From the Molecular Cardiology and ** Enzyme Research
Units, Victor Chang Cardiac Research Institute, the
Arthritis and Inflammation Research
Program, Garvan Institute of Medical Research, Darlinghurst, New South
Wales, 2010, and the § School of Biochemistry and Molecular
Genetics, University of New South Wales, New South Wales, 2054, Australia
Received for publication, December 1, 2000, and in revised form, March 22, 2001
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ABSTRACT |
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The novel G-protein,
Gh/tissue transglutaminase (TGase II), has both
guanosine triphosphatase and Ca2+-activated
transglutaminase activity and has been implicated in a number of
processes including signal transduction, apoptosis, bone ossification,
wound healing, and cell adhesion and spreading. To determine the role
of Gh in vivo, the Cre/loxP
site-specific recombinase system was used to develop a mouse line in
which its expression was ubiquitously inactivated. Despite the absence
of Gh expression and a lack of intracellular TGase activity
that was not compensated by other TGases, the
Tgm2 Transglutaminases
(TGases)1 are a family of
thiol- and Ca2+-dependent acyl transferases
that catalyze the formation of an amide bond between the
Gh/TGase II has G-protein signaling and TGase protein
cross-linking activities. It is ubiquitously expressed in mammalian tissues (6) and is found both extracellularly at the cell surface in
association with the extracellular matrix (7) and
intracellularly, where it is both membrane-associated and cytosolic.
Gh has been implicated in a variety of cellular processes
including signal transduction (8), cell adhesion, and spreading (9),
wound healing, apoptosis, and bone ossification (10).
As a G-protein Gh mediates membrane-bound phospholipase
C-activated inositol phosphate production by In the extracellular matrix, Gh cross-links and stabilizes
a number of substrates such as laminin-nidogen, fibronectin,
fibrinogen, collagen, osteonectin, osteopontin, and the cell adhesion
molecule C-CAM (16-22). Gh overexpression in fibroblasts
enhances cell attachment (9, 21) and in endothelial cells, reduced
expression, achieved by the use of antisense techniques, results in
decreased cell adhesion and spreading (23). Gh has recently
been shown to mediate cell adhesion in fibroblasts by acting as a
To evaluate the in vivo role of Gh/TGase II, we
report here the development of a Tgm2-loxP
knockin mouse, which allowed inactivation of both Tgm2
alleles after cross-breeding with animals expressing Cre-recombinase.
Generation of Floxed and Knockout Tgm2 Mice--
Clones
encoding Tgm2 were isolated from an 129SVJ mouse genomic DNA
library (Stratagene) using rat Gh cDNA as a probe (34). A binary approach based on the Cre/loxP site-specific
recombination system of bacteriophage P1 (35) was used to develop
mouse lines in which Tgm2 can either be ubiquitously
inactivated or selectively inactivated in specific tissues. A
gene-targeting construct in which loxP sites were inserted
in the same orientation into introns 5 and 8, which flank exons 6-8
(encoding the TGase catalytic core domain of Gh) was
generated. For positive selection, a hygromycin resistance gene under
the control of the phosphoglycerate kinase (PGK) promoter was inserted
in antisense orientation immediately 3' to the loxP site in
intron 5. The PGK/hygromycin cassette was also flanked by
frt sites to allow Flp-recombinase-mediated excision, should
the presence of the cassette interfere with normal mRNA splicing
(Fig. 1A). The targeting construct was transfected into W9.5
embryonic stem cells by the Genetically Modified Mouse Laboratory (GMML), Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, and homologous recombination events were confirmed by
Southern blot analysis using a 5' probe (probe 1) external to the
targeting construct and a 3' probe (probe 2) internal to the targeting
construct (Fig. 1). Chimeric mice were generated and back-crossed with
C57BL/6 mice to obtain heterozygous floxed (flanked by loxP
sites) Tgm2t/+ mice. Knockout animals
(Tgm2 Western Blot Analysis--
Recombinant rat Gh was
expressed and purified as described (37). Liver and heart tissues were
placed in a hypertonic buffer (10 mM Tris-HCl, pH 7.5, 1.4 mM EGTA, 12.5 mM MgCl2) that
included a protease inhibitor mixture (Roche Molecular Biochemicals).
Tissues were minced and homogenized in an ice bath with a mechanical
homogenizer, filtered through a 70 µm Nylon cell strainer, and
further homogenized in a Dounce homogenizer. Intact cells and
organelles were pelleted (600 × g, 10 min, 4 °C).
Membrane and cytosol fractions in the supernatant fraction were
separated by centrifugation (541,000 × g, 20 min,
4 °C). Protein concentration was determined using the Coomassie Plus
protein assay reagent (Pierce) with bovine serum albumin as a standard.
