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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 428-435, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0114OC

In Vivo Transglutaminase Type 1 Expression in Normal Lung, Preinvasive Bronchial Lesions, and Lung Cancer

Nadine Martinet, Lionel Bonnard, Véronique Regnault, Evelyne Picard, Louise Burke, Joëlle Siat, Gilles Grosdidier, Yves Martinet and Jean-Michel Vignaud

EA 3443 and Laboratoire d'Hématologie, Faculté de Médecine, Vandoeuvre-Les-Nancy, Nancy, France; Department of Histopathology, Cork University Hospital, Cork, Ireland; and Service de Chirurgie Thoracique and Laboratoire d'Anatomie Pathologique, Hôpital Central, CHU de Nancy, Nancy, France

Address correspondence to: J. M. Vignaud, Laboratoire d'Anatomie Pathologique, CHU de Nancy, Av. Maréchal de Lattre de Tassigny, 54035 Nancy Cedex, France. E-mail: jm.vignaud{at}chu-nancy.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transglutaminase type 1 (TGase 1) is a member of a class of enzymes that catalyze the cross-linking of proteins, a characteristic feature of epidermal differentiation and squamous metaplasia. The role of TGase 1 has been extensively studied in epidermis but not in the lung. Using a polyclonal anti–TGase 1 antibody prepared in our laboratory (TGase-lac), TGase 1 expression in normal bronchial epithelium, bronchial preinvasive lesions, and lung cancer was characterized. The specificity of the antibody was confirmed by the presence in squamous differentiated bronchial cells of specific 106-kD and 92-kD bands by Western blotting. In addition, immunohistochemistry displayed a recognized pattern of labeling in both normal and tumor cells beneath the cytoplasmic membrane and within the cytosol. TGase 1 was shown to be expressed by cells from bronchial epithelium and bronchial preinvasive lesions, strongly expressed in most non–small-cell lung cancer tumor cells and in apoptotic bodies, but weakly expressed in small-cell lung cancer. The distribution of TGase 1 mRNA correlated with the immunohistochemical profile. These observations suggest that TGase 1 expression is a normal feature of bronchial epithelium and is linked to the process of squamous differentiation occurring in preinvasive lesions. Its role in lung cancer remains to be clarified.

Abbreviations: adenocarcinoma, ADC • cell envelope, CE • human bronchial epithelial cells, HBE • immunohistochemistry, IHC • in situ hybridization, ISH • Lamellar Ichthyosis, LI • non–small-cell lung cancer, NSCLC • phosphate-buffered saline, PBS • squamous cell carcinoma, SCC • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE • transglutaminase 1, TGase 1 • polyclonal anti–TGase 1 antibody, TGase-1ac • soluble tissue type TGase 2, tTGase • human TGase 1 gene, TGM1 • biotin-tyramide conjugate, TSA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In vivo squamous metaplasia of bronchial mucosa is induced by carcinogens contained in tobacco smoke, as well as by severe vitamin A deficiency, and is considered to be the initial step in the multistep carcinogenic process that leads to the development of squamous cell carcinoma (1). The squamous phenotype of cells from bronchial preinvasive lesions is similar to the phenotype of differentiated keratinocytes, as illustrated by the sequential induction of the genes involved in the formation of the cornified cell envelope (CE) (2). This layer of enucleated shells is the characteristic feature of epidermal differentiation, and it contains a compact network of proteins whose structure is strongly influenced by transglutaminase (TGase). TGase is a key enzyme that catalyzes the cross-linking between the CE-precursors. Initially, it was thought to be expressed only in the skin epidermis; however, Northern as well as Western blotting analyses have indicated that TGase 1 is also expressed in tissues containing simple epithelia, such as liver and kidney. Some investigators have reported the expression of TGase 1 in cultured human bronchial epithelial cells (HBE) and lung cancer lines (3, 4), but no in vivo study has yet been conducted to our knowledge on the normal human respiratory epithelium, bronchial preinvasive lesions, or lung cancer, despite the striking similarity between some of the former's lesions and terminally differentiated epidermis. Therefore, we produced a new specific polyclonal antibody for TGase 1 so as to characterize the expression of the enzyme and corresponding mRNA in normal lung, preinvasive bronchial lesions, and lung tumor specimens.

