Interaction between fortilin and transforming growth factor‐beta stimulated clone‐22 (TSC‐22) prevents apoptosis via the destabilization of TSC‐22
2008; Wiley; Volume: 582; Issue: 8 Linguagem: Inglês
10.1016/j.febslet.2008.01.066
ISSN1873-3468
AutoresJeong Heon Lee, Seung Bae Rho, Sang‐Yoon Park, Taehoon Chun,
Tópico(s)Cell death mechanisms and regulation
ResumoFEBS LettersVolume 582, Issue 8 p. 1210-1218 Short communicationFree Access Interaction between fortilin and transforming growth factor-beta stimulated clone-22 (TSC-22) prevents apoptosis via the destabilization of TSC-22 Correction(s) for this article Corrigendum to "Interaction between fortilin and transforming growth factor-beta stimulated clone-22 (TSC-22) prevents apoptosis via the destabilization of TSC-22" [FEBS Lett. 582 (2008) 1210–1218] Jeong Heon Lee, Seung Bae Rho, Sang-Yoon Park, Taehoon Chun, Volume 583Issue 5FEBS Letters pages: 950-951 First Published online: February 11, 2009 Jeong Heon Lee, Jeong Heon Lee Department of Obstetrics and Gynecology, Chonbuk National University Medical School, Jeonju 561-712, Republic of Korea Co-first authors. Search for more papers by this authorSeung Bae Rho, Seung Bae Rho Research Institute, National Cancer Center, Goyang, Gyeonggi 411-769, Republic of Korea Co-first authors. Search for more papers by this authorSang-Yoon Park, Sang-Yoon Park Research Institute, National Cancer Center, Goyang, Gyeonggi 411-769, Republic of KoreaSearch for more papers by this authorTaehoon Chun, Corresponding Author Taehoon Chun tchun@korea.ac.kr Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of KoreaCorresponding author. Fax: +82 2 3290 3507.Search for more papers by this author Jeong Heon Lee, Jeong Heon Lee Department of Obstetrics and Gynecology, Chonbuk National University Medical School, Jeonju 561-712, Republic of Korea Co-first authors. Search for more papers by this authorSeung Bae Rho, Seung Bae Rho Research Institute, National Cancer Center, Goyang, Gyeonggi 411-769, Republic of Korea Co-first authors. Search for more papers by this authorSang-Yoon Park, Sang-Yoon Park Research Institute, National Cancer Center, Goyang, Gyeonggi 411-769, Republic of KoreaSearch for more papers by this authorTaehoon Chun, Corresponding Author Taehoon Chun tchun@korea.ac.kr Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of KoreaCorresponding author. Fax: +82 2 3290 3507.Search for more papers by this author First published: 04 March 2008 https://doi.org/10.1016/j.febslet.2008.01.066Citations: 21 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract — MINT-6173230, MINT-6173253: TSC22 (uniprotkb:Q15714) physically interacts (MI:0218) with fortilin (uniprotkb:P13693) by co-immunoprecipitation (MI:0019) — MINT-6173217: TSC22 (uniprotkb:Q15714) binds (MI:0407) fortilin (uniprotkb:P13693) by pull-down (MI:0096) — MINT-6173240, MINT-6173270: TSC22 (uniprotkb:Q15714) physically interacts (MI:0218) with fortilin (uniprotkb:P13693) by two-hybrid (MI:0018) 1 Introduction TGF-β stimulated clone 22 (TSC-22) was initially identified as one of the genes induced by TGF-β1 treatment [1, 2]. TSC-22 is evolutionarily conserved to a significant degree from fruit flies to mammals [3, 4]. The structure of TSC-22 includes a leucine zipper-like motif, thereby suggesting that TSC-22 functions as a transcriptional factor, but lacks the conventional DNA binding motif at the N-terminal region [1]. Recently, several roles of TSC-22 have been proposed, including the regulation of embryogenesis [3, 5], and the pro-apoptotic factor in several cancer cells [6-9]. TSC-22 exists within the cytoplasm in the steady state and translocates to the nucleus, and there activates signal transduction to TSC-22 [10]. After nuclear translocation, TSC-22 may form a homodimer or heterodimer with TSC-22 homologous gene-1 (THG-1), and operate as a transcriptional repressor of several genes [11]. This result suggests that TSC-22 may mediate apoptosis via a caspase-3 dependent pathway [12]. However, the precise mechanism underlying the regulation of TSC-22 activity has yet to be precisely elucidated. In order to characterize the TSC-22-dependent apoptotic pathway, we first employed a yeast two-hybrid system to screen a human ovary cDNA library for novel TSC-22 binding proteins. We identified fortilin, a recently characterized nuclear anti-apoptotic factor. The results of transient transfection analyses then demonstrated that fortilin overexpression blocks TSC-22-mediated apoptotic activity via the induction of TSC-22 degradation. Therefore, these results support the notion that a dynamic equilibrium existing between pools of TSC-22 and fortilin regulates an apoptotic homeostasis. 