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1 Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
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Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8577, Japan
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Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, UK
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Abstract |
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Introduction |
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ATF-2 can form either homodimers or heterodimers with other transcription factors including c-Jun (Benbrook & Jones 1990; Ivashkiv et al. 1990; Hai & Curran 1991). A number of target genes of ATF-2 and ATF-2/c-Jun heterodimer have been reported to date, and the signaling to activate ATF-2 may affect the selection of target genes by ATF-2. For instance, a group of target genes, including Gadd45
, whose induction causes apoptosis, is induced by ATF-2 in response to hypoxic stress (Maekawa et al., personal communication). On the other hand, serum stimulation leads to the induction of ATF-3 gene transcription via activation of ATF-2 by Ras effector pathways (Tamura et al. 2005). Thus, to understand the physiologic role of ATF-2, it is important how it is activated by specific signaling pathway.
Scaffolding proteins play an important role to determine the specificity of the signaling pathway (Morrison & Davis 2003). The JNK interacting proteins (JIPs) are typical scaffold proteins that interact with components of the JNK and p38 mitogen-activated protein kinase (MAPK) signaling modules, whereas other proteins, including ß-arrestin-2 and filamin, also act as scaffold proteins for components of the SAPK pathway. JIP1 and JIP2 can bind JNK, MKK7 (MAP kinase kinase 7), and members of the mixed-lineage kinase (MLK) group of MAP3K (Whitmarsh et al. 1998; Yasuda et al. 1999). The sites of interaction with JNK, MKK7, and MLK protein kinases correspond to separate sites on JIP1 (Whitmarsh et al. 1998). Thus, although JIP proteins possess no enzymatic activity, they potentiate the activation of JNK and p38 (Whitmarsh et al. 1998; Ito et al. 1999; Yasuda et al. 1999; Kelkar et al. 2000; Lee et al. 2002). However, all of these scaffold proteins are localized in the cytoplasm, and appear not to contribute to select specific transcription factor as a nuclear target of SAPK signaling pathway.
The fission yeast Sin1 protein interacts with the Sty1/Spc1 MAPK (a SAPK ortholog) and is required for stress-dependent phosphorylation of and transcription by Atf1, the homolog of human ATF-2 (Wilkinson et al. 1999). These observations suggest a possibility that vertebrate Sin1 could have a significant role to activate ATF-2 in intracellular signaling through interactions with SAPKs.
Here we report that human Sin1 binds to both ATF-2 and p38, and stimulates the p38-induced phosphorylation of ATF-2. Thus, Sin1 appears to function as a nuclear scaffold protein to connect p38 and ATF-2.
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Results |
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To examine whether Sin1 directly binds to ATF-2, we performed GST (glutathione S-transferase) pull-down assays using GST-Sin1, which contained the full-length Sin1, and in vitro translated ATF-2 (Fig. 1). ATF-2 efficiently bound to GST-Sin1. The results obtained using various fragments of in vitro translated ATF-2 indicate that CT91, which lacks the C-terminal 91 amino acids but still retains the b-ZIP region bound to GST-Sin1, whereas CT164, which lacks the C-terminal 164 amino acids and does not retain the bZIP region did not bind to GST-Sin1. In addition, NT341, the N-terminally truncated form of ATF-2, which lacks the N-terminal 341 amino acids bound to GST-Sin1. Furthermore, 
BR, which lacks the basic amino acid cluster of the b-ZIP region, and L34V, in which the third and fourth leucines in the leucine zipper were replaced by valines, did not bind to GST-Sin1. These results indicate that Sin1 binds to the bZIP region of the protein surface of the ATF-2 dimer, which is formed via the leucine zipper. The constitutively active form of ATF-2, in which Thr-69 and Thr-71 are replaced by Glu and Asp (T69E/T71D), bound to Sin1 with an efficiency similar to that of wild-type ATF-2, suggesting that the phosphorylation of ATF-2 by SAPKs does not affect the affinity to Sin1.
