Cystatin M/E Expression is Restricted to Differentiated Epidermal Keratinocytes and Sweat Glands: a New Skin-Specific Proteinase Inhibitor that is a Target for Cross-Linking by Transglutaminase
2001; Elsevier BV; Volume: 116; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2001.01309.x
ISSN1523-1747
AutoresPatrick L.J.M. Zeeuwen, Ivonne M.J.J. van Vlijmen‐Willems, Bas Jansen, Fred van Ruissen, Joost Schalkwijk, Georgia Sotiropoulou, Jo H. A. J. Curfs, Jacques F. Meis, J.J.M. Janssen,
Tópico(s)Proteins in Food Systems
ResumoUsing serial analysis of gene expression on cultured human keratinocytes we found high expression levels of genes putatively involved in host protection and defense, such as proteinase inhibitors and antimicrobial proteins. One of these expressed genes was the recently discovered cysteine proteinase inhibitor cystatin M/E that has not been characterized so far at the protein level with respect to tissue distribution and additional biologic properties. Here we report that cystatin M/E has a tissue-specific expression pattern in which high expression levels are restricted to the stratum granulosum of normal human skin, the stratum granulosum/spinosum of psoriatic skin, and the secretory coils of eccrine sweat glands. Low expression levels were found in the nasal cavity. The presence of cystatin M/E in skin and the lack of expression in a variety of other tissues was verified both at the protein level by immunohistochemistry or western blotting, and at the mRNA level by reverse transcriptase polymerase chain reaction or northern blotting. Using biotinylated hexapeptide probes we found that cystatin M/E is an efficient substrate for tissue type transglutaminase and for transglutaminases extracted from stratum corneum, and that it acts as an acyl acceptor but not as an acyl donor. Western blot analysis showed that recombinant cystatin M/E could be cross-linked to a variety of proteins extracted from stratum corneum. In vitro, we found that cystatin M/E expression in cultured keratinocytes is upregulated at the mRNA and protein level, upon induction of differentiation. We demonstrate that cystatin M/E, which has a putative signal peptide, is indeed a secreted protein and is found in vitro in culture supernatant and in vivo in human sweat by enzyme-linked immunosorbent assay or western blotting. Cystatin M/E showed moderate inhibition of cathepsin B but was not active against cathepsin C. We speculate that cystatin M/E is unlikely to be a physiologically relevant inhibitor of intracellular lysosomal cysteine proteinases but rather functions as an inhibitor of self and nonself cysteine proteinases that remain to be identified. Using serial analysis of gene expression on cultured human keratinocytes we found high expression levels of genes putatively involved in host protection and defense, such as proteinase inhibitors and antimicrobial proteins. One of these expressed genes was the recently discovered cysteine proteinase inhibitor cystatin M/E that has not been characterized so far at the protein level with respect to tissue distribution and additional biologic properties. Here we report that cystatin M/E has a tissue-specific expression pattern in which high expression levels are restricted to the stratum granulosum of normal human skin, the stratum granulosum/spinosum of psoriatic skin, and the secretory coils of eccrine sweat glands. Low expression levels were found in the nasal cavity. The presence of cystatin M/E in skin and the lack of expression in a variety of other tissues was verified both at the protein level by immunohistochemistry or western blotting, and at the mRNA level by reverse transcriptase polymerase chain reaction or northern blotting. Using biotinylated hexapeptide probes we found that cystatin M/E is an efficient substrate for tissue type transglutaminase and for transglutaminases extracted from stratum corneum, and that it acts as an acyl acceptor but not as an acyl donor. Western blot analysis showed that recombinant cystatin M/E could be cross-linked to a variety of proteins extracted from stratum corneum. In vitro, we