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Vol. 19, Issue 12, 5289-5295, December 2008
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*Department of Clinical and Experimental Medicine, Physiopathology Section, University School of Medicine, University of Perugia, Policlinico Monteluce, 06100 Perugia, Italy; and Dipartimento di Scienze Biopatologiche ed Igiene delle Produzioni Animali e Alimentari, University of Perugia, 06126 Perugia Italy
Submitted May 23, 2008;
Revised September 24, 2008;
Accepted October 2, 2008
Monitoring Editor: Karsten Weis
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) that can exist in equilibrium (Brown, 2006). The most studied class of microdomain are the cholesterol (CHO) and sphingomyelin (SM)-enriched lipid rafts (Edidin, 2003). However, the distinct concepts of lipid rafts, detergent-resistant membranes and liquid-ordered lipid phases are often confused in the current literature (Lichtenberg et al., 2005). Rafts are described as transient detergent-resistant membrane microdomains enriched in sphingolipids and CHO, whereas "detergent-resistant membranes" are formed after detergent treatment and do not correspond to any membrane structure. In contrast, "liquid-ordered lipid phase domains" are the result of interactions between high levels of CHO and phospholipid fatty acyl chains (Lichtenberg et al., 2005). On the other hand, several lines of investigation have supported the idea that detergent-resistant membranes are not detergent-induced artifacts, but do exist as domains in cellular membranes (Brown and London, 1997). As visualization of rafts in living cells is difficult, their existence and function rely on indirect methods such as detergent extraction (Munro, 2003). However, Schuck et al. (2003) have found that various detergents differ considerably in their ability to selectively solubilize membrane proteins and enrich the content of sphingolipids and CHO. Insolubility in detergents like Triton X-100 was observed in lipid bilayers, which exist in physical states where lipid packing is tight (London and Brown, 2000). However, Braccia et al. (2003) have demonstrated that isolation of lipid rafts from microvillar membrane with "conventional" Triton X-100 at low temperature and with Brij 98 at 37°C have essentially similar profiles of protein and lipid components, suggesting that they are not "low temperature artifacts" but do exist at physiological temperature.
Membrane microdomains are thought to act as platforms for specific proteins (Edidin, 2003; Kenworthy et al., 2004) and to have different functions for protein and lipid sorting (Ikoen, 2001) in the regulation of cell signaling (Simons and Toomre, 2000). Today, it is widely recognized that lipids are new intranuclear components (Tamiya-Koizumi et al., 1989; Martelli et al., 2004; Albi and Viola Magni, 2004; Ledeen and Wu, 2004, 2006). However, these molecules are quickly metabolized in relation to the cellular function due to the activity of neutral-sphingomyelinase (N-SMase; Tamiya-Koizumi et al., 1989; Alessenko and Chatterjee, 1995; Romanenko et al., 1998; Tsugane et al., 1999; Neitcheva and Peeva, 2001; Mizutani et al., 2001; Albi and Viola Magni, 2004; Watanabe et al., 2004), sphingomyelin-synthase (SM-synthase; Kavok et al., 2003; Albi and Viola Magni, 2004), phosphatidylcholine-specific phospholipase C (PC-PLC; Albi and Viola Magni, 2004; Ramoni et al., 2004) and reverse sphingomyelin-synthase (RSM-synthase; Albi and Viola Magni, 2003a), enzymes responsible for SM and phosphatidylcholine (PC) metabolism. Recently, it has been demonstrated that SM/PC metabolism cross-talk occurs in the nucleus, regulating the intranuclear pool of ceramide/diacylglycerol (Albi et al., 2008). Nevertheless, these nuclear lipids affect cellular functions not only as intranuclear signaling molecules but also, by modifying the subnuclear structures (Albi and Viola Magni, 2006a). In fact, during cellular proliferation induced by partial hepatectomy, the activation of SMase changes SM and CHO levels, therefore modifying the fluidity of the nuclear membrane and consequently regulating the nucleus-cytoplasm efflux of mRNA (Albi and Viola Magni, 2006a). Because nuclear-cytoplasmic exchange is a critical cellular process for eukaryotes, this highlights the importance of these lipids (Terry et al., 2007). Moreover, in the same experimental conditions, modification of the CHO-SM/PC ratio during the S phase of the cell cycle causes decreased fluidity of the nuclear matrix, suggesting that it could represent a structure involved in DNA duplication (Albi et al., 2003b). In chromatin, two pools of CHO exist: an "SM-free CHO" pool that does not change during cellular proliferation and another "SM-linked CHO" pool that can change during the S-phase of the cell cycle in relation to N-SMase activation (Albi and Viola Magni, 2002).
