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Proteinase Complex during Keratinocyte Differentiation
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
245, 263–271 (1998)
EX984241
Proteinase Inhibitor 6 (PI-6) Expression in Human Skin: Induction of PI-6 and a PI-6/Proteinase Complex during Keratinocyte Differentiation Fiona L. Scott,* Joanna E. Paddle-Ledinek,† Loretta Cerruti,* Paul B. Coughlin,* Hatem H. Salem,* and Phillip I. Bird*,1 *Department of Medicine, Monash University, Box Hill Hospital 3128, Melbourne, Australia; and †Department of Surgery, Monash University Medical School, Alfred Hospital, Melbourne, Australia
Proteinase inhibitor 6 (PI-6) is a 42-kDa intracellular protein present in epithelial cells and endothelial cells. It is capable of inhibiting a number of serine proteinases, including trypsin and chymotrypsin. In this study we examined PI-6 expression in human skin and its primary cell type, the keratinocyte. By immunohistochemical analysis, PI-6 staining is absent from the basal cells, weak in the spinous layer, and strongest in the granulosa layer of human epidermis. Immunoblotting of cultured primary keratinocytes revealed that PI-6 production increases 24-fold on differentiation. Analysis of an immortalized keratinocyte cell line, HaCat, showed a 5-fold increase in PI-6 mRNA and a 7-fold increase in PI-6 protein upon differentiation, and indirect immunofluorescence revealed that this is due to an increase in the number of differentiated cells expressing high levels of PI-6. Of particular interest is the appearance of a preformed complex between PI-6 and an endogenous serine proteinase in differentiating HaCat cells, which was detected by a monoclonal antibody demonstrated to preferentially recognize PI-6 in complex with a proteinase. This identification of a PI-6/proteinase complex is the first example of a serpin bound to a proteinase in keratinocytes. We postulate that a physiological role of PI-6 is to regulate a serine proteinase associated with keratinocyte differentiation. © 1998 Academic Press
INTRODUCTION
Serine proteinase inhibitors (serpins) are involved in the regulation of many proteolysis-dependent physiological processes, including fibrinolysis, blood coagulation, complement activation, tissue remodeling, cell-mediated cytotoxicity, cell migration, and differentiation [1]. Most are extracellular proteins that regulate extracellular serine proteinases [1], but there are 1
To whom correspondence and reprint requests should be addressed. Fax: 1161 03 98950332. E-mail: [email protected]. monash.edu.au.
recent examples of intracellular serpins that control proteolysis within cells. These intracellular serpins include viral molecules such as cytokine response modifier A, which is capable of inhibiting both granzyme B and the intracellular caspases involved in apoptosis and cytokine maturation [2, 3]. Other intracellular serpins belong to the mammalian ovalbumin serpin group (ov-serpins), which comprises proteins that lack classical N-terminal signal sequences, and display either intracellular distribution (PI-6, PI-9, PI-8), extracellular distribution (ovalbumin, maspin), or a combination of both (plasminogen activator inhibitor-2 (PAI-2) and squamous cell carcinoma antigen) [4]. Of the intracellular ov-serpins, only PI-9 has been shown to have a physiologically relevant intracellular function as a granzyme B inhibitor and may protect particular cells against granzyme B-mediated apoptosis [5]. Although no physiological role for the other intracellular ovserpins has been defined, their presence inside cells suggests a role in regulating intracellular proteolysis. The ov-serpin proteinase inhibitor 6 (PI-6) was first identified through its ability to form SDS-stable complexes with thrombin [6]. It is a 42-kDa intracellular protein that cannot be secreted through the endoplasmic reticulum–Golgi secretory pathway [7]. It contains a P1-Arg at its reactive site, suggesting inhibitory activity against tryptic proteinases, although its actual inhibitory profile is broader than this P1 residue would suggest. In vitro, PI-6 rapidly inhibits and forms SDSstable complexes with plasmin, trypsin, chymotrypsin, and thrombin and is a slower inhibitor of urokinasetype plasminogen activator and activated protein C [8, 9]. It is expressed in many human tissues but not brain [10], and immunohistochemistry experiments have shown that PI-6 is synthesized primarily in endothelial cells and epithelial cells [7]. Its broad inhibitory capacity and wide physiological distribution indicates that PI-6 may regulate the activity of intracellular proteinases in a number of physiological processes. During an immunohistochemical survey of human tissues, we noted PI-6 expression in human skin. This
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is interesting because alterations in the expression of several ov-serpins and proteases have been observed in keratinocyte differentiation and skin disease states. For example, in an in vitro model of epidermis differentiation, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are synthesized in less differentiated keratinocytes, and tissue-type plasminogen activator (t-PA) and PAI-2 are synthesized in more differentiated keratinocytes [11]. Increases in proteinases, such as u-PA and t-PA, have been noted in association with psoriasis and pemphigus [12, 13], and squamous cell carcinoma antigen-1 is upregulated 40-fold, and is located in the upper spinous layers, of psoriatic skin [14]. Other nonserpin proteinase inhibitors are also found in epidermis. Antileukoprotease is an inhibitor of stratum corneum chymotryptic enzyme (SCCE), a protease involved in desquamation [15]; elafin expression is induced during inflammatory diseases of the skin and inhibits mast cell proteinases such as proteinase-3 and elastase [16]; and tissue factor pathway inhibitor-2 is secreted into the extracellular matrix of cultured human keratinocytes [17]. In this study, we have examined PI-6 expression in human epidermis in detail using both primary human keratinocytes and an immortalized keratinocyte cell line, HaCat. PI-6 was found in differentiated but not in basal keratinocytes and formed an SDS-stable complex with an endogenous proteinase in differentiating HaCat cells. We conclude that PI-6 expression in epidermis is linked to differentiation and that PI-6 may regulate a serine proteinase associated with keratinocyte differentiation. MATERIALS AND METHODS Cell culture. Keratinocyte cells were isolated to generate “basal” or “stratified” cultures as described [18] except that the source was neonatal foreskin. HaCat cells (passage 35) [19], a spontaneously immortalized human keratinocyte cell line, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 50 mg/ml streptomycin. HaCat cells were plated at ;1.75 3 104/cm2, and medium was replenished every 3 days. COS-7 cells were cultured in DMEM with the above supplements. Monoclonal 3A hybridoma was maintained in RPMI 1640 medium with the above supplements plus 1 mM sodium pyruvate. Cells were incubated in a 5% CO2, 95% air mixture at 37°C and were maintained at subconfluent density. Antibodies. The rabbit polyclonal anti-PI-6 antiserum has been described [7, 10]. Monoclonal anti-PI-6 antibody was produced using standard methods [20]. Briefly, Balb/c mice were injected with 10 mg of purified recombinant human PI-6 (rPI-6) [8] in complete Freund’s adjuvant and then twice in incomplete Freund’s adjuvant. Spleen cells were isolated and fused with Sp-2 cells and the hybridomas producing immunoreactive antibody were identified using ELISA on immobilized rPI-6 and indirect immunofluorescence on PI-6-transfected COS cells. The hybridoma producing monoclonal 3A was further purified by two rounds of limiting dilution, and the antibody was typed using an isotyping kit (Amersham). Monoclonal anti-cytokeratin 10 (anti-CK10) antibody was from DAKO.
