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Originally published In Press as doi:10.1074/jbc.M307215200 on August 19, 2003

J. Biol. Chem., Vol. 278, Issue 48, 48066-48073, November 28, 2003
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A Novel Tumor Suppressor Protein Promotes Keratinocyte Terminal Differentiation via Activation of Type I Transglutaminase*

Michael T. Sturniolo{ddagger}, Shervin R. Dashti{ddagger}, Anne Deucher{ddagger}§, Ellen A. Rorke||, Ann-Marie Broome{ddagger}, Roshantha A. S. Chandraratna**, Tiffany Keepers{ddagger}, and Richard L. Eckert{ddagger}§{ddagger}{ddagger}§§¶¶||||

From the Departments of {ddagger}Physiology and Biophysics, {ddagger}{ddagger}Dermatology, §Biochemistry, §§Reproductive Biology, ¶¶Oncology, and ||Environmental Health Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970 and **Retinoid Research, Departments of Chemistry and Biology, Allergan Pharmaceuticals, Irvine, California 92713

Received for publication, July 7, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tazarotene-induced protein 3 (TIG3) is a recently discovered regulatory protein that is expressed in the suprabasal epidermis. In the present study, we show that TIG3 regulates keratinocyte viability and proliferation. TIG3-dependent reduction in keratinocyte viability is accompanied by a substantial increase in the number of sub-G1 cells, nuclear shrinkage, and increased formation of cornified envelope-like structures. TIG3 localizes to the membrane fraction, and TIG3-dependent differentiation is associated with increased type I transglutaminase activity. Microscopic localization and isopeptide cross-linking studies suggest that TIG3 and type I transglutaminase co-localize in membranes. Markers of apoptosis, including caspases and poly(ADP-ribose) polymerase, are not activated by TIG3, and caspase inhibitors do not stop the TIG3-dependent reduction in cell viability. Truncation of the carboxyl-terminal membrane-anchoring domain results in a complete loss of TIG3 activity. The morphology of the TIG3-positive cells and the effects on cornified envelope formation suggest that TIG3 is an activator of terminal keratinocyte differentiation. Our studies suggest that TIG3 facilitates the terminal stages in keratinocyte differentiation via activation of type I transglutaminase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TIG31 is a novel growth regulatory protein that is a member of the H-rev107 protein family (1). This family includes H-rev107 (2), RIG1/TIG3 (1, 3), H-rev107-1 (4), H-rev107-2 (4), and A-C1 (5). Immunostaining of epidermis reveals that TIG3 is expressed in the non-proliferating, suprabasal differentiated epidermal layers, but not in the proliferative basal cells (6), suggesting that TIG3 may play an important regulatory role during keratinocyte differentiation. TIG3 levels are reduced in tumor cells, consistent with the idea that the loss of TIG3 function may contribute to disease progression in cancer (1). However, the amino acid sequence of TIG3 does not reveal any particular motif that predicts a function, and no information is available regarding the TIG3 mechanism of action. Thus, identifying the mechanism of TIG3 action is an important goal that requires knowledge regarding the effects of TIG3 on cell regulatory mechanisms. In the present study, we show that TIG3 expression in cultured keratinocytes causes a cessation of cell proliferation and enhanced cornified envelope formation. Our results suggest that TIG3 associates with membranes and that the mechanism leading to reduced cell viability involves activation of type I transglutaminase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Adenovirus Infection—The tetracycline-regulated recombinant adenovirus, tAd5-TIG31–164, was constructed by cloning the TIG31–164 coding sequence, linked to an SV40 transcription terminator, into tAd5. This adenoviral vector contains a tetracycline-responsive element that includes the cytomegalovirus promoter and tetracycline operator. Activation of this promoter requires the presence of the tetracycline transactivator (TA) protein that is provided by co-infection with a second adenovirus, Ad5-TA. The ability of the TA protein to activate via the tetracycline operator can be reduced by addition of tetracycline. An empty recombinant adenovirus tAd5-EV was constructed for use as a control. A green fluorescent protein (GFP)-encoding adenovirus, Ad5-GFP, was used to determine keratinocyte infection efficiency. At greater than 10 plaque-forming units/cell, virtually 100% of the cells are infected (not shown). Human foreskin keratinocytes were cultured in keratinocyte serum-free media (KSFM, Invitrogen). Third passage cultures were plated at 15,000–20,000 cells/cm2. After 24 h, the cells were infected with adenovirus for 12 h in KSFM containing 2.5 ng of polybrene/ml. The cells were then washed and incubated in fresh virus-free KSFM for varying times prior to harvest.

