Bikunin, a Serine Protease Inhibitor, is Present on the Cell Boundary of Epidermis
1999; Elsevier BV; Volume: 113; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.1999.00655.x
ISSN1523-1747
AutoresChang‐Yi Cui, Yoshinori Aragane, Akira Maeda, Yulan Piao, Masae Takahashi, Kim Lee-Hwa, Tadashi Tezuka,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoBikunin, which is an inhibitor of serine proteases, is widely distributed in human tissues, including liver, kidney, and mucous membranes of the stomach and colon. The aim of this study was to clarify whether bikunin is expressed in human epidermis and its appendages. Immunoblot analysis using a specific polyclonal antibody to bikunin revealed that a single 43 kDa protein is present in the cell lysate from the human keratinocyte cell line HaCaT. Immunohistochemically, dotted reaction products stained with anti-bikunin antibody were localized on the cell boundary in both basal and spinous cell layers, except on the cell boundary of the basal cells facing the basal membrane. There were no reaction products in the granular-horny cell layers. Reaction products stained with anti-bikunin antibody were also observed on the hair bulb cells and eccrine sweat gland cells, but not on apocrine sweat glands. Also, reaction products were observed on the luminal surface of the renal proximal tubules and in the cytoplasm of these cells. In immunoelectron microscopy, gold particles were observed on the cell membranes close to the desmosomal structures. Reverse transcription–polymerase chain reaction and northern blot analyses showed that mRNA specific for bikunin was expressed in HaCaT cells and human epidermal keratinocytes obtained from suction blisters, and was contained in a commercially available human keratinocyte cDNA preparation. These findings indicate that bikunin is expressed in keratinocytes and may play an important part in regulating keratinocytes in either mitosis or inflammation. Bikunin, which is an inhibitor of serine proteases, is widely distributed in human tissues, including liver, kidney, and mucous membranes of the stomach and colon. The aim of this study was to clarify whether bikunin is expressed in human epidermis and its appendages. Immunoblot analysis using a specific polyclonal antibody to bikunin revealed that a single 43 kDa protein is present in the cell lysate from the human keratinocyte cell line HaCaT. Immunohistochemically, dotted reaction products stained with anti-bikunin antibody were localized on the cell boundary in both basal and spinous cell layers, except on the cell boundary of the basal cells facing the basal membrane. There were no reaction products in the granular-horny cell layers. Reaction products stained with anti-bikunin antibody were also observed on the hair bulb cells and eccrine sweat gland cells, but not on apocrine sweat glands. Also, reaction products were observed on the luminal surface of the renal proximal tubules and in the cytoplasm of these cells. In immunoelectron microscopy, gold particles were observed on the cell membranes close to the desmosomal structures. Reverse transcription–polymerase chain reaction and northern blot analyses showed that mRNA specific for bikunin was expressed in HaCaT cells and human epidermal keratinocytes obtained from suction blisters, and was contained in a commercially available human keratinocyte cDNA preparation. These findings indicate that bikunin is expressed in keratinocytes and may play an important part in regulating keratinocytes in either mitosis or inflammation. cell boundary the spontaneously transformed human epidermal keratinocyte cell line Bikunin, which is an inhibitor of