Characterization of transglutaminase activity in rabbit tracheal epithelial cells. Regulation by retinoids

Rabbit tracheal epithelial cells undergo terminal cell division, start to express a squamous phenotype, and form cross-linked envelopes when reaching the plateau phase of the growth curve. This terminal differentiation is accompanied by a 20-30-fold increase in the activity of the cross-linking enzyme transglutaminase. This activity is found almost solely in the particulate fraction of homogenized cells and can be solubilized by nonionic detergents. This transglutaminase crossreacts with a monoclonal antibody raised against type I transglutaminase, but does not react with an antiserum against type II transglutaminase. The tracheal transglutaminase contains a protein subunit of approximately 92 kDa. The omission of epidermal growth factor from the medium or the addition of fetal bovine serum, conditions that induce terminal cell division and expression of a squamous phenotype, enhance transglutaminase activity. High calcium concentrations only stimulate transglutaminase activity after the cells become committed to terminal cell division. Retinoids, which inhibit the expression of the squamous phenotype but not terminal cell division, inhibit the enhancement in transglutaminase activity induced by either confluency or serum, indicating that this enzyme activity is under the control of retinoids. Some retinoids are active at concentrations as low as 10(-12) M. The ability of retinoids to inhibit transglutaminase activity correlates well with their capacity to bind to the retinoic acid-binding protein. Our results show that the increase in transglutaminase activity correlates with the induction of the terminal differentiated phenotype and suggest that this enzyme can function as a marker for this program of differentiation of rabbit tracheal epithelial cells in culture. Our results identify the transglutaminase as type I transglutaminase and are in agreement with the concept that this transglutaminase is involved in the formation of cross-linked envelopes.     

Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes

This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.     

Expression of tissue transglutaminase in the developing chicken limb is associated both with apoptosis and endochondral ossification

The cross-linking enzyme tissue transglutaminase (tTG) participates in a variety of cellular functions. To assess its contribution to extracellular and intracellular processes during development we cloned the cDNA for chicken heart tissue transglutaminase and localized the sites of transglutaminase expression by in situ hybridization and immunohistochemistry. Compared with the chicken red blood cell transglutaminase cDNA, the heart cDNA encodes a transglutaminase with an amino-terminal truncation. The truncated enzyme retains full catalytic activity and is GTP-inhibitable. Tissue transglutaminase expression was observed in developmentally transient structures in embryonic chicken limb at day 7.5 of incubation suggesting that its expression is dynamically regulated during limb morphogenesis. The major morphogenetic events of the limb associated with transglutaminase expression were cartilage maturation during skeletal development, interdigital apoptosis, and differentiation of skeletal muscle. Maturation of the cartilage during endochondral ossification was characterized by intra- and extracellular transglutaminase accumulation in the zone of hypertrophic chondrocytes. Only intracellular enzyme could be detected in mesenchymal cells of the prospective joints, in apoptotic cells of the interdigital web, and in skeletal muscle myoblasts. An apparently constitutive expression of tissue transglutaminase was found in vascular endothelial cells corresponding to the adult expression pattern. The dynamic pattern of transglutaminase expression during morphogenesis suggests that tissue remodeling is a major trigger for transglutaminase induction.     

Expression of Transglutaminase K in Normal Cervix Tissue and Cervix Carcinomas

The localization and expression of transglutaminase K has been investigated immunohistochemically in normal cervix tissue (n=15) and in cervix carcinomas (n=23). The distribution of the transglutaminase K was compared with the staining patterns of cytokeratin 10, Ki-67, p53, and oestrogen and progesterone receptors in these tumours. Weak to strong membrane-bound immunoreactivity for transglutaminase K was detected in almost all cervix carcinomas analyzed. The immunostaining was heterogeneous, with visual differences between individual tumour cells. 66.7% of normal cervix tissues revealed no immunoreactivity for the transglutaminase K. In normal cervix tissue, the immunoreactivity was confined to upper cervix layers, predominantly to the superficial and intermediate cell layers. The intensity of both the immunostaining and the number of transglutaminase K-positive cells were upregulated in cervix carcinomas as compared to normal cervix tissue. When the coexpressions of transglutaminase K with markers of proliferation and differentiation were analyzed, no statistically significant correlation was found. Our findings indicate that (1) transglutaminase K is upregulated at the protein level in cervix carcinomas as compared to normal cervix tissue; (2) upregulation of the transglutaminase K in cervix carcinoma is not exclusively induced by alterations of epithelial differentiation or proliferation, but by different, unknown mechanisms; and (3) upregulation of transglutaminase K in cervix carcinomas may play an important role for the regulation of tumour invasive properties by modulating cell–cell interactions.     

