© 2000 American Heart Association, Inc.
Molecular Medicine |
Retinoic Acid–Induced Tissue Transglutaminase and Apoptosis in Vascular Smooth Muscle Cells
From the Center for Cardiovascular Research (H.O., J.H., B.C.B., J.M.M.), University of Rochester Medical Center, Rochester, NY, and Cardiovascular Research Center (M.R.A., L.A.K., B.C.C., G.K., A.K.-B., H.J.J.), Medical College of Wisconsin, Milwaukee, Wis.
Correspondence to Joseph M. Miano, PhD, Center for Cardiovascular Research, University of Rochester Medical Center, 601 Elmwood Ave, Box 679, Rochester, NY 14642. E-mail Joseph_Miano{at}urmc.rochester.edu
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Abstract |
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Abstract—Retinoids exert antiproliferative and prodifferentiating effects in vascular smooth muscle cells (SMCs) and reduce neointimal mass in balloon-injured blood vessels. The mechanisms through which retinoids carry out these effects are unknown but likely involve retinoid receptor-mediated changes in gene expression. Here we report the cloning, chromosomal mapping, and biological activity of the retinoid-response gene rat tissue transglutaminase (tTG). Northern blotting studies showed that tTG is rapidly and dose-dependently induced in a protein synthesis–independent manner after stimulation with the natural retinoid all-trans retinoic acid (atRA). The induction of tTG was selective for atRA and its stereoisomers 9-cis and 13-cis RA, because little or no elevation in mRNA expression was observed with a panel of growth factors. Western blotting and immunofluorescence confocal microscopy showed an accumulation of cytosolic tTG protein after atRA stimulation. Radiolabeled cross-linking studies revealed a corresponding elevation in in vitro tTG activity. The increase in tTG activity was reduced in the presence of 2 distinct inhibitors of tTG (monodansylcadaverine and cystamine). atRA-induced tTG mRNA and protein expression were followed by a significant elevation in SMC apoptosis. Such retinoid-induced programmed cell death could be partially inhibited with each tTG inhibitor and was completely blocked when both inhibitors were used simultaneously. These results establish a role for atRA in the sequential stimulation of tTG and apoptosis in cultured SMCs. atRA-mediated apoptosis in SMCs seems to require the participation of active tTG, suggesting a potential mechanistic link between this retinoid-inducible gene and programmed cell death.
Key Words: tretinoin • transcription • protein-glutamine 
-glutamyltransferase • chromosome • cDNA
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Introduction |
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Perturbations in the genetic program of smooth muscle cell (SMC) growth arrest and differentiation are central to the pathogenesis of many vascular occlusive disorders as well as diseases of the airway, gastrointestinal, and urogenital systems. The majority of research investigating the basis for changes in SMC phenotype during disease progression has focused on membrane-bound receptors that effect intracellular signaling cascades involved in SMC growth and differentiation control.1 2 In recent years, studies have been initiated to examine the role of steroid receptor-binding hormones on SMC phenotype. For example, 17ß-estradiol, which binds the estrogen receptors, suppresses SMC growth and migration, stimulates SMC vasodilation through the activation of nitric oxide synthase, and reduces neointimal formation after arterial injury.3 Ligands for the peroxisome proliferator-activated receptors exhibit similar effects on SMC phenotype.4 Another family of hormones, the retinoids,5 recognizes 6 distinct nuclear receptors designated as retinoic acid receptors (
, ß, and ) and retinoid X receptors (, ß, and
).6 Previous studies have
documented retinoid receptor expression and activity in
SMCs7 and have shown that
retinoids, principally atRA, antagonize SMC
mitogenesis7 8 9 10 11 12
and migration13 while
promoting a SMC-differentiated
phenotype.14 15 16
In a recent series of corroborative papers,
all-trans retinoic acid (atRA)
was shown to increase luminal diameter after experimental injuries to
the vessel
wall.17 18 19 20 21 22 23
Collectively, these studies point to a versatile role for retinoids in
the control of SMC phenotype and the response of the vessel wall to
insult.24 Steroid receptors act as ligand-activated transcription factors that mediate changes in a cell’s gene expression profile. For example, activated retinoid receptors, bound to discrete cis elements called retinoic acid (retinoid X) response elements, recruit critical coactivators around the preinitiation complex of a gene, stimulating the rate at which a gene is transcribed.25 Several retinoid-responsive target genes have been identified, including growth factors, cytokines, proteases and their inhibitors, cyclin-dependent kinases and their inhibitors, transcription factors (especially homeobox genes), matrix molecules, and apoptotic factors.26 Presently, there is a paucity of information relating to retinoid-response genes in SMCs and even less about the mechanisms underlying retinoid-mediated changes in SMC phenotype.
