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Signal transducer and activator of transcription 3 and sphingomyelin metabolism in intranuclear complex during cell proliferation
ABB Archives of Biochemistry and Biophysics 464 (2007) 138–143 www.elsevier.com/locate/yabbi
Signal transducer and activator of transcription 3 and sphingomyelin metabolism in intranuclear complex during cell proliferation q Graziella Rossi, Mariapia Viola Magni *, Elisabetta Albi Department of Clinical and Experimental Medicine, Physiopathology, University School of Medicine, Perugia, Policlinico Monteluce, via Brunamonti, Italy Received 6 March 2007, and in revised form 6 April 2007 Available online 24 April 2007
Abstract An ‘‘intranuclear complex’’ characterized by the presence of a new synthesised double-strand RNA stabilized by sphingomyelin and cholesterol has been recently isolated from hepatocyte nuclei. In the present research the existence of STAT3 in the intranuclear complex and its behaviour in relation to cyclin D and sphingomyelin metabolism during liver regeneration were studied with the aim to see if the sphingomyelinase could have a role in cell proliferation. Our data demonstrate that the transcription factor is present in the intranuclear complex either as unphosphorylated or phosphorylated monomeric form. After partial hepatectomy, unphosphorylated STAT3 is very low during G1/S transition of the cell cycle and it increases in correspondence of the of S-phase of the cell cycle when cyclin D1 is reduced. The phosphorylated form increases at the beginning of the S-phase when the neutral sphingomyelinase activity is stimulated with consequent enrichment of ceramide pool. In order to see if the two phenomena could be correlated, the intranuclear complex extracted from normal liver was treated with exogenous sphingomyelinase or ceramide and STAT3 was evaluated. The results show an increase of the phosphorylated transcription factor as happens in liver regeneration, suggesting that sphingomyelinase present in the intranuclear complex is responsible for the STAT3 activation during cell proliferation. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Cell proliferation; Intranuclear complex; Sphingomyelin; Sphingomyelinase; STAT3
Signal transducer and activator of transcription-3 (STAT3)1 is a component of the transcription factor family that transduces signals from cell membrane to nucleus. This molecule is present in the cytoplasm as a functionally q
Support for this work by Ministero dell’Istruzione, Universita` e Ricerca (Rome; Center of Excellence ‘‘CEMIN’’, p. CLAB01P7BR) and by Fondazione Cassa di Risparmio di Perugia is gratefully acknowledged. * Corresponding author. Fax: +39 075 5726803. E-mail addresses: [email protected] (M. Viola Magni), [email protected] (E. Albi). 1 Abbreviations used: DAG, diacylglycerol; IC, intranuclear complex; PC, phosphatidylcholine; PL, phospholipid; PMSF, phenylmethylsulfonylfluoride; PPC, phosphorylcholine; N-SMase, neutral-sphingomyelinase; STAT3, signal transducer and activator of transcription 3; SM, sphingomyelin; SM-synthase, sphingomyelin-synthase. 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.04.008
latent monomer. In response to cytokine signals STAT3 is phosphorylated at a single tyrosine (Tyr-705) by a Janus kinase [1]. This process leads to the dimerization of monomers and to the translocation of dimers to the nucleus [2,3]. STAT3 promotes cell cycle progression and cell proliferation under physiological growth conditions [4] and is able to down-regulate the expression of cyclin D [5]. On the other hand cyclin D1 inhibits STAT3 activation [6]. It was demonstrated that STAT3 may be translocated into the nucleus and back in the cytoplasm [7,8]. Recently, it has been described a basal nuclear import pathway [9]. Although the nature of this basal activity has not been well defined, a number of studies has suggested that basal level of STAT3 may serve to regulate caspase, c-myc and FAS expression in unstimulated cells [10,11]. The STAT3 import
G. Rossi et al. / Archives of Biochemistry and Biophysics 464 (2007) 138–143
pathway is active in unstimulated cells and functions independently of tyrosine phosphorylation. This pathway promotes nuclear accumulation of low levels of STAT3 in resting cells [9]. It has been shown that STAT3 phosphorylation can be stimulated by sphingomyelin (SM) hydrolysis via sphingomyelinase (SMase) activity [12]. In these years we have focused the attention on nuclear SM metabolism which changes in response to cell proliferation or apoptosis of rat hepatocytes [13]. The electron microscopy analysis demonstrated that in the rat liver sections the SM is present in nuclear domains active in DNA replication, transcription and probably also in different steps of mRNA processing (data not shown). Recently it has been isolated an ‘‘Intranuclear Complex’’ (I.C.) characterized by the presence of proteins, a small amount of DNA, a double strand RNA and lipids such as SM and cholesterol (CHO). These lipids protect RNA against RNase digestion [14]. In fact, sphingomyelinase treatment reducing the sphingomyelin level and freeing cholesterol facilitates the opening of double strand RNA and permits RNA digestion [14]. The double-strand RNA is a new-synthesised RNA and its amount increases during liver regeneration. The presence of the lamin B as a protein of the I.C. suggested that this complex could correspond to the transcription sites associated to the inner nuclear membrane. Since STAT3, which can be stimulated by SMase [12], is rapidly induced during liver regeneration [5] and the high STAT3 expression is accompanied by low cyclin D expression [6], the aim of the research was to study: (i) the presence of STAT3 in IC and its relation with SMase and (ii) the behaviour of STAT3-cyclin D1-SMase/ceramide in IC during cell proliferation. Materials and methods
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were made according to the International Regulation of National Institutes of Health.
Liver regeneration Partial hepatectomy was performed in four animals at each time and four separated experiments were made (a total of 16 animals for each timing of which four were injected with [3H]thymidine). For the control samples, two sham operated animals were used at each time and four separated experiments were performed (a total of eight animals for each timing of which four were injected with [3H]thymidine). At the different times, regenerating liver was perfused, excised and the nuclei were purified as described below. The nuclei isolated from uninjected animals were used for intranuclear complex extraction whereas those isolated from [3H]thymidine injected animals were used to extract the nucleic acids. The hydrolyzed DNA was used in part for the radioactivity evaluation and in part for amount determination [16]. The synthesis of DNA was evaluated calculating the specific activity expressed as cpm/lg of DNA.