Recently, the association between PC-SM-CHO and transcription process was demonstrated (Rossi et al., 2007a,b). During liver regeneration, an intranuclear complex was isolated in which N-SMase activity was responsible for STAT3 activation (Rossi et al., 2007b). Moreover, newly synthesized RNA was protected from RNase digestion through acquiring a double-strand structure that was stabilized by SM and CHO (Rossi et al., 2007a). As this intranuclear complex contains lamin B, it was suggested to correspond to transcription sites associated with the inner nuclear membrane (Rossi et al., 2007b), recently described as a transcription factor resting place (Heessen and Fornerod, 2007). Together, these observations led to the hypothesis that the nuclear lipids SM-CHO and PC are organized in microdomains that could regulate nuclear function.
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MATERIALS AND METHODS |
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Animals and Treatments
Sixty-day-old Sprague Dawley female rats (Harlan Nossan, Milan, Italy), kept at normal light-dark periods, were used. They had free access to pelleted food and water. [3H]uridine incorporation was studied in liver regeneration induced by partial hepatectomy corresponding to 75% of total liver, which stimulates liver cells to proliferate. Hepatectomy was performed between 8 and 10 a.m. as previously reported (Albi et al., 2002). Sham-operated animals were used as controls. Animals were injected intraperitoneally with 100 µCi [3H]uridine 23 h after operation, for 1 h and then killed. All the treatments were performed according to the international regulation of National Institutes of Health.
Preparation of Homogenate and Isolation of Nuclei and Subnuclear Fractions
The liver was homogenized in 10 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM EDTA, 0.1% ethanol, 0.1 M PMSF, and 0.2 M dithiothreitol using the Thomas homogenizer; the homogenate was filtered through two layers of surgical gauze. The hepatocyte nuclei, chromatin, nuclear membranes, and nuclear matrix were prepared as previously described (Albi et al., 2003b).
Purification of Triton X-100–insoluble Microdomains from Isolated Hepatocyte Nuclei
For the isolation of nuclear microdomains, hepatocyte nuclei were washed twice with Barnes solution (Barnes et al. 1957; 0.085 M KCl, 0.0085 M NaCl, 0.0025 M MgCl2, 0.005 M trichloroacetic acid-HCl, 0.1 M PMSF, pH 7.2) as previously reported (Albi et al., 2003b). This treatment, during which the nuclei are collected at 2000 x g avoids the presence of mitocondria and microsomes, which require a higher gravity value for sedimentation. The absence of cytoplasmic contamination was shown by electrophoretic analysis of RNA extracted from this preparation as previously reported (Albi et al., 2006a). The microdomains were prepared from normal and hepatectomized rats according to Braccia et al. (2003) with the following modifications: the extraction was carried out with Triton X-100 dissolved in distilled water (10% vol/vol), on ice. This solution was added to the purified nuclei to a final detergent concentration of 1% (vol/vol). The extract was placed in a cushion of 80% sucrose with a gradient of 15–40% sucrose on top. After centrifugation overnight, the gradients were collected in 12 1-ml fractions for subsequent analysis by SDS-PAGE and Western blotting. In other experiments floating fractions, corresponding to 3 ml, were carefully collected with a pipette, diluted five times with 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, and centrifuged at 100,000 x g for 120 min to obtain a pellet of microdomains for biochemical and electron microscopy analysis.
To test the purity of nuclear microdomain preparations, the activity of microsomal and/or mitocondria markers such as G6P and NADH-cytochrome C-reductase, respectively, was performed as previously reported (Albi et al., 2006b). Moreover, the presence of giantin as marker protein for Golgi membrane was evaluated by immunoblotting analysis to probe for contamination of Golgi apparatus (Satoh et al., 2005).
Electron Microscopy
Pellets of rat hepatocyte nuclei and nuclear microdomains were fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 2 h at 4°C according to Braccia et al. (2003). After washing in 0.1 M sodium phosphate buffer, the samples were treated with osmium tetroxide in 0.1 M sodium phosphate buffer for 1 h at 4°C, dehydrated in graded concentration of acetone, and finally embedded in Epon. Ultrathin sections were cut on a Reichert-Jung Ultracut E (ultramicrotome), stained in 1% uranyl acetate in water and lead citrate, and finally examined in Philips EM208 electron microscope (Electronic Instruments, Mahwah, NJ) equipped with a camera system at constant temperature of 18°C and 60 KW high tension.
