In vivo and in vitro induction of ‘tissue’ transglutaminase in rat hepatocytes by retinoic acid (original) (raw)
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Retinoic acid-induced modulation of rat liver transglutaminase and total polyamines in vivo
The Biochemical journal, 1988
The effect of a single intraperitoneal injection of retinoic acid on liver transglutaminase (EC 2.3.2.13) activity and total putrescine, spermidine and spermine was studied. The results demonstrate that: (1) transglutaminase activity is increased over control values as early as 4-6 h after treatment, reaching a maximum (2-fold increase) at 12 h and returning to control values at 36 h; (2) the retinoic acid-induced form of enzyme is the soluble tissue transglutaminase; (3) actinomycin D treatment does not completely inhibit the early (6 h) increase of activity, while suppressing that at 12 h; (4) the immunoassay of the soluble transglutaminase shows that, 6 h after treatment, there is no increase in the protein, whereas at 12 and 24 h a significant increase is observed; (5) putrescine, but not spermidine and spermine, increases (5-7-fold) 6 and 18 h after the retinoic acid treatment. The possibility also that the expression of soluble transglutaminase is modulated in vivo by retinoic...
Journal of Biological Chemistry, 2001
Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis. Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis. Moreover, pretreatment of HL60 cells as well as other cell lines (i.e. NIH3T3 cells) with RA blocks HPRinduced cell death. In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase. Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA. We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect. Moreover, excessive transamidation activity did not appear to be detrimental to cell viability. These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress. Tissue transglutaminase II (TGase) 1 is a member of a family of closely related thiol enzymes that are derived from a common ancestor (1, 2). The TGases function as calcium-dependent acyl * This work was supported by National Institutes of Health Grants GM61762 and GM40654 and by fellowship GM208052 (to M. A. A.
A nonradioactive dot blot assay for transglutaminase activity
Analytical Biochemistry, 2009
Aberrant transglutaminase (TG) activity has been implicated in the pathology of numerous diseases including Huntington disease and Alzheimer disease. To fully characterize the role of TGs in these disorders it is important that simple, quantifiable assays be made available. The most commonly used assay currently employed requires significant time and a radioactive substrate. The assay described here uses a biotinylated substrate in conjunction with a dot-blot apparatus to eliminate the use of radioactive substrates, and allows relative transglutaminase activity to be measured simultaneously with minimal sample preparation in a large number of samples containing purified enzyme, cell extracts or tissue homogenates. Transglutaminases (TGs) belong to a family of enzymes that modify glutaminyl residues in protein/peptide substrates usually by transamidation with a suitable amine donor, such as the ε-amino group of a lysyl residue or a polyamine. TGs, especially TG2, have been implicated in the pathology of several neurological disorders such as Huntington disease [1,2] and Alzheimer disease [3,4]. TG2 is a highly regulated enzyme that is activated by Ca 2+ and inhibited by GTP. Currently, the most widely used TG assay utilizes a radiolabeled polyamine (usually putrescine) as acyl acceptor (amine donor) (for a comprehensive review of transglutaminase assays see [5]). This substrate is covalently attached to the glutaminyl residue (acyl donor, amine acceptor) of a protein substrate. However, the use of radiolabeled polyamines requires several time-consuming steps, including denaturation of the polyaminated protein with concentrated trichloroacetic acid, followed by filtration, and counting in a liquid scintillation counter. To circumvent the use of radioactivity and problems of waste disposal, 5-(biotinamido)pentylamine has sometimes been used in place of the labeled polyamine as acyl acceptor [6-8]. In the solid-phase system described by Slaughter et al. [8], 0.5 mM 5-(biotinamido)pentylamine was used as acyl acceptor and N,N-dimethylcasein coated to polystyrene plates was used as acyl donor. Following incubation with TG, the unreacted 5-(biotinamido)pentylamine was
Inhibition of "Tissue" Transglutaminase Increases Cell Survival by Preventing Apoptosis
Journal of Biological Chemistry, 1999
with all-trans-retinoic acid (RA) commits these cells to apoptosis, which can be triggered by simply increasing intracellular calcium levels by the ionophore A23187. RA treatment of U937 cells is characterized by a decrease in Bcl-2 and marked induction of "tissue" transglutaminase (tTG) gene expression. In this study, we show that the inhibition of tTG expression in U937 cells undergoing apoptosis prevents their death. In fact, U937 cell-derived clones transfected with the human tTG gene in the antisense orientation showed a pronounced decrease in apoptosis induced by several stimuli. These findings demonstrate that the Ca 2؉ -dependent irreversible cross-linking of intracellular proteins catalyzed by tTG represents an important biochemical event in the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.