Immunohistochemistry. Preparation of 4-mm Formalin-fixed paraffin-embedded tissue samples was as described [21]. Sections were incubated in rabbit anti-PI-6 antiserum or preimmune serum diluted 1:200, washed in PBS, and then incubated for 20 min in a 1:200 dilution of biotinylated swine anti-rabbit immunoglobulins (DAKO E353). Following a further wash in PBS, streptavidin– horseradish peroxidase (DAKO K377) was added for 30 min. Sections were washed again in PBS and developed using diaminobenzidine (DAB, DAKO). COS-7 cell transfection. COS-7 cells were transfected with pSVTfPTI/P (containing PI-6 cDNA) or pSVTf (mock) [10] using the DEAE– dextran/chloroquine method as described [22]. [35S]Methionine labeling and immunoprecipitation. At 48 h posttransfection, 2 3 106 COS cells were [35S]methionine labeled as previously described [7]. Briefly, transfected cells were starved in medium lacking methionine for 30 min followed by a 3-h labeling period with 100 mCi of [35S]methionine and [35S]cysteine (Expre35s35s protein labeling mix, DuPont NEN). Cells were harvested and lysed with ice-cold 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.25% (w/v) gelatin (NETGEL) containing 1% Nonident P-40, 150 mg/ml PMSF, 1 mg/ml aprotinin, and 0.5 mM leupeptin. Cell debris was pelleted and discarded and cytosolic extract was immunoprecipitated overnight at 4°C with 100 ml of 10% (w/v) protein A–Sepharose (Pharmacia Biotech Inc.) and 5 ml rabbit polyclonal anti-PI-6 antiserum, 5 ml rabbit preimmune sera, or 50 ml monoclonal 3A anti-PI-6 hybridoma supernatant with 2 ml rabbit anti-mouse immunoglobulin (DAKO) bridging antibody. Immune complexes were pelleted and washed twice in NETGEL containing 250 mM NaCl and 0.025% (w/v) SDS and once in 10 mM Tris pH 8.0. Samples were resuspended in 30 ml of 20 mM Tris, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.1 M dithiothreitol (reducing buffer) and then analyzed by 10% SDS–PAGE according to Laemmli [23]. Gels were enhanced in Amplify (Amersham Corp.), and visualized by fluorography. Immunoblot. Primary keratinocyte cultures, HaCat cells, and COS cells were washed with ice-cold PBS and subsequently lysed with 10 mM Tris–HCl, 150 mM NaCl, 100 mM EDTA, 1% Triton X-100 containing 1 mg/ml aprotinin, 150 mg/ml PMSF, and 0.5 mM leupeptin, pH 7.6. After centrifugation, total protein concentration was determined (Bio-Rad). Protein (60 mg) was boiled with an equivalent volume of Laemmli sample buffer (20 mM Tris, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 0.1 M DTT) for 5 min and resolved on 10% SDS–PAGE [23]. In some instances, COS cell lysates were incubated with 0.2 units of thrombin (Sigma) at 37°C for 10 min before the addition of sample buffer. After electrophoresis, proteins were transferred to nitrocellulose membranes, and the membranes were incubated for 1 h in blocking buffer (5% skim milk powder in 20 mM Tris–HCl/150 mM NaCl, pH 7.6). Membranes were incubated for 1 h with rabbit polyclonal anti-PI-6 antiserum (diluted 1:5000 in 20 mM Tris–HCl/150 mM NaCl, pH 7.6) or monoclonal 3A hybridoma supernatant (neat) and washed in 20 mM Tris–HCl, 150 mM NaCl, pH 7.6. Horseradish peroxidase (HRP)-conjugated sheep anti-rabbit Ig (Silenus) or HRP-conjugated sheep anti-mouse Ig (Bio-Rad) was used as the secondary antibody and detection was with the enhanced chemiluminescence system (DuPont). In some experiments, the membrane was first probed with mouse monoclonal anti-PI-6 antibody and developed, and the same membrane was then probed with rabbit polyclonal anti-PI-6 antiserum and developed. Images of primary keratinocytes and HaCat cell immunoblots were analyzed via densitometry using the Gel-Pro Analyzer program. Indirect immunofluorescence. HaCat cells were seeded at 1.75 3 104 cells/cm2 on glass coverslips and cultured for the indicated time period. Cells were prepared for immunofluorescence as described [24]. Permeabilized cells were simultaneously incubated with rabbit anti-PI-6 diluted 1:1000 and monoclonal anti-CK10 diluted 1:50, both in PBS with 0.1 mM CaCl2 and 1 mM MgCl2. Cells were then incubated with RITC-conjugated sheep anti-mouse Ig (Silenus) and
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FITC-conjugated sheep anti-rabbit Ig (Silenus), both diluted 1:200 with PBS Ca/Mg and mounted. mRNA analysis. Total RNA was prepared from HaCat cells at various time points according to Chomczynski and Sacchi [25]. Total RNA (20 mg) was separated by electrophoresis on a 1% formaldehyde–agarose gel, transferred to Genescreen Plus (NEN Lifescience Products), and fixed to the membrane by baking at 80°C. The membrane was hybridized to a [32P]dATP-labeled (random priming kit, Promega) PI-6 cDNA probe by standard procedures [26]. The membrane was initially washed at low stringency (23 SSC at room temperature for 1 h) and exposed to film to visualize 28S ribosomal RNA. The membrane was then washed at high stringency (0.13 SSC at 68°C for 20 min) and exposed to film. Images were analyzed by densitometry using Gel-Pro Analyzer program.
RESULTS
Immunohistochemical Localization of PI-6 Protein to the Suprabasal Layers of Human Epidermis PI-6 expression in human epidermis was examined using a polyclonal anti-PI-6 antibody. Basal keratinocytes do not produce any PI-6 protein, as demonstrated by immunohistochemistry (Fig. 1A). In contrast, keratinocytes residing in the suprabasal layers do produce PI-6. Staining is weak in the cells of the early spinous layer, increases as the cells differentiate and migrate vertically through the spinous layer, and most intense at the granulosa layer. The staining observed is cytosolic, with none in the intercellular spaces. This intracellular localization of PI-6 is consistent with previous reports that it cannot be secreted from cells and that it is confined to the cytosol [7]. Positive staining of endothelial cells is also consistent with our previous report [7]. Serial sections stained with preimmune serum show no staining (Fig. 1B). PI-6 Is in Differentiated Primary Keratinocytes but Not in Basal Cells In order to examine the levels of PI-6 expression in the different stages of keratinocyte differentiation, immunoblotting of keratinocyte extracts was performed. Human keratinocytes were isolated from neonatal skin and grown in culture to generate basal keratinocytes or stratified/differentiated keratinocytes. Immunoblotting of cell extracts prepared from these cultures revealed that PI-6 is not significantly expressed in the proliferating basal cells (Fig. 2). This is consistent with the lack of staining of basal cells seen by immunohistochemistry (Fig. 1). By contrast, high levels of PI-6 expression were detected in keratinocytes grown under culture conditions that promote differentiation and stratification (Fig. 2). The increase in PI-6 protein during keratinocyte differentiation is 24-fold, as determined by densitometry. This is consistent with strong PI-6 staining in suprabasal layers of human epidermis.
FIG. 1. PI-6 in human epidermis is confined to suprabasal keratinocytes. (A) Immunostaining using polyclonal anti-PI-6 antiserum. (B) Immunostaining using preimmune sera. Serial sections were dewaxed, blocked, and immunoreacted with polyclonal antiPI-6 antibody as described under Materials and Methods. Detection was using a biotin–streptavidin–HRP system and DAB as substrate. Note strong intracellular staining for PI-6 in the spinous and granular layers of epidermis and lack of staining in basal cells. Original magnification is 380. B, basal layer; S, spinous layer; G, granular layer.