serine proteases (Gebhard and Hochstrasser, 1986Gebhard W. Hochstrasser K. Inter-α-trypsin inhibitor and its close relatives.in: Barrett A.J. Salvesen G. Proteinase Inhibitors. Elsevier, Amsterdam1986: 389-401Google Scholar), also termed ulinastatin (Kato et al., 1995Kato K. Nii J. Sakai K. Human urinary trypsin inhibitor: Its structure, biochemical properties and biosynthesis.Jpn J Med Pharm Sci. 1995; 33: 1089-1097Google Scholar) and urinary trypsin inhibitor (UTI) (Sumi et al., 1977Sumi H. Takeda Y. Takada A. Studies on human urinary trypsin inhibitor. I. Its modification on treatment of urine with acid.Thromb Res. 1977; 11: 747-754Abstract Full Text PDF PubMed Scopus (58) Google Scholar;Tanaka et al., 1982Tanaka Y. Maehara S. Sumi H. Toki N. Moriyama S. Sasaki K. Purification and partial characterization of two forms of urinary trypsin inhibitor.Biochim Biophys Acta. 1982; 703: 192-199Crossref Scopus (31) Google Scholar), was first reported byShulman, 1955Shulman N.R. Proteolytic inhibitor with anticoagulant activity separated from human urine and plasma.J Biol Chem. 1955; 213: 655-671Abstract Full Text PDF PubMed Google Scholar in the 1950s. Bikunin is first synthesized as a precursor polypeptide, which consists of bikunin and α1-microglobulin (Salier et al., 1996Salier J.P. Rouet P. Raguenez G. Daveau M. The inter-α-inhibitor: from structure to regulation.Biochem J. 1996; 315: 1-9Crossref PubMed Scopus (234) Google Scholar), and is cleaved after the completion of the chondroitin sulfate chain (Sjoberg et al., 1995Sjoberg E.M. Blom A. Larsson B.S. Alston-Smith J. Sjoquist M. Freis E. Plasma clearance of rat bikunin: evidence for receptor-mediated uptake.Biochem J. 1995; 308: 881-887Crossref PubMed Scopus (35) Google Scholar). The bikunin polypeptide has two Kunitz domains, and each domain inhibits different proteases (Hochstrasser et al., 1981Hochstrasser K. Schonberger O.L. Rossmanith I. Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-α-trypsin inhibitor.V Hoppe-Seyler's Z Physiol Chem. 1981; 362: 1357-1362Crossref PubMed Scopus (77) Google Scholar). Domain I, located in the N-terminal, inhibits chymotrypsin and leukocyte elastase, and domain II, located in the C-terminal inhibits plasmin, trypsin, and chymotrypsin (Hochstrasser et al., 1981Hochstrasser K. Schonberger O.L. Rossmanith I. Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-α-trypsin inhibitor.V Hoppe-Seyler's Z Physiol Chem. 1981; 362: 1357-1362Crossref PubMed Scopus (77) Google Scholar). In some malignant diseases, the level of bikunin is significantly increased (Yamamoto et al., 1986Yamamoto T. Sumi H. Maruyama M. Mizumoto H. Ikeda R. Yoshihara H. Mihara H. Acid stable trypsin inhibitor in bile.Clinica Chimica Acta. 1986; 158: 91-98Crossref PubMed Scopus (11) Google Scholar;Nishio et al., 1989Nishio N. Aoki K. Tokura Y. et al.Measurement of urinary trypsin inhibitor in urine, plasma, and cancer tissues of patients with stomach cancer.Haemostasis. 1989; 19: 112-119PubMed Google Scholar;Yoshida et al., 1989Yoshida E. Sumi H. Maruyama M. Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues.Cancer. 1989; 64: 860-869Crossref PubMed Scopus (34) Google Scholar). Bikunin binds to the tumor cell surface and inhibits the proteolytic enzyme plasmin, thus specifically reducing invasion and metastasis of tumor cells (Kobayashi et al., 1994aKobayashi H. Fujie M. Shinohara H. Ohi H. Sugimura M. Terao T. Effects of urinary trypsin inhibitor on the invasion of reconstituted basement membranes by ovarian cancer cells.Int J Cancer. 