Targeted Inactivation of Gh/Tissue Transglutaminase II*

Nisha NandaDagger §, Siiri E. IismaaDagger , W. Andrew OwensDagger ||, Ahsan Husain**, Fabienne MackayDagger Dagger , and Robert M. GrahamDagger §§§

From the Dagger  Molecular Cardiology and ** Enzyme Research Units, Victor Chang Cardiac Research Institute, the Dagger Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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-/- mice were viable, phenotypically normal, and were born with the expected Mendelian frequency. Absence of Gh coupling to alpha 1-adrenergic receptor signaling in Tgm2-/- mice was demonstrated by the lack of agonist-stimulated [alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Transglutaminases (TGases)1 are a family of thiol- and Ca2+-dependent acyl transferases that catalyze the formation of an amide bond between the gamma -carboxamide groups of peptide-bound glutamine residues and the primary amino groups in various compounds, including the epsilon -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

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 alpha 1B- and alpha 1D-, but not alpha 1A-adrenergic receptors (AR) (8, 11), by the TPalpha but not TPbeta 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).

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 beta 1 and beta 3 but not beta 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, beta -myosin heavy chain and alpha -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).

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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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-/-) 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.

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 (Gh7alpha , 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).

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. GTPgamma S inhibition of TGase activity is inversely proportional to Ca2+ activation and is greatest under conditions where TGase activity is minimal (37). GTPgamma S inhibition of TGase activity was therefore evaluated at ~80% of maximal Ca2+-activated TGase activity.

[alpha -32P]GTP Photolabeling of Membranes-- Liver membranes (250 µg) were prepared as described (39) and photolabeled with [alpha -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.

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-/- mice by gentle dissociation of cells between glass slides and then filtration through a 70-µm cell strainer.

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-/- 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.

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-/- 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.

Statistical Analyses-- All comparisons were performed using the unpaired Student's t test with p < 0.05 considered significant.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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, +/-; 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 (Gh7alpha ). 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.

Western blots of liver (Fig. 1F) or heart (data not shown) membrane developed with a polyclonal anti-bovine TGase II antibody Gh7alpha (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 Gh7alpha 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.

TGase Inactivation-- The absence of Gh cross-linking activity in Tgm2-/- 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 GTPgamma 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 GTPgamma S; **, p < .001 and ***, p < 0.001 versus respective responses in Tgm2+/+ animals.

The small amount of TGase activity observed in the Tgm2-/- 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 GTPgamma 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 GTPgamma S was equivalent to that of Tgm2-/-. Furthermore, the TGase activity of Tgm2-/- samples was not inhibited by GTPgamma 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.

Functional coupling of Gh to alpha 1-adrenergic receptor (alpha 1-AR) signaling was investigated in the Tgm2+/+ and Tgm2-/- mice by [alpha -32P]GTP photolabeling of purified liver membranes in the absence or presence of the alpha 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 [alpha -32P]GTP labeling of the 74-kDa Gh; a response that was completely inhibited by pretreatment with the alpha 1-antagonist, phentolamine (Fig. 3). In Tgm2-/- liver membranes, however, only labeling of a ~40 kDa G-protein, probably Galpha i, was observed. (Fig. 3). These findings indicate both that Gh is functionally coupled to the alpha 1-AR, and that other GTP-binding TGases are unable to substitute for Gh in alpha 1-AR-mediated signaling.


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Fig. 3.   Autoradiography of [alpha -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 [alpha -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.

Phenotypic Assessment of Tgm2-/- 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.

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-/- 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 GTPgamma S inhibited (by 62%, p < 0.001) the 24 h increase in TGase activity of Tgm2+/+ thymocytes, TGase activtiy in Tgm2-/- cells was GTPgamma 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).


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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.

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-/- 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.

Cardiac function in 8-10-week-old Tgm2-/- 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 beta -AR and hence a relative up-regulation of alpha 1-ARs (26). However, down-regulation of Gh has also been demonstrated in the setting of cardiac dysfunction (26). alpha 1-ARs are thought to contribute little to normal cardiac inotropy and may thus act as a reserve mechanism (for review see Ref. 45). alpha 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 alpha 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 alpha 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 alpha 1B-AR (47) or a constitutively active alpha 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.

                              
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Table I
Haemodynamic parameters in Tgm2+/+ and Tgm2-/- animals
Studies were performed in age-matched animals as described under "Experimental Procedures." Data are the means ± 1 S.E.