TPA Induces Transglutaminase C and Inhibits Cell Growth in the Colon Carcinoma Cell Line SW620
Biochemical and Biophysical Research Communications, 1997
found that the application of TPA markedly inhibited In contrast to most other systems, TPA induced TG C the increase of TG C caused by RA in mouse epidermal activity and protein in SW620 human colon carcinoma cells. Our own results also show that the activation of cells. This induction was accompanied by cell growth PKC inhibits induction of TG C by RA in NIH3T3 cells inhibition and increased apoptosis. The general pro-(manuscript in preparation). However, Keogh et al tein kinase-C inhibitor GF-109203X blocked the inducrecently reported that in a human colon tumor cell line, tion of TG C by TPA, whereas the specific inhibitor of SW620, TPA induced TG C enzyme activity. Induction the PKCa isoform, the indocarbazole Go6976, reduced of transglutaminase C by TPA is quite intriguing since, it by 40%. These PKC inhibitors had similar inhibitory in other cell lines, this agent has been shown to be effects on TPA increased apoptosis and inhibition of inhibitory. According to Birckbichler (14) inhibition of cell growth, suggesting that the observed actions of TG C resulted in an increase of proliferative markers in TPA are mediated by PKC, and a close connection behuman lung cells and our own observations (9) showed tween TG C activity, increased apoptosis and cell that induction of the enzyme paralleled a decrease in growth inhibition. We conclude that TPA may offer cell proliferation. On the basis of these results, it is new approaches in the management of colon cancer reasonable to suggest that induction of TG C is related cell growth. ᭧ 1997 Academic Press to the proliferation status of the cell, namely, transglutaminase C is induced when cell proliferation decreases. In the present study, we wanted to examine Transglutaminases are a family of enzymes that catwhether 1. the induction of TG C by TPA was paralleled alyze the transamidation of glutamine residues of proby a decrease in cell growth; 2. whether induction of teins, resulting in the formation of e-amino(g-glutamyl) the enzyme by TPA and a possible effect on cell growth lysine crosslinks (for review: (1)). Three important involved PKC; and 3. whether the activation of TG C members of this family are plasma transglutaminase was accompanied by increased apoptosis in SW620 or Factor XIII which stabilizes the fibrin clot by crosscells. linking fibrin monomers to fibrin polymers; keratinocyte transglutaminase or TG K which contributes to the formation of the protective cornified envelope of squa-MATERIALS AND METHODS mous epithelia; and tissue transglutaminase or TG C , a ubiquitous cytoplasmic enzyme involved in apoptosis, Cell culture and chemicals. SW620 cells were obtained from the cell adhesion, and nerve regeneration (2-5).
Protein Expression and Purification, 2002
of a protein/polypeptide (acyl donor, amine acceptor) to Recent evidence suggests that aberrant transglutamthe-amino group of a lysyl (K) residue of a protein/ inase activity is associated with a wide variety of dispolypeptide (acyl acceptor, amine donor) (1-3). The acyl eases. Tissue transglutaminase is the most widely donor may also be an amine, diamine, or polyamine distributed of the six well-characterized transglutami-(1-3). nases in humans. We describe a method for expressing Tissue transglutaminase (tTGase; TGase 2) is the hexahistidine-tagged human tissue transglutaminase most ubiquitous of the six human TGases that have in Escherichia coli BL21(DE3) using the pET-30 Ek/LIC been well characterized (3). Increased or defective expression vector. Purification of the expressed tTGase activity may contribute to, or be a factor in, enzyme from suspensions of E. coli cells treated with many pathological processes. They include cataracts, CelLytic B Bacterial Cell Lysis/Extraction Reagent was atherosclerosis, inflammation, fibrosis, diabetes, canaccomplished by immobilized metal (Ni 2+) affinity colcer metastases, celiac disease, autoimmune diseases, umn chromatography. The procedure typically yields lamellar ichthyosis, and psoriasis (reviewed in ref. 4). highly purified and highly active recombinant human Recent evidence suggests that TGases may also play a tissue transglutaminase in about 1 day (about 0.6 mg/ role in neurodegenerative diseases, such as Huntington from a 1-liter culture). ᭧ 2002 Elsevier Science (USA) disease, Alzheimer disease, Parkinson disease, and su-Key Words: hexahistidine tag; human tissue transglupranuclear palsy. (For recent reviews see refs. 4-10.) taminase; immobilized metal affinity chromatography.
Cell and Tissue Research, 1991
We recently reported that activation of "tissue" transglutaminase (EC 2.3.2.13; tTG) in liver cells undergoing apoptosis determines extensive cross-linking of cellular proteins resulting in the formation of SDSinsoluble shells in the so-called "apoptotic bodies". In attempt to obtain further insight into the role played by tTG in apoptosis of liver cells, we investigated its expression in primary cultures of neonatal rat liver cells stimulated with epidermal growth factor (EGF). EGFtreatment of neonatal rat liver cells induces first hyperplasia of hepatocytes, followed by involution characterized by a high incidence of apoptosis. The proliferative phase of hepatocytes is paralleled by a 10-fold increase in tTG mRNA level, which is followed, during the phase of involution, by sequential increases in enzyme activity and levels of SDS-insoluble apoptotic bodies, tTG immunostaining at both the light-and electron-microscopic levels shows that the most intensive reaction is present in globular structures showing the typical morphological appearance of mature apoptotic bodies. In early apoptotic stages, tTG protein is localized in the perinuclear region of the cell. Intense immunostaining is also found in the apoptotic bodies present inside phagosomes within the cytoplasm of neighboring cells. This evidence confirms and extends our previous findings, indicating that tTG induction and activation specifically takes place in cells undergoing apoptosis, suggesting a key role for the enzyme in the apoptotic program.
FEBS Letters, 1989
Physiological deletion of cells ensues programmed death which involves formation of apoptotic bodies with fragmented DNA. Here we report that apoptotic hepatocytes are insoluble in detergents, urea, guanidine hydrochloride, reducing agents and thereby can be isolated from rat liver following collagenase treatment. They are wrinkled, spherical structures similar to cornified envelopes of epidermis by phase-contrast microscopy and show irregular, globular morphology by scanning-electron microscopy. Part of their DNA content is cleaved into nucleosomal and oligonucleosomal fragments. Their insolubility, like that of the comified envelope, is evoked by a-(y-glutamyl)lysine and NL,1V8-bis(y-glutamyl)spermidine protein cross-linking bonds formed by transglutaminase.