Portal de Periódicos da CAPES

Expression of Transglutaminase 5 in Normal and Pathologic Human Epidermis

2002; Elsevier BV; Volume: 119; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2002.01853.x

1523-1747

Eleonora Candi, Sergio Oddi, Andrea Paradisi, Alessandro Terrinoni, Marco Ranalli, Patrizia Teofoli, Gennaro Citro, Silvia Scarpato, Pietro Puddu, Gerry Melino,

Blood properties and coagulation

To explore the expression and gain more information on the function of transglutaminase 5 enzyme in normal and defective human epidermis, we generated a rat antihuman transglutaminase 5 antiserum elicited against a purified active recombinant protein expressed in the baculovirus system. By use of Western blotting and immunofluorescence methods, the immunospecificity of the antibodies for transglutaminase 5 was tested; no crossreactivity with other transglutaminases (types 1, 2, and 3) was observed, thus allowing histochemistry studies. By indirect immunofluorescence analysis the antibodies decorated the upper layers of normal human epidermis, with consistent staining in the spinous and granular layers. We evaluated transglutaminase 5 expression in comparison with proliferating (keratin 14) and differentiating (transglutaminase 3) markers in different diseases, such as psoriasis, ichthyosis vulgaris, lamellar ichthyosis, and Darier's disease. We observed that transglutaminase 5 contributes, as a secondary effect, to the hyperkeratotic phenotype in ichthyosis (both vulgaris and lamellar) and in psoriasis. In Darier's disease, transglutaminase 5 expression, as well as transglutaminase 3, is completely missregulated, being overexpressed or totally absent in different areas of the same lesion. To explore the expression and gain more information on the function of transglutaminase 5 enzyme in normal and defective human epidermis, we generated a rat antihuman transglutaminase 5 antiserum elicited against a purified active recombinant protein expressed in the baculovirus system. By use of Western blotting and immunofluorescence methods, the immunospecificity of the antibodies for transglutaminase 5 was tested; no crossreactivity with other transglutaminases (types 1, 2, and 3) was observed, thus allowing histochemistry studies. By indirect immunofluorescence analysis the antibodies decorated the upper layers of normal human epidermis, with consistent staining in the spinous and granular layers. We evaluated transglutaminase 5 expression in comparison with proliferating (keratin 14) and differentiating (transglutaminase 3) markers in different diseases, such as psoriasis, ichthyosis vulgaris, lamellar ichthyosis, and Darier's disease. We observed that transglutaminase 5 contributes, as a secondary effect, to the hyperkeratotic phenotype in ichthyosis (both vulgaris and lamellar) and in psoriasis. In Darier's disease, transglutaminase 5 expression, as well as transglutaminase 3, is completely missregulated, being overexpressed or totally absent in different areas of the same lesion. Terminal differentiation of stratified squamous epithelia, including human epidermis, ends with the formation of a specialized protein structure deposited just beneath the inner surface of the cell plasma membrane, called the cornified cell envelope (CE). CE components are several epidermal specific structural proteins [involucrin, cystatin A, loricrin, several small proline-rich proteins (SPRs), trichohyalin, keratins;Rice and Green, 1979Rice R.H. Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.Cell. 1979; 11: 681-694Abstract Full Text PDF Scopus (647) Google Scholar;Kartasova et al., 1987Kartasova T. Cornelissen B.J. Belt P. van de Putte P. Effects of UV, 4-NQO and TPA on gene expression in cultured human epidermal keratinocytes.Nucleic Acids Res. 1987; 15: 5945-5962Crossref PubMed Scopus (82) Google Scholar;Mehrel et al., 1990Mehrel T. Hohl D. Rothnagel J.A. et al.Identification of a major keratinocyte cell envelope protein, loricrin.Cell. 1990; 61: 1103-1112Abstract Full Text PDF PubMed Scopus (383) Google Scholar; Takahashi et al., 1992Takahashi M. Tezuka T. Katunuma N. Phosphorylated cystatin alpha is a natural substrate of epidermal transglutaminase for formation of skin cornified envelope.FEBS Lett. 1992; 308: 79-82Abstract Full Text PDF PubMed Scopus (71) Google Scholar assembled by the catalytic action of transglutaminase (TGase) enzymes. TGases (EC 2.3.2.13) are Ca2+-dependent crosslinking enzymes that catalyze an acyl-transfer reaction between the γ-carboxamide group of protein-bound glutamine and various primary amines, most commonly the ε-amino group of lysine residues, thus forming isopeptide bonds between proteins that generate insoluble macromolecular assemblies (Folk and Finlayson, 1977Folk J.E. Finlayson J.S. The epsilon-(gamma-glutamyl) lysine crosslink and the catalytic role of transglutaminases.Adv Protein Chem. 1977; 31: 1-133Crossref PubMed Scopus (784) Google Scholar;Thacher and Rice, 1985Thacher S.M. Rice R.H. Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation.Cell. 1985; 40: 685-695Abstract Full Text PDF PubMed Scopus (400) Google Scholar). Although the order of deposition of the structural proteins in the CE has not yet been established unambiguously, much evidence suggests that they are crosslinked by three of the seven known TGases: TGase 1, TGase 3, and TGase 5 (Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar; for review on TGases seeMelino et al., 2000Melino G. Candi E. Steinert P.M. Assay for transglutaminases in cell death.Meth Enzymol. 2000; 322: 433-473Crossref PubMed Google Scholar). Even though the expression pattern of TGase 5 enzyme is not restricted to the epidermis (Aeschlimann et al., 1998Aeschlimann D. Koeller M.K. Allen-Hoffmann B.L. Mosher D.F. Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers.J Biol Chem. 1998; 273: 3452-3460Crossref PubMed Scopus (86) Google Scholar;2001;Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar), previous studies, by us and others (Aeschlimann et al., 1998Aeschlimann D. Koeller M.K. Allen-Hoffmann B.L. Mosher D.F. Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers.J Biol Chem. 1998; 273: 3452-3460Crossref PubMed Scopus (86) Google Scholar;Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar), suggested that TGase 5 probably plays a role in keratinocyte differentiation and CE assembly. Indeed, TGase 5 transcript is induced by differentiating agents in normal keratinocytes, and kinetic constants (Kcat/Km and Km) showed high affinity for TGase 5 in using loricrin, involucrin, and SPRs as substrate. In an attempt to investigate further the function of the novel enzyme TGase 5 in epidermal differentiation, and to gain more information on CE assembly, we developed an antihuman TGase 5 antiserum immunizing two rats by injection of the purified active recombinant protein expressed in the baculovirus system. TGase 5 is detected in the upper layers of normal human epidermis, following a gradient of expression stretching from the spinous layer to the horny layer. We have also evaluated TGase 5 expression in pathologic conditions such us psoriasis, ichthyosis (lamellar and vulgaris), and Darier's disease (DD), comparing proliferating (keratin 14) and differentiating (TGase 3) markers. Taken together, these data indicated that TGase 5 is expressed in differentiating keratinocytes in vivo, and its expression is upregulated, as a secondary effect, in many of the defects studied, and contributes to the hyperkeratotic phenotype observed in ichthyosis. Purified TGase 5 recombinant enzyme (Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar) was used to produce polyclonal antibodies in rats. The enzyme was purified to homogeneity using metal affinity chromatography and 200 µg of the purified enzyme was used to raise specific rat polyclonal antibodies. All procedures involving patients, animals, and their care followed institutional guidelines in compliance with national and international laws and policies (EEC Council Directive 86/109, OJL 358, December 1, 1987, and D.L. January 1992, n.116). Two Sprague-Dawley male rats, weighing 150–180 g, were subcutaneously injected with 25 µg (50 µl) of antigen adsorbed onto 20 µl of aluminum hydroxide (Pierce). The mixture was injected weekly for three consecutive weeks. The booster dose was administered 15 d after the last inoculation. Four days after the booster injection the antiserum was collected from the animals by tail bleed and tested to determine the strength of the specific antibody response. Cryopreserved normal human epidermal keratinocytes were obtained from BioWhittaker and grown in bovine-skin-collagen-coated dishes in serum-free keratinocyte medium at 0.05 mM Ca2+, supplemented with 7.5 (µg per ml) bovine pituitary extract, insulin (0.5 mg per ml), hydrocortisone (0.5 mg per ml), and human epidermal growth factor (0.1 UG per ml). Third passage cells were used for transfection