Intracellular trafficking of vitamin E in hepatocytes: the role of tocopherol transfer protein
2005; Elsevier BV; Volume: 46; Issue: 10 Linguagem: Inglês
10.1194/jlr.m500143-jlr200
ISSN1539-7262
AutoresJinghui Qian, Samantha Morley, Kathleen H. Wilson, Phil Nava, Jeffrey Atkinson, Danny Manor,
Tópico(s)Free Radicals and Antioxidants
ResumoThe term vitamin E denotes a family of tocopherols and tocotrienols, plant lipids that are essential for vertebrate fertility and health. The principal form of vitamin E found in humans, RRR-α-tocopherol (TOH), is thought to protect cells by virtue of its ability to quench free radicals, and functions as the main lipid-soluble antioxidant. Regulation of vitamin E homeostasis occurs in the liver, where TOH is selectively retained while other forms of vitamin E are degraded. Through the action of tocopherol transfer protein (TTP), TOH is then secreted from the liver into circulating lipoproteins that deliver the vitamin to target tissues. Presently, very little is known regarding the intracellular transport of vitamin E. We utilized biochemical, pharmacological, and microscopic approaches to study this process in cultured hepatocytes.We observe that tocopherol-HDL complexes are efficiently internalized through scavenger receptor class B type I. Once internalized, tocopherol arrives within ∼30 min at intracellular vesicular organelles, where it colocalizes with TTP, and with a marker of the lysosomal compartment (LAMP1), before being transported to the plasma membrane in a TTP-dependent manner. We further show that intracellular processing of tocopherol involves a functional interaction between TTP and an ABC-type transporter. The term vitamin E denotes a family of tocopherols and tocotrienols, plant lipids that are essential for vertebrate fertility and health. The principal form of vitamin E found in humans, RRR-α-tocopherol (TOH), is thought to protect cells by virtue of its ability to quench free radicals, and functions as the main lipid-soluble antioxidant. Regulation of vitamin E homeostasis occurs in the liver, where TOH is selectively retained while other forms of vitamin E are degraded. Through the action of tocopherol transfer protein (TTP), TOH is then secreted from the liver into circulating lipoproteins that deliver the vitamin to target tissues. Presently, very little is known regarding the intracellular transport of vitamin E. We utilized biochemical, pharmacological, and microscopic approaches to study this process in cultured hepatocytes. We observe that tocopherol-HDL complexes are efficiently internalized through scavenger receptor class B type I. Once internalized, tocopherol arrives within ∼30 min at intracellular vesicular organelles, where it colocalizes with TTP, and with a marker of the lysosomal compartment (LAMP1), before being transported to the plasma membrane in a TTP-dependent manner. We further show that intracellular processing of tocopherol involves a functional interaction between TTP and an ABC-type transporter. Vitamin E is a neutral plant lipid that is an essential nutrient in vertebrates. It is generally accepted that by virtue of its antioxidant activity, vitamin E is able to scavenge free radicals, alleviate oxidative stress, and thus promote normal cell function. Indeed, reduced vitamin E levels are associated with a plethora of pathologies, including infertility, hemolysis, and neuronal degeneration. Thus, adequate vitamin E intake is considered critical for health, and supplementation with the vitamin has become widespread in humans and companion animals (1Traber M.G. Arai H. Molecular mechanisms of vitamin E transport.Annu. Rev. Nutr. 1999; 19: 343-355Google Scholar, 2Sokol R.J. Vitamin E deficiency and neurologic disease.Annu. Rev. Nutr. 1988; 8: 351-373Google Scholar). There are eight chemically distinct forms of vitamin E, among which RRR-α-tocopherol (TOH) is considered the most biologically active and is selectively retained in the body (3Traber M.G. Determinants of plasma vitamin E concentrations.Free Radic. Biol. Med. 1994; 16: 229-239Google Scholar). Despite the strict physiological requirement for vitamin E, our understanding of the mechanisms that regulate its levels in the body is limited. Dietary TOH is absorbed by enterocytes, and secreted to the circulation together with other lipids incorporated in chylomicra (4Traber M.G. Ingold K.U. Burton G.W. Kayden H.J. Absorption and transport of deuterium-substituted 2R,4'R,8'R-alpha-tocopherol in human lipoproteins.Lipids. 