Intranuclear complex extraction The IC was extracted from hepatocyte nuclei isolated and purified according to Rossi et al. [14]. Hepatocyte nuclei were washed in 10 mM Tris, containing 0.25 M sucrose, 2.5 mM MgCl2, 0.5 mM PMSF and 1% Triton X-100, pH 7.5, at 0 °C, in order to remove the external nuclear membrane preserving the inner nuclear membrane, and were centrifuged at 1000g for 15 min at 4 °C. The pellet was re-suspended in 0.1 M Barnes et al. solution [17] at pH 7.4, and was digested according to Herman et al. [18] with DNase I (120 lg/ml) and RNase cocktail (500 lg/ml) for 15 min at 37 °C. The reaction was stopped on ice with cold Barnes et al. solution [17] (1:1 by vol.). After centrifugation at 5000 rpm for 5 min, the pellet was re-suspended in 30 mM Tris containing 0.4 M ammonium sulphate, which facilitates the RNA precipitation, and 1 mM PMSF, pH 8.4, and then was centrifuged at 10,000 rpm for 15 min. The pellet was re-suspended in Barnes et al. solution [17] pH 8.4, added up sodium orthovanadate (2 mg/ ml), sodium fluoride (2 mg/ml), leupeptin (1 lg/ml), pepstatin A (1 lg/ml), put on in the dialysis tube and dialyzed against 150 vol. of 0.1 M Tris– HCl, pH 8.4, in the presence of the same inhibitors overnight with stirring at 4 °C. The suspension dialyzed was recovered and centrifuged at 14,000 rpm for 10 min at 4 °C and the pellet was suspended in 10 mM Tris–HCl, pH 8.4.
Reagents Biochemical determination Chemicals: Bovine serum albumin (BSA), phenylmethylsulfonylfluoride (PMSF), PC, SM, ceramides, sphingomyelinase, non-hydroxy fatty acids ceramide and polyclonal anti-STAT3, and anti-cyclin D1 were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). AntiphosphoSTAT3 was from Calbiochem (La Jolla, CA, USA), SDS–PAGE Molecular Weight Standard (Bio-Rad Laboratories, Hercules, CA, USA). Radioactive SM (choline-methyl [14C], 54.5 Ci/mol), PC (L-3-phosphatidyl N-[3H]methyl-choline-1, 2 dipalmitoyl, 81.0 Ci/mmol) and thymidine (746 Bq/mM) were obtained from Amersham–Pharmacia Biotech (Rainham, Essex, UK); Ecoscint A from National Diagnostic (Atlanta, Georgia, USA).
Protein, DNA and RNA contents were determined as reported by Viola Magni et al. [21]. After lipid extraction, PC and SM were separated by thin layer chromatography and measured evaluating inorganic phosphorous as reported by Albi et al. [13]. The ceramide content was evaluated according to Previati et al. [19], as reported by Albi et al [20].
Sphingomyelinase and sphingomyelin-synthase assay The SMase and SM-synthase activities were detected according to Albi et al. methods [22,23].
Animals
Sphingomyelinase or ceramide treatment
Thirty-day-old Sprague–Dawley rats of either sex (Harlan Nossan, Milan, Italy) kept at normal light–dark periods were used. They had free access to pelleted food and water. Hepatectomy was performed, after anaesthesia, between 8 and 10 a.m. as previously reported [15]. To stimulate liver regeneration, partial hepatectomy corresponding to 75% of rat liver was performed. Sham operated animal were used as controls. Hepatectomised and sham operated animals were killed at 0, 6, 12, 18, 24 and 30 h from operation. For the study of the DNA synthesis, animals were injected with 100 lCi [3H]thymidine 1 h prior to killing. All treatments
The IC was treated with exogenous sphingomyelinase. The reaction mixture contained 0.1 M Tris–HCl, pH 8.4, 6 mM MgCl2, 0.1% Triton X-100, 1.3 U SMase and IC equivalent to 1 mg protein to a final volume of 1 ml. For the ceramide treatment, the reaction mixture contained 0.1 M Tris–HCl, pH 8.4, 0.1% Triton X-100, 0.15 mM ceramide, 80 lg/ml Na taurodeoxycholate and IC equivalent to 1 mg protein to a final volume of 1 ml. The lower concentration of ceramide did not determine any effect. For the control samples, the same mixtures were used without SMase or ceramide. Incubation was performed at 37 °C for 90 min. The suspensions
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were centrifuged at 14,000 rpm for 10 min at 4 °C and the pellets were suspended in 10 mM Tris–HCl, pH 8.4.
Electrophoresis and Western blot analysis About 30 lg of IC proteins were submitted to SDS–PAGE electrophoresis in 12% polyacrylamide slab gel according to Laemmli [24]. The transfer of protein was carried out into nitrocellulose in 75 min according to Towbin et al. [25]. The membranes were blocked for 30 min with 0.5% no fat-dry milk in PBS, pH 7.5, and incubated over night at 4 °C with antibody anti-STAT3 (diluted 1:1000), anti-phosphoSTAT3 (diluted 1:1000), and anti-cyclin D1 (diluted 1:1000). The blots were treated with horseradish-conjugated secondary antibodies for 90 min. Visualization was performed with the enhanced chemiluminescence’s kit from Amersham. The quantification of the immunoblot bands were performed by Scion Image. In the experiments of liver regeneration and of SMase or ceramide treatment, the blot of STAT3 was probed first with anti-STAT3 antibody and then, after stripping, with anti-phosphoSTAT3.
Results Intranuclear complex composition The IC is constituted by proteins, DNA, RNA and lipids as previously reported [14]; the content of ceramide is 59% of that of SM. Also the activities of SMase and SM-synthase are similar to those previously reported [26] (Table 1).
