Modulation of the in Situ Activity of Tissue Transglutaminase by Calcium and GTP

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situtransglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r 2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly lessin situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTGin situ. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situtransglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r 2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly lessin situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTGin situ. Tissue transglutaminase (tTG) 1The abbreviations used are: tTG, tissue transglutaminase; TG, transglutaminase; BSA, bovine serum albumin; Cbz-LLY-DMK,N-benzyloxycarbonyl-l-leucyl-l-leucyl-l-tyrosine diazomethyl ketone; ER, endoplasmic reticulum; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MTX, maitotoxin; RA, retinoic acid; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; BBS, borate-buffered saline; HPLC, high pressure liquid chromatography.1The abbreviations used are: tTG, tissue transglutaminase; TG, transglutaminase; BSA, bovine serum albumin; Cbz-LLY-DMK,N-benzyloxycarbonyl-l-leucyl-l-leucyl-l-tyrosine diazomethyl ketone; ER, endoplasmic reticulum; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MTX, maitotoxin; RA, retinoic acid; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; BBS, borate-buffered saline; HPLC, high pressure liquid chromatography. is a novel, dual function protein having both transamidating activity and a role as a signal-transducing GTP-binding protein (1Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (926) Google Scholar, 2Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar). As a member of the transglutaminase family, tTG catalyzes a calcium-dependent acyl transfer reaction between the γ-carboxamide of a peptide-bound glutamine residue and the ε-amino group of a peptide-bound lysine, or the primary amino group of a polyamine, yielding either an isopeptide bond or a (γ-glutamyl)polyamine bond, respectively (1Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (926) Google Scholar). This transamidating activity of tTG is inhibited by GTP, an effect that is reversed by an intrinsic GTPase activity of tTG (3Lee K.N. Birckbichler P.J. Patterson M.K. Biochem. Biophys. Res. Commun. 1989; 162: 1370-1375Crossref PubMed Scopus (93) Google Scholar, 4Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar). GTP-bound tTG was subsequently shown to function as a signal-transducing GTP-binding protein (Gαh), which couples activated receptors to phospholipase Cδ, resulting in stimulation of this effector enzyme (2Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar, 5Feng J.-F. Rhee S.G. Im M.-J. J. Biol. Chem. 1996; 271: 16451-16454Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Thus, this intriguing protein can serve the cell in two apparently unrelated capacities, its role apparently determined by incompletely characterized intracellular regulators. tTG is found in many different mammalian cells and tissues and has been implicated as a participant in a vast array of physiological and pathological processes. In its capacity as a transamidating enzyme, tTG has been proposed to play an important role in bone development (6Aeschlimann D. Wetterwald A. Fleisch H. Paulsson M. J. Cell Biol. 1993; 120: 1461-1470Crossref PubMed Scopus (166) Google Scholar), axonal growth and regeneration (7Eitan S. Schwartz M. Science. 1993; 261: 106-108Crossref PubMed Scopus (126) Google Scholar, 8Eitan S. Solomon A. Lavie V. Yoles E. Hirschberg D.L. Belkin M. Schwartz M. Science. 1994; 264: 1764-1768Crossref PubMed Scopus (109) Google Scholar), modulation of cell adhesion (9Gentile V. Thomazy V. Piacentini M. Fesus L. Davies P.J.A. J. Cell Biol. 1992; 119: 463-474Crossref PubMed Scopus (229) Google Scholar,10Borge L. Demignot S. Adolphe M. Biochim. Biophys. Acta. 1996; 1312: 117-124Crossref PubMed Scopus (33) Google Scholar), differentiation and apoptosis (11Hand D. Campoy F.J. Clark S. Fisher A. Haynes L.W. J. Neurochem. 1993; 61: 1064-1072Crossref PubMed Scopus (35) Google Scholar, 12Amendola A. Gougeon M.