1988; 23: 791-797Google Scholar). Following endothelial catabolism of the chylomicra, vitamin E is taken up by parenchymal cells of the liver. It is in the liver that the key reactions that regulate vitamin E status take place. Specifically, the cytochrome P450 isoform CYP4F2 catabolizes tocols other than RRR-α-tocopherol into water-soluble products that are excreted in urine (5Sontag T.J. Parker R.S. Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status.J. Biol. Chem. 2002; 277: 25290-25296Google Scholar). The remaining vitamin form, RRR-α-tocopherol (TOH), is then secreted from the hepatocytes and delivered to peripheral tissues complexed to circulating lipoproteins. Hepatic tocopherol secretion is facilitated by the tocopherol transfer protein (TTP), a soluble polypeptide that binds TOH with high affinity and selectivity and catalyzes its transfer between lipid vesicles (6Sato Y. Hagiwara K. Arai H. Inoue K. Purification and characterization of the alpha-tocopherol transfer protein from rat liver.FEBS Lett. 1991; 288: 41-45Google Scholar, 7Arita M. Sato Y. Miyata A. Tanabe T. Takahashi E. Kayden H.J. Arai H. Inoue K. Human alpha-tocopherol transfer protein: cDNA cloning, expression and chromosomal localization.Biochem. J. 1995; 306: 437-443Google Scholar, 8Hosomi A. Arita M. Sato Y. Kiyose C. Ueda T. Igarashi O. Arai H. Inoue K. Affinity for alpha-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs.FEBS Lett. 1997; 409: 105-108Google Scholar). In support of a critical role for TTP in regulating vitamin E status are the observations that humans who carry mutations in the ttpA gene display low plasma tocopherol levels and neurological disorders associated with elevated oxidative stress termed ataxia with vitamin E deficiency (AVED) (2Sokol R.J. Vitamin E deficiency and neurologic disease.Annu. Rev. Nutr. 1988; 8: 351-373Google Scholar, 3Traber M.G. Determinants of plasma vitamin E concentrations.Free Radic. Biol. Med. 1994; 16: 229-239Google Scholar, 9Ouahchi K. Arita M. Kayden H. Hentati F. Hamida M. Ben Sokol R. Arai H. Inoue K. Mandel J.L. Koenig M. Ataxia with isolated vitamin E deficiency is caused by mutations in the alpha-tocopherol transfer protein.Nat. Genet. 1995; 9: 141-145Google Scholar, 10Hentati A. Deng H.X. Hung W.Y. Nayer M. Ahmed M.S. He X. Tim R. Stumpf D.A. Siddique T. Ahmed A. Human alpha-tocopherol transfer protein: gene structure and mutations in familial vitamin E deficiency.Ann. Neurol. 1996; 39: 295-300Google Scholar). Similarly, TTP−/− mice display low vitamin E levels, are infertile, and exhibit an AVED-like pathology (11Terasawa Y. Ladha Z. Leonard S.W. Morrow J.D. Newland D. Sanan D. Packer L. Traber M.G. V. Farese Jr, R. Increased atherosclerosis in hyperlipidemic mice deficient in alpha -tocopherol transfer protein and vitamin E.Proc. Natl. Acad. Sci. USA. 2000; 97: 13830-13834Google Scholar, 12Yokota T. Igarashi K. Uchihara T. Jishage K. Tomita H. Inaba A. Li Y. Arita M. Suzuki H. Mizusawa H. Arai H. Delayed-onset ataxia in mice lacking alpha-tocopherol transfer protein: model for neuronal degeneration caused by chronic oxidative stress.Proc. Natl. Acad. Sci. USA. 2001; 98: 15185-15190Google Scholar).Details regarding the intracellular transport of tocopherol are scarce. In many cell types, the vitamin accumulates primarily in lysosomes (13Rupar C.A. Albo S. Whitehall J.D. Rat liver lysosome membranes are enriched in alpha-tocopherol.Biochem. Cell Biol. 1992; 70: 486-488Google Scholar) and mitochondria (14Mayne S.T. Parker R.S. Subcellular distribution of dietary beta-carotene in chick liver.Lipids. 