PI-6 mRNA Increases in Differentiating HaCat Cells HaCat cells are an immortalized human keratinocyte cell line of basal cell phenotype that retain the potential to undergo keratinocyte differentiation [19]. After seeding, they undergo a proliferative phase until the cells reach confluence (;7 days), and this is followed by differentiation [27–29]. On in vitro differentiation, HaCat cells express markers of mature keratinocytes such as cytokeratins 1 and 10 [19, 30]. Stratification of cells also occurs in conventional submerged culture, although not to the same extent as when HaCat cells are transplanted onto nude mice, where complete formation of a highly ordered epider-
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pected for normal expression of this protein. PI-6 expression is also seen, although at substantially lower levels, in cells that are not producing detectable CK10. This indicates that induction of PI-6 expression occurs earlier than CK10 expression during HaCat differentiation. This is not surprising as it has been reported that the induction of CK10 during HaCat differentiation is slightly delayed compared to that observed in vivo [30]. Finally, by day 10 in culture, the number of cells expressing CK10 has increased and they are confined to the upper cellular layers (Fig. 4). CK10-positive HaCat cells of the upper layer express PI-6 protein more strongly than those cells found in the lower layers that do not express CK10 (Fig. 4). FIG. 2. PI-6 expression is increased in differentiated human keratinocytes. Primary keratinocyte extracts were prepared, resolved on 10% SDS–PAGE, and transferred to nitrocellulose according to Materials and Methods. Immunoblotting with polyclonal antiPI-6 antiserum demonstrates a 42-kDa PI-6 band which increases 24-fold in stratified keratinocytes.
Induction of a PI-6/Proteinase Complex in Differentiating HaCat Cells Immunoblotting of HaCat cell extracts with polyclonal anti-PI-6 antiserum detects the 42-kDa PI-6 pro-
mis is observed [19]. To determine whether the increase in PI-6 protein observed in primary keratinocyte differentiation is due to a rise in PI-6 mRNA, Northern analysis of total RNA extracted from differentiating HaCat was performed. Figure 3B shows that the 1.4-kb PI-6 mRNA [10] is detected in HaCat cells as early as day 2 in culture. Scanning densitometry analysis of the PI-6 signal, when corrected for loading of RNA, reveals that the level of PI-6 mRNA increases approximately fivefold after the cells have been in culture for 21 days (Fig. 3A). Localization of PI-6 in Differentiating HaCat Cells To establish the profile of PI-6 expression in HaCat cells, indirect immunofluorescence analysis of cultures at different stages of differentiation was performed using polyclonal anti-PI-6 antisera. Staining for the keratinocyte differentiation marker cytokeratin 10 was done simultaneously. After 2 days in culture, HaCat cells are highly proliferative and show no morphological evidence of differentiation or stratification by phase-contrast microscopy (Fig. 4). By indirect immunofluorescence, these basal-like cells show no cytokeratin 10 (CK10) expression and little PI-6 expression. By day 5, the HaCat cells have formed clusters, with some cells residing on top of those attached to the tissue culture dish (in Fig. 4 at day 5, the phasecontrast photo is focused on cells in the upper layer). These stratified cells express high levels of CK10, indicating that they are differentiating. When the same field is stained with polyclonal anti-PI-6 antiserum, those cells expressing CK10 also express high levels of PI-6. The PI-6 staining is cytosolic, as would be ex-
FIG. 3. PI-6 mRNA increases in differentiating HaCat cells. (A) Fold increase of PI-6 mRNA as determined by scanning densitometry and corrected for equal loading of total RNA as determined via the amount of 28S ribosomal RNA. (B) Autoradiograph of PI-6 mRNA species and 28S ribosomal RNA species. PI-6 mRNA increases approximately fivefold by day 21 of HaCat differentiation. Total RNA (20 mg) was resolved on a 1% agarose gel containing formaldehyde, transferred to Genescreen Plus membrane, and hybridized with [32P]dATP-labeled PI-6 cDNA probe. The membrane was initially washed in 23 SSC at room temperature and exposed to film and then 0.13 SSC at 68°C and exposed to film. Images were analyzed via densitometry.
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FIG. 4. Immunolocalization of PI-6 to the cytosol of differentiating HaCat cells. HaCat cells grown on glass coverslips were fixed and permeabilized as described under Materials and Methods at various time points. Cells were double stained with monoclonal mouse anti-CK10 antibody and polyclonal rabbit anti-PI-6 antiserum. Images are of the same field. (Phase) Phase-contrast microscopy; (CK10) RITC fluorescence microscopy of mouse anti-cytokeratin 10 staining; (PI-6) FITC fluorescence microscopy of polyclonal rabbit anti-PI-6 antiserum staining. Day 2 and day 5 micrographs were photographed at 3200 magnification; day 10 at 3100 magnification.
tein after 4 days of culture (Fig. 5A). At this time, the cells are of a basal-like phenotype and have begun to form “islands,” with those in the center beginning to differentiate and stratify (Fig. 4). As the HaCat cells differentiate further in culture, expression of PI-6 increases 1.9-fold by day 8, 4.3-fold by day 10, and 7.1fold by day 14 (Fig. 5B). Also, by day 10 in culture, when there is a well-formed sheet of cells and the development of a more complete stratified layer, a species of approximately 55 kDa is detected that increases in abundance from days 10 to 14, correlating with a
state of further differentiation. This 55-kDa species may represent either a distinct non-PI-6 protein that cross-reacts with the PI-6 antiserum or PI-6 complexed to an endogenous proteinase. In order to determine whether this 55-kDa species contains PI-6, we raised monoclonal anti-PI-6 antibodies. Of several monoclonal antibodies produced, clone 3A (IgG2b/k) had the highest affinity for PI-6 (data not shown) and was used in subsequent experiments. Immunoprecipitation of 35S-labeled PI-6 from transfected COS cells with the monoclonal 3A detected a 42-kDa
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FIG. 5. PI-6 protein and a PI-6/proteinase complex is induced during HaCat cell differentiation. (A) Immunoblot of differentiating HaCat cells with polyclonal and monoclonal anti-PI-6 antibodies. HaCat cell extracts were prepared at indicated time points as described under Materials and Methods. Equal loadings of protein were resolved on 10% SDS–PAGE, transferred to nitrocellulose, blocked, and immunoblotted first with monoclonal anti-PI-6 and developed and then with polyclonal anti-PI-6. (B) Fold increase of the 42-kDa PI-6 species as determined by scanning densitometry.
