human fetal lung fibroblast cells, TIG-3, by heat shock, and we found the heat-induced expression of steryl glycoside. From structural analyses of purified steryl glycoside, it was identified as a cholesteryl glucoside. These findings suggest the involvement of steryl glucoside in heat shock responses in mammalian cells.

RESULTS

Expression of glycolipids in the heat shocked TIG-3 cells

TIG-3 cells were heat treated at 42°C for various periods, and total lipid fractions were extracted to be analyzed using high-performance thin-layer chromatography (HPTLC). A certain glycolipid designated as Hsl-X (heat shock lipid X; Fig 1), of which the Rf value is slightly lower than that of poriferasterol monoglucoside, appeared 15 and 30 minutes after the temperature shift, whereas the lipid band was scarcely detectable at 0, 5, and 60 minutes after the temperature shift. Because Hsl-X was visualized with both orcinol reagent (Fig 1A) and ferric chloride reagent (Fig 1B), Hsl-X was thought to be composed of hexose and sterol.

Fig 1.

 Expression of glycolipids in TIG-3 cells 0 min (lane 2). TIG-3 cells, human fetal lung fibroblasts (Health Science Research Bank, Osaka, Japan), were cultured in Eagle's minimum essential medium (Nissui Pharmaceutical Co, Tokyo, Japan) containing 10% fetal bovine serum (Moregate, Australia) in 5% CO₂/95% air at 37°C. For heat shock, TIG-3 cells growing in 10-cm-diameter culture dishes at 37°C were exposed to 42°C prewarmed Eagle's minimum essential medium containing 10% fetal bovine serum and were incubated for the indicated periods after the temperature shift. After heat treatment, the medium was removed and the cells were harvested in cold methanol. Control cells (lane 2) were not exposed to high temperature and were harvested in the same manner. Cells were sonicated for 10 minutes and centrifuged at 1 400 × g for 10 minutes; then the lipids were extracted with the mixture of chloroform/methanol (1:2, 1:1, 1:1, and 2:1) successively by sonication and centrifuged at 1 400 × g for 10 minutes. The supernatants were combined and evaporated to dryness. Then the lipids were dissolved in chloroform/methanol (1:1) and used as a total lipid fraction. After phosphorus determination (Gerlach and Deuticke 1963), the same amount of total lipids from heat-shocked and control cells were analyzed using high-performance thin-layer chromatography plates of Silica Gel 60 (E Merck, Darmstadt) with solvent system I, chloroform/methanol/water (60:40:9, v/v). Lipids were visualized by spraying with 2% orcinol in 2N H₂SO₄ for glycosides (A) and 0.05% ferric chloride in 5% each of acetic and sulfuric acid for sterols (B) followed by heating at 100°C. Poriferasterol monoglucoside from Physarum polycephalum was used as a TLC standard (lane 1 and 8). The heat-induced glycolipid that is asterisked was designated as Hsl-X

Purification and structural analysis of Hsl-X

Hsl-X was extracted and purified as described in Fig 1. The purified fraction showed a single spot on the 2-dimensional TLC in solvent systems I and II, by visualizing with orcinol-H₂SO₄ and ferric chloride (data not shown).

To determine components of Hsl-X, the purified lipid was methanolyzed, and trimethylsilyl derivatives of methyl glycoside and sterol were analyzed using gas-liquid chromatography (GLC) as shown in Figure 2. These results indicate that the sugar moiety (Fig 2A) and sterol moiety (Fig 2B) of Hsl-X were glucose and cholesterol, respectively.

Fig 2.

 Gas liquid chromatogram of O-trimethylsilyl derivatives of methyl glyoside (A) and sterol (B). Lipid extracts from cells exposed to 42°C for 30 minutes were used for purification of a heat-expressed steryl glucoside. Partial purification was performed using preparative thin-layer chromatography (TLC) using Silica Gel 60 plates (E Merck, Darmstadt) in the solvent systems I and II successively: solvent system I, chloroform/methanol/water (60:40:9, v/v); solvent system II, chloroform/methanol/acetone/acetic acid/water (10:2:4:2:1, v/v). Then TLC-purified glycolipid was methanolyzed and resultant methyl glycoside and sterol were trimethylsilylated and analyzed using gas-liquid chromatography (GLC) on a column of 3% OV-101 on Shimalite W (80-100 mesh; Shimadzu, Kyoto, Japan), respectively, as described (Murakami-Murofushi et al 1987). GLC analysis was carried out at 150-250°C for glycoside and at 250°C for sterol. Fucose, galactose, mannitol, N-acetylgalactosamine, cholesterol, ergosterol, and stigmasterol (Sigma Chemical Co, St Louis, MO, USA) were used as GLC standards

For more structural analyses, Hsl-X was further purified using HPLC with a C18 reversed-phase column to remove a trace of contaminants. Synthetic α–cholesteryl glucoside was eluted slightly slower than a synthetic β-isomer on a C18 column of HPLC with acetonitrile/methanol (5:1) at a flow rate of 0.7 mL/min. The purified Hsl-X was eluted at the same retention time as chemically synthesized β–cholesteryl glucoside (data not shown), suggesting a β configuration of a glucose moiety.