Cell Viability and Proliferation—Trypan blue exclusion and colorimetric XTT-based assays were used to measure cell viability and proliferation. For dye exclusion, cells, suspended in 200 µl of divalent cation-free phosphate-buffered saline (PBS), were added to 500 µl of 0.4% (w/v) trypan blue solution (Sigma catalog numberT8154) and maintained for 5 min at 37 °C prior to counting. Alternatively, cell viability and proliferation were measured using an assay based on the cleavage of the tetrazolium salt XTT. In this assay, only viable cells are able to convert the XTT to an orange product. Cells growing in 96-well plates are incubated with the XTT salt for 4–24 h, and the orange cleavage product is detected using an enzyme-linked immunosorbent assay reader.

Fluorescence Microscopy—Keratinocytes growing on coverslips were fixed in 2% paraformaldehyde, permeabilized with methanol, and incubated with rabbit anti-TIG3 (1:100) (7) followed by CY3-conjugated goat anti-rabbit IgG (1:1000, Sigma). After washing, cells were treated with 1 µg/ml Hoechst 33258 to stain nuclei. The cells were washed, placed onto slides, and sealed with N-propyl galate for microscopic visualization by epifluorescence.

Immunoblot Methods—Equivalent quantities of protein were electrophoresed on 10% acrylamide gels, and separated proteins were transferred to a nitrocellulose membrane for immunoblot as described previously (7). The blots were then incubated overnight at 4 °C with primary antibody. Antibody binding was detected by incubation for 1 h with horseradish peroxidase-conjugated secondary antibody, and the antibody complex was visualized using chemiluminescence detection reagents.

Flow Cytometry—Keratinocytes were trypsinized, washed twice with cold PBS, and resuspended at 2 x 106 cells/50 µl of PBS containing 0.05% formaldehyde (37 °C for 10 min and 4 °C for 10 min). The cells were permeabilized by addition of 450 µl of methanol and stored at –20 °C. After methanol treatment, cells were incubated for 10 min at 4 °C in 500 µl of PBS containing 50 µg/ml RNase followed by incubation for 30 min in 500 µl of PBS containing 100 µg/ml propidium iodide. The cells were then sorted.

Fluorescent Detection of Transglutaminase Substrate Incorporation in Situ—Human keratinocytes, growing on coverslips, were treated with either 20 m.o.i. tAd5-TIG31–164 or 20 m.o.i. tAd5-EV with 5 m.o.i. Ad5-TA in KSFM containing 2.5 µg/ml polybrene for 12 h followed by addition of fresh virus-free medium. At 48 h after infection, fresh KSFM containing 100 µM fluorescein cadaverine (FC) (catalog number A-10466, Molecular Probes) was added for 4 h. The cells were then washed with PBS and fixed with 100% methanol at –20 °C, washed twice with cold methanol, and washed twice more with PBS prior to placing the samples onto slides using N-propyl galate for visualization by fluorescent microscopy using a Nikon Optiphot fluorescence microscope.

Cell Fractionation and Transglutaminase Activity Assay—Cells (1 x 50 cm2 dish) were washed with PBS, collected by scraping, and homogenized in 1 ml of lysis buffer (10 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 10 µg/ml phenylmethylsulfonyl fluoride). The extract was centrifuged at 100,000 x g for 30 min to yield the supernatant fraction (cytosol) containing soluble type II transglutaminase. The pellet was homogenized in lysis buffer containing 0.2% Triton X-100 followed by centrifugation at 100,000 x g for 30 min at 4 °C to yield membrane-associated type I transglutaminase. An equivalent amount of protein from each fraction was added to the reaction tube in a total volume of 125 µl of homogenization buffer (100 mM Tris-HCl, pH 7.6, 4 mM CaCl2, 5 mM dithiothreitol, 10 µg/ml phenylmethylsulfonyl fluoride, 0.5 mg casein, and 0.5 µCi of [3H]putrescine). Samples were incubated at 37 °C for 1 h, precipitated, washed with trichloroacetic acid, washed with ethanol, and air-dried. [3H]Putrescine incorporation into the pellet was assayed by liquid scintillation counting.

Caspase 3 Activity Assay—Human keratinocytes (one 10-cm2 dish/point) were washed twice with cold PBS and lysed in 50 µl of caspase buffer (25 mM HEPES, pH 7.4, 10% sucrose, 0.1% CHAPS, 2 mM EDTA, 5 mM dithiothreitol) (8). Cell lysates were centrifuged for 3 min at 15,000 x g, and the supernatant was saved for analysis. Supernatant (15 µg of protein in 50 µl of lysis buffer) was added to 50 µl of 2x assay buffer (25 mM HEPES, pH 7.4, 5 mM dithiothreitol, 100 µM Ac-DEVD-AFC) and incubated at 37 °C for 3 h. The caspase 3 substrate, Ac-DEVD-AFC, was obtained from Enzyme Systems. Caspase 3-dependent release of fluorogenic AFC product was monitored at 360-nm excitation and 530-nm emission using a Shimadzu RF-5301 PC spectrofluorophotometer. Values were corrected for background and expressed as pmols of converted substrate/mg of protein/h.