protein (Fig. 6A). This agrees with the previously reported size of PI-6 [6] and is similar to that precipitated by the rabbit polyclonal anti-PI-6 antiserum (Fig. 6A). Endogenous PI-6 in the mock-transfected COS cells was also detected by the polyclonal anti-PI-6 antiserum but not the monoclonal 3A. This indicates that the 3A antibody is species-specific and does not crossreact with monkey PI-6. The polypeptides of ;35 and ;30 kDa precipitated by both the polyclonal and monoclonal anti-PI-6 antibodies are most likely rPI-6 degradation products. The monoclonal 3A failed to detect denatured PI-6 via immunoblotting of extracts from transfected COS cells (Fig. 6B). This suggests that the monoclonal rec-
ognizes a conformational epitope on native PI-6 that is sensitive to SDS. However, when the extracts were incubated with thrombin prior to SDS–PAGE, a PI-6/ thrombin complex of ;67 kDa was detected by immunoblotting. Thus binding to a proteinase apparently results in a conformational change that stabilizes the SDS-sensitive epitope on PI-6 that is recognized by monoclonal 3A. This is not surprising, considering the large conformational change that serpins undergo on binding of a proteinase [31], and there are several well-characterized precedents for monoclonal antibodies recognizing serpin/proteinase complexes better than the native serpin. For example, the monoclonal anti-PAI-2 antibody, 2H5, only recognizes PAI-2 in
FIG. 6. Monoclonal 3A recognizes native PI-6 and denatured PI-6/thrombin complexes but not denatured PI-6 alone. (A) Immunoprecipitation of 35S-labeled PI-6 by monoclonal 3A anti-PI-6 antibody. Mock, pSVTf transfected; PI-6, pSVTfPTI/P transfected. At 48 h posttransfection COS cells were starved for 30 min in media lacking methionine, labeled for 3 h in media containing 100 mCi [35S]methionine, lysed, and immunoprecipitated according to Materials and Methods. Immune complexes were collected, reduced, resolved on 10% SDS– PAGE, and analyzed by fluorography. (B) Immunoblot of P-6/thrombin complexes with monoclonal 3A anti-PI-6 antibody, rabbit polyclonal anti-PI-6 antiserum, and rabbit preimmune serum. Extracts of PI-6-transfected COS cells were prepared 48 h posttransfection and were incubated with (1) or without (2) thrombin at 37°C for 10 min. Equal loadings of protein were resolved on 10% SDS–PAGE, transferred to nitrocellulose, blocked, immunoblotted with the appropriate antibody, and analyzed by fluorography.
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complex with u-PA or t-PA [32], and the monoclonal anti-PAI-1 antibodies, MA-13C1 and MA-14D5, have stronger affinity for PAI-1 complexed with t-PA than for PAI-1 alone [33]. Immunoblotting of the HaCat extracts with monoclonal 3A demonstrated that the 55-kDa species is also recognized by the monoclonal 3A antibody (Fig. 5A). Because the monoclonal antibody preferentially recognizes PI-6/proteinase complexes, this species probably represents PI-6 complexed to an endogenous proteinase present in HaCat cells. The HaCat cell extracts were prepared with lysis buffer containing EDTA and the proteinase inhibitors aprotinin, leupeptin, and PMSF. These inhibitors ablate proteinase activity and prevent serine proteinases and serpins forming SDSstable complexes during, or after, cell lysis. The 55-kDa PI-6/proteinase complex detected in HaCat cell extracts occurred in the presence of these compounds. This suggests the PI-6/proteinase complex is already present in the cell prior to cell lysis and does not form as a consequence of the extract preparation. The protein of approximately 82 kDa also seen in Fig. 5A is not a PI-6-specific protein as it is recognized by the preimmune sera (data not shown). Given that PI-6 is 42 kDa, the size of the PI-6/ proteinase complex suggests that the proteinase component is approximately 20 –30 kDa. This may represent either the entire proteinase or a subchain of a larger enzyme released due to disulphide bond disruption with reducing SDS–PAGE conditions. The stability of the complex in SDS, which is a hallmark of serpin–serine proteinase interactions [1], strongly suggests that the enzyme is a serine proteinase. It is possible that PI-6 regulates a process involving a serine proteinase in keratinocyte differentiation or desquamation. So far two proteinases have been described that are thought to play a role in the process of desquamation. These are the SCCE and an unidentified trypsin-like proteinase [34 –37]. The ability of PI-6 to inhibit chymotryptic- and tryptic-like proteinases suggests that it may be able to regulate either or both of these proteinases. However, both of these enzymes are extracellular [38], whereas PI-6 is located intracellularly. Since PI-6 can not be released from cells through the endoplasmic reticulum–Golgi network [7], and PI-6 protein has never been detected in plasma or in the media of cultured cells, PI-6 is unlikely to regulate the extracellular functions of these proteinases. Perhaps PI-6 resides in keratinocytes to protect them from exposure to their own desquamation proteinases, either during their production and packaging into secretory vesicles or resulting from inadvertent endocytosis from the intercellular space. This role would be similar to that proposed for PI-9, which resides in the cytosol of cytotoxic lymphocytes to protect them from inadvertent expo-
sure to their own granzyme B during proteinase packaging or degranulation [5]. Another process that occurs during keratinocyte differentiation is the maturation of profilaggrin to filaggrin. Filaggrin is involved in the keratin rearrangements necessary for cornified envelope formation during terminal differentiation. Profilaggrin is stored in non-membrane-bound cytosolic keratohyalin granules as an insoluble precursor. At terminal differentiation, granules disperse and profilaggrin is processed by phosphatases, endopeptidases, and exopeptidases [39 – 42]. In the rat, processing involves profilaggrin endoproteinase-1 (PEP-1), an intracellular chymotryptic-like serine proteinase. PEP-1 is thought to act in the cytosol [43], and PI-6, due to its ability to inhibit chymotryptic-like proteinases and its cytosolic location, may be a suitable regulator of this protease. At present a human counterpart of PEP-1 has not been described, although profilaggrin processing does occur in human keratinocytes. In conclusion, we have shown that PI-6 expression is induced during primary keratinocyte and HaCat cell differentiation. We have also demonstrated that PI-6 interacts with an endogenous protease in differentiating HaCat cells. To our knowledge, this is the first demonstration of a serpin interacting with a target proteinase in human keratinocyte cells. At present, it is unclear whether this proteinase (and its interaction with PI-6) is keratinocyte-specific or whether it also occurs during differentiation of other PI-6 expressing epithelial cells [7]. REFERENCES 1.
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SCOTT ET AL. teinase inhibitor 6 expressed in Pichia pastoris. Biochim. Biophys. Actal. 1252, 28 –34. Riewald, M., and Schleef, R. R. (1996). Human cytoplasmic antiproteinase neutralizes rapidly and efficiently chymotrypsin and trypsin-like proteases using distinct reactive site residues. J. Biol. Chem. 271, 14526 –14532. Coughlin, P. B., Sun, J., Cerruti, L., Salem, H. H., and Bird, P. (1993). Cloning and molecular characterization of a human intracellular serine proteinase inhibitor. Proc. Natl. Acad. Sci. USA 90, 9417–9421. Chen, C., Lyons-Giordano, B., Lazarus, G. S., and Jensen, P. (1993). Differential expression of plasminogen activators and their inhibitors in an organotypic skin coculture system. J. Cell Science 106, 45–53. Baird, J., Lazaruus, G. S., Belin, D., Vassali, J. D., Busso, N., Gubler, P., and Jensen, P. J. (1990). mRNA for tissue-type plasminogen activator is present in lesional epidermis from patients with psoriasis, pemphigus, or bullous pemphigoid, but is not detected in normal epidermis. J. Invest. Dermatol. 95, 548 –552. Grondahl-Hansen, J., Ralfkiaer, E., Nielsen, L. S., Kristensen, P., Frentz, G., and Dano, K. (1987). Immunohistochemical localization of urokinase- and-tissue-type plasminogen activators in psoriatic skin. J. Invest. Dermatol. 88, 28 –32. Rivas, M. V., Jarvis, E. D., Morisaki, S., Carbonaro, H., Gottlieb, A. B., and Krueger, J. G. (1997). Identification of abberantly regulated genes in diseased skin using the cDNA differential display technique. J. Invest. Dermatol. 108, 188 –194. Franzke, C., Baici, A., Bartels, J., Christophers, E., and Wiedow, O. (1996). Antileukoprotease inhibits stratum corneum chymotryptic enzyme. J. Biol. Chem. 271, 21886 –21890. Alkemade, J. A., Molhuizen, H. O., Pones, M., Kempenaar, J. A., Zeeuwen, P. L., de Jonh, G. J., van Vlijmen-Wilems, I. M., van Erp, P. E., van de Kerkhof, P. C., and Schalkwijk, J. (1994). SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes. J. Cell Sci. 107, 2335–2342. Rao, C. N., Reddy, P., Liu, Y., O’Toole, E., Reeder, D., Foster, D. C., Kisiel, W., and Woodley, D. T. (1996). Extracellular matrix-associated serine protease inhibitors (Mr 33,000, 31,000, and 27,000) are single-gene products with differential glycosyltion: cDNA cloning of the 33-kDa inhibtor reveals its identity to tissue factor pathway inhibitor-2. Arch. Biochem. Biophys. 335, 82–92. Paddle-Ledinek, J. E., Cruickshank, D. G., and Masterton, J. P. (1997). Skin replacement by cultured keratinocyte grafts: an Australian experience. Burns 23, 204 –211. Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., and Fusenig, N. E. (1988). Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761–771. Harlow, E., and Lane, D. (1988). “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Jackson, D. E., Mitchell, C. A., Bird, P., Salem, H. H., and Hayman, J. (1995). Immunohistochemical localization of thrombomodulin in normal human skin and skin tumours. J. Pathol. 175, 421– 432. Teasdale, M. S., Bird, C., and Bird, P. (1994). Internalization of the anticoagulant thrombomodulin is constitutive and does not require a signal in the cytoplasmic domain. Immunol. Cell Biol. 72, 480 – 488. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685.
