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Originally published In Press as doi:10.1074/jbc.M308479200 on December 11, 2003

J. Biol. Chem., Vol. 279, Issue 10, 8715-8722, March 5, 2004
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Intracellular Localization and Activity State of Tissue Transglutaminase Differentially Impacts Cell Death*

Tamara Milakovic, Janusz Tucholski, Eric McCoy, and Gail V. W. Johnson{ddagger}

From the Department of Psychiatry, University of Alabama at Birmingham, Alabama 35294-0017

Received for publication, August 1, 2003 , and in revised form, December 10, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue transglutaminase (tTG) is a unique member of the transglutaminase family as it is both a transamidating enzyme and a GTPase. In the cell tTG is mostly cytosolic, however it is also found in the nucleus and associated with the plasma membrane. tTG can be proapoptotic, however anti-apoptotic activities of the enzyme have also been reported. To determine how the intracellular localization and transamidating activity of tTG modulates its effects on apoptosis, HEK293 cells were transiently transfected with tTG or [C277S]tTG (which lacks transamidating activity) constructs that were targeted to different intracellular compartments. Apoptosis was induced by thapsigargin treatment, which results in increased intracellular calcium concentrations. Cytosolic tTG was pro-apoptotic, while nuclear localization of [C277S]tTG attenuated apoptosis. Membrane-targeted tTG had neither pro- nor anti-apoptotic functions. This finding indicates for the first time that intracellular localization is an important determinant of the effect of tTG on apoptosis. Previous studies have suggested that tTG may modulate retinoblastoma (Rb) protein, an important suppressor of apoptosis. tTG interacted with Rb and after induction of apoptosis, the interaction of nuclear-targeted [C277S]tTG with Rb was increased significantly concomitant with an attenuation of apoptosis. In contrast, the interaction of nuclear-targeted tTG with Rb was significantly decreased and apoptosis was not attenuated. These data suggest that tTG protects cells against apoptosis in response to stimuli that do not result in increased transamidating activity by translocating to the nucleus, and that complexing with Rb may be an important aspect of the protective effects of tTG.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue transglutaminase (tTG)1 is a unique member of the transglutaminase family of proteins (for a review see Refs. 1 and 2). Transglutaminases catalyze a calcium dependent transamidation reaction that results in protein cross-linking, polyamination, or deamination (1, 3, 4). Tissue TG is a multifunctional enzyme as in addition to transamidating activity it also has GTPase activity and may act as a signal transducing G protein (5–7). Tissue TG has been implicated in numerous cellular processes such as wound healing (8), differentiation (9–11), apoptosis (12, 13), and cell survival (14–16). Further, although tTG is found mostly in the cytosol, it is also present in the nucleus (17, 18) and associated with the plasma membrane (19). Since intracellular localization affects protein function by determining what interactions are likely to occur, it is possible that by having access to different substrates in different intracellular pools the multiplicity of tTG effects in different cellular pathways may be due to both its localization and which enzymatic activity of tTG is favored (transamidating or GTP binding/hydrolyzing).

There is significant evidence that tTG plays a regulatory role in the process of apoptosis. Several studies have reported that tTG levels and tTG transamidating activity are elevated when apoptosis is induced (12, 20), and there is evidence suggesting that tTG facilitates apoptosis (21, 22). However recent studies have provided evidence that tTG can also attenuate apoptosis (14–16). Interestingly, it has been recently demonstrated that tTG differentially modulates apoptosis in a stimuli-dependent manner (15). This study demonstrated that when the stressor increased the transamidating activity of the enzyme, tTG was pro-apoptotic. However, tTG ameliorated apoptosis when the stressor did not result in an increase in the transamidating activity (15). These findings suggest that the effects of tTG on apoptosis are complex and likely due in part to its multifunctional character.