UVB Treatment—Near confluent (70%) keratinocytes (one 50-cm2 dish) were exposed to 60 mJ/cm2 of Kodacel-filtered UVB light and harvested at 20 h after treatment.

Inhibition of Caspase Activity Using Z-VAD-FMK—Keratinocytes were infected with 5 m.o.i. of Ad5-TA and 20 m.o.i. of either tAd5-EV or tAd5-TIG31–164. At 5 h after infection, fresh KSFM was added containing 50 µM of the pan-caspase inhibitor Z-VAD-FMK. The medium was changed every 24 h with fresh addition of Z-VAD-FMK. At 48 h, cell viability was assessed by trypan blue exclusion. Parallel dishes were treated with UVB (60 mJ/cm2) in the presence or absence of 50 µM Z-VAD-FMK and then harvested and assayed for viability at 48 h.

Antibodies—Rabbit anti-caspase 3 (AHZ0052, 1:2000) was obtained from Biosource International. Goat anti-caspase 8 (sc-8355, 1:2000) and rabbit anti-caspase 9 (sc-8355, 1:2000) were obtained from Santa Cruz Biotechnology. Mouse anti-poly(ADP-ribose) polymerase (PARP) (55494, 1:1000) was obtained from Pharmingen. Mouse anti-human {beta}-actin (A5441, 1:10,000) was obtained from Sigma. Rabbit anti-human involucrin (1:2000), rabbit preimmune serum, and rabbit anti-TIG3 (1:2000) were produced in our laboratory (7). Mouse anti-transglutaminase type 1 was obtained from Biomedical Technologies, Inc. (BT-621, 1:50). Alexa Fluor 488-conjugated goat anti-mouse IgG (A11001, 1:1000) was from Molecular Probes. Cy3-conjugated sheep anti-rabbit IgG (c-2306, 1:1000) was obtained from Sigma. Peroxidase-conjugated donkey anti-rabbit IgG (NA934, 1:8000) and peroxidase-conjugated sheep anti-mouse IgG (NA931, diluted 1:8000) were purchased from Amersham Biosciences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TIG3 Reduces Keratinocyte Cell Number—To study the effects of TIG3 on keratinocyte function, we infected cultured normal human keratinocytes with an empty adenovirus, tAd5-EV, or an adenovirus encoding full-length TIG3, tAd5-TIG31–164. As shown in Fig. 1A, TIG31–164 expression can be detected as early as 12 h after adenovirus infection, and expression is maximal between 24 and 36 h. The appearance of TIG3 is associated with a reduction in keratinocyte cell number beginning at 24 h. At 60 h, the viable cell number is reduced to 10% of the initial density, suggesting that TIG31–164 promotes cell death. To assess the effects of TIG31–164 level on cell viability, cells were infected with tAd5-EV or tAd5-TIG31–164 in the presence of increasing concentrations of tetracycline. TIG3 expression is reduced with increasing tetracycline. As shown in Fig. 1B, reduced keratinocyte cell number is correlated with increased TIG31–164 expression. To characterize cell status, we sorted cells at various times after tAd5-EV or tAd5-TIG31–164 infection. As shown in Fig. 1C, at 24 h after infection, the sub-G1 cell population, i.e. cells with less than a full complement of DNA due to partial apoptotic degradation, is 2.4% in tAd5-EV-infected cells. This number does not increase out to 72 h (not shown). In contrast, TIG31–164 expression increases the sub-G1 cell population from 1.4% at 24 h to 7.2% at 48 h and 21% at 72 h. As a control, we show that UVB treatment of keratinocytes, which promotes apoptosis and accumulation of cells in sub-G1, increases the sub-G1 population to 6.2%.