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PI-6 IN HUMAN SKIN AND KERATINOCYTES sion of profilaggrin to filaggrin in mammalian epidermis. J. Biol. Chem. 264, 1837–1845. 41. Resing, K. A., Johnson, R. S., and Walsh, K. A. (1995). Characterization of protease processing sites durng conversion of rat profilaggrin to filaggrin. Biochemistry 32, 10036 –10045. 42. Resing, K. A., al-Alawi, N., Blomquist, C., Fleckman, P., and Dale, B. A. (1993). Independent regulation of two cytoplasmic Received March 30, 1998 Revised version received June 9, 1998
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Proteinase Inhibitor 6 (PI-6) Expression in Human Skin: Induction of PI-6 and a PI-6/Proteinase Complex during Keratinocyte Differentiation Fiona L. Scott,* Joanna E. Paddle-Ledinek,† Loretta Cerruti,* Paul B. Coughlin,* Hatem H. Salem,* and Phillip I. Bird*,1 *Department of Medicine, Monash University, Box Hill Hospital 3128, Melbourne, Australia; and †Department of Surgery, Monash University Medical School, Alfred Hospital, Melbourne, Australia
Proteinase inhibitor 6 (PI-6) is a 42-kDa intracellular protein present in epithelial cells and endothelial cells. It is capable of inhibiting a number of serine proteinases, including trypsin and chymotrypsin. In this study we examined PI-6 expression in human skin and its primary cell type, the keratinocyte. By immunohistochemical analysis, PI-6 staining is absent from the basal cells, weak in the spinous layer, and strongest in the granulosa layer of human epidermis. Immunoblotting of cultured primary keratinocytes revealed that PI-6 production increases 24-fold on differentiation. Analysis of an immortalized keratinocyte cell line, HaCat, showed a 5-fold increase in PI-6 mRNA and a 7-fold increase in PI-6 protein upon differentiation, and indirect immunofluorescence revealed that this is due to an increase in the number of differentiated cells expressing high levels of PI-6. Of particular interest is the appearance of a preformed complex between PI-6 and an endogenous serine proteinase in differentiating HaCat cells, which was detected by a monoclonal antibody demonstrated to preferentially recognize PI-6 in complex with a proteinase. This identification of a PI-6/proteinase complex is the first example of a serpin bound to a proteinase in keratinocytes. We postulate that a physiological role of PI-6 is to regulate a serine proteinase associated with keratinocyte differentiation. © 1998 Academic Press
INTRODUCTION
Serine proteinase inhibitors (serpins) are involved in the regulation of many proteolysis-dependent physiological processes, including fibrinolysis, blood coagulation, complement activation, tissue remodeling, cell-mediated cytotoxicity, cell migration, and differentiation [1]. Most are extracellular proteins that regulate extracellular serine proteinases [1], but there are 1
To whom correspondence and reprint requests should be addressed. Fax: 1161 03 98950332. E-mail: [email protected]. monash.edu.au.
recent examples of intracellular serpins that control proteolysis within cells. These intracellular serpins include viral molecules such as cytokine response modifier A, which is capable of inhibiting both granzyme B and the intracellular caspases involved in apoptosis and cytokine maturation [2, 3]. Other intracellular serpins belong to the mammalian ovalbumin serpin group (ov-serpins), which comprises proteins that lack classical N-terminal signal sequences, and display either intracellular distribution (PI-6, PI-9, PI-8), extracellular distribution (ovalbumin, maspin), or a combination of both (plasminogen activator inhibitor-2 (PAI-2) and squamous cell carcinoma antigen) [4]. Of the intracellular ov-serpins, only PI-9 has been shown to have a physiologically relevant intracellular function as a granzyme B inhibitor and may protect particular cells against granzyme B-mediated apoptosis [5]. Although no physiological role for the other intracellular ovserpins has been defined, their presence inside cells suggests a role in regulating intracellular proteolysis. The ov-serpin proteinase inhibitor 6 (PI-6) was first identified through its ability to form SDS-stable complexes with thrombin [6]. It is a 42-kDa intracellular protein that cannot be secreted through the endoplasmic reticulum–Golgi secretory pathway [7]. It contains a P1-Arg at its reactive site, suggesting inhibitory activity against tryptic proteinases, although its actual inhibitory profile is broader than this P1 residue would suggest. In vitro, PI-6 rapidly inhibits and forms SDSstable complexes with plasmin, trypsin, chymotrypsin, and thrombin and is a slower inhibitor of urokinasetype plasminogen activator and activated protein C [8, 9]. It is expressed in many human tissues but not brain [10], and immunohistochemistry experiments have shown that PI-6 is synthesized primarily in endothelial cells and epithelial cells [7]. Its broad inhibitory capacity and wide physiological distribution indicates that PI-6 may regulate the activity of intracellular proteinases in a number of physiological processes. During an immunohistochemical survey of human tissues, we noted PI-6 expression in human skin. This
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is interesting because alterations in the expression of several ov-serpins and proteases have been observed in keratinocyte differentiation and skin disease states. For example, in an in vitro model of epidermis differentiation, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are synthesized in less differentiated keratinocytes, and tissue-type plasminogen activator (t-PA) and PAI-2 are synthesized in more differentiated keratinocytes [11]. Increases in proteinases, such as u-PA and t-PA, have been noted in association with psoriasis and pemphigus [12, 13], and squamous cell carcinoma antigen-1 is upregulated 40-fold, and is located in the upper spinous layers, of psoriatic skin [14]. Other nonserpin proteinase inhibitors are also found in epidermis. Antileukoprotease is an inhibitor of stratum corneum chymotryptic enzyme (SCCE), a protease involved in desquamation [15]; elafin expression is induced during inflammatory diseases of the skin and inhibits mast cell proteinases such as proteinase-3 and elastase [16]; and tissue factor pathway inhibitor-2 is secreted into the extracellular matrix of cultured human keratinocytes [17]. In this study, we have examined PI-6 expression in human epidermis in detail using both primary human keratinocytes and an immortalized keratinocyte cell line, HaCat. PI-6 was found in differentiated but not in basal keratinocytes and formed an SDS-stable complex with an endogenous proteinase in differentiating HaCat cells. We conclude that PI-6 expression in epidermis is linked to differentiation and that PI-6 may regulate a serine proteinase associated with keratinocyte differentiation. MATERIALS AND METHODS Cell culture. Keratinocyte cells were isolated to generate “basal” or “stratified” cultures as described [18] except that the source was neonatal foreskin. HaCat cells (passage 35) [19], a spontaneously immortalized human keratinocyte cell line, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 50 mg/ml streptomycin. HaCat cells were plated at ;1.75 3 104/cm2, and medium was replenished every 3 days. COS-7 cells were cultured in DMEM with the above supplements. Monoclonal 3A hybridoma was maintained in RPMI 1640 medium with the above supplements plus 1 mM sodium pyruvate. Cells were incubated in a 5% CO2, 95% air mixture at 37°C and were maintained at subconfluent density. Antibodies. The rabbit polyclonal anti-PI-6 antiserum has been described [7, 10]. Monoclonal anti-PI-6 antibody was produced using standard methods [20]. Briefly, Balb/c mice were injected with 10 mg of purified recombinant human PI-6 (rPI-6) [8] in complete Freund’s adjuvant and then twice in incomplete Freund’s adjuvant. Spleen cells were isolated and fused with Sp-2 cells and the hybridomas producing immunoreactive antibody were identified using ELISA on immobilized rPI-6 and indirect immunofluorescence on PI-6-transfected COS cells. The hybridoma producing monoclonal 3A was further purified by two rounds of limiting dilution, and the antibody was typed using an isotyping kit (Amersham). Monoclonal anti-cytokeratin 10 (anti-CK10) antibody was from DAKO.