1994; 57: 378-384Crossref PubMed Scopus (48) Google Scholar,Kobayashi et al., 1994bKobayashi H. Shinohara H. Ohi H. Sugimura M. Terao T. Fujie M. Urinary trypsin inhibitor (UTI) and fragments derived from UTI by limited proteolysis efficiently inhibit tumor cell invasion.Clin Exp Metastasis. 1994; 12: 117-128Crossref PubMed Scopus (37) Google Scholar,Kobayashi et al., 1994cKobayashi H. Gotoh J. Fujie M. Terao T. Characterization of the cellular binding site for the urinary trypsin inhibitor.J Biol Chem. 1994; 269: 20642-20647Abstract Full Text PDF PubMed Google Scholar, Kobayashi et al., 1994dKobayashi H. Shinohara H. Takeuchi K. Itoh M. Fujie M. Saitoh M. Terao T. Inhibition of the soluble and the tumor cell receptor-bound plasmin by urinary trypsin inhibitor and subsequent affects on tumor cell invasion and metastasis.Cancer Res. 1994; 54: 844-849PubMed Google Scholar 1996). Although the expression of the mRNA of bikunin was shown to be derived from the α1-microglobulin/bikunin gene (Salier et al., 1996Salier J.P. Rouet P. Raguenez G. Daveau M. The inter-α-inhibitor: from structure to regulation.Biochem J. 1996; 315: 1-9Crossref PubMed Scopus (234) Google Scholar) and bikunin mRNA expression was reported only in the liver (Kaumeyer et al., 1986Kaumeyer J.F. Polazzi J.O. Kotick M.P. The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-α-trypsin inhibitor also encodes α1-microglobulin (protein HC).Nucleic Acids Res. 1986; 14: 7839-7850Crossref PubMed Scopus (190) Google Scholar;Salier et al., 1987Salier J.P. Diarra-Mehrpour M. Sesbou R. Isolation and characterization of cDNAs encoding the heavy chain of human urinary trypsin inhibitor (Iα1): Unambiguous evidence for multipolypeptide chain structure of IαT1.Proc Natl Acad Sci USA. 1987; 84: 8272-8276Crossref PubMed Scopus (77) Google Scholar), immunohistochemical staining revealed that this inhibitor is widely distributed in body tissues such as the brain, lung, liver, stomach, and kidney, and in body fluids (Kaumeyer et al., 1986Kaumeyer J.F. Polazzi J.O. Kotick M.P. The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-α-trypsin inhibitor also encodes α1-microglobulin (protein HC).Nucleic Acids Res. 1986; 14: 7839-7850Crossref PubMed Scopus (190) Google Scholar;Salier et al., 1987Salier J.P. Diarra-Mehrpour M. Sesbou R. Isolation and characterization of cDNAs encoding the heavy chain of human urinary trypsin inhibitor (Iα1): Unambiguous evidence for multipolypeptide chain structure of IαT1.Proc Natl Acad Sci USA. 1987; 84: 8272-8276Crossref PubMed Scopus (77) Google Scholar;Nishio et al., 1989Nishio N. Aoki K. Tokura Y. et al.Measurement of urinary trypsin inhibitor in urine, plasma, and cancer tissues of patients with stomach cancer.Haemostasis. 1989; 19: 112-119PubMed Google Scholar). Recently,Itoh et al., 1996Itoh H. Tomita M. Kobayashi T. Uchino H. Maruyama H. Nawa Y. Expression of inter-α-trypsin inhibitor light chain (bikunin) in human pancreas.J Biochem. 1996; 120: 271-275Crossref PubMed Scopus (39) Google Scholar reported expression of mRNA of bikunin in the human pancreas, suggesting that bikunin may be expressed in tissues other than the liver; however, there has been no evidence showing the presence of bikunin in human skin (Yoshida et al., 1989Yoshida E. Sumi H. Maruyama M. Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues.Cancer. 1989; 64: 860-869Crossref PubMed Scopus (34) Google Scholar). In human epidermis, keratinocytes are bound to each other by desmosomes, the adhesion and dissociation of which occurs repeatedly during mitosis and differentiation (Wolf and Wolf-Schreiner, 1976Wolf K. Wolf-Schreiner E.C. Trends in electron microscopy of skin.J Invest Dermatol. 