1986; 21: 164-169Google Scholar), where high levels of oxidative stress are presumed to exist. Especially enigmatic are the paths for intracellular transport of vitamin E in hepatocytes, the cells that regulate whole-body distribution of tocopherol. In attempts to identify intracellular vitamin E transporters, multiple groups have reported that the only specific tocopherol binding activity in liver is that of TTP (6Sato Y. Hagiwara K. Arai H. Inoue K. Purification and characterization of the alpha-tocopherol transfer protein from rat liver.FEBS Lett. 1991; 288: 41-45Google Scholar, 15Rajaram O.V. Fatterpaker P. Sreenivasan A. Occurrence of -tocopherol binding protein in rat liver cell sap.Biochem. Biophys. Res. Commun. 1973; 52: 459-465Google Scholar, 16Catignani G.L. An alpha-tocopherol binding protein in rat liver cytoplasm.Biochem. Biophys. Res. Commun. 1975; 67: 66-72Google Scholar, 17Kuhlenkamp J. Ronk M. Yusin M. Stolz A. Kaplowitz N. Identification and purification of a human liver cytosolic tocopherol binding protein.Protein Expr. Purif. 1993; 4: 382-389Google Scholar). Arai and colleagues reported that when TTP is overexpressed in a cultured rat hepatocyte cell line, tocopherol secretion is markedly facilitated (18Arita M. Nomura K. Arai H. Inoue K. alpha-tocopherol transfer protein stimulates the secretion of alpha-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway.Proc. Natl. Acad. Sci. USA. 1997; 94: 12437-12441Google Scholar, 19Horiguchi M. Arita M. Kaempf-Rotzoll D.E. Tsujimoto M. Inoue K. Arai H. pH-dependent translocation of alpha-tocopherol transfer protein (alpha-TTP) between hepatic cytosol and late endosomes.Genes Cells. 2003; 8: 789-800Google Scholar). These authors concluded, on the basis of pharmacological sensitivity, that hepatocytes possess a novel and specific pathway for tocopherol secretion, independent of the route utilized for secretion of nascent very low density lipoproteins. Presently, nothing is known regarding the molecular mechanisms that underlie this activity.We aim to delineate the pathway of vitamin E transport in liver cells, and the role that TTP plays in this process. Toward this goal, we report here our studies on the uptake, transport, and secretion of vitamin E in cultured hepatocyte cell lines that are capable of inducible expression of TTP.EXPERIMENTAL PROCEDURESCell cultureMcA-RH7777 cells and HepG2/C3A cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and additional 10% horse serum for McA-RH7777 cells. All transfections were done using Fugene6 (Roche Biochemicals Co.).Inducible expression of TTPCell lines were generated using the TetOn system according to the manufacturer's instructions (Clontech). Briefly, HepG2 or McA-RH7777 cells were first transfected with the tetracycline regulatory element pTetOn and stable transfectants selected with neomycin (G418; 400 μg/ml). Then the cells were transfected with the pTRE2 vector containing the human TTP gene fused to a 5′ hemagglutin (HA) tag to aid in immunodetection. Double stable clones were selected with both neomycin and hygromycin (400 and 200 μg/ml, respectively), screened for inducible expression of HA-TTP by immunoblotting, and positive clones maintained in the presence of both antibiotics (200 μg/ml each). To induce TTP expression, doxycycline (Calbiochem, 1 μg/ml) was added to the growth media for 48 h.Tocopherol complexesTocopherol-serum complexes were prepared according to the method of Asmis (20Asmis R. Physical partitioning is the main mechanism of alpha-tocopherol and cholesterol transfer between lipoproteins and P388D1 macrophage-like cells.Eur. J. Biochem. 1997; 250: 600-607Google Scholar). Briefly, a glass scintillation vial was coated with a film containing the required amount of [14C]α-tocopherol (0.18 μCi/ml) or NBD-tocopherol, and the solvent was evaporated under vacuum. After addition of FBS (tetracycline-free, Clontech), the vial was purged under nitrogen, sealed, and rotated at 4°C for 1 h at 10 rpm. DMEM was then added to the vial, to obtain a final serum concentration of 10% (v/v). The final concentration of [14C]α-tocopherol and NBD-α-tocopherol was 3 μM and 50 μM, respectively.To compare the efficacy of HDL and LDL in delivering tocopherol to hepatocytes (Fig. 2A), the amount of endogenous tocopherol in each preparation was determined by gas chromatography/mass spectroscopy (5Sontag T.J. Parker R.S. Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status.J. Biol. Chem. 2002; 277: 25290-25296Google Scholar) and found to be 7.4 and 15.6 nmol/mg protein of HDL and LDL, respectively. These preparations were then loaded with [14C]α-tocopherol (Amersham, 55 mCi/mmol) as described above, and an equal amount of total tocopherol (endogenous plus labeled) from each preparation was presented to the cultured cells, as described by Sattler and colleagues (21Goti D. Reicher H. Malle E. Kostner G.M. Panzenboeck U. Sattler W. High-density lipoprotein (HDL3)-associated alpha-tocopherol is taken up by HepG2 cells via the selective uptake pathway and resecreted with endogenously synthesized apo-lipoprotein B-rich lipoprotein particles.Biochem. J. 1998; 332: 57-65Google Scholar).Tocopherol accumulationHepG2-TetOn-TTP cells were seeded into each well in 24-well plates (4 × 105 cells/well), and TTP expression was induced with doxycycline. Serum or lipoprotein complexes of [14C]RRR-α-tocopherol were then added to the culture media for the indicated time periods. At the end of each incubation, media was removed, and the cells were washed with DMEM and lysed in 20 mM HEPES (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% NP40, 20 mM sodium fluoride, 20 mM β-glycerophosphate, 1 mM sodium vanadate, and 200 μM PMSF. After measurement of the radioactivity in a scintillation counter, tocopherol accumulation was calculated as follows:tocopherol accumulation (%)=CPMcellsCPMcells+CPMmedium×100To block uptake through the SR-BI receptor, cells were pretreated with the anti-SR-BI antibody (KKB-1, (22Gu X. Kozarsky K. Krieger M. Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor.J. Biol. Chem. 2000; 275: 29993-30001Google Scholar), generous gift of Karen Kozarsky, 1:200 dilution) for 3 h prior to loading of the cells with [14C]tocopherol-HDL as described above.Tocopherol secretionHepG2-TetOn-TTP cells were grown in 24-well plates, and TTP expression was induced by doxycycline treatment. FBS complexed to [14C]α-tocopherol was then added to the induction media (10% FBS, 0.18 μCi/ml; 3 μM tocopherol) for 36 h (loading period). The cells were then washed three times in complete medium and once in DMEM, and incubated with DMEM for the indicated duration (secretion period). The media was then collected, and the cells were washed twice in DMEM and lysed as described above. After measurement of the radioactivity in a scintillation counter, tocopherol secretion was calculated as follows:tocopherol secretion (%)=CPMmediumCPMcells+CPMmedium×100At the end of a typical secretion experiment, secreted radioactivity was ∼8,400 dpm per well.Fluorescence microscopyCells, cultured on dual-chamber microscope slides (Labtek), were rinsed three times with PBS, fixed in 4% formaldehyde, and permeabilized with 0.2% Triton X-100 before processing with the indicated stains or antibodies. Actin cytoskeleton and cell nuclei were visualized with direct fluorescence using Texas Red phalloidin and To-Pro3, respectively (Molecular Probes, Inc.). TTP protein was visualized using the AT-R1 mouse monoclonal antibody (generous gift of H. Arai, University of Tokyo, Japan) or anti-HA antibodies (monoclonal HA.11, Covance Inc.; or rabbit polyclonal, Santa Cruz, Inc.). Organelle markers were as follows: for endosomes, the early endosome antigen (anti-EEA1; Calbiochem); for lysosomes, lysosome-associated membrane protein (anti-LAMP1; Stressgen, Inc.); and for Golgi and endoplasmic reticulum, mannosidase (anti-mannosidase II; kindly supplied by Dr. K. Moremen, University of Georgia) and calnexin (anti-calnexin; Molecular Probes). Confocal images were collected with the appropriate excitation laser lines on a Leica TCS-SP2 microscope at the Cornell BioResource Center.Fluorescent tocopherolView Large Image Figure ViewerDownload (PPT)Analytical details are provided below for the silyl-protected material.