Biochimie (1991) 73, 1317-1320 © Soci6t6 franqaise de biochimie et biologic ~ol6culaire / Elsevier, Paris
1317
Oxysterois: biological activities and physicochemical studies B Luu, C Moog Laboratoire de Chimie Organique des Substances Naturelles, URA3 !, CNRS, 5, rue Blaise-Pascal, 67084 Strasbourg. France
(Received 14 June 1991; accepted 15 September 1991)
Summary - - To improve the understanding of the various biological activities of oxysterols (oxygenated derivatives of cholesterol), studies of their physicochemical properties have been undertaken. Oxysterols modify membrane dynamic properties which consequently trigger several biological effects. Despite the presence of at least one oxygenated group in addition to the C3~i-hydroxyl, oxysterols insert perfectly into the lipidic bilayer of the membrane inducing a condensing effect similar to, but less potent than, that of cholesterol. In biological membranes oxysterols probably interact with membrane components as they are not easily exchanged after their incorporation into the cell membrane. These lipid-protein interactions are probably crucial for the expression of the biological activities of the oxysterols.
oxysterois / membrane affinity / cytotoxicity / immunomodulating effect / membrane dynamic properties
Introduction Oxysterols, oxygenated derivatives of cholesterol, have been known to exhibit a number of biological activities [1-3]. Some of these widely occurring compounds e g 7 ketocholesteroL 7-hydroxycho!esterol or 25-hydroxycholesterol are generated by simple autoxidation of cholesterol when the sterol is exposed to air, heat, ionic radiation or a combination of these factors [4]. Some other oxysterols such as 26-hydroxycholesterol, 22R-hydroxycholesterol and 24S-hydroxycholesterol (cerebrosterol) are p~oduced by enzymatic reaction i n s i t u [5]. Thus, it is tempting to suppose that some of them arise by accident whereas metabolic role may be attributed to other oxysterols. The presence of a second or a third oxygenated chemical group in the skeleton of the sterols makes these oxysterols less likely to interact with the lipid part of the cell membrane and subsequently renders their incorporation more difficult. Thus, the nature of the interactions leading to the different biological activities of oxysterols should be the subject of a major study.
Membrane dynamic properties of oxysterols Oxysterols are well known for their inhibitory effect on the biosynthesis of cholesterol in mammalian cells
[1]. This inhibition is related to the inactivation of HMGCoA reductase, an enzyme which converts HMGCoA to mevalonate, the rate limiting step in cholesterol biosynthetic pathway. The mechanism of action is not yet precisely known. It has been reported t. h n t . ~vyet,~.,~l~ . . . .,~ ... i : - protein: . . . h. ; n d o,,,~,.;~;...dl v , . . . . . . . . . j. . . .t,, the oxysterol binding protein [6, 7] but this interaction has no influence either on the synthesis of HMGCoA reductase or on its degradation rate [8]. Bucher e t a l observed an insertion of oxysterols in microsomal membranes [9] where this enzyme is located, suggesting a relationship between the inhibitory effect of oxysterol and a modification of dynamic properties of the cell membrane [10]. To test the validity of this hypothesis, a fluorescence polarization study using the 1,6-diphenyl 1,3,5hexatriene fluorescence probe incorporated into the membrane of rapidly proliferating hepatoma cells has been carried out [ 11]. During division, the cells need cholesterol to form the new membrane. In fact, in relation to growth rate, an increase in HMGCoA reductase activity correlated with a decrease of fluorescence polarization in cultured cells can be observed. Such a relationship between membrane dynamic properties and HMGCoA reductase activity was similarly assessed using various other oxysterols. 71~-Hydroxycholesterol and 25-hydroxycholesterol both increase the fluorescence polarization of treated cells. These oxysterols also inhibit the HMGCoA reductase activity. Within the range of dose de~...a