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  		FIG. 1.
TIG3-mediated cell death is time and dosage dependent. As shown in A, ieratinocytes were infected with 5 m.o.i. of Ad5-TA and 20 m.o.i. of tAd5-TIG3 or empty vector (tAd5-EV) for 12 h. Cells were harvested at 0, 12, 24, 36, 48, and 60 h after the start of infection, and viability was evaluated using trypan blue exclusion. Immunoblot analysis (inset) shows the TIG31–164 expression level at each time point. KER, keratinocytes. B, TIG31–164 dose response. Cells were infected with Ad5-TA and tAd5-TIG31–164 as above, except that the level of tetracycline in the medium was varied from 0–125 ng/ml. After 48 h, the cells were harvested and assayed for the ability to exclude trypan blue. TIG31–164 level was monitored by immunoblot (inset). As shown in C, cells were infected with TIG31–164-encoding virus or empty virus. At the indicated times after infection the cells were stained with propidium iodide and analyzed by flow cytometry. As a positive control for activation of apoptosis, one group of cells was treated with 50 mJ of UVB/cm2 and sorted 16 h later. The percentage of sub-G1 cells is indicated in each panel.

 
TIG3 Regulates Keratinocyte Cell Morphology—TIG3 expression is also associated with altered cell morphology. The photographs, taken 48 h after infection with empty or TIG3-encoding vector (Fig. 2A), indicate that TIG31–164-expressing cells display a cornified envelope-like morphology and retention of cell nuclei (arrows). To determine whether these structures display cornified envelope-like properties, we assessed their ability to survive boiling in detergent and reducing agent. As shown in Fig. 2B, TIG31–164 expression reduces cell number and markedly increases the percentage of cells, from 3.2 to 33%, that are resistant to boiling in detergent and reducing agent, a characteristic of cornified envelopes. To correlate time of TIG31–164 expression with morphological change, cells were infected with tAd5-TIG31–164 and harvested at various times after infection. As shown in Fig. 2C, limited morphology change is observed at 24, 30, and 40 h after infection, but marked changes are observed at 48 h. Nuclear staining (blue) reveals that TIG3 expression (red) is associated with nuclear shrinkage (arrows).



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  		FIG. 2.
TIG3 expression results in morphologic change and cornified envelope formation. As shown in A, cells grown on glass coverslips were infected with 20 m.o.i. of either tAd5-TIG31–164 or tAd5-EV in the presence of 5 m.o.i. of Ad5-TA for 12 h. At 48 h after infection, the cells were fixed and visualized by light microscopy. The arrows indicate cornified structures that have retained nuclei. KER, keratinocytes. As shown in B, TIG31–164 promotes cornified envelope formation. Cells treated as in panel A were harvested at 48 h and assayed for cell number and cornified envelope formation. Cornified envelopes are defined as cells that survive boiling in detergent and reducing agent (16). Cell number and cornified envelope number were evaluated by counting with a hemocytometer. As shown in C, TIG3 overexpression causes nuclear condensation. Keratinocytes, growing on coverslips, were treated with virus as indicated and harvested at the listed times after infection. The cells were then incubated with anti-TIG31–164 in conjunction with a CY3-labeled secondary antibody (red) and stained for nuclear morphology using Hoechst 33258 (blue). The arrows indicate condensed nuclei, which appear at 48 h in tAd5-TIG3-infected cells.

 
TIG3 Regulates Transglutaminase Activity—Transglutaminases (TGs) are a family of enzymes that assemble differentiation- and cell death-associated structures, including the cornified envelope (9–14). To determine whether TIG3 influences TG activity, cells were treated with tAd5-EV or tAd5-TIG31–164 for 48 h. During the last 4 h of treatment, the medium was supplemented with 100 µM FC, a cell-permeable fluorescent transglutaminase substrate. Fig. 3A shows that FC incorporation (green) is greatly increased in TIG3-expressing (red) cells. As a second method of detecting TIG3-dependent transglutaminase activity, Fig. 3B shows that total transglutaminase activity, measured by monitoring [3H]putrescene incorporation, is increased 3-fold by TIG31–164. Separation of lysate into soluble and particulate fractions reveals that most of the increase in transglutaminase activity is observed in the particulate (membrane) fraction, suggesting that membrane-associated type I transglutaminase is activated. The increase in type I transglutaminase (TG1) activity could be due to increased expression of TG1. The immunoblot of total extract prepared from tAd5-EV and tAd5-TIG31–164-infected cells (inset) shows that TG1 levels are not visibly altered by TIG3 treatment. Consistent with activating a membrane-associated target, the cell fractionation study shown in Fig. 3C indicates that TIG3 is also associated with the membrane (Part, particulate) fraction. In these studies, cells were infected with TIG3 and then fractionated into cytosol and particulate fractions. As a control, we demonstrate that involucrin, a cytosolic protein, is retained in the soluble fraction. {beta}-actin is present in both the soluble and the particulate fractions. The membrane co-localization of TIG31–164 and TG1 suggests that TIG31–164 may localize in the vicinity of TG1. Indeed, as shown in Fig. 3D, a fraction of the TIG31–164 present in the particulate fraction migrates at the stacking/separating gel interface (asterisk). This type of migration in TG-regulated systems is indicative of covalent cross-link formation to form high molecular weight species (15–17). This suggests that some of the TIG3 serves as a substrate for transglutaminase, suggesting a close juxtaposition. As expected, the high molecular weight material is also present in the total extract lane but is not present in the cytosol (Sol, soluble). The above results suggest that TIG3 and TG1 co-localize. As an additional method of monitoring this localization, cells were infected with TIG31–164 and, after 48 h, the cells were immunostained with anti-TIG3 (red) and anti-TG1 (green). The epifluorescent images (Fig. 3E) show that TIG3 and TG1 accumulate in punctate aggregates. The yellow color in the combined image shows that TIG3 and TG1 co-localize in these structures, further suggesting that the two proteins are in close proximity.



