Cell Type-specific Activation of Intracellular Transglutaminase 2 by Oxidative Stress or Ultraviolet Irradiation

Abstract

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 αB-crystallin, βB2-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. 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 αB-crystallin, βB2-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. Transglutaminases (TGases) 1The abbreviations used are: TGase, transglutaminase; BP, biotinylated pentylamine; HRP, horseradish peroxidase; PBS, phosphate-buffered saline. are a family of enzymes that mediate the post-translational modification of proteins by catalyzing acyl transfer reaction between the γ-carboxamide group of glutamine residues and the ϵ-amino group of lysine residues or polyamines (1Lornad L. Graham R.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 140-156Crossref PubMed Scopus (1213) Google Scholar). The calcium-dependent transamidation activity of TGases produces cross-linked protein polymers that are resistant to mechanical stress and proteolytic degradation (2Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (935) Google Scholar). Thus, TGases are recognized to play critical roles in the formation of highly insoluble structures such as cornified cell envelope of the terminally differentiated keratinocyte (TGases 1 and 3, fibrin aggregates (coagulation factor XIII), and the post-copulatory plug of rodents (TGase 4) (3Kalinin A. Marekov L.N. Steinert P.M. J. Cell Sci. 2001; 117: 3069-3070Google Scholar, 4Takahashi N. Takahashi Y. Putnam F.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8019-8023Crossref PubMed Scopus (133) Google Scholar, 5Mukherjee D.C. Agrawal A.K. Manjunath R. Mukherjee A.B. Science. 1983; 219: 989-991Crossref PubMed Scopus (94) Google Scholar). TGase 2 exhibits a similar protein structure and catalyzes transamidation reaction following a common mechanism as other TGases (6Liu S. Cerione R. Clardy J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2743-2747Crossref PubMed Scopus (280) Google Scholar). However, TGase 2 is distinguished from other TGases by its widespread expression pattern, GTP hydrolyzing activity, and intracellular distribution. Although TGase 2 is mainly distributed in the cytosol, TGase 2 can translocate into the nucleus by binding to importin-α3 in response to an elevated intracellular calcium concentration (7Lesort M. Attanavanich K. Zhangm J. Johnson G.V. J. Biol. Chem. 1998; 273: 11991-11994Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 8Peng X. Zhang Y. Zhang H. Graner S. Williams J.F. Levitt M.L. Lokshin A. FEBS Lett. 1999; 446: 35-39Crossref PubMed Scopus (73) Google Scholar). A significant portion of TGase 2 is found in the plasma membrane or secreted into the extracellular matrix (9Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (530) Google Scholar, 10Akimov S.S. Krylov D. Fleischman L.F. Belkin A.M. J. Cell Biol. 2000; 148: 825-838Crossref PubMed Scopus (421) Google Scholar). Because of its ability to cross-link proteins at distinct subcellular location, TGase 2 is implicated in a wide range of physiological processes, including apoptosis, cell adhesion, and differentiation (11Melino G. Piacentini M. FEBS Lett. 1988; 430: 59-63Crossref Scopus (170) Google Scholar, 12Sporn M.B. Roberts A.B. Cancer Res. 1983; 43: 3034-3040PubMed Google Scholar). However, the overexpression of TGase 2 did not change cell survival, indicating that the intracellular transamidation activity of TGase 2 is tightly regulated (13Gentile V. Thomazy V. Piacentini M. Fesus L. Davies P.J.A. J. Cell Biol. 1992; 119: 463-474Crossref PubMed Scopus (230) Google Scholar). The inappropriate cross-linking or polyamination of various proteins within cells may induce pathological consequences. Because TGase 2 catalyzes transamidation of the glutamine residues of amine acceptors, TGase 2 may contribute to the aggregation of huntingtin molecules with the expanded polyglutamine domain in Huntington's disease (14Green H. Cell. 1993; 74: 955-956Abstract Full Text PDF PubMed Scopus (215) Google Scholar), the modification of the glutamine-rich gliadin peptides in celiac disease (15Anderson R.P. Degano P. Godkin A.J. Jewell D.P. Hill A.V. Nat. Med. 2000; 6: 337-342Crossref PubMed Scopus (488) Google Scholar), or the formation of crystallin dimers in age-related cataracts (16Lorand L. Hsu L.K.H. Siefring Jr., G.E. Rafferty N.S. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1356-1360Crossref PubMed Scopus (127) Google Scholar). Indeed, in the study of Huntington's disease the administration of cystamine, an inhibitor of TGase, prolonged the survival period of the mouse model (17Karpuj M.V. Becher M.W. Springer J.E. Chabas D. Youssef S. Pedotti R. Mitchell D. Steinman L. Nat. Med. 2002; 8: 143-149Crossref PubMed Scopus (348) Google Scholar). These findings suggest that an aberrant activation of TGase 2 is crucial for some pathologic processes. However, the molecular mechanism by which TGase 2 is activated has not been determined. In this report, we show that oxidative stress or UV irradiation can stimulate the in situ transamidation activity of TGase 2, and the elevated level of activity depends on the cell type and the nature of stress. Among the various cell lines examined, human lens epithelial cells (HLE-B3) were found to be most sensitive to the oxidative stress or UV irradiation. We present some evidence that the activation of intracellular TGase 2 may play a significant role in the development of human cataracts. Cell Culture and Transfection Experiments—Human cancer cell lines, A549 (lung), MCF7 (breast), and K562 (leukemia) cells, were maintained in RPMI 1640 medium containing 10% fetal bovine serum (Hyclone). NIH 3T3 (mouse fibroblast), HeLa (human cervical carcinoma), PC3M (human prostate carcinoma), and Du145 (human prostate carcinoma) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. HLE-B3 (human lens epithelial cell line) cells were grown in modified Eagle's medium containing 20% fetal bovine serum. Penicillin (100 units/ml), streptomycin sulfate (100 μg/ml), and glutamine (2 mm) were supplemented to all cell culture media. All of the cell lines were maintained in a humidified atmosphere with 5% CO2 at 37 °C. To investigate the effects of UV irradiation or oxidative stress, the cells (2× 106) were exposed to UVC (UVC-508, ULTRA-LUM Inc.; 100, 200, 300, and 400 J/m2, respectively) or treated with H2O2 (200, 400, 600, and 800 μm, respectively) for various times indicated and maintained in culture until analysis. Cystamine (Sigma) at 0.5 mm was added to culture media to inhibit the TGase 2 activity. Expression of TGase 2 in NIH 3T3 and MCF7 cells was carried out by transfection of pSG5 vector (Stratagene) containing full-length human TGase 2 or control vector using LipofectAMINE (Invitrogen) as previously described (18Jeon J.H. Kim C.W. Shin D.M. Kim K. Cho S.Y. Kwon J.C. Choi K.H. Kang H.S. Kim I.G. FEBS Lett. 2003; 534: 180-184Crossref PubMed Scopus (28) Google Scholar). In Vitro Transamidation Assays—In vitro TGase 2 activity was determined by measuring the incorporation of [14C] putrescine (PerkinElmer Life Sciences) into N,N′-dimethylcasein (Sigma) as previously described (19Jeon J.H. Cho S.Y. Kim C.W. Shin D.M. Kweon J.C. Choi K.H. Park S.C. Kim I.G. Biochem. Biophys. Res. Commun. 2002; 294: 818-822Crossref PubMed Scopus (16) Google Scholar). In brief, the cell extracts were prepared by disrupting the cells with lysis buffer (50 mm Tris-Cl, pH 7.5, 50 mm KCl, 0.5 mm EDTA, 5 mm MgCl2, 0.5% Nonidet P-40, 5 mm dithiothreitol, and protease inhibitor mixture) followed by centrifugation (14,000 × g, 10 min at 4 °C). The cell extracts in 100 μg were incubated with substrate solution (1 mg of N,N′-dimethylcasein, 500 nCi of [14C] putrescine, 50 mm Tris acetate, pH 7.5, 10 mm CaCl2, 5 mm dithiothreitol, 150 mm NaCl, 0.5% Triton X-100, and protease inhibitor mixture) for 1 h at 37 °C. The reaction was stopped by adding 7.5% trichloroacetic acid, and the radioactivity bound to N,N′-dimethylcasein was measured using liquid scintillation counter (Packard Bioscience). The background radioactivity with bovine serum albumin was used as a control. In Situ Transamidation Assays—In situ TGase 2 activity was measured by three different assay methods (18Jeon J.H. Kim C.W. Shin D.M. Kim K. Cho S.Y. Kwon J.C. Choi K.H. Kang H.S. Kim I.G. FEBS Lett. 2003; 534: 180-184Crossref PubMed Scopus (28) Google Scholar, J.H. Cho S.Y. Kim C.W. Shin D.M. Kweon J.C. Choi K.H. Park S.C. Kim I.G. Biochem. Biophys. Res. Commun. 