1976; 67: 39-57Crossref PubMed Scopus (33) Google Scholar;Jones et al., 1986Jones J.C.R. Yokoo K.M. Goldman R.D. Further analysis of pemphigus autoantibodies and their use in studies on the heterogeneity, structure, and function of desmosomes.J Cell Biol. 1986; 102: 1109-1117Crossref PubMed Scopus (72) Google Scholar;Kitajima et al., 1986Kitajima Y. Inoue S. Yaoita H. Effects of pemphigus antibody on the organization of microtubules and keratin-intermediate filaments in cultured human keratinocytes.Br J Dermatol. 1986; 114: 171-179Crossref PubMed Scopus (22) Google Scholar,Kitajima et al., 1987Kitajima Y. Inoue S. Yaoita H. Effects of pemphigus antibody on the regeneration on cell-cell contact in keratinocyte cultures grown in low to normal Ca++ concentration.J Invest Dermatol. 1987; 89: 167-171Abstract Full Text PDF PubMed Google Scholar;Koch et al., 1990Koch P.J. Walsh M.J. Schmelz M. Goldschmidt M.D. Zimbelmann R. Franke W.W. Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules.Eur J Cell Biol. 1990; 53: 1-12PubMed Google Scholar;Buxton et al., 1993Buxton R.S. Cowin P. Franke W.W. Nomenclature of the desmosomal cadherins.J Cell Biol. 1993; 121: 481-483Crossref PubMed Scopus (256) Google Scholar). Although it is considered that desmosomes are digested by proteases like plasmin during keratinocyte dissociation (Hennings et al., 1980Hennings H. Michael D. Chang C. Steinert P. Holbrook K.A. Yuspa S.H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1502) Google Scholar,Hennings and Holbrook, 1983Hennings H. Holbrook K.A. Calcium regulation of cell–cell contact and differentiation of epidermal cells in culture: an ultrastructural study.Exp Cell Res. 1983; 143: 127-142Crossref PubMed Scopus (300) Google Scholar;Boyce and Ham, 1983Boyce S.T. Ham R.G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.J Invest Dermatol. 1983; 81: 33s-40sAbstract Full Text PDF PubMed Scopus (958) Google Scholar), the mechanisms of inhibition of plasmin or other proteases are not completely understood and the mechanisms of dissociation under normal conditions have not been completely clarified yet. In this study, we used reduced bikunin as the immunizing antigen, and succeeded in producing specific antibody. Using this antibody, we demonstrated that bikunin is expressed on the keratinocyte cell membrane in normal human skin, mainly in epidermis, hair follicles, and eccrine sweat glands. The antibody was obtained by injection of 0.5 mg of bikunin (the purified bikunin was kindly provided by Mochida Pharmaceutical Corp.), which had been previously reduced by 2-mercaptoethanol, with 0.5 ml of Freund complete adjuvant into the subcutaneous tissue of two rabbits. The immunization was repeated six times within 2 mo, and then the anti-serum was obtained. The anti-serum was further purified using a bikunin affinity column which was prepared as follows: 10 mg of purified bikunin was dissolved in 1 ml of 0.2 M NaHCO3 buffer containing 0.5 M NaCl, pH 8.3, and gently added to a 1 ml volume Hitrap affinity column (Pharmacia, Tokyo, Japan). After washing according to the manufacturer's recommendations, bikunin was fixed to the column with 0.5 M ethanolamine containing 0.5 M NaCl, pH 8.3, washed with a 0.1 M acetic acid solution containing 0.5 M NaCl, pH 4.0, and equilibrated with 0.05 M phosphate buffer containing 0.1% NaN3, 0.5 M NaCl, pH 7.4, at 4°C. The anti-serum, which had been previously equilibrated with 0.05 M phosphate buffer containing 0.5 M NaCl, pH 8.3, by PD-10 column chromatography, was gently added to the bikunin affinity column, and then eluted with 0.2 M glycine–HCl buffer containing 0.5 M NaCl, pH 2.5. The eluate was immediately neutralized with 2 M Tris–HCl, pH 8.5, and then the buffer was changed to phosphate-buffered saline by PD-10 