Regulation of Type I and Type II Transglutaminase in Normal Human Bronchial Epithelial and Lung Carcinoma Cells Thomas M. Vollberg, Margaret D. George, Clara Nervi, and Anton M. Jetten Cell Biology Section, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
In cultured, undifferentiated normal human bronchial epithelial (HBE) cells, transglutaminase activity was localized predominantly in the cytosolic fraction of cell lysates. Upon squamous differentiation, this cytosolic activity declined and was replaced by a 40-fold increase in the activity of particulate (membraneassociated) transglutaminase. Immunoblot analysis demonstrated that the cytosolic transglutaminase was Type II (tissue) transglutaminase and that squamous differentiation shifted gene expression to the Type I (epidermal) transglutaminase. Retinoic acid, an inhibitor of squamous cell differentiation, suppressed the increase in Type I transglutaminase. The decrease in Type II transglutaminase activity was unaffected by retinoic acid. Transforming growth factor-S, (TGF-{3J) enhanced Type II transglutaminase activity about lO-fold in the undifferentiated cells but did not increase Type I transglutaminase or cholesterol sulfate, two early markers of squamous differentiation. TGF-{32 was equivalent to TGF-{3J in inducing Type II transglutaminase and in inhibiting the growth of HBE cells. The differentiation-related and TGF-{3-induced changes in trans glutaminase activity were reflected in the level of transglutaminase Type I and Type II protein and mRNA. Expression oftransglutaminases in lung carcinoma cell lines was variable. No correlation was observed between the expression of Type I transglutaminase and the classification of the cells as squamous cell carcinoma. Several lung carcinoma cell lines exhibited high levels of Type II transglutaminase activity that were increased several-fold by TGF-{3J treatment. Retinoic acid was ineffective in altering transglutaminase expression in most cell lines but induced Type II transglutaminase in a time- and dosedependent manner in NCI-HUT-460 cells. Our results demonstrate that expression of transglutaminases is differentially regulated during squamous differentiation of HBE cells and that TGF-{3 and retinoic acid can affect the expression of transglutaminases in normal and neoplastic epithelial cells derived from the human airways.
Several in vitro cell culture models have been developed to study the proliferation and differentiation of tracheobronchial epithelial cells (for review, see references 1 and 2). In culture, these cells can undergo a pathway Of either mucociliary differentiation or squamous cell differentiation (3-7). The proliferation and/or differentiation of tracheobronchial epithelial cells has been shown to be influenced by retinoids, phorbol esters, and a variety of growth factors including epidermal growth factor, insulin-like growth factor I, and transforming growth factor-S (TGF-13) (1-12). Our labora(Received in original form August 13,1991 and in revised form December 26,1991) Address correspondence to: Anton M. Jetten, Ph.D., Cell Biology Section, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O.Box 12233, Research Triangle Park, NC 27709. Abbreviations: cytosolic retinoic acid binding protein, CRABP; human bronchial epithelial cells, HBE cells; retinoic acid receptors, RARs; sodium dodecyl sulfate, SDS; transforming growth factor-S, TGF-I3. Am. J. Respir. CeU Mol. BioI. \UI. 7. pp. 10-18, 1992
tory has been interested in the mechanisms by which squamous differentiation in tracheobronchial epithelial cells is regulated. Study of such mechanisms will increase the understanding of the repair processes that occur after injury and those processes that lead to squamous cell carcinomas of the lung. Recently, a multistage model for squamous differentiation of tracheobronchial epithelial cells has been proposed (2, 13, 14). Transglutaminases (Rglutamylpeptide.amine-j-glutarnyl transferase) are a group of Ca2+-dependent enzymes that catalyze the formation of E-(-y-glutamyl)lysine-protein crosslinks and the incorporation of primary amines into proteinbound glutamine residues (2, 15-17). Transglutaminases have been characterized based on differences in their cellular distribution, immunoreactivity, amino acid sequence, and enzymatic properties (17-23). In addition, different transglutaminases promote different biologic events. The polymerization of fibrin during hemostasis is catalyzed by plasma Factor XIIIa, a transglutaminase formed from the zymogen Factor XIII by thrombin action (24). A soluble trans glutaminase has been found in many cell types and has
Vollberg, George, Nervi et al.: Regulation of Type I and Type II Transglutaminase
been termed tissue or Type II transglutaminase (15, 16,21). This transglutaminase may play a role in regulating cell growth or cellular morphology. Epidermal or Type I transglutaminase is membrane bound and catalyzes the formation of the crosslinked envelope during squamous differentiation of epithelial cells in several tissues (5, 17, 25). Recently, cDNAs coding for Type I and Type II transglutaminase and Factor XIIIa have been cloned and sequenced (19, 20, 22-24). In this report, we examine the expression of Type I and Type II transglutaminase in normal human bronchial epithelial (HBE) cells in relation to squamous differentiation and in several human lung carcinoma cell lines. We report that both Type I and Type II transglutaminase are differentially expressed during squamous differentiation of HBE cells and that their expression is influenced by retinoic acid and
TGF-IJ.