experiments. Transient transfections of pCDNA3.1-TGase 5 were performed using Effectene (Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar) as described previously. Briefly, 8 × 105 keratinocytes were plated in collagen-coated 100 mm dishes 1 d before transfection. Transfected cells were kept for 2 d in culture. For confocal microscopy, keratinocytes were plated on collagen-coated cover glass, transfected with an equivalent ratio cell/DNA/lipid reagent, and kept in culture for 2 d. Cells were then collected and analyzed by Western blot and confocal microscopy. Western blots were performed using the whole cells or insoluble extracts from infected Sf9 insect cells or normal human epidermal keratinocytes; the blots (polyvinylidene difluoride membrane) were kept in blocking solution (10% dried milk, 5% bovine serum albumin) for 2 h at room temperature. Blots were incubated, shaking, for 2 h at room temperature with the anti-TGase 5 rat serum (dilution 1:200 or 1:1000, to detect native or overexpressed TGase 5). After three washes in PBS-Tween 20 (0.05%) secondary antibody was added (1:20,000 dilution in blocking solution). Proteins were detected using the enhanced chemiluminescence method. Antibody-competition experiments were performed by pretreating anti-TGase 5 antiserum with 200 µg of insoluble normal human epidermal keratinocyte (grown in differentiating conditions) cell extract for 1 h at 4°C, in the presence of a protease inhibitor cocktail to avoid degradation. For immuno-crossreactivity evaluation with other TGases, we used baculovirus expressed TGase 1 (Candi et al., 1998Candi E. Melino G. Lahm A. et al.Transglutaminase 1 mutations in lamellar ichthyosis. Loss of activity due to failure of activation by proteolytic processing.J Biol Chem. 1998; 273: 13693-13702Crossref PubMed Scopus (78) Google Scholar), purified guinea-pig liver TGase 2 (Sigma Chemical), and the baculovirus expressed TGase 3 (a kind gift of Dr. P.M. Steinert). Normal and defective adult trunk skin samples were paraffin embedded and cut into sections 6 µm thick. Human skin biopsies were performed for diagnostic purposes and five sections were used for research following a well-informed consent by the patient. The ethical committee of the hospital approved the study. Sections were permeabilized with 0.1% Triton X-100 for 3 min at room temperature and blocked using 10% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) overnight at +4°C. Sections were then incubated with rat anti-TGase 5 antibodies diluted 1:20 in 10% BSA solution and antikeratin 14 (Babco) and anti-TGase 3 antibodies (kind gift of Dr. Soo Kim) diluted, respectively, 1:100 and 1:1000. Lamellar ichthyosis (LI) skin sections were also stained with anti-TGase 1 antibody (kind gift of Dr. Soo Kim) following the conditions described inKim et al., 1995Kim S.Y. Chung S.I. Yoneda K. Steinert P.M. Expression of transglutaminase 1 in human epidermis.J Invest Dermatol. 1995; 104: 211-217Crossref PubMed Scopus (93) Google Scholar. Antibody-competition experiments were performed by pretreating anti-TGase 5 antiserum with 200 µg of insoluble normal human epidermal keratinocyte (grown in differentiating conditions) cell extract for 1 h or overnight at 4°C, in the presence of a protease inhibitor cocktail to avoid degradation. After washing three times in PBS, sections were incubated for 1 h with secondary antiserum (goat antirat Alexa Fluor 488 or Alexa Fluor 568) diluted in 10% BSA solution 1:1000. Sections were then mounted using Prolong Antifade kit. Fluorescence was evaluated with a confocal microscope (Nikon Instruments Spa, Eclipse TE200), exciting at 488 nm with an Ar laser and at 542 nm with an He laser. The software used was EZ2000 for PCM2000. To assess anti-TGase 5 antibody specificity, we transfected normal human epidermal keratinocytes (Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar) with the pCDNA3.1 vector, bearing c-myc tagged TGase 5 cDNA. TGase 5 protein was then detected in transfected cells by confocal microscopy, using anti-c-myc antibody and our novel anti-TGase 5 antiserum. Figure 1(a) shows that anti-TGase 5 and anti-c-myc antibodies recognized the same protein as indicated by the appearance of yellow in image superimposition. The signal detected by anti-TGase 5 antibody that did not overlap perfectly (see green staining, Figure 1a) with the signal detected by anti-c-myc antibody (see red staining, Figure 1a) is probably due to endogenous TGase 5 in