2 Materials and methods 2.1 Yeast two-hybrid screening For bait construction with human TSC-22, cDNA encoding for full-length human TSC-22 was subcloned into the EcoRI and XhoI restriction sites of the pGilda cloning vector. The resultant plasmid, pGilda-TSC-22, was then introduced into the EGY48 yeast strain [MATa, his3, trp1, ura3-52, leu2::pLeu2-LexAop6/pSH18-34 (LexAop-lacZ reporter)] via a modified lithium acetate technique [13]. The ovary cDNA library was then constructed by cloning the cDNA fragments into the EcoRI and XhoI restriction sites of pJG4-5, thus generating B42 fusion proteins (Clontech, Palo Alto, CA, USA). The cDNAs encoding for the B42 fusion proteins were introduced into competent yeast cells that already harbored pGilda-TSC-22, and the transformants were selected for tryptophan prototrophy (plasmid marker) on synthetic medium (Ura, His, Trp) containing 2% (w/v) glucose. 2.2 Quantitation of interaction The activity of the interaction between TSC-22 and fortilin was verified via measurements of the relative levels of β-galactosidase expression. The β-galactosidase assay was conducted in accordance with the previously described protocols [14], with slight modification. In brief, yeast cells (EGY48 strain) containing each construct were cultured in yeast synthetic media (Ura, His, Trp) with 2% (w/v) glucose until they achieved mid-growth phase. The cells were then transferred to a yeast medium (Ura, His, Trp) containing 2% (w/v) galactose and 0.2% dimethyl sulfoxide (Me2SO). After transformation, equivalent numbers of cells were lysed in 0.7 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM β-mercaptoethanol, pH 7.0) containing 50 μl of 0.1% SDS and 50 μl of chloroform for 30 s at 30 °C. β-Galactosidase activity was then measured via the addition of 140 μl of 4 mg/ml σ-nitrophenyl β-d-galactopyranoside (ONPG). The reaction was conducted at 30 °C until a yellow color formed, and was then quenched via the addition of 0.4 ml of 1 M Na2CO3. The samples were then briefly centrifuged to remove any remaining cell debris, and the absorbance was measured at wavelengths of 420 and 550 nm. β-Galactosidase activity was calculated using the formula units = [1000 × (A 420 − 1.75 × A 550)]/(time × volume × A 600). 2.3 In vitro pull-down assay cDNA encoding for human TSC-22 was subcloned into the EcoRI and XhoI restriction sites of the pGEX4T-1 vector (GE Healthcare, Piscataway, NJ, USA) to generate the glutathione S-transferase (GST) fusion protein (GST-TSC-22). Human fortilin cDNA was ligated into pET28a (Novagen, Madison, WI, USA) using EcoRI and XhoI, thereby generating a histidine fusion protein (His-fortilin). The GST and histidine fusion proteins were purified using either a GST column (GE Healthcare) or an Ni2+ column (Novagen), in accordance with the manufacturer's protocols. The purified GST-TSC-22 (2 μg) was then mixed with His-fortilin (2 μg). The mixtures were subsequently added to 20 μl of a GST column matrix (glutathione sepharose 4B, GE Healthcare) and incubated for 30 min at 25 °C. The slurry was pelleted via centrifugation and washed. The pellet of the gel matrix was resuspended in 20 ml of elution buffer (10 mM glutathione, 50 mM Tris–HCl, pH 8.0) and incubated for 20 min at 25 °C in order to elute the bound GST fusion proteins. The eluted proteins were separated via gel electrophoresis, and the proteins were detected by Coomassie staining. 2.4 Co-immunoprecipitation For co-immunoprecipitation, SKOV-3 ovarian cancer cells were washed in PBS and resuspended in cell lysis solution (50 mM Tris–HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1 μg/ml leupeptin ml, 1 μg/ml pepstatin ml, 2 μg/ml aprotinin, 200 μg/ml PMSF). The lysates were then incubated with goat anti-fortilin antibody (sc-20426, Santa Cruz Biotechnology), goat anti-TSC-22 antibody (sc-27844, Santa Cruz Biotechnology) or goat IgG (sc-2028, Santa Cruz Biotechnology), and precipitated with protein A-agarose (GE Healthcare). The precipitated proteins were resolved via SDS gel electrophoresis, transferred onto Immobilon P membranes (Millipore Corporation, Billerica, MA, USA), and immunoblotted with anti-TSC-22 antibody or anti-fortilin antibody using the ECL system (GE Healthcare). β-Actin antibody (sc-69879, Santa Cruz Biotechnology) was employed as an internal control. 