    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-beta ,gamma -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; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; DAPI, 4,6-diamidino-2-phenylindole; Gh, high molecular weight G-protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Folk, J. E. (1980) Annu. Rev. Biochem. 49, 517-531[CrossRef][Medline] [Order article via Infotrieve]
2. Aeschlimann, D., and Paulsson, M. (1994) Thromb. Haemost. 71, 402-415[Medline] [Order article via Infotrieve]
3. Greenberg, C. S., Birckbichler, P. J., and Rice, R. H. (1991) FASEB J. 5, 3071-3077[Abstract/Free Full Text]
4. Chen, J. S. K., and Mehta, K. (1999) Int. J. Biochem. Cell Biol. 31, 817-836[CrossRef][Medline] [Order article via Infotrieve]
5. Aeschlimann, D., Koeller, M. K., Allen-Hoffmann, B. L., and Mosher, D. F. (1998) J. Biol. Chem. 273, 3452-3460[Abstract/Free Full Text]
6. Thomazy, V., and Fesus, L. (1989) Cell Tiss. Res. 255, 215-224[Medline] [Order article via Infotrieve]
7. Gaudry, C. A., Verderio, E., Aeschlimann, D., Cox, A., Smith, C., and Griffin, M. (1999) J. Biol. Chem. 274, 30707-30714[Abstract/Free Full Text]
8. Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Misono, K., Im, M.-J., and Graham, R. M. (1994) Science 264, 1593-1596[Medline] [Order article via Infotrieve]
9. Gentile, V., Thomazy, V., Piacentini, M., Fesus, L., and Davies, P. J. A. (1992) J. Cell Biol. 119, 463-474[Abstract]
10. Upchurch, H. F., Conway, E., Patterson, M. K., Jr., and Maxwell, M. P. (1991) J. Cell. Physiol. 149, 375-382[Medline] [Order article via Infotrieve]
11. Chen, S., Lin, F., Iismaa, S., Lee, K. N., Birckbichler, P. J., and Graham, R. M. (1996) J. Biol. Chem. 271, 32385-32391[Abstract/Free Full Text]
12. Vezza, R., Habib, A., and FitzGerald, G. A. (1999) J. Biol. Chem. 274, 12774-12779[Abstract/Free Full Text]
13. Baek, K. J., Kwon, N. S., Lee, H. S., Kim, M. S., Muralidhar, P., and Im, M.-J. (1996) Biochemistry 315, 739-744
14. Lee, M.-Y., Chung, S., Bang, H.-W., Baek, K. J., and Uhm, D.-Y. (1997) Eur. J. Physiol. 433, 671-673[CrossRef][Medline] [Order article via Infotrieve]
15. Gentile, V., Porta, R., Chiosi, E., Spina, A., Valente, F., Pezone, R., Davies, P. J. A., Alaadik, A., and Illiano, G. (1997) Biochim. Biophys. Acta 1357, 115-122[Medline] [Order article via Infotrieve]
16. Barsigian, C., Stern, A. M., and Martinez, J. (1991) J. Biol. Chem. 266, 22501-22509[Abstract/Free Full Text]
17. Aeschlimann, D., and Paulsson, M. (1991) J. Biol. Chem. 266, 15308-15317[Abstract/Free Full Text]
18. Aeschlimann, D., Wetterwald, A., Fleisch, H., and Paulsson, M. (1993) J. Cell Biol. 120, 1461-1470[Abstract]
19. Klenman, J. P., Aeschlimann, D., Paulsonn, M., and van de Rest, M. (1995) Biochemistry 34, 13768-13775[Medline] [Order article via Infotrieve]
20. Kaartinen, M. T., Pirhonen, A., Linnala-Kankkunen, A., and Mäenpää, P. H. (1997) J. Biol. Chem. 272, 22736-22741[Abstract/Free Full Text]
21. Verderio, E., Nicholas, B., Gross, S., and Griffin, M. (1998) Exp. Cell Res. 239, 119-138[CrossRef][Medline] [Order article via Infotrieve]
22. Hunter, I., Sigmundsson, K., Beuchemin, N., and Öbrink, B. (1998) FEBS Lett. 425, 141-144[CrossRef][Medline] [Order article via Infotrieve]
23. Jones, R. A., Nicholas, B., Mian, S., Davies, P. J. A., and Griffin, M. (1997) J. Cell Sci. 110, 2461-2472[Abstract/Free Full Text]
24. Akimov, S. S., Krylov, D., Fleischmann, L. F., and Belkin, A. M. (2000) J. Cell Biol. 148, 825-838[Abstract/Free Full Text]