o~.~a~.la,.=
uaJL
lwytV31Jlli.,
13 i 8
B Luu, C Moog
pendency, there is a correlatic~e between this reductase activity and the degree of fluorescence polarization in cells treated with oxysterols. This study clearly demonstrates the membrane dynamic property of oxysterols: oxysterols increase the degree of fluorescence polarization in treated cells and at the same time inhibit HMGCoA reductase activity. The membrane dynamic property of oxysterols is also expected to account for their immunomodulating effect, as it has been shown by Del Bueno et al [ 12]. These authors reported that the stimulation of lymphocyte #1 vitro and in vivo is characterized by the transition from an ordered state to a disordered state in plasma membrane lipids. Indeed, at submicromolar concentrations, oxysterols are not toxic towards lymphocytes, although they depress lymphocyte proliferation induced by mitogenesis [13]. This immunosuppressive effect, which is different from those induced by dexamethasone and cyclosporine A [14, 15], is antagonized by high K ÷ concentration [16] and is triggered by phorbol ester plus calcium ionophore stimulation. These results strongly suggest that 713hydroxycholesterol acts at the membrane level by impairing the early steps in cell activation. To assess the mechanism of oxysterol action on immune cells, we have studied the effect of 713,25dihydroxycholesterol on protein kinase C (PKC) activity in cytosolic and particulate fraction of spleen cells [17]. Cells treated by 713,25-dihydroxycholesterol showed a decrease of the relative PKC activity on the particulate fraction compared to control R_a 24
t3
[ - ~
H O ~ X ' - ~ . . . / / ~ / ~--~ R
Membrane affinity properties of oxysterols Apart from their membrane dynamic effect, another property of oxysterols concerns their membrane affinity. The interest of this study has been reinforced by the results obtained during the study of the cytotoxic effect of oxysterols [18]. Oxysterols are toxic towards rapidly proliferating cells; for example, at micromolar concentrations, 713-hydroxycholesterol is more toxic towards hepatoma cells (HTC, ZHC, etc) than towards normal hepatocytes [ 19] and 25-hydroxycholesterol is more toxic towards lymphoma cells than towards lymphocytes [20]. 713-Hydroxycholesterol is also more toxic towards transformed astrocytes than towards normal ones [21]. In any case, oxysterols exert their cytotoxic effect preferentially towards rapidly proliferating cells. However, the potency of biological activities depends mm'kedly on the chemical structure and the type of cells which are used. The first sign of this cytotoxicity is an alteration of the cell membrane [22]. Morphological studies using the electronic microscope showed that the formation of microvilli is dose-dependent. This formation of these microvilli is caused only by toxic oxystet'ols: 25hydroxycholesterol, which is not cytotoxic towards hepatoma cells, does not induce any modification of the membrane morphology of the surface of th.,:se cells 123]. Interaction o f oxysterols with an artificial membrane
7i
1
R 1 = OH, R2 = R3 = H : 7[3 hydroxycholesterol R 1 = R2 = OH, R3 = H : 7[3, 25 dihydroxycholesterol R1 = R2 = H, R3 = OH : 22R hydroxycholesterol
Fig 1.
cells. These results are confirmed by the observation that 713,25-dihydroxycholesterol also significantly reduced the phosphorylation of the endogenous PKC substrates and suggest an inhibitory effect of oxysterol on the active form of the membrane bound PKC. This phenomenon where oxysterols disturb the transduction of the signal at the membrane level is probably amplified by the extensive changes occurring in the organization of membrane lipids during the differentiation of lymphocytes and their activation by mitogen.