Fig. 1. Presence of STAT3 and cyclin D1 in intranuclear complex. Intranuclear complex was prepared from unstimulated rat hepatocyte nuclei. Immunoblots of proteins (30 lg) were probed with anti-STAT3 (1, apparent molecular weight 90 kDa), anti-phosphoSTAT3 (2, apparent molecular weight 90 kDa), and anti-cyclin D1 (3, apparent molecular weight 36 kDa) antibodies and visualized by ECL (a) The area density was evaluated by densitometry scanning and analysis with Scion Image; the data represent means ± SD of four separated experiments (b). Significance *P < 0.01 versus anti-STAT3.
STAT3 and cyclin D1 in intranuclear complex
Rat liver regeneration
The existence of STAT3 in IC isolated from unstimulated hepatocyte nuclei was evaluated by anti-STAT3 and anti-phosphoSTAT3 following SDS–PAGE. It is evident from Fig. 1a that primary antibody shows a strong immunopositivity at about 90 kDa. An other protein band, corresponding to apparent molecular weight of 200 kDa, is revealed with polyclonal STAT3 antibody, but it represents a non-specific band and its value is 29% of that 90 kDa (Fig. 1b). Therefore only the band corresponding to the apparent molecular weight of STAT3 monomeric form was considered and the results show that it is present in IC either as unphosphorylated or as phosphorylated form. The quantification of the immunoblot bands has demonstrated that the STAT3/phosphoSTAT3 ratio is 1.56 (Fig. 1b). The anti-cyclin D1 antibodies shows the strong immunopositivity at apparent molecular weight of 36 kDa corresponding to cyclin D1 and two very little evident bands corresponding to apparent molecular weight of 19 and 66 kDa that were not considered (Fig. 1a).
The hepatic regeneration was induced as above reported and the animals were killed between 0 and 30 h at regular intervals after operation. The kinetics of the DNA synthesis was studied by [3H]thymidine incorporation. The specific activity of the DNA, calculated as cpm/lg DNA, is very low at 6 and 12 h after partial hepatectomy, it increases strongly at 18 and 24 h corresponding to the S-phase of the cell cycle supporting previously results [27]. The percentage variation of the specific activity in hepatectomised and sham operated animals is represented in the Fig. 2. The IC was isolated from hepatocyte nuclei as above reported and either STAT3 or cyclin D1 were analysed with specific antibodies. The Fig. 3a shows that after hepatectomy the band corresponding to apparent molecular weight 90 kDa, highlighted by anti-STAT3 antibody, is reduced by almost 94% immediately after surgery with respect to that showed in IC isolated from normal rat liver (Fig. 1a) and begins to recover slowly after 6 h. A similar effect is observed in sham operated animals thus indicating
Table 1 Intranuclear complex composition Proteins
DNA
RNA
PC
SM
Ceramide
SMase
SM-synthase
1100.46 ± 98.44
4.20 ± 1.12
18.70 ± 2.03
1.20 ± 0.06
0.99 ± 0.08
0.58 ± 0.10
20.00 ± 3.51
14.87 ± 2.61
The values are expressed in lg/g of liver weight for proteins, DNA, RNA, PC and SM and pmol/mg protein/min for SMase and SM-synthase activity. The data represent the media ± SD of five separated experiments. PC, phosphatidylcholine; SM, sphingomyelin; SMase, sphingomyelinase; SM-synthase, sphingomyelin-synthase.
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Fig. 2. DNA synthesis in the hepatocyte nuclei during rat liver regeneration. The rat liver was stimulated to proliferate by partial hepatectomy corresponding to 75% of rat liver. The rats were killed at regular intervals of time between 0 and 30 h after hepatectomy. The specific activity (cpm/ lg DNA) was evaluated and the percentage variations were calculated taking in account the minimum value at 0 h and the maximum value of [3H]thymidine incorporation at 24 h. The data represent the media ± SD of four separated experiments performed in duplicate. Significance *P < 0.01 versus 0 h.
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strated an increase of 105% at 18 h and 99% at 24 h with respect to 0 h, in correspondence of the S-phase of the cell cycle, whereas the percentage of the increase is between 11 and 34% at the other hours considered (Fig. 3b). The cyclin D1 area quantification has revealed that at only 6 h the immunopositivity increased 80% with respect to the control and the value was similar at 30 h; these steps correspond to the beginning of the G1/S-phase transition of the two mitotic waves (Fig. 3b). During the S-phase of the cell cycle, at 12 and 18 h, the value is increased with respect to 0 h, but it is lower with respect to 6 and 30 h. At 24 h, when the S-phase stopped, the level of cyclin D1 was reduced by 41% with respect to 0 h. No variations of STAT3 and cyclin D1 are observed in sham operated animals. The STAT3 stripped blot was probed by anti-phosphoSTAT3 antibodies. The results show that after hepatectomy the phosphorylated STAT3 increases at the beginning of the S-phase of the cell cycle with respect to 0 h (Fig. 4a). The area quantification reveals that the increase value is 3.98 times and 5.63 times at 12 and 18 h, respectively (Fig. 4b). The IC SMase activity increases at 12 h and forms a peak at 18 h after hepatectomy when the SM-synthase activity is very low. This activity increased strongly at 24 and 30 h when the SMase activity was drastically reduced (Fig. 5a). The ceramide content increases 1.31 times and 1.6 times at 12 and 18 h, respectively, compared to the value obtained at 0 h (Fig. 5b). Sphingomyelinase-ceramide and STAT3 In order to study the relationship between the STAT3, SMase activity and ceramide increase during liver regeneration, the IC isolated from normal liver was treated with exogenous SMase or ceramide. The proteins were analysed first with anti-STAT3 antibodies and then, after
Fig. 3. Levels of STAT3 and cyclin D1 in intranuclear complex during rat liver regeneration. The intranuclear complex was isolated from rat liver stimulated to proliferate by partial hepatectomy. The rats were killed at regular intervals of time between 0 and 30 h after hepatectomy. Immunoblots of proteins (30 lg) were probed by anti-STAT3 (apparent molecular weight 90 kDa) and anti-cyclin D1 (apparent molecular weight 36 kDa) antibodies and visualized by ECL (a) The area density was evaluated by densitometry scanning and analysis with Scion Image; the data represent means ± SD of four separated experiments (b). Significance *P < 0.01 versus 0 h.