25. Haroon, Z. A., Hettasch, J. M., Lai, T.-S., Dewhirst, M. W., and Greenberg, C. S. (1999) FASEB J. 13, 1787-1795[Abstract/Free Full Text]
26. Hwang, K.-C., Gray, C. D., Sweet, W. E., Moravec, C. S., and Im, M.-J. (1996) Circulation 94, 718-726[Abstract/Free Full Text]
27. Small, K., Feng, J.-F., Lorenz, J., Donnelly, E. T., Yu, A., Im, M.-J., Dorn, G. W., II, and Liggett, S. B. (1999) J. Biol. Chem. 274, 21291-21296[Abstract/Free Full Text]
28. Melino, G., Annicchiarico-Petruzzelli, M., Piredda, L., Candi, E., Gentile, V., Davies, P. J. A., and Piacentini, M. (1994) Mol. Cell. Biol. 14, 6584-6596[Abstract]
29. Piredda, L., Amendola, A., Colizzi, V., Davies, P. J. A., Farrace, M. G., Maurizio, F., Gentile, V., Uray, I., Piacentini, M., and Fesus, L. (1997) Cell Death Diff. 4, 463-472[CrossRef]
30. Oliverio, S., Amendola, A., Rodolfo, C., Spinedi, A., and Piacentini, M. (1999) J. Biol. Chem. 274, 34123-34128[Abstract/Free Full Text]
31. Thomazy, V. A., and Davies, P. J. A. (1999) Cell Death Diff. 6, 146-154[CrossRef][Medline] [Order article via Infotrieve]
32. Stevens, H. Y., Reeve, J., and Noble, B. S. (2000) J. Anat. 196, 181-191[CrossRef][Medline] [Order article via Infotrieve]
33. Piacentini, M., and Autuori, F. (1994) Differentiation 57, 51-61[CrossRef][Medline] [Order article via Infotrieve]
34. Nanda, N., Iismaa, S. E., Copeland, N. G., Gilbert, D. J., Jenkins, N., Graham, R. M., and Sutrave, P. (1999) Arch. Biochem. Biophys. 366, 151-156[CrossRef][Medline] [Order article via Infotrieve]
35. Sauer, B. (1993) Methods Enzymol. 225, 890-900[Medline] [Order article via Infotrieve]
36. Schwenk, F., Baron, U., and Rajewsky, K. (1995) Nucleic Acids Res. 23, 5080-5081[Medline] [Order article via Infotrieve]
37. Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265[Abstract/Free Full Text]
38. Fesus, L., and Arato, G. (1986) J. Immunol. Methods 94, 131-136[Medline] [Order article via Infotrieve]
39. Prpic, V., Green, K. C., Blackmore, P. F., and Exton, J. H. (1984) J. Biol. Chem. 259, 1382-1385[Abstract/Free Full Text]
40. Im, M.-J., and Graham, R. M. (1990) J. Biol. Chem. 265, 18944-18951[Abstract/Free Full Text]
41. Szondy, Z., P., M., Nemes, Z., M., B., Kedei, N., Tóth, R., and Fésüs, L. (1997) FEBS Lett. 404, 307-313[CrossRef][Medline] [Order article via Infotrieve]
42. Baek, K. J., Das, T., Gray, C., Antar, S., Murugesan, G., and Im, M.-J. (1993) J. Biol. Chem. 268, 27390-27397[Abstract/Free Full Text]
43. Hitomi, K., Kanehiro, S., Ikura, K., and Maki, M. (1999) J. Biochem. (Tokyo) 125, 1048-1054[Abstract]
44. Platt, N., Suzuki, H., Kurihara, Y., Kodama, T., and Gordon, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12456-12460[Abstract/Free Full Text]
45. Brodde, O., and Michel, M. (1999) Pharmacol. Rev. 51, 651-689[Abstract/Free Full Text]
46. Cavalli, A., Lattion, A. L., Hummler, E., Nenniger, M., Pedrazzini, T., Aubert, J. F., Michel, M. C., Yang, M., Lembo, G., Vecchione, C., Mostardini, M., Schmidt, A., Beermann, F., and Cotecchia, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11589-11594[Abstract/Free Full Text]
47. Akhter, S. A., Milano, C. A., Shotwell, K. F., Cho, M. C., Rockman, H. A., Lefkowitz, R. J., and Koch, W. J. (1997) J. Biol. Chem. 272, 21253-21259[Abstract/Free Full Text]
48. Milano, C. A., Dolber, P. C., Rockman, H. A., Bond, R. A., Venable, M. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10109-10113[Abstract/Free Full Text]


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