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Binds to the C-terminal region of Sin1
To examine whether Sin1 directly binds to p38, we performed GST pull-down assays using GST-Sin1, which contains the full-length Sin1, and HA-tagged p38 expressed in 293T cells (Fig. 3). 293T cells were transfected with the HA-p38 expression plasmid, and whole-cell lysates were prepared. The GST-Sin1 resin was mixed with the transfected cell lysates, and bound HA-p38 was detected by Western blotting. p38 bound to GST-Sin1, but only weakly interacted with GST-Sin1ß, the alternatively spliced form. The Sin1 mutant lacking the C-terminal 55 amino acids failed to bind p38, whereas the C-terminal 55 amino acid-fragment alone was able to bind p38. Thus, p38 binds to the C-terminal region of Sin1. The internal deletion of a small portion of Sin1ß may affect protein conformation, leading to decreased affinity to p38.
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in vivo
To confirm the in vivo interaction between Sin1 and ATF-2, co-immunoprecipitation assays of over-expressed proteins were performed. 293T cells were transfected with plasmids to express FLAG-tagged Sin1 and HA-tagged ATF-2, and the cell lysates were immunoprecipitated with anti-HA antibodies or control IgG. The immunocomplexes obtained using anti-HA contained FLAG-Sin1, but those obtained using control IgG did not (Fig. 4A). To confirm the in vivo interaction of Sin1 with p38 or JNK1, similar co-immunoprecipitation assays were performed. 293T cells were transfected with the FLAG-Sin1 expression plasmid and the HA-p38 or HA-JNK1 expression plasmid, and the cell lysates were used for co-immunoprecipitation. Anti-HA co-precipitated FLAG-Sin1 with HA-p38 or with HA-JNK1, but control IgG did not (Fig. 4B,C). We also co-immunoprecipitated endogenous Sin1 and ATF-2. Anti-Sin1 serum co-immunoprecipitated endogenous ATF-2 in cell lysates prepared from 293T cells, whereas control pre-immune serum did not (Fig. 4D). Thus, Sin1 binds to both ATF-2 and p38/JNK1.
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expression plasmids (Fig. 4E, right). Thus, all of three proteins, p38, Sin1, and ATF-2, were localized primarily in the nucleus when they were over-expressed in 293T cells. Sin1 enhances osmotic stress-induced phosphorylation and the trans-activating capacity of ATF-2
We then asked whether Sin1 enhances the p38-induced phosphorylation of ATF-2. We analyzed the effect of Sin1 over-expression on the osmotic stress-induced phosphorylation of ATF-2. 293T Cells were transfected with the HA-ATF-2 expression plasmid together, with or without, the FLAG-Sin1 expression plasmid. The transfected cells were then treated with 0.5 M sorbitol, and Thr-60/Thr-71 phosphorylated ATF-2 was detected using the phospho-ATF-2-specific antibody. In the absence of Sin1 co-expression, phosphorylated ATF-2 was detected within 15 min after sorbitol addition, its levels were further increased at 30 min, and decreased thereafter (Fig. 5A, left). In contrast, the amount of ATF-2 was essentially unaffected by osmotic stress, although it slightly increased at 15 min after sorbitol addition, and then decreased. When Sin1 was co-expressed, the levels of phospho-ATF-2 increased by 15 min after sorbitol addition, and remained elevated up to 2 h (Fig. 5A, right). The amount of ATF-2 was essentially unaffected by osmotic stress in the presence of over-expressed Sin1. Thus, Sin1 strongly enhances the osmotic stress-induced phosphorylation of ATF-2.