that the decrease is not due to hepatectomy but probably it is a consequence of the operation. This effect is evident also for the bands 36 kDa (Fig. 3a) revealed by anti-cyclin D1 which is reduced, immediately after surgery, four times with respect to that present in normal liver (Fig. 1a). The band 90 kDa is strongly evident at 18 and 24 h after hepatectomy whereas the band 36 kDa is strongly coloured at 6 and 30 h. The quantification of STAT3 area has demon-
Fig. 4. Phospho-STAT3 in intranuclear complex during rat liver regeneration. Immunoblots of proteins (30 lg) were probed with anti-phosphoSTAT3 antibodies after stripping of those, reported in the Fig. 3, probed with anti-STAT3 antibodies (a). The area density was evaluated by densitometry scanning and analysis with Scion Image; the data represent means ± SD of four separated experiments (b). Significance *P < 0.01 versus 0 h.
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tein concentration is equal. The bands of the phosphoSTAT3 present in the samples treated with SMase or ceramide are more coloured with respect to the control (Fig. 6a). The Western blot analysis indicated that SMase and ceramide treatment results in 2.8- and 1.73fold increase of phospho-STAT3, respectively. Discussion
Fig. 5. Sphingomyelinase (SMase) and sphingomyelins-synthase (SMsynthase) activities (a) and ceramide content (b) in intranuclear complex during rat liver regeneration. The values of the enzymatic activities are expressed as pmol/mg protein/min and represent the median and the range of three independent experiments. The values of the ceramide content are expressed as lg/g liver and represent the media ± SD of four separated experiments performed in duplicate. Significance *P < 0.01 versus 0 h.
Fig. 6. Modifications of STAT3 in intranuclear complex after sphingomyelinase or ceramide treatment. Intranuclear complex prepared from unstimulated rat hepatocyte nuclei was treated with exogenous sphingomyelinase or ceramide as reported in experimental procedures and the immunoblots of proteins (30 lg) was probed first with anti-STAT3 and then, after stripping, with anti-phosphoSTAT3 antibodies (a). AntiphosphoSTAT3 area was evaluated by densitometry scanning and analysis with Scion Image; the values represent the media ± SD of four separated experiments (b). Significance *P < 0.01 versus control. 1, control sample; 2, SMase treated sample; 3, ceramide treated sample.
stripping, with anti-phosphoSTAT3 antibodies. The similarity of the three bands of the STAT3 in the control, SMase and ceramide sample demonstrates that the pro-
It has been recently demonstrated by confocal microscopy that after IL-6 stimulation of HepG2 cells STAT3 protein transiently accumulates in intranuclear bodies and this accumulation is correlated with tyrosine phosphorylation [28]. We demonstrate for the first time by biochemical techniques that STAT3 is present in an IC, containing the new-synthesised RNA, which has been recently isolated from hepatocyte nuclei. This RNA has a double-strand structure maintained by SM and cholesterol (CHO) and it is associated to the proteins and a small amount of DNA [14]. Since IC contains lamin B, it has been supposed that it could be a transcription site associated to the inner nuclear membrane. The demonstration of the presence of STAT3 in IC supports this observation. STAT3 is activated in a wide variety of signalling systems and mediates a bewildering complexity of responses [29]. It has been recently shown that tumours with high STAT3 expression have low cyclin D1 expression [30]. Matsui et al. [5] report that cyclin D1 gene is an important target of STAT3 in hepatocytes and that this relation depends on the cell stage; in fact, STAT3 acts as a negative regulator of cyclin D1 transcription during foetal liver development, whereas it positively regulates cyclin D1 expression in hepatoma cells and at the initial of liver regeneration. Our results demonstrate a co-existence of STAT3 and cyclin D1 in IC. During rat liver regeneration, in the IC cyclin D1 increases during the G1/S-phase transition of the cell cycle when STAT3 is very low. It is possible that the increase of STAT3 at 18 and 24 h, during the S-phase of the cell cycle, is responsible for the down-regulation of cyclin D1. At the beginning of the S-phase of the cell cycle, when the major molecular events of the cell proliferation start, the phosphorylated STAT3 present in IC increases in parallel with the increase of SMase activity and production of ceramide. The relationship between these modifications has been demonstrated by SMase and ceramide treatment in vitro. The reason of the lower increase of phosphoSTAT3 obtained by ceramide incubation with respect to that obtained with SMase treatment is not clear at the moment. It is possible that endogenous and exogenous ceramide have the different effects on STAT3 phosphorylation. Moreover, since it is difficult to think that SMase and/or ceramide act directly on this process, it can hypothesise that other proteins are present in IC and that they are activated during the incubation time of the reaction.