TGase 1 is a member of a class of Ca2+-dependent enzymes that catalyze the cross-linking of proteins by acyl transfer (5). In this reaction, {gamma}-carboxamide groups of peptide-bound glutamine residues and primary amines of peptide-bound lysine serve as acyl donor and acceptor substrates, respectively, to form -({gamma}-glutamyl)lysine bonds, which are covalent, stable, and resistant to proteolysis. The resulting stable cell structures are composed of polymerized substrate proteins that participate in the maintenance of skin tissue integrity and impermeability. To date, at least seven distinct types of TGases have been identified, including five intracellular forms (TGases 1–5 and band 4.2 protein) and one extracellular form (plasma coagulation factor XIIIa) (5, 6). Four are expressed during terminal differentiation of stratified squamous epithelia: the so-called "keratinocyte-type" TGase 1, the ubiquitous soluble tissue type TGase 2 (tTGase) of ~ 80 -D, the soluble proenzyme TGase 3 of ~ 67 kD, and the recently discovered TGase 5 (5). TGase 1 is expressed predominantly in the upper spinous and granular layer of the epidermis, and is required for the formation of the cornified cell envelope (7) and the hair shaft (8). TGase 1 exists in keratinocytes as multiple soluble forms which can be proteolytically processed at conserved sites (9) and which have varied specific activities. The 106-kD TGase 1 protein corresponds to the soluble native enzyme, whereas the 92-kD protein corresponds to the membrane-anchored form. The major functionally active form of TGase 1 results from the proteolytic processing of the native enzyme (10), and consists of, at least in keratinocytes, a membrane-bound 67/33/10-kD complex with a myristoylated and palmitoylated amino-terminal 10 kD membrane anchorage fragment. In addition to the cross-linking of the CE-precursor proteins, such as involucrin and loricrin (11), TGase 1 also participates in the formation of the lipid envelope on the surface of the epidermal keratinocytes with the establishment of ester bonds between involucrin and omega-hydroxyceramides. In liver and kidney epithelia, immunofluorescence and immunoelectron microscopy revealed that endogenous TGase 1 colocalized mostly with -E cadherin and was concentrated, although not exclusively, at adherens-junctions, suggesting that TGase 1 is also directly involved in the formation and maintenance of intercellular junctions in simple epithelial cells (12). The human TGase 1 gene (TGM1) is located on chromosome 14q11.2. Several mutations of TGM1 gene have been identified in individuals with Lamellar Ichthyosis (LI), and studies of this disorder have confirmed the importance of the gene and proteins (13). LI is an autosomal recessive skin disorder which presents with a characteristic skin scaling. The point mutations, deletions, and truncations found in LI affect the structure and function of TGase 1 (14). Gene function restoration in primary keratinocytes from patients with LI has the effect of normalizing the epidermal architecture and expression of the differentiation marker filaggrin, thus confirming a major role for TGase 1 during normal epidermal differentiation (15). The transcriptional regulation of TGase 1 is poorly understood. Regulation by signaling systems such as protein kinase C, retinoic acid and X receptors, and Jun/Fos has been suggested in keratinocytes (16, 17). Recently, DNA elements involved in its transcriptional control were identified in tracheobronchial epithelial cells (18), but enzyme substrates and cellular function have not yet been fully defined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of TGase 1 Polyclonal Antibody
Polyclonal purified antibodies were prepared against the synthetic peptide (Y-K-H-P-E-G-S-D-A-E-R-K-A-V-E) which was chosen for its potential specific TGase 1 antigenicity by Neosystem SA (Strasbourg, France). The antibodies were purified by affinity chromatography with a Hitrap protein A column (Pharmacia, Les Ulis, France), aliquoted, and stored at -20°C until the validation experiments.

To validate the specificity of the TGase 1 antibodies, TGase affinity chromatography was performed with extracts of human epidermal callus collected from the soles of staff members. For this purpose, a TGase affinity column was prepared grafting the TGase 1 antibodies onto a cyanogen activated sepharose 4B resin according to our previously published protocol (19). Epidermal callus was homogenized in 0.5 M Tris acetate buffer (pH 6.0), 1 mM EDTA, 2 mM benzamidine, and 10 mg/ml aprotinin. The supernatant was collected by centrifugation for 30 min at 17,600 x g and was loaded on the TGase 1 affinity column. The absorbent was washed with phosphate-buffered saline (PBS), pH 7.0. Elution was conducted in 0.3 M glycine (pH 2.8). Collected samples were dialyzed in PBS. One hundred microliters of each dialyzed fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Fractions demonstrating antigenicity were separated by preparative SDS-PAGE and transferred onto PVP membrane for sequencing on a microsequencing system 476 A from Applied Biosystems (Lincoln Centre Drive, Foster City, CA) equipped with an on-line reverse-phase HPLC, in accordance with the manufacturer's instructions. Among anti–TGase 1 antibodies prepared, TGase-1ac antibody was retained and utilized for the present study due to its specificity and high affinity.