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alone, or both p38 and the active form of MKK6 (MKK6D/D), an upstream constitutively activator of p38, were co-expressed with ATF-2, the ATF-2-dependent luciferase expression from the CRE-containing promoter was stimulated approximately 1.6-fold or twofold, respectively. When all three components, Sin1, p38, and MKK6DD, were co-expressed with ATF-2, the luciferase expression level from the CRE-containing promoter was enhanced approximately fourfold. Sin1 over-expression enhanced the basal activity of CRE-containing promoter approximately fourfold, suggesting that Sin1 stimulates endogenous ATF-2-dependent transcription. Thus, Sin1 enhances the trans-activating capacity of ATF-2. A decrease in Sin1 inhibits osmotic stress-induced phosphorylation of ATF-2 and trans-activation by ATF-2
We next asked whether a decrease in Sin1 levels affects the stress-induced phosphorylation of and trans-activation by ATF-2. 293T cells were transfected with an siRNA against Sin1 mRNA, and the Sin1 protein levels were examined by Western blotting. Sin1 siRNA decreased the levels of both wild-type Sin1 and the alternative spliced form Sin1ß (Fig. 6A). When 293T cells transfected with control siRNA were treated with 0.5 M sorbitol, Thr-69/Thr-71 phosphorylated ATF-2 was detected within 15 min of sorbitol addition, and its levels further increased by 30 min (Fig. 6B). However, when 293T cells were transfected with Sin1 siRNA, the levels of phosphorylated ATF-2 were low, although the levels of total ATF-2 protein were not affected by Sin1 siRNA.
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alone, or in the presence of both p38 and MKK6D/D. In the presence of over-expressed p38 and MKK6D/D, phosphorylation of ATF-2 and ATF-2-induced transcription may occur efficiently without Sin1. A decrease in Sin1 inhibits osmotic stress-induced apoptosis
We also investigated the effect of Sin1 siRNA on osmotic stress-induced apoptosis. When 293T cells were not treated with osmotic stress, the fraction of apoptotic cells that were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), was only 4% of the total population (Fig. 7A). When cells were treated with 0.5 M sorbitol, 31% of the cells were apoptotic. Transfection of cells with Sin1 siRNA dramatically decreased the number of sorbitol-induced apoptotic cells to 6%, whereas Sin1 siRNA did not affect the number of apoptotic cells in the absence of osmotic stress. Thus, a decrease in Sin1 strongly inhibits osmotic stress-induced apoptosis.
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Recently, we performed a microarray analysis to identify ATF-2 target genes using RNA prepared from 293T cells treated with ATF-2 siRNA or control siRNA, and one of the ATF-2 target genes we identified was Gadd45ß (unpublished data), which has been reported to control SAPK-mediated apoptosis (Yoo et al. 2003; Gupta et al. 2005). Therefore we investigated the effect of Sin1 and ATF-2 siRNA on osmotic stress-induced expression of Gadd45ß in 293T cells. Treatment of 293T cells with 0.5 M sorbitol resulted in a 75-fold induction of Gadd45ß mRNA (Fig. 7C). When cells were transfected with Sin1 siRNA, the degree of Gadd45ß mRNA induction by sorbitol treatment was reduced to 25-fold. A similar reduction was observed with ATF-2 siRNA treatment. Thus, Sin1 is important for ATF-2-dependent induction of Gadd45ß transcription in response to osmotic stress.
Sin1 does not enhance the serum-induced phosphorylation of ATF-2 by ERK
Serum stimulation can lead to phosphorylation of ATF-2 at Thr-71 via the Raf-MEK-ERK pathway, followed by subsequent phosphorylation at Thr-69 via the Ral-RalGDS-Src-p38 pathway (Ouwens et al. 2002). We examined whether Sin1 is involved in this serum-induced phosphorylation of ATF-2. When 293T cells transfected with control siRNA were treated with 20% serum, Thr-69/Thr-71 phosphorylated ATF-2 was detected within 15 min after serum stimulation, and its levels remained elevated at 30 min (Fig. 8). However, transfection of 293T cells with Sin1 siRNA did not affect the Thr-69/Thr-71 phosphorylation of ATF-2 induced by serum stimulation (Fig. 8). These results suggest that Sin1 is not involved in the ERK-mediated phosphorylation of ATF-2.
