From the Department of Integrative Biology, Pharmacology and Physiology, The University of Texas Medical School, Houston, Texas 77030
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ABSTRACT |
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Tissue transglutaminase is a
calcium-dependent, protein cross-linking enzyme that is
highly expressed in cells undergoing apoptosis. The expression of
tissue transglutaminase is regulated by a variety of molecules
including retinoids, interleukin-6, and transforming growth factor-1
(TGF-
1). Retinoid and interleukin-6 inductions of tissue
transglutaminase expression are mediated by specific
cis-regulatory elements located within the first 4.0 kilobase pairs of the promoter of the gene. The present studies were
designed to identify the molecular mechanisms mediating the regulation
of tissue transglutaminase gene expression by TGF-
family members.
Transient transfection of Mv1Lu cells with transglutaminase promoter
constructs demonstrated that 0.2 nM TGF-
1 maximally induced the activation of the promoter through a 10-base pair TGF-
1
response element (TRE; GAGTTGGTGC) located 868 base pairs upstream of
the transcription start site. This same element mediated an inhibitory
activity of TGF-
1 on the transglutaminase promoter in MC3T3 E1
cells. The TRE through which TGF-
1-regulated the activity of the
transglutaminase promoter was necessary and sufficient for bone
morphogenetic protein 2- (BMP) and BMP4-dependent
inhibition of the tissue transglutaminase promoter. The TGF-
1, BMP2,
and BMP4 regulation of the transglutaminase promoter activity was similar to the responses we observed for the endogenous
transglutaminase activity of Mv1Lu and MC3T3 E1 cells. For BMP2 and
BMP4, this regulation was paralleled by a decrease in tissue
transglutaminase mRNA in MC3T3 E1 cells. The results of these
experiments suggest that TGF-
1, BMP2, and BMP4 regulation of mouse
tissue transglutaminase gene expression requires a composite TRE
located in the 5'-flanking DNA.
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INTRODUCTION |
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Tissue transglutaminase (transglutaminase type II, EC 2.3.2.13) is
a calcium-dependent, acyltransferase that catalyzes the formation of covalent -(
-glutaminyl)lysyl isopeptide cross-links between and within proteins (1-4). The enzyme has been implicated in
the cross-linking of extracellular matrix proteins during osteogenesis and wound healing (5, 6) as well as in the cross-linking of
intracellular proteins in cells undergoing apoptotic cell death (2). In
addition, tissue transglutaminase has recently been identified as a
GTP-dependent regulator (G
-h) of
receptor-dependent phospholipase C activity in hepatocytes
(7).
As can be expected for an enzyme involved in diverse physiological
responses, the expression of tissue transglutaminase is regulated by
multiple factors. Retinoids act as acute regulators of tissue
transglutaminase expression in many cells and tissues (8, 9). Animals
rendered vitamin A (all-trans-retinol)-deficient show a
generalized decrease in transglutaminase activity suggesting that
expression of the enzyme is physiologically controlled by retinoids
(10). Transglutaminase activity is frequently increased in tissues
following injury or during acute inflammation (11). Several
pro-inflammatory cytokines, including interleukin-6 and TNF, have
been implicated in the induction of the enzyme during these processes
(12, 13). Additionally,
TGF-
11 has been shown to
induce tissue transglutaminase in hepatoma cells and keratinocytes (14,
15). In hepatoma cells, the TGF-
1-dependent induction of
tissue transglutaminase is associated with the induction of
apoptosis. Previously, we isolated the mouse tissue
transglutaminase promoter and demonstrated that cis-elements
located within the proximal 4.0 kilobase pairs of the 5'-flanking DNA
regulate the activity of the promoter in both regions of chondrogenesis
and in cells undergoing apoptotic cell death (16). Detailed analysis of
the transglutaminase promoter identified discrete regions linked to
retinoid- and TNF
-regulated expression (8, 12).