column chromatography. Thus purified anti-bikunin antibody (Ig G fraction) was used for further experiments. The specificity of the anti-bikunin antibody for the purified, human bikunin was confirmed by immunoblot analysis. The epidermis obtained from suction blisters, HaCaT cells, and human serum was examined by immunoblot analysis. HaCaT cells were cultured in the following conditions: (i) normal culture medium; (ii) in normal medium and then cultured in serum-free medium for an additional 48 h before becoming confluent; (iii) at a low calcium concentration, then at a high calcium concentration; and (iv) low calcium concentration and harvested. Both the epidermis obtained from suction blisters and HaCaT cells were washed in phosphate-buffered saline three times to remove the serum components, and the epidermis was homogenized in RIPA buffer [phosphate-buffered saline containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 0.234 trypsin inhibitor unit aprotinin per ml] and stirred for 1 h at room temperature, and HaCaT cells were extracted by vortexing in RIPA buffer, and both were finally centrifuged at 15,000 × g for 30 min. The cell lysates, the culture supernatants, concentrated 100 times, and human serum were then subjected to SDS–polyacrylamide gel electrophoresis, transferred on to Immobilon (Millipore, Bedford, MA) membranes, and allowed to react with anti-bikunin antibody diluted 1:500 for 16 h at 4°C. The reaction products were further allowed to react with anti-rabbit IgG goat IgG labeled with peroxidase for 90 min at room temperature, and finally visualized by diaminobenzidine and H2O2. Either the indirect immunofluorescence method (fluorescein isothiocyanate method) or labeled streptavidin–biotin method (Gueston et al., 1979Gueston J.L. Ternynck T. Avrameras S. The use of avidin–biotin interaction in immunoenzymatic techniques.J Histochem Cytochem. 1979; 27: 1131-1139Crossref PubMed Scopus (1426) Google Scholar) was used to examine the localization of the antigen in the epidermis. Each of more than six samples of paraffin-embedded sections from normal human epidermis, oral mucosa, tongue and scalp skin, and paraffin-embedded sections from needle biopsies of the kidney were deparaffinized and treated for 10 min at 37°C in phosphate-buffered saline containing 0.1% trypsin before immunostaining, and were further incubated with either our anti-bikunin antibody diluted 1:500 or the anti-serums produced byYoshida et al., 1989Yoshida E. Sumi H. Maruyama M. Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues.Cancer. 1989; 64: 860-869Crossref PubMed Scopus (34) Google Scholar, or Mochida Pharmaceutical, diluted 1:500 for 1 h at 37°C. Normal human forearm skin obtained from a surgical operation for a benign skin tumor was minced into 1 mm cubes and immediately fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C for 1 h without glutaraldehyde, then rinsed thoroughly in 0.1 M phosphate buffer, pH 7.4, and embedded in Unicryl K4M resin (BioCell, Cardiff, U.K.) after dehydration, and used to prepare ultrathin sections, which were incubated with the purified, anti-bikunin antibody diluted 1:500 at 37°C for 1 h. After being rinsed, they were further incubated with anti-rabbit IgG goat IgG labeled with 15 nm gold particles, diluted 1:100 (Amersham Japan, Tokyo), at 37°C for 30 min. Finally, after being stained with uranyl acetate and lead acetate, these ultrathin sections were observed using an HU-7100 electron microscope (Hitachi, Tokyo, Japan). Total RNA was isolated from the human keratinocyte cell line HaCaT (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breikreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3464) Google