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  		FIG. 3.
TIG3 overexpression is associated with increased transglutaminase activity. As shown in A, keratinocytes, grown on coverslips, were infected for 12 h with 20 m.o.i. of either tAd5-EV or tAd5-TIG31–164 encoding adenovirus and 5 m.o.i. of Ad5-TA. At 48 h after infection, the cells were pulsed for 4 h with 100 µM FC (green). The cells were then fixed and stained with rabbit anti-TIG3 and Cy3-conjugated anti-rabbit IgG (red) (7) and visualized by fluorescent light microscopy. The combined image (yellow) indicates co-localization of TIG3 and FC incorporation. As shown in B, transglutaminase activity was measured in vitro by monitoring [3H]putrescine incorporation. Cells, growing in 50-cm2 dishes, were infected with either tAd5-EV or tAd5-TIG31–164 for 12 h as in panel A. After 48 h, whole cell and particulate and soluble fractions were isolated and assayed for transglutaminase activity. The open bars indicate activity in cells infected with tAd5-EV, and the hatched bars represent activity in TIG31–164-expressing cells. The error bars represent standard errors of the mean from three experiments. The inset depicts an immunoblot showing that TG1 level is not significantly altered in TIG3-producing cells. A {beta}-actin immunoblot is included as a protein loading control. C, immunoblot analysis of TIG3 distribution in cell fractions. Cells were infected as outlined above. Total, soluble (Sol), and particulate (Part) cell fractions were prepared, and equal cell number equivalents were assayed for TIG3, involucrin (15, 36), and {beta}-actin by immunoblot. D, high molecular weight TIG3-immunoreactive complex. This experiment was performed exactly as in panel C, except that the higher molecular weight region of the gel is shown. The membrane was incubated with anti-TIG3 (7). The asterisk indicates the high molecular weight anti-TIG3 immunoreactive complex. E, TG1 and TIG3 co-localization. Keratinocytes were grown on coverslips and then infected for 48 h with tAd5-TIG31–164. The cells were then fixed, permeabilized, and stained with rabbit anti-TIG3 (1:100)/Cy3-conjugated goat anti-rabbit IgG (1:1000, Sigma) and mouse anti-transglutaminase type 1 (1:50)/Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000). The red (anti-TIG3) and green (anti-TG1) fluorescence was detected by using a Nikin epifluorescent microscope.

 
TIG3 Regulation of Apoptosis—The above results suggest that TIG3 activates events associated with keratinocyte differentiation. To determine whether TIG3 also regulates apoptotic processes, untreated cells (control) and cells treated for 48 h with empty vector (EV) or TIG31–164-encoding vector were harvested and assayed for caspase 3 activity using the caspase 3-specific substrate, Ac-DEVD-AFC. Uniformly low caspase activity is observed in control, EV- or TIG3-treated cells (Fig. 4A). As a positive control, we show that UVB treatment, a treatment that causes extensive cell apoptosis and caspase activation, markedly increases apoptosis-associated caspase 3 activity. Caspase 3 has been shown to be activated by UVB treatment of keratinocytes (8, 18–21). Caspases are expressed as pro-forms that require cleavage for activation (22). To confirm the caspase assay result, we assayed procaspase 3 protein level by immunoblot. Fig. 4B shows that procaspase 3 is not degraded by TIG31–164, suggesting that it is not activated. However, levels are markedly reduced by UVB treatment, consistent with the known ability to UVB light to promote caspase-associated cell death (8, 19, 20). The middle panel confirms that TIG3 is expressed in tAd5-TIG31–164 vector-infected cells, and the {beta}-actin blot confirms appropriate protein loading. We next determined whether activity of the initiator caspases (caspase 8 and 9) and PARP are increased by TIG3. These enzymes are also expressed as pro-forms that require cleavage for activation (22). As shown in Fig. 4, C and D, expression of TIG31–164 does not enhance cleavage of these enzymes. In contrast, UVB treatment does promote cleavage. We next determined whether the pan-caspase inhibitor, Z-VAD-FMK, halts the reduction in TIG3-associated cell number. As shown in Fig. 5, TIG31–164 reduces viable cell number to 30%, but the reduction is not reversed by addition of 50 µM Z-VAD-FMK. In contrast, the UVB-associated reduction in viable cell number is substantially reversed by the inhibitor.