Materials and Methods Cell Culture Human bronchial tissue was obtained from donors at the time of autopsy and provided by Dr. G. D. Stoner (Medical College of Ohio, Toledo, OH). Explant cultures were established according to the procedures described by Lechner and LaVeck (26). Epithelial outgrowths from the explants were trypsinized, and cells subcultured in 60-mm dishes precoated with a solution of fibronectin/albumin/Vitrogen (Collagen Corp., Palo Alto, CA) as described (26). HBE cells were cultured in HBE medium consisting of a modified MCDB 153 medium containing 0.15 mM CaCI 2 , amphotericin, gentamycin, and 10 ng/ml epidermal growth factor, 5 J-tg/ml insulin, 1.4 J-tM hydrocortisone, and 50 J-tg/ml bovine pituitary extract (Keratinocyte Growth Medium; Clonetics, San Diego, CA) and supplemented with 10 J-tg/ml transferrin, 10 nM 3,3',5-triiodothyronine and 3 J-tM epinephrine. Medium was changed every other day. The lung carcinoma cell lines NCI-HUT-460, NCI-HUT-125, NCIHUT-69, NCI-HUT-596, NCI-HUT-292, NCI-HUT-520, and NCI-HUT-647 were obtained from Dr. A. Gazdar (National Cancer Institute, Bethesda, MD). The cell lines Calu-l, A549, SK-LU-l, and SK-MES-l were from ATCC (Rockville, MD). The cell lines were maintained in RPMI 1640 containing 10% fetal calf serum but cells were tested in HBE medium. Cell counts were determined with a hemacytometer. Crosslinked envelope formation was measured in the absence of calcium ionophore as described previously (27). Assay of Transglutaminase Cells grown in 60-mm dishes were washed twice in phosphate-buffered saline containing EDTA (1 mM) and phenylmethylsulfonyl fluoride (1 mM), and dishes were stored at -70° C until assay was performed. Cells were lysed by three freeze/thaw cycles, and the homogenate was scraped in 200 J-tl of the same phosphate-buffered saline solution. Aliquots were taken to measure protein, and 10 mM dithiothreitol was added to the remaining homogenate. The homogenate was centrifuged for 15 min at 10,000 x g, yielding the soluble and particulate fractions. Transglutaminase assays were performed on the total homogenate to determine total transglutaminase activity and on the particulate and soluble frac-
11
tions to determine the activity of Type I and Type II transglutaminase, respectively. Transglutaminase activities were determined in duplicate or triplicate by measuring the incorporation of ['Hlputrescine (16.2 Ci/mmol; Dupont, Boston, MA) into casein as described previously (27). Assay of Cholesterol Sulfate Cells were grown in 60-mm dishes in KGM medium containing Nal5S04 (carrier-free; ICN, Irvine, CA) for 24 h, harvested by trypsinization, pelleted by centrifugation, and extracted with chloroform/methanol as described previously (28). Western Blot Analysis Cells were harvested, collected by centrifugation, and solubilized in sample buffer. The cellular proteins were separated by sodium dodecyl sulfate (SDS)-lO% polyacrylamide slab gel electrophoresis according to Laemmli (29). The proteins were transferred electrophoretically to a Nitroplus 2000 membrane (Enprotech, Hyde Park, MA) as described (30). Immunoreactivity of the monoclonal antibody Cub-7401 or RCI with antigen was examined by immunogold/silver staining with the Auroprobe BLplus and IntenSE BL silver enhancement kit (Janssen Biotech, Olen, Belgium) according to the manufacturer's protocol. The monoclonal antibody Cub-7401 immunoreacts only with transglutaminase Type II (18) and was a gift from Dr. P. 1. Birckbichler (Samuel Robert Noble Foundation, Ardmore, OK). The antibody RCI recognizes Type I transglutaminase (17) and was kindly provided by Dr. S. M. Thacher (University of Texas, College Station, TX). Northern Blot Analysis Total RNA was isolated via the guanidinium isothiocyanate method (31). RNA was electrophoresed through a 1.2 % agarose-formaldehyde gel and then transferred to Nytran using 1.5 M NaCl/0.15 M sodium citrate, pH 7.0, as the transfer buffer in a Vacublot apparatus (American Bionetics, Hayward, CA) according to the manufacturer's protocol. After transfer, the RNA was crosslinked to the blot by UV irradiation. Northern blots were prehybridized for 4 to 24 h at 42° C in a buffer containing 50% formamide, 5x SSPE, 2 X Denhardt's, 1% SDS, and 250 J-tg/ml sheared salmon sperm DNA. After addition of the probe (3 to 15 ng/ml), hybridization was allowed to proceed for 24 to 48 h at 42° C. Blots were washed at a final stringency of 60° C in O.1x SSC, 0.1% SDS. The recombinant cDNA clone pTG3400 contains a 3,348-bp fragment of the coding region of mouse Type II transglutaminase cloned into the Eco RI site of the plasmid vector pGEM3Z (20); the Eco RI insert was used as a probe. The cDNA clone pTG-7 contains a 2.7-kb insert encoding the full-length coding region of the rabbit Type I transglutaminase cloned into the Eco RI site of the vector Bluescript SK( - )(22); this insert was used as a probe. The recombinant cDNA clone pGAD-28, which contains a 1,261-bp cDNA insert of chicken glyceraldehyde-3-phosphate dehydrogenase (32), was digested with Pstl and an approximately 1,120-bp fragment used as a probe. The cDNA probes were labeled with [o:-32P]dCTP ("'3,000 Ci/mmol; Amersham Corp., Arlington Heights, IL) via random priming using-the kit and
12
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
protocols supplied by Boehringer Mannheim Biochemicals (Indianapolis, IN). Materials TGF-{3\ and TGF-{32 and antibodies against TGF-{3} and TGF-{32 were purchased from R&D Systems (Minneapolis, MN). All-trans-retinoic acid was provided by Hoffman-La Roche (Nutley, NJ).
Results The subcellular localization of transglutaminase activity in cultured HBE cells was dependent on the differentiated state of these cells (Figure 1). Transglutaminase activity was ex101 ........- - - - - - - - - - - - - - - - - .
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Figure 6. Effects of TGF-I3, on cell proliferation and transglutaminase Type II activity of human lung carcinoma HUT-647 (closed triangles), A549 (open triangles), SK-LU-l (closed circles), HUT-460 (open squares), and HUT-292 (closed squares) cells. Cells were grown in HBE medium and treated with TGF-131 at the concentrations indicated. Cell number/dish was determined after 5 days (panel A). Transglutarninase activity was assayed after 3 days of treatment (panel B).
Type II transglutaminase expression. In HUT-460 cells, retinoic acid increased Type II transglutaminase in a concentration- and time-dependent manner (Figure 7). The growth of the HUT-460 cells was not affected by the retinoic acid treatment (data not shown). Northern analysis demonstrated that the increase in Type II transglutaminase by TGF-~, in the HUT-292 and A549 cells was due to an increase in the level of the corresponding mRNA (Figure 8). In A549 cells, the stimulation of Type II transglutaminase by retinoic acid was, at least in part, related to the increased levels of Type II transglutaminase mRNA. Untreated HUT-460 cells expressed Type II transglutaminase mRNA at a level
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Figure 7. Induction of Type II transglutaminase activity in HUT-460 cells by retinoic acid. Cells were grown in KGM medium in the absence (open squares) or presence (closed squares) of retinoic acid. Panel A: Induction as a function of time of retinoic acid (l p.M) exposure. Panel B: Induction as a function of the retinoic acid concentration. Type II transglutarninase activity was assayed 48 h after retinoic acid exposure.