The 5-carboxyfluorescence loaded liposomes prepared according to Gerst et al [24] and treated with 713hydroxycholesterol up to millimolar concentrations do not show any leakiness (Gerst and Schubert, personal communication). This observation indicates that the nature of the lysis is not the consequence of a detergent effect. The data are in agreement with those of Theunissen et al [25] who demonstrated a reduction of glucose permeability induced by 713-hydroxycholesterol in egg-PC liposome. Theunissen et al [25] showed that this phenomenon is compatible with a
Oxysterols: biological and physicochemical studies condensing effect of 713-hydroxycholesterol which is, however, less extensive than that of cholesterol. In fact, they studied the transfer of radiolabelled oxysterols and cholesterol from monolayers to lipoproteins or vesicles and showed that cholesterol does not give high transfer rates, thus demonstrating a marked condensing effect. In the presence of high density lipoproteins or small unilamellar vesicles, this transfer rate is considerable for oxysterols and increases in the following order: 7-ketocholesterol < 7~-hydroxycholesterol < 7¢t-hydroxycholesterol and < 25-hydroxycholesterol. The difference in rate between cholesterol and 7-ketocholesterol is 40-fold and that between 7-ketocholesterol and 25-hydroxycholesterol 20-fold. This diminution in the condensing effect is consistent with the more polar nature of these oxygenated derivatives of cholesterol. Oxysterols and affinity on biological membrane
In biological membranes, the oxysterol affinity m a y be completely different due to possible interactions between oxysterols and proteins. Streuli et al [26] showed that even the more polar oxysterols could not be removed from red blood cells despite repeated washing of the cells in buffer. The study by Lelong et al using Mycoplasma capricolum supported these data [27]. Mycoplasma growing in a mixture of [14C]cholesterol and [3H]-713-hydroxycholesterol incorporate both sterols. The exchange studies with lipid vesicles showed that whereas most of the cholesterol underwent exchange, only about 25% of 7~-hydroxycholesterol was released. The authors interpreted the difficulty of this exchange by assuming an interaction between oxysterols and Mycoplasma capricolum membrane component. This study is also in agreement with the recent study of Lin et al [28] who showed a positive correlation between the antiproliferative effects of oxysterols and the binding affinities for the anti-estrogen-binding sites.
Conclusion It is highly probable that oxysterols affect membrane fluidity by modifying cholesterol biosynthesis within the cells. Oxysterols are inserted into the cell membrane and exert a condensing effect similar to that of cholesterol. However, the interaction of oxysterols with protein components in the cell membrane m a y be a crucial step in the expression of oxysterol activities.
! 3 !9
References 1 Kandutsch AA, Chen HW, Heininger ILl (1978) Biological activity of some oxygenated sterols. Science 201,498-501 2 Parish EJ, Nanduri VBB, Kohi HH, Taylor FR (1986) Oxysterols: chemical synthesis, biosynthesis and biological activities. Lipids 21, 27-30 3 Ji YH, Moog C, Schmitt G, Luu B (1990) Polyoxygenated sterols and triterpenes: chemical structures and biological activities. J Steroid Biochem 35, 741-744 4 Van Lier JE, Smith LL (1970) Autoxidation of cholesterol via hydroperoxide intermediates. J Org Chem 35, 2627-2632 5 Koch P, Luu B, Beck JP, Ourisson G (1985) Occurrence and biological activity of brain oxygenated sterols. Bull Soc Chim Fr II, 779-782 6 Taylor FR, Kandutsch AA (1985) Oxysterol binding protein. Chem Phys Lipids 38, 187-194 7 Beseme F, Astruc ME, Defay R, Crastes de Paulet A (1987) Rat liver cytosol oxysterol binding protein. Characterization and comparison with the HTC cell protein. FEBS Lett 210, 97-103 8 Saucier SE, Kandutsch AA (1979) Inactive 3-hydroxy-3methylglutaryl-coenzyme A reductase in broken cell preparation of various mammalian tissues and cell cultures. Biochim Biophys Acta 572, 541-556 9 Bucher LR, Overath P, Lynen F (1960) ~-Hydroxy-I~methylglutaryl coenzyme A reductase, cleavage and condensing enzyme in relation to cholesterol formation in rat liver. Biochim Biophys Acta 40, 491-501 10 Lichtenstein A, Brecher P (1983) Esterification of cholesterol and 25-hydroxycholesterol by rat liver microsomes. Biochim Biophys Acta 751,340-348 11 Richert L, Castagna M, Beck JP, Rong SH, Luu B, Ourisson G (1984) Growth rate-related and hydroxysterol-induced changes in membrane fluidity of cultured hepatoma cells: correlation with 3-hydroxy-3-methylgiutaryi CoA reductase activity. Biochem Biophys Res Commun 120, 192-198 12 Del Buono BJ, Williamson PL, Schlegel RA (1986) Alteration in plasma membrane lipid