Extracts and Immunoblotting
Human bronchial tissue was obtained from donors at the time of autopsy. Bronchial epithelium was separated and explanted in LH9 medium. HBE cells at confluence were switched to LH9 medium containing 2 mM CaCl2, without retinoic acid, for squamous differentiation. Normal epidermal layers were microdissected from breast cutaneous tissue obtained after surgery and stored at -70°C. Extracts (5 µg) were prepared from HBE cells and skin epidermis as previously described (20, 21), fractionated by SDS-PAGE (8% polyacrylamide gel containing 1% SDS), and electrotransferred onto nitrocellulose membranes. After blocking in PBS containing 3% nonfat powdered milk, the membranes were immunoprobed with the specific rabbit polyclonal antibody TGase-1ac (diluted 1:1,000) for 2 h at 37°C, extensively washed in PBS containing 0.05% Tween 20, and then incubated for 30 min at room temperature with peroxidase-conjugated Protein A dilued 1:10,000 (Amersham, Saclay, France). Specific complexes were revealed by chemiluminescence detection according to the manufacturer's protocol (Pierce, Rockford, IL)

Surgical Specimens
Patients with non–small-cell lung cancer (NSCLC) (n = 25; squamous cell carcinoma [SCC], n = 13; and adenocarcinoma [ADC], n = 12; 21 males and 4 females; mean age, 61 ± 9 yr) and small-cell lung cancer (SCLC) (n = 13) not subjected to preoperative radiotherapy and/or chemotherapy were consecutively enrolled in this study after informed consent. All of these patients had a prior history of smoking. Lung cancer histologic subtypes were defined according to the 1999 WHO criteria. Disease staging was defined after surgery for NSCLC, according to the recommendations for NSCLC staging given at the Fifth World Conference of Lung Cancer. There were 8 stage I NSCLC tumors, 6 stage II tumors, 10 stage III tumors, and 1 stage IV tumor. Concerning SCLC there were 6 limited stage diseases and 7 extensive stage diseases.

Preneoplastic lesions were obtained, when present, in the free resection margin from the main bronchus of the resected lung. The lesions procured consisted of: 2 carcinomas in situ, 3 squamous metaplasia with dysplasia, and 18 foci of squamous metaplasia without dysplasia.

Normal control lung specimens were obtained from a lobe free of any pathologic evidence of malignancy, and normal human epidermis from breast tissue was also used.

Immunohistochemical Procedures
Two techniques of signal amplification were used, so as to appreciate the relative level of expression of TGase 1 enzyme: a standard biotin–streptavidin procedure and the ultrasensitive peroxidase-mediated deposition of a biotin-tyramide conjugate (TSA biotin system; NEN Life Science, Boston, MA) as previously described (20). Briefly, immunohistochemistry (IHC) was performed on 5-µm paraffin sections that were dewaxed, and antigen retrieval was performed in a pressure cooker in citrate buffer (0.1 M, pH 6.0) for 10 min. Sections were incubated overnight at 4°C with the specific polyclonal or monoclonal antibodies. The TGase-1ac antibody was used at a dilution of either 1:400 (standard biotin–streptavidin procedure) or 1:15,000 to 1:40,000 (TSA); the monoclonal anti-active caspase 3 antibody (R&D Systems, Abingdon, UK) was diluted at 1:400, and the polyclonal anti–von Willebrand factor VIII (Dako, Globstrup Denmark) was diluted at 1:80. Anti-caspase and anti–von Willebrand antibodies were used to identify respectively apoptotic cells and endothelial cells. After washing in TBS-Tween (Tris-HCl 0.05 M, pH 7; NaCl 150 mM; 0.1% Tween) the bound antibodies were detected using biotinylated goat anti-rabbit or anti-mouse antibodies (Dako). The sections were then successively incubated either only with streptavidin–peroxidase complex (Dako), for the standard biotin–streptavidin procedure or with the biotin-tyramide substrate solution for 10 min, followed by a last incubation in streptavidin–peroxidase complex, for the TSA biotin system. The sections were finally incubated in a substrate solution of 0.6 mg/ml 3,3'-diaminobenzidine in 0.05 M Tris-HCl buffer containing 0.01% H2O2.