16
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
near the limit of detection. This level of expression was enhanced after retinoic acid or lGF-,B, treatment.
Discussion Our laboratory has been using cultured human tracheobronchial epithelial cells as a model cell system to study the mechanisms that regulate the proliferation and differentiation of the tracheobronchial epithelium (2, 12, 33). HBE cells growing in the exponential phase are relatively undifferentiated and undergo squamous differentiation upon reaching confluence. The pathway leading to squamous differentiation is characterized by cells initially undergoing irreversible growth arrest (terminal cell division) followed by the expression of several squamous cell markers (2, 13, 14). In this study, we showed that exponentially growing HBE cells expressed almost solely the soluble, Type II transglutaminase activity. In contrast, confluent, squamous-differentiated cultures expressed high levels of the membraneassociated, Type I transglutaminase activity. These findings are in agreement with studies of squamous differentiation in other cell systems (17, 26, 34). lGF-,B has been shown previously to inhibit growth and to induce squamous differentiation in normal human tracheobronchial epithelial cells (8, 9). In the present study, the growth inhibition of normal HBE cells by lGF-,B, and lGF-,Bz was in agreement with these reports. However, lGF-,B, did not increase the level of Type I transglutaminase activity or cholesterol sulfate, two early biochemical markers of squamous differentiation (32). These observations suggest that lGF-,B does not induce the expression of a squamous cell phenotype in HBE cells. This discrepancy with previous reports (8, 9) appears to be related to the dependency of the lGF-,B, responsiveness on the density of the tracheobronchial epithelial cell culture (35). At the culture density used in our experiments, addition of lGF-,B, or lGF-,B2 caused a to-fold stimulation in Type II transglutaminase activity. This increase in activity was related to an enhancement in the level of Type II transglutaminase protein and corresponding mRNA. Recently, we reported that lGF-,B increases Type II transglutaminase activity in human epidermal keratinocytes (36). In other cell systems, lGF-,B has been reported to stimulate the expression of several other gene products including fibronectin and collagen type I (37, 38). lGF-,B regulates the expression of these genes at both the transcriptional and post-transcriptional level, Efforts are presently under way to elucidate the mechanism by which lGF-,B regulates the expression of the Type II transglutaminase mRNA in HBE cells and carcinoma cells. In vivo and in vitro studies have shown that retinoids are effective inhibitors of squamous cell differentiation (2, 13). We have reported previously that retinoic acid inhibits the induction of the squamous cell marker cholesterol sulfate. In this study, we showed that retinoic acid very effectively suppressed the induction of Type I transglutaminase in HBE cells. This suppression was related to a decrease in the level of transglutaminase Type I protein and corresponding mRNA. The suppression of squamous cell differentiation by retinoids occurs at nanomolar concentrations and specific structural requirements are critical for the activity of retinoids, suggesting that the action of retinoic acid is mediated by specific
retinoic acid-responsive pathways (2). Several nuclear retinoic acid receptors (RARs), which act as retinoic acid-dependent transcriptional factors (39-41), are expressed by HBE cells (42). Based on structure-activity relationships, binding of a retinoid to RARs appears to be related to the inhibition of squamous differentiation in tracheobronchial epithelial cells (42). Whether RARs mediate the suppression of squamous cell-specific marker genes by directly altering their transcription or by altering the expression of another transcriptional factor is a matter of speculation. Presently, we are determining which regions of the Type I transglutaminase promoter are required for the retinoic acid-dependent downregulation of transcription. Examination of the expression of transglutaminase Type I and Type II in the various lung cell carcinoma cell lines showed that the level of these enzymes varied from cell line to cell line in agreement with the findings of another recently published study (43). No correlation could be established between histologic classification and the expression of a specific transglutaminase. Several of the carcinoma cell lines were very responsive to lGF-,B. TGF-,B effectively inhibited their growth and enhanced Type II transglutaminase activity. The high levels of Type II transglutaminase in some untreated cells could be due to the endogenous production and secretion of lGF-,B. It has been reported that many carcinoma cell lines including HUT-292 and A549 secrete lGF-,B (44). However, these cell lines secrete only the latent form of TGF-,B (44). Moreover, addition of antibodies against lGF-,B, and lGF-,Bz to the culture medium did not affect transglutaminase Type II activity (A. M. Jetten, unpublished observations). These findings suggest that the high level of transglutaminase Type II activity in these cell lines does not result from the paracrine effects of lGF-,B but probably is increased through a different mechanism. Two cell lines, HUT460 and A549, responded to retinoic acid with a substantial increase in trans glutaminase Type II activity. Recently, it has been reported that retinoic acid increases the synthesis of TGF-,B2 in mouse epidermal keratinocytes (45). The retinoic acid-induced increase in Type II transglutaminase activity in HUT-460 cells could be due to an increase in the levels of lGF-,B by retinoic acid. However, addition of anti-TGF-,B, and/or anti-lGF-,B2 antibodies to the medium did not reduce the increase in transglutaminase activity by retinoic acid in these cells (not shown). These findings suggest that retinoic acid-dependent induction of transglutaminase Type II in HUT-460 cells may not be mediated by increased secretion or activation of lGF-,B. It is interesting to note that the EC so for the increase in Type II transglutaminase in HUT-460 cells is 3 nM retinoic acid, about to- to 20-fold higher than the ECsos for the inhibition of cholesterol sulfate and transglutaminase Type I in normal tracheobronchial epithelial cells (27, 28). The difference in EC so values might be due to the different mechanisms affecting these biochemical markers or could be related to the presence of relatively high levels of cytosolic retinoic acid binding protein (CRABP) in HUT-460 cells andlow levels of CRABP in tracheobronchial epithelial cells (42). CRABP has been postulated to regulate the concentration of retinoic acid in cells; increasing expression of CRABP would reduce the effective concentration of retinoic acid and cause a shift of the EC so to a higher concentration (46).