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  		FIG. 4.
TIG3 regulation of cell death markers. Keratinocytes were mock-infected (Control) or infected with 5 m.o.i. of Ad5-TA with 20 m.o.i. tAd5-EV or 20 m.o.i. tAd5-TIG3 for 12 h and harvested at 48 h after infection. Parallel cultures were treated with UVB (50 mJ/square cm) and harvested at 16 h after treatment. As shown in A, cell extracts were prepared and assayed for the ability to cleave a fluorescent caspase 3 substrate, Ac-DEVD-AFC. Caspase activity is expressed as nmoles of substrate cleaved/mg protein/h. Control cells were not treated. As shown in B, TIG3 does not regulate caspase 3 activation. Extracts from the experiment shown in panel A were electrophoresed, and procaspase 3, TIG3, and {beta}-actin levels were assayed by immunoblot. As shown in C and D, TIG3 does not activate procaspase 8, procaspase 9, or PARP. Extracts were prepared as outlined above, and the level of each protein was measured by immunoblot. The position of known molecular mass markers are indicated in each panel.

 



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  		FIG. 5.
Inhibition of caspase activity does not prevent TIG3-mediated responses. Cells were infected with 5 m.o.i. of Ad5-TA with 20 m.o.i. of tAd5-EV or tAd5-TIG3 for 12 h. Cells were grown during and after infection in the presence or absence of 50 µM Z-VAD-FMK. Parallel cultures were treated with 50 mJ of UVB in the presence or absence of 50 µM Z-VAD-FMK. After 48 h, the cells were harvested, and viable cell number was assayed by trypan blue exclusion. The means ± S.E. of mean is presented as n = 3.

 
TIG3 Mutant Lacking Carboxyl Hydrophobic Domain Is Inactive—To begin identifying TIG3 functional domains and to show that the cell response is not an artifact of protein overexpression, we tested the ability of TIG31–134, which lacks the carboxyl-terminal hydrophobic domain, to reduce cell viability. The immunoblot shown in Fig. 6A confirms that TIG31–164 and TIG31–134 are expressed at comparable levels. The {beta}-actin blot confirms that protein loading is appropriately normalized. Fig. 6B compares TIG1–164 and TIG31–134 subcellular localization. The left panel confirms that TIG31–164 is preferentially distributed in the particulate fraction and that a fraction of the particulate-localized TIG3 is present in a high molecular weight, presumably cross-linked form. In contrast, unlike TIG31–164, most TIG31–134 immunoreactivity distributes in the cytosol (right panel).



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  		FIG. 6.
The TIG3 carboxyl terminus is required for function. As shown in A, keratinocytes were infected with 5 m.o.i. Ad5-TA in the presence of 20 m.o.i. of tAd5-EV, tAd5-TIG31–164, or tAd5-TIG31–134. After 48 h, cell extracts were prepared and assayed for TIG3 expression by immunoblot. {beta}-actin levels were detected to normalize protein loading. B, immunoblot analysis of TIG31–164 and TIG31–134 distribution in cell fractions. Cells were infected as outlined above. Total, soluble, and particulate cell fractions were prepared, and equal cell number cells equivalents were electrophoresed and assayed for TIG3 expression by immunoblot. As shown in C, cells were infected as outlined as above. After 48 h, viable cell number was assayed by trypan blue exclusion. KER, keratinocytes. D, intracellular localization of TIG31–164 and TIG1–134. Cells were infected with 20 m.o.i. of TIG31–164- or TIG31–134-encoding adenoviruses for 12 h. At 48 h after the start of infection, the cells were harvested, fixed, co-incubated with antibodies specific for TIG3 and TG1 followed by the appropriate secondary antibody, and examined by epifluorescent microscopy. The yellow image combines the red and green signals.