organization during lymphocyte differentiation. J Cell Physiol 126, 379-388 13 Defay R, Astruc ME, Roussillon S, Descomp B, Crastes de Paulet A (1982) DNA synthesis and 3-hydroxy-3methylglutaryl CoA reductase activity in PHA stimulated human lymphocytes: a comparative study of the inhibitory effects of some oxysterols with special reference to side chain hydroxylatcd derivatives. Biochem Biophys Res Commun 106, 362-372 14 Moog C, Luu B, Altmeyer A, Bischoff P (1989) Studies on the immunosuppressive properties of 7,25-dihydroxycholesterol. II. Effect on early steps of T cell activation. lnt J Immunopharm 11,559-565 15 Moog C, Ji YH, Waltzinger C, Luu B, Bischoff P (1990) Studies on the immunological properties of oxysterols:, in vivo actions of 7,25-dihydroxycholesterol upon munnc peritoneal cells. Immunology 70, 344 350 16 Hietter H, Luu B, Moog C, Bischoff P (1988) ActivitE immunosuppressive des oxystErols. I. Inhibition de la blastogEn~se par le 71]-hydroxycholestErol et reversion partielle de cet effet par le serum, les LDL et le KCI. In: ActiviMs biologiques des oxyst~rols (Beck JP, Crastes de Paulet A, eds) Editions Inserm 166, 23-46
1. , , 0 17
18
19
20
21
22
B Luu, C Moog
Mtrog C, Deloulme JC, Baudier J, Revel MO, Bischoff P, Hietter H, Luu B (1991) Membrane-related oxysterol /'unction: preliminary results on the modification of protein kinase C activity and substrate phosphorylation by 7~,25-dihydroxycholesterol. Biochimie 73, 1321-1326 Christ M, Ji YH, Moog C, Pannecoucke X, Schmitt G, Bischoff P, Luu B (1991) Antitumor activity of oxysterols. Effect of two water-soluble monophosphoric acid diesters of 7~-hydroxycholesteroi on mastocytoma P-815 in vivo. Anticancer Res ! 1,359--364 Nordman P, Diez-lbanez M, Chessebeuf-Padieu M, Luu B, Mack G, Mersel M (1989) Toxic effects of 7~-hydroxycholesterol on rat liver primary cultures, epithelial lines and co-cultures. Cell Bioi Toxicology 5, 261-270 Hietter H, Bischoff P, Beck JP, Ourisson G, Luu B (1986) Comparative effects of 713-hydroxycholesterol towards murine lymphomas, lymphoblasts and lymphocytes: selective cytotoxicity and blastogenesis inhibition. Cancer Biochem Biophys 9, 75--83 Kupferberg A, Cremel G, Behr P, Van Dorsselaer A, Luu B, Mersei M (1991) Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 7l$-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity, and on cell surface protein expression. Moll Cell Biochem 101, 11-12 Rong S, Bergmann C, Luu B, Beck JP, Ourisson G (1985) Activit6 antitumorale in vivo de d6riv6s hydrosolubles de
7[3-hydroxycholest6rol. CR Acad Sci Paris 300 (III) 89-94 23 Richert L (1987) Effets de d6riv6s st6roliques au niveau cellulaire. Importance physiologique de la dynamique membranaire. PhD Dissertation Thesis 24 Gerst N, Schuber F, Viola F, Cattel L (1986) Inhibition of cholesterol biosynthesis in 3T3 fibroblasts by 2 Aza,2,3dihydrosqualene, a rationally designed 2,3-oxidosqualene cyclase inhibitor. Biochem Pharmacology 35, 42434250 25 Theunissen JJH, Jackson R J, Kempen HJM, Demel RA (1986) Membrane properties of oxysterols. Interfacial orientation, influence on membrane permeability and redistribution between membrane. Biochim Biophys Acta 860, 66-74 26 Streuli RA, Kanofsky JR, Gunn RB, Yachnin S (1981) Diminished osmotic fragility of human-erythrocytes following the membrane insertion of oxygenated sterol compounds. Blood 58, 317-325 27 Lelong I, Luu B, Mersel M, Rottem S (1988) Effect of 7 6hydroxycholesterol on growth and membrane composition of Mycoplasma capricolum. FEBS Lett 232, 354-358 28 Lin L, Hwang PL (1991) Antiproliferative effects of oxygenated sterols: positive correlation with binding affinities for the antiestrogen-binding sites. Biochim Biophys Acta 1082, 177-184
1317
Oxysterois: biological activities and physicochemical studies B Luu, C Moog Laboratoire de Chimie Organique des Substances Naturelles, URA3 !, CNRS, 5, rue Blaise-Pascal, 67084 Strasbourg. France
(Received 14 June 1991; accepted 15 September 1991)
Summary - - To improve the understanding of the various biological activities of oxysterols (oxygenated derivatives of cholesterol), studies of their physicochemical properties have been undertaken. Oxysterols modify membrane dynamic properties which consequently trigger several biological effects. Despite the presence of at least one oxygenated group in addition to the C3~i-hydroxyl, oxysterols insert perfectly into the lipidic bilayer of the membrane inducing a condensing effect similar to, but less potent than, that of cholesterol. In biological membranes oxysterols probably interact with membrane components as they are not easily exchanged after their incorporation into the cell membrane. These lipid-protein interactions are probably crucial for the expression of the biological activities of the oxysterols.