Biochemical Analysis
Protein and RNA content were determined as previously described (Rossi et al., 2007a). [3H]uridine incorporation in homogenate, nuclei, and nuclear microdomains isolated from either regenerating liver of hepatectomized animals or normal liver of sham-operated animals was evaluated in counting vials containing 2 ml of Ecoscint A and 0.2 ml distilled water. Radioactivity was measured with a Packard liquid scintillation analyzer. Phospholipids (PLs) and CHO content, N-SMase, SM-synthase, PC-PLC (Albi et al., 2003) and RSM-synthase (Albi et al., 2003a) activity were evaluated as previously described.
Electrophoresis and Western Blot Analysis
Thirty micrograms of protein from homogenate, nuclei, Barnes washing nuclei, and microdomains were submitted to SDS-PAGE electrophoresis in 8% polyacrylamide slab gel according to Laemmli (1970). For the electrophoresis image analysis, the gel was stained by Coomassie blue. The transfer of protein was carried out into nitrocellulose for 90 min according to Towbin et al. (1979). The membranes were blocked for 30 min with 5% nonfat dry milk in PBS, pH 7.5, and incubated over night at 4°C with mAb anti-lamin B (diluted 1:1000), polyclonal antibody anti-STAT3 (diluted 1:1000), or polyclonal antibody anti-giantin (diluted 1: 5000). The blots were treated with horseradish-conjugated secondary antibodies for 90 min (diluted 1:100,000). Visualization was performed with the enhanced chemiluminescence kit from Amersham.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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16% of that present in homogenate (Table 1). After Barnes treatment, the activity of G6P decreased to 4%, whereas NADH cytocrome C reductase was undetectable. No activity of either enzyme was detected in the nuclear microdomains (Table 1), indicating the absence of cytoplasmic contamination. These data are strongly supported by immunoblot analysis with giantin antibodies, a marker protein for Golgi membrane. The results showed that the band for giantin corresponding to apparent molecular weight of 367 kDa was very high in homogenate, but was reduced in the nuclei and absent in nuclei after Barnes treatment and in the microdomains (Figure 1).
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17.00 mg/g liver and 3.0 mg/g liver in both the homogenate and the nuclei (Table 2). Further analysis in different subnuclear fractions showed that 27% of the total nuclear proteins was localized in the nuclear membrane, 44% in the chromatin, 22% in the nuclear matrix, and only 0.09% in the nuclear microdomains. The PL content was 0.45 mg/g liver in the homogenate and 0.12 mg/g liver in the nuclei, of which 79% belongs to nuclear membranes, 8% to chromatin, 8% to nuclear matrix, and 2.5% to nuclear microdomains. The ratio PL to protein of the nuclear microdomains was very similar to that of nuclear membranes, suggesting that it could correspond to a specific section. To verify this, analysis of lipid composition was carried out by TLC. Nuclear microdomains had a specific ratio among CHO, PC, and SM that equals to 1:1:1 (Figure 2), characteristic of the lipid microdomains enriched in CHO and SM content (Kenworthy et al., 2004). Therefore, the lipid composition of the microdomains was different from that of the nuclear membranes where the CHO, PC, and SM ratio was 1:1.5:0.6. Similar values for SM and CHO but higher for PC were present in either chromatin or the nuclear matrix (Figure 2).
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) as probes for nuclear microdomains as they are not present in the nuclei. Therefore, lamin B was used as specific marker of nuclear membrane and STAT3, the nuclear protein involved in the transcription process, was used to highlight a nuclear microdomain marker. The specific distribution in the density gradient of the two proteins described above was revealed by Western blotting. As shown in Figure 4, top, the band of lamin B, corresponding to 68 kDa apparent molecular weight, appeared in the fractions 1 to 7. Lamim B was also partially present in nuclear microdomains, indicating that these domains are specific parts of nuclear membrane. Differently, STAT3, corresponding to 92 kDa apparent molecular weight, was exclusively present in the raft fractions (Figure 4, bottom), confirming STAT3 being a nuclear microdomain marker.