Hsl-X purified by HPLC was analyzed using electrospray ionization time of flight/mass spectrometer. A characteristic ion peak at m/z 593.4, which corresponds to [M+HCOO]⁻, was observed (Fig 3B). Synthetic β–cholesteryl glucoside gave an identical ion peak at m/z 593.4 under the same conditions (Fig 3A). From these results, the structure of Hsl-X was assigned as β–cholesteryl glucoside (Fig 4).

Fig 3.

 Electrospray ionization time of flight/mass spectrometer (ESI/TOF-MS) spectra of chemically synthesized β-cholesteryl glucoside (A) and a purified steryl glucoside (B). For further purification, the partially purified lipid was applied to high-performance liquid chromatography (HPLC) with a Capcell Pak C18 column (10 × 250 mm). Samples dissolved in methanol were injected, and lipids were eluted with methanol at a flow rate of 1.0 mL/min and the elution was monitored by measuring absorbance at 205 nm with an SPD-6AV ultraviolet spectrometer (Shimadzu, Kyoto, Japan). Then HPLC-purified steryl glucoside was analyzed using the ESI/TOF-MS Mariner (PE Biosystems Japan, Tokyo). The samples were dissolved in acetonitrile/methanol (1:1) containing 1% ammonium formate at a concentration of 5–10 pmol/μL, and were injected to the instrument. MS analysis was performed in the negative mode with a nozzle potential of −180 eV at a nozzle temperature of 180°C. Optimization of ion spray was carried out with chemically synthesized β-cholesteryl glucoside (basically according to Alivisatos et al 1981) (A). (B) heat shock lipid X (Hsl-X) from TIG-3 cells. An ion peak at m/z 593.4 was assigned as [M+HCOO]⁻

Fig 4.

. Structure of heat shock lipid X (Hsl-X)

DISCUSSION

In this study, we demonstrated an expression of cholesteryl glucoside by heat shock at 42°C in human cultured fibroblasts. We previously indicated that poriferasterol monoglucoside was also expressed immediately after heat shock treatment in myxoamoebae of a true slime mold, P. polycephalum, with rapid activation of sterol glucosyltransferase. Preliminarily, we observed the activation of sterol glucosyltransferase in heat shocked homogenate from TIG-3 cells and are now trying to clarify this reaction and the enzyme that catalyzes it. These findings suggest that the expression of steryl glycoside by heat shock is a phenomenon common to mold and to humans and may have some important roles in a variety of organisms.

Steryl glucoside and its 6’-O-acyl derivatives are known as common constituents of higher plants (Bolt and Clarke 1970; Lepaga 1964; Bush and Grunwald 1972). Cholesteryl glucosides are also found in fungi (Kastelic-Suhadolc 1980) and bacteria (Haque et al 1996; Livermore et al 1978; Mayberry and Smith 1983; Patel et al 1978; Rothblat and Smith 1961; Smith 1971; Hirai et al 1995). Their possible functions have included metabolically active components of plant membrane structure (Gunwald 1971), intercellular transporters of sterols (Evans 1972), or glucose carriers through cell membranes (Patel et al 1978; Wojciechowski et al 1976). Recently, a steryl glucoside was shown to act as a glucose donor in the formation of glucosyl ceramide in bean hypocotyl microsomes (Lynch et al 1997). A soybean-derived steryl glucoside showed an enhancement of insulin permeation through the nasal mucosa in rabbits (Ando et al 1998), and cholesteryl glucoside of Helicobacter pylori showed a hemolytic activity (Smith 1971). However, the existence of steryl glucoside has not yet been reported in animal cells or tissues, and this is the first report to show the natural occurrence of this substance in animal cells.

As steryl glucoside in human fibroblasts and in Physarum myxoamoebae (Murakami-Murofushi et al 1997) expressed after heat shock treatment, this phenomenon may be an important step in stress responses of the cell. We also reported the succeeding activation of Ca²⁺-dependent protein kinase followed by Hsp induction in Physarum cells (Maruya et al 1997). Further, we observed that Hsp70 was induced when cholesteryl glucosides were added to the culture medium of TIG-3 cells without heat stress (data not shown). Based on these results, steryl glucoside is considered to act to enhance tolerance to high temperatures and to participate in an activation of Hsfs. That is, when cells are exposed to heat stress, membrane lipids would become more fluid and transferase in the membrane would be activated, causing steryl glucoside to be synthesized. Steryl glucoside may then activate Hsfs via protein kinase activation or may activate Hsfs directly. Synthesized steryl glucoside may be released to the inside or the outside of the cell. In a recent report, plasma membrane cholesterol in a caveola-like domain was shown to be a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase (Park et al 1998). Cholesteryl glucoside can exist in caveola or caveolae-like domains and may act in the same manner. Further investigations are necessary to clarify the physiological functions of steryl glucoside, subsequent reactions involving the activation of protein kinase and Hsfs resulting in Hsp formation.