In a directed screen for retinoid-inducible genes that may mediate the biological effects of atRA in SMCs, we cloned a portion of the rat tissue transglutaminase (tTG) cDNA. tTG is a retinoid-inducible, multifunctional gene implicated in substrate-specific isopeptide bond cross-linking, GTP-mediated signaling, and apoptosis.27 Although its expression in vascular SMCs has been documented both in vitro and in vivo,28 29 30 the precise role of tTG in SMC biology is unclear. Here, we report the cDNA sequence and chromosomal mapping of rat tTG. We show that tTG is induced selectively by retinoids and that such gene activation occurs in the absence of de novo protein synthesis. We additionally demonstrate that the activation of tTG precedes retinoid-induced apoptosis, which can be completely blocked with inhibitors of tTG. These results suggest that one component of atRA’s growth antagonism in SMCs may involve tTG-mediated apoptosis.
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Material and Methods |
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Cell Culture
Cultured rat aortic SMCs (RASMCs) were obtained from the thoracic aorta of male Sprague-Dawley rats by a modified explant protocol, as described.31 These cells and the PAC-1 SMC line32 were grown in DMEM supplemented with 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Both cell types express many of the major SMC-restricted markers.33
Cloning and Chromosomal Mapping of Rat
tTG
A first-strand cDNA library was generated (Amersham
Pharmacia) from cultured RASMCs treated with
all-trans retinoic acid (kindly
provided by Dr Louise Foley, Hoffmann-La Roche, Nutley, NJ). The
library was then PCR-screened using a high-fidelity polymerase (TaKaRa;
Panvera Corp) with primers to the mouse tTG cDNA (GenBank accession
number M55154). Three independent tTG clones were sequenced on both
strands (Biotech Thermo Sequenase dye terminator cycle sequencing kit;
Amersham Pharmacia) using a Perkin Elmer Applied Biosystems 377
automated sequencing machine. Sequence analysis was performed using the
FASTA, pileup, and pretty algorithms within Genetic Computer Group’s
Wisconsin Package (version 10). All three clones were aligned and
analyzed using the GeneDoc program (online at
http://www.cris.com/~ketchup/genedoc/shtml). Chromosomal mapping of
the rat tTG gene was accomplished with a rat-hamster radiation hybrid
(RH) panel (Research Genetics), as
described.34
Northern Blotting
Cells stimulated with atRA
(2x10-6
mol/L), 9-cis RA
(2x10-6
mol/L), 13-cis RA
(2x10-6
mol/L), 10% FBS, -thrombin (2 U/mL), basic fibroblast growth factor
(20 ng/mL), transforming growth factor-ß1 (2
ng/mL), epidermal growth factor (10 ng/mL), or an equal volume of
dimethylsulfoxide (DMSO control) were harvested for total RNA
isolation, as described,33
and probed with an 800-bp fragment of the rat tTG cDNA. A
glyceraldehyde phosphate dehydrogenase cDNA was used as an internal
control. In some experiments, we used cycloheximide (CHX; 2.5 µg/mL;
Sigma) to assess the requirement for de novo protein synthesis on tTG
expression. This concentration of CHX was shown to have minimal
toxicity over a 24-hour time period and reduced new protein synthesis
by >90% (data not shown).