oxysterois / membrane affinity / cytotoxicity / immunomodulating effect / membrane dynamic properties
Introduction Oxysterols, oxygenated derivatives of cholesterol, have been known to exhibit a number of biological activities [1-3]. Some of these widely occurring compounds e g 7 ketocholesteroL 7-hydroxycho!esterol or 25-hydroxycholesterol are generated by simple autoxidation of cholesterol when the sterol is exposed to air, heat, ionic radiation or a combination of these factors [4]. Some other oxysterols such as 26-hydroxycholesterol, 22R-hydroxycholesterol and 24S-hydroxycholesterol (cerebrosterol) are p~oduced by enzymatic reaction i n s i t u [5]. Thus, it is tempting to suppose that some of them arise by accident whereas metabolic role may be attributed to other oxysterols. The presence of a second or a third oxygenated chemical group in the skeleton of the sterols makes these oxysterols less likely to interact with the lipid part of the cell membrane and subsequently renders their incorporation more difficult. Thus, the nature of the interactions leading to the different biological activities of oxysterols should be the subject of a major study.
Membrane dynamic properties of oxysterols Oxysterols are well known for their inhibitory effect on the biosynthesis of cholesterol in mammalian cells
[1]. This inhibition is related to the inactivation of HMGCoA reductase, an enzyme which converts HMGCoA to mevalonate, the rate limiting step in cholesterol biosynthetic pathway. The mechanism of action is not yet precisely known. It has been reported t. h n t . ~vyet,~.,~l~ . . . .,~ ... i : - protein: . . . h. ; n d o,,,~,.;~;...dl v , . . . . . . . . . j. . . .t,, the oxysterol binding protein [6, 7] but this interaction has no influence either on the synthesis of HMGCoA reductase or on its degradation rate [8]. Bucher e t a l observed an insertion of oxysterols in microsomal membranes [9] where this enzyme is located, suggesting a relationship between the inhibitory effect of oxysterol and a modification of dynamic properties of the cell membrane [10]. To test the validity of this hypothesis, a fluorescence polarization study using the 1,6-diphenyl 1,3,5hexatriene fluorescence probe incorporated into the membrane of rapidly proliferating hepatoma cells has been carried out [ 11]. During division, the cells need cholesterol to form the new membrane. In fact, in relation to growth rate, an increase in HMGCoA reductase activity correlated with a decrease of fluorescence polarization in cultured cells can be observed. Such a relationship between membrane dynamic properties and HMGCoA reductase activity was similarly assessed using various other oxysterols. 71~-Hydroxycholesterol and 25-hydroxycholesterol both increase the fluorescence polarization of treated cells. These oxysterols also inhibit the HMGCoA reductase activity. Within the range of dose de~...a
o~.~a~.la,.=
uaJL
lwytV31Jlli.,
13 i 8
B Luu, C Moog
