Mol Cell Biol 21(1): 148-155

Gene Disruption of Tissue Transglutaminase

IDI-IRCCS Biochemistry Lab, Department of Experimental Medicine, University Tor Vergata, Rome, Italy

^{Corresponding author. Mailing address: IDI-IRCCS, Biochemistry Lab, c/o Dep. Experimental Medicine, D26/F153, University of Rome Tor Vergata, Via Tor Vergata 135, 00133 Rome, Italy. Phone: 39 6 20427299. Fax: 39 6 20427290. E-mail:}ti.2amorinu@onilem.yrreg.

Received 2000 Jul 20; Revisions requested 2000 Sep 12; Accepted 2000 Sep 28.

Abstract

Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either ɛ-(γ-glutamyl)lysine or N^,N^{-(γ-glutamyl)spermidine isopeptide bonds. TGase 2 expression has been associated with apoptosis, and it has been proposed that its activation should lead to the irreversible assembly of a cross-linked protein scaffold in dead cells. Thus, TGase 2-catalyzed protein polymerization contributes to the ultrastructural changes typical of dying apoptotic cells; it stabilizes the integrity of the apoptotic cells, preventing the release of harmful intracellular components into the extracellular space and, consequently, inflammation and scar formation. In order to perform a targeted disruption of the enzyme, we prepared a construct deleting part of exons 5 and 6, containing the active site, and intron 5. Complete absence of TGase 2 was demonstrated by reverse transcription-PCR and Western blot analysis. TGase activity measured on liver and thymus extracts showed, however, a minimal residual activity in TGase 2}^{mice. PCR analysis of mRNA extracted from the same tissues demonstrated that at least TGase 1 (normally present in the skin) is also expressed in these tissues and contributes to this residual activity. TGase 2}^{mice showed no major developmental abnormalities, and histological examination of the major organs appeared normal. Induction of apoptosis ex vivo in TGase 2}^{thymocytes (by CD95, dexamethasone, etoposide, and H}₂O₂) and in vitro on TGase 2^{mouse embryonal fibroblasts (by retinoids, UV, and H}₂O₂) showed no significant differences. A reduction in cross-linked apoptotic bodies with a modestly increased release of lactate dehydrogenase has been detected in some cases. Together our results show that TGase 2 is not a crucial component of the main pathway of the apoptotic program. It is possible that the residual enzymatic activity, due to TGase 1 or redundancy of other still-unidentified TGases, can compensate for the lack of TGase 2.

Abstract

Transglutaminase 2 (TGase 2; also called tissue transglutaminase or TG C) belongs to the transglutaminase (EC 2.3.2.13) family, which includes intracellular and extracellular enzymes catalyzing Ca^{-dependent reactions resulting in the formation of ɛ-(γ-glutamyl)lysine cross-links and/or in the covalent incorporation of di- and polyamines and histamine (}25, 26). The establishment of these covalent cross-links leads to the posttranslational modification and, in many instances, the oligomerization of substrate proteins. The resulting protein polymers are resistant to breakage and chemical attack and can release polypeptides only through the proteolytic degradation of protein chains. At least seven distinct types of TGases in mammals have been characterized: TGase 1 (or TG K), TGase 2, TGase 3 (or TG E), TGase X, coagulation factor XIII, band 4.2., and prostate TGase. At least four transglutaminases (TGases 1, 2, 3, and X) are expressed and synthesized during terminal differentiation and death of human epidermal keratinocytes (44, 45), where they contribute to the formation of the cornified envelope.

The TGase 2 gene is constitutively expressed both during development (29, 48) and in adult tissues (for a review, see reference 36). In both cases a tight correlation between TGase 2 expression and occurrence of apoptosis has been found. This includes, for example, interdigital web formation (29), implantation of the embryo in utero (35), and mammary gland regression (31, 46). In addition, the presence and activity of the enzyme have been shown to increase in cells undergoing apoptosis in several models (2, 9, 10, 21, 23–25, 32–34, 37, 40). Indeed, during apoptosis de novo transcription of the TGase 2 gene is induced by several factors (e.g., retinoic acid [RA], prostaglandin E2, interleukin 6, and tumor growth factor β). Moreover, in addition to transcriptional regulation (24, 28, 41), TGase 2 can also be modulated posttranscriptionally (1, 23, 50) during apoptosis. TGase 2 activation leads to the assembly of intracellular cross-linked protein polymers, which irreversibly modifies cell organization, contributing to the wide ultrastructural changes occurring in cells undergoing apoptosis (9, 10, 39). This extensive TGase 2-dependent protein polymerization stabilizes apoptotic cells before their clearance by phagocytosis, thus contributing to the prevention of inflammation in the surrounding tissues (39).

In addition to its cross-linking activity, TGase 2 acts as the Gαh subunit, associated with the 50-kDa β subunit (Gβh), of the GTP-binding protein (Gh) in a ternary complex associated with the rat liver α1-adrenergic receptor (30). Thus, TGase 2-Gαh is a multifunctional protein, which by binding GTP in a Gαh-GTP complex can modulate receptor-stimulated phospholipase C activation.

In order to clarify the role of TGase 2 in apoptosis we have generated mice lacking TGase 2 by homologous recombination techniques. Our results, however, show that the disruption of TGase 2 does not produce a major phenotype and that apoptosis still occurs normally in the absence of TGase 2.

ACKNOWLEDGMENTS

We thank Mauro Piacentini, Gennaro Ciliberto, and Richard A. Knight for generous support, critical discussions, and helpful suggestions. This work could not have been completed without the generous help of Francesca Bernassola, Eleonora Candi, Marco Corazzari, Daniela Barcaroli, and Marco Ranalli. We thank Giuseppe Bertini, Giancarlo Cortese, and Pierino Piccoli (S.S.D. SAFU, Instituto Fisioterapici Ospedalieri, Rome, Italy) for technical assistance and mouse husbandry.

The work was partially supported by grants from MURST, MinSan, Associazione Neuroblastoma, AIRC, Telethon (E 872 and E 1257), and EU (QLG1-1999-00739).

ACKNOWLEDGMENTS

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