Induction of keratinocyte type-I transglutaminase in epithelial cells of the rat

Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: during the differentiation of keratinocytes, during pregnancy, being evident principally in the endometrium but detectable elsewhere as well, and during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.     

Two types of transglutaminase in the PC12 pheochromocytoma cell line. Stimulation by sodium butyrate

Transglutaminases are a class of enzymes capable of covalently cross-linking both intracellular and extracellular proteins. The activity of tissue transglutaminase is known to decrease precipitously following neoplastic transformation, and it has been hypothesized that transglutaminase may be involved in growth regulation. We have found that the differentiation promoter sodium butyrate is able to cause a marked increase in transglutaminase activity in PC12 pheochromocytoma cells in a time- and dose-dependent manner. This increased transglutaminase activity is associated with growth arrest, as well as with striking morphological changes including increased cell adhesion. The transglutaminase induced by sodium butyrate appears to be tissue transglutaminase, based on its cytosolic localization, thermal lability at basic pH, and elution profile on anion-exchange chromatography. Untreated PC12 cells contain only small amounts of transglutaminase which resembles epidermal transglutaminase, an enzyme previously described only in skin. In contrast to sodium butyrate, nerve growth factor did not stimulate tissue transglutaminase in PC12 cells, although it, too, caused growth arrest. It is hypothesized that transglutaminase may be involved in certain morphological changes accompanying cellular differentiation and neoplastic transformation, rather than in growth regulation per se.     

Activation and de novo synthesis of transglutaminase in cultured glioma cells

The activity of transglutaminase (TGase) was measured in cultured C6 glioma cells after their stimulation by either isoproterenol and isobutyl-methylxanthine or by a serum-containing medium. The activity fluctuated in a biphasic manner, with the peaks at 2-3 hr and 7-8 hr poststimulation. The first peak of TGase activity was affected neither by cycloheximide nor by actinomycin D, which inhibited protein synthesis. The second peak, on the other hand, was completely eliminated by cycloheximide and was reduced by actinomycin D. Immunological procedures were employed to find out whether or not the activity of TGase corresponded with the presence of the TGase antigen in the cultured cells. Indirect immunofluorescent staining and radioimmunoblot techniques suggested that unstimulated cells contained an inactive enzyme. This inactive, or cryptic, enzyme had the same molecular weight as its active counterpart. Activation of the enzyme was mediated by cell stimulation, probably by its release from the membrane. This step did not require protein synthesis, unlike the second step, which was dependent on de novo protein synthesis.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutamine activity (-(γ-glutamyl)lysine crosslinking enzme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of the soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20–70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Expression of the cytosolic and particulate forms of transglutaminase during chemically induced rat liver carcinogenesis

Transglutaminase (EC 2.3.2.13) activity in chemically induced rat hepatocellular carcinomas was reduced by some 65% when compared to normal rat livers. The majority of the remaining activity (approx. 85%) was found in the particulate fraction. The use of non-ionic detergent to extract the transglutaminase activity present in both normal and tumour tissue followed by its separation on a Mono-Q column revealed two distinct peaks of activity. These peaks of activity were equivalent to those previously identified as a membrane-bound transglutaminase and the more characteristic cytosolic or tissue transglutaminase. The ratio of the activity of the cytosolic enzyme to that of the membrane-bound enzyme in normal liver was calculated as 5:1. In hepatocellular carcinomas, this ratio was reduced to 0.4:1. No significant change in the activity of the membrane-bound enzyme was detectable in tumour tissue. Comparison of the cytosolic enzyme found in hepatocellular carcinomas with that found in normal liver indicated no change in its molecular weight, Km,app for putrescine incorporation into N,N'-dimethylcasein and sensitivity to activation by Ca2+. These observations suggest that the reduction in transglutaminase activity observed in the hepatocellular carcinoma is due to a selective reduction in the expression of the cytosolic transglutaminase.     