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It can be concluded that IC is an active chromatin complex present in the nucleus in which an interaction between the sphingomyelin/ceramide pathway and STAT3-Cyclin D1 occurs in vivo to regulate cell proliferation. Acknowledgments This work is partially supported by MIUR (PRIN2006). We thank R. Lazzarini for the technical assistance and S. Pagnotta for figure preparation. References [1] D. Guschin et al., EMBO J. 14 (1995) 1421–1429. [2] T. Kisimoto, T. Taga, S. Akira, Cell 76 (1994) 235–262. [3] M.A. Wishingrad, S. Koshlukova, S.W. Halvorsen, J. Biol. Chem. 272 (1997) 19752–19757. [4] W. Li, X. Liang, C. Kellendonk, V. Poli, R. Taub, J. Biol. Chem. 277 (2002) 28411–28417. [5] T. Matsui, T. Kinoshita, T. Hirano, T. Yokota, A. Miyajima, J. Biol. Chem. 277 (2002) 36167–36173. [6] F. Bienvenu, H. Gascan, O. Coqueret, J. Biol. Chem. 276 (2001) 16840–16847. [7] I. Strehlow, C. Schindler, J. Biol. Chem. 273 (1998) 28049–28056. [8] K.M. McBride, C. McDonald, N.C. Reich, EMBO J. 19 (2000) 6196– 6206. [9] S. Bhattacharya, C. Schindler, J. Clin. Invest. 111 (2003) 553–559. [10] C.V. Ramana, N. Grammatikakis, M. Chernov, H. Nguyen, K.C. Goh, B.R. Williams, G.R. Stark, EMBO J. 19 (2000) 263–272. [11] A. Kumar, M. Commane, T.W. Flicknger, C.M. Horvarth, G.R. Stark, Science 278 (1997) 1630–1632. [12] C. Maziere, M.A. Conte, J.C. Maziere, FEBS Lett. 507 (2001) 163–168.
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Signal transducer and activator of transcription 3 and sphingomyelin metabolism in intranuclear complex during cell proliferation q Graziella Rossi, Mariapia Viola Magni *, Elisabetta Albi Department of Clinical and Experimental Medicine, Physiopathology, University School of Medicine, Perugia, Policlinico Monteluce, via Brunamonti, Italy Received 6 March 2007, and in revised form 6 April 2007 Available online 24 April 2007
Abstract An ‘‘intranuclear complex’’ characterized by the presence of a new synthesised double-strand RNA stabilized by sphingomyelin and cholesterol has been recently isolated from hepatocyte nuclei. In the present research the existence of STAT3 in the intranuclear complex and its behaviour in relation to cyclin D and sphingomyelin metabolism during liver regeneration were studied with the aim to see if the sphingomyelinase could have a role in cell proliferation. Our data demonstrate that the transcription factor is present in the intranuclear complex either as unphosphorylated or phosphorylated monomeric form. After partial hepatectomy, unphosphorylated STAT3 is very low during G1/S transition of the cell cycle and it increases in correspondence of the of S-phase of the cell cycle when cyclin D1 is reduced. The phosphorylated form increases at the beginning of the S-phase when the neutral sphingomyelinase activity is stimulated with consequent enrichment of ceramide pool. In order to see if the two phenomena could be correlated, the intranuclear complex extracted from normal liver was treated with exogenous sphingomyelinase or ceramide and STAT3 was evaluated. The results show an increase of the phosphorylated transcription factor as happens in liver regeneration, suggesting that sphingomyelinase present in the intranuclear complex is responsible for the STAT3 activation during cell proliferation. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Cell proliferation; Intranuclear complex; Sphingomyelin; Sphingomyelinase; STAT3
Signal transducer and activator of transcription-3 (STAT3)1 is a component of the transcription factor family that transduces signals from cell membrane to nucleus. This molecule is present in the cytoplasm as a functionally q
Support for this work by Ministero dell’Istruzione, Universita` e Ricerca (Rome; Center of Excellence ‘‘CEMIN’’, p. CLAB01P7BR) and by Fondazione Cassa di Risparmio di Perugia is gratefully acknowledged. * Corresponding author. Fax: +39 075 5726803. E-mail addresses: [email protected] (M. Viola Magni), [email protected] (E. Albi). 1 Abbreviations used: DAG, diacylglycerol; IC, intranuclear complex; PC, phosphatidylcholine; PL, phospholipid; PMSF, phenylmethylsulfonylfluoride; PPC, phosphorylcholine; N-SMase, neutral-sphingomyelinase; STAT3, signal transducer and activator of transcription 3; SM, sphingomyelin; SM-synthase, sphingomyelin-synthase. 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.04.008
latent monomer. In response to cytokine signals STAT3 is phosphorylated at a single tyrosine (Tyr-705) by a Janus kinase [1]. This process leads to the dimerization of monomers and to the translocation of dimers to the nucleus [2,3]. STAT3 promotes cell cycle progression and cell proliferation under physiological growth conditions [4] and is able to down-regulate the expression of cyclin D [5]. On the other hand cyclin D1 inhibits STAT3 activation [6]. It was demonstrated that STAT3 may be translocated into the nucleus and back in the cytoplasm [7,8]. Recently, it has been described a basal nuclear import pathway [9]. Although the nature of this basal activity has not been well defined, a number of studies has suggested that basal level of STAT3 may serve to regulate caspase, c-myc and FAS expression in unstimulated cells [10,11]. The STAT3 import
G. Rossi et al. / Archives of Biochemistry and Biophysics 464 (2007) 138–143
pathway is active in unstimulated cells and functions independently of tyrosine phosphorylation. This pathway promotes nuclear accumulation of low levels of STAT3 in resting cells [9]. It has been shown that STAT3 phosphorylation can be stimulated by sphingomyelin (SM) hydrolysis via sphingomyelinase (SMase) activity [12]. In these years we have focused the attention on nuclear SM metabolism which changes in response to cell proliferation or apoptosis of rat hepatocytes [13]. The electron microscopy analysis demonstrated that in the rat liver sections the SM is present in nuclear domains active in DNA replication, transcription and probably also in different steps of mRNA processing (data not shown). Recently it has been isolated an ‘‘Intranuclear Complex’’ (I.C.) characterized by the presence of proteins, a small amount of DNA, a double strand RNA and lipids such as SM and cholesterol (CHO). These lipids protect RNA against RNase digestion [14]. In fact, sphingomyelinase treatment reducing the sphingomyelin level and freeing cholesterol facilitates the opening of double strand RNA and permits RNA digestion [14]. The double-strand RNA is a new-synthesised RNA and its amount increases during liver regeneration. The presence of the lamin B as a protein of the I.C. suggested that this complex could correspond to the transcription sites associated to the inner nuclear membrane. Since STAT3, which can be stimulated by SMase [12], is rapidly induced during liver regeneration [5] and the high STAT3 expression is accompanied by low cyclin D expression [6], the aim of the research was to study: (i) the presence of STAT3 in IC and its relation with SMase and (ii) the behaviour of STAT3-cyclin D1-SMase/ceramide in IC during cell proliferation. Materials and methods
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were made according to the International Regulation of National Institutes of Health.