TGF-1 and transglutaminase activity are associated with
chondrogenesis and certain forms of apoptotic cell death. A cooperative interaction between TGF-
1 and tissue transglutaminase is suggested by the observations that TGF-
1 increases tissue transglutaminase expression, and in turn, tissue transglutaminase catalyzes the conversion of latent TGF-
1 to its active form (17, 18). The studies
presented here investigated the molecular mechanisms by which TGF-
1
induces the expression of tissue transglutaminase. The results of these
studies demonstrate that in Mv1Lu and MC3T3 E1 cells TGF-
1 and BMP2
and BMP4 regulate the activity of the tissue transglutaminase promoter
via a composite TGF-
response element (TRE) located 868 bp upstream
of the transcription start site. This regulatory activity of TGF-
1
was cell-specific; in Mv1Lu cells TGF-
1 was stimulatory on the
tissue transglutaminase gene promoter, and in MC3T3 E1 cells it was
inhibitory. Thus, our results indicate that TGF-
-family members can
regulate the transcriptional activity of tissue transglutaminase via a
TRE embedded within the promoter of the gene.
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EXPERIMENTAL PROCEDURES |
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Materials
Mv1Lu cells, an epithelial-like cell line derived from normal
mink lungs, were purchased from the American Type Culture Collection (ATCC, Rockville, MD). MC3T3 E1 cells, a mouse preosteoblastic cell
line, were a gift from Dr. Renee Franchesci (University of Cincinnati).
Recombinant human TGF-1 (rhTGF-
1) was purchased from R & D
Systems (Minneapolis, MN), and recombinant human BMP2 and BMP4 were
generous gifts from Genetics Institute, Inc. (Cambridge, MA). The
pCAT-Basic, SV2-
-gal, and pRL-SV40 vectors were
purchased from Promega Corp. (Madison, WI). The Quick Change
mutagenesis kit was purchased from Stratagene Cloning Systems (La
Jolla, CA). Oligonucleotide primers were purchased from Genosys
(Houston, TX).
Cell Cultures
Mv1Lu cells were maintained in Eagle's MEM supplemented with
10% heat-inactivated fetal bovine serum. MC3T3 E1 cells were maintained in -MEM containing 10% heat-inactivated calf serum. Both
cell lines were cultured in the presence of 100 units/ml penicillin and
50 mg/ml streptomycin.
Plasmid Constructs and Site-directed Mutagenesis
The regulation of transglutaminase gene expression by TGF-
family members was investigated in transient transfection experiments with mouse tissue transglutaminase promoter constructs (pmTG4.0-Luc, pmTG1.8-CAT, pmTG0.2-CAT, and pmTG1.8-
SmaI-CAT; Fig.
1A) (8). PmTG1.65-CAT, pmTG1.3-CAT, and pmTG1.0-CAT were
constructed using a Quick Change Site-directed Mutagenesis Kit
(Stratagene, La Jolla, CA) to introduce a HindIII
restriction site at positions
1647,
1287, and
1006, respectively,
followed by excision of the DNA fragment with HindIII
(
1647,
1287, or
1006 to
1842; Boehringer Mannheim) and
religation with T4-DNA ligase (Boehringer Mannheim). PmTG1.8-
Mut-CAT
was generated by site-directed mutagenesis of the TGF-
1 regulatory
element (TRE) within pmTG1.8-CAT using the Quick Change Site-directed
Mutagenesis Kit (Stratagene, La Jolla, CA) and complementary primers
that converted the TTGG (5 '
3 ')
tetranucleotide sequence at position
871 to
CTAG.
The nucleotide sequence for each of the mouse tissue transglutaminase gene promoter constructs was determined. Samples were processed according to the standard protocol for the T7 Sequenase Quick-Denature Plasmid Sequencing Kit (Amersham Pharmacia Biotech). Following the sequencing reaction, samples were resolved using a 6% denaturing polyacrylamide gel and a GenomyxLR programmable DNA sequencer (Genomyx Corp., Foster City, CA).
Transient Transfections
Co-transfection assays with test and control plasmids were
conducted using approximately 70% confluent Mv1Lu or MC3T3 E1 cells and a 12-well dish format. Mv1Lu cells were washed with serum-free Eagle's MEM prior to Lipofectin-mediated transfection (Life
Technologies, Inc.) of 1 µg of test plasmid along with 0.1 µg of
pRL-SV40. Cells were transfected for 3 h prior to addition of
Eagle's MEM plus 10% heat-inactivated fetal bovine serum containing
treatments described within figure legends. MC3T3 E1 cells were
co-transfected using 3 µg of DOSPER (Boehringer Mannheim), 1 µg of
test plasmid, and 0.05 µg of pRL-SV40. MC3T3 E1 cells were
transfected for 6 h prior to addition of -MEM plus 10%
heat-inactivated calf serum containing treatments. Mv1Lu and MC3T3 E1
cells were cultured for 48 h in the presence of the treatment(s),
washed once with phosphate-buffered saline and cell extracts were
prepared as described for enzyme assays.