2.5 Subcloning of deletion mutants of fortilin and TSC-22 Three deletion mutants (Met1-Asp52, Asn53-Phe110, Gln111-Ala144) of TSC-22 were isolated by PCR using the combination of the following primers (TSC-22-F1, 5′-CGGGAATTCATGAAATCCCAATGGTGT-3′; TSC-22-R1, 5′-ATTCTCGAGTCAGTCAATAGCTACCAC-3′; TSC-22-F2, 5′-CGGGAATTCAACAAAATCGAGCAAGCT-3′; TSC-22-R2, 5′-ATTCTCGAGTCAAAACTGGGCAAGCTG-3′; TSC-22-F3, 5′-CGGGAATTCCAGGCCCAGCTGCAGACT- 3′; TSC-22-R3, 5′-CGGCTCGAGCTATGCGGTTGGTCCTGA-3′). PCR products spanning each fragment were cloned into the EcoRI and XhoI restriction sites of the pGilda. Three deletion mutants (Met1-Gly69, Val70-Ala119, Glu120-Cys172) of fortilin were isolated by polymerase chain reaction (PCR) using the combination of the following primers (fortilin-F1, 5′-CGGGAATTCATGATTATCTACCGGGAC-3′; fortilin-R1, 5′-CGGCTCGAGTCAACCAGTGATTACTGT-3′; fortilin-F2, 5′-CGGGAATTCGTCGATATTGTCATGAAC-3′; fortilin-R2, 5′-ATTCTCGAGTCATGC AGCCCCTGTCAT-3′; fortilin-F3, 5′-CGGGAATTCGAACAAATCAAGCACATC-3′; fortilin-R3, 5′-CGGCTCGAGTTAACATTTTTCCATTTC-3′). PCR products spanning each fragment were cloned into the EcoRI and XhoI restriction sites of the pJG4-5. Each constructed plasmid was introduced into yeast EGY48 expressing either fortilin or TSC-22 hybrid protein. 2.6 Small interfering RNA (siRNA) construction The siRNA oligonucleotide sequence targeting TSC-22 (5′-GGCGATGGATCTAGGAGTT-3′) corresponded to nucleotides 27–45 in the human sequence, and the siRNA oligonucleotide sequence targeting fortilin (5′-AAGGTACCGAAAGCACAGT-3′) corresponded to nucleotides 179–197 in the human sequence. siRNA was synthesized using an siRNA construction kit (Ambion, Austin, TX, USA) and was then transfected by oligofectamine (Invitrogen), in accordance with the manufacturer's protocols. After transfection of siRNA, the mRNA and protein expression levels of both fortilin and TSC-22 were compared to transfectants with either cDNA constructs of pEGFPC1-fortilin or pEGFPC1-TSC-22 by RT-PCR and immunoblot as described above. 2.7 Cell viability assay SKOV-3 ovarian cancer cells were plated in six-well plates and transfected with the indicated cDNA and/or siRNA. Three days after transfection, the cells were harvested, stained with trypan blue, and counted under a light microscope. Cell viability assay was also assessed with the presence of caspase-3 specific inhibitor, z-DEVD-fmk (50 μM; BDPharmingen, San Diego, CA, USA). 2.8 Apoptosis assay SKOV-3 ovarian cancer cells were plated onto six-chamber slides and transfected with the indicated cDNA and/or siRNA. For the evaluation of nuclear morphology, the nuclei were fixed in methanol and stained with 4,6′-diamidino-2-phenylindole (DAPI, 1 μg/mL, Boehringer Mannheim, Mannheim, Germany) for 15 min and rinsed twice with PBS, then examined under a fluorescence microscope. 2.9 In vitro caspase-3 activity assay Caspase-3 enzymatic activity in the cell lysates was evaluated using acetyl-DEVD-7-amino-4-trifluoromethyl coumarin, in accordance with the manufacturer's protocols (BDPharmingen). Activity was assessed using a Spectramax 340 microplate reader (Molecular Devices, Sunnyvale, USA) in fluorescence mode with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Enzyme activity was calculated and indicated as fluorescence in accordance with the formula provided by the manufacturer (BDPharmingen). Caspase-3 enzymatic activity in the cell lysates was also visualized by immunoblot using anti-caspase-3 antibody (610324, BDPharmingen). 2.10 Determination of TSC-22 protein stability SKOV-3 ovarian cancer cells were transfected with mock (an expression vector only without insert), fortilin siRNAs, or co-transfected with TSC-22 and the indicated pEGFPC1-fortilin cDNA mutant. Three days after transfection, the cells were treated with cycloheximide (Sigma) at a final concentration of 100 μg/ml. The cell lysates were subsequently harvested at sequential time points after treatment, resolved via SDS gel electrophoresis, transferred onto Immobilon P membranes (Millipore Corporation, Billerica, MA, USA), and immunoblotted with anti-TSC-22 antibody or anti-GFP antibody (sc-8334, Santa Cruz Biotechnology) using the ECL system (GE Healthcare). β-Actin antibody was utilized as an internal control. 