Cytosolic samples (30-100 µg) were separated on an 8%
SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride
membrane. Membranes were blocked (5% nonfat dry milk/Tris-buffered
saline (TBS); 16 h, 4 °C), incubated with a rabbit polyclonal
anti-bovine TGII antibody (Gh7 Northern Blot Hybridization and Reverse Transcription
(RT)-PCR--
Total RNA was extracted from heart and liver tissue
using the Totally RNA kit (Ambion) according to the manufacturer's
instructions. A 1-kb fragment encoding exon 13 and the 3'-untranslated
region of Tgm2 was used as a probe in Northern blot
analyses. Superscript One-Step RT-PCR system (Life Technologies) was
used to perform RT-PCR with the following primers: exon 3 forward
primer, 5'-GCTTCATCTACCAAGGC-3' and exon 11 reverse primer,
5'-GCTGGTTCGATGAGAAGGC-3'.
Transglutaminase Assay--
Fibroblast and thymocyte cell
lysates were prepared as described (38). Hearts and livers were
harvested as follows. Mice were anesthetized with a mixture of xylazine
(20 mg/kg) and ketamine (100 mg/kg) and anticoagulated with a single
bolus of heparin (5000 units/kg) administered intravenously into the
right internal jugular vein. After 5 min, the left internal jugular
vein was cannulated, and the animals were perfused with heparinized
saline at a rate of 4 ml/min following transection of the right common carotid artery, until all blood was cleared. TGase activity (37) of
cytosol and membrane preparations from heart and liver, as well as cell
lysates from fibroblasts and thymocytes, was assayed 40 min after
addition of 0 mM (basal), 300 µM (80%
maximal), or 2 mM (maximal) CaCl2. The
specificity of the TGase assay was confirmed by addition of the
competitive substrate inhibitor, monodansylcadaverine. GTP [ Primary Fibroblast and Thymocyte Cultures--
Fibroblasts were
isolated from finely minced neonatal heart and lung tissues following
digestion (0.6 mg/ml collagenase (Worthington), 1× pancreatin (Life
Technologies, Inc.) in 116 mM NaCl, 18.3 mM HEPES, 5.5 mM glucose, 5.4 mM KCl, 1 mM MgCl2, and 0.96 mM
NaH2PO4, pH 7.4, 20 min, 37 °C),
centrifugation (8 × g, 10 min, room temperature) and
plating for adherent cells (high glucose-Dulbeccos's modified Eagle's
medium supplemented with 10% fetal calf serum, and 0.1% penicillin/streptomycin, 30 min, 37 °C). Passages 3-8 were used in
experiments. Thymocytes were isolated in RPMI 1640 supplemented with
10% fetal calf serum, 2 mM glutamine, and 0.1%
penicillin/streptomycin from the thymuses of 4-6-week-old
Tgm2+/+ and Tgm2 Apoptosis Assays--
Freshly isolated thymocytes (1 × 106) were analyzed by cell sorting using a FACScalibur
(Becton Dickinson) after incubating cells with fluorescein
isothiocyanate (FITC)-conjugatedanti-CD4 and
phycoerythrin(PE)-conjugated-anti-CD8 antibodies (PharMingen International). Isolated thymocytes (1 × 106/ml in
high glucose-Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum, 2 mM glutamine, and 0.1%
penicillin/streptomycin) were cultured in the presence or absence of
dexamethasone (1 µM) for 8 or 24 h. Cell viability
was analyzed using a flow cytometer after both
FITC-conjugated-Annexin-V and propidium iodide staining according to
the manufacturer's instructions (PharMingen International). The
thymuses from 4-6-week-old Tgm2+/+ and
Tgm2 Fibroblast Adherence Assays--
Cells (2 × 105/ml) were plated onto poly-L-coated slides (Nalge Nunc
International) and non-adherent cells removed after 2 or 16 h.
Adhered cells were washed (PBS, pH 7.4), fixed (4% (w/v) paraformaldehyde/PBS, 10 min, room temperature) and permeabilized (0.1% Triton X-100/PBS, 10 min, room temperature). Actin stress fibers
were stained with FITC-labeled phalloidin (1:1000 in PBS, 30 min, room
temperature) and then washed with PBS. Cells were viewed by
fluorescence microscopy after mounting with Vectashield and DAPI (1.5 µg/ml) staining. Cells in ten fields of 1.27 mm2 were
counted and expressed as a percentage ± S.E. of initial plating density.
Cardiac Hemodynamic Assessment--
Age-matched
Tgm2+/+ and Tgm2 Statistical Analyses--
All comparisons were performed using
the unpaired Student's t test with p < 0.05 considered significant.
Targeted Disruption of Tgm2--
The Cre/loxP
site-specific recombination system of bacteriophage P1 was used to
develop mouse lines in which Tgm2 can either be ubiquitously
inactivated or selectively inactivated only in specific tissues.