The focus of the present study was on determining how both tTG localization and transamidating activity modulated apoptosis. To accomplish this goal, HEK293 cells were transiently transfected with wild-type tTG (tTG) or tTG without transamidating activity ([C277S]tTG) that were tagged with a nuclear localization sequence (NLS), or a signal sequence that directed proteins to the plasma membrane (Myr/Pal) or with tTG or [C277S]tTG that lacked any added cellular localization sequences and therefore are predominantly cytosolic (17). Apoptosis was subsequently induced by treatment with thapsigargin, an inhibitor of the Ca2+-ATPase of endoplasmic reticulum that has been shown to induce apoptosis in different cell models (23–25). After induction of cell death differential effects of the tTG constructs on the apoptotic process were observed: (i) wild-type tTG without any added cellular localization signals facilitated apoptosis, (ii) transamidating inactive tTG localized in the nucleus (NLS-C277S) ameliorated apoptosis, and (iii) localizing tTG to the membrane had no effect on apoptosis. These findings show that the effects of tTG on the apoptotic process are dependent on the localization of the enzyme in the cell, and its multiple functions. Further, we demonstrated that tTG interacts with retinoblastoma (Rb) protein, a primarily nuclear protein that plays a key role in the regulation of apoptosis (26). However, after apoptosis was induced and the transamidating activity of tTG activated, the interaction between nuclear-localized wild-type tTG (NLS-tTG) and Rb was decreased, while the interaction of NLS-C277S with Rb was increased. Overall, our data demonstrate for the first time that tTG differentially modulates apoptosis in an intracellular localization and transamidating activity dependent manner. We also provide evidence that tTG binds to Rb both in the cytosol and nucleus, however induction of apoptosis impacts only nuclear tTG-Rb interactions. Given importance of Rb in the regulation of apoptosis (26), this interaction may be crucial for elucidating the mechanism by which tTG can protect cells against apoptosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs—Six different tTG constructs were used in this study. The pcDNA3.1-tTG and pcDNA3.1-C277S-tTG constructs containing cDNA for human tTG or transglutaminase inactive tTG mutant respectively have been described previously (27). To make the NLS-tagged constructs, the cDNA for human tTG or [C277S]tTG was amplified from the pcDNA3.1 vectors by Pfu DNA polymerase-catalyzed PCR using a pair of DNA primers, the forward (5'-CCC GAC CAT GGC CGA GGA GCT GGT CTT AG-3') containing an NcoI site and reverse (5'-CCG CTC GAG GGC GGG GCC AAT GAT GAC ATT C-3') containing an XhoI site, digested with NcoI and XhoI and subsequently ligated into the NcoI/XhoI cloning sites of pShooter vector pCMV/myc/nuc (Invitrogen). The resulting DNA constructs, pCMV-tTG-NLS, pCMV-C277S-tTG-NLS contained cDNA for tTG or [C277S]tTG fused in-frame C-terminally with the 3x NLS from SV40 large T antigen (28).

To generate DNA constructs that localize tTG and [C277S]tTG to the plasma membrane a two-step procedure was used. In the first step cDNA for tTG or C277StTG was amplified by PCR using the forward primer (5'-CGC GAA TTC TGA TGG CCG AGG AGC TGG TC-3') containing an EcoRI site and the reverse primer (5'-CCG CGG TAC CGT GGC GGG GCC AAT GAT GAC-3') containing a KpnI site, digested with EcoRI and KpnI and subsequently ligated into the EcoRI/KpnI cloning sites of pcDNA3.1(–)/myc-His B vector (Invitrogen). The above step resulted in generation of pcDNA-tTG-myc-His and pcDNA-C277S-myc-His constructs in which cDNA for tTG or [C277S]tTG was fused in-frame with a C terminus myc epitope and polyhistidine tag. In the second step a double-strand 67-base synthetic oligonucleotide (top strand: 5'-CCC GCT AGC ATG GGC TGT GTG CAA TGT AAG GAT AAA GAA GCA ACA AAA CTG ACG GAG GGA ATT CGC C-3') encoding the 17-amino acid N terminus of Fyn that is both myristylated and palmitoylated and thus constitutively targets the protein to the plasma membrane (29) and containing NheI and EcoRI restriction sites was used. It was inserted into the NheI/EcoRI cloning sites of pcDNA-tTG-myc-His and pcDNA-C277S-myc-His constructs resulting in a Myr/Pal tag at the N terminus of tTG or [C277S]tTG. All DNA constructs were verified by automated sequencing.

Cell Culture—Human embryonic kidney (HEK) 293 cells were cultured in Ham's F-12/Dulbecco's modified Eagle's medium (Irvine Scientific) supplemented with 5% fetal bovine serum (HyClone), 2 mM L-glutamine (Invitrogen, Life Technologies, Inc.), 100 µg/ml streptomycin (Invitrogen, Life Technologies, Inc.) and 100 units/ml penicillin (Invitrogen, Life Technologies, Inc.). Cells were grown in humidified atmosphere containing 5% CO2 at 37 °C. Transient transfections were carried out using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions.

Cell Treatment Paradigm—Twenty-four hours after transfection, cells (50–60% confluency) were transferred to serum-free media and 12 h later the cells were treated with 5–8 µM thapsigargin (Alexis) (due to the fact that there was lot to lot variation in the potency of the drug, slightly different concentrations had to be used in different experiments) in serum-free medium for 4 h at 37 °C. The thapsigargin was prepared in Me2SO, and the final concentration of Me2SO in the media was 0.04%. Control cells were treated with 0.04% Me2SO under the same conditions.