normal keratinocytes. The anti-TGase 5 antibody specificity has been confirmed by Western blot analysis of Sf9 cells infected with recombinant baculovirus bearing c-myc tagged TGase 5 cDNA (Figure 1b). Western blots were performed on total cellular extract using antitag antibody (Figure 1b, lane 1, control; lane 2, anti-c-myc) and anti-TGase 5 antibodies (Figure 1b, lane 3, control; lane 4, anti-TGase 5). As already described by the confocal analysis, results indicate that the same bands are recognized by the two antibodies, one of 84 kDa, which is the monomeric form of the enzyme, and a 200 kDa band that is obtained by the autocatalytic activity of TGase 5 (Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar). The high molecular weight band is probably due to autocatalytic activity of TGases, as already described for TGase 2 and factor XIIIa (Barry and Mosher, 1990Barry E.L. Mosher D.F. Binding and degradation of blood coagulation factor XIII by culture fibroblasts.J Biol Chem. 1990; 265: 9302-9307Abstract Full Text PDF PubMed Google Scholar). In addition, another less intense 30 kDa band appears. Identical results were obtained using insoluble keratinocyte extract. A Western blot of transfected TGase 5 detected using anti-TGase 5 antibody (Figure 1c, lane 2) and anti-c-myc antibody (Figure 1c, lane 3) showed a similar pattern, with the control of nontransfected normal human epidermal keratinocytes shown in lane 1. Note that native TGase 5 is not detected by Western blot in keratinocytes grown in proliferating conditions (lane 1). A faint band of 84 kDa is detected in keratinocytes grown in differentiating conditions (data not shown). This suggests that, as already described for TGase 3, TGase 5 is poorly expressed in in vitro differentiating keratinocytes. The additional bands present in lane 2 are probably due to degradation as they are not observed in other experimental conditions. To verify the specificity of the bands detected by Western blot, we performed antigen-competition experiments. The anti-TGase 5 antiserum was incubated with an insoluble keratinocyte extract (which contained native TGase 5) for 1 h at +4°C, prior to TGase 5 detection by Western blotting. Results showed that insoluble keratinocyte extract competes with the recognition of all three bands detected (84 kDa, 200 kDa, and 30 kDa, Figure 1d), reducing them by between 3-fold and 11-fold as indicated by quantitative scanning analysis of the bands. In human epidermis, at least four TGases are expressed. Whereas TGase 2 has been detected only in the basal layer, the others (TGase 1, TGase 3, and TGase 5) are expressed in the upper layers. To evaluate whether our TGase 5 antiserum crossreacts with the other TGases we performed a Western blot using purified native or recombinant TGase 1, TGase 2, TGase 3, and TGase 5 as positive control. The staining, shown in Figure 1(e), revealed that anti-TGase 5 antiserum does not crossreact with the other TGases known to be expressed in human skin but recognizes only recombinant TGase 5 as an 84 kDa band (full length) and a higher molecular weight band of about 200 kDa that probably represents the auto-crosslinking product of TGase 5 (Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar). In our previous studies we have shown that TGase 5 plays a role in in vitro keratinocyte differentiation (Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar). To confirm this, we performed confocal analysis on cultured human keratinocytes grown in proliferating and differentiating conditions. We observed that keratinocytes grown in vitro in proliferating conditions (0.05 mM calcium) expressed a very low level of TGase 5 (Figure 1f), whereas the addition of calcium to the growth medium (1.2 mM) enhances TGase 5 expression (Figure 1g), with some cells intensively stained compared to other cells. Pretreated antiserum (incubated for 1 h at +4°C with native TGase 5) shows a consistent reduction of the signal (Figure 1h), confirming the specificity of anti-TGase 5 antibody in the experimental conditions used. To verify the specificity of the anti-TGase 5 antiserum in paraffin-embedded sections, we stained skin sections using anti-TGase 5 antiserum (diluted 1:200; Figure 2a), preimmune rat antiserum (diluted 1:200; Figure 2b), and pretreated anti-TGase 5 antiserum (final dilution 1:200). As shown in Figure 2(a), (d), results indicated that the immunofluorescences performed using anti-TGase 5 antiserum are