Heterozygous (t/+) and homozygous (t/t) "Tgm2-floxed (flanked by loxP sites)" mouse lines were generated using
a gene-targeting vector in which loxP sites, for
Cre-mediated excision, were inserted to flank exons 6-8, which encode
the TGase catalytic core domain of Gh. mRNA splicing
after Cre-mediated excision, results in a frameshift that introduces a
number of downstream stop codons, thereby ensuring disruption of
Gh. Tgm2-floxed mice were crossed with mice
expressing Cre ubiquitously under the control of the human
cytomegalovirus (CMV) minimal promoter to generate Tgm2 knockouts (heterozygous, +/
Western blots of liver (Fig. 1F) or heart (data not shown)
membrane developed with a polyclonal anti-bovine TGase II antibody Gh7 TGase Inactivation--
The absence of Gh
cross-linking activity in Tgm2
The small amount of TGase activity observed in the
Tgm2
Functional coupling of Gh to Phenotypic Assessment of Tgm2
In the thymuses of young mice, immature thymocytes that are
not selected to differentiate into T-cells, are cleared by apoptotic cell death. The percentage and absolute numbers of the different thymocyte populations in 4-6-weekold
Tgm2+/+ and Tgm2
Previous studies of Swiss 3T3 fibroblasts and endothelial cells
indicate that Gh has an extracellular role in cell
attachment (21, 23) and cell spreading (23). In agreement with these studies, it was more difficult to establish primary fibroblast cultures
from Tgm2
Cardiac function in 8-10-week-old Tgm2/
mice were viable, phenotypically
normal, and were born with the expected Mendelian frequency. Absence of
Gh coupling to
1-adrenergic receptor
signaling in Tgm2
/
mice was demonstrated by
the lack of agonist-stimulated [
-32P]GTP photolabeling
of a 74-kDa protein in liver membranes. Annexin-V positivity observed
with dexamethasone-induced apoptosis was not different in
Tgm2
/
thymocytes compared with
Tgm2+/+ thymocytes. However, with this
treatment there was a highly significant decrease in the viability
(propidium iodide negativity) of Tgm2
/
thymocytes. Primary fibroblasts isolated from
Tgm2
/
mice also showed decreased adherence
with culture. These results indicate that Gh may be
importantly involved in stabilizing apoptotic cells before
clearance, and in responses such as wound healing that require
fibroblast adhesion mediated by extracellular matrix cross-linking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-carboxamide groups of peptide-bound glutamine residues and the
primary amino groups in various compounds, including the
-amino
group of lysine in certain proteins (1). Seven distinct
transglutaminases have been described (reviews in Refs. 2-4 and 5). In
addition to Gh, also known as tissue TGase (TGase II,
74-80 kDa), these include, the enzymatically inactive band 4.2 (72-77
kDa), involved in the cytoskeletal network; plasma factor XIIIA
(fXIIIA, 75 kDa) involved in catalyzing formation of the fibrin clot at
sites of blood coagulation; keratinocyte TGase (TGase I, 90 kDa), which
plays a major role in terminal differentiation of epithelia, and in the
formation of the cornified cell envelope of the epidermis; epidermal
TGase (TGase III, 77 kDa), involved in differentiating epidermal and
hair follicle cells; prostate TGase (TGase IV, 65-77 kDa), which, in
rodents results in the formation of the copulatory plug through
cross-linking of proteins in the seminal vesicle secretion (1); and
TGase X (TGase 5, 80 kDa), a novel TGase gene isolated from human
keratinocytes. Two new TGases (VI and VII) have recently been
identified.2
1B- and
1D-, but not
1A-adrenergic receptors (AR)
(8, 11), by the TP
but not TP
thromboxane A2 receptor (12), and
by oxytocin receptors (13). It also modulates conductance of the
Maxi-K+ ion channel in smooth muscle (14) and adenylyl
cyclase in Balb-C 3T3 fibroblasts and bovine aortic endothelial cells
(15).
1 and
3 but not
2
integrin-associated coreceptor for fibronectin; an action that is
independent of its TGase activity (24). A rat punch biopsy wound
healing model, followed over 6 days, demonstrated increased Gh expression and activity at sites of neovascularization
and invasion of the fibrin matrix and then in the granulation tissue matrix during healing (25). An intracellular role for Gh is suggested by the finding that its activity is down-regulated in the
myocardium of humans with cardiac failure (26). Also, transgenic overexpression of Gh (~37-fold) in the heart, results in
ventricular remodelling with elevated expression of the
hypertrophy-associated genes,
-myosin heavy chain and
-skeletal
actin, and diffuse interstitial fibrosis (27). Various mammalian cells
(human neuroblastoma cells (SK-N-BE, Ref. 28); Balb-C 3T3, Ref. 9; and
L929 fibroblasts, Ref. 29) transfected with full-length Gh
cDNA showed an increase in both spontaneous and induced apoptosis.