2002; 294: 818-822Crossref PubMed Scopus (16) Google Scholar). The cells were with 1 mm biotinylated for 1 h to and the cell extracts were prepared by followed by centrifugation (14,000 × g, 10 min at 4 °C). cell extracts 50 in buffer (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 5 mm 5 mm were added to of a In situ TGase 2 activity was evaluated by the using horseradish followed by the reaction with The were by measuring the at on was by the cell extracts to using and the proteins were to The proteins with were with followed by The protein level of TGase 2 or was by with for TGase 2 J.H. Choi K.H. Cho S.Y. Kim C.W. Shin D.M. Kweon J.C. Park S.C. Kim I.G. J. 2003; PubMed Scopus Google or To the cells were in a and cultured for to UV at the of or with H2O2 at 400 μm, the cells were maintained in culture for The cells were with 1 mm for 1 h to with in phosphate-buffered for and the cellular membrane was by with Triton in for 5 min at The cells were incubated in bovine serum albumin in at for and the of biotinylated polyamines into the cellular proteins was using The cells were and with a TGase of situ TGase assay was carried out by HLE-B3 cells to UV or by the cells with H2O2 and the and water-insoluble as previously described S. J.H. L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In brief, the cell extracts were prepared by in buffer (50 mm Tris-Cl, pH 7.5, 20% 5 mm dithiothreitol, and protease inhibitor mixture) and centrifugation × g, 5 min at 4 °C). The extracts were into and water-insoluble by centrifugation at × g, min at 4 °C. The were with buffer and by in buffer (50 mm Tris-Cl, pH mm dithiothreitol, and protease inhibitor mixture) for min at 37 °C. The proteins of were in and with for J. J. M. Sci. 2000; Google Scholar), and vimentin assay of TGase HLE-B3 cells were by in buffer followed by centrifugation × g, 10 min at 4 °C). The cell extracts were incubated with buffer containing various of for 2 h at 37 °C. Cystamine at 1 mm or GTP at 100, 200, and 400 was added to the reaction the cell extracts were into and water-insoluble and was by as described was in cells as protein and by to the A expression was to TGase 2 to the In brief, protein expression was by cells with that a full-length human TGase 2 TGase 2 was using a to the and TGase 2 activity was in of [14C] putrescine incorporation (19Jeon J.H. Cho S.Y. Kim C.W. Shin D.M. Kweon J.C. Choi K.H. Park S.C. Kim I.G. Biochem. Biophys. Res. Commun. 2002; 294: 818-822Crossref PubMed Scopus (16) Google Scholar). The of TGase 2 was by followed by of protein bovine and were from and was from of were with and vimentin 100 in for 2 h at and with of 5% bovine serum albumin with for 2 h at TGase 2 was added to which of in 50 of reaction buffer (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 5 mm CaCl2, 0.5% Triton X-100, 0.5 mm EDTA, pH 5 mm dithiothreitol, and protease inhibitor and the were incubated for 1 h at 37 °C. To study the of calcium or GTP on the of the a of calcium or GTP was added to the reaction to the The of reaction was by measuring the of using between in Situ and the Expression of TGase been on the role of transamidation activity of TGase 2 in cellular S. A. A. Piacentini M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. Cerione J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). To the nature of the transamidation activity of TGase 2, we the expression level of TGase 2 with its in situ transamidation activity. NIH 3T3 and MCF7 cell lines that TGase 2 at were in the transfection The expression level of TGase 2 in cell lines, as in appeared the in vitro transamidation activity of the cell was measured by incorporation NIH 3T3 cells exhibited a increase of transamidation activity with the MCF7 cells showed significant we the in situ activity of TGase 2 by the cells with and by measuring the concentration in the cell extracts using the in situ transamidation activity was not in NIH 3T3 and MCF7 cells These findings indicate that in vitro or in situ transamidation activity of TGase 2 not correlate with its expression We the of in the transfection study by MCF7 cells with to induce the expression of TGase of MCF7 cells with for to h an increase in TGase 2 the increase in vitro nor in situ transamidation activity not To that the of between the in situ activity and the expression level of TGase 2 may also in other cell lines as we the expression and transamidation activity of TGase 2 in various cell lines The in vitro activity of cell appeared to be with its TGase 2 expression elevated in situ TGase 2 activity was in HeLa and Du145 cells of the other cell lines examined, of TGase 2 expression indicating that the in situ TGase 2 activity on the type of cell and To the of the observed in situ TGase 2 activity the of BP, we measured intracellular concentration of cell (18Jeon J.H. Kim C.W. Shin D.M. Kim K. Cho S.Y. Kwon J.C. Choi K.H. Kang H.S. Kim I.G. FEBS Lett. 2003; 534: 180-184Crossref PubMed Scopus (28) Google Scholar). we did not significant in intracellular concentration in the various of cell not of TGase 2 by or UV of in vitro TGase 2 activity in MCF7 cells the of cellular factor(s) that may regulate TGase 2 activity. Because TGase 2 expression is C.W. P.J. 2000; PubMed Scopus Google or by that K. J. 1994; PubMed Scopus Google Scholar), we that oxidative stress may this cellular We evaluated the of oxidative stress on the in situ activity and in vitro activity as as the expression level of TGase 2 in a variety of cells were treated with of H2O2 and maintained in culture for h to significant change of in vitro TGase 2 activity was observed with all of the cell lines However, K562 and HLE-B3 cells showed a increase of in situ TGase 2 activity with that of the the in degradation or of TGase 2 did not to analysis. These suggest that the elevation of the in situ TGase 2 activity in K562 and HLE-B3 cells may be by activation of a We also the of type of oxidative stress, UV on the TGase 2 activity. The cell lines were with 100 of UVC and maintained in culture for Among the cell lines examined, HeLa and HLE-B3 cells a increase in in situ TGase 2 activity with These indicate that activation of TGase 2 on cell type and also the type of stress on the We with cell that the observed change in the in situ TGase 2 activity is not a of cell in response to oxidative not of TGase 2 in HLE-B3 cells, human lens epithelial cells, exhibited a significant activation of intracellular TGase 2 in response to H2O2 or UV irradiation These are of of the that lens epithelial cells are to into cells that the human and that cross-linking of protein molecules from environmental may to We the between the stress level and the in situ TGase 2 activity in HLE-B3 or HLE-B3 cells showed a in the in situ TGase 2 activity on the of irradiation or H2O2 concentration The of irradiation or the concentration of H2O2 was and μm, a of UV irradiation or H2O2 the cells to from the culture Thus, the UV of and the H2O2 concentration of 400 were for the following The of proteins with catalyzed by TGase 2 the at h and was by using degradation of TGase 2 the was not to 3, A and To we the in the of as an inhibitor to showed that the of to the HLE-B3 cells the elevated in situ TGase 2 activity to that of the control. was by using and also by for in situ TGase 2 activity using of by TGase the that the TGase 2 activated by UV irradiation or H2O2 to cross-linking lens HLE-B3 cells were exposed to UV or H2O2 and maintained in culture for h in the or of The cells were and into and water-insoluble the proteins were by methods using the for αB-crystallin, βB2-crystallin, and to or of αB-crystallin, βB2-crystallin, and vimentin appeared in the water-insoluble The or were in the nor in the from cells treated with in vitro showed that the lens proteins be cross-linked by the of calcium into lens (16Lorand L. Hsu L.K.H. Siefring Jr., G.E. Rafferty N.S. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1356-1360Crossref PubMed Scopus (127) Google Scholar, S. J.H. L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, P. Y. D. FEBS Lett. 2001; PubMed Scopus Google Scholar, H. J. Biochem. 1983; PubMed Scopus Google Scholar). To our from in situ assay with from in vitro HLE-B3 cell was incubated in buffer containing or on the of to HLE-B3 cell cross-linking of αB-crystallin, βB2-crystallin, and vimentin These the effects of oxidative on HLE-B3 cells as described and that oxidative can the formation of water-insoluble polymers of lens proteins by intracellular TGase of of and into water-insoluble was These suggest that TGase 2 catalyzes some other of modification such as polyamination or (1Lornad L. Graham R.