-L. Poccia F. Bondurand A. Fesus L. Piacentini M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11057-11062Crossref PubMed Scopus (123) Google Scholar), and tumor growth and metastasis (13Johnson T.S. Knight C.R. el-Alaoui S. Mian S. Rees R.C. Gentile V. Davies P.J. Griffin M. Oncogene. 1994; 9: 2935-2942PubMed Google Scholar, 14Hettasch J.M. Bandarenko N. Burchette J.L. Lai T.S. Marks J.R. Haroon Z.A. Peters K. Dewhirst M.W. Iglehart J.D. Greenberg C.S. Lab. Invest. 1996; 75: 637-645PubMed Google Scholar). Recent studies have begun to elucidate the specific roles of tTG in these different biochemical processes. For example, tTG is likely to be involved in the activation of both midkine, a heparin-binding growth/differentiation factor, and interleukin 2 by catalyzing the formation of stable dimers (7Eitan S. Schwartz M. Science. 1993; 261: 106-108Crossref PubMed Scopus (126) Google Scholar, 8Eitan S. Solomon A. Lavie V. Yoles E. Hirschberg D.L. Belkin M. Schwartz M. Science. 1994; 264: 1764-1768Crossref PubMed Scopus (109) Google Scholar, 15Kojima S. Inui T. Muramatsu H. Suzuki Y. Kadomatsu K. Yoshizawa M. Hirose S. Kimura T. Sakakibara S. Muramatsu T. J. Biol. Chem. 1997; 272: 9410-9416Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). It has also been suggested that tTG contributes to the transforming growth factor-β activation process by cross-linking the large latent transforming growth factor-β complex to the extracellular matrix (16Nunes I. Gleizes P.-E. Metz C.N. Rifkin D.B. J. Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (345) Google Scholar). Additionally, there are data to indicate that tTG is involved in stabilizing tissue during wound healing by cross-linking anchoring fibrils and, more specifically, collagen VII (17Raghunath M. Hopfner B. Aeschlimann D. Luthi U. Meuli M. Altermatt S. Gobet R. Bruckner-Tuderman L. Steinmann B. J. Clin. Invest. 1996; 98: 1174-1184Crossref PubMed Scopus (88) Google Scholar). In addition to catalyzing the formation of isodipeptide bonds, tTG in its role as a transamidating enzyme covalently incorporates polyamines into substrate proteins. Protein-polyamine conjugates have been detected in several tissues and cell lines (18Piacentini M. Martinet N. Beninati S. Folk J.E. J. Biol. Chem. 1988; 263: 3790-3794Abstract Full Text PDF PubMed Google Scholar, 19Beninati S. Folk J.E. Adv. Exp. Med. Biol. 1988; 250: 411-422Crossref PubMed Scopus (17) Google Scholar), and in vitro tTG incorporates polyamines into numerous proteins (20Hohenadl C. Mann K. Mayer U. Timpl R. Paulsson M. Aeschlimann D. J. Biol. Chem. 1995; 270: 23415-23420Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Miller M.L. Johnson G.V.W. J. Neurochem. 1995; 65: 1760-1770Crossref PubMed Scopus (106) Google Scholar, 22Ballestar E. Abad C. Francos L. J. Biol. Chem. 1996; 271: 18817-18824Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Although functional changes resulting from tTG-catalyzed incorporation of polyamines into proteins have not been well defined, previous studies have shown that in vitro the covalent incorporation of polyamines into phospholipase A2 increases activity (23Cordella-Miele E. Miele L. Beninati S. Mukherjee A.B. J. Biochem. (Tokyo). 1993; 113: 164-173Crossref PubMed Scopus (67) Google Scholar). In its capacity as a signal transducing GTP-binding protein tTG has been designated Gαh, a protein that forms noncovalent heterodimers with a 50-kDa protein (2Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar, 24Baek K.J. Das T. Gray C.D. Desai S. Hwang K.-C. Gacchui R. Ludwig M. Im M.-J. Biochem. J. 1996; 35: 2651-2657Crossref Scopus (34) Google Scholar). Gαh has been shown to couple to α1-adrenoreceptors and thereby mediate the activation of phospholipase Cδ (2Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar, 5Feng J.-F. Rhee S.G. Im M.-J. J. Biol. Chem. 1996; 271: 16451-16454Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Recent data indicate that in the heart the α1-adrenoreceptor couples to Gαh, and in cardiomyopathic heart tissue the intrinsic activity of Gαh is decreased (25Hwang K.-C. Gray C.D. Sweet W.E. Moravec C.S. Im M.-J. Circulation. 1996; 94: 718-726Crossref PubMed Scopus (56) Google Scholar). The reason for the decrease in Gαh activity and GTP binding in the failing heart is unknown; however, it has been suggested that other proteins, such as the 50-kDa protein that binds Gαh in a GTP-dependent manner, may be involved (24Baek K.J. Das T. Gray C.D. Desai S. Hwang K.