Immunohistochemistry. Preparation of 4-mm Formalin-fixed paraffin-embedded tissue samples was as described [21]. Sections were incubated in rabbit anti-PI-6 antiserum or preimmune serum diluted 1:200, washed in PBS, and then incubated for 20 min in a 1:200 dilution of biotinylated swine anti-rabbit immunoglobulins (DAKO E353). Following a further wash in PBS, streptavidin– horseradish peroxidase (DAKO K377) was added for 30 min. Sections were washed again in PBS and developed using diaminobenzidine (DAB, DAKO). COS-7 cell transfection. COS-7 cells were transfected with pSVTfPTI/P (containing PI-6 cDNA) or pSVTf (mock) [10] using the DEAE– dextran/chloroquine method as described [22]. [35S]Methionine labeling and immunoprecipitation. At 48 h posttransfection, 2 3 106 COS cells were [35S]methionine labeled as previously described [7]. Briefly, transfected cells were starved in medium lacking methionine for 30 min followed by a 3-h labeling period with 100 mCi of [35S]methionine and [35S]cysteine (Expre35s35s protein labeling mix, DuPont NEN). Cells were harvested and lysed with ice-cold 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.25% (w/v) gelatin (NETGEL) containing 1% Nonident P-40, 150 mg/ml PMSF, 1 mg/ml aprotinin, and 0.5 mM leupeptin. Cell debris was pelleted and discarded and cytosolic extract was immunoprecipitated overnight at 4°C with 100 ml of 10% (w/v) protein A–Sepharose (Pharmacia Biotech Inc.) and 5 ml rabbit polyclonal anti-PI-6 antiserum, 5 ml rabbit preimmune sera, or 50 ml monoclonal 3A anti-PI-6 hybridoma supernatant with 2 ml rabbit anti-mouse immunoglobulin (DAKO) bridging antibody. Immune complexes were pelleted and washed twice in NETGEL containing 250 mM NaCl and 0.025% (w/v) SDS and once in 10 mM Tris pH 8.0. Samples were resuspended in 30 ml of 20 mM Tris, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.1 M dithiothreitol (reducing buffer) and then analyzed by 10% SDS–PAGE according to Laemmli [23]. Gels were enhanced in Amplify (Amersham Corp.), and visualized by fluorography. Immunoblot. Primary keratinocyte cultures, HaCat cells, and COS cells were washed with ice-cold PBS and subsequently lysed with 10 mM Tris–HCl, 150 mM NaCl, 100 mM EDTA, 1% Triton X-100 containing 1 mg/ml aprotinin, 150 mg/ml PMSF, and 0.5 mM leupeptin, pH 7.6. After centrifugation, total protein concentration was determined (Bio-Rad). Protein (60 mg) was boiled with an equivalent volume of Laemmli sample buffer (20 mM Tris, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 0.1 M DTT) for 5 min and resolved on 10% SDS–PAGE [23]. In some instances, COS cell lysates were incubated with 0.2 units of thrombin (Sigma) at 37°C for 10 min before the addition of sample buffer. After electrophoresis, proteins were transferred to nitrocellulose membranes, and the membranes were incubated for 1 h in blocking buffer (5% skim milk powder in 20 mM Tris–HCl/150 mM NaCl, pH 7.6). Membranes were incubated for 1 h with rabbit polyclonal anti-PI-6 antiserum (diluted 1:5000 in 20 mM Tris–HCl/150 mM NaCl, pH 7.6) or monoclonal 3A hybridoma supernatant (neat) and washed in 20 mM Tris–HCl, 150 mM NaCl, pH 7.6. Horseradish peroxidase (HRP)-conjugated sheep anti-rabbit Ig (Silenus) or HRP-conjugated sheep anti-mouse Ig (Bio-Rad) was used as the secondary antibody and detection was with the enhanced chemiluminescence system (DuPont). In some experiments, the membrane was first probed with mouse monoclonal anti-PI-6 antibody and developed, and the same membrane was then probed with rabbit polyclonal anti-PI-6 antiserum and developed. Images of primary keratinocytes and HaCat cell immunoblots were analyzed via densitometry using the Gel-Pro Analyzer program. Indirect immunofluorescence. HaCat cells were seeded at 1.75 3 104 cells/cm2 on glass coverslips and cultured for the indicated time period. Cells were prepared for immunofluorescence as described [24]. Permeabilized cells were simultaneously incubated with rabbit anti-PI-6 diluted 1:1000 and monoclonal anti-CK10 diluted 1:50, both in PBS with 0.1 mM CaCl2 and 1 mM MgCl2. Cells were then incubated with RITC-conjugated sheep anti-mouse Ig (Silenus) and
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FITC-conjugated sheep anti-rabbit Ig (Silenus), both diluted 1:200 with PBS Ca/Mg and mounted. mRNA analysis. Total RNA was prepared from HaCat cells at various time points according to Chomczynski and Sacchi [25]. Total RNA (20 mg) was separated by electrophoresis on a 1% formaldehyde–agarose gel, transferred to Genescreen Plus (NEN Lifescience Products), and fixed to the membrane by baking at 80°C. The membrane was hybridized to a [32P]dATP-labeled (random priming kit, Promega) PI-6 cDNA probe by standard procedures [26]. The membrane was initially washed at low stringency (23 SSC at room temperature for 1 h) and exposed to film to visualize 28S ribosomal RNA. The membrane was then washed at high stringency (0.13 SSC at 68°C for 20 min) and exposed to film. Images were analyzed by densitometry using Gel-Pro Analyzer program.