3 Results and discussion 3.1 Identification of fortilin as a TSC-22 binding protein The human ovary cDNA library fused to the gene for the transcription activator, pJG4-5, was introduced into yeast cells harboring pGilda-TSC-22 as bait. Approximately, 3 × 106 independent transformants were pooled and spread again onto the selection media (Ura−, His−, Trp−, Leu−) containing 2% (w/v) galactose in order to induce cDNA expression. If a B42-tagged protein interacts with the TSC-22, the transcription of the LEU2 gene is activated, thus allowing the host cells to grow on a leucine-deficient synthetic medium. Among the seven colonies obtained on the selection media, a total of five colonies evidenced galactose dependency. The plasmids were isolated from the selected yeast cells and introduced into Escherichia coli KC8 in order to isolate the plasmids harboring the pJG4-5-cDNA inserts. The plasmids were then isolated by the plasmid marker, trp, in the E. coli host, and the purified plasmids were sequenced. A GenBank homology search using the BLAST program showed that all five of the plasmids encoded for human fortilin (GenBank accession number: NM_003295). In order to verify this result, we determined the binding activity between TSC-22 and fortilin via measurements of the relative β-galactosidase expression level. As is shown in Fig. 1 A, the β-galactosidase activity between TSC-22 and fortilin was clearly observed, whereas very little β-galactosidase activity was observed between TSC-22 and the empty vector (vector only). Figure 1Open in figure viewerPowerPoint Interaction analysis between human fortilin and TSC-22. (A) Binding activity of TSC-22 and fortilin measured by σ-nitrophenyl β-d-galactopyranoside (ONPG) assays. β-Galactosidase activity was measured via the addition of ONPG. Data are expressed as the means ± S.E.M. (B) Interactions between TSC-22 (GST-TSC-22) and fortilin (His-fortilin) were confirmed via in vitro pull-down assays. For GST pull-down analysis, the purified GST-TSC-22 (2 μg) was mixed with His-E-fortilin (2 μg). The protein mixtures were then subjected to GST affinity purification. The eluted proteins were separated via gel electrophoresis, and the proteins were detected by Coomassie staining. Lanes: 1, no input; 2, GST-TSC-22; 3, His-fortilin; 4, GST-TSC-22 and His-fortilin, respectively. The results are representative of three separate experiments. (C) Co-immunoprecipitation of fortilin with TSC-22 in human ovarian carcinoma cells, SKOV-3. IP means immunoprecipitation and WB means immunoblotting with indicated antibodies. The results are representative of three separate experiments. The interaction between TSC-22 and fortilin was further evaluated via in vitro pull-down assays and co-immunoprecipitation. For the in vitro pull-down analysis, the GST–TSC-22 fusion protein was mixed with His–fortilin, and the mixture was purified using a GST column matrix. The eluted proteins were separated via gel electrophoresis and the proteins were detected by Coomassie staining. As is shown in Fig. 1B, the His–fortilin fusion protein, which measured approximately 25 kDa, was co-purified with the GST–TSC-22 fusion protein (lane 4). No protein bands were detected when no input was added to the GST column matrix (lane 1). A single band of approximately 45 kDa, equivalent to the GST–TSC-22 fusion protein, was observed when the GST–TSC-22 fusion protein was added to the GST column matrix (lane 2). However, no bands were observed when the His–fortilin fusion protein was added to the GST column matrix (lane 3). For co-immunoprecipitation, SKOV-3 ovarian cancer cells were washed in PBS and resuspended in cell lysis solution. Immunoprecipitation was subsequently conducted using anti-fortilin antibody or anti-TSC-22 antibody with whole cell lysates. After immunoprecipitation, the precipitated proteins were immunoblotted with anti-TSC-22 antibody or anti-fortilin antibody. As is shown in Fig. 1D, TSC-22 was co-immunoprecipitated with fortilin (upper left panel), whereas no interactions were observed between isotype control (goat IgG) and TSC-22 (upper left panel). Also, fortilin was co-immunoprecipitated with TSC-22 (upper right panel), whereas no interactions were observed between isotype control (goat IgG) and fortilin (upper right panel). Immunoblotting using anti-TSC-22 antibody and anti-fortilin antibody verified that an equal quantity of both fortilin and TSC-22 was present in the whole cell lysates. The whole cell lysates from both samples harbored the equivalent proteins when immunoblotted using anti-β-actin antibody (lower panel). These results clearly demonstrated that TSC-22 and fortilin interact with each other on the physiological level in SKOV-3 ovarian cancer cells. 