pendency, there is a correlatic~e between this reductase activity and the degree of fluorescence polarization in cells treated with oxysterols. This study clearly demonstrates the membrane dynamic property of oxysterols: oxysterols increase the degree of fluorescence polarization in treated cells and at the same time inhibit HMGCoA reductase activity. The membrane dynamic property of oxysterols is also expected to account for their immunomodulating effect, as it has been shown by Del Bueno et al [ 12]. These authors reported that the stimulation of lymphocyte #1 vitro and in vivo is characterized by the transition from an ordered state to a disordered state in plasma membrane lipids. Indeed, at submicromolar concentrations, oxysterols are not toxic towards lymphocytes, although they depress lymphocyte proliferation induced by mitogenesis [13]. This immunosuppressive effect, which is different from those induced by dexamethasone and cyclosporine A [14, 15], is antagonized by high K ÷ concentration [16] and is triggered by phorbol ester plus calcium ionophore stimulation. These results strongly suggest that 713hydroxycholesterol acts at the membrane level by impairing the early steps in cell activation. To assess the mechanism of oxysterol action on immune cells, we have studied the effect of 713,25dihydroxycholesterol on protein kinase C (PKC) activity in cytosolic and particulate fraction of spleen cells [17]. Cells treated by 713,25-dihydroxycholesterol showed a decrease of the relative PKC activity on the particulate fraction compared to control R_a 24
t3
[ - ~
H O ~ X ' - ~ . . . / / ~ / ~--~ R
Membrane affinity properties of oxysterols Apart from their membrane dynamic effect, another property of oxysterols concerns their membrane affinity. The interest of this study has been reinforced by the results obtained during the study of the cytotoxic effect of oxysterols [18]. Oxysterols are toxic towards rapidly proliferating cells; for example, at micromolar concentrations, 713-hydroxycholesterol is more toxic towards hepatoma cells (HTC, ZHC, etc) than towards normal hepatocytes [ 19] and 25-hydroxycholesterol is more toxic towards lymphoma cells than towards lymphocytes [20]. 713-Hydroxycholesterol is also more toxic towards transformed astrocytes than towards normal ones [21]. In any case, oxysterols exert their cytotoxic effect preferentially towards rapidly proliferating cells. However, the potency of biological activities depends mm'kedly on the chemical structure and the type of cells which are used. The first sign of this cytotoxicity is an alteration of the cell membrane [22]. Morphological studies using the electronic microscope showed that the formation of microvilli is dose-dependent. This formation of these microvilli is caused only by toxic oxystet'ols: 25hydroxycholesterol, which is not cytotoxic towards hepatoma cells, does not induce any modification of the membrane morphology of the surface of th.,:se cells 123]. Interaction o f oxysterols with an artificial membrane
7i
1
R 1 = OH, R2 = R3 = H : 7[3 hydroxycholesterol R 1 = R2 = OH, R3 = H : 7[3, 25 dihydroxycholesterol R1 = R2 = H, R3 = OH : 22R hydroxycholesterol
Fig 1.
cells. These results are confirmed by the observation that 713,25-dihydroxycholesterol also significantly reduced the phosphorylation of the endogenous PKC substrates and suggest an inhibitory effect of oxysterol on the active form of the membrane bound PKC. This phenomenon where oxysterols disturb the transduction of the signal at the membrane level is probably amplified by the extensive changes occurring in the organization of membrane lipids during the differentiation of lymphocytes and their activation by mitogen.