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To examine the tissue distribution of Sin1 expression, we performed Northern blot analyses. The mouse Sin1 mRNA is expressed in most tissues examined and two transcripts of 3.1 and 2.5 kb were detected. Expression of Sin1 mRNA is relatively high in the testis, kidney, and liver (Fig. 9A). Sin1 expression in the kidney was confirmed by immunostaining using the anti-Sin1 polyclonal antibody. The Sin1 signals were detected specifically in the renal tubule cells (Fig. 9B). Furthermore, the Sin1 signals were also detected in the cerebellar Purkinje cells and in some hepatocytes.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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and is required for p38-induced phosphorylation of ATF-2 in response to osmotic stress (Fig. 10). This is consistent with previous report that yeast Sin1 binds to Sty1, a SAPK ortholog, and is necessary for the stress-induced phosphorylation of the transcription factor Atf1, a homolog of human ATF-2. In mammalian cells, both ATF-2 and Sin1 are localized in the nucleus (Fig. 4E). Binding of Sin1 to both ATF-2 and p38 suggests that Sin1 functions as a scaffold to stimulate the phosphorylation of ATF-2 by p38. Thus far, a number of scaffold proteins have been identified and characterized, and Sin1 may function as a nuclear scaffold protein. A decrease in Sin1 levels using siRNA drastically reduced osmotic stress-induced apoptosis and ATF-2-dependent induction of Gadd45ß gene transcription (Fig. 7), indicating that Sin1 plays an important role in the SAPK signaling pathway (Fig. 10).
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We have observed that ATF-2 is localized in the nucleus of all cells examined, including 293T cells (Fig. 4E), and also in various types of cells in the brain (Takeda et al. 1991). p38 was previously demonstrated to be in both the nucleus and cytoplasm of NIH3T3 cells and rat cardiomyocytes (Seternes et al. 2002; Lu et al. 2006). We observed that p38 is localized primarily in the nucleus of 293T cells, although minor signals were detected in the cytoplasm. The ratio of nuclear and cytosolic p38 may differ depending on the cell type or culture condition. Similarly, we observed that Sin1 is primarily in the nucleus of 293T cells (Fig. 4E), although Schroder et al. (2005) reported that Sin1 is found primarily in the cytoplasm of the Burkitt's lymphoma cell line DG75. Thus, the subcellular localization of Sin1 may also vary depending on the cell type. However, at least in 293T cells, all three proteins, p38, Sin1, and ATF-2, are mainly found in the nucleus, and Sin1 may act as a nuclear scaffold protein by binding to both p38 and ATF-2. p38 binds to the C-terminal 55 amino acids (Fig. 3), whereas ATF-2 binds to both the N- and C-terminal regions of Sin1. The structure of the p38- and ATF-2-interacting regions in Sin1 is unknown at present because no significant homology with other proteins has been observed. Further analysis to identify the minimal Sin1 elements required for interactions with ATF-2, p38, and other signaling molecules is necessary to understand the molecular mechanism of Sin1 action.
We have found that Sin1 is highly expressed in mouse renal tubule cells (Fig. 9B). Because the kidney produces urine of highly variable osmolarity depending on the hydration status of the individual, renal tubular epithelial cells are exposed to highly variable concentrations of extracellular NaCl, organic osmolytes, and urea. Therefore, high levels of Sin1 expression may play a role in activating the p38-induced phosphorylation of ATF-2 in renal tubular cells under conditions of high osmolarity. We have found that ATF-2 induces transcription not only of apoptosis-related genes, but also of various heat shock proteins (unpublished data). These heat shock proteins may be required for renal tubular cell tolerance of high osmotic stress. In addition to the renal tubule cells, we also observed that Sin1 was highly expressed in the cerebellar Purkinje cells and in some hepatocytes. Understanding the role of Sin1 in these tissues may reveal the roles of p38-ATF-2 signaling pathway in these tissues.