Immunoblotting—The cells were washed twice with ice-cold PBS, harvested in SDS buffer (2% SDS, 5 mM EGTA, 5 mM EDTA, 10% glycerol, 0.25 M Tris-Cl, pH 6.8) with protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of each of aprotinin, leupeptin, pepstatin A) or in lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.05% Nonidet P-40, 1 mM EGTA) with protease inhibitors and sonicated on ice. Lysates were clarified, and the protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce). Lysates (20 µg) were diluted with SDS stop buffer (SDS buffer with 10 mM dithiothreitol and 0.01% bromphenol blue), incubated in a boiling water bath for 5 min, electrophoresed on 8% SDS-polyacrylamide gels, transferred to nitrocellulose and probed with one of the following antibodies: anti-tTG (TG100) (NeoMarkers), anti-histone H1 (Upstate), anti-actin (Chemicon), anti-{beta}-tubulin (gift from Dr. L. Binder), anti-insulin receptor {beta} (BD Biosciences), or anti-Rb (IF8) (Santa Cruz Biotechnology). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, the immunoblots were developed using enhanced chemiluminescence (ECL) (Amersham Biosciences).

Caspase-3 Assay—After treatment, cells were harvested in media, rinsed once with ice-cold PBS, and resuspended in lysis buffer. Caspase-3 activity was measured as described previously (15).

Nuclear Fractionation—Cells were washed twice, harvested in ice-cold PBS and the cell pellets were resuspended in lysis buffer by triturating followed by centrifugation at 380 x g for 5 min at 4 °C. The supernatants were collected and used as the cytosolic fractions. The pellets were washed once in lysis buffer and twice in wash buffer (30 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 1 mM EGTA, 25 mM NaCl) with protease inhibitors. The crude nuclei were overlaid on the top of 1 M sucrose with protease inhibitors, and spun at 1200 x g for 10 min at 4 °C. The pellets were collected and resuspended in buffer B (300 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 1 mM EGTA, 25 mM NaCl, 0.5% Triton X-100) with protease inhibitors and used as the nuclear fractions.

Plasma Membrane Fractionation—Plasma membrane fractions were prepared as described previously (30). Briefly, cell surfaces were labeled with sulfosuccinimidyl 2-(biotinamide) ethyl-1,3-dithiopropionate (Pierce), cells were then incubated with previously prepared streptavidin-coupled microspheres (Sigma) and harvested. A magnetic bead attractor was used to separate bound fractions (plasma membrane) from unbound fractions (cytosol, organelles). Plasma membranes were then separated from the microspheres using buffer with 0.1 M {beta}-mercaptoethanol (Sigma). Cytosolic fractions were separated from organelles by high-speed centrifugation of the unbound fractions. Plasma membrane fractions and cytosol fractions were precipitated separately using 2% sodium deoxycholate and 24% trichloroacetic acid, and pellets were resuspended in SDS buffer.

In Situ Transglutaminase Activity Assay—In situ transglutaminase activity was measured as described previously (31). Briefly, cells were labeled with 2 mM 5-(biotinamido)pentylamine (BAP) (Pierce) for 2 h and then treated with thapsigargin for 4 h. Cells were collected, washed, and pelleted. Pellets were resuspended in homogenization buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA), sonicated on ice, and protein concentrations were determined using the BCA assay. The incorporation of BAP into proteins was quantified as described previously (31).

Luciferase Assay—HEK293 cells were transiently transfected with the indicated tTG or C277S expression vector or an espy vector, E2F-pTA-Luc reporter vector (Clontech, BD Biosciences) After 24 h cells were serum-starved overnight, and subsequently treated with thapsigargin or Me2SO for 4 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System Kit (Promega) and a TD-2021 luminometer (Turner Designs). For each sample, the Firefly luciferase data were normalized to the Renilla luciferase values.

Immunoprecipitation—Cells were collected in medium and centrifuged at 1800 x g for 10 min. Pellets were rinsed once with ice-cold PBS and resuspended in IP extraction buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Triton X-100, 2 mM EGTA, 2 mM EDTA, 1 mM Na3VO4, 100 nM okadaic acid, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin, aprotinin, pepstatin A). Cell lysates were sonicated, precleared with protein G Sepharose 4 Fast Flow (Amersham Biosciences) for 1 h, and protein concentrations were determined using the BCA assay. Cell lysates (200 µg of protein) were then incubated with anti-Rb (IF8) (0.6 µg) antibody (Santa Cruz Biotechnologies) overnight at 4 °C. Cell lysates were then incubated with protein G Sepharose 4 Fast Flow (Amersham Biosciences) for 2 h at 4 °C. Beads were washed twice with IP extraction buffer, resuspended in SDS stop buffer and incubated in a boiling water bath for 10 min to extract all the immunoprecipitated proteins. After spinning, supernatants were electrophoresed on 8% SDS-polyacrylamide gels, and subsequently immunoblotted with anti-TG (TG 100) (NeoMarkers), anti-Rb (IF 8) (Santa Cruz Biotechnology), anti-E2F-1 (KH 95) (Santa Cruz Biotechnology), anti-histone deacetylase (HDAC) 1 (Oncogene) or anti-histone H2B (Cell Signaling) antibodies.