RESULTS
Immunohistochemical Localization of PI-6 Protein to the Suprabasal Layers of Human Epidermis PI-6 expression in human epidermis was examined using a polyclonal anti-PI-6 antibody. Basal keratinocytes do not produce any PI-6 protein, as demonstrated by immunohistochemistry (Fig. 1A). In contrast, keratinocytes residing in the suprabasal layers do produce PI-6. Staining is weak in the cells of the early spinous layer, increases as the cells differentiate and migrate vertically through the spinous layer, and most intense at the granulosa layer. The staining observed is cytosolic, with none in the intercellular spaces. This intracellular localization of PI-6 is consistent with previous reports that it cannot be secreted from cells and that it is confined to the cytosol [7]. Positive staining of endothelial cells is also consistent with our previous report [7]. Serial sections stained with preimmune serum show no staining (Fig. 1B). PI-6 Is in Differentiated Primary Keratinocytes but Not in Basal Cells In order to examine the levels of PI-6 expression in the different stages of keratinocyte differentiation, immunoblotting of keratinocyte extracts was performed. Human keratinocytes were isolated from neonatal skin and grown in culture to generate basal keratinocytes or stratified/differentiated keratinocytes. Immunoblotting of cell extracts prepared from these cultures revealed that PI-6 is not significantly expressed in the proliferating basal cells (Fig. 2). This is consistent with the lack of staining of basal cells seen by immunohistochemistry (Fig. 1). By contrast, high levels of PI-6 expression were detected in keratinocytes grown under culture conditions that promote differentiation and stratification (Fig. 2). The increase in PI-6 protein during keratinocyte differentiation is 24-fold, as determined by densitometry. This is consistent with strong PI-6 staining in suprabasal layers of human epidermis.
FIG. 1. PI-6 in human epidermis is confined to suprabasal keratinocytes. (A) Immunostaining using polyclonal anti-PI-6 antiserum. (B) Immunostaining using preimmune sera. Serial sections were dewaxed, blocked, and immunoreacted with polyclonal antiPI-6 antibody as described under Materials and Methods. Detection was using a biotin–streptavidin–HRP system and DAB as substrate. Note strong intracellular staining for PI-6 in the spinous and granular layers of epidermis and lack of staining in basal cells. Original magnification is 380. B, basal layer; S, spinous layer; G, granular layer.
PI-6 mRNA Increases in Differentiating HaCat Cells HaCat cells are an immortalized human keratinocyte cell line of basal cell phenotype that retain the potential to undergo keratinocyte differentiation [19]. After seeding, they undergo a proliferative phase until the cells reach confluence (;7 days), and this is followed by differentiation [27–29]. On in vitro differentiation, HaCat cells express markers of mature keratinocytes such as cytokeratins 1 and 10 [19, 30]. Stratification of cells also occurs in conventional submerged culture, although not to the same extent as when HaCat cells are transplanted onto nude mice, where complete formation of a highly ordered epider-
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pected for normal expression of this protein. PI-6 expression is also seen, although at substantially lower levels, in cells that are not producing detectable CK10. This indicates that induction of PI-6 expression occurs earlier than CK10 expression during HaCat differentiation. This is not surprising as it has been reported that the induction of CK10 during HaCat differentiation is slightly delayed compared to that observed in vivo [30]. Finally, by day 10 in culture, the number of cells expressing CK10 has increased and they are confined to the upper cellular layers (Fig. 4). CK10-positive HaCat cells of the upper layer express PI-6 protein more strongly than those cells found in the lower layers that do not express CK10 (Fig. 4). FIG. 2. PI-6 expression is increased in differentiated human keratinocytes. Primary keratinocyte extracts were prepared, resolved on 10% SDS–PAGE, and transferred to nitrocellulose according to Materials and Methods. Immunoblotting with polyclonal antiPI-6 antiserum demonstrates a 42-kDa PI-6 band which increases 24-fold in stratified keratinocytes.
Induction of a PI-6/Proteinase Complex in Differentiating HaCat Cells Immunoblotting of HaCat cell extracts with polyclonal anti-PI-6 antiserum detects the 42-kDa PI-6 pro-
mis is observed [19]. To determine whether the increase in PI-6 protein observed in primary keratinocyte differentiation is due to a rise in PI-6 mRNA, Northern analysis of total RNA extracted from differentiating HaCat was performed. Figure 3B shows that the 1.4-kb PI-6 mRNA [10] is detected in HaCat cells as early as day 2 in culture. Scanning densitometry analysis of the PI-6 signal, when corrected for loading of RNA, reveals that the level of PI-6 mRNA increases approximately fivefold after the cells have been in culture for 21 days (Fig. 3A). Localization of PI-6 in Differentiating HaCat Cells To establish the profile of PI-6 expression in HaCat cells, indirect immunofluorescence analysis of cultures at different stages of differentiation was performed using polyclonal anti-PI-6 antisera. Staining for the keratinocyte differentiation marker cytokeratin 10 was done simultaneously. After 2 days in culture, HaCat cells are highly proliferative and show no morphological evidence of differentiation or stratification by phase-contrast microscopy (Fig. 4). By indirect immunofluorescence, these basal-like cells show no cytokeratin 10 (CK10) expression and little PI-6 expression. By day 5, the HaCat cells have formed clusters, with some cells residing on top of those attached to the tissue culture dish (in Fig. 4 at day 5, the phasecontrast photo is focused on cells in the upper layer). These stratified cells express high levels of CK10, indicating that they are differentiating. When the same field is stained with polyclonal anti-PI-6 antiserum, those cells expressing CK10 also express high levels of PI-6. The PI-6 staining is cytosolic, as would be ex-
FIG. 3. PI-6 mRNA increases in differentiating HaCat cells. (A) Fold increase of PI-6 mRNA as determined by scanning densitometry and corrected for equal loading of total RNA as determined via the amount of 28S ribosomal RNA. (B) Autoradiograph of PI-6 mRNA species and 28S ribosomal RNA species. PI-6 mRNA increases approximately fivefold by day 21 of HaCat differentiation. Total RNA (20 mg) was resolved on a 1% agarose gel containing formaldehyde, transferred to Genescreen Plus membrane, and hybridized with [32P]dATP-labeled PI-6 cDNA probe. The membrane was initially washed in 23 SSC at room temperature and exposed to film and then 0.13 SSC at 68°C and exposed to film. Images were analyzed via densitometry.
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FIG. 4. Immunolocalization of PI-6 to the cytosol of differentiating HaCat cells. HaCat cells grown on glass coverslips were fixed and permeabilized as described under Materials and Methods at various time points. Cells were double stained with monoclonal mouse anti-CK10 antibody and polyclonal rabbit anti-PI-6 antiserum. Images are of the same field. (Phase) Phase-contrast microscopy; (CK10) RITC fluorescence microscopy of mouse anti-cytokeratin 10 staining; (PI-6) FITC fluorescence microscopy of polyclonal rabbit anti-PI-6 antiserum staining. Day 2 and day 5 micrographs were photographed at 3200 magnification; day 10 at 3100 magnification.