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Experimental procedures |
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Human Sin1 and Sin1ß cDNA clones (Schroder et al. 2004) were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using RNA prepared from HEK293 cells. To express Sin1 in mammalian cells, Sin1 cDNA was inserted downstream of the chicken cytoplasmic ß-actin promoter, with or without an N-terminal FLAG tag (pact-FLAG-Sin1 and pact-Sin1). The plasmids used to express the GST-Sin1 fusion proteins containing various fragments of Sin1 were constructed using appropriate enzyme sites or by the PCR-based method with pGEX vectors (Amersham Pharmacia Biotechnologies). For in vitro translation of ATF-2, plasmids encoding a series of mutants of ATF-2 were constructed using the pSPUTK vector (Stratagene). The ATF-2 expression plasmids that were used in this study have been previously described (Sano et al. 1999). The p38 and JNK1 expression plasmids containing the SR promoter and the MKK6D/D expression vector, in which the CMV promoter is linked to the cDNA encoding a constitutively active form of MKK6, were obtained from Dr T. Sudo.
In vitro binding assays
GST pull-down assays were performed as previously described (Nomura et al. 2004), except for the 20 µg of GST fusion protein per assay used. To avoid nonspecific interactions with the DNA, the resin with bound GST fusion proteins was washed with phosphate-buffered saline (PBS) containing 0.7 M NaCl and PBS containing 0.1% Triton X-100 to remove bacterial DNA. Buffer containing 20 mM HEPES, 75 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1.25 mM MgCl2, 1 mM dithiothreitol, and 0.05% NP-40 was used for binding assays with GST-Sin1 and in vitro-translated ATF-2.
To investigate in vitro binding between GST-Sin1 and p38, p38 was expressed in 293T cells. 293T cells (1 x 106 cells per 10-cm dish) were transfected with 6 µg of the HA-tagged p38 expression plasmid, and approximately 48 h after transfection, whole cell lysates were prepared using lysis buffer containing 20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10% glycerol, and a protease inhibitor cocktail. The concentration of Triton X-100 in the lysates was decreased to 0.1% by diluting with lysis buffer lacking Triton X-100, and the lysates were then mixed with GST-Sin1.
Co-immunoprecipitation
To investigate the in vivo interaction of over-expressed proteins, 293T cells were transfected using Lipofectamine 2000 (Invitrogen) with the FLAG-tagged Sin1 expression plasmid (pact-FLAG-Sin1, 3 µg) and either the HA-tagged ATF-2 expression plasmid (pact-HA-ATF2, 3 µg), the HA-tagged p38 expression plasmid (pSR-HA-p38, 3 µg) or the HA-tagged JNK1 expression plasmid (pSR-HA-JNK1, 3 µg). Forty hours after transfection, the cell lysates were prepared with lysis buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, and a protease inhibitor cocktail (for interactions between Sin1 and ATF-2 or JNK), or lysis buffer containing 50 mM HEPES, pH 7.5, 250 mM NaCl, 0.2 mM EDTA, 10 µM NaF, 0.5% NP-40 and a protease inhibitor cocktail (for interactions between Sin1 and p38). Anti-HA (12CA5, Roche Diagnostics, Indianapolis, IN) or nomal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were used for immunoprecipitaion. The immunocomplexes were washed 3 times with lysis buffer and separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and analyzed by Western blotting with an anti-FLAG M2 antibody (Sigma).
Co-immunoprecipitation of endogenous ATF-2 and Sin1 was performed as follows. The anti-Sin1-specific rabbit polyclonal antibody was generated using a Pseudomonas exotoxin-Sin1 fusion protein that contains full-length human Sin1, as the antigen, and the antibody was then purified using protein G-sepharose. 293T cell lysates were prepared with lysis buffer containing 50 mM HEPES, pH 7.5, 200 mM NaCl, 0.2 mM EDTA, 10 µM NaF, 0.1% NP-40, 10% glycerol, and a protease inhibitor cocktail. The concentrations of NaCl and NP-40 were decreased to 100 mM and 0.05% by adding an equal volume of lysis buffer lacking NaCl and NP-40. Immunoprecipitation was performed using the anti-Sin1 serum or control pre-immune serum. The immunocomplexes were washed 5 times with buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 10 µM NaF, and 0.05% NP-40 and then separated on 10% SDS-PAGE gels and analyzed by Western blotting with an anti-ATF-2 monoclonal antibody (F2BR-1, Santa Cruz Biotechnology, Santa Cruz, CA).