Western Blotting
Extracts of RASMCs treated with atRA or DMSO were
isolated for total protein synthesis and Western blotting, as
described.33 A polyclonal
goat antibody raised against rabbit tTG (Upstate Biotechnology) was
applied to immobilized proteins at a dilution of 1:1000. An antibody to
smooth muscle calponin (clone hCP, Sigma) was used as an internal
control. Secondary antibodies conjugated to horseradish peroxidase
(1:2000 dilution) were used to reveal immunoreactive protein with ECL
Plus reagents (Amersham Pharmacia).
Confocal Microscopy
RASMCs were cultured in chamber slides in the
presence of DMSO or atRA for 48 hours. After fixation and
permeabilization with acetone and methanol, the cells were incubated
with anti-tTG (1:100 dilution) for 1 hour, washed in PBS with 0.1%
tween 20, incubated with rabbit antigoat IgG conjugated to biotin
(1:100 dilution, Pierce), washed and incubated with streptavidin-FITC
(1:100 dilution, Pierce) for an additional 30 minutes, and then washed
2 times with PBS. Cells were then stained with the DNA fluorochrome
TO-PRO-3 iodide (Molecular Probes). Slides were cover-slipped under
aqueous mounting medium and viewed with an Olympus Fluoview FV 300
confocal microscope. Voltage settings were kept constant between
atRA-treated and control cells during imaging.
tTG Activity Assay
tTG activity was measured indirectly in an in vitro
assay as the Ca2+-dependent incorporation of
3H-putrescine (0.832 Ci/mmol; New England
Nuclear) into
N,N-dimethyl
casein (Calbiochem), as
described.35
Monodansylcadaverine (MDC) (Sigma), a competitive substrate
inhibitor,36 and cystamine
(Sigma), an active site
inhibitor,37 were tested at
varying doses to optimize studies aimed at assessing their inhibitory
effects on tTG activity and apoptosis. Trypan blue exclusion was used
as a measure of cell viability. tTG activity was expressed as pmol of
3H-putrescine incorporation into
N,N-dimethyl
casein per minute per milligram of total protein after subtraction of
background levels obtained by substituting EDTA for
Ca2+.
Apoptosis Assays
SMCs stimulated for 22 hours with atRA
(2x10-6 mol/L)
or an equal volume of DMSO were fixed and either stained with DAPI or
processed for TUNEL, as
described.38
Statistics
All graphical data are presented as mean±SEM and are
representative of at least 2 independent studies. ANOVA with
appropriate post hoc testing (GraphPad Prism) was carried out on data
pertaining to the tTG activity and the DAPI staining assay for
apoptosis. Statistical significance was assumed if
P<0.05.
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Results |
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Sequence and Amino Acid Homology of Rat tTG
The rat tTG cDNA is at least 3393 nt in length, with 2058 nt of coding sequence yielding a 686 amino acid protein (GenBank accession number AF106325). As anticipated, the rat tTG cDNA is highly homologous within the coding region to the orthologous mouse (92.4%), human (83%), and guinea pig (82.2%) cDNA sequences (data not shown). The deduced amino acid sequence encompassing the active site domain of rat tTG is identical to mouse and evolutionarily conserved across several species (Figure 1
). Nucleotide sequence homologies diverge
considerably within the 3' untranslated regions of mouse (77.5%),
guinea pig (49.6%), and human (43.7%) tTG (data not shown). We
exploited this sequence divergence in the design of PCR primers for RH
mapping studies. Rat tTG was RH-mapped to chromosome 3, between
D3Got123 and D3Rat4 at >1000:1 odds (data not shown). This region,
corresponding roughly to 3q41 to 3q42, is syntenic with human
chromosome 20q12, which is where the human tTG has been mapped
previously.39 Information
regarding the RH framework maps can be found online at
http://goliath.ifrc.mcw.edu.