specific. Indeed, the staining obtained using the preimmune rat serum shows very little background (Figure 2b), whereas the staining obtained with the anti-TGase 5 antiserum is strong (Figure 2a). The latter is almost completely abolished using pretreated anti-TGase 5 antiserum (incubated before staining with native and/or recombinant TGase 5 for 1 h or overnight at +4°C; Figure 2c), indicating the specificity of the staining shown in Figure 2(a), (d). As we observed very low background in Figure 2(b) and specificity in the staining shown in Figure 2(a), demonstrated by the blocking experiments, we decided not to affinity-purify the antiserum. In normal epidermis, TGase 5 antibody decorated the upper layers, with consistent staining in the spinous and granular layers (Figure 2d, g), and the enzyme was also detected in the basal layer at very low levels (Figure 2a, d, g). TGase 3 is expressed in the upper granular layer (Figure 2e) whereas TGase 5 expression follows a gradient from the spinous to the granular layers (Figure 2a, d) and therefore appears in the early stage of epidermal differentiation. TGase 3 and TGase 5 expression in normal skin overlapped mainly in a restricted area of the granular layer (Figure 2d–f) as shown by the appearance of yellow staining in Figure 2(f). In human dermis, positive immunofluorescent staining is observed in the erythrocytes, suggesting that the novel anti-TGase 5 antibody might crossreact with band 4.2, which is another member of the TGase family. This crossreactivity it is not crucial for our studies, however; in fact band 4.2 is exclusively present in the red cells. Psoriasis is a chronic skin disorder characterized by marked hyperproliferation and altered differentiation of keratinocytes in the context of activated cell-mediated immunity and inflammation (Barker, 1991Barker J.N. The pathophysiology of psoriasis.Lancet. 1991; 338: 227-230Abstract PubMed Scopus (208) Google Scholar). Histopathologic features consist of achantosis, parakeratosis, papillomatosis, and inflammatory infiltrates. In Figure 3(a), (b) we can observe some of these features in hematoxylin-eosin stained psoriatic sections taken from one of the patients analyzed. The pathophysiology of this disease remains unknown, however, although an alteration in cell-cell and cell-matrix adhesion versus an autoimmune process has been proposed as the primary defect (Fleischmajer et al., 2000Fleischmajer R. Kuroda K. Hazan R. et al.Basement membrane alterations in psoriasis are accompanied by epidermal overexpression of MMP-2 and its inhibitor TIMP-2.J Invest Dermatol. 2000; 115: 771-777Crossref PubMed Scopus (59) Google Scholar). Figure 3 shows a series of sections of psoriatic human skin examined using indirect immunofluorescence. We analyzed five unrelated psoriasis patients and obtained a very similar expression pattern in all of them for the analyzed molecules (TGase 3, TGase 5, and keratin 14). Immunofluorescence staining using anti-keratin-14 antibody indicated that keratinocyte differentiation is indeed abnormal in the skin of the analyzed patients: keratin 14 can be detected in every layer (Figure 3g), including the horny layer, where parakeratosis is also evident. TGase 5 expression in psoriatic lesions appears similar to normal (Figure 3c–e, f); in fact it is possible to observe the gradient of expression from the lower to the upper layers. TGase 5 seems overexpressed in some areas (Figure 3c, f), however, whereas the same is not true for TGase 3. This observation could also be due to the increased number of layers in the psoriatic lesion studied, and suggests that the involvement of TGase 5 in this pathogenesis is mainly secondary. TGase 3 expression appears downregulated in all sections analyzed, confirming the altered differentiation program in psoriatic keratinocytes. Ichthyosis vulgaris is an autosomal dominant skin disorder of keratinization characterized histologically by absent or reduced keratohyaline granules in the epidermis and mild hyperkeratosis (Anton-Lamprecht and Hofbauer, 1972Anton-Lamprecht I. Hofbauer M. Ultrastructural distinctive features of autosomal dominant ichthyosis vulgaris and X-linked recessive ichthyosis.Dermatologica. 1972; 145: 60-64Crossref PubMed Google Scholar;Sybert et al., 1985Sybert V.P. Dale B.A. Holbrook K.A. Ichthyosis vulgaris: identification of a defect in synthesis of filaggrin correlated with an absence of keratohyaline granules.J Invest Dermatol. 