Furthermore, inhibition of neuroblastoma and human promonocytic cell
Gh expression results in decreased susceptibility to
retinoic acid-induced apoptosis (30). In vivo, the
expression of Gh coincides with apoptosis during formation
of the interdigital web (31) and is observed in hypertrophic
chondrocytes during endochondral ossification (18, 31, 32), in
myoblasts during differentiation of skeletal muscle (31), and during
embryo implantation and postpartum involution of the uterine epithelium
(33).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
) were generated by crossing
heterozygous or homozygous floxed mice with transgenic mice expressing
Cre recombinase in the germline under the control of the human
cytomegalovirus minimal promoter (36). Mice were initially genotyped by
Southern blot analysis of BamHI-digested tail genomic DNA.
PCR analysis of tail DNA using 3 primers (P1: forward primer,
5'-CATGAATCAGGATGCATCTG-3'; P2: forward primer,
5'-TAGGGATACAAGAAGCATTG-3'; P3: reverse primer, 5'-GACAAAGGAGCAAGTGTTAC-3') was performed to genotype the animals in
the successive generations.
, kindly provided by Dr
Mie-Jae Im, Cleveland Clinic, Ohio; 1:500 dilution in 5% nonfat dry
milk/TBS, 16 h, 4 °C), a goat polyclonal anti-guinea pig TGII
antibody (Upstate Biotech, 06-471, 1:1000 dilution in 5% nonfat dry
milk/TBS, 1 h, room temperature) or a monoclonal anti-human TGII
antibody (CUB7402, Neomarkers, 1:100 dilution in 5% nonfat dry
milk/TBS, 16 h, 4 °C). Blots were washed (three times with
TBS/0.1% Triton, 10 min), incubated with anti-rabbit IgG-HRP (Amersham
Pharmacia Biotech), anti-goat IgG-HRP (Santa Cruz Biotechnologies) or
anti-mouse IgG-HRP (Amersham Pharmacia Biotech), respectively at 1:1500
dilution in 5% nonfat dry milk/TBS for 1 h at room temperature,
washed (three times with TBS/Triton) and analyzed using an enhanced
chemiluminescence Western blot detection kit (Amersham Pharmacia Biotech).
S
inhibition of TGase activity is inversely proportional to Ca2+ activation and is greatest under conditions where
TGase activity is minimal (37). GTP
S inhibition of TGase activity
was therefore evaluated at ~80% of maximal
Ca2+-activated TGase activity.
-32P]GTP Photolabeling of Membranes--
Liver
membranes (250 µg) were prepared as described (39) and photolabeled
with [
-32P]GTP as described (40) in the presence or
absence of 10
5 M (
)epinephrine or after
preincubation with 10
4 M phentolamine. After
autoradiography, signal intensity was quantitated by densitometry.
/
mice by gentle dissociation of cells between glass slides and then
filtration through a 70-µm cell strainer.
/
mice were removed 0, 8, or 24 h
after intraperitoneal injection with 0.5 mg/animal
dexamethasone-21-acetate and fixed for immunofluorescence (41),
weighed, or used for TGase assays. Fragmented DNA of apoptotic cells
was detected by TUNEL assay (Apoptosis Detection System, Fluorescein,
Promega Corporation) and visualized by fluorescence microscopy after
mounting with Vectashield (Vector Laboratories) and DAPI (1.5 µg/ml) staining.
/
mice were anesthetized with xylazine (20 mg/kg) and ketamine (100 mg/kg) given intraperitoneally, connected to a rodent ventilator after
endotracheal intubation and placed on a thermostatically controlled
heating pad. The right carotid artery was cannulated with a 1.4 F pressure transducer (Millar Instruments, Houston, TX), which
was advanced into the ascending aorta and then left ventricular cavity.
Pressure measurements were recorded in both the left ventricle and the
ascending aorta at a sampling frequency of 2000 Hz with a Biopac MP-100
data acquisition system (Biopac Systems Inc., Santa Barbara, CA). Data
was subsequently analyzed to determine aortic and left ventricular
pressures and heart rate; maximum rates of pressure development
(dP/dtmax) and relaxation (dP/dtmin) were
calculated from the first derivative of the left ventricular pressure.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
; homozygous,
/
). Genotyping of progeny by Southern blot analysis of BamHI-digested genomic
tail DNA using probe 1 allowed Tgm2-floxed (t, 10 kb), wild
type (+, 8 kb) and Cre-deleted (
, 4 kb) alleles to be identified
(Fig. 1B). Successive
generations were genotyped using primers, P1, P2, and P3. P2 and P3
amplifies the wild type (100 bp) and/or floxed (140 bp) alleles, and P1
and P3 amplifies a 180-bp product after Cre-mediated deletion (Fig.