found that cystatin M/E expression in cultured keratinocytes is upregulated at the mRNA and protein level, upon induction of differentiation. We demonstrate that cystatin M/E, which has a putative signal peptide, is indeed a secreted protein and is found in vitro in culture supernatant and in vivo in human sweat by enzyme-linked immunosorbent assay or western blotting. Cystatin M/E showed moderate inhibition of cathepsin B but was not active against cathepsin C. We speculate that cystatin M/E is unlikely to be a physiologically relevant inhibitor of intracellular lysosomal cysteine proteinases but rather functions as an inhibitor of self and nonself cysteine proteinases that remain to be identified. cornified cell envelope glutathione S-transferase human acidic ribosomal phosphoprotein P0 immunohistochemistry serial analysis of gene expression stratum corneum chymotryptic enzyme stratum corneum thiol proteinase skin-derived antileukoproteinase secretory leukocyte proteinase inhibitor transglutaminase Cystatins are natural and specific inhibitors of endogenous mammalian lysosomal cysteine proteinases, such as cathepsins B, L, H, and S (Turk and Bode, 1991Turk V. Bode W. The cystatins: protein inhibitors of cysteine proteinases.FEBS Lett. 1991; 285: 213-219Abstract Full Text PDF PubMed Scopus (696) Google Scholar;Abrahamson, 1994Abrahamson M. Cystatins.Meth Enzymol. 1994; 244: 685-700Crossref PubMed Scopus (171) Google Scholar), and exogenous microbial cysteine proteinases (Bjorck, 1990Bjorck L. Proteinase inhibition, immunoglobulin-binding proteins and a novel antimicrobial principle.Mol Microbiol. 1990; 4: 1439-1442Crossref PubMed Scopus (27) Google Scholar). Several studies have indicated that cystatins provide important regulatory and protective functions against uncontrolled proteolysis by cysteine proteinases from host, bacterial, and viral origin (Bobek and Levine, 1992Bobek L.A. Levine M.J. Cystatins – inhibitors of cysteine proteinases.Crit Rev Oral Biol Med. 1992; 3: 307-332Crossref PubMed Scopus (130) Google Scholar). A disturbed balance between proteinases and their inhibitors can lead to irreversible damage as in chronic inflammatory reactions (Henskens et al., 1996Henskens Y.M. Veerman E.C. Nieuw Amerongen A.V. Cystatins in health and disease.Biol Chem Hoppe Seyler. 1996; 377: 71-86Crossref PubMed Google Scholar) and tumor metastasis (Calkins and Sloane, 1995Calkins C.C. Sloane B.F. Mammalian cysteine protease inhibitors: biochemical properties and possible roles in tumor progression.Biol Chem Hoppe Seyler. 1995; 376: 71-80PubMed Google Scholar). In addition to their proteinase inhibitory activity, some of the cystatins (C and S) were shown to have antimicrobial activity against bacteria (Bjorck et al., 1989Bjorck L. Akesson P. Bohus M. Trojnar J. Abrahamson M. Olafsson I. Grubb A. Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor.Nature. 1989; 337: 385-386Crossref PubMed Scopus (165) Google Scholar;Blankenvoorde et al., 1998Blankenvoorde M.F. van'T Hof W. Walgreen-Weterings E. van Steenbergen T.J. Brand H.S. Veerman E.C. Nieuw Amerongen A.V. Cystatin and cystatin-derived peptides have antibacterial activity against the pathogen Porphyromonas gingivalis.Biol Chem. 1998; 379: 1371-1375PubMed Google Scholar) and viruses (Korant et al., 1986Korant B.D. Towatari T. Ivanoff L. Petteway S. Brzin J. Lenarcic B. Turk V. Viral therapy: prospects for protease inhibitors.J Cell Biochem. 1986; 32: 91-95Crossref PubMed Scopus (40) Google Scholar;Bjorck et al., 1990Bjorck L. Grubb A. Kjellen L. Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus.J Virol. 