Activation of the Ras-ERK pathway inhibits retinoic acid-induced stimulation of tissue transglutaminase expression in NIH3T3 cells

Retinoic acid (RA) is a potent activator of tissue transglutaminase (TGase) expression, and it was recently shown that phosphoinositide 3-kinase (PI3K) activity was required for RA to increase TGase protein levels. To better understand how RA-mediated TGase expression is regulated, we considered whether co-stimulation of NIH3T3 cells with RA and epidermal growth factor (EGF), a known activator of PI3K, would facilitate the induction or increase the levels of TGase expression. Instead of enhancing these parameters, EGF inhibited RA-induced TGase expression. Activation of the Ras-ERK pathway by EGF was sufficient to elicit this effect, since continuous Ras signaling mimicked the actions of EGF and inhibited RA-induced TGase expression, whereas blocking ERK activity in these same cells restored the ability of RA to up-regulate TGase expression. However, TGase activity is not antagonistic to EGF signaling. The mitogenic and anti-apoptotic effects of EGF were not compromised by TGase overexpression, and in fact, exogenous TGase expression promoted basal cell growth and resistance to serum deprivation-induced apoptosis. Moreover, analysis of TGase expression and GTP binding activity in a number of cell lines revealed high basal TGase GTP binding activity in tumor cell lines U87 and MDAMB231, indicating that constitutively active TGase may be a characteristic of certain cancer cells. These findings demonstrate that TGase may serve as a survival factor and RA-induced TGase expression requires the activation of PI3K but is antagonized by the Ras-ERK pathway.     

Monocyte-fibronectin interactions, via alpha(5)beta(1) integrin, induce expression of CXC chemokine-dependent angiogenic activity.

Monocyte-derived macrophages are important sources of angiogenic factors in cancer and other disease states. Upon extravasation from vasculature, monocytes encounter the extracellular matrix. We hypothesized that interaction with extracellular matrix proteins leads monocytes to adopt an angiogenic phenotype. We performed endothelial cell chemotaxis assays on conditioned medium (CM) from monocytes that had been cultured in vitro on various matrix substrates (collagen I, laminin, Matrigel, fibronectin), in the presence of autologous serum, or on tissue culture plastic alone. Monocytes cultured on Matrigel and on fibronectin were the most potent inducers of angiogenic activity compared with tissue culture plastic or autologous serum-differentiated monocytes. This increased angiogenic activity was associated with increased expression of angiogenic CXC chemokines (IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene alpha, and growth-related oncogene gamma) but not of vascular endothelial growth factor. Additionally, CM from monocytes cultured on fibronectin-depleted Matrigel (MG(FN-)) induced significantly less angiogenic activity than CM from monocytes cultured on control-depleted Matrigel. ELISA analysis of CM from monocytes cultured on MG(FN-) revealed a significant decrease in GRO-alpha and GRO-gamma compared with CM from monocytes cultured on MG. Incubation of monocytes before adherence on fibronectin with PHSCN (a competitive peptide inhibitor of the PHSRN sequence of fibronectin binding via alpha(5)beta(1) integrin) results in diminished expression of angiogenic activity and CXC chemokines compared with control peptide. These data suggest that fibronectin, via alpha(5)beta(1) integrin, promotes CXC chemokine-dependent angiogenic activity from monocytes.     

Amphotericin B inhibits the serum-induced expression of tissue transglutaminase in murine peritoneal macrophages

Culture of mouse resident peritoneal macrophages (PM) in serum-containing medium causes a rapid and marked induction of the enzyme tissue transglutaminase (tissue TGase). Coculture of PM with amphotericin B (AmpB) inhibited the serum-induced expression and accumulation of tissue TGase. The AmpB-mediated inhibition of tissue TGase was specific and was due to inhibition of enzyme synthesis. The serum-dependent induction of tissue TGase was inhibited in a dose-dependent fashion, and a complete inhibition was observed at 1.5 microgram/ml dose of AmpB. The inhibition was reversible; however, the time of recovery depended on the dose and time of exposure of the cells to AmpB. The present studies suggest that AmpB-mediated inhibition of tissue TGase is due to inhibition of the uptake of serum retinoids by PM.     

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.     