1985; 84: 191-194Crossref PubMed Scopus (243) Google Scholar). Affected skin appears dry and scaly. The basic molecular defect of ichthyosis vulgaris is unknown. Immunohistochemistry of skin samples taken from patients affected by ichthyosis vulgaris revealed that filaggrin, and its precursor pro-filaggrin, may have a role in the pathogenesis. In fact, filaggrin is not present in the more severely affected patients (Sybert et al., 1985Sybert V.P. Dale B.A. Holbrook K.A. Ichthyosis vulgaris: identification of a defect in synthesis of filaggrin correlated with an absence of keratohyaline granules.J Invest Dermatol. 1985; 84: 191-194Crossref PubMed Scopus (243) Google Scholar). In order to evaluate TGase 5 expression in this disease, we analyzed samples from four different patients affected by ichthyosis vulgaris and found that in one patient TGase 5 expression was similar to normal epidermis (Figure 4d–f), being expressed as a gradient that stained more intensively in the upper layer. In other patients, however, TGase 5 seems to contribute to the hyperkeratotic phenotype observed in ichthyosis vulgaris (Figure 4a, b, white arrows). This observation is even more interesting if we consider TGase 3 expression (Figure 4b); in fact, in the patients studied, it was observed that, whereas TGase 3 expression is not altered (Figure 4b), TGase 5 is clearly overexpressed and several keratinocytes in the spinous layer present a very intense staining (Figure 4a, arrows). Figure 4(g), (h) shows hematoxylin-eosin staining of serial sections of the patient corresponding to Figure 3(d)–(f). LI is part of congenital recessive ichthyosis, which includes the heterogeneous class of disorders of cornification that affect epidermis and hair. The clinical phenotype can range from generalized large brownish plate-like scales to fine white scales with underlying erythroderma. Many patients also exhibit ectropion and eclabian due to tautness of the facial skin. Histopathologic findings show orthokeratotic hyperkeratosis and mild to moderate acanthosis (Traupe et al., 1984Traupe H. Kolde G. Happle R. Autosomal dominant lamellar ichthyosis: a new skin disorder.Clin Genet. 1984; 26: 457-461Crossref PubMed Scopus (47) Google Scholar;Williams and Elias, 1985Williams M.L. Elias P.M. Heterogeneity in autosomal recessive ichthyosis. Clinical and biochemical differentiation of lamellar ichthyosis and nonbullous congenital ichthyosiform erythroderma.Arch Dermatol. 1985; 121: 477-488Crossref PubMed Scopus (157) Google Scholar;Williams and Elias, 1993Williams M.L. Elias P.M. From basket weave to barrier. Unifying concepts for the pathogenesis of the disorders of cornification.Arch Dermatol. 1993; 129: 626-629Crossref PubMed Scopus (73) Google Scholar;Williams, 1992Williams M.L. Ichthyosis: mechanisms of disease.Pediatr Dermatol. 1992; 9: 365-368Crossref PubMed Scopus (45) Google Scholar). Mutations in the gene coding for TGase 1 enzyme, TGM1, have been reported in LI patients, providing compelling evidence for the importance of TGases for the correct assembly of the CE and therefore for the homeostasis of the epidermis (Huber et al, 1995;Russell et al., 1995Russell L.J. DiGiovanna J.J. Rogers G.R. Steinert P.M. Hashem N. Compton J.G. Bale S.J. Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyosis.Nat Genet. 1995; 9: 279-283Crossref PubMed Scopus (323) Google Scholar). Since then, several different mutations of TGM1 have been identified (Parmentier et al., 1995Parmentier L. Blanchet-Bardon C. Nguyen S. Prud'homme J.F. Dubertret L. Weissenbach J. Autosomal recessive lamellar ichthyosis: identification of a new mutation in transglutaminase 1 and evidence for genetic heterogeneity.Hum Mol Genet. 1995; 4: 1391-1395Crossref PubMed Scopus (87) Google Scholar;Huber et al., 1997Huber M. Yee V.C. Burri N. Vikerfors E. Lavrijsen A.P. Paller A.S. Hohl D. Consequences of seven novel mutations on the expression and structure of keratinocyte transglutaminase.J Biol Chem. 1997; 272: 21018-21026Crossref PubMed Scopus (66) Google Scholar;Laiho et al., 1997Laiho E. Ignatius J. Mikkola H. et al.Transglutaminase 1 mutations in autosomal recessive congenital ichthyosis: private and recurrent mutations in an isolated population.Am J Hum Genet. 1997; 61: 529-538Abstract Full Text PDF PubMed Scopus (93) Google Scholar;Petit et al., 1997Petit E. Huber M. Rochat A. et al.Three novel point mutations in the keratinocyte transglutaminase (TGK) gene in lamellar ichthyosis: significance for mutant transcript level, TGK immunodetection and activity.Eur J Hum Genet. 