(2R)-((9-(4-nitrobenzo[c][1,2,3]oxadiazole-7-ylamino)nonyl)-3,4-dihydro-2,5,7,8-tetramethylchroman-6-yloxy)(tert-butyl)dimethylsilane.Dark orange oil, Rf = 0.36 (Et2O-Hex, 1:1); 1H NMR (CDCl3) δ 8.46 (d, 1H, J = 9 Hz), 6.18 (br, 1H), 6.14 (d, 1H, J = 9 Hz), 3.45 (q, 2H, J = 6 Hz), 2.53 (t, 2H, J = 7 Hz), 2.07 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.77 (m, 4H), 1.39 (m, 6H), 1.28 (br, 8H), 1.20 (s, 3H), 1.02 (s, 9H), 0.09 (s, 6H); 13C NMR (CDCl3) δ 145.8, 144.2, 144.0, 143.8, 143.8, 136.5, 125.8, 124.0, 123.5, 122.6, 117.4, 98.5, 74.4, 43.9, 39.5, 31.5, 30.0, 29.4, 29.3, 29.1, 28.5, 26.9, 26.0, 23.8, 23.5, 20.8, 18.5, 14.3, 13.4, 11.9, −3.3; MS (EI) m/z 624 (M+). HRMS (EI): calculated for C34H52N4O5Si: 624.37069; found: 624.36959.Uptake of NBD-tocopherolMcA-RH7777-TetOn-TTP cells in two-chamber slides were incubated with 50 μM NBD-tocopherol (complexed to FBS) for 2 h at 4°C, followed by chasing at 37°C with fresh DMEM-10% FBS for the indicated time periods prior to fixing and staining. NBD fluorescence was excited with the 453 nm line from an Ar2+ laser.RESULTSGeneration of hepatocyte cell lines capable of inducible TTP expressionDetailed investigations of the biochemical functions of TTP in cells are hindered by the fact that the protein is not expressed in any known cell lines of hepatic origin. Furthermore, expression of TTP in freshly prepared primary hepatocytes declines precipitously following isolation (24Kim H.S. Arai H. Arita M. Sato Y. Ogihara T. Tamai H. Inoue K. Mino M. Age-related changes of alpha-tocopherol transfer protein expression in rat liver.J. Nutr. Sci. Vitaminol. (Tokyo). 1996; 42: 11-18Google Scholar). To facilitate studies of TTP in a physiologically relevant system, we generated human and rat cell lines that stably express TTP under the regulation of a tetracycline-inducible promoter. Figure 1Ashows that the human hepatoblastoma HepG2-TetOn-TTP and the rat hepatoma McA-RH7777-TetOn-TTP clones that we isolated indeed exhibit strong expression of TTP upon treatment with doxycycline. Importantly, no "leaky" expression of the protein is observed in the absence of induction. To assess the functionality of TTP in these cell lines, we turned to the only known physiological activity of this protein, namely, the facilitation of tocopherol secretion (18Arita M. Nomura K. Arai H. Inoue K. alpha-tocopherol transfer protein stimulates the secretion of alpha-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway.Proc. Natl. Acad. Sci. USA. 1997; 94: 12437-12441Google Scholar). Indeed, upon induction of TTP expression in the HepG2-TetOn-TTP cell line, a pronounced (∼3-fold) increase in secretion of tocopherol from the cells to the media is observed (Fig. 1B). Both the extent and the kinetic characteristics of the observed facilitation are similar to those reported by Arita et al. (18Arita M. Nomura K. Arai H. Inoue K. alpha-tocopherol transfer protein stimulates the secretion of alpha-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway.Proc. Natl. Acad. Sci. USA. 1997; 94: 12437-12441Google Scholar). Essentially identical results were observed in the McA-RH7777-TetOn-TTP cells (data not shown). We conclude that the TTP cell lines we have developed provide a proper experimental system for studying the biochemical functions of TTP under physiologically relevant conditions. Due to their clearer morphology, the McA-RH7777 cells are particularly suitable for microscopy studies. However, because McA-RH7777 cells are difficult to grow for the extended periods required for uptake and secretion assays (3–4 days past confluence), we utilize the HepG2-TetOn-TTP cells for these purposes.Fig. 1Inducible expression of tocopherol transfer protein (TTP) in cultured hepatocytes. A: The indicated cell lines were generated as described in Experimental Procedures and cultured for 48 h in the presence or absence of doxycycline (dox) (1 μg/ml). TTP expression was assayed in lysates after 48 h of induction using anti-HA Western blotting. B: TTP-induced α-tocopherol secretion in HepG2-TetOn-TTP cells. Cells were loaded with [14C]RRR-α-tocopherol for 36 h and washed, and the appearance of radioactivity in the media was assayed at the indicated times by scintillation counting. Shown are averages and standard deviation of quadruplicate wells. Data are representative of 10 independent experiments. When those data are fitted to linear functions (dashed lines), their slopes differ by 3.3-fold.View Large Image Figure ViewerDownload (PPT)Vitamin E accumulation by TTP-expressing cellsWe first investigated the process of vitamin E accumulation, using radioactively labeled [14C]α-RRR-tocopherol as a tracer. To mimic the physiological scenario, tocopherol was delivered to the cells complexed to serum lipoproteins (20Asmis R. Physical partitioning is the main mechanism of alpha-tocopherol and cholesterol transfer between lipoproteins and P388D1 macrophage-like cells.Eur. J. Biochem. 