Intracellular distribution of active and inactive transglutaminase in stimulated cultured C6 glioma cells

The cellular distribution of active and inactive transglutaminase (TGase) was studied in C6 glioma cells before and during stimulation by a serum-containing medium. The activity of the enzyme was determined in the soluble and insoluble fractions obtained by freezing and thawing the cells, followed by centrifugation at 12,000g for 5 min. In the soluble fractions, the activity of TGase decreased 2.5 h post-stimulation and increased after 5 and 8 h. In the corresponding insoluble fractions, no significant changes in the activity of the enzyme were noted up to 8 h after stimulating the cells with fresh medium. An immunological approach was next used to determine the quantity of TGase antigen during the stimulation of the cultured glioma cells. In the soluble fraction, the quantity of the antigen decreases significantly at 2.5, 5, and 8 h. In contrast, in the insoluble fraction, a significant increase in TGase antigen was detected 8 h after the addition of fresh medium. Cycloheximide completely inhibited the increase in the quantity of TGase antigen in the insoluble fraction, 8 h post-stimulation, while actinomycin D caused a partial inhibition. Trypsin, neuraminidase, or Sendai viruses increased the activity of TGase significantly, when added to nonstimulated cells. Trypsin had no effect on TGase activity when added to the cells 2 h after stimulation with a serum-containing medium. These findings suggest that an inactive form of the enzyme is present in the insoluble cellular fraction. A model has been proposed to explain the variations in TGase activity, its distribution and translocation during cellular stimulation.     

Transglutaminase activity in testicular homogenates and serum-free Sertoli cell cultures

Transglutaminase (EC 2.3.2.13) (TGase) activity has been localized in homogenates of rat Leydig cells and seminiferous tubules and is present in cytosol and membrane fractions. The enzyme has a requirement for Ca2+ and when the acceptor substrate casein was deleted from the assay mixture, incorporation of [14C]putrescine into cytosolic and membrane fractions occurred. Transglutaminase was also detected in Sertoli cells cultured in serum-free medium. Sertoli cells reside within the seminiferous tubule and are involved in normal spermatogenesis. Sertoli cell TGase has a strict requirement for Ca2+ and is not activated by Mg2+. Activation of the enzyme occurs with as little as 0.3 microM Ca2+; however, consistent with intracellular calcium levels, maximum stimulation occurred at 1.9 mM Ca2+. Sertoli cell TGase activity is markedly stimulated if the cells are cultured in 10% fetal bovine serum rather than in serum-free medium. Inhibition of Sertoli cell TGase by monodansylcadaverine concomitantly decreased the response of the cells to follicle-stimulating hormone (FSH)-induced secretion of cAMP but did not change basal cAMP levels. These data suggest that TGase may play a facilitative rather than an absolute role in activation of Sertoli cells by FSH and the resultant secretion of cellular products. This may occur through modulation of activities of membrane and cytosolic components by TGase.     

Transglutaminase activity increases in HL60 cells induced to differentiate with retinoic acid and TPA but not with DMSO

HL60 cells induced to differentiate into myeloid cells by retinoic acid exhibited a 300-fold increase in transglutaminase (TGase) activity which peaked on day 5. HL60 cells induced to differentiate into monocytes by a phorbol ester tetradecanoylphorbol-12-myristate-13-acetate (TPA) had a greater than 840-fold increase in TGase activity on day 7. In contrast, cells induced to differentiate along the myeloid pathway by dimethyl sulfoxide (DMSO) exhibited no increase in TGase activity. Elevation of TGase activity appears to be characteristic of monocyte differentiation and retinoic acid-induced myeloid differentiation but not of myeloid differentiation in response to DMSO.     