1C). Northern blots of total RNA isolated from +/+, +/
,
/
, t/
, and t/t mice were analyzed using either full-length rat
Gh cDNA (data not shown) or a 1-kb probe encoding exon
13 and the 3'-untranslated region of Tgm2 (Fig.
1D). This demonstrated Tgm2 transcripts of both
the appropriate size and abundance in Tgm2-floxed mice,
indicating normal Tgm2 transcription/splicing despite intron
manipulation and the absence of Tgm2 transcripts
(full-length or truncated) in the knockout mice. These results were
confirmed by RT-PCR (Fig. 1E) using primers directed to
exons 3 and 11.
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Fig. 1.
Targeted disruption of
Tgm2. A, targeted locus showing exons
4-10: PGK, phosphoglycerate kinase I promoter;
hygro, hygromycin resistance gene; PA, SV40
poly(A) tail; Sc, ScaI; B,
BamHI; H, HindII; C,
ClaI; loxP and frt, sequences
recognized by Cre and Flp recombinases, respectively. The location of
probes and primers used for genotyping is indicated. B,
genotyping by Southern blot analysis of BamHI-digested
genomic tail DNA using probe 1. C, genotyping by PCR
screening of genomic tail DNA using primers P1, P2, and P3.
D, Northern blot analysis of 20 µg of heart total RNA
using Tgm2 3'-untranslated region probe. E,
RT-PCR of 0.5-1 µg of heart total RNA using exons 3 and 11 forward
and reverse primers, respectively. F, Western blot analysis
of liver membranes (100 µg) using an anti-bovine TGII polyclonal
antibody (Gh7 ). G, left panel;
Western blot analysis of heart cytosol (100 µg) using an anti-human
TGII monoclonal antibody (CUB 7402) and right panel, heart
cytosol (30 µg) using an anti-guinea pig TG II polyclonal antibody
(06-471). P (0.1 µg), purified recombinant rat
Gh; +/+, wild type; +/
, heterozygous knockout;
/
,
homozygous knockout; t/+, heterozygous floxed; t/t, homozygous floxed;
t/
, heterozygous floxed, and heterozygous knockout.
(42) showed a 74-kDa band in
Tgm2+/+ mice that was of lesser intensity in
Tgm2+/
mice and absent in
Tgm2
/
mice. Westerns blots of heart (Fig.
1G) and liver (data not shown) cytosols or membranes (data
not shown) were developed using commercial monoclonal anti-human TGase
II (CUB7402) or polyclonal anti-guinea pig TGase II (Upstate
Biotechnology, 06-471) antibodies, respectively. The monoclonal
antibody (Fig. 1G, left panel) recognized a
74-kDa band corresponding to Gh in
Tgm2+/+ mice, which was less intense in
Tgm2+/
and absent in
Tgm2
/
mice (Fig. 1F). The
polyclonal anti-guinea pig TGase II antibody (Fig. 1G,
right panel) recognized a single band of 74 kDa in
Tgm2+/+ mice that was progressively weaker, but
nonetheless still present, in Tgm2+/
and
Tgm2
/
mice, indicating that this commercial
polyclonal anti-guinea pig TGase II antibody (06-471) recognizes two
or more proteins of ~74 kDa, only one of which is Gh. It
has been suggested (25) that the three additional bands (~60, 50, and
20 kDa) recognized by the monoclonal antibody are proteolytically
degraded Gh products. However, the absence of the
full-length Gh band in Tgm2
/
animals developed with the Gh7
polyclonal antibody or
the monoclonal antibody (Fig. 1F), and the absence of
smaller molecular size bands in blots developed with both polyclonal
antibodies (Fig. 1, G and F, left
panel), make this unlikely. These findings indicate a lack of
specificity of the commercial antibodies and question previously
reported data generated using these antibodies.
/
mice was
demonstrated by TGase activity assays. To minimize the contribution to
TGase activity by fXIIIA and other TGases in blood, animals were
anticoagulated and perfused with heparinized saline before tissue
collection. TGase activity of liver and heart cytosol (Fig. 2, A and B) and
membrane preparations (data not shown) was markedly decreased in
Tgm2
/
as compared with
Tgm2+/+ mice. There was no significant
difference in activity between the Tgm2+/+ and
Tgm2+/
animals. The competitive substrate
inhibitor, monodansylcadavarine, although used at a concentration (40 µM) that was submaximal, reduced the
Ca2+-stimulated TGase activity in all samples from 100 to
23-40% (data not shown), confirming the specificity of the assay.
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Fig. 2.
Inactivation of TGase activity.