Vollberg, George, Nervi et al.: Regulation of Type I and Type II Transglutaminase
The function of transglutaminase Type I has been well established (17). This transglutaminase plays an important role in catalyzing the formation of covalent links between the protein precursors, such as involucrin, that will form the crosslinked envelope in the final stages of squamous differentiation (17). Much less is known about the function of the Type II transglutaminase. It has been suggested that this transglutaminase may be involved in apoptosis (programmed cell death) (47). This process may provide an alternative mechanism to control the size of a tissue that is initiated during hormone-induced atrophy, involution of hyperplasia, or regression of tumors. Other studies have implicated transglutaminase Type II in the regulation of cell proliferation and cell-substratum adhesion. However, in H460 cells, TGF-{31 caused an increase in Type II transglutaminase activity but had no effect on cell growth (Figure 7), indicating that in these cells no direct correlation exists between an increase in this enzyme activity and growth inhibition. A recent study has provided evidence that Type II transglutaminase can be found covalently linked to fibronectin in hepatocytes (48). The investigators in this study suggest that Type II transglutaminase may playa role in modulating cell-cell interactions or in the covalent restructuring of extracellular matrix proteins. If this is a common function of Type II transglutaminase in all tissues, the elevated expression of Type II trans glutaminase in certain lung carcinoma cells could be relevant to the altered architecture seen in tumorous tissue. The upregulation of transglutaminase Type II and fibronectin as well as several other extracellular matrix proteins by TGF-{3 may also be important to the invasive character of each carcinoma. Further studies are necessary to establish the function of this enzyme in normal and tumorigenic tracheobronchial epithelial cells. References 1. Lechner, J. F., G. D. Stoner, A. Haugen etal. 1985. In vitro human bronchial epithelial model systems for carcinogenesis studies. In In Vitro Models for Cancer Research. Vol. 6. M. M. Webber, Editor. CRC Press, Boca Raton, FL. 3-17. 2. Jetten, A. M. 1991. Growth and differentiation factors in tracheobronchial epithelium. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 260:L361-L373. 3. Lechner, J. F., A. Haugen, L. A. McClendon, and A. M. Shamsuddin. 1984. Induction of squamous differentiation of normal human bronchial epithelial cells by small amounts of serum. Differentiation 25:229-237. 4. Adler, K. B., J. E. Schwarz, M. J. Whitcutt, and R. Wu. 1987. A new chamber system for maintaining differentiated guinea pig respiratory epithelial cells between air and liquid phases. Biotechniques 5:462-465. 5. Jetten, A. M., A. R. Brody, M. A. Deas, G. E. R. Hook, J. I. Rearick, and S. M. Thacher. 1987. Retinoic acid and substratum regulate the differentiation of rabbit tracheal epithelial cells into squamous and secretory phenotype. Lab. Invest. 56:654-664. 6. Rearick, J. I., M. Deas, and A. M. Jetten. 1987. Synthesis of mucous glycoproteins by rabbit tracheal epithelial cells in vitro. Modulation by substratum, retinoids and cyclic AMP. Biochem. J. 242: 19-25. 7. Van Scott, M. R., J. R. Yankaskas, and R. C. Boucher. 1986. Culture of airway epithelial cells: research techniques. Exp. Lung Res. 11:75-94. 8. Masui, T., L. M. Wakefield, J. F. Lechner, M. A. LaVeck, M. B. Sporn, and C. C. Harris. 1986. Type (3 transforming growth factor is the primary differentiation inducing serum factor for normal human bronchial epithelial cells. Proc. Natl. Acad. Sci. USA 83:2438-2442. 9. Jetten, A. M., J. E. Shirley, and G. Stoner. 1986. Regulation of prolifer ation and differentiation of respiratory tract epithelial cells by TGF(3. Exp. Cell Res. 167:539-549. 10. Willey, J. C., C. E. Moser, J. F. Lechner, and C. C. Harris. 1984. Differential effects of 12-0-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells. Cancer Res. 44:5124-5126. 11. Sanchez, J. If., C. J. Boreiko, J. Furlong, and T. W. Hesterberg. 1987. Differential effects of tumour promoters upon the growth of normal bron-
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transcriptional activation of a type I col1agen promoter by transforming growth factor-S, Cell 52:405-414. Petkovich, M., N. Brand, A. Krust, and P. Chambon. 1987. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 330:444-450. Giguere, V., E. S. Ong, P. Segui, and R. M. Evans. 1987. Identification of a receptor for the morphogen retinoic acid. Nature 330:620-629. Zelent, A., A. Krust, M. Petkovich, P. Kastner, and P. Chambon. 1989. Cloning of murine Ci and 13 retinoic acid receptors and a novel receptor 'Y predominantly expressed in the skin. Nature 339:714-717. Nervi, c., T. M. Vollberg, M. D. George, A. Zelent, P. Chambon, and A. M. Jetten. 1991. Expression of nuclear retinoic acid receptors in normal tracheobronchial epithelial cel1sand lung carcinoma cel1s. Exp. Cell Res. 195:163-170. Levitt, M. L., A. F. Gazdar, H. 0. Oie, H. Schuller, and S. C. Thacher. 1990. Cross-linked envelope-related markers for squamous differentiation in human lung cancer cell lines. Cancer Res. 50:120-128.
44. Wakefield, L. M., D. M. Smith, T. Masui, C. C. Harris, and M. B. Sporn. 1987. Distribution and modulation of the cellular receptor for transforming growth factor-beta. J. Cell BioI. 105:967-975. 45. Glick, A. B., K. C. Flanders, D. Danielpour, S. H. Yuspa, and M. B. Sporn. 1989. Retinoic acid induces transforming growth factor-132 in cultured keratinocytes and mouse epidermis. Cell Regulation 1:87-97. 46. Jetten, A. M. 1990. Multi-stage program of differentiation in human epidermal keratinocytes: regulation by retinoids. J. Invest. Dermatol. 95:44S-46S. 47. Fesus, L., V. Thomazy, and A. Falus. 1987. Induction and activation oftissue transglutaminase during programmed cell death. FEBS Lett. 224:104-108. 48. Barsigian, c., A. M. Stern, andJ. Martinez. 1991. Tissue (Type Il) transglutaminase covalently incorporates itself, fibrinogen, or fibronectin into high molecular weight complexes on the extracellular surface of isolated hepatocytes. J. BioI. Chern. 266:22501-22509.