-C. Gacchui R. Ludwig M. Im M.-J. Biochem. J. 1996; 35: 2651-2657Crossref Scopus (34) Google Scholar). Because tTG is apparently involved in multiple cellular processes, its expression and activation are likely to be tightly regulated processes. Interleukin 6 has been shown to induce tTG expression in hepatocytes (26Suto N. Ikura K. Sasaki R. J. Biol. Chem. 1993; 268: 7469-7473Abstract Full Text PDF PubMed Google Scholar), and cAMP induces expression in cerebellar granule cells (27Perry M.J.M. Mahoney S.-A. Haynes L.W. Neuroscience. 1995; 65: 1063-1076Crossref PubMed Scopus (56) Google Scholar). In many, but not all, cell types, retinoids are effective inducers of tTG expression (28Davies P.J. Murtaugh M.P. Moore Jr., W.T. Johnson G.S. Lucas D. J. Biol. Chem. 1985; 260: 5166-5174Abstract Full Text PDF PubMed Google Scholar, 29Piacentini M. Annicchiarico-Petruzzelli M. Oliverio S. Piredda L. Biedler J.L. Melino G. J. Cancer. 1992; 52: 271-27820Crossref PubMed Scopus (110) Google Scholar, 30Benedetti L. Grignani F. Scicchitano B.M. Jetten A.M. Diverio D. Lo Coco F. Avvisati G. Gambacorti-Passerini C. Adamo S. Levin A.A. Pelicci P.G. Nervi C. Blood. 1996; 87: 1939-1950Crossref PubMed Google Scholar, 31Kosa K. Jones C.S. De Luca L.M. Cancer Res. 1995; 55: 4850-4854PubMed Google Scholar). Nagy et al. (32Nagy L. Saydak M. Shipley N. Lu S. Basilion J.P. Yan Z.H. Syka P. Chandraratna R.A.S. Stein J.P. Heyman R.A. Davies P.J.A. J. Biol. Chem. 1996; 271: 4355-4365Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) have demonstrated that the mouse tTG promoter is activated by retinoid activation of either retinoic acid receptor-retinoid X receptor heterodimers or retinoid X receptor homodimers. Retinoic acid receptor and retinoid X receptor are highly regulated and exhibit specific temporal, spatial, and tissue-specific expression patterns (33Chambon P. Semin. Cell Biol. 1994; 5: 115-125Crossref PubMed Scopus (497) Google Scholar) and therefore are likely to be important determinants in tTG expression. The in vitroregulation of tTG transglutaminating activity by calcium and GTP has been well documented (4Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar, 34Folk J.E. Ann. N. Y. Acad. Sci. 1972; 202: 59-76Crossref PubMed Scopus (19) Google Scholar, 35Folk J.E. Finlayson J.S. Adv. Protein Chem. 1977; 31: 1-133Crossref PubMed Scopus (780) Google Scholar). However, the in situmodulation of tTG by these and other factors has not been thoroughly examined. Therefore, the purpose of this study was to examine the modulation of tTG by calcium and GTP in situ. N,N-Dimethylated casein, bovine serum albumin (BSA), retinoic acid (RA), ionomycin, carbachol, putrescine dihydrochloride, Tween 20, Bisbenzamide (Hoescht), poly-d-lysine (30,000–70,000 daltons), ando-phenylenediamine dihydrochloride were purchased from Sigma; thapsigargin was from Alexis; phenylmethylsulfonyl fluoride, SDS, and FITC-conjugated streptavidin were purchased from Boehringer Mannheim; [1,4-3H]putrescine dihydrochloride (1 mCi/ml) and the enhanced chemiluminescence (ECL) reagents were purchased from Amersham Corp.; 5-(biotinamido)pentylamine, horseradish peroxidase (HRP)-conjugated streptavidin, and BCA protein assay reagents were purchased from Pierce; andN-benzyloxycarbonyl-l-leucyl-l-leucyl-l-tyrosine diazomethyl ketone (Cbz-LLY-DMK) was from Molecular Probes. Calpain I, Nonidet P-40, and Bay K8644 were purchased from Calbiochem, and Tiazofurin was a gift from NCI, National Institutes of Health. RPMI 1640 was purchased from Cellgro; penicillin/streptomycin and horse serum were from Life Technologies, Inc.; and fetal clone II was purchased from Hyclone. The tTG monoclonal antibody 4C1 was produced by the Hybridoma Core Facility at the University of Alabama at Birmingham (36Johnson G.V.W. Cox T.M. Lockhart J.P. Zinnerman M.D. Miller M.L. Powers R.E. Brain Res. 1997; 751: 323-329Crossref PubMed Scopus (173) Google Scholar); the monoclonal antibody to type I TG was from Biomedical Technologies; the monoclonal tTG antibody CUB 7402 was from Neomarkers; and HRP-conjugated goat anti-mouse IgG was purchased from Bio-Rad. Texas