3.2 Mapping of the interaction region between TSC-22 and fortilin To identify the fortilin binding region of TSC-22, cDNA constructs containing three TSC-22 deletion mutants were designed as shown in Fig. 2 A. These truncated regions were predicted to be predominantly α-helices. In the two-hybrid system, the full-length human fortilin cDNA and either plasmid containing a full-length human TSC-22 cDNA (left panel in Fig. 2A, full) or plasmids containing three truncation mutant forms (left panel in Fig. 2A, Met1-Asp52, Asn53-Phe110, Gln111-Ala144) of cDNAs were co-transformed into EGY48 yeast cells. Cells containing full-length TSC-22 cDNA and also one deletion mutant (Asn53-Phe110) grew on the Ura, His, Trp and Leu deficient plates. Yeast cells transformed with the other deletion mutants (Met1-Asp52 and Gln111-Ala144) failed to grow (right panel in Fig. 2A). To confirm this result, we determined the binding activity of these constructs by measuring the relative expression level of β-galactosidase. As shown in right panel in Fig. 2A, β-galactosidase assay results confirmed that either of these mutants (Met1-Asp52 and Gln111-Ala144) cannot bind to fortilin. However, we cannot exclude that the fortilin binding site of TSC-22 is located near the junction of these mutants since β-galactosidase activity in one deletion mutant (Asn53-Phe110) is lower than full-length TSC-22 (right panel in Fig. 2A). Figure 2Open in figure viewerPowerPoint Mapping of the critical interaction region between TSC-22 and fortilin using the yeast two-hybrid system. The cDNA constructs were co-transformed into EGY48 yeast cells to test a protein–protein interaction within the yeast two-hybrid system. The results are representative of three separate experiments. Data are shown as means ± S.E. (A) Left panel shows the schematic representation of cDNA constructs for each TSC-22 deletion mutant and full-length TSC-22 fusion proteins in the yeast two-hybrid system. Right panel shows the result of protein–protein interaction determined in the yeast two-hybrid system. The values of β-galactosidase activity (U) measured by ONPG assays are indicated below the corresponding lanes. The values of β-galactosidase activity in negative controls (vector only) of each construct were below 1.05 ± 0.05. (B) Left panel shows the schematic representation of cDNA constructs for each fortilin deletion mutant and full-length fortilin fusion proteins in the yeast two-hybrid system. Right panel shows the result of protein–protein interaction determined in the yeast two-hybrid system. The values of β-galactosidase activity (U) measured by ONPG assays are indicated below the corresponding lanes. The values of β-galactosidase activity in negative controls (vector only) of each construct were below 1.56 ± 0.25. (C) Interaction between cDNA constructs for a fortilin deletion mutant (1Met-Gly69) and three TSC-22 deletion (1Met-Asp52, 53Asn-Phe110 and 111Gln-Ala144) fusion proteins in the yeast two-hybrid system. Subsequently, cDNA constructs containing three fortilin truncation mutants were designed to localize the TSC-22 binding region of fortilin (left panel in Fig. 2B, full). These truncated regions were predicted to be predominantly α-helices. In the two-hybrid system, full-length human TSC-22 cDNA and either full-length human fortilin cDNA (left panel in Fig. 2B, full) or three truncation mutants (left panel in Fig. 2B, Met1-Gly69, Val70-Ala119, Glu120-Cys172) were co-transformed into EGY48 yeast cells. Cells containing full-length fortilin cDNA and also the one deletion mutant (Met1-Gly69) grew on the Ura, His, Trp and Leu deficient plates. Yeast cells transformed with the other deletion mutants (Val70-Ala119, Glu120-Cys172) failed to grow (right panel in Fig. 2B). We also quantitated the binding activity of these constructs by measuring relative expression level of β-galactosidase. Results on β-galactosidase assay also indicated that the critical fortilin region