In a preliminary study, normal lung and cutaneous tissues were processed with TGase-1ac antibody, using in parallel the standard biotin–streptavidin and TSA–biotin amplification methods. Both techniques stained the epithelia, but TSA–biotin amplification procedure showed a clear cut increased sensitivity compared with the standard biotin–streptavidin method. Thus it was used for an accurate semiquantitative evaluation of TGase 1 expression in normal and pathologic lung tissue samples. To appreciate the relative level of expression of the enzyme in carcinoma cells compared with normal surrounding bronchial or lung tissue cells, we used the TGase-1ac antibody at two optimized dilutions: 1:15,000 and 1:40,000. The former dilution was found to be the most sensitive for the detection of the protein and the identification of all tumors expressing TGase 1, whereas the latter dilution allowed us to discriminate between tumors with high or low expression of the enzyme. The surrounding normal bronchial and bronchiolar cells expressing the TGase 1 at constant levels in all samples studied were used as an internal standard, and the cellular staining of bronchial epithelial cells was scored as ++. Therefore in cells under analysis no expression was scored as 0, a decreased expression was scored as +, and an overexpression was as scored +++.

Controls used to test the specificity of the labeling protocols for each antibody involved, omission of the incubation step with the primary antibody; substitution of a non-immune serum in place of the primary antibody; and omission of the incubation step with both primary and secondary antibodies. Furthermore the specificity of the TGase-1ac antibody was controlled in all tissue samples analyzed. This was assessed as follows: before the IHC procedure, the TGase-1ac antibody was preincubated with an excess of 10 times of the synthetic peptide that was used for raising the antibody. Under these conditions, no labeling was observed in all tissue samples analyzed.

In Situ Hybridization Procedures
Total RNA from squamous differentiated HBE cells was isolated by the TRIZOL reagent (Sigma, L'isle d'Abeau, France) and reversely transcribed with the use of First Strand cDNA Kit (Amersham) and the following primers: 5'-GCAGTAGAGACAGCAGCAGCCCA and 5'-CTGTACTTCACACTCCTGGCCAA. A DNA probe was generated by PCR, employing high-fidelity Taq polymerase and the same primers. The 465-bp PCR products were subcloned in the Topo II PCR cloning vector (Invitrogen, Groningen, Netherlands). The resulting construct was verified by DNA sequencing at the cloning sites. The human TGase 1 riboprobe was prepared, as previously detailed (22), according to Melton's transcription protocol using 35S-labeled nucleotides (Amersham). Probe length was reduced to an average of 150 nucleotides by limited alkaline hydrolysis. Sections (5 µm thick) from tissue samples fixed in 4% paraformaldehyde were deparaffinized and acetylated in anhydrous acetic acid (0.5% in 0.1 M triethanolamine, pH 8.0) for 10 min. The 35S-labeled sense or antisense probes diluted to 60,000 cpm/µl were applied to each section in 20 µl of the hybridization buffer (50% deionized formamide; 2x SSC [300 mM NaCl, 30 mM Na citrate, pH 7.0], 100 mM dithiotreitol; 1 mg/ml yeast tRNA; 1 mg/ml sonicated salmon sperm DNA, 2 mg/ml BSA). Hybridization was performed overnight at 60°C. Then, the sections were rinsed with formamide (50%, 2x SSC) at 60°C for 1 h, and digested with RNase (RNase A, 10 mg/ml; RNase T1, 500 U/ml; in 2x SSC) for 30 min at 37°C . The sections were washed again in formamide for 1 h at 50°C, with a final wash in 1x SSC for 30 min at room temperature. Hybridized slides were autoradiographed with NTB2 emulsion (Kodak, Rochester, NY) and exposed at 4°C. Triplicate sections from each specimen were developed at weekly intervals over a period of 3 wk with D19 Kodak.

Control experiments included pretreatment of sections with RNase for 1 h at 37°C (RNase A, 10 mg/ml; RNase T1, 500 U/ml; in 2x SSC) and hybridization with sense probes.

Statistics
Statistical analysis was performed using Student's t test and P < 0.01 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The specificity of the polyclonal TGase-1ac antibody was extensively confirmed before screening normal and pathologic lung tissue samples for TGase 1 presence. In addition, obtained results were confirmed by in situ hybridization (ISH) analysis of corresponding mRNA.