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tTG Is an Immediate-Early Retinoid Response
Gene
tTG is induced in a variety of cell types on exposure
to retinoids, such as
atRA.27
Figure 2A shows similar activation of tTG when SMCs are
stimulated with atRA. Increasing steady-state transcript levels of tTG
emerge as early as 3 hours after atRA addition, with peak expression at
24 hours. Induction of tTG in SMCs seems to be highly restricted to
atRA, because little or no activation is observed on treatment with
serum and a panel of growth factors
(Figure 2B). 13-cis
RA, which has more favorable pharmacokinetic and toxicological profiles
than atRA and readily isomerizes to both atRA and
9-cis
RA,24 induces tTG expression
(Figure 2B), as does the
9-cis RA stereoisomer itself
(data not shown). The induction of tTG with atRA is dose-dependent
(Figure 2C) and occurs in the absence of de novo protein
synthesis
(Figure 2D). A consistent superinduction of tTG mRNA is
observed in the presence of CHX, suggesting the existence of a labile
mRNA binding protein involved in the destabilization of the tTG
transcript
(Figure 2D). The mRNA kinetics, protein
synthesis–independent expression, and superinduction with CHX indicate
that tTG is an immediate-early retinoid response
gene.
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Protein Expression and Activity of tTG
To determine whether atRA-induced tTG mRNA led to its
enhanced translation, we measured steady-state protein levels by
Western blotting. tTG is clearly increased within 24 hours after atRA
stimulation
(Figure 3). tTG protein remains elevated up to 4 days, with
levels returning toward baseline at 7 days
(Figure 3). Shorter time-course studies reveal increases in
tTG protein as early as 6 hours after atRA administration (data not
shown). Little, if any, tTG protein was detected in the culture medium,
indicating that the protein is not actively secreted under our culture
conditions (data not shown). Confocal microscopy demonstrates low
levels of immunoreactive tTG in the cytosol of control RASMCs
(Figure 4A). At the same voltage setting, cultured RASMCs
show a dramatic increase in cytosolic tTG after 48 hours of atRA
stimulation
(Figure 4B). Consistent with previous reports in other cell
types,40 a small percentage
of tTG is noted within the nucleus
(Figures 4A and 4B).
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We next used a well-defined in vitro assay to assess 2 inhibitors of tTG cross-linking activity. We used an indirect assay that measures the amount of cross-linking activity on addition of Ca2+ (see Materials and Methods). Peak tTG protein levels (48 hours) coincided with a 6-fold elevation in tTG cross-linking activity (control, 0.0105±0.001 versus atRA, 0.0625±0.009; P<0.001). This induced activity could be partially inhibited with the active site inhibitor cystamine (0.0395±0.003); however, inclusion of both cystamine and MDC caused a consistent and significant inhibition of in vitro tTG activity (0.0625±0.009 versus 0.009±0.003, P<0.001) with no evidence of overt cytotoxicity. Similar tTG expression and activity results were found in the PAC-1 SMC line (data not shown). These results establish tTG protein induction with atRA treatment and demonstrate the utility of combining 2 inhibitors to block tTG cross-linking activity in cultured RASMCs.
atRA-Induced SMC Apoptosis Is
tTG-Dependent
Several studies (mainly in cancer cell lines) have
shown that atRA stimulates the expression of tTG and elicits
apoptosis.27 41
To determine whether a mechanistic link exists between atRA-stimulated
tTG expression and programmed cell death, we evaluated the tTG
inhibitors studied above for their effects on atRA-induced apoptosis in
SMCs. The results depicted in
Figure 5 show a 7-fold increase in apoptotic SMCs (DAPI
stained nuclear morphology) with exposure to atRA. The number of
atRA-induced apoptotic SMCs ranged from 5% to 8% of the cell
population. This level of apoptosis is considerably less than the 20%
observed in RASMCs stimulated with 200 µmol/L
H2O2 (data not shown).
Each tTG inhibitor shows partial, but statistically insignificant,
inhibition of apoptosis when added individually
(Figure 5). However, consistent with the in vitro activity
data above, combining both tTG inhibitors (nontoxic by trypan blue
exclusion) results in near complete inhibition of atRA-stimulated
apoptosis
(Figure 5). TUNEL staining confirmed the inhibition of
atRA-induced apoptosis with both tTG inhibitors
(Figure 6). These results document atRA-mediated apoptosis in
RASMCs and suggest that tTG is an important determinant of such
programmed cell death.