for binding TSC-22 resided within Met1-Gly69 (right panel in Fig. 2B). To confirm these results, one deletion mutant of fortilin (Met1-Gly69) and either vector cDNA or three truncation mutants of TSC-22 (Met1-Asp52, Asn53-Phe110, Gln111-Ala144) were co-transformed into EGY48 yeast cells (Fig. 2C). Consistent with results from Fig. 2A and B, cells containing fortilin deletion mutant (Met1-Gly69) with one TSC-22 deletion mutant (Asn53-Phe110) only grew on the Ura, His, Trp and Leu deficient plates whereas the other deletion mutants (Val70-Ala119, Glu120-Cys172) failed to grow. Subsequent results on β-galactosidase assay were also agreed with these results (Fig. 2C). 3.3 Overexpression of TSC-22 can induce apoptosis in SKOV-3 ovarian cancer cells via caspase-3 dependent pathway To evaluate the functional consequences of the interaction taking place between TSC-22 and fortilin, we conducted a couple of experiments involving mammalian cell apoptosis. First, we attempted to construct siRNA system that inhibits TSC-22 and fortilin since both proteins endogenously express in SKOV-3 ovarian cancer cells. To test whether siRNA transfection efficiently blocks endogenous TSC-22 or fortilin, we measured both mRNA and protein expression levels of siRNA and overexpression co-transfectants as well as overexpression transfectants. As shown in Fig. 3 , the mRNA and protein expression levels of both TSC-22 and fortilin in siRNA and overexpression co-transfectants (lane 2 in Fig. 3) were significantly decreased comparing with overexpression transfectants (lane 1 in Fig. 3). Figure 3Open in figure viewerPowerPoint The construction of siRNA system that inhibits TSC-22 and fortilin. SKOV-3 ovarian cancer cells were transfected with each GFP fused cDNA alone or co-transfected each GFP fused cDNA with each siRNA. After transfection, the cells were visualized under fluorescence microscope, and analyzed the mRNA or protein expression levels by RT-PCRs and immunoblots. Size bar, 20 μm. The results are representative of three separate experiments. Next, we determine whether TSC-22 overexpression induces apoptosis in SKOV-3 cells. As is shown in Fig. 4 A, the viability of the SKOV-3 cells is gradually reduced in a transfected TSC-22 cDNA dose-dependent manner, and nearly 50% of cells died when 1 μg of TSC-22 cDNAs was transfected. This result is also supported by immunoblot analysis using caspase-3 antibody. Only activated form of caspase-3 was observed in TSC-22 transfectant, not in both mock and siTSC-22 (siRNA of TSC-22) transfectants (Fig. 4B). To confirm these results, we treated z-DEVD-fmk, a specific inhibitor of caspase-3, in TSC-22 transfectant. As a result, z-DEVD-fmk treatment definitively restores viability of TSC-22 transfectant (Fig. 4C). Thus, 1 μg of TSC-22 cDNAs was utilized as the treatment concentration in all of the following experiments. Figure 4Open in figure viewerPowerPoint TSC-22 mediated apoptosis via caspase-3 dependent pathway in human ovarian carcinoma cells, SKOV-3. (A) SKOV-3 ovarian cancer cells were transfected with mock (an expression vector only without insert) or TSC-22 cDNA. Three days after transfection, the cells were stained with trypan blue and quantitated to analyze cell viability. Data are expressed as the means ± S.E.M. (B) SKOV-3 ovarian cancer cells were transfected with mock (an expression vector only without insert), siRNA of TSC-22 (siTSC-22) or TSC-22 cDNA. The cells were harvested and caspase-3 was detected by immunoblot using anti-caspase-3 antibody. The results are representative of three separate experiments. (C) A caspase-3 specific inhibitor, z-DEVD-fmk, was treated in mock or TSC-22 transfectants. The cells were stained with trypan blue and quantitated to analyze cell viability. Data are expressed as the means ± S.E.M. 3.4 Fortilin prevents TSC-22 mediated apoptosis via the destabilization of TSC-22 Fortilin is a highly conserved hydrophilic nuclear protein, and is expressed ubiquitously in a variety of cells [15]. The results of a recent study showed that fortilin overexpression can prevent stress-induced mammalian cell apoptosis [15, 16]. Conversely, the inhibition of fortilin in malignant human carcinoma cells induced an increase in apoptosis [15, 16]. Thus, fortilin may be relevant to cell survival as a negative apoptotic regulator. However, the exact mechanism underlying the anti-apoptotic effects