Specificity and properties of TGase-1ac antibody
The specificity of the antibody was assessed as follows:

By the Western blotting method (Figure 1) the polyclonal antibody recognizes, in protein extracts from both human callus and squamous differentiated HBE cells, two major bands of ~ 92 and 106 kD. The 92-kD band is considered to be the membrane-associated TGase 1 (7, 9) whereas the 106-kD band is reflective of the soluble and true full-length size of the TGase 1 enzyme in keratinocytes (9). The 15% increase in size may by due to postsynthetic modifications of a basic core protein of 92 kD (9). In addition to the major 106- and 92-kD bands, the antibody recognizes inconstantly, in callus, a minor band of lower molecular mass (67 kD), which has also been reported earlier (9, 23), and possibly results from the proteolytic processing of the TGase 1 enzyme. HBE proliferating cells express the mRNA for TGase 1, but the encoded protein is difficult to detect by Western blotting. However, it becomes detectable in HBE cells switched to 2 mM CaCl2 for squamous-like differentiation.



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  		Figure 1. Immunoblotting detection of TGase 1 in normal human epidermis and normal human bronchial cells (HBE). Cultured HBE cells were switched to 2 mM CaCl2 for squamous differentiation. Whole cell extracts (5 µg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose filters, and immunoprobed with a rabbit polyclonal antibody raised against TGase 1, followed by peroxidase-conjugated Protein A and chemiluminescence detection. The polyclonal antibody TGase-1ac recognizes two major bands of ~ 92 and 106 kD, in both cellular extracts from epidermis (lanes 3 and 4) and HBE cells (lanes 5 and 6), corresponding respectively to the membrane-associated and cytosolic forms of TGase-1. Lane 1: molecular weight standard; lane 2: corresponding Coomassie blue staining of whole cell extract from epidermis.

 
Purification of TGase 1 from human callus by immunoaffinity with the TGase-1ac was confirmed by amino acid sequencing, for which the following amino acid sequence was obtained: XMDGPRSDVGRWGG.

Finally, immunohistochemical analysis of lung and skin tissue samples showed no labeling when performed after a preincubation of the antibody with an excess of 10 times of the synthetic peptide used to prepare the antibody.

TGase 1 Expression in Epidermis
Using the standard biotin–streptavidin IHC technique, TGase-1ac stained the suprabasal layers of the epidermis, with frequent accentuation in the granular layer (Figure 2a). However, the TSA technique clearly showed that TGase 1 was expressed throughout the human epidermis, including the basal cell layer. TGase 1 was also expressed in hair follicles, sebaceous glands, and sweat glands. Furthermore, endothelial cells from the dermal microvasculature, positively stained for von Willebrand factor, were also decorated with the TGase-1ac antibody. Within the keratinocytes the antigen was located beneath the cytoplasmic membrane and within the cytosolic compartment (Figure 2b,c). ISH confirmed that TGase 1 mRNA distribution paralleled the protein presence in epithelial cells. As demonstrated immunohistochemically, the signal was observed throughout the epidermis (Figures 2d and 2e) and within endothelial cells.



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  		Figure 2. Immunohistochemical and in situ hybridization (ISH) analysis of TGase 1 expression in human normal epidermis (a–e) and bronchial epithelium (f–i). TGase 1 is expressed throughout the human epidermis (a). The smear of keratinocytes from the upper layers of the epidermis clearly shows that part of the protein is associated to the cytoplasmic membrane (b). ISH shows a parallel distribution of TGase 1 mRNA (d); (c and e) corresponding respective controls. Membranous and cytosolic staining for TGase 1 of each cell type in normal bronchial epithelium (f); mRNA distribution correlates with the distribution of the protein (h); (g and i): corresponding respective controls.

 
TGase 1 Expression in Normal Respiratory Epithelium
All cell types in normal bronchial epithelium, except for basal cells, stained with the TGase-1ac antibody. Staining distribution was mostly observed beneath the cytoplasmic membrane; however, cytosolic compartment staining was also noted (Figures 2f and 2g). With the standard biotin–streptavidin IHC procedure, the staining intensity was weaker than that observed in the epidermis, but was clearly positive and clear-cut when the TSA technique was used. Normal pneumocytes were negative using the standard biotin–streptavidin IHC procedure, with only a low level signal detected using TSA, suggesting a low level of expression of TGase 1 in pneumocytes. Interestingly, however, an intense labeling was observed in reactive hyperplastic pneumocytes surrounding lung carcinomas. Endothelial cells of some vessels within the submucosa were stained, as were pleural mesothelial cells. ISH confirmed that the distribution of TGase 1 mRNA correlated with the distribution of the protein by IHC techniques (Figures 2h and 2i).