Enzyme Assays
Chloramphenicol Acetyltransferase Assay-- Chloramphenicol acetyltransferase (CAT) activity was determined using the method of Gorman et al. (19). Mv1Lu or MC3T3 E1 cells were scraped into 250 µl of 0.25 M Tris-HCl (pH 7.5) and lysed by three rounds of freeze-thawing. The extracts were briefly centrifuged, and the supernatant was used for CAT and renilla-luciferase (LucRen) assays. Duplicate 100-µl cell extracts were incubated with 200 µCi of [14C]chloramphenicol, 32 µl of 1 M Tris-HCl (pH 7.5), 10 µl of 8 mM acetyl-CoA for 20 h at 37 °C. The reaction was terminated with 300 µl of xylenes and extracted with 500 µl of ethyl acetate. 700 µl of the organic phase was removed, dried, and separated by thin layer chromatography (Whatman silica gel) in chloroform:methanol (97:3) for 1 h. Acetylated products were detected by exposure to a phosphorimaging screen and developed using a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Di-acetylated chloramphenicol was quantified using ImageQuant version 1.1 software (Molecular Dynamics). CAT activity was normalized to LucRen activity to correct for any difference in transfection efficiency or generalized alterations in transcription.
Luciferase and Renilla Assay-- Cells were lysed with 250 µl of cell lysis buffer (Promega Corp., Madison, WI), and 20 µl of the extract was assayed for firefly-luciferase (LucFf) activity using a Luciferase Assay kit (Promega Corp., Madison, WI) according to the manufacturer's suggested protocol. LucRen activity was determined in 20 µl of the Mv1Lu cell extract using a Dual Luciferase Assay kit (Promega Corp., Madison, WI) according to the manufacturer's suggested protocol. The total protein concentration of each extract was determined using a Bio-Rad Protein Assay Kit (Bio-Rad), and luciferase activity was normalized to the total protein concentration of the cell extract.
Tissue Transglutaminase Assay-- Tissue transglutaminase activity in Mv1Lu and MC3T3 E1 cell extracts was determined as described previously (20). Cells were washed once with phosphate-buffered saline, scraped into 250 µl of Tris-HCl (pH 7.5), and lysed with three cycles of freeze-thawing. Cell lysates were incubated in the presence of [3H]putrescine and N,N'-dimethylcasein. Ca2+-dependent conjugation of [3H]putrescine to N,N'-dimethylcasein was determined by trichloroacetic acid precipitation followed by scintillation counting.
Tissue Transglutaminase mRNA Measurement
Total RNA Preparation--
Total RNA was prepared from MC3T3 E1
cells treated with either TGF-1, BMP2, or BMP4 according to the
QIAShredder and RNeasy protocols (Qiagen, Inc., Santa Carita, CA) and
quantified by measuring the absorbance at 260 nm.
Quantification of Selected mRNA-- 50 ng of total RNA from MC3T3 E1 cells was reverse-transcribed according to a standard protocol (Current Protocols in Molecular Biology) using Superscript Reverse Transcriptase (Life Technologies, Inc.) with an oligonucleotide primer (300 nM) for either mouse tissue transglutaminase (CCAAGATCCCTCCTCCACAT; Genosys, Houston, TX) or mouse 36B4 (ATATGAGGCAGCAGTTTCTCCAG; Genosys, Houston, TX) in the presence of 4 mM MgCl2 and 2.5 mM dNTPs. Following reverse transcription, the cDNA product was PCR-amplified using 300 nM forward primers for either mouse tissue transglutaminase (AGACTCACGTTCGGTGCT; Genosys, Houston, TX) or human 36B4 (AGATGCAGCAGATCCGCAT; Genosys, Houston, TX) and 100 nM of an oligonucleotide probe for either tissue transglutaminase (ACCGGCCCAGATCCCAGTGAAGA; Synthetic Genetics, San Diego, CA) or mouse 36B4 (AGGCTGTGGTGCTGATGGGCAAGAAC; Applied Biosystems, Foster City, CA). Probes were labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine (5' and 3', respectively). The PCR amplification was accomplished with Taq DNA-polymerase (Boehringer Mannheim) in an ABI Prisim 7700 "real-time" PCR analysis instrument (Applied Biosystems, Foster City, CA), monitoring product generation throughout each of 40 cycles. Synthetic RNA was used to generate standard concentration curves for either mouse tissue transglutaminase (20 pg to 0.2 fg) or human 36B4 (20 pg to 2 fg). The point at which the fluorescent signal rose above the background (Ct; 10 S.D. above the base-line values) was used to quantitate the amount of the specified mRNA in the starting sample. Quantification of the sample template from the determined Ct was accomplished by comparison with cRNA standards run in parallel.