1990; 64: 941-943Crossref PubMed Google Scholar). Cystatins are members of a superfamily of evolutionary related proteins and can be divided into three major families (Rawlings and Barrett, 1990Rawlings N.D. Barrett A.J. Evolution of proteins of the cystatin superfamily.J Mol Evol. 1990; 30: 60-71Crossref PubMed Scopus (259) Google Scholar): family 1 cystatins A and B (or steffins A and B), family 2 cystatins (C, D, S, SN, and SA), and the kininogens, which belong to family 3 cystatins. A new member of the human cystatin superfamily, named cystatin M, was recently identified and characterized (Sotiropoulou et al., 1997Sotiropoulou G. Anisowicz A. Sager R. Identification cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.J Biol Chem. 1997; 272: 903-910Crossref PubMed Scopus (145) Google Scholar). The gene was identified by differential display, comparing mRNAs from primary and metastatic breast tumor cells, and was found to be downregulated in metastatic cells. The same cystatin was independently found by expressed sequence tag sequencing in epithelial-cell-derived cDNA libraries and reported as cystatin E (Ni et al., 1997Ni J. Abrahamson M. Zhang M. et al.Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins.J Biol Chem. 1997; 272: 10853-10858Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Cystatin M/E is a 14 kDa protein that shares a 35% homology with the human family 2 cystatins, has similar overall structures, such as signal peptide and two intrachain disulfide bonds, but possesses the unusual characteristic of being a glycoprotein. Cystatin M/E proved to be a potent inhibitor of papain and cathepsin B (Ni et al., 1997Ni J. Abrahamson M. Zhang M. et al.Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins.J Biol Chem. 1997; 272: 10853-10858Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar;Sotiropoulou et al., 1997Sotiropoulou G. Anisowicz A. Sager R. Identification cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.J Biol Chem. 1997; 272: 903-910Crossref PubMed Scopus (145) Google Scholar), although specific physiologic functions or target molecules have not been reported so far. Cystatin M/E was reported to have a broad tissue distribution at the mRNA level, although the reports of Ni et al and Sotiropoulou et al gave conflicting results. No tissue localization studies at the protein level have been reported to date. Epithelial tissues serve as a first line of defense between the host and the environment. Disturbance of this barrier can lead to the invasion of microbial pathogens and subsequent inflammation. Previous studies have shown that epithelia that are subjected to continuous microbial stress or inflammatory stimuli (e.g., oral epithelia, trachea, skin) express several proteins that provide protection against microbial pathogens and excessive proteolysis by endogenous and exogenous proteinases. These proteins have been assigned as a key component of the innate immune system (Boman, 1998Boman H.G. Gene-encoded peptide antibiotics and the concept of innate immunity: an update review.Scand J Immunol. 1998; 48: 15-25Crossref PubMed Scopus (200) Google Scholar;Lehrer and Ganz, 1999Lehrer R.I. Ganz T. Antimicrobial peptides in mammalian and insect host defence.Curr Opin Immunol. 1999; 11: 23-27https://doi.org/10.1016/s0952-7915(99)80005-3Crossref PubMed Scopus (0) Google Scholar), a system that can be triggered by infection and protects the host organism against invading microorganisms (Medzhitov and Janeway, 1998Medzhitov R. Janeway C.A. Innate immune recognition and control of adaptive immune responses.Semin Immunol. 1998; 10: 351-353https://doi.org/10.1006/smim.1998.0136Crossref PubMed Scopus (286) Google Scholar). Recently, several proteins that are involved in protection against tissue damage were found to be expressed in human skin in response to inflammatory events. These proteins include two well-characterized serine proteinase inhibitors with antimicrobial properties, secretory leukocyte proteinase inhibitor (SLPI) (Hiemstra et al., 1996Hiemstra P.S. Maassen R.J. Stolk J. Heinzel-Wieland R. Steffens G.J. Dijkman J.H. Antibacterial activity of antileukoprotease.Infect Immun. 1996; 64: 4520-4524Crossref PubMed Google Scholar;Wiedow et al., 1998Wiedow O. Harder J. Bartels J. Streit V. Christophers E. Antileukoprotease in human skin: an antibiotic peptide constitutively produced by keratinocytes.Biochem Biophys Res Commun. 1998; 248: 904-909https://doi.org/10.1006/bbrc.1998.9069Crossref PubMed Scopus (141) Google Scholar;Wingens et al., 1998Wingens M. van Bergen B.H. Hiemstra P.S. et al.Induction of SLPI (ALP/HUSI-I) in epidermal keratinocytes.J Invest Dermatol. 