A, liver cytosols (30 µg) were assayed at ~80% of
maximal TGase activity in the absence (filled bars) or
presence (open bars) of 500 µM GTP S as
detailed under "Experimental Procedures." Maximal
Ca2+-activated TGase activities (pmol
[3H]putrescine incorporated/µg of protein/min) were:
Tgm2+/+, 1.3 ± 0.17;
Tgm2+/
, 1.1 ± .18, and
Tgm2
/
, 0.13 ± 0.04. B,
heart cytosols (30 µg) were assayed as in A. Maximal
Ca2+-activated TGase activities (pmol
[3H]putrescine incorporated/µg of protein/min) were:
Tgm2+/+, 1.1 ± 0.04;
Tgm2+/
, 1.0 ± 0.18, and
Tgm2
/
, 0.3 ± 0.05. C,
maximal Ca2+-activated TGase activity of whole cell lysates
from fibroblasts (30 µg) was determined as described under
"Experimental Procedures." Data represent means ± 1 S.E. of
three experiments performed in triplicate; ##, p < 0.001 and ###, p < 0.0001 versus respective
responses in the absence of GTP
S; **, p < .001 and
***, p < 0.001 versus respective responses
in Tgm2+/+ animals.
/
heart and liver preparations may
reflect residual blood (and therefore fXIIIA) contamination of the
samples or compensation by other intracellular TGases. To address the
issue of compensation, TGase activity was evaluated in the presence of
GTP
S, which inhibits the TGase activities of Gh and
TGase III (43), but not that of other TGases. As shown in Fig. 2,
A and B, the TGase activity of both
Tgm2+/+ and Tgm2+/
samples in the presence of GTP
S was equivalent to that of
Tgm2
/
. Furthermore, the TGase activity of
Tgm2
/
samples was not inhibited by GTP
S,
indicating the TGase activity observed in the
Tgm2
/
mice is not contributed by a
GTP-sensitive TGase and is most likely contributed by an extracellular
TGase. This was confirmed by performing TGase assays on primary
fibroblast cultures established from Tgm2+/+ and
Tgm2
/
heart and lung tissue. No residual
TGase activity was observed in cell lysates from knockout primary
cultured fibroblasts, whereas robust TGase activity was evident in
Tgm2+/+ cells (Fig. 2C). This
indicates that the small amount of activity observed in the
Tgm2
/
hearts and livers can be attributed to
a small amount of blood contamination, and therefore residual activity
of fXIIIA or other blood-borne TGases.
1-adrenergic
receptor (
1-AR) signaling was investigated in the
Tgm2+/+ and Tgm2
/
mice by [
-32P]GTP photolabeling of purified liver
membranes in the absence or presence of the
1-AR
agonist, (
)epinephrine, or in the presence of (
)epinephrine plus
the antagonist, phentolamine (Fig. 3). In
Tgm2+/+ liver membranes, (
)epinephrine
treatment resulted in a significant enhancement (2-fold,
p < 0.01) in [
-32P]GTP labeling of
the 74-kDa Gh; a response that was completely inhibited by
pretreatment with the
1-antagonist, phentolamine (Fig.
3). In Tgm2
/
liver membranes, however, only
labeling of a ~40 kDa G-protein, probably G
i, was
observed. (Fig. 3). These findings indicate both that Gh is
functionally coupled to the
1-AR, and that other GTP-binding TGases are unable to substitute for Gh in
1-AR-mediated signaling.
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Fig. 3.
Autoradiography of
[ -32P]GTP-photolabeled liver
membrane preparations from Tgm2+/+
(lanes 1, 2, and 3)
and Tgm2
/
(lanes 4,
5, and 6) mice. Purified liver
membranes (250 µg) were incubated at 30 °C for 10 min with 10 µCi [
-32P]GTP, 2 mM MgCl2,
100 mM NaCl, and 0.5 mM App(NH)p in HEGD buffer
(20 mM HEPES, 1 mM EGTA, 0.5 mM
dithiothreitol, 10% glycerol). Samples were incubated in the absence
(lanes 1 and 4) or presence (lanes 2 and 5) of (
)epinepherine (10
5
M), or were preincubated (lanes 3 and
6) with phentolamine (10
4 M) for
10 min before addition of (
)epinephrine (10
5
M). Reactions were irradiated with UV light (254 nm) in an
ice bath for 10 min, size fractionated by SDS-polyacrylamide gel
electrophoresis (8%) and subjected to autoradiography on Kodak XAR
film. Note the enhancement of the labeled 74-kDa Gh species
with (
)epinephrine-treatment of the Tgm2+/+
samples, which is prevented by phentolamine, and the absence of a
labeled 74-kDa species in the Tgm2
/
samples.
/
Mice--
Tgm2 knockout mice are viable, of normal size and
weight, and are born at the expected Mendelian frequency. The
Tgm2
/
animals have normal separation of their digits
and open eyelids, indicating that developmental apoptosis is not
impaired. In addition, homozygous knockouts breed normally and have no
problems with parturition. These findings indicate that Gh
is not critically involved in reproduction or in maturational apoptosis.