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.     

Subcellular localization of a membrane-associated transglutaminase activity in rat liver

Fractionation of rat liver by homogenization and differential centrifugation revealed that only about 83% of the transglutaminase activity in the tissue is in a soluble form, and that the remainder is associated with the particulate fraction. This latter activity remained with the membranes even after they were extensively washed to remove 99% of such soluble enzymes as lactate dehydrogenase and aldolase. Subsequent fractionation of the membranes by isopycnic density gradient centrifugation in sucrose resulted in a single band of transglutaminase activity at a density of 1.194 g/cm3. This activity was coincident with the major band of plasma membranes, which was identified by its content of 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and leucine aminopeptidase activities. After treatment with digitonin and fractionation on sucrose gradients, the transglutaminase activity and the plasma membrane marker enzyme activities were found at a new density of 1.210 g/cm3, while the enzyme markers for the other membrane fractions remained unchanged. From these data, we conclude that approximately 17% of the transglutaminase activity in rat liver is specifically associated with the plasma membranes.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutaminase activity (epsilon-(gamma-glutamyl)lysine crosslinking enzyme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20--70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Retinoic acid-induced expression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.     

Tissue transglutaminase in the small intestine of the mouse as a marker for apoptotic cells. Colocalization with DNA fragmentation

Besides the morphological changes in cells undergoing apoptosis, such as chromatin condensation and cell shrinkage, histological demonstration of DNA fragmentation by in situ end labeling (ISEL) has been widely used for the demonstration of apoptotic cells in tissue sections. Although DNA fragmentation can be demonstrated in apoptotic cells and apoptotic bodies in most cases, there is no clear correlation of ISEL staining with apoptosis. It has often been demonstrated that, in many morphologically intact cells, nuclei with fragmented DNA can be found. Thus staining with ISEL for the detection of apoptosis is useful only in connection with other markers for apoptosis as, for example, characteristic morphological changes. Here we show that tissue transglutaminase protein is unequivocally expressed in apoptotic enterocytes as shown by DNA fragmentation and morphology. Tissue transglutaminase is not expressed in enterocytes with healthy morphology, although DNA fragmentation can be demonstrated in these cells. Thus the immunohistochemical demonstration of tissue transglutaminase may serve as a simple marker for apoptotic epithelial cells in tissue sections.     

Rat gastrointestinal transglutaminase: demonstration of enzyme activity and cell and tissue distributions

The properties, tissue and cellular distribution of intestinal transglutaminase have been investigated. Transglutaminase was assayed with dimethylcasein and [14C]putrescine as substrates. The enzyme has maximum activity at pH 10, although more reliable assays are made at pH 9. Transglutaminase showed an absolute requirement for Ca2+ and exhibited linear assay kinetics. The Km for putrescine was approx. 0.15 mmol/l. Tissue distribution studies suggest transglutaminase is more active in the more muscular segments of the gut. The cellular localization in jejunum was investigated by sequential cell release techniques. Approximately 2 per cent of the total activity was found in the enterocytes and crypt cells. Most of the activity was in the submucosa and serosa suggesting an interstitial cell localization. Acute hypoplastic enteropathy induced by methotrexate was accompanied by a striking decrease in mucosal transglutaminase but the activity returned to control values by 72 h. There was no significant increase in activity during the period of intense crypt cell hyperplasia and it is concluded that intestinal transglutaminase is not implicated in crypt cell proliferation.     

Expression of tissue transglutaminase in Balb-C 3T3 fibroblasts: effects on cellular morphology and adhesion

Tissue transglutaminase is a cytosolic enzyme whose primary function is to catalyze the covalent cross-linking of proteins. To investigate the functions of this enzyme in physiological systems, we have established lines of Balb-C 3T3 fibroblasts stably transfected with a constitutive tissue transglutaminase expression plasmid. Several cell lines expressing high levels of catalytically active tissue transglutaminase have been isolated and characterized. Transglutaminase-transfected cells showed morphologic features quite distinct from their nontransfected counterparts. Many of the cells showed an extended and very flattened morphology that reflected increased adhesion of the cells to the substratum. Other cells, particularly those showing the highest levels of intracellular transglutaminase expression, showed extensive membrane blebbing and cellular fragmentation. The results of these experiments suggest that the induction and activation of tissue transglutaminase may contribute both to changes in cellular morphology and adhesiveness.     

Induction of keratinocyte type-I transglutaminase in epithelial cells of the rat

Abstract. Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: (1) during the differentiation of keratinocytes, (2) during pregnancy. being evident principally in the endometrium but detectable elsewhere as well, and (3) during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.     

Rapid increases in the transglutaminase activity of A431 cells following treatment with epidermal growth factor

Transglutaminase activity was detected in lysates of A431 cells, a human epidermal carcinoma cell line. Enzyme activity was increased 1.5-2.5-fold in lysates prepared from cells pretreated with epidermal growth factor (EGF) relative to untreated control cells. Half-maximal activation of the transglutaminase activity occurred at 3-5 nM EGF, a concentration in good agreement with the Kd for EGF binding to its receptor in these cells. The increase in transglutaminase activity could be detected as early as 2 min after the addition of EGF, with the maximal response attained by 30 min. The activation was not blocked by pretreatment of the cells with cycloheximide, suggesting that the increased activity was not the result of an induction of transglutaminase synthesis. Fractionation of A431 cell lysates by centrifugation at 100000g for 30 min demonstrated that 90% of the transglutaminase activity was present in the soluble fraction and that this soluble transglutaminase activity was increased after treatment of the cells with EGF. The demonstration that EGF acutely increases the activity of a soluble, intracellular transglutaminase defines a novel pathway of growth factor action and provides a useful model system for identifying and comparing the mechanism(s) by which growth factors activate soluble enzymes.     

Cellular transglutaminase. The particulate-associated transglutaminase from chondrosarcoma and liver: partial purification and characterization

A transglutaminase from the malignant chondrocytes, rat swarm chondrosarcoma cells, was partially purified and characterized in an effort to understand transformation-induced changes in its activity. This enzyme separated by DE52 column chromatography after extraction from the particulate fraction of cell lysate was found to be distinct from previously characterized transglutaminases in its electrophoretic mobility, molecular size, substrate specificity, and immunologic reactivity. This enzyme was identified as a transglutaminase by its catalysis of amine (putrescine, spermine) incorporation at the carboxamide group of protein-bound gamma-glutamyl residues, and accordance of its kinetic data with the modified double displacement mechanism described for other transglutaminases. Limited proteolysis of the isolated enzyme resulted in a 3-4-fold increase of catalytic activity and a concomitant reduction of molecular size by approximately one-half. Incubation of labeled amine with chondrosarcoma cell lysate resulted in labeling of only a few proteins that appeared to be extensively cross-linked and that were located mostly in the particulate fraction of the cells. Transglutaminase extracted from the rat liver particulate fraction displayed enzymatic and structural properties closely resembling those of the enzyme from chondrosarcoma cells.     

Induction of tissue transglutaminase in mouse peritoneal macrophages

Tissue transglutaminase accumulates rapidly and to very high levels (1-2% of cellular protein) in mouse peritoneal macrophages cultured in mouse serum. The induction is due to accelerated synthesis of the enzyme (150-fold increase) that occurs within 90 min of exposure of the cells to a heat-labile constituent of serum or plasma. The induction is reversible and is not reproduced by known activators of macrophage function such as lipopolysaccharide, muramyl dipeptide, and tuftsin. In animals, elevated levels of tissue transglutaminase are also found in inflammatory macrophages elicited by thioglycolate broth.