1997; 5: 218-228PubMed Google Scholar;Bichakjian et al., 1998Bichakjian C.K. Nair R.P. Wu W.W. Goldberg S. Elder J.T. Prenatal exclusion of lamellar ichthyosis based on identification of two new mutations in the transglutaminase 1 gene.J Invest Dermatol. 1998; 110: 179-182Crossref PubMed Scopus (25) Google Scholar;Esposito et al., 2001Esposito G. Auricchio L. Rescigno G. Paparo F. Rinaldi M. Salvatore F. Transglutaminase 1 gene mutations in Italian patients with autosomal recessive lamellar ichthyosis.J Invest Dermatol. 2001; 116: 809-812Crossref PubMed Scopus (15) Google Scholar;Yang et al., 2001Yang J.M. Ahn K.S. Cho M.O. et al.Novel mutations of the transglutaminase 1 gene in lamellar ichthyosis.J Invest Dermatol. 2001; 117: 214-218Crossref PubMed Google Scholar). Biochemical data clearly showed that about 50%-60% of LI patients have normal TGase activity (Huber et al., 1995aHuber M. Rettler I. Bernasconi K. Wyss M. Hohl D. Lamellar ichthyosis is genetically heterogeneous – cases with normal keratinocyte transglutaminase.J Invest Dermatol. 1995; 105: 653-654Crossref PubMed Scopus (75) Google Scholar), however, suggesting a genetic heterogeneity of the disorder. Genetic heterogeneity is further supported by genetic mapping studies identifying, beside chromosome 14q11 where TGM1 is located, a second locus at chromosome 2q33–35 (Parmentier et al., 1996Parmentier L. Lakhdar H. Blanchet-Bardon C. Marchand S. Dubertret L. Weissenbach J. Mapping of a second locus for lamellar ichthyosis to chromosome 2q33–35.Hum Mol Genet. 1996; 5: 555-559Crossref PubMed Scopus (75) Google Scholar) and the existence of a third gene apparently unlinked to the above chromosomes (14 and 2;Bale et al., 1996Bale S.J. Russell L.J. Lee M.L. Compton J.G. DiGiovanna J.J. Congenital recessive ichthyosis unlinked to loci for epidermal transglutaminases.J Invest Dermatol. 1996; 107: 808-811Crossref PubMed Scopus (25) Google Scholar;Parmentier et al., 1996Parmentier L. Lakhdar H. Blanchet-Bardon C. Marchand S. Dubertret L. Weissenbach J. Mapping of a second locus for lamellar ichthyosis to chromosome 2q33–35.Hum Mol Genet. 1996; 5: 555-559Crossref PubMed Scopus (75) Google Scholar). We analyzed five patients affected by LI. Before starting our studies we investigated whether the diseases were due to mutations in the TGM1 gene by biochemical measurement of the TGase activity in patient keratinocytes kept in culture (data not shown). We found that two out of five patients had 80%-90% reduction in TGase activity indicating that mutations in TGM1 are responsible for LI in these patients, whereas the remaining three had normal TGase activity comparing to normal control suggesting that TGM1 is not the gene involved in the pathogenesis. In all samples analyzed, from five LI patients, TGase 5 expression is upregulated (Figure 5a, d) independently of the involvement of TGase 1 enzyme in the pathogenesis. In Figure 5 we show the immunofluorescence of two patients with normal TGase activity in cultured keratinocytes. TGase 1 immunofluorescent staining for one of them is shown in Figure 5(g) and appears similar to the normal pattern described previously in normal human epidermis (Kim et al., 1995Kim S.Y. Chung S.I. Yoneda K. Steinert P.M. Expression of transglutaminase 1 in human epidermis.J Invest Dermatol. 1995; 104: 211-217Crossref PubMed Scopus (93) Google Scholar). Most of the keratinocytes in the upper layers appear intensively stained (Figure 5a, d, arrows) in LI patients compared to keratinocytes in the upper layers of normal skin (Figure 2a, d, g). TGase 3 expression is also upregulated (Figure 5b) in all the five patients studied, and staining is detected in granular and spinous layers (Figure 5d–f). Keratin 14 expression appears similar to normal skin; very faint not specific staining appeared in the stratum corneum. Figure 4(h), (i) shows hematoxylin-eosin staining of a patient affected by LI. Finally, we investigated patients affected by DD. DD is an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization (Burge and Wilkinson, 1992Burge S.M. Wilkinson J.D. Darier-White disease: a review of the clinical features in 163 patients.J Am Acad Dermatol. 