Subcellular localization
293T cells were transfected with a mixture of the FLAG-tagged Sin1 expression plasmid pact-FLAG-Sin1 (0.5 µg) and the HA-tagged ATF-2 expression plasmid pact-HA-ATF-2 (0.5 µg), or the HA-tagged p38 expression plasmid SR-HA-p38 (0.5 µg), using Lipofectamine 2000. Forty hours after transfection, cells were fixed on cover glasses with 2% paraformaldehyde/PBS for 45 min at room temperature and permeabilized by 0.2% Triton X-100/PBS for 12 min. After blocking with 3% skim milk/PBS, cells were immunostained with anti-FLAG M2 and anti-HA antibodies (3F10, Roche) followed by the appropriate secondary antibodies conjugated to Alexa488 or TRITC. Cells were counterstained for 10 min with 1 µM TOPRO3 (Molecular Probes, Inc.) and analyzed by laser scanning confocal microscopy (Zeiss LSM510).
Detection of phosphorylated ATF-2
To examine the phosphorylation of endogenous ATF-2 protein, 293T cells were serum-starved and incubated with sorbitol (0.5 M) or 20% FBS for the indicated times. The cells were then lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 50 mM NaF, 2 mM Na3VO4, 25 mM ß-glycerophosphate, and protease inhibitors). After centrifugation, the supernatant was analyzed by 10% SDS-PAGE, followed by Western blotting. The phosphorylation of ATF-2 was examined using a phospho-ATF-2 (Thr69/71) antibody (Cell Signaling). ATF-2 and -tubulin used as loading control were detected using an anti-ATF-2 polyclonal antibody (C-19, Santa Cruz Biotech.) and an anti--tubulin monoclonal antibody (SIGMA), respectively. To analyze the phosphorylated state of ATF-2 expressed from the transfected DNA, 293T cells were transfected using Lipofectamine 2000 with a mixture of 1.3 µg of the HA-ATF-2 expression plasmid and 1.3 µg of the FLAG-Sin1 expression plasmid, or the control vector expressing no protein, and 0.4 µg of the internal control plasmid pRL-TK (Promega). Approximately 40 h after transfection, cell lysates were prepared, and the phosphorylated state of ATF-2 was examined as described in previous discussions. Aliquots of the cells were used to determine the transfection efficiency by measuring luciferase activity and the amounts of the lysates used for Western blotting were normalized to the luciferase activity.
Luciferase reporter assay
Using LipofectAMINE PLUS (Invitrogen), 293T cells (1 x 105 cells per six-well dish) were transfected with a mixture containing 0.16 µg of the CRE-containing luciferase reporter plasmid, 0.3 µg of the ATF-2 expression plasmid, 0.15 µg of the p38 expression plasmid, 0.15 µg of the MKK6D/D expression plasmid, 0.04 µg of the internal control plasmid pRL-TK, various amounts (0.1, 0.2, or 0.4 µg) of the Sin1 expression plasmid. The total amount of DNA was adjusted to 1.2 µg by adding empty control vector DNA. Approximately 44 h after transfection, lysates were prepared and luciferase activity was measured using the dual luciferase assay system (PG-DUAL-SP, TOYO Ink).
RNAi
The sense-strand sequences used for each siRNA (Qiagen) are as follows (the siRNA consists of a complementary oligonucleotide): Sin1 siRNA 5'-GAUUAGAACGACUCCGAAAdTdT-3': ATF-2 siRNA 5'-AAUGAAGUGGCACAGCUGAdTdT-3'. siRNA specific for green fluorescent protein and luciferase were used as controls. Oligonucleotide siRNAs (100 nM) were transfected using the Oligofectamine (Invitrogen) reagent according to the manufacturer's instructions. At the indicated intervals following transfection, cell lysates were assayed for gene-silencing effects by Western blotting.