/
animals, identified by expression of CD4 and CD8 receptors, was determined by flow cytometry. There was no difference between the
Tgm2+/+ and Tgm2
/
mice with respect to the percentage (± 1 S.E.) of
CD4
CD8
(Tgm2+/+
3.1 ± 0.3%, Tgm2
/
2.66 ± 0.17%), CD4+CD8
(Tgm2+/+ 4.2 ± 0.2%,
Tgm2
/
4.8 ± 0.27%),
CD4
CD8+ (Tgm2+/+
10.9 ± 0.5%, Tgm2
/
10.5 ± 0.6%), or CD4+CD8+
(Tgm2+/+ 81.9 ± 1.0%,
Tgm2
/
81.9 ± 0.6%) thymocytes. This
indicates that normal thymocyte apoptotic turnover of immature T-cells
is not affected by the lack of Gh. Because dexamethasone
induces apoptosis and increases TGase activity in the thymus
in vivo (41), the effect of intraperitoneal dexamethasone administration was assessed. In
Tgm2+/+ thymocytes, TGase activity was
marginally increased at 8 h and markedly increased at 24 h
after dexamethasone treatment, and the latter was significantly greater
than the 24 h value in Tgm2
/
cells
(p < 0.05; Fig.
4A). In contrast to the
effects of dexamethasone in Tgm2+/+ cells, in
Tgm2
/
thymocytes, TGase activity at 24 h was slightly but not significantly increased over that observed at
8 h (Fig. 4A). Moreover, whereas GTP
S inhibited (by
62%, p < 0.001) the 24 h increase in TGase activity of Tgm2+/+ thymocytes, TGase activtiy
in Tgm2
/
cells was GTP
S-insensitive (not
shown). This lack of increase in thymic TGase activity in the
Tgm2
/
animals was associated with smaller
thymuses and less TUNEL positivity (Fig. 4, B and
C). To ascertain if this was because of decreased cell death
or an increased rate of clearance of dead cells from the thymus by
macrophages (44), flow cytometry analysis was performed on isolated
thymocytes. Cells were cultured for 8 or 24 h in the absence or
presence of 1 µM dexamethasone, although only the cells
at 8 h were further evaluated because of the marked loss of
Tgm2
/
cells with 24 h of dexamethasone
treatment. Early apoptosis results in membrane exposure of
phosphatidylserine that is recognized by the phospholipid-binding
protein, Annexin-V. As seen in Fig. 4D, although Annexin-V
positivity of both Tgm2+/+ and
Tgm2
/
thymocytes increased with
dexamethasone treatment (p < 0.0001), there was no
significant difference between the Tgm2
/
and
Tgm2+/+ cells. However, propidium iodide
staining of dead cells (Fig. 4E), an index of late-stage
cell death, showed a small but highly statistically significant
decrease (p < 0.0001) in the viability of
Tgm2
/
thymocytes. This indicates that
Tgm2
/
thymocytes are more susceptible to
dexamethasone-induced cell death, and that the decreased TUNEL
positivity of Tgm2
/
thymuses is the result
of increased clearance of dead cells. This, in turn, likely contributes
to the decreased size of dexamethasone-treated Tgm2
/
thymuses compared with those from
Tgm2+/+ animals. Thus,
Gh-dependent cross-linking is likely
importantly involved in stabilizing apoptotic cells before clearance,
as suggested by Piredda et al. (29).
View larger version (25K):
[in a new window]
Fig. 4.
Dexamethasone-induced thymocyte
apoptosis. A, dexamethasone-induced increases in
TGase activity of thymus gland cell lysates from 4-6-week-old
Tgm2+/+ and Tgm2 /
animals sacrificed 8 h (open bars) or 24 h
(hatched bars) after intraperitoneal dexamethasone (0.5 mg/animal) treatment. Maximal Ca2+-activated TGase
activities (pmol [3H]putrescine incorporated/µg of
protein/min) at 0 h were: 0.09 ± 0.003 for
Tgm2+/+ and 0.07 ± 0.004 for
Tgm2
/
animals. B, weights of
Tgm2+/+ (open bars) and
Tgm2
/
(filled bars) thymuses
harvested from 4-week-old animals 0, 8, or 24 h after
dexamethasone (0.5 mg, intraperitoneal) administration
(n = 3). C, TUNEL of thymus glands from
4-week-old animals harvested 24 h after dexamethasone (0.5 mg,
intraperitoneal) administration (magnification × 2.5).
D and E, isolated thymocytes from 4-6-week-old
Tgm2+/+ (+/+, open bars) and
Tgm2
/
(
/
, filled bars)
animals were cultured for 8 h with (+ Dex) or without
(
Dex) dexamethasone (1 µM) and analyzed by
FACS for the-fold-increase in Annexin-V-positive cells (D)
and the percentage of viable (propidium iodide-negative) cells
(E). Data are the means ± 1 S.E. of experiments
performed in triplicate using 3-6 mice/group; *, p < 0.05; **, p < 0.01; ***, p < 0.0001;
NS, not significant.