TGase 1 Expression in Lung Cancer
The level of TGase 1 expression in lung cancer samples was evaluated using the semiquantitative TSA–biotin immunohistochemical amplification procedure, as described in MATERIALS AND METHODS. Using low dilution of TGase 1ac antibody showed that all the 25 NSCLC tumor samples analyzed expressed TGase 1, but only 10 out of 13 SCLC. Using this antibody at high dilution allowed discrimination between NSCLC with high or low expression of the enzyme (Table 1): 69% of SCC (Figure 3a) and 66% of ADC (Figure 3b) demonstrated a strong staining (+++) pattern in tumor cells. The signal was more intense in tumor cells than in the normal surrounding lung parenchyma, and indeed this difference in TGase 1 expression between normal cells and carcinoma cells was statistically significant (P < 0.0001). Staining was observed predominantly beneath the cytoplasmic membrane, but scattered cells, including squamous perl cells, also showed a strong cytosolic labeling. The other NSCLC analyzed (31% of SCC and 34% of ADC) showed either a normal (++) or low level (+) of TGase 1 expression (Figure 3d). None of the NSCLC analyzed showed an abscence of expression of TGase 1. In addition, all apoptotic bodies stained with the anti-active caspase 3 antibody were also intensely stained with the TGase-1ac antibody (Figure 3d). By contrast, labeling of tumor cells in ischemic foci was not observed. Endothelial cells from the tumor stromal microvasculature were stained with TGase-1ac antibody, but this labeling was observed in limited areas of the tumor only (Figure 3b), without an obvious specific topography. Finally, focal weak staining of stromal fibroblasts was observed without labeling in the extracellular matrix. A correlation between the expression of TGase 1 and the classification of the tumors as SCC or ADC was not observed. This contrasts with the statistical significance observed on comparison of NSCLC and SCLC (Table 1) whereby strong staining of the majority of NSCLC tumor was observed as compared with an almost constant low level or absence of expression of TGase 1 (0/+) (92%) of SCLC (P < 0.0001) (Figure 3c). One SCLC sample showed a normal level of enzyme expression. ISH showed that TGase 1 mRNA distribution paralleled the protein immunohistochemical presence in tumor cells (Figures 3e and 3f). No substantial relationship could be found between the level of TGase 1 expression and either NSCLC or SCLC disease staging, and smoking background (data not shown).


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  		TABLE 1 Semiquantitative immunohistochemical evaluation of TGase 1 expression in lung cancer

 


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  		Figure 3. Immunohistochemical and in situ hybridization analysis of TGase 1 expression in lung cancer (a–f) and bronchial epithelial lung cancer precursor lesions (g–i). Strong expression of TGase 1 in tumor cells from squamous cell carcinoma (SCC) (a) and adenocarcinoma (ADC) (b) as well as in endothelial cells (b, arrowhead); no expression of TGase 1 in a small-cell lung cancer tumor sample versus normal surrounding bronchial epithelium (c); TGase 1 expression in apoptotic bodies within a SCC sample otherwise weakly expressing TGase 1 (d, arrowhead). TGase 1 mRNA in SCC (e) and ADC (f). Squamous metaplastic (g) and dysplastic (h) foci express TGase 1 throughout; (i) corresponding TGase 1 mRNA expression in the former sample.

 
TGase 1 Expression in Bronchial Preinvasive Lesions
Preinvasive lesions (squamous metaplasia, dysplasia, and carcinoma in situ) were stained throughout with the antibody (Figures 3g and 3h). The intensity of the signal was not significantly different from the one observed in the surrounding normal bronchial epithelium. No significant differences in the staining were observed between low- and high-grade lesions. ISH showed a homogeneous distribution of the corresponding mRNA throughout the lesions (Figure 3i). Neither the TGase 1 antibody nor the anti–caspase 3 antibody detected apoptotic bodies in the preinvasive lesions. Focal staining of endothelial cells was also observed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to revive research about TGase 1 in bronchial epithelium and especially to evaluate its potential role in the development of bronchial preinvasive lesions, which so much resemble the normal epidermis and precancerous cutaneous lesions. For this purpose, a new antibody was produced and characterized because we observed that commercially available antibodies were unspecific, whereas the number of different TGases was increasing. Before this study, TGase 1 expression has been reported mainly in keratinocytes, and its cross-linking activity is thought to play a key role in the formation of the CE (2). In this study, IHC, ISH, and Western blotting analyses, however, have showed that in vivo, TGase 1 is also expressed in the normal respiratory epithelium, as well as in bronchial preinvasive lesions and lung carcinomas.