red-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch. Fura-2 was from TefLabs, andN-succinyl-l-leucyl-l-leucyl-l-tyrosine-7-amido-4-methylcoumarin was from Bachem. For these experiments, maitotoxin (MTX) was purchased from Calbiochem. However, it should be noted that the potency of MTX varies significantly depending on the vendor; therefore, initial experiments to determine the effective doses of the drug should be carried out. All other reagents were from Sigma. Human neuroblastoma SH-SY5Y cells were maintained on Corning dishes in RPMI 1640 medium supplemented with 20 mm glutamine, 10 units/ml penicillin, 100 μg/ml streptomycin, 5% fetal clone II serum, and 10% horse serum. For differentiation, the percentage of fetal clone II serum and horse serum in the media were reduced to 1 and 4%, respectively. To initiate differentiation, cells were grown in the low serum medium containing 20 μm RA. The differentiating medium supplemented with 20 μm RA was replaced every 48 h. All experiments were carried out on subconfluent cultures. To evaluate the expression level of tTG in cells during differentiation, extracts from cells were prepared and quantitatively immunoblotted. Cells were harvested in cold phosphate-buffered saline (PBS), collected by centrifugation, resuspended in a homogenizing buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1 mmphenylmethylsulfonyl fluoride, and a 10 μg/ml concentration each of aprotinin, leupeptin, and pepstatin) and sonicated on ice. Protein concentrations of the homogenates were determined using the BCA method and diluted to a final concentration of 1 mg/ml with 2 × reducing stop buffer (0.25 m Tris-HCl, pH 6.8, 5 mmEDTA, 5 mm EGTA, 25 mm dithiothreitol, 2% SDS, and 10% glycerol with bromphenol blue as the tracking dye). Samples (25 μg of protein) were resolved on 8% SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were blocked in 5% nonfat dry milk in TBST (20 mm Tris-HCl, pH 7.6, 137 mmNaCl, 0.05% Tween 20) for 1 h at room temperature. The blots were then incubated with 1–2 μg/ml anti-tTG monoclonal antibody 4C1 in the same buffer for 2 h at room temperature. The blots were rinsed once with TBST and incubated with HRP-conjugated goat anti-mouse IgG (1:3000) in TBST for 1 h at room temperature. The blots were rinsed three times for 30 min with TBST, followed by four quick rinses with distilled water, and developed with ECL. The immunoblots were analyzed using a Bio-Rad GS-670 imaging densitometer and normalized to an internal standard to eliminate blot to blot variation in staining intensity. Data were expressed as a percentage of the maximal level of tTG ± S.E. In vitrotTG activity was measured in cell extracts using a modification of the procedure of Hand et al. (11Hand D. Campoy F.J. Clark S. Fisher A. Haynes L.W. J. Neurochem. 1993; 61: 1064-1072Crossref PubMed Scopus (35) Google Scholar) as described previously (36Johnson G.V.W. Cox T.M. Lockhart J.P. Zinnerman M.D. Miller M.L. Powers R.E. Brain Res. 1997; 751: 323-329Crossref PubMed Scopus (173) Google Scholar). For in situ tTG activity measurements, SH-SY5Y cells were preincubated with 5-(biotinamido)pentylamine, a biotinylated polyamine, and incorporation of the reagent into proteins was determined (27Perry M.J.M. Mahoney S.-A. Haynes L.W. Neuroscience. 1995; 65: 1063-1076Crossref PubMed Scopus (56) Google Scholar). Prior to treatment with the indicated drugs or appropriate vehicles (controls), cells were incubated for 1 h with 2 mm 5-(biotinamido)pentylamine which was prepared as a 100 mm stock in 50 mmTris-HCl, 150 mm NaCl, pH 7.5. MTX, carbachol, and tiazofurin were dissolved in water; ionomycin and thapsigargin were dissolved in Me2SO; and Bay K8644 was dissolved in ethanol. The maximal Me2SO or ethanol concentration to which the cells were exposed was 0.1%. Cells were treated with the drugs as indicated and then harvested and lysed as described above. Ten micrograms of homogenate protein was diluted to 50 μl with coating buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EGTA, 5 mm EDTA) and added to each well of a 96-well microtiter plate (Falcon), and the plates were incubated overnight at 4 °C. Two hundred microliters of 5% BSA, 0.01% SDS, 0.01% Tween 20 in borate-buffered saline (BBS; 