In cultured, undifferentiated normal human bronchial epithelial (HBE) cells, transglutaminase activity was localized predominantly in the cytosolic fraction of cell lysates. Upon squamous differentiation, this cytosolic activity declined and was replaced by a 40-fold increase in the activity of particulate (membraneassociated) transglutaminase. Immunoblot analysis demonstrated that the cytosolic transglutaminase was Type II (tissue) transglutaminase and that squamous differentiation shifted gene expression to the Type I (epidermal) transglutaminase. Retinoic acid, an inhibitor of squamous cell differentiation, suppressed the increase in Type I transglutaminase. The decrease in Type II transglutaminase activity was unaffected by retinoic acid. Transforming growth factor-S, (TGF-{3J) enhanced Type II transglutaminase activity about lO-fold in the undifferentiated cells but did not increase Type I transglutaminase or cholesterol sulfate, two early markers of squamous differentiation. TGF-{32 was equivalent to TGF-{3J in inducing Type II transglutaminase and in inhibiting the growth of HBE cells. The differentiation-related and TGF-{3-induced changes in trans glutaminase activity were reflected in the level of transglutaminase Type I and Type II protein and mRNA. Expression oftransglutaminases in lung carcinoma cell lines was variable. No correlation was observed between the expression of Type I transglutaminase and the classification of the cells as squamous cell carcinoma. Several lung carcinoma cell lines exhibited high levels of Type II transglutaminase activity that were increased several-fold by TGF-{3J treatment. Retinoic acid was ineffective in altering transglutaminase expression in most cell lines but induced Type II transglutaminase in a time- and dosedependent manner in NCI-HUT-460 cells. Our results demonstrate that expression of transglutaminases is differentially regulated during squamous differentiation of HBE cells and that TGF-{3 and retinoic acid can affect the expression of transglutaminases in normal and neoplastic epithelial cells derived from the human airways.
Several in vitro cell culture models have been developed to study the proliferation and differentiation of tracheobronchial epithelial cells (for review, see references 1 and 2). In culture, these cells can undergo a pathway Of either mucociliary differentiation or squamous cell differentiation (3-7). The proliferation and/or differentiation of tracheobronchial epithelial cells has been shown to be influenced by retinoids, phorbol esters, and a variety of growth factors including epidermal growth factor, insulin-like growth factor I, and transforming growth factor-S (TGF-13) (1-12). Our labora(Received in original form August 13,1991 and in revised form December 26,1991) Address correspondence to: Anton M. Jetten, Ph.D., Cell Biology Section, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O.Box 12233, Research Triangle Park, NC 27709. Abbreviations: cytosolic retinoic acid binding protein, CRABP; human bronchial epithelial cells, HBE cells; retinoic acid receptors, RARs; sodium dodecyl sulfate, SDS; transforming growth factor-S, TGF-I3. Am. J. Respir. CeU Mol. BioI. \UI. 7. pp. 10-18, 1992
tory has been interested in the mechanisms by which squamous differentiation in tracheobronchial epithelial cells is regulated. Study of such mechanisms will increase the understanding of the repair processes that occur after injury and those processes that lead to squamous cell carcinomas of the lung. Recently, a multistage model for squamous differentiation of tracheobronchial epithelial cells has been proposed (2, 13, 14). Transglutaminases (Rglutamylpeptide.amine-j-glutarnyl transferase) are a group of Ca2+-dependent enzymes that catalyze the formation of E-(-y-glutamyl)lysine-protein crosslinks and the incorporation of primary amines into proteinbound glutamine residues (2, 15-17). Transglutaminases have been characterized based on differences in their cellular distribution, immunoreactivity, amino acid sequence, and enzymatic properties (17-23). In addition, different transglutaminases promote different biologic events. The polymerization of fibrin during hemostasis is catalyzed by plasma Factor XIIIa, a transglutaminase formed from the zymogen Factor XIII by thrombin action (24). A soluble trans glutaminase has been found in many cell types and has
Vollberg, George, Nervi et al.: Regulation of Type I and Type II Transglutaminase
been termed tissue or Type II transglutaminase (15, 16,21). This transglutaminase may play a role in regulating cell growth or cellular morphology. Epidermal or Type I transglutaminase is membrane bound and catalyzes the formation of the crosslinked envelope during squamous differentiation of epithelial cells in several tissues (5, 17, 25). Recently, cDNAs coding for Type I and Type II transglutaminase and Factor XIIIa have been cloned and sequenced (19, 20, 22-24). In this report, we examine the expression of Type I and Type II transglutaminase in normal human bronchial epithelial (HBE) cells in relation to squamous differentiation and in several human lung carcinoma cell lines. We report that both Type I and Type II transglutaminase are differentially expressed during squamous differentiation of HBE cells and that their expression is influenced by retinoic acid and
TGF-IJ.