Statistics—All data were analyzed using analysis of variance. The values were considered significantly different when p < 0.05. Results were expressed as mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of tTG Constructs in HEK293 cells—To determine the relative expression levels of the different tTG constructs, HEK293 cells were transiently transfected with each tTG construct, collected 24 h later and immunoblotted for tTG. The results shown in Fig. 1 demonstrate that the expression levels of all the constructs were approximately equivalent. The untagged tTG and C277S migrated at ~77 kDa, while the addition of the NLS and Myr/Pal tags resulted in a slight decrease in electrophoretic mobility of the tagged proteins (Fig. 1). Actin blots are shown as loading controls (Fig. 1).



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  		FIG. 1.
Representative immunoblots of expression levels of wild-type (A) and C277S (B) tTG constructs in transiently transfected HEK293 cells. HEK293 cells were transiently transfected with vector only (pc) or wild-type (A) or C777S (B) tTG constructs with no subcellular localization signal (tTG); the nuclear localization signal (NLS) or the membrane localization signal (MP). Twenty-four hours after transfection cells were collected and lysates were immunoblotted for tTG or actin. These data demonstrate that the tTG constructs are expressed at similar levels. The positions at which molecular mass standards (kDa) migrated are indicated at the left.

 
Subcellular Localization of tTG in Transfected Cells—The subcellular localization of the differentially tagged tTG constructs was verified by performing fractionation experiments of transiently transfected HEK293 cells. To determine the subcellular localization of the NLS-tagged tTG constructs (NLS-tTG, NLS-C277S), cells were separated into cytosolic and nuclear fractions. As expected, small amounts of tTG and C277S were found in the nucleus (Fig. 2A) as it has been previously reported for endogenous tTG (17, 18), however NLS-tTG and NLS-C277S were found predominantly in the nucleus (Fig. 2A). The lighter lower band in the cytosolic fractions from the cells transfected with NLS-tTG or NLS-C277S corresponds to endogenous tTG (Fig. 2A). To confirm the purity of the cytosolic and nuclear fractions, the fractions were immunoblotted for histone, a nuclear protein or {beta}-tubulin, a cytosolic protein. Histone was found only in the nuclear fractions, while {beta}-tubulin was only in the cytosolic fractions (Fig. 2A). To determine the subcellular localization of the Myr/Pal-tagged tTGs (Myr/Pal-tTG, Myr/Pal-C277S), transfected cells were separated into plasma membrane and cytosolic fractions. These results demonstrate that tTG and C277S were found only in the cytosol, while Myr/Pal-tTG and Myr/Pal-C277S were found predominantly in the membrane fraction (Fig. 2B). The purity of isolated fractions was confirmed by immunoblotting for the insulin receptor {beta}-subunit, a plasma membrane protein and {beta}-tubulin. As expected, the insulin receptor {beta}-subunit was present only in the membrane fractions and {beta}-tubulin in the cytosolic fractions (Fig. 2B).



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  		FIG. 2.
Differentially tagged tTG proteins localize to different cellular compartments. A, nuclear fractionation: HEK293 cells were transiently transfected with wild-type tTG or [C277S]tTG constructs with no localization signal (tTG) or with the NLS signal (NLS). Cell lysates were separated into nuclear (n) and cytosolic (c) fractions. Fractions were immunoblotted for tTG, histone or {beta} tubulin. B, membrane fractionation: HEK293 cells were transiently transfected with wild-type or C277S constructs with no localization signal (tTG) or with a Myr/Pal signal (MP). Cell lysates were separated into membrane (m) and cytosolic (c) fractions. Fractions were immunoblotted for tTG, insulin receptor {beta}-subunit (Insulin rec. {beta}) or {beta}-tubulin. These data demonstrate that the NLS tag localizes tTG to the nucleus, and the Myr/Pal tag results in tTG localizing to the membrane.

 
In Situ Transglutaminase Activity in Transfected Cells—To determine how the transamidating activity of the different tTG constructs was affected in the thapsigargin treated cells an in situ transglutaminase assay was used (31). Thapsigargin, an inhibitor of the Ca2+-ATPase of endoplasmic reticulum, has been shown to produce an increase in intracellular calcium levels in numerous cell types (32–34). Additionally, prolonged thapsigargin treatment has been shown to induce apoptotic cell death in various cell models (23–25). Our results clearly demonstrate that thapsigargin treatment significantly increased in situ transglutaminase activity in the cells transfected with tTG or Myr/Pal-tTG, but not in cells transfected with the C277S mutants when compared with cells transfected with vector only (Fig. 3A). When whole cell lysates were used in the assay, no significant increase in transglutaminase activity was observed in the NLS-tTG-transfected cells compared with vector-transfected cells after thapsigargin treatment (Fig. 3A). However, when transglutaminase activity was measured in crude nuclear fractions, a significant increase was observed in the NLS-tTG-transfected cells in response to thapsigargin treatment, with no significant increase occurring in the NLS-C277S-transfected cells (Fig. 3B).



