Production of tissue inhibitor of metalloproteinase-1 and -2 by cultured keratinocytes

Journal of Dermatological Science 22 (2000) 107 – 116 www.elsevier.com/locate/jdermsci

Production of tissue inhibitor of metalloproteinase-1 and -2 by cultured keratinocytes Yasushi Sugita, Eishin Morita *, Toshihiko Tanaka, Kohji Nakamura, Shoso Yamamoto Department of Dermatology, Hiroshima Uni6ersity School of Medicine, Kasumi 1 -2 -3, Minami-ku, Hiroshima 734 -8551, Japan Received 24 March 1999; received in revised form 19 April 1999; accepted 21 April 1999

Abstract The imbalance between metalloproteinases and their inhibitors in tissue remodelling is likely to play an important role in various pathologic conditions. In order to understand the role of keratinocytes in regulating extracellular matrix degradation in skin, we analyzed the production of metalloproteinase inhibitors in keratinocytes. Tissue inhibitor of metalloproteinase-1 (TIMP-1) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA were detected in cultured human keratinocytes and mouse transformed keratinocyte cell line (KCMH-1) cells by RT-PCR. On several column chromatography separation steps of the KCMH-1 conditioned medium, two specific inhibitors for mammalian collagenase were purified showing an Mr of 29 kDa and an Mr of 22 kDa, respectively. The analysis of their N-terminal amino acid sequence revealed that two inhibitors were TIMP-1 and TIMP-2. The final preparation of TIMP-1 had a specific activity of 56 000 U/mg and that of TIMP-2 had a specific activity of 26 200 U/mg. Our results suggest that keratinocytes take part in tissue remodelling in skin in secreting both TIMP-1 and TIMP-2. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Collagenase; TIMP-1; TIMP-2; Keratinocyte

1. Introduction Restructuring of the extracellular matrix (ECM) occurs throughout embryonic development and during wound healing of the organisms. Metalloproteinases including collagenases, stromelysins and gelatinases, play a central role in degrading ECM and their activity is regulated by * Corresponding author. Tel.: +81-082-257-5236; fax: + 81-082-257-5237. E-mail address: [email protected] (E. Morita)

activators such as serine proteinases [1] and by inhibitors, including tissue inhibitor of metalloproteinases (TIMPs) [2] and a2-macroglobulin [3]. The imbalance between metalloproteinases and their inhibitors is likely to play an important role in various pathologic conditions associated with either excess connective tissue deposition, such as scleroderma and fibrosis, or excess connective tissue destruction, such as arthritis and bullous dermatoses [4]. Originally, TIMP was purified from culture media of rabbit bone explants [5]. Recently, two

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additional inhibitors with high homology with TIMP, TIMP-2 [6] and TIMP-3 [7], have been purified and now these TIMPs are considered to form a family. Subsequent studies have revealed that TIMPs are produced from many types of cells. Leco et al. have investigated the expression pattern of TIMP mRNA in adult mouse tissues and demonstrated a characteristic expression pattern in organs [8]. TIMP-1 expression is seen at high level in bone, lung and ovary and at low level in thymus, heart and uterus. The highest levels of TIMP-3 mRNA were found in kidney and lung and lesser level in bone, brain, muscle, uterus and ovary [8]. These data suggest that TIMPs production is regulated in tissue specific manners. Although the dermis has been thought to be the primary source of metalloproteinase inhibitors as well as metalloproteinases in skin, keratinocytes have also been demonstrated to produce collagenolytic enzyme and TIMP [9], indicating that keratinocytes also take part in ECM remodelling. Now keratinocytes are known to produce a series of proteolytic enzymes [10], but little is known about their ability to secrete inhibitors for these proteolytic enzymes. In the present study, in order to understand the role of keratinocytes on tissue remodelling in skin, we analyzed the production of inhibitors for mammalian collagenase in cultured keratinocytes and a transformed keratinocyte cell line.

2. Materials and methods

2.1. Cell preparation Normal human epidermal keratinocytes were prepared from surgically obtained skin and cultured in K-GM medium (Kurabo, Osaka, Japan) as previously described [11]. A mouse transformed keratinocyte cell line, KCMH-1 [12] was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin G and 100 mg/ml streptomycin.