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Discussion |
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Three major findings emerge from this study. First, we have shown that atRA rapidly induces the expression of tTG mRNA with subsequent elevations in immunoreactive tTG protein and in vitro cross-linking activity. The activation of tTG in SMCs is a relatively specific response to retinoids, because no other reagent tested induced the expression of this multifunctional enzyme. Its rapid kinetics of expression and independence of de novo protein synthesis indicate that tTG is an immediate-early retinoid response gene. Second, using 2 independent assays (DAPI and TUNEL staining), we have shown that atRA can induce apoptosis in vascular SMCs. Finally, we provide evidence for a mechanistic link between atRA-induced tTG activity and apoptosis by showing the near complete inhibition of apoptosis in the presence of MDC (a tTG substrate inhibitor) and cystamine (a tTG active site inhibitor). These results identify tTG as a critical target of retinoid action in cultured SMCs and suggest that atRA-mediated effects on vascular SMC phenotype and vessel remodeling after injury may, in part, involve the action of this multifunctional enzyme.
tTG (EC 2.3.2.13) and other members of the transglutaminase
gene family exert calcium-dependent posttranslational modifications of
substrate-specific proteins through an acyl transferase reaction
involving -carboxamide groups of glutamine residues (donor peptide)
with 
-amino groups of lysine residues (acceptor peptide). The
resulting covalent modification imparts an irreversible cross-link that
stabilizes proteins, conferring structural integrity to intracellular
and extracellular
microenvironments.42 tTG
protein is present at high levels in disease-free blood
vessels28 and is restricted
to adult rat aorta by Northern blotting (unpublished data, July 2000),
suggesting that tTG is part of the SMC differentiation program of gene
expression. In this context, levels of tTG have been shown to drop
precipitously in RASMCs after enzymatic dispersion and cell passaging,
indicating that the loss of tTG coincides with the phenotypic
modulation of these cells in
vitro.43 The dramatic
upregulation of tTG after atRA administration is consistent with this
retinoid’s prodifferentiation effects in cultured
SMCs.24 44
In a previous study, atRA was shown to modestly elevate tTG mRNA in human venous SMCs but did not elevate tTG activity in these cells. Human arterial SMCs showed a 3-fold increase in tTG activity with atRA stimulation, but the mRNA expression of tTG was not reported in these cells.29 Interestingly, tTG colocalized with stress fibers in human arterial and venous SMCs and was shown to coimmunoprecipitate with myosin, suggesting that codistribution of tTG with stress fibers was attributable to tTG’s interaction with myosin.29 Although we did observe some evidence of stress fiber formation in atRA-treated SMCs, which is consistent with atRA’s prodifferentiation effects in these cells, extensive confocal microscopy failed to reveal any colocalization of tTG with the stress fiber apparatus (data not shown). A likely explanation for these varying results may relate to the species differences in vascular SMCs.
Although tTG-mediated cross-linking reactions have been implicated to play a role in diverse biological processes, the precise physiological function of the enzyme remains unclear, particularly with respect to vascular biology. Previous studies have shown atRA to antagonize growth factor–stimulated SMC proliferation.7 8 9 10 11 12 The findings reported here suggest that one component of this growth-inhibitory effect may be related to apoptosis. In support of this concept are several studies carried out in various non-SMC lines. For example, cystamine inhibited tTG activity while elevating cell growth in cultured WI-38 human lung cells.45 atRA-stimulated human epithelial prostate cells showed elevated tTG and apoptosis, both of which correlated with reduced cell growth.41 A similar correlation was observed in rat tracheobronchial epithelial cells.46 Melino et al47 showed increased cell death and a marked reduction in proliferative capacity of several tumor cell lines that were engineered to overexpress tTG. Gentile et al48 showed comparable effects of overexpressing tTG in Balb-c 3T3 fibroblasts. Conversely, cells transfected with tTG in the antisense orientation resulted in a reduction of both spontaneous and atRA-induced cell death.47 49 Collectively, these studies point to an important role for activated tTG in mediating cellular apoptosis and, consequently, modulating growth responses. Although the mechanisms remain to be clarified, evidence suggests that tTG-mediated cross-linking of intracellular proteins, such as the retinoblastoma protein and histones, may be critical in signaling an apoptotic program.50 However, we cannot rule out effects of tTG on SMC apoptosis that are independent of its cross-linking activity.