Statistics
Differences within and between data sets were determined using
SigmaStat for Windows (SPSS Inc., Chicago, IL). One-way analysis of
variance and subsequent Tukey's or Dunnett's mean separation procedure were performed at a significance level of p 0.05.
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RESULTS |
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TGF-1--
TGF-
1 has been reported to increase the
transglutaminase activity of several cell types (14, 15). To assess the
ability of TGF-
1 to regulate the activity of the tissue
transglutaminase gene promoter, we co-transfected mink lung epithelial
cells line, Mv1Lu, and mouse preosteoblastic cells, MC3T3 E1, with
reporter constructs that included segments of the mouse tissue
transglutaminase promoter linked to a reporter gene (either CAT or
LucFf) and a normalization plasmid (LucRen).
Following transfection, cells were treated with recombinant human
TGF-
1 or control media, and 48 h later cells were lysed and
assayed for reporter gene (CAT or LucFf) and
normalization gene (LucRen) activities. In each assay,
differences in transfection efficiency were corrected by normalizing
reporter gene activity to LucRen activity. Previous studies
from our laboratory have demonstrated that pmTG3.8-Luc, a reporter gene
construct containing 3.8 kilobase pairs of the mouse tissue
transglutaminase promoter, possesses all the necessary information to
direct physiological expression of tissue transglutaminase in
vivo (16). We therefore transfected Mv1Lu cells with pmTG3.8-Luc (Fig. 1A) and measured the
effect of TGF-
1 on the activity of the promoter. The results with
Mv1Lu cells (Fig. 1B) indicate that TGF-
1 induces a
dose-dependent increase in pmTG3.8-Luc activity.
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BMP2 and BMP4--
To determine if the effects of TGF-1
on the transglutaminase promoter activity could be extended to other
members of the TGF-
superfamily, we examined the effects of BMP2 and
BMP4 on the activity of the tissue transglutaminase promoter.
Expression of BMP2 and BMP4 in the developing limb coincides with
tissue transglutaminase, occurring in regions such as the interdigital web and the apical ectodermal ridge (16, 30, 31). Mv1Lu cells,
co-transfected with pmTG3.8-Luc and SV2-
-galactosidase, were treated with recombinant human BMP2, BMP4, or control media for
48 h. After 48 h, the luciferase and
-galactosidase
activities were determined in the cell extracts. Both BMP2 and BMP4
inhibited the activity of the transglutaminase promoter to a level that was 9 and 13%, respectively, of control (Fig.
2A; p
0.001, n = 2).
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MC3T3 E1 Cells--
Since inhibition of transcription for
several genes is known to require the presence of the TRE sequence
(23-28), we investigated whether our observation of this element
mediating TGF-1 induction and BMP2 and BMP4 inhibition of tissue
transglutaminase promoter activity was specific for the Mv1Lu cell
line. To examine this question, mouse calvarial pre-osteoblasts (MC3T3
E1 cells) were transfected with the intact tissue transglutaminase gene
promoter linked to a CAT reporter gene (pmTG1.8-CAT) or the tissue
transglutaminase gene promoter containing the mutated TRE
(pmTG1.8-
mut-CAT) and treated with 0.2 nM TGF-
1, 2.5 nM BMP2, or 2.5 nM BMP4 for 48 h. In
contrast to what we observed in Mv1Lu cells, the activity of the
pmTG1.8-CAT promoter construct was inhibited by TGF-
1 to
approximately 34% of control in MC3T3 E1 cells (Fig.