of fortilin remains to be clearly elucidated. Therefore, we attempted to determine whether TSC-22 and fortilin evidence an additive effect or antagonize each other upon apoptosis. As shown in Fig. 5 A, nearly 50% of the cells perished after transfection with TSC-22 cDNA alone as compared to the mock transfectant, whereas no change in cell viability was observed when the cells were transfected with fortilin cDNA alone (Fig. 5A). Consistent with previous observation [15, 16], the fortilin siRNA transfectant shows decreased cell viability, similar level as that of TSC-22 transfectant (Fig. 5A). Interestingly, cell viability largely recovered upon co-transfection with TSC-22 and fortilin cDNAs (Fig. 5A). This suggests that fortilin may antagonize the TSC-22-mediated apoptosis of SKOV-3 cells. In order to verify this result, we inhibited fortilin expression by siRNA when we transfected the cells with TSC-22 cDNA. As is shown in Fig. 5A, the viability of TSC-22 and the sifortilin (siRNA of fortilin) co-transfectant was greatly reduced as compared to the TSC-22 and fortilin co-transfectant. Figure 5Open in figure viewerPowerPoint Fortilin inhibits TSC-22 mediated apoptosis in human ovarian carcinoma cells, SKOV-3. Cell viability assay (A), DAPI staining (B) and caspase-3 activity assay (C) were performed on SKOV-3 ovarian cancer cells transfected with mock (an expression vector only without insert), fortilin, sifortilin, TSC-22 or siTSC-22, or co-transfected with TSC-22 and fortilin cDNAs, co-transfected with TSC-22 and sifortilin, or co-transfected with TSC-22 and sifortilin, or triple-transfected with fortilin, sifortilin and TSC-22. (A) Cell viability assay was quantified via tryptophan blue staining. Data are expressed as the means ± S.E.M. (B) Cells were stained with DAPI to visualize DNA fragmentation for the apoptosis assay. Arrows indicate the observed DNA fragmentations. Size bar, 20 μm. The results are representative of three separate experiments. (C) Caspase-3 activity was measured using a microplate reader in fluorescence mode with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Enzyme activity was calculated and indicated as fluorescence in accordance with the formula provided by the manufacturer. Data are expressed as the means ± S.E.M. To verify that the reduction in cell numbers was reflective of apoptosis, we employed DAPI staining to confirm the observed reduced proliferation in SKOV-3 cells overexpressing mock (expression vector only), fortilin, sifortilin, TSC-22, or siTSC-22, or co-transfected with TSC-22 and fortilin cDNAs, co-transfected with TSC-22 and sifortilin, or co-transfected with TSC-22 and sifortilin, or triple-transfected with fortilin, sifortilin and TSC-22. As had been expected, after transfection with TSC-22 cDNA alone compared to mock transfectant, nearly 60% of the cells evidenced fragmented nuclei, whereas no change in cell viability was observed when the cells were transfected with mock and fortilin cDNA alone. Cells transfected with a combination of TSC-22 and fortilin evidenced a significantly reduced number of cells harboring fragmented DNA, but cells transfected with fortilin-siRNA did not (Fig. 5B). We subsequently assessed the caspase-3 activity in those transfectants. As is shown in Fig. 5C, no significant changes in caspase-3 activity were observed in the cells transfected with fortilin cDNA alone, as compared to the mock-transfected cells. A significant upregulation of caspase-3 activity was detected in the cells transfected with TSC-22 cDNA alone compared to the mock-transfected cells (Fig. 5C). However, caspase-3 activity was reduced by almost half in the co-transfectant harboring both TSC-22 and fortilin cDNAs as compared to the transfectant harboring TSC-22 cDNA alone (Fig. 5C). Conversely, caspase-3 activity in the co-transfectant containing both TSC-22 cDNA and sifortilin was restored markedly, to almost the same levels observed in the cells transfected with TSC-22 cDNA alone. These results clearly showed that fortilin inhibits TSC-22 mediated apoptosis in SKOV-3 human ovarian carcinoma cells. To define the mechanism by which fortilin inhibits TSC-22 mediated apoptosis, we assessed the stability of the TSC-22 protein in the presence or absence of fortilin in SKOV-3 cells. In brief, mock transfectant and sifortilin transfectant were treated with cycloheximide. Then, the cell