Scholar) and epidermis from suction blisters according to standard procedures (Luduena et al., 1992Luduena R.F. Banerjee A. Khan I.A. Tubulin structure and biochemistry.Curr Opin Cell Biol. 1992; 4: 53-57Crossref PubMed Scopus (70) Google Scholar). Briefly, the cells or the epidermal sheet were lysed in 4 M guanidine isothiocyanate, 3 M sodium acetate (pH 4.8), 1% lauroylsarcosin, and 100 mM 2-mercaptoethanol for 10 min, mixed for 1 min by vortexing, and frozen at –20°C until use. Cell lysates were extracted twice with acidic phenol (pH 4.8) and chloroform (5:1 vol/vol). Precipitation was performed with 2.5 volumes of cold ethanol and one-third volume of 3 M sodium acetate buffer, pH 4.7, at –20°C overnight. The pellet was rinsed with 70% ethanol, evaporated in a vacuum desiccator and dissolved in doubly distilled water. The amounts of RNA in samples were determined photometrically at 260 nm and the RNA was then used for reverse transcription–polymerase chain reaction (reverse transcription–PCR). Five micrograms of total RNA was reverse-transcribed. Reaction buffer (5 × 250 mM Tris–HCl, pH 8.3, 250 mM KCl, 25 mM MgCl2) and 4 μl of 25 mM dNTP (each), 1 μl of RNase inhibitor (Promega, WI) or and 0.8 μl of dT17 (Takara, Tokyo, Japan) were mixed and diluted to 38 μl with RNase-free water, heated to 65°C for 5 min in a water bath, and chilled on ice. After the addition of 2 μl of MuLV reverse transcriptase (Promega), samples were incubated at 37°C for 90 min and the cDNA was then precipitated with 2.5 volumes of cold ethanol and one-third volume of 3 M sodium acetate buffer, pH 7.8, at –20°C overnight. The pellet was rinsed with 70% ethanol, evaporated in a vacuum desiccator and dissolved in doubly distilled water. To perform PCR amplification on a semiquantitative basis, we amplified all cDNA samples with primers specific for β-actin using various cycle numbers and dilution factors, and thereby determined the optimal concentration of each sample and a cycle number showing a logarithmic increase in amplification. Samples were then amplified using primers specific for CD95. For PCR amplification, we used a 50 μl reaction mixture containing 1 μl cDNA, 200 μM dNTP (each), and 20 pmol of each primer pair. The PCR primers for bikunin were as follows: sense primer: 5′-TGGGCAACTGGTAACT GAAG-3′ anti-sense primer: 5′-GCAGCTCCTCATCACCATCA-3′. These primers correspond to exon 7 and the junction between exons 9 and 10 (from 7020 to 7038 in exon 7 and from 8802 in exon 9–9471 in exon 10;Yetr and Gebhard, 1990Yetr H. Gebhard W. Structure of the human α1-microglobulin-bikunin gene.Biol Chem Hoppe-Seyler. 1990; 371: 1185-1196Crossref PubMed Scopus (60) Google Scholar). The PCR primers for β-actin were as follows: sense primer: 5′-CACCTTCTACAATGAGCTGC-3′ anti-sense: 5′-TTCATGAGGTAGTCCGTCAG-3′. Positive controls (human liver cDNA and human keratinocyte cDNA) (Clontech, CA) and a negative control (without cDNA) were included in each experiment. For northern blot analysis (Aragane et al., 1994Aragane Y. Riemann H. Bhardwaj R.S. et al.IL-12 is expressed and released by human keratinocytes and epidermoid carcinoma cell lines.J Immunol. 