The 5-carboxyfluorescence loaded liposomes prepared according to Gerst et al [24] and treated with 713hydroxycholesterol up to millimolar concentrations do not show any leakiness (Gerst and Schubert, personal communication). This observation indicates that the nature of the lysis is not the consequence of a detergent effect. The data are in agreement with those of Theunissen et al [25] who demonstrated a reduction of glucose permeability induced by 713-hydroxycholesterol in egg-PC liposome. Theunissen et al [25] showed that this phenomenon is compatible with a
Oxysterols: biological and physicochemical studies condensing effect of 713-hydroxycholesterol which is, however, less extensive than that of cholesterol. In fact, they studied the transfer of radiolabelled oxysterols and cholesterol from monolayers to lipoproteins or vesicles and showed that cholesterol does not give high transfer rates, thus demonstrating a marked condensing effect. In the presence of high density lipoproteins or small unilamellar vesicles, this transfer rate is considerable for oxysterols and increases in the following order: 7-ketocholesterol < 7~-hydroxycholesterol < 7¢t-hydroxycholesterol and < 25-hydroxycholesterol. The difference in rate between cholesterol and 7-ketocholesterol is 40-fold and that between 7-ketocholesterol and 25-hydroxycholesterol 20-fold. This diminution in the condensing effect is consistent with the more polar nature of these oxygenated derivatives of cholesterol. Oxysterols and affinity on biological membrane
In biological membranes, the oxysterol affinity m a y be completely different due to possible interactions between oxysterols and proteins. Streuli et al [26] showed that even the more polar oxysterols could not be removed from red blood cells despite repeated washing of the cells in buffer. The study by Lelong et al using Mycoplasma capricolum supported these data [27]. Mycoplasma growing in a mixture of [14C]cholesterol and [3H]-713-hydroxycholesterol incorporate both sterols. The exchange studies with lipid vesicles showed that whereas most of the cholesterol underwent exchange, only about 25% of 7~-hydroxycholesterol was released. The authors interpreted the difficulty of this exchange by assuming an interaction between oxysterols and Mycoplasma capricolum membrane component. This study is also in agreement with the recent study of Lin et al [28] who showed a positive correlation between the antiproliferative effects of oxysterols and the binding affinities for the anti-estrogen-binding sites.
Conclusion It is highly probable that oxysterols affect membrane fluidity by modifying cholesterol biosynthesis within the cells. Oxysterols are inserted into the cell membrane and exert a condensing effect similar to that of cholesterol. However, the interaction of oxysterols with protein components in the cell membrane m a y be a crucial step in the expression of oxysterol activities.
! 3 !9
References 1 Kandutsch AA, Chen HW, Heininger ILl (1978) Biological activity of some oxygenated sterols. Science 201,498-501 2 Parish EJ, Nanduri VBB, Kohi HH, Taylor FR (1986) Oxysterols: chemical synthesis, biosynthesis and biological activities. Lipids 21, 27-30 3 Ji YH, Moog C, Schmitt G, Luu B (1990) Polyoxygenated sterols and triterpenes: chemical structures and biological activities. J Steroid Biochem 35, 741-744 4 Van Lier JE, Smith LL (1970) Autoxidation of cholesterol via hydroperoxide intermediates. J Org Chem 35, 2627-2632 5 Koch P, Luu B, Beck JP, Ourisson G (1985) Occurrence and biological activity of brain oxygenated sterols. Bull Soc Chim Fr II, 779-782 6 Taylor FR, Kandutsch AA (1985) Oxysterol binding protein. Chem Phys Lipids 38, 187-194 7 Beseme F, Astruc ME, Defay R, Crastes de Paulet A (1987) Rat liver cytosol oxysterol binding protein. Characterization and comparison with the HTC cell protein. FEBS Lett 210, 97-103 8 Saucier SE, Kandutsch AA (1979) Inactive 3-hydroxy-3methylglutaryl-coenzyme A reductase in broken cell preparation of various mammalian tissues and cell cultures. Biochim Biophys Acta 572, 541-556 9 Bucher LR, Overath P, Lynen F (1960) ~-Hydroxy-I~methylglutaryl coenzyme A reductase, cleavage and condensing enzyme in relation to cholesterol formation in rat liver. Biochim Biophys