Materials and Methods Cell Culture Human bronchial tissue was obtained from donors at the time of autopsy and provided by Dr. G. D. Stoner (Medical College of Ohio, Toledo, OH). Explant cultures were established according to the procedures described by Lechner and LaVeck (26). Epithelial outgrowths from the explants were trypsinized, and cells subcultured in 60-mm dishes precoated with a solution of fibronectin/albumin/Vitrogen (Collagen Corp., Palo Alto, CA) as described (26). HBE cells were cultured in HBE medium consisting of a modified MCDB 153 medium containing 0.15 mM CaCI 2 , amphotericin, gentamycin, and 10 ng/ml epidermal growth factor, 5 J-tg/ml insulin, 1.4 J-tM hydrocortisone, and 50 J-tg/ml bovine pituitary extract (Keratinocyte Growth Medium; Clonetics, San Diego, CA) and supplemented with 10 J-tg/ml transferrin, 10 nM 3,3',5-triiodothyronine and 3 J-tM epinephrine. Medium was changed every other day. The lung carcinoma cell lines NCI-HUT-460, NCI-HUT-125, NCIHUT-69, NCI-HUT-596, NCI-HUT-292, NCI-HUT-520, and NCI-HUT-647 were obtained from Dr. A. Gazdar (National Cancer Institute, Bethesda, MD). The cell lines Calu-l, A549, SK-LU-l, and SK-MES-l were from ATCC (Rockville, MD). The cell lines were maintained in RPMI 1640 containing 10% fetal calf serum but cells were tested in HBE medium. Cell counts were determined with a hemacytometer. Crosslinked envelope formation was measured in the absence of calcium ionophore as described previously (27). Assay of Transglutaminase Cells grown in 60-mm dishes were washed twice in phosphate-buffered saline containing EDTA (1 mM) and phenylmethylsulfonyl fluoride (1 mM), and dishes were stored at -70° C until assay was performed. Cells were lysed by three freeze/thaw cycles, and the homogenate was scraped in 200 J-tl of the same phosphate-buffered saline solution. Aliquots were taken to measure protein, and 10 mM dithiothreitol was added to the remaining homogenate. The homogenate was centrifuged for 15 min at 10,000 x g, yielding the soluble and particulate fractions. Transglutaminase assays were performed on the total homogenate to determine total transglutaminase activity and on the particulate and soluble frac-
11
tions to determine the activity of Type I and Type II transglutaminase, respectively. Transglutaminase activities were determined in duplicate or triplicate by measuring the incorporation of ['Hlputrescine (16.2 Ci/mmol; Dupont, Boston, MA) into casein as described previously (27). Assay of Cholesterol Sulfate Cells were grown in 60-mm dishes in KGM medium containing Nal5S04 (carrier-free; ICN, Irvine, CA) for 24 h, harvested by trypsinization, pelleted by centrifugation, and extracted with chloroform/methanol as described previously (28). Western Blot Analysis Cells were harvested, collected by centrifugation, and solubilized in sample buffer. The cellular proteins were separated by sodium dodecyl sulfate (SDS)-lO% polyacrylamide slab gel electrophoresis according to Laemmli (29). The proteins were transferred electrophoretically to a Nitroplus 2000 membrane (Enprotech, Hyde Park, MA) as described (30). Immunoreactivity of the monoclonal antibody Cub-7401 or RCI with antigen was examined by immunogold/silver staining with the Auroprobe BLplus and IntenSE BL silver enhancement kit (Janssen Biotech, Olen, Belgium) according to the manufacturer's protocol. The monoclonal antibody Cub-7401 immunoreacts only with transglutaminase Type II (18) and was a gift from Dr. P. 1. Birckbichler (Samuel Robert Noble Foundation, Ardmore, OK). The antibody RCI recognizes Type I transglutaminase (17) and was kindly provided by Dr. S. M. Thacher (University of Texas, College Station, TX). Northern Blot Analysis Total RNA was isolated via the guanidinium isothiocyanate method (31). RNA was electrophoresed through a 1.2 % agarose-formaldehyde gel and then transferred to Nytran using 1.5 M NaCl/0.15 M sodium citrate, pH 7.0, as the transfer buffer in a Vacublot apparatus (American Bionetics, Hayward, CA) according to the manufacturer's protocol. After transfer, the RNA was crosslinked to the blot by UV irradiation. Northern blots were prehybridized for 4 to 24 h at 42° C in a buffer containing 50% formamide, 5x SSPE, 2 X Denhardt's, 1% SDS, and 250 J-tg/ml sheared salmon sperm DNA. After addition of the probe (3 to 15 ng/ml), hybridization was allowed to proceed for 24 to 48 h at 42° C. Blots were washed at a final stringency of 60° C in O.1x SSC, 0.1% SDS. The recombinant cDNA clone pTG3400 contains a 3,348-bp fragment of the coding region of mouse Type II transglutaminase cloned into the Eco RI site of the plasmid vector pGEM3Z (20); the Eco RI insert was used as a probe. The cDNA clone pTG-7 contains a 2.7-kb insert encoding the full-length coding region of the rabbit Type I transglutaminase cloned into the Eco RI site of the vector Bluescript SK( - )(22); this insert was used as a probe. The recombinant cDNA clone pGAD-28, which contains a 1,261-bp cDNA insert of chicken glyceraldehyde-3-phosphate dehydrogenase (32), was digested with Pstl and an approximately 1,120-bp fragment used as a probe. The cDNA probes were labeled with [o:-32P]dCTP ("'3,000 Ci/mmol; Amersham Corp., Arlington Heights, IL) via random priming using-the kit and
12
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
protocols supplied by Boehringer Mannheim Biochemicals (Indianapolis, IN). Materials TGF-{3\ and TGF-{32 and antibodies against TGF-{3} and TGF-{32 were purchased from R&D Systems (Minneapolis, MN). All-trans-retinoic acid was provided by Hoffman-La Roche (Nutley, NJ).
Results The subcellular localization of transglutaminase activity in cultured HBE cells was dependent on the differentiated state of these cells (Figure 1). Transglutaminase activity was ex101 ........- - - - - - - - - - - - - - - - - .
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Figure 6. Effects of TGF-I3, on cell proliferation and transglutaminase Type II activity of human lung carcinoma HUT-647 (closed triangles), A549 (open triangles), SK-LU-l (closed circles), HUT-460 (open squares), and HUT-292 (closed squares) cells. Cells were grown in HBE medium and treated with TGF-131 at the concentrations indicated. Cell number/dish was determined after 5 days (panel A). Transglutarninase activity was assayed after 3 days of treatment (panel B).
Type II transglutaminase expression. In HUT-460 cells, retinoic acid increased Type II transglutaminase in a concentration- and time-dependent manner (Figure 7). The growth of the HUT-460 cells was not affected by the retinoic acid treatment (data not shown). Northern analysis demonstrated that the increase in Type II transglutaminase by TGF-~, in the HUT-292 and A549 cells was due to an increase in the level of the corresponding mRNA (Figure 8). In A549 cells, the stimulation of Type II transglutaminase by retinoic acid was, at least in part, related to the increased levels of Type II transglutaminase mRNA. Untreated HUT-460 cells expressed Type II transglutaminase mRNA at a level
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Figure 7. Induction of Type II transglutaminase activity in HUT-460 cells by retinoic acid. Cells were grown in KGM medium in the absence (open squares) or presence (closed squares) of retinoic acid. Panel A: Induction as a function of time of retinoic acid (l p.M) exposure. Panel B: Induction as a function of the retinoic acid concentration. Type II transglutarninase activity was assayed 48 h after retinoic acid exposure.
16
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
near the limit of detection. This level of expression was enhanced after retinoic acid or lGF-,B, treatment.