Regulation of the dual function tissue transglutaminase/Galpha(h) during murine neuromuscular development: gene and enzyme isoform expression

Coagulation Factor XIII (F. VIII), a member of the transglutaminase (TGase) superfamily, is activated by thrombin, cross-links fibrin and stabilizes clots. Another member of this family, tissue TGase (tTG), having similar enzymatic activity, is implicated in neural development and synapse stabilization. Our previous studies indicated that synapse formation and maintenance at the neuromuscular junction (NMJ) involved components of the coagulation cascade in development. Others then showed that either F. XIII or tTG were localized at NMJs in a developmentally-regulated fashion. In the current studies, we addressed the temporal course of skeletal muscle tTG gene expression and found maximal expression at birth and continuing into the immediate postnatal period. Subcellular fractionation revealed a relatively constant particulate isoform of TGase activity which predominated in early embryonic muscle development. In contrast, cytosolic TGase specific activity became the major isoform in the postnatal period. The timing of muscle TGase activity correlated well with expression of tTG mRNA and we now present novel data of Tgm 2 gene expression for tTG in skeletal muscle. Confirming and extending the previous studies, TGase becomes localized at NMJs in the early, further ramifying in the late, neonatal period. These data suggest that the early pulse of particulate activity could coincide with the period of myoblast cell death in embryonic muscle. On the other hand, the peak cytosolic TGase activity occurs in the neonatal period, correlating temporally with muscle prothrombin expression during activity-dependent synapse elimination and possibly the source of the enzyme localized to the NMJ extracellular matrix resulting in synaptic stabilization.     

Implication of tissue transglutaminase and desmoplakin in cell adhesion mechanism in human epidermis

The distribution patterns of both tissue and keratinocyte transglutaminases (TGase), as well as that of desmoplakin (DP), have been immunohistochemically investigated in human skin cultured in the absence or presence of cystamine and enalapril, two acantholytic agents. In the control samples, tissue TGase is predominantly expressed in lower layers of the epidermis and is located intercellularly. Conversely, in tissues cultured with cystamine or enalapril, a diffuse cytoplasmatic staining was observed. Similarly, DP, detected on the cell membrane in the control, shifts into the cytosol of the keratinocytes following treatment. The distribution pattern of the keratinocyte enzyme in the acantholytic epidermis was identical to that observed in the normal one. Since cystamine and enalapril are TGase inhibitors and DP was shown to act as a TGase substrate in vitro, we suggest that DP and tissue enzyme may participate in cell adhesion at the intraepidermal level.     

Suppression of macrophage cytostatic activation by serum retinoids: a possible role for transglutaminase

Mouse resident peritoneal macrophages, activated in vitro with murine recombinant interferon-gamma and lipopolysaccharide in the presence of sera from different sources, showed marked differences in their abilities to inhibit murine adenocarcinoma cell growth, and in induced activity of the enzyme, tissue transglutaminase. The extraction of lipids from the serum abolished its ability to induce tissue TGase activity and to inhibit cytostatic activity, but these capabilities were fully restored by readdition of all trans-retinol or all trans-retinoic acid at physiological concentrations. Addition of dansylcadaverine, a competitive inhibitor of TGase, resulted in complete recovery of macrophages from retinoid-induced suppression of cytostatic activity. These results suggest that endogenous retinoids play an important role in the regulation of macrophage-mediated cytostatic activity in a process that is independent of prostaglandin secretion but seems to involve the protein cross-linking enzyme, tissue transglutaminase.     

Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.     

The porcine 2A10 antigen is homologous to human CD163 and related to macrophage differentiation

The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5-50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10- monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10- monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10- cells. LPS-stimulated 2A10+ and 2A10- monocytes both produce proinflammatory cytokines (TNF-alpha and IL-1alpha), but antiinflammatory IL-10 is only detected on the latter population. When 2A10- monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10- circulating monocytes.     

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.     

Sporadic inclusion body myositis correlates with increased expression and cross-linking by transglutaminases 1 and 2

Sporadic inclusion body myositis (SIBM) is characterized by vacuolar degeneration of muscle fibers and intrafiber clusters of paired helical filaments with abnormal amyloid deposition. Because of their potential involvement in other degenerative disorders, we have examined the expression of transglutaminases (TGases) in normal and SIBM tissues. We report that at least two different enzymes, the ubiquitous TGase 2 as well as the TGase 1 enzyme, are present in muscle tissues. However, in comparison with normal tissue, the expression of TGases 1 and 2 was increased 2.5- and 4-fold in SIBM, accompanied by about a 20-fold higher total TGase activity. By immunohistochemical staining, in normal muscle, TGase 2 expression was restricted to some endomysial connective tissue elements, whereas TGase 1 and beta-amyloid proteins were not detectable. In SIBM muscle, both TGases 1 and 2 as well as amyloid proteins were brightly expressed and co-localized in the vacuolated muscle fibers, but none of these proteins colocalized with inflammatory cell markers. Next, we isolated high molecular weight insoluble proteins from SIBM muscle tissue and showed that they were cross-linked by about 6 residues/1000 residues of the isopeptide bond. Furthermore, by amino acid sequencing of solubilized tryptic peptides, they contain amyloid and skeletal muscle proteins. Together, these findings suggest that elevated expression of TGases 1 and 2 participate in the formation of insoluble amyloid deposits in SIBM tissue and in this way may contribute to progressive and debilitating muscle disease.     