1997; 250: 600-607Google Scholar) rather than from organic solvent stocks (25Nalecz K.A. Nalecz M.J. Azzi A. Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.Eur. J. Biochem. 1992; 209: 37-42Google Scholar) or liposome preparations (18Arita M. Nomura K. Arai H. Inoue K. alpha-tocopherol transfer protein stimulates the secretion of alpha-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway.Proc. Natl. Acad. Sci. USA. 1997; 94: 12437-12441Google Scholar). We found that tocopherol accumulates in the cells in a time- and dose-dependent manner, reaching saturation after ∼36 h (data not shown). In serum, vitamin E is distributed between LDLs and HDLs (26Bjornson L.K. Kayden H.J. Miller E. Moshell A.N. The transport of alpha-tocopherol and beta-carotene in human blood.J. Lipid Res. 1976; 17: 343-352Google Scholar, 27Behrens W.A. Thompson J.N. Madere R. Distribution of alpha-tocopherol in human plasma lipoproteins.Am. J. Clin. Nutr. 1982; 35: 691-696Google Scholar, 28Ogihara T. Miki M. Kitagawa M. Mino M. Distribution of tocopherol among human plasma lipoproteins.Clin. Chim. Acta. 1988; 174: 299-305Google Scholar). To compare the relative contributions of these fractions to vitamin E uptake by hepatocytes, we complexed purified lipoprotein preparations with radiolabeled vitamin E, and monitored its accumulation by the cells in the absence or presence of TTP expression. Figure 2Ashows the time-dependent tocopherol import by HepG2 cells when the vitamin is presented as an LDL or as an HDL complex, and the effect of TTP expression on this process. Although tocopherol is efficiently taken up from both lipoprotein preparations (5–10% of available tocopherol internalized in 4 h), the rate of accumulation from HDL complexes exceeds that from LDL particles by ∼2-fold. This finding is in agreement with the observations of Sattler and colleagues, who reported HDL to be the main delivery route for vitamin E in endothelial cells (29Goti D. Hammer A. Galla H.J. Malle E. Sattler W. Uptake of lipoprotein-associated alpha-tocopherol by primary porcine brain capillary endothelial cells.J. Neurochem. 2000; 74: 1374-1383Google Scholar) and hepatocytes (21Goti D. Reicher H. Malle E. Kostner G.M. Panzenboeck U. Sattler W. High-density lipoprotein (HDL3)-associated alpha-tocopherol is taken up by HepG2 cells via the selective uptake pathway and resecreted with endogenously synthesized apo-lipoprotein B-rich lipoprotein particles.Biochem. J. 1998; 332: 57-65Google Scholar). Further support for this conclusion is the finding that treatment of cells with antibodies directed against HDL's cellular "port of entry," the scavenger receptor class B type I (SR-BI) (30Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science. 1996; 271: 518-520Google Scholar), leads to pronounced inhibition of tocopherol accumulation (Fig. 2B). Importantly, induction of TTP expression does not increase tocopherol accumulation. Rather, expression of TTP leads to a small (10–20%) yet significant reduction in the accumulation of tocopherol from either LDL or HDL (Fig. 2A). This apparent attenuation of tocopherol accumulation is likely to result from TTP's activity in facilitating tocopherol efflux (Fig. 1B). The data indicate that a significant portion of vitamin E accumulation by hepatocytes involves interactions between HDL-bound tocopherol and the SR-BI receptor. We conclude further that TTP does not participate in tocopherol uptake from either HDL or LDL.To better characterize the cellular compartments that are involved in vitamin E uptake, we utilized a novel fluorescent analog of vitamin E, NBD-tocopherol (see Experimental Procedures). The 7-nitrobenz-2-oxa-1,3-diazol-4-yl-substituted tocopherol emits green fluorescence (λmax emission = 530 nm) upon excitation with blue light (λmax excitation = 466 nm). Furthermore, NBD-tocopherol resembles natural vitamin E, in