Liver regeneration Partial hepatectomy was performed in four animals at each time and four separated experiments were made (a total of 16 animals for each timing of which four were injected with [3H]thymidine). For the control samples, two sham operated animals were used at each time and four separated experiments were performed (a total of eight animals for each timing of which four were injected with [3H]thymidine). At the different times, regenerating liver was perfused, excised and the nuclei were purified as described below. The nuclei isolated from uninjected animals were used for intranuclear complex extraction whereas those isolated from [3H]thymidine injected animals were used to extract the nucleic acids. The hydrolyzed DNA was used in part for the radioactivity evaluation and in part for amount determination [16]. The synthesis of DNA was evaluated calculating the specific activity expressed as cpm/lg of DNA.
Intranuclear complex extraction The IC was extracted from hepatocyte nuclei isolated and purified according to Rossi et al. [14]. Hepatocyte nuclei were washed in 10 mM Tris, containing 0.25 M sucrose, 2.5 mM MgCl2, 0.5 mM PMSF and 1% Triton X-100, pH 7.5, at 0 °C, in order to remove the external nuclear membrane preserving the inner nuclear membrane, and were centrifuged at 1000g for 15 min at 4 °C. The pellet was re-suspended in 0.1 M Barnes et al. solution [17] at pH 7.4, and was digested according to Herman et al. [18] with DNase I (120 lg/ml) and RNase cocktail (500 lg/ml) for 15 min at 37 °C. The reaction was stopped on ice with cold Barnes et al. solution [17] (1:1 by vol.). After centrifugation at 5000 rpm for 5 min, the pellet was re-suspended in 30 mM Tris containing 0.4 M ammonium sulphate, which facilitates the RNA precipitation, and 1 mM PMSF, pH 8.4, and then was centrifuged at 10,000 rpm for 15 min. The pellet was re-suspended in Barnes et al. solution [17] pH 8.4, added up sodium orthovanadate (2 mg/ ml), sodium fluoride (2 mg/ml), leupeptin (1 lg/ml), pepstatin A (1 lg/ml), put on in the dialysis tube and dialyzed against 150 vol. of 0.1 M Tris– HCl, pH 8.4, in the presence of the same inhibitors overnight with stirring at 4 °C. The suspension dialyzed was recovered and centrifuged at 14,000 rpm for 10 min at 4 °C and the pellet was suspended in 10 mM Tris–HCl, pH 8.4.
Reagents Biochemical determination Chemicals: Bovine serum albumin (BSA), phenylmethylsulfonylfluoride (PMSF), PC, SM, ceramides, sphingomyelinase, non-hydroxy fatty acids ceramide and polyclonal anti-STAT3, and anti-cyclin D1 were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). AntiphosphoSTAT3 was from Calbiochem (La Jolla, CA, USA), SDS–PAGE Molecular Weight Standard (Bio-Rad Laboratories, Hercules, CA, USA). Radioactive SM (choline-methyl [14C], 54.5 Ci/mol), PC (L-3-phosphatidyl N-[3H]methyl-choline-1, 2 dipalmitoyl, 81.0 Ci/mmol) and thymidine (746 Bq/mM) were obtained from Amersham–Pharmacia Biotech (Rainham, Essex, UK); Ecoscint A from National Diagnostic (Atlanta, Georgia, USA).
Protein, DNA and RNA contents were determined as reported by Viola Magni et al. [21]. After lipid extraction, PC and SM were separated by thin layer chromatography and measured evaluating inorganic phosphorous as reported by Albi et al. [13]. The ceramide content was evaluated according to Previati et al. [19], as reported by Albi et al [20].
Sphingomyelinase and sphingomyelin-synthase assay The SMase and SM-synthase activities were detected according to Albi et al. methods [22,23].
Animals
Sphingomyelinase or ceramide treatment
Thirty-day-old Sprague–Dawley rats of either sex (Harlan Nossan, Milan, Italy) kept at normal light–dark periods were used. They had free access to pelleted food and water. Hepatectomy was performed, after anaesthesia, between 8 and 10 a.m. as previously reported [15]. To stimulate liver regeneration, partial hepatectomy corresponding to 75% of rat liver was performed. Sham operated animal were used as controls. Hepatectomised and sham operated animals were killed at 0, 6, 12, 18, 24 and 30 h from operation. For the study of the DNA synthesis, animals were injected with 100 lCi [3H]thymidine 1 h prior to killing. All treatments
The IC was treated with exogenous sphingomyelinase. The reaction mixture contained 0.1 M Tris–HCl, pH 8.4, 6 mM MgCl2, 0.1% Triton X-100, 1.3 U SMase and IC equivalent to 1 mg protein to a final volume of 1 ml. For the ceramide treatment, the reaction mixture contained 0.1 M Tris–HCl, pH 8.4, 0.1% Triton X-100, 0.15 mM ceramide, 80 lg/ml Na taurodeoxycholate and IC equivalent to 1 mg protein to a final volume of 1 ml. The lower concentration of ceramide did not determine any effect. For the control samples, the same mixtures were used without SMase or ceramide. Incubation was performed at 37 °C for 90 min. The suspensions
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were centrifuged at 14,000 rpm for 10 min at 4 °C and the pellets were suspended in 10 mM Tris–HCl, pH 8.4.