1998; 111: 996-1002https://doi.org/10.1046/j.1523-1747.1998.00425.xCrossref PubMed Scopus (103) Google Scholar) and skin-derived antileukoproteinase (SKALP)/elafin (Simpson et al., 1999Simpson A.J. Maxwell A.I. Govan J.R. Haslett C. Sallenave J.M. Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens.FEBS Lett. 1999; 452: 309-313https://doi.org/10.1016/s0014-5793(99)00670-5Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), a member of the trappin gene family (Zeeuwen et al., 1997Zeeuwen P.L. Hendriks W. de Jong W.W. Schalkwijk J. Identification and sequence analysis of two new members of the SKALP/elafin and SPAI-2 gene family. Biochemical properties of the transglutaminase substrate motif and suggestions for a new nomenclature.J Biol Chem. 1997; 272: 20471-20478Crossref PubMed Scopus (51) Google Scholar;Schalkwijk et al., 1999Schalkwijk J. Wiedow O. Hirose S. The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core.Biochem J. 1999; 340: 569-577https://doi.org/10.1042/0264-6021:3400569Crossref PubMed Scopus (169) Google Scholar). Both proteins are originally described as inhibitors of polymorphonuclear leukocyte derived enzymes, like human neutrophil elastase, that are secreted under inflammatory conditions of the skin (e.g., wound healing, psoriasis) (Thompson and Ohlsson, 1986Thompson R.C. Ohlsson K. Isolation properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase.Proc Natl Acad Sci USA. 1986; 83: 6692-6696Crossref PubMed Scopus (417) Google Scholar;Schalkwijk et al., 1990Schalkwijk J. Chang A. Janssen P. de Jongh G.J. Mier P.D. Skin-derived antileucoproteases (SKALPs): characterization of two new elastase inhibitors from psoriatic epidermis.Br J Dermatol. 1990; 122: 631-641Crossref PubMed Scopus (91) Google Scholar;Wiedow et al., 1990Wiedow O. Schroder J.M. Gregory H. Young J.A. Christophers E. Elafin an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence.J Biol Chem. 1990; 265: 14791-14795Abstract Full Text PDF PubMed Google Scholar;Molhuizen et al., 1993Molhuizen H.O. Alkemade H.A. Zeeuwen P.L. de Jongh G.J. Wieringa B. Schalkwijk J. SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking.J Biol Chem. 1993; 268: 12028-12032Abstract Full Text PDF PubMed Google Scholar;Pfundt et al., 1996Pfundt R. van Ruissen F. Vlijmen-Willems I.M. et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (135) Google Scholar). Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE) (Robinson et al., 1997Robinson N.A. Lapic S. Welter J.F. Eckert R.L. S100A11, S100A10, annexin I, desmosomal proteins, small proline-rich proteins, plasminogen activator inhibitor-2, and involucrin are components of the cornified envelope of cultured human epidermal keratinocytes.J Biol Chem. 1997; 272: 12035-12046Crossref PubMed Scopus (198) Google Scholar;Nemes and Steinert, 1999Nemes Z. Steinert P.M. Bricks and mortar of the epidermal barrier.Exp Mol Med. 1999; 31: 5-19Crossref PubMed Scopus (415) Google Scholar), which is assembled by transglutaminase (TGase) cross-linking of several structural proteins, like small proline-rich proteins, loricrin, cytokeratins, filaggrin, and involucrin (Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar). Two epidermal proteinase inhibitors were found to be anchored to structural proteins of the stratum corneum. SKALP/elafin was found in foreskin by direct protein sequencing (Steinert and Marekov, 1997Steinert P.M. Marekov L.N. Direct evidence that involucrin is a major early isopeptide cross- linked component of the keratinocyte cornified cell envelope.J Biol Chem. 1997; 272: 2021-2030Crossref PubMed Scopus (193) Google Scholar), and cystatin A, the only member of the cystatin family so far that has been found in human epidermis, was shown to be part of the CE and acts as a TGase substrate (Takahashi et al., 1996Takahashi M. Tezuka T. Katunuma N. Filaggrin linker segment peptide and cystatin alpha are parts of a complex of the cornified envelope of epidermis.Arch Biochem Biophys. 