that it binds reversibly and with high affinity to the TTP (Kd = 15 ± 3 nM as compared with 30 ± 4 nM for RRR-α-tocopherol; unpublished observations). We utilized NBD-tocopherol in conjunction with confocal fluorescence microscopy to monitor the temporal and spatial fate of tocopherol upon internalization by cultured hepatocytes. NBD-tocopherol was complexed to serum lipoproteins, and incubated with the McA-RH7777-TetOn-TTP cells on microscope slides at 4°C. To visualize the uptake process, cells were then transferred to 37°C, "chased" with plain media for a predetermined period, prepared for microscopy, and observed under a confocal fluorescence microscope. Figure 3shows a typical time-course, in which fluorescence from NBD-tocopherol and from Texas red-labeled phalloidin (staining cellular actin structures) were imaged after different incubation times. Following a 5 min incubation at 37°C, NBD-tocopherol was observed in bright fluorescent spots, ∼20 nm in diameter, associated with the plasma membrane (Fig. 3). At longer incubation times, NBD fluorescence progressively translocated to the cell interior, reaching a perinuclear compartment after ∼30 min (Fig. 3). The morphologic and kinetic characteristics of the internalization process suggest that the mechanism underlying vitamin E internalization involves the endocytic pathway (31Kim J.H. Lingwood C.A. Williams D.B. Furuya W. Manolson M.F. Grinstein S. Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor.J. Cell Biol. 1996; 134: 1387-1399Google Scholar).Fig. 3Uptake of NBD-tocopherol cultured hepatocytes. McA-RH7777-TetOn-TTP cells cultured on microscope chamber slides were incubated with NBD-tocopherol (as serum complex) for 2 h at 4°C. The cells were then incubated at 37°C for the indicated times before processing for fluorescence microscopy. Red: Texas Red phalloidin (actin stain); green: NBD-tocopherol. Bar = 16 μm.View Large Image Figure ViewerDownload (PPT)Intracellular localization of TTPTo determine the intracellular localization of TTP, we employed confocal fluorescence microscopy utilizing monoclonal antibodies directed against either TTP (AT-R1 antibodies) or the amino-terminal hemaglutinin tag (anti-HA antibodies). As shown in Fig. 4A, B, both antibodies reveal that TTP is expressed in a punctate pattern that resembles vesicular structures surrounding the cell nucleus. To confirm that this intracellular distribution pattern is not an overexpression artifact, we also determined the localization of TTP in freshly isolated primary mouse hepatocytes. As can be seen in Fig. 4C, both the overall intensity and the spatial distribution pattern of endogenous TTP in primary hepatocytes are very similar to the pattern observed in transfected cell lines. To determine the intracellular organelle with which TTP is associated, we used antibody markers for the Golgi compartment (anti-mannosidase II) and the endoplasmic reticulum (anti-calnexin), as well as fluorescent probes for mitochondria (Mitotracker). We found that TTP is not associated with these compartments (data not shown). However, TTP exhibits partial colocalization with EEA1, a marker for the early endosome (data not shown), and significant colocalization with LAMP-1, a resident protein of the lysosome (Fig. 4D, F). The localization pattern of TTP did not change upon treatment of the cells with vitamin E, nor upon depletion of vitamin E by culturing in delipidated serum for 14 days (data not shown). Importantly, the intracellular localization of TTP strongly overlapped with that of NBD-tocopherol 30 min after internalization (Fig. 5). Taken together, these data suggest that vitamin E is internalized into vesicles that end up in the endocytic compartment (endosomes and lysosomes), where TTP is localized.Fig. 4Intracellular localization of TTP. A–C: McA-RH7777-TetOn-TTP cells treated with doxycycline for 48 h (A, B) or freshly isolated mouse hepatocytes (C) were cultured on microscope chamber slides and prepared for microscopy as detailed in Experimental Procedures. TTP was visualized with either anti-HA or anti-TTP antibodies (green), and the actin cytoskeleton was visualized with T
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