tein after 4 days of culture (Fig. 5A). At this time, the cells are of a basal-like phenotype and have begun to form “islands,” with those in the center beginning to differentiate and stratify (Fig. 4). As the HaCat cells differentiate further in culture, expression of PI-6 increases 1.9-fold by day 8, 4.3-fold by day 10, and 7.1fold by day 14 (Fig. 5B). Also, by day 10 in culture, when there is a well-formed sheet of cells and the development of a more complete stratified layer, a species of approximately 55 kDa is detected that increases in abundance from days 10 to 14, correlating with a
state of further differentiation. This 55-kDa species may represent either a distinct non-PI-6 protein that cross-reacts with the PI-6 antiserum or PI-6 complexed to an endogenous proteinase. In order to determine whether this 55-kDa species contains PI-6, we raised monoclonal anti-PI-6 antibodies. Of several monoclonal antibodies produced, clone 3A (IgG2b/k) had the highest affinity for PI-6 (data not shown) and was used in subsequent experiments. Immunoprecipitation of 35S-labeled PI-6 from transfected COS cells with the monoclonal 3A detected a 42-kDa
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FIG. 5. PI-6 protein and a PI-6/proteinase complex is induced during HaCat cell differentiation. (A) Immunoblot of differentiating HaCat cells with polyclonal and monoclonal anti-PI-6 antibodies. HaCat cell extracts were prepared at indicated time points as described under Materials and Methods. Equal loadings of protein were resolved on 10% SDS–PAGE, transferred to nitrocellulose, blocked, and immunoblotted first with monoclonal anti-PI-6 and developed and then with polyclonal anti-PI-6. (B) Fold increase of the 42-kDa PI-6 species as determined by scanning densitometry.
protein (Fig. 6A). This agrees with the previously reported size of PI-6 [6] and is similar to that precipitated by the rabbit polyclonal anti-PI-6 antiserum (Fig. 6A). Endogenous PI-6 in the mock-transfected COS cells was also detected by the polyclonal anti-PI-6 antiserum but not the monoclonal 3A. This indicates that the 3A antibody is species-specific and does not crossreact with monkey PI-6. The polypeptides of ;35 and ;30 kDa precipitated by both the polyclonal and monoclonal anti-PI-6 antibodies are most likely rPI-6 degradation products. The monoclonal 3A failed to detect denatured PI-6 via immunoblotting of extracts from transfected COS cells (Fig. 6B). This suggests that the monoclonal rec-
ognizes a conformational epitope on native PI-6 that is sensitive to SDS. However, when the extracts were incubated with thrombin prior to SDS–PAGE, a PI-6/ thrombin complex of ;67 kDa was detected by immunoblotting. Thus binding to a proteinase apparently results in a conformational change that stabilizes the SDS-sensitive epitope on PI-6 that is recognized by monoclonal 3A. This is not surprising, considering the large conformational change that serpins undergo on binding of a proteinase [31], and there are several well-characterized precedents for monoclonal antibodies recognizing serpin/proteinase complexes better than the native serpin. For example, the monoclonal anti-PAI-2 antibody, 2H5, only recognizes PAI-2 in
FIG. 6. Monoclonal 3A recognizes native PI-6 and denatured PI-6/thrombin complexes but not denatured PI-6 alone. (A) Immunoprecipitation of 35S-labeled PI-6 by monoclonal 3A anti-PI-6 antibody. Mock, pSVTf transfected; PI-6, pSVTfPTI/P transfected. At 48 h posttransfection COS cells were starved for 30 min in media lacking methionine, labeled for 3 h in media containing 100 mCi [35S]methionine, lysed, and immunoprecipitated according to Materials and Methods. Immune complexes were collected, reduced, resolved on 10% SDS– PAGE, and analyzed by fluorography. (B) Immunoblot of P-6/thrombin complexes with monoclonal 3A anti-PI-6 antibody, rabbit polyclonal anti-PI-6 antiserum, and rabbit preimmune serum. Extracts of PI-6-transfected COS cells were prepared 48 h posttransfection and were incubated with (1) or without (2) thrombin at 37°C for 10 min. Equal loadings of protein were resolved on 10% SDS–PAGE, transferred to nitrocellulose, blocked, immunoblotted with the appropriate antibody, and analyzed by fluorography.
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complex with u-PA or t-PA [32], and the monoclonal anti-PAI-1 antibodies, MA-13C1 and MA-14D5, have stronger affinity for PAI-1 complexed with t-PA than for PAI-1 alone [33]. Immunoblotting of the HaCat extracts with monoclonal 3A demonstrated that the 55-kDa species is also recognized by the monoclonal 3A antibody (Fig. 5A). Because the monoclonal antibody preferentially recognizes PI-6/proteinase complexes, this species probably represents PI-6 complexed to an endogenous proteinase present in HaCat cells. The HaCat cell extracts were prepared with lysis buffer containing EDTA and the proteinase inhibitors aprotinin, leupeptin, and PMSF. These inhibitors ablate proteinase activity and prevent serine proteinases and serpins forming SDSstable complexes during, or after, cell lysis. The 55-kDa PI-6/proteinase complex detected in HaCat cell extracts occurred in the presence of these compounds. This suggests the PI-6/proteinase complex is already present in the cell prior to cell lysis and does not form as a consequence of the extract preparation. The protein of approximately 82 kDa also seen in Fig. 5A is not a PI-6-specific protein as it is recognized by the preimmune sera (data not shown). Given that PI-6 is 42 kDa, the size of the PI-6/ proteinase complex suggests that the proteinase component is approximately 20 –30 kDa. This may represent either the entire proteinase or a subchain of a larger enzyme released due to disulphide bond disruption with reducing SDS–PAGE conditions. The stability of the complex in SDS, which is a hallmark of serpin–serine proteinase interactions [1], strongly suggests that the enzyme is a serine proteinase. It is possible that PI-6 regulates a process involving a serine proteinase in keratinocyte differentiation or desquamation. So far two proteinases have been described that are thought to play a role in the process of desquamation. These are the SCCE and an unidentified trypsin-like proteinase [34 –37]. The ability of PI-6 to inhibit chymotryptic- and tryptic-like proteinases suggests that it may be able to regulate either or both of these proteinases. However, both of these enzymes are extracellular [38], whereas PI-6 is located intracellularly. Since PI-6 can not be released from cells through the endoplasmic reticulum–Golgi network [7], and PI-6 protein has never been detected in plasma or in the media of cultured cells, PI-6 is unlikely to regulate the extracellular functions of these proteinases. Perhaps PI-6 resides in keratinocytes to protect them from exposure to their own desquamation proteinases, either during their production and packaging into secretory vesicles or resulting from inadvertent endocytosis from the intercellular space. This role would be similar to that proposed for PI-9, which resides in the cytosol of cytotoxic lymphocytes to protect them from inadvertent expo-
sure to their own granzyme B during proteinase packaging or degranulation [5]. Another process that occurs during keratinocyte differentiation is the maturation of profilaggrin to filaggrin. Filaggrin is involved in the keratin rearrangements necessary for cornified envelope formation during terminal differentiation. Profilaggrin is stored in non-membrane-bound cytosolic keratohyalin granules as an insoluble precursor. At terminal differentiation, granules disperse and profilaggrin is processed by phosphatases, endopeptidases, and exopeptidases [39 – 42]. In the rat, processing involves profilaggrin endoproteinase-1 (PEP-1), an intracellular chymotryptic-like serine proteinase. PEP-1 is thought to act in the cytosol [43], and PI-6, due to its ability to inhibit chymotryptic-like proteinases and its cytosolic location, may be a suitable regulator of this protease. At present a human counterpart of PEP-1 has not been described, although profilaggrin processing does occur in human keratinocytes. In conclusion, we have shown that PI-6 expression is induced during primary keratinocyte and HaCat cell differentiation. We have also demonstrated that PI-6 interacts with an endogenous protease in differentiating HaCat cells. To our knowledge, this is the first demonstration of a serpin interacting with a target proteinase in human keratinocyte cells. At present, it is unclear whether this proteinase (and its interaction with PI-6) is keratinocyte-specific or whether it also occurs during differentiation of other PI-6 expressing epithelial cells [7]. REFERENCES 1.
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