4). Consistent with our observations in
Mv1Lu cells, both BMP2 and BMP4 inhibited the activity of pmTG1.8-CAT.
However, as we observed in Mv1Lu cells, the response of all three
cytokines was eliminated by mutation of two nucleotides contained
within the core of the identified tissue transglutaminase TRE
(pmTG1.8-
mut-CAT, Fig. 4).
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DISCUSSION |
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TGF- superfamily members are important regulators of apoptosis
in a number of tissues including liver, mammary gland, prostate gland,
and the developing limb (31-38). In these tissues, the apoptotic processes are associated with a marked induction of tissue
transglutaminase expression (1). A link between TGF-
superfamily
members and tissue transglutaminase is suggested by the fact that
TGF-
1 can induce tissue transglutaminase expression in apoptotic
cells (14). The studies reported here were initiated to gain further
insight into the molecular basis for TGF-
superfamily member
regulation of tissue transglutaminase expression. The results we
obtained indicate that BMP2 and BMP4, members of the TGF-
family of
cytokines, inhibit the activity of the tissue transglutaminase gene
promoter in both Mv1Lu and MC3T3 E1 cells. In contrast, our data
suggest that TGF-
1 can directly increase the transcriptional
activity of the tissue transglutaminase promoter in Mv1Lu cells and
inhibit this transcriptional activity in MC3T3 E1 cells. Thus, the
effect of TGF-
superfamily members on tissue transglutaminase
promoter activity is specific for the cytokine and the cellular
context.
BMPs--
During limb development, tissue transglutaminase is
expressed in the same regions (interdigital webs and apical ectodermal ridge) as BMP2 and BMP4 (16, 30, 31). This observation prompted us to
examine whether these TGF- superfamily members could regulate the
activity of the tissue transglutaminase promoter. The results of our
studies indicate that both BMP2 and BMP4 can negatively regulate the
tissue transglutaminase gene promoter activity. This inhibition was
discernible in both Mv1Lu and MC3T3 E1 cells, suggesting a ubiquitous
activity for these cytokines on tissue transglutaminase activity. In
both cell lines, the BMP-mediated inhibition of the tissue
transglutaminase gene promoter required the presence of a canonical TRE
located 868 bp upstream of the transcription start site. Thus, the
effects of BMPs on transglutaminase expression are quite similar to
those of retinoids (8) and TNF
(12), both of which regulate tissue
transglutaminase expression via activation of specific
cis-regulatory elements embedded within the promoter of the
gene. For retinoids, the regulation is mediated via a complex retinoid
response element located 1703 bp upstream of the transcription start
site (8). For TNF
, regulation is mediated through an NF-
B site
located in the proximal 5'-flanking DNA (
1338 bp) (12). Thus, factors
such as BMPs, retinoids, and TNF
that can regulate apoptosis in
different cell types control tissue transglutaminase expression via
distinct cis-regulatory elements embedded within the
promoter of the gene.
TGF-1--
Our studies with TGF-
1 were prompted by the
observations that TGF-
1 increases both the activity and mRNA
levels for tissue transglutaminase in NHEK cells (14, 15) and that
tissue transglutaminase, in turn, cooperates to promote the conversion
of latent TGF-
1 to its active form (17, 18). This positive
cooperation appears to be restricted to the TGF-
1-transglutaminase
interaction since our studies with other members of the TGF-
superfamily, specifically BMP2 and BMP4, demonstrated a negative
regulation of the tissue transglutaminase gene promoter.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Integrative
Biology, Pharmacology and Physiology, the University of Texas Medical
School, P. O. Box 20708, Houston, TX 77030. Tel.: 713-500-7480; Fax: 713-500-7455; E-mail: pdavies{at}farmr1.med.uth.tmc.edu.
1
The abbreviations used are: TGF-,
transforming growth factor-
; BMP, bone morphogenetic protein; CAT,
chloramphenicol acetyltransferase; TRE, transforming growth factor-beta
response element; bp, base pair(s); PAI-1, plasminogen activator
inhibitor-1; PCR, polymerase chain reaction; Luc, luciferase; MEM,
minimum Eagle's medium; NaB, sodium butyrate.
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REFERENCES |
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