 
We next determined whether TIG31–134 reduces cell viability. Cells were infected with TIG31–164- or TIG31–134-encoding virus. After 48 h, cells were harvested and counted. As shown in Fig. 6C, TIG31–134 does not reduce keratinocyte viability. The microscopy data presented in Fig. 3E indicate that TIG31– 164 is preferentially associated with the particulates. We therefore used microscopic methods to monitor TIG31–134 subcellular localization. Cells were infected with TIG31–134 and then stained with anti-TIG3 (red) and anti-TG1 (green). As shown in Fig. 6D, in contrast to TIG31–164, TIG31–134 is diffusely distributed throughout the cell. In addition, the combined image shows only a partial and weak co-localization of TIG31–134 and TG1.

TIG31–134 Does Not Activate Type I Transglutaminase—To evaluate whether the TIG3 truncation mutant can regulate biochemical responses, we expressed TIG31–134 in keratinocytes and then determined whether the cells incorporate FC in situ. The immunohistology (anti-TIG3) shown in Fig. 7 indicates that the cells produce TIG31–164 and TIG31–134. The green fluorescence, which indicates the transglutaminase-dependent incorporation of FC substrate, is absent in cells expressing TIG31–134. Thus, TIG31–134 does not cause transglutaminase activation.



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  		FIG. 7.
TIG31–134 does not activate transglutaminase activity. Keratinocytes, grown on coverslips, were infected with 20 m.o.i. of TIG31–164- or TIG31–134-encoding adenoviruses and 5 m.o.i. of Ad5-TA for 15 h. At 48 h after the start of infection, the cells were pulsed for 4 h with 100 µM FC. The cells were then fixed and stained with anti-TIG3 (red). The green signal indicates covalently cross-linked FC.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TIG3 is a member of a recently discovered H-rev family of proteins (1, 2, 4). These proteins are important because they function to inhibit cell proliferation (2, 3, 5, 23). Moreover, their expression is reduced in cells that have escaped growth regulation (1, 4). Each family member displays a distinct pattern of tissue-specific expression. For example, RIG1 is expressed in human gastric cancer cells, and expression is increased by treatment with retinoic acid (3). H-REV107-2, a 216-aminoacid protein, is highly expressed in leukocytes and in the thymus (4). TIG3 is expressed in several cell types, including keratinocytes (1, 6), and TIG3 levels are increased by agents that inhibit cell proliferation (1, 3). A common feature shared by all of these proteins is a carboxyl-terminal hydrophobic domain. Studies with H-rev107 (2) and TIG3 (7) suggest that this domain may function as a membrane anchor. However, apart from these observations, very little information is available regarding their mechanism of action. The present studies were designed to gain new insight regarding the mechanism whereby TIG3 regulates keratinocyte function.

TIG3 Halts Keratinocyte Proliferation and Enhances Differentiation—TIG3 is expressed in the differentiated, suprabasal epidermal layers in vivo (6). Consistent with the expression in epidermis, we also detected TIG3 mRNA in cultured keratinocytes. However, we could not detect TIG3 protein, an observation consistent with the finding that H-rev family members are generally expressed at barely detectable levels. To study TIG3 effects on keratinocyte proliferation, apoptosis, and differentiation, we expressed exogenous TIG3 using adenovirus. Our studies show that TIG3 expression markedly reduces keratinocyte proliferation/survival and that this is associated with accumulation of cells in the sub-G1 growth phase. The reduction in viable cell number is associated with a 10-fold increase in accumulation of structures resembling cornified envelopes.

TIG3 Promotes Cell Death by Enhancing Transglutaminase Activity—There is virtually no information available regarding how members of the H-rev family cause cell death. Our studies, however, indicate that TIG3-dependent cell death is associated with cornified envelope formation. Cornified envelopes are cross-linked arrays of protein that are assembled during the terminal stages of normal keratinocyte differentiation (24). These structures are assembled via formation of covalent {epsilon}-({gamma}-glutamyl)lysine protein-protein bonds (24, 25). Since cornified envelopes are assembled via action of transglutaminases, we explored the possibility that TIG3 may activate these enzymes. Transglutaminases exist in multiple forms, are expressed in a tissue-specific manner, and are involved in formation of differentiated structures (14, 26, 27). They are also involved in assembly of apoptosis-associated structures (9, 11, 28–30). Keratinocytes produce several types of transglutaminase (31–34). However, the major type involved in corneocyte assembly is TG1. TG1 is anchored to the plasma membrane via a palmitate or myristate linkage (31, 35). Biochemical and in situ substrate incorporation assays reveal a substantial increase in membrane-associated transglutaminase activity in TIG3-expressing keratinocytes, suggesting that TIG3 acts to increase TG1 activity. Type 2 transglutaminase (TG2) is also expressed at low levels in keratinocytes and other surface epithelial cells and can be induced under specific conditions (32, 33). It is possible that TIG3 could induce TG2, as TG2 activity is known to facilitate apoptosis. TG2 activity is assayed based on changes in cross-linking activity in the soluble phase. As shown in Fig. 3B, TIG3 expression does not alter TG activity in this compartment. These results confirm that TG2 is not involved in the TIG3-dependent response. Moreover, we suspect that the soluble TG activity may actually be TG1 that is contaminating the soluble phase.