Electrophoresis and Western blot analysis About 30 lg of IC proteins were submitted to SDS–PAGE electrophoresis in 12% polyacrylamide slab gel according to Laemmli [24]. The transfer of protein was carried out into nitrocellulose in 75 min according to Towbin et al. [25]. The membranes were blocked for 30 min with 0.5% no fat-dry milk in PBS, pH 7.5, and incubated over night at 4 °C with antibody anti-STAT3 (diluted 1:1000), anti-phosphoSTAT3 (diluted 1:1000), and anti-cyclin D1 (diluted 1:1000). The blots were treated with horseradish-conjugated secondary antibodies for 90 min. Visualization was performed with the enhanced chemiluminescence’s kit from Amersham. The quantification of the immunoblot bands were performed by Scion Image. In the experiments of liver regeneration and of SMase or ceramide treatment, the blot of STAT3 was probed first with anti-STAT3 antibody and then, after stripping, with anti-phosphoSTAT3.
Results Intranuclear complex composition The IC is constituted by proteins, DNA, RNA and lipids as previously reported [14]; the content of ceramide is 59% of that of SM. Also the activities of SMase and SM-synthase are similar to those previously reported [26] (Table 1).
Fig. 1. Presence of STAT3 and cyclin D1 in intranuclear complex. Intranuclear complex was prepared from unstimulated rat hepatocyte nuclei. Immunoblots of proteins (30 lg) were probed with anti-STAT3 (1, apparent molecular weight 90 kDa), anti-phosphoSTAT3 (2, apparent molecular weight 90 kDa), and anti-cyclin D1 (3, apparent molecular weight 36 kDa) antibodies and visualized by ECL (a) The area density was evaluated by densitometry scanning and analysis with Scion Image; the data represent means ± SD of four separated experiments (b). Significance *P < 0.01 versus anti-STAT3.
STAT3 and cyclin D1 in intranuclear complex
Rat liver regeneration
The existence of STAT3 in IC isolated from unstimulated hepatocyte nuclei was evaluated by anti-STAT3 and anti-phosphoSTAT3 following SDS–PAGE. It is evident from Fig. 1a that primary antibody shows a strong immunopositivity at about 90 kDa. An other protein band, corresponding to apparent molecular weight of 200 kDa, is revealed with polyclonal STAT3 antibody, but it represents a non-specific band and its value is 29% of that 90 kDa (Fig. 1b). Therefore only the band corresponding to the apparent molecular weight of STAT3 monomeric form was considered and the results show that it is present in IC either as unphosphorylated or as phosphorylated form. The quantification of the immunoblot bands has demonstrated that the STAT3/phosphoSTAT3 ratio is 1.56 (Fig. 1b). The anti-cyclin D1 antibodies shows the strong immunopositivity at apparent molecular weight of 36 kDa corresponding to cyclin D1 and two very little evident bands corresponding to apparent molecular weight of 19 and 66 kDa that were not considered (Fig. 1a).
The hepatic regeneration was induced as above reported and the animals were killed between 0 and 30 h at regular intervals after operation. The kinetics of the DNA synthesis was studied by [3H]thymidine incorporation. The specific activity of the DNA, calculated as cpm/lg DNA, is very low at 6 and 12 h after partial hepatectomy, it increases strongly at 18 and 24 h corresponding to the S-phase of the cell cycle supporting previously results [27]. The percentage variation of the specific activity in hepatectomised and sham operated animals is represented in the Fig. 2. The IC was isolated from hepatocyte nuclei as above reported and either STAT3 or cyclin D1 were analysed with specific antibodies. The Fig. 3a shows that after hepatectomy the band corresponding to apparent molecular weight 90 kDa, highlighted by anti-STAT3 antibody, is reduced by almost 94% immediately after surgery with respect to that showed in IC isolated from normal rat liver (Fig. 1a) and begins to recover slowly after 6 h. A similar effect is observed in sham operated animals thus indicating
Table 1 Intranuclear complex composition Proteins
DNA
RNA
PC
SM
Ceramide
SMase
SM-synthase
1100.46 ± 98.44
4.20 ± 1.12
18.70 ± 2.03
1.20 ± 0.06
0.99 ± 0.08
0.58 ± 0.10
20.00 ± 3.51
14.87 ± 2.61
The values are expressed in lg/g of liver weight for proteins, DNA, RNA, PC and SM and pmol/mg protein/min for SMase and SM-synthase activity. The data represent the media ± SD of five separated experiments. PC, phosphatidylcholine; SM, sphingomyelin; SMase, sphingomyelinase; SM-synthase, sphingomyelin-synthase.
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Fig. 2. DNA synthesis in the hepatocyte nuclei during rat liver regeneration. The rat liver was stimulated to proliferate by partial hepatectomy corresponding to 75% of rat liver. The rats were killed at regular intervals of time between 0 and 30 h after hepatectomy. The specific activity (cpm/ lg DNA) was evaluated and the percentage variations were calculated taking in account the minimum value at 0 h and the maximum value of [3H]thymidine incorporation at 24 h. The data represent the media ± SD of four separated experiments performed in duplicate. Significance *P < 0.01 versus 0 h.