Transglutaminase activity is present in highly purified nonsynaptosomal mouse brain and liver mitochondria

Several active transglutaminase (TGase) isoforms are known to be present in human and rodent tissues, at least three of which, namely, TGase 1, TGase 2 (tissue transglutaminase), and TGase 3, are present in the brain. TGase activity is known to be present in the cytosolic, nuclear, and extracellular compartments of the brain. Here, we show that highly purified mouse brain nonsynaptosomal mitochondria and mouse liver mitochondria and mitoplast fractions derived from these preparations possess TGase activity. Western blotting and experiments with TGase 2 knock-out (KO) mice ruled out the possibility that most of the mitochondrial/mitoplast TGase activity is due to TGase 2, the TGase isoform responsible for the majority of the activity ([14C]putrescine-binding assay) in whole brain and liver homogenates. The identity of the mitochondrial/mitoplast TGase(s) is not yet known. Possibly, the activity may be due to one of the other TGase isoforms or perhaps to a protein that does not belong to the classical TGase family. This activity may play a role in regulation of mitochondrial function both in normal physiology and in disease. Its nature and regulation deserve further study.     

拉达克轮丝菌TGase在大肠杆菌中的分子改造

【背景】谷氨酰胺转氨酶是一种能够催化酰基转移反应的酶,催化各种蛋白质分子之间或之内发生交联反应,在食品、化妆品、医药等领域具有重要的潜在价值。【目的】克隆来自拉达克轮丝菌(Streptoverticillium ladakanum) B1的谷氨酰胺转氨酶(TGase)基因并对其进行分子改造,使其在大肠杆菌中获得高效异源表达。【方法】分别克隆来自拉达克轮丝菌谷氨酰胺转氨酶的自身前导肽(pro)和除前导肽以外的成熟谷氨酰胺转氨酶(TGase)基因,以pET-22b为表达载体构建pro、TGase共表达和融合表达两种表达模式,在这两种表达模式的基础上进一步用定点突变的方法对成熟TGaseN端前4个氨基酸进行改造,检测不同表达模式以及突变对酶活的影响。【结果】当采用前导肽与TGase共表达时,可以直接得到活性形式的TGase,比酶活达到37.71 U/mg。在融合表达的基础上,将TGaseN端前3个氨基酸DSD突变为AAA,比酶活达到14.04U/mg,相较于原始表达模式提高了14.05%。【结论】前导肽与TGase共表达可以直接产生活性TGase,对于融合表达模式,合适位点的突变有利于提高TGase酶活。     

Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation: implications of transglutaminase 2 in age-related cataractogenesis

Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of alphaB-crystallin, betaB(2)-crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.     

Dexamethasone inhibition of DMSO-induced transglutaminase activity and differentiation of leukemic cells

Treatment of the Friend erythroleukemic (FL) cell line GM979 with dimethyl sulfoxide (DMSO) or n-butyric acid induced erythroid differentiation. Transglutaminase (TGase) activity also increased in these treated cells. Glucocortical steroids, i.e., dexamethasone (DEX) and triamcinolone acetonide, when added to the cultured medium, inhibited the DMSO-induced hemoglobin synthesis but not n-butyric acid-induced hemoglobin synthesis. Similarly, these steroids inhibited DMSO-increased TGase activity but not n-butyric acid-increased TGase activity in intact FL cells. Neither the differentiation-inducing agents nor the steroids had any effect on TGase activity when they were directly added to cell lysates. These results support the view that the increase of TGase activity may be related to erythroid differentiation of FL cells and of its possible role of this enzyme in FL cell-induced differentiation.