1994; 153: 5366-5372PubMed Google Scholar), total cellular RNA was denatured in 50% formamide, 6% formaldehyde, 1 × 0.02 M 3-[N-morpholino] propane-sulfonic acid (MOPS), 0.05 M sodium acetate (pH 7.0), and 0.01 M Na2 ethylenediamine tetraacetic acid at 65°C for 5 min. Ten micrograms of total RNA was separated by gel electrophoresis using 1% agarose gels containing 6.3% formaldehyde and 1 × MOPS. Gels were run at 100 V for 1 h. To monitor equal loading of RNA samples, membranes were stained with 1 × MOPS solution containing 0.05 μg per ml ethidium bromide and ribosomal RNA was photographed under an ultraviolet lamp. Subsequently, RNA samples were transferred to a nylon filter by capillary blotting. Filters were baked at 80°C for 2 h. After prehybridization at 42°C in 50% formamide, 10% dextran sulfate, 1% SDS, and 1 M MaCl for at least 15 min. Hybridization was conducted using deoxyadenosine-5′-[32P]triphosphate-labeled cDNA probes encoding bikunin which was obtained by PCR amplification of cDNA derived from human normal keratinocytes and their sequences were ascertained as 100% identical to established sequences of bikunin as listed in GenBank by a sequencing analysis. The labeled probe and heat-denatured salmon sperm DNA (100 μg per ml) were added to the prehybridization solution and incubated at 42°C at least 12 h in a water bath. After hybridization, filters were washed in 2 × sodium citrate/chloride buffer at room temperature for 10 min, 2 × sodium citrate/chloride buffer containing 1% SDS at 60°C for 1 h, and 0.1 × sodium citrate/chloride buffer for 1 h at room temperature. Filters were exposed to X-ray films at –70°C. Cell lysates were prepared from normal human keratinocytes obtained from suction blister epidermis and cultured HaCaT cells. HaCaT cells were cultured in various conditions which were described in Materials and Methods. The culture supernatants were collected and concentrated 100-fold before use. Cell lysates, culture supernatants, and normal human serum were subjected to SDS–polyacrylamide gel electrophoresis, blotted on to Immobilon membranes, and reacted with the purified anti-bikunin antibody. The purified anti-bikunin antibody specifically recognized antigenic bikunin (Figure 1a, lane a), serum inter-α-trypsin inhibitor (225 kDa) and pre-α inhibitor (125 kDa) in human serum (Figure 1a, lane b). This antibody also reacted with 43 kDa, 36 kDa, and 19 kDa epidermal proteins of human, normal epidermis (Figure 1b, lane b), and with a 43 kDa protein in all cell lysates of cultured HaCaT cells (Figure 1c, lanes k–n); however, no 43 kDa protein was detected in the culture supernatants (Figure 1c, lanes o–r). Purified bikunin showed a broad band in the immunoblots ranging from 30 kDa to 45 kDa, which was in good agreement with the previous reports of other researchers (Balduyck et al., 1986Balduyck M. Mizon C. Loutfi H. Richet C. Roussel P. Mizon J. The major human urinary trypsin inhibitor is a proteoglycan.Eur J Biochem. 1986; 158: 417-422Crossref PubMed Scopus (44) Google Scholar;Laroui et al., 1988Laroui S. Balduyck M. Mizon C. Selloum L. Mizon J. Plasma proteins immunologically related to inter-α-trypsin inhibitor.Biochim Biophys Acta. 1988; 953: 263-268Crossref PubMed Scopus (15) Google Scholar;Itoh et al., 1996Itoh H. Tomita M. Kobayashi T. Uchino H. Maruyama H. Nawa Y. Expression of inter-α-trypsin inhibitor light chain (bikunin) in human pancreas.J Biochem. 1996; 120: 271-275Crossref PubMed Scopus (39) Google Scholar;Kobayashi et al., 1994bKobayashi H. Shinohara H. Ohi H. Sugimura M. Terao T. Fujie M. Urinary trypsin inhibitor (UTI) and fragments derived from UTI by limited proteolysis efficiently inhibit tumor cell invasion.Clin Exp Metastasis. 