lysates were harvested at 1 h, 3 h and 5 h after treatment, and immunoblotted with anti-TSC-22 antibody or anti-fortilin antibody. As is shown in Fig. 6 , the level of TSC-22 protein evidenced a significant reduction in a time-dependent manner in the presence of fortilin. Conversely, the level of TSC-22 protein evidenced no significant change for over 5 h in the absence of fortilin (Fig. 6A). To confirm this result, we co-transfected TSC-22 cDNA with the each pEGFPC1-fortilin cDNA mutant (Met1-Gly69, Val70-Ala119, Glu120-Cys172) and performed TSC-22 stabilization assay and cell viability test using these co-transfectants. For TSC-22 stabilization assay, we harvested cell lysates at 5 h after cycloheximide treatment, and immunoblotted with anti-TSC-22 antibody or anti-GFP antibody. As shown in Fig. 6B, the level of TSC-22 protein was significant decreased in co-transfectant containing TSC-22 cDNA and one deletion fortilin mutant (Met1-Gly69). Also, cell viability in co-transfectant containing TSC-22 cDNA and one deletion fortilin mutant (Met1-Gly69) was recovered, compared to transfectant containing TSC-22 cDNA alone (Fig. 6C). However, neither the level of TSC-22 protein nor cell viability was changed in co-transfectants containing TSC-22 cDNA and the other deletion mutant (Val70-Ala119, Glu120-Cys172). These results clearly showed that the interaction between fortilin and TSC-22 accelerates the destabilization of TSC-22 in SKOV-3 human ovarian carcinoma cells. Figure 6Open in figure viewerPowerPoint Stability of TSC-22 is reduced by fortilin. (A) SKOV-3 ovarian cancer cells were transfected with mock (an expression vector only without insert) or fortilin siRNAs. After transfection, the cells were treated with cycloheximide (100 μg/ml). At each time point, the whole cell lysates were prepared and analyzed via immunoblot analysis using antibodies against TSC-22, fortilin or β-actin to measure the amounts of TSC-22 protein remaining at the respective times. The 'h' refers to hours after cycloheximide treatment. The results are representative of three experiments. (B) SKOV-3 ovarian cancer cells were co-transfected TSC-22 cDNA with the each pEGFPC1-fortilin cDNA mutant (Met1-Gly69, Val70-Ala119, Glu120-Cys172). The cells were treated with cycloheximide (100 μg/ml). At 5 h after cycloheximide treatment, the whole cell lysates were prepared and analyzed via immunoblot analysis using antibodies against TSC-22, GFP or β-actin to measure the amounts of TSC-22 and fortilin. The results are representative of three experiments. (C) SKOV-3 ovarian cancer cells were transfected with mock (an expression vector only without insert) or TSC-22 cDNA, or co-transfected TSC-22 cDNA with the each pEGFPC1-fortilin cDNA mutant (Met1-Gly69, Val70-Ala119, Glu120-Cys172). Three days after transfection, the cells were stained with trypan blue and quantitated to analyze cell viability. Data are expressed as the means ± S.E.M. In this study, we have described the specific interaction occurring between TSC-22 and fortilin, a highly conserved anti-apoptotic protein [15, 16]. The interaction between TSC-22 and fortilin appears to be physiologically relevant, as this interaction occurs in both yeast and mammalian cells. Our results also show that fortilin binds to and destabilizes TSC-22. TSC-22 and fortilin are both localized predominantly within the nucleus. During apoptosis, activated TSC-22 moves from the cytosol to the nucleus and functions as a transcriptional repressor of several genes [10]. Fortilin is a known nuclear-residing protein, and myeloid cell leukemia 1 protein is known to stabilize fortilin via direct interaction [17]. Thus, it is likely that fortilin destabilizes the activity of TSC-22 within the nucleus. Conversely, the weakening activity of fortilin induced by treatment with sifortilin reversed the apoptotic activity of TSC-22. Therefore, the interaction between TSC-22 and fortilin appears crucial for the regulation of TSC-22-mediated apoptosis. The results of this study provide valuable information as to the mechanisms by which fortilin exerts its anti-apoptotic protein properties. Further studies in this regard will focus on the specific mechanism by which fortilin destabilizes TSC-22 in the nucleus, and will also concern the possible influence of fortilin on other pro-apoptotic proteins. 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