1996; 329: 123-126https://doi.org/10.1006/abbi.1996.0199Crossref PubMed Scopus (33) Google Scholar). Recently we have started with the construction of a transcriptome of human epidermis using serial analysis of gene expression (SAGE). Using SAGE we have made an extensive expression profile of cultured human keratinocytes, comprising over 25,000 sequenced tags (Jansen et al., 2001Jansen B.J. van Ruissen F. de Jongh G.J. Zeeuwen P.L. Schalkwijk J. Serial analysis of gene expression (SAGE) in human epidermal keratinocytes reveals high expression levels of genes involved in host defense and protection.J Invest Dermatol. 2001; 116: 12-22https://doi.org/10.1046/j.1523-1747.2001.00218.xCrossref PubMed Scopus (30) Google Scholar). From this analysis it was clear that genes involved in host protection and host defense (proteinase inhibitors and antimicrobial proteins) were expressed at high levels. Cystatin M/E, not previously known to be expressed by adult human keratinocytes, was found at considerable levels in these analyses. In this paper we report the skin-specific expression of cystatin M/E, and its induction by serum in vitro using cultured human keratinocytes. We demonstrate that cystatin M/E is a secreted protein and, in addition to its reported antiproteinase activity, has at least one additional biologic property by acting as an acyl acceptor in TGase-mediated reactions, thereby becoming a structural part of the CE. Human keratinocytes were obtained from shave biopsies of adult epidermis (lower back) and were primary cultured according to the Rheinwald-Green system (Rheinwald and Green, 1975Rheinwald J.G. Green H. Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma.Cell. 1975; 6: 317-330Abstract Full Text PDF PubMed Scopus (397) Google Scholar). First passage cells were seeded in keratinocyte growth medium (KGM; Biowhittaker, Walkersville, MD) as described byvan Ruissen et al., 1996van Ruissen F. de Jongh G.J. Zeeuwen P.L. van Erp P.E. Madsen P. Schalkwijk J. Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.J Cell Physiol. 1996; 168: 442-452Crossref PubMed Scopus (59) Google Scholar. Differentiation of keratinocytes was induced by switching confluent keratinocyte cultures for 72 h to KGM supplemented with 5% fetal bovine serum (FBS). Based on the cDNA sequence of cystatin M/E (Sotiropoulou et al., 1997Sotiropoulou G. Anisowicz A. Sager R. Identification cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.J Biol Chem. 1997; 272: 903-910Crossref PubMed Scopus (145) Google Scholar) we designed two oligonucleotide primers that could amplify a partial cDNA, encoding amino acid residue Pro23 to the stop codon at position 150 (cystatin M/E forward primer, 5′-GAAGATCTCCACGCGATGCCCG-3′; cystatin M/E reverse primer, 5′-CGGAATTCTCACATCTGCACACAG-3′), excluding the amino acid residues corresponding to the hydrophobic signal peptide (Sotiropoulou et al., 1997Sotiropoulou G. Anisowicz A. Sager R. Identification cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.J Biol Chem. 1997; 272: 903-910Crossref PubMed Scopus (145) Google Scholar). Polymerase chain reactions (PCR) were carried out using a DNA thermal cycler (PTC-200, Biozym, Landgraaf, The Netherlands) in 25 µl mixtures. The following buffer conditions were used: 10 mM Tris-HCl, pH 9.0, 1.5 mM magnesium chloride, 50 mM potassium chloride, 0.1% Triton X-100, all four dNTPs (each at 200 µM), 1 unit of Taq DNA Polymerase (Promega, Madison, WI), and 20 pmol of each primer. After an initial incubation of 6 min at 94°C amplification was conducted for one cycle of 1 min at 94°C, 1 min at 47°C, and 2 min at 72°C, followed by 34 cycles with an annealing temperature of 60°C. An additional 10 min at 72°C was used for the last cycle. As template for the PCR we used cystatin M cDNA (Sotiropoulou et al., 1997Sotiropoulou G. Anisowicz A. Sager R. Identification cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.J Biol Chem. 1997; 272: 903-910Crossref PubMed Scopus (145) Google Scholar). The amplified fragment was cloned in frame into the pFastbac-HTb baculovirus expression vector, a component of the Bac-to-Bac Baculovirus Expression System (Life Technologies). After the recombinant pFastbac-HTb donor plasmid has been determined