1992; 27: 40-50Abstract Full Text PDF PubMed Scopus (383) Google Scholar). DD patients develop warty papules and plaques on the central trunk, scalp, forehead, and flexure. The typical histologic features include focal areas of separation between suprabasal epidermal cells and abnormal keratinization; some of these features can be observed in Figure 6(a), (b), which shows hematoxylin-eosin staining of affected skin. Immunohistopathology and electron microscopy reveal loss of desmosomal attachments and perinuclear aggregations of keratin filaments (Burge and Garroa, 1991Burge S.M. Garroa D.R. An immunohistological study of desmosorres in Darier's disease and Hailey-Hailey disease.B J Dermatol. 1991; 124: 242-251Crossref PubMed Scopus (95) Google Scholar). Recently, mutations in a specific Ca2+ pump ATPase (ATP2A2) have been indicated as the defective gene for DD (Anavaj et al, 1999). In all the patients that we analyzed (four) we observed that both TGase 3 and TGase 5 expression altered (Figure 6c–f). TGase 5, for instance, appeared upregulated in some areas of the lesion (Figure 6f) but was almost absent in others (Figure 6c). Keratin 14 expression appeared normal; in addition to the normal keratin 14 expression observed in the proliferative layer, low level keratin 14 expression was observed in the upper layers (Figure 6g), probably due to nonspecific staining. Although the expression pattern of the TGase 5 enzyme is not exclusively restricted to the epidermis (Aeschlimann et al., 1998Aeschlimann D. Koeller M.K. Allen-Hoffmann B.L. Mosher D.F. Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers.J Biol Chem. 1998; 273: 3452-3460Crossref PubMed Scopus (86) Google Scholar;Grenard et al., 2001Grenard P. Bates M.K. Aeschlimann D. Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15.J Biol Chem. 2001Google Scholar;Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar), previous in vitro studies, by us and others (Aeschlimann et al., 1998Aeschlimann D. Koeller M.K. Allen-Hoffmann B.L. Mosher D.F. Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers.J Biol Chem. 1998; 273: 3452-3460Crossref PubMed Scopus (86) Google Scholar;Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar), have indicated that TGase 5 contributes to keratinocyte differentiation and CE assembly. To confirm these studies, we made an anti-TGase 5 antiserum and we demonstrated the specificity of this antibody by Western blot and immunofluorescence techniques both on cultured keratinocytes and in paraffin-embedded skin sections. We performed indirect immunofluorescence on normal human skin, and the results obtained indicated that TGase 5 is expressed in vivo in human epidermis in the spinous and granular layers, but it is also detectable at very low levels in the basal layer. This observation is very important and supports our early in vitro data (Candi et al., 2001Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro′ A. Melino G. Transglutaminase 5 cross-links loricrin, involucrin and SPRs in vitro.J Biol Chem. 2001; 276: 35014-35023Crossref PubMed Scopus (92) Google Scholar), which showed that TGase 5 enzyme is efficient in crosslinking specific epidermal substrates such as loricrin, SPRs, and involucrin to the same extent as TGase 1 and TGase 3. In addition, we investigated skin diseases where the keratinization process is impaired such as ichthyosis (ichthyosis vulgaris and LI) and DD. We also analyzed samples from patients affected by psoriasis, where hyperproliferation and altered differentiation of keratinocytes are both present. Our data suggested that TGase 5 is not directly involved in the pathogenesis of these diseases, and although its expression is altered, it seems to contribute consistently, even though as a secondary event, to the hyperkeratotic phenotype observed. This is evident in skin samples from patients affected by LI and ichthyosis vulgaris, as well as for some samples from psoriatic lesions. In DD, TGase 5 expression, as well as TGase 3, is completely altered, being overexpressed or totally absent in different areas of the same lesion. These results strongly indicate that TGase 5 plays an important role in the homeostasis of the epidermis, and when the function of the epithelia is compromised (as occurs in the diseases studied), overexpression of TGase 5 contributes to the hyperkeratotic phenotype, possibly as a compensation mechanism of the abnormal epidermis. We thank G. Bertini, G. Cortese, and P. Piccoli (SSD-SAFU Regina Elena Institute) for technical assistance. This work was mainly supported by Telethon grant 417/bi to EC and partially supported by AIRC, Telethon E872, and EU Grant QLG1-1999-00739 to GM.