The observation that our prepared TGase-1ac labeled both the cytosolic compartment and the cytoplasmic membrane in keratinocytes is in agreement with the known subcellular distribution of the two major forms of the protein as determined by Western blotting analysis. The 106-kD band corresponds to the soluble/cytosolic form of the protein and the 92-kD band to the membrane-anchored form. The 67-kD minor band is a membrane-bound functionally active fragment resulting from the proteolytic processing of the native form of the enzyme. The immunohistochemical labeling of the entire epidermis with accentuation in the granular layer is also in agreement with the labeling observed in a previous study using another polyclonal specific TGase 1 antibody (24) and with the TGase 1 mRNA distribution observed here by ISH. These results differ, however, from the staining restricted to the granular layer observed when a commercially available TGase 1 monoclonal antibody (B-C1) is used (25). In our hands and using our antibody, the cytosolic compartment and the cytoplasmic membrane of bronchial cells were labeled, similarly to previous studies on keratinocytes. In addition, Western blotting analysis of squamous differentiated HBE cultured cells extracts revealed the 106- and 92-kD bands as expected. This strongly suggests that in bronchial epithelium, TGase 1 is present as both a soluble and a membrane-anchored form. The protein substrates of TGase 1 in normal respiratory epithelium are unknown, and as yet the exact cellular function of the protein in these cells is also unknown. The subcellular distribution of the enzyme, identical to that observed in keratinocytes, could suggest a role in the cohesiveness of this epithelium through cell-to-cell attachment, in line with previous studies in human epidermis and mouse liver that showed how TGase 1 was in part concentrated at the intercellular junctions (15). The recently developed TGase 1–null mice (26) could perhaps provide the answer to TGase 1 cellular function. These mice have erythrodermic skin with abnormal keratinization and an impaired skin function, and die within a few hours after birth. This demonstrates that TGase 1 is essential for the development of the epidermis and that TGases 2 or 3 are not compensating for the phenotype of the TGase 1–null mice. It was speculated that these mice died of dehydration due to their skin barrier dysfunction, but as the expression of TGase1 activity in other tissues had not been analyzed one cannot absolutely say that this was the sole reason. Indeed, in line with this it is interesting to note that TGase 1–deficient mice are smaller than controls, and preliminary studies identified histologic changes in the lung (15). It is obvious, therefore, that these animals will be a very useful model to precisely identify the role of TGase 1 in normal differentiation and function of the lung.

The role of TGase 1 in cancer biology also has been poorly investigated. In lung cancer, the most striking feature that we observed was the frequent overexpression of TGase 1 by tumor cells from NSCLC. This is in agreement with two previous reports that reported the high incidence of expression of the enzyme in NSCLC cell lines (3, 4). We suggest that in line with previous studies postulating the function of TGase 1, the high expression in NSCLC of TGase 1 could favor stable cell-to-cell attachment of tumor cells. The strong expression and preferential distribution of the enzyme beneath the plasma membrane in tumor cells would support this hypothesis. In addition, TGase 1 probably participates in the differentiation process of squamous cell carcinomas, due to the presence of TGase 1, as demonstrated by the immunohistochemical staining, in tumor keratinized cells. In contrast, SCLC are characterized by a poor cohesiveness of tumor cells with an early metastatic potential as distinct to NSCLC, and interestingly and perhaps not surprinsingly show a very low level of expression of TGase 1. Also as TGase 1 expression is linked to the differentiation process, it is not surprising that it is only weakly or unexpressed within the undifferentiated actively replicating cells of SCLC. This observation is in line with the fact that proliferating undifferentiated cell lines has been shown not to express TGase 1 (27). This observation suggests that absent or low-level TGase 1 activity favors tumor progression as proposed for tTGase. An inverse relationship between tTGase activity and metastatic potential has been reported by several investigators in tumor cells of murine origin (28). Further studies comparing TGase 1 expression in metastatic and nonmetastatic lung cancer would help to evaluate this hypothesis further. We also observed that apoptotic bodies within tumor sheets were strongly stained both with the anti-active caspase 3 antibody, an effector of the apoptotic process, and TGase-1ac antibody, whereas in terminally differentiated keratinocytes only TGase 1 is expressed. The covalent cross-linking of cellular proteins by TGases is one of the effector elements of various cell death pathways, and there is accumulating data relating the induction of tTGase in the molecular program of cell death (29); however, the role of TGase 1 has been poorly investigated in this process. The accumulation of the enzyme in apoptotic bodies suggests that TGase 1 participates, in conjunction with tTGase, to the apoptotic process and thus possibly to the control of lung cancer progression. The role of TGase 1 extensive protein cross-linking may be directly related to the act of killing and/or devoted to prevent leakage of macromolecules and enhancing phagocytosis of the dead cells. It is noteworthy that we did not observe a labeling of tumor cells in ischemic foci, suggesting that if TGase 1 is one the effector elements of apoptosis, it is not induced or activated in the cells following ischemia. Thus TGase 1 participates in different cell death mechanisms: the cell death of epithelial cells from stratified epithelia such as the epidermis and from keratinized cells of SCC, a process mostly relevant to differentiation programs, and apoptotic processes. Thus TGase-1ac could be included in the panel of antibodies useful to characterize the cell death signaling pathways.