Additional results further support the idea that TIG3 activates TG1. First, TIG3 and TG1 are located in the same subcellular location, in membranes. Second, as determined by monitoring of sites of incorporation of TG fluorescent substrate, the intracellular sites of TG1 activity correspond with site of TIG3 localization. Finally, a fraction of TIG3 is cross-linked to form high molecular weight structures. The final point suggests a close juxtaposition, as TG1 and TIG3 must actually make close contact for TIG3 to serve as a TG substrate. The covalent cross-linking of TG-associated proteins, either by design or incidentally, is known to occur. Thus, it is not surprising that a regulatory protein such as TIG3 can become cross-linked by TG1. Overall, these results strongly suggest that TG1 activation is required for the cell to manifest the TIG3-assocated effects.

TIG3 Does Not Activate Killer Caspases—Keratinocyte cell death can occur via at least two distinct pathways: normal differentiation and activation of apoptotic processes (37, 38). These processes are distinguished by the differential activation of killer caspases. Ultraviolet light treatment of epidermis and cultured keratinocytes activates caspases, and the cells undergo apoptosis (8, 21). This is in contrast to normal differentiation, which does not involve killer caspase activity (39, 40). Thus, in contrast to normal differentiation, caspase-involved apoptosis is usually a response to trauma. Our present studies show that the killer caspases (caspases 3, 8, and 9) are not activated as part of the cell death program in TIG3-positive keratinocytes. PARP is a nuclear enzyme that protects against genome damage and increases during apoptosis (41, 42). TIG3 expression does not induce PARP cleavage. In addition, TIG3-dependent cell death is not inhibited by the pan-caspase inhibitor, Z-VAD-FMK. These observations, together with the finding that TIG3 enhances differentiation-associated responses, increased TG1 activity, and cornified envelope formation, suggest that TIG3 is primarily an activator of keratinocyte differentiation.

TIG3 Activity Requires the Carboxyl-terminal Hydrophobic Domain—Our previous report identified homologous domains shared between TIG3 and other H-rev family proteins (7). This comparison revealed the presence of a conserved 30-amino-acid hydrophobic carboxyl-terminal domain. It is hypothesized that this domain may be required for membrane localization (7). To test this hypothesis, we expressed the carboxyl-terminal truncation mutant. To examine whether the TIG3 carboxyl-terminal domain is necessary for function, we determined whether the truncation mutant has activity. Our studies show that TIG31–134 does not inhibit cell proliferation, reduce cell viability, or promote cornified envelope formation. This loss of activity is associated with a distribution in the soluble phase, reduced localization with TG1, and a loss of the ability to activate transglutaminase. These findings strongly suggest that membrane localization, and perhaps localization with or near TG1, is required for TIG3-dependent cell differentiation. Future studies, using additional mutants, will be necessary to identify additional functional domains.

Mechanism of TIG3 Action—Our studies have identified a new regulator and a new mechanism regulating keratinocyte differentiation. We hypothesize that TIG3 promotes keratinocyte differentiation via activation of membrane-associated transglutaminase. TIG3 is produced and inserted into various membranous structures where it functions to activate type I transglutaminase. Activation may be via direct interaction with TG1 or by facilitating the local release of calcium that, in turn, activates the calcium-dependent enzyme. Additional studies are underway to test these hypotheses.


   FOOTNOTES
 
* This work utilized the facilities of the Skin Diseases Research Center of Northeast Ohio (National Institutes of Health Grant AR41456) and was supported by the American Institute for Cancer Research and the National Institutes of Health (to R. L. E.). 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

A Medical Scientists Training Program Fellow (National Institutes of Health Grant T32GM07250). Back

|||| To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Rm. E532, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106-4970. Tel.: 216-368-5530; Fax: 216-368-5586; E-mail: rle2{at}po.cwru.edu.

1 The abbreviations used are: TIG3, tazarotene-induced protein 3; TG, transglutaminase; TG1, type I transglutaminase; TG2, type II transglutaminase; m.o.i., multiplicity of infection; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; AFC, 7-aminotrifluoromethylcoumarin; TA, transactivator; EV, empty vector; KSFM, keratinocyte serum-free medium; PBS, phosphate-buffered saline; FC, fluorescein cadaverine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PARP, anti-poly(ADP-ribose) polymerase; XTT, 2,3-bis[2-methoxy-4nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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