Discussion Our laboratory has been using cultured human tracheobronchial epithelial cells as a model cell system to study the mechanisms that regulate the proliferation and differentiation of the tracheobronchial epithelium (2, 12, 33). HBE cells growing in the exponential phase are relatively undifferentiated and undergo squamous differentiation upon reaching confluence. The pathway leading to squamous differentiation is characterized by cells initially undergoing irreversible growth arrest (terminal cell division) followed by the expression of several squamous cell markers (2, 13, 14). In this study, we showed that exponentially growing HBE cells expressed almost solely the soluble, Type II transglutaminase activity. In contrast, confluent, squamous-differentiated cultures expressed high levels of the membraneassociated, Type I transglutaminase activity. These findings are in agreement with studies of squamous differentiation in other cell systems (17, 26, 34). lGF-,B has been shown previously to inhibit growth and to induce squamous differentiation in normal human tracheobronchial epithelial cells (8, 9). In the present study, the growth inhibition of normal HBE cells by lGF-,B, and lGF-,Bz was in agreement with these reports. However, lGF-,B, did not increase the level of Type I transglutaminase activity or cholesterol sulfate, two early biochemical markers of squamous differentiation (32). These observations suggest that lGF-,B does not induce the expression of a squamous cell phenotype in HBE cells. This discrepancy with previous reports (8, 9) appears to be related to the dependency of the lGF-,B, responsiveness on the density of the tracheobronchial epithelial cell culture (35). At the culture density used in our experiments, addition of lGF-,B, or lGF-,B2 caused a to-fold stimulation in Type II transglutaminase activity. This increase in activity was related to an enhancement in the level of Type II transglutaminase protein and corresponding mRNA. Recently, we reported that lGF-,B increases Type II transglutaminase activity in human epidermal keratinocytes (36). In other cell systems, lGF-,B has been reported to stimulate the expression of several other gene products including fibronectin and collagen type I (37, 38). lGF-,B regulates the expression of these genes at both the transcriptional and post-transcriptional level, Efforts are presently under way to elucidate the mechanism by which lGF-,B regulates the expression of the Type II transglutaminase mRNA in HBE cells and carcinoma cells. In vivo and in vitro studies have shown that retinoids are effective inhibitors of squamous cell differentiation (2, 13). We have reported previously that retinoic acid inhibits the induction of the squamous cell marker cholesterol sulfate. In this study, we showed that retinoic acid very effectively suppressed the induction of Type I transglutaminase in HBE cells. This suppression was related to a decrease in the level of transglutaminase Type I protein and corresponding mRNA. The suppression of squamous cell differentiation by retinoids occurs at nanomolar concentrations and specific structural requirements are critical for the activity of retinoids, suggesting that the action of retinoic acid is mediated by specific
retinoic acid-responsive pathways (2). Several nuclear retinoic acid receptors (RARs), which act as retinoic acid-dependent transcriptional factors (39-41), are expressed by HBE cells (42). Based on structure-activity relationships, binding of a retinoid to RARs appears to be related to the inhibition of squamous differentiation in tracheobronchial epithelial cells (42). Whether RARs mediate the suppression of squamous cell-specific marker genes by directly altering their transcription or by altering the expression of another transcriptional factor is a matter of speculation. Presently, we are determining which regions of the Type I transglutaminase promoter are required for the retinoic acid-dependent downregulation of transcription. Examination of the expression of transglutaminase Type I and Type II in the various lung cell carcinoma cell lines showed that the level of these enzymes varied from cell line to cell line in agreement with the findings of another recently published study (43). No correlation could be established between histologic classification and the expression of a specific transglutaminase. Several of the carcinoma cell lines were very responsive to lGF-,B. TGF-,B effectively inhibited their growth and enhanced Type II transglutaminase activity. The high levels of Type II transglutaminase in some untreated cells could be due to the endogenous production and secretion of lGF-,B. It has been reported that many carcinoma cell lines including HUT-292 and A549 secrete lGF-,B (44). However, these cell lines secrete only the latent form of TGF-,B (44). Moreover, addition of antibodies against lGF-,B, and lGF-,Bz to the culture medium did not affect transglutaminase Type II activity (A. M. Jetten, unpublished observations). These findings suggest that the high level of transglutaminase Type II activity in these cell lines does not result from the paracrine effects of lGF-,B but probably is increased through a different mechanism. Two cell lines, HUT460 and A549, responded to retinoic acid with a substantial increase in trans glutaminase Type II activity. Recently, it has been reported that retinoic acid increases the synthesis of TGF-,B2 in mouse epidermal keratinocytes (45). The retinoic acid-induced increase in Type II transglutaminase activity in HUT-460 cells could be due to an increase in the levels of lGF-,B by retinoic acid. However, addition of anti-TGF-,B, and/or anti-lGF-,B2 antibodies to the medium did not reduce the increase in transglutaminase activity by retinoic acid in these cells (not shown). These findings suggest that retinoic acid-dependent induction of transglutaminase Type II in HUT-460 cells may not be mediated by increased secretion or activation of lGF-,B. It is interesting to note that the EC so for the increase in Type II transglutaminase in HUT-460 cells is 3 nM retinoic acid, about to- to 20-fold higher than the ECsos for the inhibition of cholesterol sulfate and transglutaminase Type I in normal tracheobronchial epithelial cells (27, 28). The difference in EC so values might be due to the different mechanisms affecting these biochemical markers or could be related to the presence of relatively high levels of cytosolic retinoic acid binding protein (CRABP) in HUT-460 cells andlow levels of CRABP in tracheobronchial epithelial cells (42). CRABP has been postulated to regulate the concentration of retinoic acid in cells; increasing expression of CRABP would reduce the effective concentration of retinoic acid and cause a shift of the EC so to a higher concentration (46).