to be correct, the DNA is transformed into competent DH10Bac Escherichia coli cells for transposition into a baculovirus shuttle vector (bacmid), according to the protocol provided by the manufacturer. High molecular weight mini-prep DNA is prepared from selected E.coli clones containing the recombinant bacmid. These clones were screened for the presence of the desired sequences, and then used to transfect insect cells. Recombinant bacmid was transfected into Spodoptera frugiperda (Sf9) cells using Celfectin Reagent (Life Technologies) according to the protocol provided by the manufacturer, followed by infection of insect cells with recombinant baculovirus particles. The infected cells were harvested and lyzed, the cell debris was removed by centrifugation, and the supernatant, which contains the recombinant (His)6-tagged cystatin M/E protein, was stored at 4°C. We purified recombinant cystatin M/E with a Tris-based buffer system according to the protocol provided by the manufacturer (Bac-to-Bac Baculovirus Expression System, Life Technologies), with the exception that 2-mercaptoethanol and glycerol were omitted from all buffer solutions. To obtain recombinant cystatin M/E without the polyhistidine tag, the baculovirus expressed protein was digested with tobacco etch virus (TEV) proteinase according to the protocol provided by the manufacturer (Life Technologies). The recombinant plasmid pGEX-2T/cystatin M/E was used to produce and purify recombinant GST-cystatin M/E fusion protein as described previously (Sotiropoulou et al., 1997Sotiropoulou G. Anisowicz A. Sager R. Identification cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.J Biol Chem. 1997; 272: 903-910Crossref PubMed Scopus (145) Google Scholar). To obtain recombinant cystatin M/E without the GST tag, the bacterially expressed fusion protein was digested with thrombin according to the protocol provided by the manufacturer (Sigma). The purified GST-cystatin M/E fusion protein was used to immunize a New Zealand White rabbit and a Duncan Hartley guinea pig, which was carried out at the Central Animal Laboratory, University of Nijmegen, The Netherlands. Antisera raised against the GST-cystatin M/E fusion protein were purified by affinity chromatography using recombinant fusion protein coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech). Autopsic material was obtained from the Department of Pathology, University of Nijmegen, The Netherlands. The following tissues were studied with respect to cystatin M/E expression: skin, tongue, gingiva, palatum, pharynx, nasal cavity, sole of the foot, esophagus, ileum, colon, stomach, bronchus, lung, trachea, ureter, kidney, bladder, pancreas, liver, heart, spleen, skeleton muscle, lymph node, aorta, cartilage, mammary gland, and uterus. Biopsies of normal skin and from psoriatic lesions were taken under local anesthesia with a keratome as described byAlkemade et al., 1994Alkemade J.A. Molhuizen H.O. Ponec M. et al.SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes.J Cell Sci. 1994; 107: 2335-2342Crossref PubMed Google Scholar. Human skin biopsies and autopsic material were processed for IHC as previously described (Latijnhouwers et al., 1996Latijnhouwers M.A. Bergers M. van Bergen B.H. Spruijt K.I. Andriessen M.P. Schalkwijk J. Tenascin expression during wound healing in human skin.J Pathol. 1996; 178: 30-35Crossref PubMed Scopus (70) Google Scholar). The sections were subsequently incubated with affinity-purified polyclonal rabbit anticystatin M/E antibodies at a 1:50 dilution followed by IHC staining as described previously (Latijnhouwers et al., 1996Latijnhouwers M.A. Bergers M. van Bergen B.H. Spruijt K.I. Andriessen M.P. Schalkwijk J. Tenascin expression during wound healing in human skin.J Pathol. 1996; 178: 30-35Crossref PubMed Scopus (70) Google Scholar). Total RNA from human cultured keratinocytes and human tissues was isolated with RNA extraction solution (Wingens et al., 1999Wingens M. Pfundt R. Vlijmen-Willems I.M. van Hooijdonk C.A. van Erp P.E. Schalkwijk J. Sequence-specific inhibition of gene expression in intact human skin by epicutaneous application of chimeric antisense oligodeoxynucleotides.Lab Invest. 