1994; 12: 117-128Crossref PubMed Scopus (37) Google Scholar,Kobayashi et al., 1994cKobayashi H. Gotoh J. Fujie M. Terao T. Characterization of the cellular binding site for the urinary trypsin inhibitor.J Biol Chem. 1994; 269: 20642-20647Abstract Full Text PDF PubMed Google Scholar,Kobayashi et al., 1996Kobayashi H. Gotoh J. Hirashima Y. Terao T. Inter-α-trypsin inhibitor bound to tumor cells is cleaved into the heavy chains and the light chain on the cell surface.J Biol Chem. 1996; 271: 11362-11367Crossref PubMed Scopus (47) Google Scholar). In immunohistochemical staining, dotted reaction products were observed on the cell boundary (CB) of keratinocytes from basal cells to the upper spinous cells, except for the cell membrane facing the basal membrane (Figure 2a), and on the CB of oral mucosa (Figure 2b), but there were no reaction products in the granular-horny cell layers (Figure 2a). There was also positive staining on the endothelial cells of capillaries in the dermis (Figure 2a). This purified, anti-bikunin antibody as well as the antibody from Mochida Pharmaceutical reacted with the luminal surface and intracytoplasmic materials of renal proximal tubules (Figure 2c). In addition, the antibody provided by Mochida Pharmaceutical also reacted with the epidermis. Dotted reaction products were observed on the membrane of eccrine sweat ducts and the secretory portion (Figure 3a), but there was no positive reaction in apocrine sweat glands (Figure 3b). There were also dotted reaction products on the cell membrane of the outermost cells of the outer root sheath at the level of the hair supra-bulge region (Figure 4a), and at the level of the hair supra-bulbar region (Figure 4b), and on hair bulb cells (Figure 4c). In immunoelectron microscopy, the gold particles appeared to be located on the cell membrane close to desmosomes (Figure 5).Figure 3Bikunin is located on the membrane of eccrine sweat gland cells but not on apocrine sweat gland cells. Paraffin-embedded sections of normal human scalp skin were pretreated with 0.1% trypsin and reacted with anti-bikunin antibody. Fluorescence was observed on the cell membrane of the eccrine sweat gland cells and some dermal cells. A, eccrine sweat gland; B, apocrine sweat gland; x, luminal area of sweat gland. Scale bar: 40 μm.View Large Image Figure ViewerDownload (PPT)Figure 4Bikunin is located on outer root sheath cells and hair bulb cells. Paraffin-embedded sections of normal human scalp skin were pretreated with 0.1% trypsin and reacted with anti-bikunin antibody. (A) Positive reaction was observed on the outer root sheath cell membranes at the level of the hair supra-bulge region. (B) Positive reaction was observed on the outer root sheath cells at the level of the hair supra-bulbar region. (C) Positive reaction was observed on the hair bulb cells. Some dermal cells are also positively stained, as shown in B. Scale bar: 40 μmView Large Image Figure ViewerDownload (PPT)Figure 5Bikunin is located on the cell membrane close to the desmosomes of spinous cells. Normal human forearm skin was fixed in 4% paraformaldehyde and reacted with anti-bikunin antibody. Arrows indicate desmosomes. Gold particles were seen on the cell membrane close to the desmosomes. N, nucleus. Scale bar: 0.5 μm.View Large Image Figure ViewerDownload (PPT) In order to examine whether mRNA specific for bikunin is expressed in human epidermal keratinocytes, we performed semiquantitative reverse transcription–PCR using primers specific for bikunin and northern blot analysis. Reverse transcribed cDNA samples derived from a human keratinocyte-derived cell line, HaCaT, epidermal cells of suction blister samples, and neonatal foreskin-derived cultured human keratinocytes were amplified with primer pairs specific either for bikunin or for β-actin. As a positive control, cDNA derived from human liver and keratinocytes were also amplified, as it is known that human liver cells express bikunin mRNA (Kaumeyer et al., 1986Kaumeyer J.F. Polazzi J.O. Kotick M.P. The mRNA for a proteinase inhibitor relat
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