TGase 1 is also expressed in endothelial cells from the stromal vessels of lung cancer. The expression of tTGase by endothelial cells has also been previously reported in vessels from breast cancer and glioblastoma cases (30, 31), but the role of TGases in endothelial cell biology and possibly in the angiogenesis process is unknown.

TGase 1 was also expressed in bronchial preinvasive lesions. The lesions are characterized by the transformation of the ciliated pseudostratified epithelium into the squamous phenotype; and when additional dysplastic changes occur they are definitively considered as high-risk precancerous lesions. The induction of the squamous metaplasia phenotype is marked by the sequential induction of many squamous-specific genes such as specific keratins (5 and 6) and TGase 1. When the process is fully completed, an insoluble layer of cross-linked protein is deposited just beneath the plasma membrane. TGase 1 appears to be the major enzyme catalyzing the cross-linking of proteins in human epidermis and very probably does so in the preinvasive bronchial lesions. This is suggested by the constant expression of TGase 1 mRNA and protein in all of the bronchial preinvasive lesions analyzed here, together with the demonstration that squamous differentiated HBE cells also expressed TGase 1 (27).

Although TGase 1 was identified 15 yr ago as one of the genes differentially expressed in squamous differentiating tracheobronchial cells, there is still a paucity of data pertaining to signal transduction pathways controlling TGase 1 expression (18, 32). The enzyme was identified from the junctional fraction of the mouse liver as a tyrosine-phosphorylated protein (15), suggesting that TGase1 activity is regulated through tyrosine phosphorylation. However, the exert effect of tyrosine phosphorylation on the enzymatic activity of TGase1 remains to be elucidated. Interferon-ß and retinoids are two demonstrated effectors of TGase 1 expression. Interferon-ß stimulates squamous differentiation and programmed cell death in human lung cancer cell lines NCI-H 596 and NCI-H 226, and causes an increase in TGase 1 activity and DNA fragmentation (33). In contrast, retinoids prevent TGase 1 expression in rabbit tracheal epithelial cells, as well as downregulate preexisting TGase 1 mRNA expression in squamous differentiated rabbit bronchial epithelial cells (34).

In conclusion, this study indicates that in vivo TGase 1 is synthesized by cells from normal bronchial epithelia, bronchial preinvasive lesions, strongly expressed in most NSCLC tumors and in apoptotic cells, but weakly expressed in SCLC. Although the role of TGase 1 is still questionable in the normal respiratory epithelium and the lung carcinogenic process, our observation suggests that TGase 1 expression is linked to the differentiation program of normal lung and lung tumors and plays a role in different cell death pathways. Thus TGase 1 may participate in the control of lung cancer progression in a variety of methods. Further studies looking for the characterization of the transduction pathways controlling TGase 1 expression, and the identification of enzyme substrates in respiratory epithelium, will determine whether the TGase 1 enzyme is indispensable to maintain the integrity of the respiratory epithelium and will provide insight into the control of squamous metaplasia and the development of lung carcinoma.


   Acknowledgments
 
This work was supported by grants from the Ligue Nationale contre le Cancer de Meuse, Moselle, Vosges et Meurthe et Moselle and the Wittner Fondation. E.P. was a fellow from "Region Lorraine."

Received in original form July 12, 2002

Received in final form October 21, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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