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  		FIG. 3.
In situ transglutaminase activity of HEK293 cells transiently transfected with different tTG constructs. HEK293 cells were transiently transfected with the different tTG constructs, followed by BAP labeling and treatment with thapsigargin. In situ transglutaminase (TGase) activity was determined as described under "Experimental Procedures." The data are expressed as a percent of activity in the vector-transfected group treated with thapsigarin. A, in situ transglutaminase activity in the whole cell lysates: cells transfected with wild-type Myr/Pal (MP) or untagged (tTG) constructs showed significantly greater transglutaminase activities than the vector-transfected control group. In contrast none of the C277S mutant-transfected cells (C277S) showed significant increases in the transglutaminase activity compared with the vector-transfected group. B, in situ transglutaminase activity in crude nuclear fractions: cells transfected with pcDNA vector (pc), or NLS-tagged (NLS) wild-type (tTG) or [C277S]tTG (C277S) constructs were labeled with BAP and subsequently treated with thapsigargin. Crude nuclear fractions were prepared and in situ transglutaminase (TGase) activity was measured. Results were expressed as a percent of the values obtained with the vector-transfected group treated with thapsigargin (pc). Crude nuclei from cells expressing wild-type NLS-tTG showed significantly greater transglutaminase activity compared with the pc or NLS-C277S-transfected cells. Results are expressed as mean ± S.E., n = 3 separate experiments *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with pc-transfected cells.

 
Intracellular Localization and Transamidating Activity of tTG Differentially Impacts Thapsigargin-induced Apoptosis— There is increasing evidence for the involvement of tTG in the process of apoptosis. Several studies have suggested that tTG is pro-apoptotic (21, 22) while other studies have shown that tTG attenuates apoptosis (14–16). Interestingly, it has been shown that the effects of tTG on apoptosis may depend on the type of apoptotic inducer and how the stress impacts the activity of tTG (15). To investigate how the localization and activity of tTG affects apoptosis, HEK293 cells were transiently transfected with the different tTG constructs and subsequently treated with thapsigargin (or vehicle only) prior to measuring caspase activity as an indicator of apoptosis. Thapsigargin treatment resulted in a significant increase in caspase-3 activation in all transfected cells. However, caspase-3 activity was significantly greater in cells transfected with tTG, which is predominantly cytosolic, compared with cells transfected with vector only (Fig. 4B), while expression of NLS-tTG or Myr/Pal-tTG did not significantly affect caspase-3 activity compared with the vector (pc) control groups (Fig. 4A). Expression of C277S without any subcellular localization signal or Myr/Pal-C277S also did not have any effect on caspase-3 activation in response to thapsigargin treatment relative to the vector (pc)-transfected cells (Fig. 4B). However, thapsigargin-induced caspase-3 activation was significantly attenuated in the cells transiently expressing transamidation inactive tTG mutant, localized in the nuclear compartment (NLS-C277S) (Fig. 4B). These data indicate that tTG localized to the cytosol that is active as a transamidating enzyme is pro-apoptotic, while nuclear-localized tTG that is only active as a GTP binding protein is protective for the cell.



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  		FIG. 4.
Caspase-3 activity is significantly increased in cells expressing wild-type tTG and significantly decreased in cells expressing NLS-C277S. HEK293 cells were transiently transfected with vector only (pc) or the indicated wild-type (A) or C277S (B) tTG constructs, followed by treatment with thapsigargin (+Thap, closed bars) or Me2SO (–Thap, open bars). All data are expressed as a percent of vector control (pc, –Thap). A, after thapsigargin treatment cells expressing wild-type tTG (tTG) showed significantly increased caspase-3 activity compared with the pc-transfected cells. Caspase activity in cells transfected with Myr/Pal-tagged tTG (MP) or NLS-tagged tTG (NLS) was not different from the vector-transfected group (pc). B, in contrast, expression of NLS-tagged [C277S]tTG (NLS) significantly attenuated caspase-3 activation compared with pc-transfected cells (pc). Untagged C277S (tTG) and Myr/Pal-tagged [C277S]tTG (MP) did not significantly affect caspase activity compared with vector controls. Results are expressed as mean ± S.E., n = 3 separate experiments, **, p < 0.01; ***, p < 0.001.