Measurement of apoptosis
293T cells were transfected with siRNA twice, 48 h apart, serum starved, and incubated with sorbitol (0.5 M) for 24 h. The cells were washed twice with PBS, resuspended in 200 µL PBS, and then 3 mL of 1% paraformaldehyde solution was added dropwise for fixation. The fixation was allowed to continue for 20 min on ice. Cells were then washed with PBS and finally resuspended in 70% ethanol, and incubated at –20 °C for at least 4 h until FACS analysis using a TUNEL assay (APO-BRDU kit, BD Pharmingen, San Diego, CA) was performed after a final washing step with PBS. To examine proteolytic cleavage of PARP, 293T cells transfected with siRNA were serum-starved and incubated with sorbitol (0.5 M) for 12 h and lysed in RIPA buffer. After centrifugation, the supernatant was analyzed using 10% SDS-PAGE, followed by Western blotting with an anti-PARP rabbit polyclonal antibody (BIOMOL). Apoptosis was indicated by the cleavage of 116-kDa PARP into 85-kDa peptide product.
Real-time quantitative RT-PCR and Northern blotting
293T cells were transfected with siRNA, serum-starved, and incubated with sorbitol (0.5 M) for 6 h. Total RNA was extracted from the transfected cells with Isogen (Nippon Gene, Tokyo, Japan). GADD45ß expression was examined by real-time RT-PCR using an ABI Prism 7500 instrument. All samples were run in triplicate in each experiment using a QuantiTect Probe RT-PCR kit (QIAGEN) according to the manufacturer's instructions. Primers were custom designed and obtained from NIPPON EGT. GADD45ß primers were 5'-CGGCCAAGTTGATGAATGTG-3' and 5'-TCCTCGTCAATGGCCAAGAG-3', and the internal TaqMan probe was 5'-6FAM-ACCCAGACAGCGTGGTCCTCTG-TAMRA-3'. GAPDH primers were 5'-CAACGGATTTGGTCGTATTGG-3' and 5'-GGCAACAATATCCACTTTACCAGAGT-3', with TaqMan probe 5'-6FAM-CCTGGTCACCAGGGCTGCTT-TAMRA-3'. The QuantiTect Probe RT-PCR Master Mix (Qiagen) was used for all the reaction components, except primers, probe, and template. The final primer concentrations were 400 nM for GADD45ß and GAPDH. The final probe concentration for both GADD45ß and GAPDH was 160 nM. Cycling conditions were 50 °C for 30 min, 95 °C for 15 min, and 45 cycles of 94 °C for 15 s, 60 °C for 1 min. The data were collected and analyzed using the ABI PRISM 7500 Sequence Detection System Software, Version1.2.2. The relative gene expression was quantified by the comparative Ct method.
A multiple tissue mouse mRNA blot was purchased from Clontech (Palo Alto, CA) and used for Northern blotting. The probe was the mouse Sin1 coding sequence.
Immunohistochemistry
Paraffin sections (6 µm thick) were used for immunohistochemistry. For Sin1 protein staining, sections were deparaffinized and incubated in 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase. Specimens were microwaved for 5 min in the presence of 10 mM sodium citrate pH 6.0, treated with 0.3% Triton X-100 in PBS for 10 min at room temperature and then incubated for 1 h at 37 °C with anti-Sin1 antibody (1 : 100). EnVision peroxidase Rabbit (DAKO) was used as secondary antibody and 3,3'-diaminobenzadine tetrahydrochloride as a chromogen.
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Acknowledgements |
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and MKK6D/D expression plasmids. This work was supported in part by Grants-in-Aid for Scientific Research and a grant from the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan. |
Footnotes |
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* Correspondence: E-mail: sishii{at}rtc.riken.jp
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Received: 11 July 2006
Accepted: 2 August 2006
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