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 140-156Crossref PubMed Scopus (1213) Google Scholar), to a change in in To the mechanism for cross-linking lens we evaluated the with which TGase 2 catalyzes the cross-linking of in determined by incorporation assay for and vimentin were about μm, the for was μm, indicating that and vimentin for TGase 2 The of calcium for transamidation reaction was evaluated with lens The in revealed that is most sensitive to calcium and is sensitive protein the lens proteins vimentin in of calcium GTP was in the activity of TGase 2 in incorporation into lens proteins TGase 2 is an enzyme with function transamidation and GTP The calcium-dependent transamidation activity of TGase 2 catalyzes cross-linking or the polyamination of which may induce Thus, has been that TGase 2 catalyzes aggregation or of proteins in various human diseases such as disease of its expression S.Y. P. J.H. Steinert P.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we found that activity or activation of intracellular TGase 2 is cell and that the activity not correlate with the TGase 2 expression in to and the in vitro assay of TGase 2, the in situ activity of TGase 2 must be evaluated to the between elevated expression of TGase 2 and that intracellular TGase 2 is activated by calcium and by GTP J. M. R.P. Johnson J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the molecular mechanism that the intracellular TGase 2 activity physiological has not been elucidated. We in this report, the that that in situ TGase 2 activity is cells are exposed to oxidative stress or UV irradiation and that the level of activation to be on cell type and the nature of stress. These suggest that cellular factor(s) that specifically regulate TGase 2 activity in response to oxidative stress may on this may be that TGase 2 activation is in the mechanism of the stress of the cellular factor that TGase 2 activation and the nature of the proteins modified by TGase 2 stress in the intracellular of TGase The of this study also for the role of TGase 2 in age-related cataractogenesis. The showed between the of cataracts and the level of UV K.J. R. J. 1992; PubMed Scopus Google Scholar). Because is the of lens Res. PubMed Scopus Google Scholar), formation may from of lens proteins from oxidative stress by UV in vitro showed that and vimentin be cross-linked by TGase 2 (16Lorand L. Hsu L.K.H. Siefring Jr., G.E. Rafferty N.S. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1356-1360Crossref PubMed Scopus (127) Google Scholar, S. J.H. L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, P. Y. D. FEBS Lett. 2001; PubMed Scopus Google Scholar). In our UV irradiation on various of cell significant in the expression or in in vitro activity of TGase However, we showed that UV irradiation intracellular TGase 2 of HLE-B3 cells, which the formation of cross-linked αB-crystallin, βB2-crystallin, and Thus, our to a between the on the role of UV in age-related cataracts and the evidence that cross-linking of proteins as an mechanism of age-related cataracts. has been that of including a increase in calcium may be of with lens Res. PubMed Scopus Google Scholar). However, significant in the GTP level was between and GTP level with A. H. Res. PubMed Scopus Google Scholar). show that GTP the protein cross-linking which is The activation of intracellular TGase 2 may other cellular from oxidative an increase in calcium a of lens and epithelial cells, to the protein family that and aggregation of proteins in cells that are G. FEBS Lett. PubMed Scopus Google Scholar). lens D. Y. Jr., A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), indicating that is for of Because may its function and be into the water-insoluble P. Y. D. FEBS Lett. 2001; PubMed Scopus Google Scholar), TGase of may be a critical to protein the present study the evidence that TGase 2 may play an role in age-related expression is to be elevated in the of diseases such as disease T. A. J.E. Cell. Full Text PDF PubMed Scopus Google Scholar), which is to protein In this activation of intracellular TGase 2 in response to oxidative stress is an that with various cell We Y. D. Kim for critical on the and for providing bovine