100 mmboric acid, 20 mm sodium borate, 80 mm NaCl) was added to each well, and the incubation continued for 2 h at 37 °C. The mixture was removed, and each well was rinsed once with 1% BSA, 0.01% Tween 20 in BBS. One hundred microliters of HRP-conjugated streptavidin (1:1000) in 1% BSA, 0.01% Tween 20 was added to each well and incubated at room temperature for 1 h. The wells were rinsed four times with 1% BSA, 0.01% Tween 20 in BBS, and then 200 μl of substrate solution (0.4 mg ofo-phenylenediamine dihydrochloride/ml of 0.05 msodium citrate phosphate buffer, pH 5.0) was added to each well. After incubating 10–20 min at room temperature, the reactions were stopped by the addition of 50 μl of 3 n HCl to each well, and the presence of proteins into which 5-(biotinamido)pentylamine had been incorporated was quantitated by measuring the absorbance at 492 nm on a microplate spectrophotometer (Molecular Devices). All measurements were done in triplicate and repeated at least three times. The activity of tTG in situ was calculated as a percentage of basal activity (i.e. no drug additions) within a given group of samples. Data were analyzed using Student's t test, and values were considered significantly different when p < 0.05. To visualize the proteins into which the 5-(biotinamido)pentylamine had been incorporated, 2 μg of the homogenates was resolved on a 8% polyacrylamide SDS gel; transferred to nitrocellulose; blocked in 5% BSA, 0.05% SDS, 0.01% Nonidet P-40 in BBS for at least 2 h at room temperature; and incubated with HRP-conjugated streptavidin (1:2000) in 1% BSA, 0.05% SDS, 0.01% Nonidet P-40 in BBS for 2 h at room temperature. The blots were rinsed three times for 30 min each with the same buffer followed by four quick rinses with distilled water. The blots were then developed following the standard ECL protocol. Except where indicated, studies were carried out on cells that had been treated with RA for 6 days. For cytochemical analysis, SH-SY5Y cells were replated onto poly-d-lysine-coated coverslips in 24-well plates. Cells were preincubated for 1 h at 37 °C in the presence of 2 mm 5-(biotinamido)pentylamine and subsequently treated with 1 nm MTX for 20 min. Cells not exposed to MTX served as controls. After treatment, SH-SY5Y cells were fixed in paraformaldehyde (4% in PBS) for 30 min at room temperature, incubated for 1.5 min with 0.2% Triton X-100 in PBS, and rinsed three times for 5 min each with PBS, prior to incubation with 3% BSA in PBS for 30 min to reduce the background. Cells were then incubated for 1 h with the tTG monoclonal antibody CUB 7402 (diluted 1:10 in PBS containing 0.1% BSA), rinsed three times for 5 min each with PBS, and incubated for 1 h at room temperature with FITC-conjugated streptavidin diluted 1:100 in PBS and Texas red-conjugated goat anti-mouse IgG diluted 1:50 in PBS. Cells were counterstained with the nuclear dye Hoescht (5 μg/ml) for the evaluation of the cell number, and coverslips were washed extensively in PBS prior to mounting. Controls contained either no primary antibody or no 5-(biotinamido)pentylamine. Cells were viewed with a Nikon Diaphot 300 epifluorescence microscope, and images were captured with a Photometric CH250 CCD camera; digitally stored images were combined and displayed with the IP Lab Spectrum software. The levels of GTP in the cells were determined using reverse phase HPLC as described previously (37Payne S.M. Ames B.N. Anal. Biochem. 1982; 123: 151-161Crossref PubMed Scopus (95) Google Scholar). In brief, cells were rinsed once with PBS and collected into 1m formic acid. The samples were vortexed and spun at 16,000 × g for 4 min at 4 °C. The supernatants were lyophilized, and the pellets were used for protein determinations. The lyophilized samples were resuspended in HPLC grade water, injected onto a C-18 column (Vydac, 250 × 4.6 mm, 5 μm), and eluted with a 4–42% acetonitrile gradient. Nucleotide standards were used to determine the position at which GTP eluted. All data were normalized to mg of protein in each sample. Intracellular calcium levels were measured in cultured cells using Fura-2 essentially as described previously (38Mattson M.P. Cheng B. Davis D. Bryant K. Lieberburg I. Rydel R.E. J. Neurosci. 