Significant inhibition of atRA-induced SMC apoptosis
required the simultaneous addition of both tTG inhibitors
(Figures 5
and 6). Several factors may explain why,
individually, each inhibitor was ineffective in significantly blocking
atRA-mediated apoptosis. First, our data cannot distinguish the effects
of each inhibitor on tTG activity versus other, undefined effects.
Thus, it is possible that tTG-independent pathways of apoptosis are
blocked in the presence of both inhibitors. Second, concentrations used
for each inhibitor in this study (150 µmol/L) were comparatively
lower than in previous cell culture studies (>250
µmol/L).45 51
Another explanation for the nonsignificant effect of each inhibitor on
atRA-induced apoptosis may relate to their distinct modes of
inhibition; although cystamine seems to modify the active site cysteine
residue
(Figure 1), MDC acts as a pseudosubstrate inhibitor. Thus,
there seems to be a requirement for both modes of inhibition to effect
significant blockage of atRA-induced apoptosis. In this context, tTG
inhibitors only partially attenuated in vitro tTG activity but, when
combined, always elicited near complete inhibition of in vitro tTG
activity (see text and Reference 4545 ). Clearly, more work in this area
is required, including the use of more specific reagents (eg, antisense
tTG) and an analysis of apoptosis in atRA-treated SMCs derived from tTG
knockout mice.
It is important to stress that although virtually all SMCs showed elevated immunoreactivity for tTG after atRA treatment, only a subpopulation (<8%) of such cells actually underwent apoptosis. This finding is consistent with in vivo immunohistochemistry studies showing substantial levels of tTG protein in blood vessels28 30 where apoptosis of contractile SMCs is low or near absent. As calcium is a critical determinant of tTG activity,27 we hypothesize that those tTG-positive cells undergoing apoptosis achieve a critical threshold of intracellular calcium levels that activate the cross-linking activity of the enzyme. Alternatively, the 8% apoptotic index may be explained by the heterogeneity of SMC subtypes in these cultures having variable apoptotic pathways or tTG substrate profiles. The function of tTG in cells that do not undergo apoptosis is unclear but could relate to signal transduction events involving its GTP-binding domain.52 Future studies will be necessary to determine the rules governing tTG activity and its downstream biological effects in atRA-treated SMCs.
In summary, the data presented here are consistent with the hypothesis that tTG activation mediates SMC apoptosis after atRA stimulation. However, we emphasize that atRA likely orchestrates myriad changes in gene expression that could impinge on the apoptotic program independent of tTG expression and activity. Future studies should be directed toward a more complete understanding of atRA-induced gene expression in SMC as well as tTG signaling in SMC after atRA stimulation (eg, GTP-binding and phospholipase C–mediated signaling52 ). Finally, an appraisal of the proposed atRA-tTG-apoptosis axis in the setting of vascular remodeling after injury warrants investigation.
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Acknowledgments |
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This work was supported by grants from the American Heart Association (SDG No. 9730145N to J.M.M.) and the National Institutes of Health (HL55795 to B.C.B.). J.H. was supported by a grant from the Deutsche Forschungsgesellschaft Ha 2868/1-1. We thank Peter J.A. Davies for antisera to tTG as well as the protocol for carrying out tTG activity assays.
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Footnotes |
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1 Both authors contributed equally to this study.
Received August 10, 2000; revision received October 3, 2000; accepted October 4, 2000.
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