Vollberg, George, Nervi et al.: Regulation of Type I and Type II Transglutaminase
The function of transglutaminase Type I has been well established (17). This transglutaminase plays an important role in catalyzing the formation of covalent links between the protein precursors, such as involucrin, that will form the crosslinked envelope in the final stages of squamous differentiation (17). Much less is known about the function of the Type II transglutaminase. It has been suggested that this transglutaminase may be involved in apoptosis (programmed cell death) (47). This process may provide an alternative mechanism to control the size of a tissue that is initiated during hormone-induced atrophy, involution of hyperplasia, or regression of tumors. Other studies have implicated transglutaminase Type II in the regulation of cell proliferation and cell-substratum adhesion. However, in H460 cells, TGF-{31 caused an increase in Type II transglutaminase activity but had no effect on cell growth (Figure 7), indicating that in these cells no direct correlation exists between an increase in this enzyme activity and growth inhibition. A recent study has provided evidence that Type II transglutaminase can be found covalently linked to fibronectin in hepatocytes (48). The investigators in this study suggest that Type II transglutaminase may playa role in modulating cell-cell interactions or in the covalent restructuring of extracellular matrix proteins. If this is a common function of Type II transglutaminase in all tissues, the elevated expression of Type II trans glutaminase in certain lung carcinoma cells could be relevant to the altered architecture seen in tumorous tissue. The upregulation of transglutaminase Type II and fibronectin as well as several other extracellular matrix proteins by TGF-{3 may also be important to the invasive character of each carcinoma. Further studies are necessary to establish the function of this enzyme in normal and tumorigenic tracheobronchial epithelial cells. References 1. Lechner, J. F., G. D. Stoner, A. Haugen etal. 1985. In vitro human bronchial epithelial model systems for carcinogenesis studies. In In Vitro Models for Cancer Research. Vol. 6. M. M. Webber, Editor. CRC Press, Boca Raton, FL. 3-17. 2. Jetten, A. M. 1991. Growth and differentiation factors in tracheobronchial epithelium. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 260:L361-L373. 3. Lechner, J. F., A. Haugen, L. A. McClendon, and A. M. Shamsuddin. 1984. Induction of squamous differentiation of normal human bronchial epithelial cells by small amounts of serum. Differentiation 25:229-237. 4. Adler, K. B., J. E. Schwarz, M. J. Whitcutt, and R. Wu. 1987. A new chamber system for maintaining differentiated guinea pig respiratory epithelial cells between air and liquid phases. Biotechniques 5:462-465. 5. Jetten, A. M., A. R. Brody, M. A. Deas, G. E. R. Hook, J. I. Rearick, and S. M. Thacher. 1987. Retinoic acid and substratum regulate the differentiation of rabbit tracheal epithelial cells into squamous and secretory phenotype. Lab. Invest. 56:654-664. 6. Rearick, J. I., M. Deas, and A. M. Jetten. 1987. Synthesis of mucous glycoproteins by rabbit tracheal epithelial cells in vitro. Modulation by substratum, retinoids and cyclic AMP. Biochem. J. 242: 19-25. 7. Van Scott, M. R., J. R. Yankaskas, and R. C. Boucher. 1986. Culture of airway epithelial cells: research techniques. Exp. Lung Res. 11:75-94. 8. Masui, T., L. M. Wakefield, J. F. Lechner, M. A. LaVeck, M. B. Sporn, and C. C. Harris. 1986. Type (3 transforming growth factor is the primary differentiation inducing serum factor for normal human bronchial epithelial cells. Proc. Natl. Acad. Sci. USA 83:2438-2442. 9. Jetten, A. M., J. E. Shirley, and G. Stoner. 1986. Regulation of prolifer ation and differentiation of respiratory tract epithelial cells by TGF(3. Exp. Cell Res. 167:539-549. 10. Willey, J. C., C. E. Moser, J. F. Lechner, and C. C. Harris. 1984. Differential effects of 12-0-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells. Cancer Res. 44:5124-5126. 11. Sanchez, J. If., C. J. Boreiko, J. Furlong, and T. W. Hesterberg. 1987. Differential effects of tumour promoters upon the growth of normal bron-
17
chial epithelial cells and human lung tumor cell lines. Toxicol. In Vitro 4: 183-188. 12. Jetten, A. M., M. A. George, G. R. Pettit, and J. I. Rearick. 1989. Effects of bryostatins and retinoic acid on phorbol ester- and diacylglycerolinduced squamous differentiation in human tracheobronchial epithelial cells. Cancer Res. 49:3990-3995. 13. Jetten, A. M. 1989. Multistep process of squamous differentiation in tracheobronchial epithelial cells in vitro: analogy with epidermal differentiation. Environ. Health Perspect. 80:149-/60. 14. Jetten, A. M., C. Nervi, and T. M. Vollberg. 1992. Control of squamous differentiation in tracheobronchial and epidermal epithelial cells. Role of retinoids. J. Natl. Cancer Inst. In press. 15. Folk, J. E. 1980. Transglutaminases. Annu. Rev. Biochem. 49:517-531. 16. Lorand, L., and S. M. Conrad. 1984. Transglutaminases. Mol. Cell. Biochern. 58:9-35. 17. Thacher, S. M., and R. H. Rice. 1985. Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell 40:685-695. 18. Birckbichler, P. J., H. F. Upchurch, M. K. Patterson, and E. Conway. 1985. A monoclonal antibody to cellular transglutaminase. Hybridoma 4:179-186. 19. Ikura, K., T. Nasu, H. Yokota, Y. Tsuchiya, R. Sasaki, and H. Chiba. 1988. Amino acid sequence of guinea pig liver transglutaminase from its eDNA sequence. Biochemistry 27:2898-2905. 20. Chiocca, E. A., P. J. A. Davies, andJ. P. Stein. 1988. Regulationoftissue transglutaminase gene expression as a molecular model for retinoid effects on proliferation and differentiation. J. Cell. Biochem. 39:293-304. 21. Moore, W. T., M. P. Murtaugh, and P. J. A. Davies. 1984. Retinoic acid-induced expression of tissue transglutaminase in mouse peritoneal macrophages. J. BioI. Chem. 259:12794-12802. 22. Floyd, E. E. R., and A. M. Jetten. 1989. Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down regulation by retinoids. Mol. Cell. BioI. 9:4846-4851. 23. Phillips, M. A., B. E. Stewart, Q. Qin et al. 1990. Primary structure of keratinocyte transglutaminase. Proc. Natl. Acad. Sci. USA 87:93339337. 24. Grundmann, U., E. Amann, G. Zettlmeissl, and H. A. Kupper. 1986. Characterization of eDNA coding for human factor XIIIa. Proc. Natl. Acad. Sci. USA 83:8024-8028. 25. Ichinose, A., and E. W. Davie. 1988. Characterization of the gene for the a subunit of human factor XIII (plasma transglutaminase), a blood coagulation factor. Proc. Natl. Acad. Sci. USA 85:5829-5833. 26. Lechner, J. F., and M. A. LaVeck. 1985. A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J. Tissue Cult. Methods 9:45-52. 27. Jetten, A. M., and J. E. Shirley. 1986. Characterization of transglutaminase activity in rabbit tracheal epithelial cells. J. BioI. 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