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). Importantly, PC-PLC activity was absent (Figure 5), whereas, in addition, SM-synthase activity was 41% lower with respect to the intact nuclei. In contrast, nuclear microdomain N-SMase and RSM-synthase activities were 1.9 and 63 times higher of those present in the whole nuclei. N-SMase and RSM-synthase activity in nuclear microdomains was also significantly higher than that present in the chromatin and nuclear matrix. Moreover, SM-synthase activity in nuclear microdomains was lower than that present in the chromatin but higher than that present in nuclear matrix. In comparison to the whole nuclear membrane, N-SMase and SM-synthase activities were lower in nuclear microdomains, whereas RSM-synthase activity was higher.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Sun et al., 2007; Manfiolli et al., 2008). Braccia et al. (2003) showed that cold Triton X-100 extraction results in the isolation of microdomains rich in lipid content. This detergent preserves vesicle-like structures whereas extraction with Brij 98 at 37°C largely breaks up microvillar vesicles into open membrane fragments. Therefore, we have isolated the lipid microdomains from highly purified hepatocyte nuclei with Triton X-100 at low temperature. Our results show that 1) microdomains isolated from nuclei are characterized by specific lipid composition; 2) lipid composition of nuclear microdomains changes during cellular proliferation in regenerating liver; and 3) nuclear microdomains are enriched in labeled uridine when RNA synthesis increases.
The possibility that the isolated microdomains are artifacts due to detergent treatment was reported in the literature (Munro 2003; Schuck et al., 2003). Nevertheless, we obtained by Triton X-100 treatment, a sample with properties similar to other microdomains isolated from cells (Braccia et al., 2003) but corresponding in composition to the purified intranuclear complex previously reported (Rossi et al., 2007a,b). In fact, we have previously demonstrated that it is possible to isolate from the nucleus a complex that is associated to inner nuclear membrane and constituted by proteins, PC-SM-CHO, enzymes for lipid metabolism and newly synthesized RNA (Rossi et al., 2007a,b). In the present work, we obtained through biochemical isolation of microdomains, a sample that presents similar biochemical characteristics of intranuclear complex and morphological properties of lipid microdomains.
The lipid composition is characterized by an exact ratio of PC:SM:CHO maintained by N-SMase, SM-synthase, and RSM-synthase activities: N-SMase degrades SM to form phosphocholine and ceramide; SM-synthase resynthesizes SM using ceramide and phosphocholine derived from PC freeing diacylglycerol and finally, RSM-synthase synthesizes PC using diacylglycerol and phosphocholine derived from SM producing ceramide. Therefore, these enzymes regulate the balance of PC and SM levels. This becomes very important considering the dynamic of nuclear membrane in interphase and mitosis cells (Ellenberg et al., 1997).
As a strong relationship between structure and function exists in the nuclear membrane (Jacobson et al., 1984) and as microdomains are proposed to act as platforms for protein segregation and cell signaling (Simons and Ikonen, 1997; Simons and Toomre, 2000; Hancock, 2003; Gómez-Moutón et al., 2004), we have studied nuclear lipid microdomains during cell proliferation. Our data have demonstrated that, during liver regeneration, nuclear microdomains are characterized by an increased SM content due to higher activity of SM-synthase. Because recent studies provide evidence that the presence of SM and CHO in rafts leads to strongly packed and rigid bilayers (Niemelä et al., 2007), this possibly results in a more rigid structure that could act as an anchor for RNA synthesis, as demonstrated by labeled uridine incorporation during the S phase of the cell cycle. This observation induces to think over the nuclear plasticity in relation to cell function as indicated for nuclear bodies during mitosis (Chen et al., 2008). On the other hand, electron microscopy analysis on the rat liver sections performed with a complex of N-SMase conjugated to colloidal gold particles showed that SM is present in nuclear domains active in DNA replication, transcription, and possibly in different steps of mRNA processing as well (unpublished data). Therefore, on the basis of our current data it is supposed that the nuclear lipid microdomains act as platform for the transcription process.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Elisabetta Albi (ealbi{at}unipg.it)
Abbreviations used: N-SMase, neutral-sphingomyelinase; PC, phosphatidylcholine; PC-PLC, phosphatidylcholine-specific phospholipase C; PPC, phosphocholine; SM, sphingomyelin; SM-synthase, sphingomyelin-synthase; RSM-synthase, reverse sphingomyelin-synthase.
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