 
Tissue TG Differentially Interacts with Rb in an Activity- and Localization-dependent Manner—Further studies were carried out to determine the mechanism by which NLS-C277S was anti-apoptotic. Previous reports have provided evidence that tTG may modify Rb via transamidation, although a direct interaction of tTG and Rb was not demonstrated (14, 35). Since Rb is a nuclear phosphoprotein that has important functions in the regulation of apoptosis (26), we decided to investigate if there was an interaction between tTG and Rb in our cell model. Although, the primary site of action of Rb is the nucleus, it is also found in the cytosol (36–40). Therefore, HEK293 cells were transfected with cytosolic-localized (tTG or C277S) or nuclear-localized (NLS-tTG or NLS-C277S) constructs and subsequently incubated in the absence or presence of thapsigargin. Cell lysates were prepared, immunoprecipitated for Rb and immunoblotted for tTG. These findings clearly demonstrate that tTG complexes with Rb in situ both in the cytosol and nucleus (Fig. 5). More interestingly, in the nucleus this interaction is dynamic and dependent upon the transamidating activity of tTG, as after induction of apoptosis nuclear-localized wild-type tTG (NLS-tTG) is released from the complex with Rb, while the binding of the nuclear-localized transamidating inactive mutant (NLS-C277S) tTG to Rb increases (Fig. 5B). In contrast, the interaction of tTG with Rb protein in the cytosol is unaffected by thapsigargin treatment. To determine if this effect was specific for Rb, the interaction between nuclear-localized tTGs (NLS-tTG and NLS-C277S) and histone H2B, another nuclear protein reported to interact with tTG and be its substrate (41, 42) was investigated. Histone H2B complexed with both NLS-tTG and NLS-C277S but there was no changes in this interaction after apoptosis was induced (data not shown). This suggests that the dynamic character of tTG interaction with Rb in the nucleus after induction of apoptosis is specific and that this interaction may be significant for tTG effects on apoptosis when localized in the nucleus.



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  		FIG. 5.
tTG differentially interacts with Rb in an activity and localization-dependent manner. HEK293 cells were transiently transfected with untagged (A) or NLS-tagged (B) tTG or C277S constructs, followed by treatment with thapsigargin (+ Thap) or Me2SO (– Thap). Cell lysates were immunoprecipitated (IP) with the Rb antibody and immunoprecipitates were immunoblotted (Blot) for tissue transglutaminase (tTG) and then stripped and reprobed for Rb. Cell lysates were also immunoblotted (WB) for tTG. A, after induction of cell death there were no changes in interactions of Rb with cytosolic-localized tTGs. B, in contrast the binding of Rb with nuclear-localized tTGs was altered after induction of cell death. Binding of Rb to NLS (wild-type) was decreased, while binding of Rb to NLS (C277S) was increased after thapsigargin treatment.

 
The Interaction of tTG with Rb in the Nucleus Does Not Alter the Ability of Rb to Bind and Repress E2F Transcriptional Activity or the Stability of Rb—Although Rb interacts with over a hundred different proteins, the E2F transcription factor remains the most investigated of the Rb protein partners (43). Rb binding to E2F suppresses E2F driven transcription. Therefore, Rb-E2F interactions play a pivotal role in regulating the expression of genes that are important for the execution of the apoptotic cell death process such as p53, Apaf1, and others (44). Given the importance of Rb-E2F interactions in the regulation of apoptosis, we investigated if tTG binding to Rb affects Rb-E2F interactions and/or E2F transcriptional activity. Cells were transfected with pcDNA, NLS-tTG, or NLS-C277S and subsequently incubated in the absence or presence of thapsigargin. Cell lysates were immunoprecipitated for Rb and immunoblotted for E2F. No changes in Rb-E2F interaction were detected between the groups (Fig. 6A). To measure transcriptional activity of E2F, cells were transfected with an E2F reporter vector and Renilla vector as an internal control, together with the expression vectors (pcDNA, NLS-tTG, or NLS-C277S), and subsequently incubated in the absence or presence of thapsigargin prior to the measurement of luciferase activity. These data demonstrate that treatment with thapsigargin did not result in significant changes in E2F activity, although E2F transcriptional activity was slightly, although statistically decreased (~30%) in the NLS-tTG expressing cells compared with vector-transfected (pc) cells (Fig. 6B). Rb also interacts with and regulates HDAC activity (45), and this may have important effects on the apoptotic process through transcriptional repression (46). Therefore we also investigated the effects of NLS-tTG and NLS-C277S on Rb-HDAC interactions using the same protocol as for Rb-E2F interactions. Neither NLS-tTG nor NLS-C277S had any effect on Rb-HDAC interactions in the absence or presence of thapsigargin (data not shown).