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strated an increase of 105% at 18 h and 99% at 24 h with respect to 0 h, in correspondence of the S-phase of the cell cycle, whereas the percentage of the increase is between 11 and 34% at the other hours considered (Fig. 3b). The cyclin D1 area quantification has revealed that at only 6 h the immunopositivity increased 80% with respect to the control and the value was similar at 30 h; these steps correspond to the beginning of the G1/S-phase transition of the two mitotic waves (Fig. 3b). During the S-phase of the cell cycle, at 12 and 18 h, the value is increased with respect to 0 h, but it is lower with respect to 6 and 30 h. At 24 h, when the S-phase stopped, the level of cyclin D1 was reduced by 41% with respect to 0 h. No variations of STAT3 and cyclin D1 are observed in sham operated animals. The STAT3 stripped blot was probed by anti-phosphoSTAT3 antibodies. The results show that after hepatectomy the phosphorylated STAT3 increases at the beginning of the S-phase of the cell cycle with respect to 0 h (Fig. 4a). The area quantification reveals that the increase value is 3.98 times and 5.63 times at 12 and 18 h, respectively (Fig. 4b). The IC SMase activity increases at 12 h and forms a peak at 18 h after hepatectomy when the SM-synthase activity is very low. This activity increased strongly at 24 and 30 h when the SMase activity was drastically reduced (Fig. 5a). The ceramide content increases 1.31 times and 1.6 times at 12 and 18 h, respectively, compared to the value obtained at 0 h (Fig. 5b). Sphingomyelinase-ceramide and STAT3 In order to study the relationship between the STAT3, SMase activity and ceramide increase during liver regeneration, the IC isolated from normal liver was treated with exogenous SMase or ceramide. The proteins were analysed first with anti-STAT3 antibodies and then, after
Fig. 3. Levels of STAT3 and cyclin D1 in intranuclear complex during rat liver regeneration. The intranuclear complex was isolated from rat liver stimulated to proliferate by partial hepatectomy. The rats were killed at regular intervals of time between 0 and 30 h after hepatectomy. Immunoblots of proteins (30 lg) were probed by anti-STAT3 (apparent molecular weight 90 kDa) and anti-cyclin D1 (apparent molecular weight 36 kDa) antibodies and visualized by ECL (a) The area density was evaluated by densitometry scanning and analysis with Scion Image; the data represent means ± SD of four separated experiments (b). Significance *P < 0.01 versus 0 h.
that the decrease is not due to hepatectomy but probably it is a consequence of the operation. This effect is evident also for the bands 36 kDa (Fig. 3a) revealed by anti-cyclin D1 which is reduced, immediately after surgery, four times with respect to that present in normal liver (Fig. 1a). The band 90 kDa is strongly evident at 18 and 24 h after hepatectomy whereas the band 36 kDa is strongly coloured at 6 and 30 h. The quantification of STAT3 area has demon-
Fig. 4. Phospho-STAT3 in intranuclear complex during rat liver regeneration. Immunoblots of proteins (30 lg) were probed with anti-phosphoSTAT3 antibodies after stripping of those, reported in the Fig. 3, probed with anti-STAT3 antibodies (a). The area density was evaluated by densitometry scanning and analysis with Scion Image; the data represent means ± SD of four separated experiments (b). Significance *P < 0.01 versus 0 h.
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tein concentration is equal. The bands of the phosphoSTAT3 present in the samples treated with SMase or ceramide are more coloured with respect to the control (Fig. 6a). The Western blot analysis indicated that SMase and ceramide treatment results in 2.8- and 1.73fold increase of phospho-STAT3, respectively. Discussion
Fig. 5. Sphingomyelinase (SMase) and sphingomyelins-synthase (SMsynthase) activities (a) and ceramide content (b) in intranuclear complex during rat liver regeneration. The values of the enzymatic activities are expressed as pmol/mg protein/min and represent the median and the range of three independent experiments. The values of the ceramide content are expressed as lg/g liver and represent the media ± SD of four separated experiments performed in duplicate. Significance *P < 0.01 versus 0 h.
Fig. 6. Modifications of STAT3 in intranuclear complex after sphingomyelinase or ceramide treatment. Intranuclear complex prepared from unstimulated rat hepatocyte nuclei was treated with exogenous sphingomyelinase or ceramide as reported in experimental procedures and the immunoblots of proteins (30 lg) was probed first with anti-STAT3 and then, after stripping, with anti-phosphoSTAT3 antibodies (a). AntiphosphoSTAT3 area was evaluated by densitometry scanning and analysis with Scion Image; the values represent the media ± SD of four separated experiments (b). Significance *P < 0.01 versus control. 1, control sample; 2, SMase treated sample; 3, ceramide treated sample.
stripping, with anti-phosphoSTAT3 antibodies. The similarity of the three bands of the STAT3 in the control, SMase and ceramide sample demonstrates that the pro-
It has been recently demonstrated by confocal microscopy that after IL-6 stimulation of HepG2 cells STAT3 protein transiently accumulates in intranuclear bodies and this accumulation is correlated with tyrosine phosphorylation [28]. We demonstrate for the first time by biochemical techniques that STAT3 is present in an IC, containing the new-synthesised RNA, which has been recently isolated from hepatocyte nuclei. This RNA has a double-strand structure maintained by SM and cholesterol (CHO) and it is associated to the proteins and a small amount of DNA [14]. Since IC contains lamin B, it has been supposed that it could be a transcription site associated to the inner nuclear membrane. The demonstration of the presence of STAT3 in IC supports this observation. STAT3 is activated in a wide variety of signalling systems and mediates a bewildering complexity of responses [29]. It has been recently shown that tumours with high STAT3 expression have low cyclin D1 expression [30]. Matsui et al. [5] report that cyclin D1 gene is an important target of STAT3 in hepatocytes and that this relation depends on the cell stage; in fact, STAT3 acts as a negative regulator of cyclin D1 transcription during foetal liver development, whereas it positively regulates cyclin D1 expression in hepatoma cells and at the initial of liver regeneration. Our results demonstrate a co-existence of STAT3 and cyclin D1 in IC. During rat liver regeneration, in the IC cyclin D1 increases during the G1/S-phase transition of the cell cycle when STAT3 is very low. It is possible that the increase of STAT3 at 18 and 24 h, during the S-phase of the cell cycle, is responsible for the down-regulation of cyclin D1. At the beginning of the S-phase of the cell cycle, when the major molecular events of the cell proliferation start, the phosphorylated STAT3 present in IC increases in parallel with the increase of SMase activity and production of ceramide. The relationship between these modifications has been demonstrated by SMase and ceramide treatment in vitro. The reason of the lower increase of phosphoSTAT3 obtained by ceramide incubation with respect to that obtained with SMase treatment is not clear at the moment. It is possible that endogenous and exogenous ceramide have the different effects on STAT3 phosphorylation. Moreover, since it is difficult to think that SMase and/or ceramide act directly on this process, it can hypothesise that other proteins are present in IC and that they are activated during the incubation time of the reaction.
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