/
mice than from
Tgm2+/+ animals. Thus, despite plating
fibroblasts at equal density, fewer Tgm2
/
fibroblasts were adherent after 2 h (20 ± 3%
versus 79 ± 8% for Tgm2+/+,
n = 10 fields ± 1 S.E.). This was not because of
increased death of non-adherent Tgm2
/
fibroblasts, as confirmed by trypan blue exclusion. Similar results were obtained with cells grown on fibronectin-coated plates (data not
shown). Thus, Gh may be importantly involved in various
physiological and pathological responses, such as wound healing and
scar formation, which are mediated by the interaction of fibroblasts
with the extracellular matrix.
/
and
Tgm2+/+ animals was evaluated by micromanometry
(Table I). Systolic and diastolic blood
pressures were measured in the ascending aorta, and maximum rates of
pressure development (dP/dtmax) and of relaxation
(dP/dtmin) were calculated from the left ventricular
pressure. The Tgm2
/
mice showed no
statistically significant differences compared with
Tgm2+/+ mice, for any of the measured
parameters. The role of Gh in the maintenance of normal
cardiovascular function is unclear. Cardiac failure is associated with
both a down-regulation and uncoupling of
-AR and hence a relative
up-regulation of
1-ARs (26). However, down-regulation of
Gh has also been demonstrated in the setting of cardiac
dysfunction (26).
1-ARs are thought to contribute little
to normal cardiac inotropy and may thus act as a reserve mechanism (for
review see Ref. 45).
1B-ARs, however, do have a
significant role in maintaining normal vascular tone, and hence in
blood pressure homeostasis (46). The lack of change in either blood
pressure or parameters of left ventricular function in this study
suggests either that Gh in the vasculature contributes
minimally to
1B-AR-mediated vasoconstrictor responses,
or that in the absence of Gh, vascular tone can be
maintained by compensatory signaling pathways. Alternatively, impaired
inotropic drive in the hearts of the knockout animals may be
counterbalanced by a reduced afterload that results from the impaired
ability of the
1B-AR to mediate vasoconstriction in the
absence of Gh. Interestingly, cardiac-specific overexpression of Gh results in mild hypertrophy and
ventricular fibrosis, as well as impaired cardiac function (27), a
phenotype that is consistent with that obtained by overexpression of
either the wild-type
1B-AR (47) or a constitutively
active
1B-AR mutant (48). Thus, elucidating the
potential in vivo role of Gh in cardiovascular
homeostasis may require additional evaluations in animals in which
Gh is selectively inactivated only in the heart or only in
the vasculature, or may only be revealed by subjecting the knockout
animals to a pathophysiological stress, such as thoracic aortic
constriction. Such studies are currently in progress.
Haemodynamic parameters in Tgm2+/+ and Tgm2/
animals
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ACKNOWLEDGEMENTS |
---|
We thank Frank Köntgen and Michelle Swift of the GMML, Walter Eliza Hall Institute of Medical Research, Melbourne, Australia for expert assistance in the development of the floxed Tgm2 line; Professor Richard Harvey, Victor Chang Cardiac Research Institute, Sydney, Australia, for the CMV/Cre line, Dr. Kieran Scott for helpful discussions, and Dr. David Humphreys for advice with flow cytometry.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant 980199 from the National Health and Medical Research Council.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.
¶ Recipient of an Australian Postgraduate Award.
Recipient of a Joint Royal Australasian College of
Surgeons/Royal College of Surgeons of England Research Exchange
Fellowship and National Heart Foundation of Australia Postgraduate
Medical Research Scholarship.
§§ To whom correspondence should be addressed: 384 Victoria St., Darlinghurst, NSW, 2010, Australia. Tel.: 61 2 9295 8500; Fax: 61 2 9295 8501; E-mail: b.graham@victorchang.unsw.edu.au.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M010846200
2 D. Aeschlimann, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGase, transglutaminase;
AR, adrenergic receptor;
fXIIIA, factor XIIIA;
RT-PCR, reverse transcription-polymerase chain reaction, App(NH)p,
5'-adenylyl-,
-imidodiphosphate;
PBS, phosphate-buffered saline;
HRP, horseradish peroxidase;
FITC, fluorescein isothiocyanate, TUNEL,
TdT-mediated dUTP nick-end labeling;
bp, base pairs;
FACS, fluorescence-activated cell sorter;
PGK, phosphoglycerate kinase I;
GTP
S, guanosine 5'-3-O-(thio)triphosphate;
DAPI, 4,6-diamidino-2-phenylindole;
Gh, high molecular weight
G-protein.
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