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  		FIG. 6.
The interaction of tTG with Rb in the nucleus did not affect the interaction of Rb with E2F (A) or E2F transcriptional activity (B). A, HEK293 cells were transiently transfected with pcDNA vector (pc) or NLS (wild-type [tTG] or C277S) tTG constructs, followed by treatment with thapsigargin (+ Thap) or Me2SO (– Thap). Cell lysates were immunoprecipitated (IP) with Rb antibody and immunoblotted (Blot) for E2F1, and then the blots were stripped and reprobed for Rb. Cell lysates were also blotted (WB) for tTG. Levels of Rb binding to E2F1 were equivalent in all groups. B, HEK293 cells were transiently transfected with pcDNA vector (pc) or the wild-type (tTG) or C277S NLS-tagged constructs, E2F-pTA-Luc (Firefly luciferase) reporter construct and an internal control pRL-TK vector (Renilla luciferase), followed by treatment with thapsigargin (+ Thap) or Me2SO (– Thap) and luciferase activity was measured in cell lysates. Luciferase activity was significantly suppressed in cells expressing the NLS-tagged wild-type tTG compared with vector-transfected cells (pc). However, there were no differences in the levels of induction of E2F transcription, after thapsigargin treatment between the groups. Results are expressed as mean ± S.E., n = 4 separate experiments; ***, p < 0.001.

 
It has been proposed that the levels of Rb are important in regulating its affects on apoptosis and several studies have shown that cleavage of Rb occurs early in apoptosis (47–49). It has also been shown that cells expressing caspase-resistant Rb are protected from apoptosis (50). Therefore, we investigated if NLS-tTG or NLS-C277S affected Rb stability during thapsigargin-induced apoptosis. HEK293 cells were transfected with either NLS-tTG or NLS-C277S followed by treatment with thapsigargin. Although Rb was degraded 24 h after treatment with thapsigargin, the results of these studies revealed the extent of Rb degradation was not different between NLS-tTG (0.58 ± 0.15, n = 3 experiments) and NLS-C277S (0.69 ± 0.21, n = 3 experiments)-transfected cells, indicating that NLS-C277S is not attenuating apoptosis by increasing the stability of Rb.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Tissue TG has been shown to be both pro- and anti-apoptotic (15, 16, 21). Indeed tTG is increased both in conditions such as neurodegenerative disorders where there is increased cell death in specific brain regions (51–53), and in some tumors which are characterized by a lack of apoptosis (54, 55). Therefore it is of critical importance to investigate the role of tTG in the process of apoptosis. The present study demonstrates for the first time that the effects of tTG on the apoptotic cell death process are strongly dependent upon its localization in the cell. Wild-type tTG localized predominantly in the cytosolic compartment facilitated apoptosis, while both membrane and nuclear-localized wild-type tTGs did not significantly affect the apoptotic cell death process. Further, our study emphasizes the importance of the transamidating activity of tTG in the cytosol for the facilitation of the apoptotic cell death process since the tTG pro-apoptotic effect in the cytosolic compartment was completely abolished in cells expressing the transglutaminase inactive mutant tTG-C277S. In contrast to the pro-apoptotic function of wild-type tTG, the transglutaminase inactive mutant [C277S]tTG significantly protected cells against apoptosis when it was shuttled to the nuclear compartment. Therefore this study provides significant evidence that the nuclear space is the place where tTG exerts its anti-apoptotic effects. Overall, the findings presented in this paper begin to delineate the seemingly contradictory roles of tTG as both a facilitator and suppressor of apoptosis.

In the cell, tTG is present mostly in the cytosol, although it is also in the nucleus and associated with the plasma membrane (17, 19). In this study we demonstrate that when tTG is localized mostly in the cytosol, it facilitates apoptosis. Further, and in agreement with a previous study (15), we found that the transamidating activity of tTG is necessary for its pro-apoptotic activity in the cytosol. Both membrane and nuclear-localized wild-type tTG did not affect apoptosis, even though thapsigargin treatment resulted in a significant increase in the transamidating activity of these constructs. These data suggest that tTG must modify specific substrates in the cytosol to be proapoptotic, and sequestering tTG at the membrane or in the nucleus prevents these interactions.