1992; 12: 376-389Crossref PubMed Google Scholar, 39Guttmann R.P. Elce J.S. Bell P.D. Isbell J.C. Johnson G.V.W. J. Biol. Chem. 1997; 272: 2005-2012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) with a few modifications. In brief, cells were grown on coverslips, and prior to experimentation they were loaded with a 5 μm concentration of the acetoxymethyl ester form of Fura-2. Coverslips were placed in an imaging chamber (Warner Instrument Co.) and mounted in a heater platform on the stage of a Nikon Diaphot. The cells were maintained at 37 °C in a Ringer's solution for the duration of the experiments. Images were obtained using an Ionoptix ICCD camera (Ionoptix Corp., Milton, MA) and processed with the IonWizard software program. Intracellular calcium concentrations were determined from the ratio of fluorescence using excitation wavelengths of 340 and 380 nm. Background fluorescence was taken from regions not containing cells and subtracted from the cell images at each wavelength. The ratio of fluorescence in the digitized images is a direct indicator of intracellular calcium concentrations. The system was calibrated following standardized protocols (40Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Calpain activity was measured in SH-SY5Y using the membrane-permeable, calpain-selective fluorescent peptide,N-succinyl-l-leucyl-l-leucyl-l-tyrosine-7-amido-4-methylcoumarin as described previously (41Xie H.Q. Johnson G.V.W. J. Neurochem. 1997; 69: 1020-1030Crossref PubMed Scopus (37) Google Scholar). The release of the intracellular enzyme lactate dehydrogenase (LDH) into the media was used as a quantitative measure of cell viability. The measurement of LDH was carried out as described previously (42Decker T. Lohmann-Matthes M.L. J. Immunol. Methods. 1988; 115: 61-69Crossref PubMed Scopus (1062) Google Scholar). The percentage of LDH released was defined by LDH activity in the media divided by total LDH activity. Previously, it had been demonstrated that treatment of human neuroblastoma SK-N-BE cells with RA increased tTG gene expression and tTG protein levels (29Piacentini M. Annicchiarico-Petruzzelli M. Oliverio S. Piredda L. Biedler J.L. Melino G. J. Cancer. 1992; 52: 271-27820Crossref PubMed Scopus (110) Google Scholar). To determine if this was also the case for SH-SY5Y human neuroblastoma cells, the cells were treated with 20 μm RA, and the level of tTG expression was determined at various times for 12 days. The levels of tTG increased rapidly after RA treatment. In Fig. 1, a representative immunoblot shows that prior to RA treatment the levels of tTG are virtually undetectable, but after RA treatment tTG levels increase rapidly for 3 days and remain elevated for 12 days. A quantitation of the increase in tTG levels in RA-treated SH-SY5Y cells is shown in Fig.2. Type 1 (keratinocyte) TG was not detected in naive or RA-treated cells (data not shown). In vitro measurements of TG activity were carried out to determine if the increased levels of tTG correlated with increased activity (Fig.2). RA treatment of SH-SY5Y cells also resulted in a rapid increase inin vitro TG activity that correlated (r 2 = 0.959) with the increased expression levels of tTG. The TG activity in cells treated with RA for 9 days was approximately 10-fold higher than the activity measured in untreated cells.Figure 2Quantitative analysis of the effects of RA treatment on the expression of tTG (•) and in vitro TG activity (▴). Treatment of SH-SY5Y cells with RA for the times indicated resulted in a concomitant increase in both the expression of tTG and in vitro TG activity. The graph is logarithmic.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine if basal in situTG activity also correlated with the increases in tTG expression, 5-(biotinamido)pentylamine was used as a probe for endogenous tTG activity. TGs react with free glutamine residues in substrate proteins releasing ammonia, and then the enzyme-substrate intermediate reacts with an appropriate nearby primary amine. This can be either the ε-amino group of lysine in an adjacent protein, resulting in an ε-(γ-glutamyl)lysine isodipeptide bond, or the primary amino group of a polyamine, resulting in the covalent incorporation of the polyamine into the protein by a (γ-glutamyl)polyamine bond (1Gre