1999; 79: 1415-1424PubMed Google Scholar). mRNA was isolated using the Quickprep Micro mRNA Purification Kit (Pharmacia Biotech), according to the manufacturer's protocol. First strand cDNA was generated from mRNA as described previously (Zeeuwen et al., 1997Zeeuwen P.L. Hendriks W. de Jong W.W. Schalkwijk J. Identification and sequence analysis of two new members of the SKALP/elafin and SPAI-2 gene family. Biochemical properties of the transglutaminase substrate motif and suggestions for a new nomenclature.J Biol Chem. 1997; 272: 20471-20478Crossref PubMed Scopus (51) Google Scholar). The reverse transcriptase reaction products were used for PCR amplification to obtain the cDNA of cystatin M/E and the housekeeping gene human acidic ribosomal phosphoprotein P0 (hARP). We designed oligonucleotide primers that could amplify the cystatin M/E cDNA including the ATG start codon and the TGA stop codon. These oligonucleotide sequences (first sense then antisense) for the cystatin M/E PCR were 5′-TCCGACGGCACTGACGGC-3′ and 5′-CCAATGGCCTTCGCCCTCG-3′. The oligonucleotide sequences (first sense then antisense) to generate hARP cDNA were 5′-ATGTGAAGTCACTGTGCC-3′ and 5′-ACCAAATCCCATATCCTC-3′. PCR were carried out using a DNA thermal cycler (TB1, Biometra, Göttingen, Germany) in 25 µl mixtures. The following buffer conditions were used: 10 mM Tris-HCl, pH 9.0, 1 mM magnesium chloride, 50 mM potassium chloride, 0.1% Triton X-100, all four dNTPs (each at 200 µM), 1 unit of Taq DNA Polymerase (Promega), and 20 pmol of each primer. After an initial incubation of 6 min at 94°C amplification was conducted for 35 cycles as follows: 1 min at 94°C, 1 min at annealing temperature, and 2 min at 72°C. An additional 10 min at 72°C was used for the last cycle. Annealing temperatures were 58°C when using the cystatin M/E primers and 47°C for the hARP primers. PCR products were analyzed by agarose gel electrophoresis. Northern blot analysis was carried out as described byvan Ruissen et al., 1996van Ruissen F. de Jongh G.J. Zeeuwen P.L. van Erp P.E. Madsen P. Schalkwijk J. Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.J Cell Physiol. 1996; 168: 442-452Crossref PubMed Scopus (59) Google Scholar. Cystatin M/E cDNA (Sotiropoulou et al., 1997Sotiropoulou G. Anisowicz A. Sager R. Identification cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.J Biol Chem. 1997; 272: 903-910Crossref PubMed Scopus (145) Google Scholar) and hARP cDNA were labeled with (α-32P)dCTP using the Oligolabeling Kit (Pharmacia Biotech) according to the protocol provided by the manufacturer. Proteinase inhibitory activity of recombinant cystatin M/E was determined by measuring the inhibition of papain (Sigma) and cathepsin B (ICN Pharmaceuticals, Costa Mesa, CA) essentially described byAbrahamson, 1994Abrahamson M. Cystatins.Meth Enzymol. 1994; 244: 685-700Crossref PubMed Scopus (171) Google Scholar, using the fluorogenic synthetic substrate Z-Phe-Arg-AMC (Sigma). Papain/cathepsin B was titrated in the absence and presence of increasing concentrations of recombinant cystatin M/E. Extracts of normal human skin were tested for cathepsin C activity by measuring the hydrolysis of fluorogenic substrate H-Gly-Phe-AMC (Bachum, Bubendorf, Switzerland) using a modified protocol (Toomes et al., 1999Toomes C. James J. Wood A.J. et al.Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis.Nat Genet. 1999; 23: 421-424https://doi.org/10.1038/70525Crossref PubMed Scopus (373) Google Scholar). The amount of fluorescence signal produced from the substrate by skin extract was measured in sodium phosphate buffer (0.1 M, pH 5.5) containing 100 mM NaCl and 2 mM dithiothreitol. Substrate hydrolysis was measured after an incubation of 30 min at 37°C. Skin extract (containing cathepsin C) was titrated in the absence and presence of increasing concentrations of recombinant cystatin M/E. Cystatin M/E concentrations were measured in supernatant of cultured keratinocytes and human sweat and nasal mucus, using a
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