It has been shown that tTG migrates to the nucleus in response to specific stressors (17), and importin-3 has been identified as a possible transporter responsible for the translocation of tTG into the nucleus (56). Here, we demonstrate that tTG localized in the nucleus attenuates apoptosis when it is not active as a transamidating enzyme. Therefore, translocation of tTG into the nucleus may be protective for the cell. So why is nuclear tTG without transamidating activity anti-apoptotic? Previous studies have suggested that tTG may regulate Rb activity (14, 35). Rb is a nuclear phosphoprotein that is important regulator of proliferation, differentiation and apoptosis (26, 57, 58). Therefore, we investigated the interaction of tTG with Rb in our apoptosis model. We found that tTG interacts with Rb both in cytosol and nucleus. Further, the C277 site that is critical for transamidating activity is not necessary for this interaction. Interestingly, after apoptosis was induced, nuclear-localized tTG with transamidating activity (NLS-tTG) was released from Rb, while the interaction between the transamidating inactive mutant (NLS-C277S) and Rb in the nucleus increased. The interaction between tTG and Rb in the cytosol was not altered after apoptosis was induced. This is the first demonstration that tTG interacts with Rb in an intracellular localization and transamidating activity dependent manner in response to an apoptotic stimulus. These findings indicate that tTG is protective against apoptosis only when it is in the nucleus and is not active as a transamidating enzyme and that its interaction with Rb may play a central role in this process. These studies differ somewhat from a previous report (14) in which it was postulated that Rb was modified by tTG and that this resulted in decreased proteolysis of Rb, which was antiapoptotic. However in this previous study (14) there was no direct demonstration that Rb was modified by tTG and therefore the anti-apoptotic effects of tTG maybe due to the interaction of tTG and Rb, rather than a modification of Rb. It is important to mention that we also investigated the interaction of tTG with another nuclear protein, histone H2B, which has been reported to be a tTG substrate and to interact with tTG (41, 42) and no differences in this interaction were observed before or after induction of apoptosis with any of the tTG constructs. Therefore the dynamic character of tTG interaction with Rb is specific, substantiating the hypothesis that this interaction may be significant for the effect of tTG on apoptosis when localized in the nucleus.

Rb likely plays an important role in apoptosis as Rb-deficient mice die very early, and show increased susceptibility to apoptosis in several developing tissues (59, 60). The question is if the binding of tTG to Rb affects Rb function or its stability. Rb is a phosphoprotein and when Rb is hypophosphorylated it binds to E2F and keeps E2F transcriptionally inactive, and therefore is anti-apoptotic (44). Also, when bound to E2F, Rb recruits HDAC to repress transcription (46). Although NLS-C277S interacted with Rb and was anti-apoptotic, this interaction had no effect on E2F activity or Rb interaction with HDAC. However, it has been suggested that its not the phosphorylation state of Rb that is important for regulation of apoptosis, but the overall level of Rb in the cell (26). Through interaction with E2F Rb can suppress transcription of different apoptotic genes. However, apoptosis in Rb-null embryos is not always suppressed by E2F gene disruption (61). These findings indicate that Rb can be anti-apoptotic through mechanisms other than suppression of E2F activity. Therefore we examined the overall levels of Rb before and after apoptosis was induced. Although, Rb was degraded in response to treatment with thapsigargin, there were no differences in the extent of proteolysis between NLS-C277S and NLS-tTG-expressing cells. Therefore, further studies are required to elucidate the mechanisms by which NLS-C277S attenuates the apoptotic process.

How is it decided if tTG will be pro-apoptotic, anti-apoptotic, or have no effect on apoptosis? It has been shown that tTG modulates apoptosis in a stimulus dependent manner (15). It is well known that for tTG to exhibit transamidating activity, an increase in intracellular calcium levels is necessary (31). Since different apoptotic stimuli activate different signaling pathways and differentially effect intracellular calcium levels, it can be postulated that increases in the levels of calcium in certain cellular compartments in response to a specific stressor is one of the factors that determines the effects of tTG on the apoptotic process. Indeed, tTG potentiated apoptosis in response to osmotic stress, a stimulus that significantly increases in situ TG activity (15), most likely due to an increase in intracellular calcium (62, 63). In contrast tTG protected against heat shock induced apoptosis, a stressor that did not increase tTG activity (15). Another deciding factor would be how a certain stressor affects tTG transport to the nucleus. Therefore it can be hypothesized that the combined effects of the concentration of calcium within a given intracellular compartment, and the location of tTG within the cell likely determine how tTG modulates apoptosis.

In conclusion, we believe that the present findings contribute to our understanding of the complex role of tTG in apoptosis. These findings clearly demonstrate that the transamidating activity of tTG is essential for its pro-apoptotic effects, while the anti-apoptotic effects of tTG only occur in the absence of transamidating activity. Further, we demonstrate for the first time that nuclear localization of tTG is protective against apoptosis when it is not active as a transamidating enzyme. The anti-apoptotic effects of tTG in the nucleus may due to the increased interaction of tTG with Rb. Overall, it is clear that tTG plays an important, but complex regulatory role in the process of apoptosis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant AG12396. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Psychiatry, University of Alabama at Birmingham, 1720 7th Ave. South, SC1061, Birmingham, AL 35294-0017. Tel.: 205-934-2465; Fax: 205-934-3709; E-mail: gvwj{at}uab.edu.

1 The abbreviations used are: tTG, tissue transglutaminase; Rb, retinoblastoma protein; HDAC, histone deacetylase; PBS, phosphate-buffered saline; NLS, nuclear localization signal; Pipes, 1,4-piperazinediethanesulfonic acid; Me2SO, dimethyl sulfoxide; BAP, 5-(biotinamido)pentylamine; BCA, bicinchoninic acid. Back



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