Acta 40, 491-501 10 Lichtenstein A, Brecher P (1983) Esterification of cholesterol and 25-hydroxycholesterol by rat liver microsomes. Biochim Biophys Acta 751,340-348 11 Richert L, Castagna M, Beck JP, Rong SH, Luu B, Ourisson G (1984) Growth rate-related and hydroxysterol-induced changes in membrane fluidity of cultured hepatoma cells: correlation with 3-hydroxy-3-methylgiutaryi CoA reductase activity. Biochem Biophys Res Commun 120, 192-198 12 Del Buono BJ, Williamson PL, Schlegel RA (1986) Alteration in plasma membrane lipid organization during lymphocyte differentiation. J Cell Physiol 126, 379-388 13 Defay R, Astruc ME, Roussillon S, Descomp B, Crastes de Paulet A (1982) DNA synthesis and 3-hydroxy-3methylglutaryl CoA reductase activity in PHA stimulated human lymphocytes: a comparative study of the inhibitory effects of some oxysterols with special reference to side chain hydroxylatcd derivatives. Biochem Biophys Res Commun 106, 362-372 14 Moog C, Luu B, Altmeyer A, Bischoff P (1989) Studies on the immunosuppressive properties of 7,25-dihydroxycholesterol. II. Effect on early steps of T cell activation. lnt J Immunopharm 11,559-565 15 Moog C, Ji YH, Waltzinger C, Luu B, Bischoff P (1990) Studies on the immunological properties of oxysterols:, in vivo actions of 7,25-dihydroxycholesterol upon munnc peritoneal cells. Immunology 70, 344 350 16 Hietter H, Luu B, Moog C, Bischoff P (1988) ActivitE immunosuppressive des oxystErols. I. Inhibition de la blastogEn~se par le 71]-hydroxycholestErol et reversion partielle de cet effet par le serum, les LDL et le KCI. In: ActiviMs biologiques des oxyst~rols (Beck JP, Crastes de Paulet A, eds) Editions Inserm 166, 23-46
1. , , 0 17
18
19
20
21
22
B Luu, C Moog
Mtrog C, Deloulme JC, Baudier J, Revel MO, Bischoff P, Hietter H, Luu B (1991) Membrane-related oxysterol /'unction: preliminary results on the modification of protein kinase C activity and substrate phosphorylation by 7~,25-dihydroxycholesterol. Biochimie 73, 1321-1326 Christ M, Ji YH, Moog C, Pannecoucke X, Schmitt G, Bischoff P, Luu B (1991) Antitumor activity of oxysterols. Effect of two water-soluble monophosphoric acid diesters of 7~-hydroxycholesteroi on mastocytoma P-815 in vivo. Anticancer Res ! 1,359--364 Nordman P, Diez-lbanez M, Chessebeuf-Padieu M, Luu B, Mack G, Mersel M (1989) Toxic effects of 7~-hydroxycholesterol on rat liver primary cultures, epithelial lines and co-cultures. Cell Bioi Toxicology 5, 261-270 Hietter H, Bischoff P, Beck JP, Ourisson G, Luu B (1986) Comparative effects of 713-hydroxycholesterol towards murine lymphomas, lymphoblasts and lymphocytes: selective cytotoxicity and blastogenesis inhibition. Cancer Biochem Biophys 9, 75--83 Kupferberg A, Cremel G, Behr P, Van Dorsselaer A, Luu B, Mersei M (1991) Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 7l$-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity, and on cell surface protein expression. Moll Cell Biochem 101, 11-12 Rong S, Bergmann C, Luu B, Beck JP, Ourisson G (1985) Activit6 antitumorale in vivo de d6riv6s hydrosolubles de
7[3-hydroxycholest6rol. CR Acad Sci Paris 300 (III) 89-94 23 Richert L (1987) Effets de d6riv6s st6roliques au niveau cellulaire. Importance physiologique de la dynamique membranaire. PhD Dissertation Thesis 24 Gerst N, Schuber F, Viola F, Cattel L (1986) Inhibition of cholesterol biosynthesis in 3T3 fibroblasts by 2 Aza,2,3dihydrosqualene, a rationally designed 2,3-oxidosqualene cyclase inhibitor. Biochem Pharmacology 35, 42434250 25 Theunissen JJH, Jackson R J, Kempen HJM, Demel RA (1986) Membrane properties of oxysterols. Interfacial orientation, influence on membrane permeability and redistribution between membrane. Biochim Biophys Acta 860, 66-74 26 Streuli RA, Kanofsky JR, Gunn RB, Yachnin S (1981) Diminished osmotic fragility of human-erythrocytes following the membrane insertion of oxygenated sterol compounds. Blood 58, 317-325 27 Lelong I, Luu B, Mersel M, Rottem S (1988) Effect of 7 6hydroxycholesterol on growth and membrane composition of Mycoplasma capricolum. FEBS Lett 232, 354-358 28 Lin L, Hwang PL (1991) Antiproliferative effects of oxygenated sterols: positive correlation with binding affinities for the antiestrogen-binding sites. Biochim Biophys Acta 1082, 177-184