2.2. Re6erse transcriptase-mediated polymerase chain reaction (RT-PCR) Polyadenylated RNA was directly isolated from

cells using a Fast Track mRNA Isolation Kit (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Briefly, the cells were lyzed by incubating with 15 ml SDS buffer (2% SDS, 200 mM NaCl, 200 mM Tris–HCl, pH 7.5, 1.5 mM MgCl2) containing 0.3 ml RNase/protein degrader for 20 min at 45°C. The NaCl concentration of the lysate was then adjusted to 0.5 M, and 75 mg of oligo(dT)-cellulose was added. After the mixture was rocked gently at room temperature for 20 min, the oligo(dT)-cellulose was pelleted and dissolved in binding buffer (500 mM NaCl, 10 mM Tris–HCl, pH 7.5). The DNA, dissolved membranes, proteins and cell debris were washed out with binding buffer by repeating this process. The non-polyadenylated RNAs were washed out with low-salt wash buffer (250 mM NaCl, 10 mM Tris–HCl, pH 7.5). The oligo(dT)-cellulose was packed into a spin column. The polyadenylated mRNA was then eluted with 400 ml elution buffer (10 mM Tris–HCl, pH 7.5), precipitated in ethanol and quantitated by spectrophotometry. cDNA was synthesized from 0.2 mg of polyadenylated mRNA using a cDNA Cycle Kit (Invitrogen) according to the manufacturer’s instructions. Briefly, 8 ml of mRNA solution was mixed with 2 ml of 100 mM methyl mercuric hydroxide, 2.5 ml of 0.7 M b-mercaptoethanol, 1 ml of placental RNase inhibitor, 4 ml of 5×RT buffer (250 mM Tris–HCl, pH 8.3, 500 mM KCl, 50 mM MgCl2, 50 mM dithiothreitol), 1 ml of 25 mM dNTPs (dATP, dCTP, dTTP, dGTP) and 0.5 ml reverse transcriptase (10 U/ml). The reaction mixture was incubated in water bath at 45°C for 60 min, then terminated by heating to 95°C for 3 min and then the mixture was quick-chilled on ice. The PCR mixture (in a volume of 50 ml) contained PCR buffer (10 mM Tris–HCl, pH 9.0, 50 mM KCl, 0.1% Triton-X, 2.5 mM MgCl2) and 200 mM dNTPs, 1 mM of each specific primer (listed in Table 1), 1 ml of cDNA solution and 2.5 U Taq DNA polymerase (Toyobo, Osaka, Japan). The reaction conditions were: denaturation at 94°C for 1 min; annealing at 55°C for 2 min; extention at 72°C for 3 min for 35 cycles in a DNA thermal cycler (Astec PC700, Fukuoka, Japan). The PCR

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products were electrophoresed in 2.5% agarose gels in 1×TAE buffer (40 mM Tris, 20 mM acetic acid, 2 mM Na3EDTA) at a constant 100 V for 2 h. Products were visualized by ethidium bromide over a UV transilluminator (CSF-20B, CosmoBio, Tokyo, Japan) and photographed.

2.3. Assay for collagenase inhibitory acti6ity Collagen-coated 96 multi-well plates were prepared as described [13]. Briefly, aliquots (50 ml) of ice-cold type-I collagen solution (600 mg/ml) in neutralizing buffer (100 mM Tris – HCl, 200 mM NaCl, 0.04% NaN3, pH 7.8) were added to the 96 multi-well plates (30-mg collagen per well), incubated at 37°C in a humidified atmosphere for 16 h to gel the collagen and the plates were further incubated at 37°C in a dry atmosphere until the collagen dried. The wells were then

Table 1 Specific primer sequences for amplifying TIMP cDNAsa hTIMP-l-A 5%-TGC ACC TGT GTC CCA CCC CAC CCA CAG ACG-3% hTIMP-l-B 5%-GGC TAT CTG GGA CCG CAG GGA CTG CCA GGT-3% hTIMP-2-A 5%-TGC AGC TGC TCC CCG GTG CAC CCG CAA CAG-3% hTIMP-2-B 5%-TGG GTC CTC GAT GTC CAG AAA CTC CTG CTT-3% hTIMP-3-A 5%-TGC ACA TGC TCG CCC AGC CAC CCC CAG GAC-3% hTIMP-3-B 5%-GGG TCT GTG GCA TTG ATG ATG CTT TTA TCC-3% mTIMP-l-A 5%-TGT AGC TGT GCC CCA CCC CAC CCA CAG ACA-3% mTIMP-l-B 5%-TCG GGC CCC AAG GGA TCT CCA GGT GCA CAA-3% mTIMP-2-A 5%-TGC AGC TGC TCC CCG GTG CAC CCG CAA CAG-3% mTIMP-2-B 5%-CGG GTC CTC GAT GTC AAG AAA CTC TTG CTT-3% mTIMP-3-A 5%-TGC ACA TGC TCT CCC AGC CAT CCC CAG GAT-3% mTIMP-3-B 5%-GGG GTC TGT GGC GTT GCT GAT GCT CTT GTC-3% a

h, human; m, mouse; A, sense primer; B, antisense primer.

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rinsed three times with reagent-grade water and left to dry at room temperature prior to use. Collagenase solution was prepared from human polymorphonuclear leukocytes (PMN) isolated from venous blood as previously described [14]. Briefly, PMN suspended at 1× 108 cells/ml in assay buffer (50 mM Tris–HCl, 100 mM NaCl, 10 mM CaCl2, 0.02% NaN3, pH 7.5) were lyzed by adding one tenth volume of 0.1% Triton X-100, the insoluble residue was removed by centrifugation at 1000×g for 10 min and the supernatant containing procollagenase was collected. Procollagenase in the supernatant was activated by incubation with trypsin (Sigma, St. Louis, MO) at a final concentration of 100 mg/ ml for 10 min at 37°C and the reaction was terminated by adding soybean trypsin inhibitor (Wako Pure Chemicals, Osaka, Japan) at final concentration of 500 mg/ml. The collagenase solution showing an activity to degrade 1 mg type I collagen per 1 h was defined as 1 U of collagenase. For collagenase inhibitory activity assay, aliquots of sample solution or assay buffer alone were incubated with 100 ml of 10 U collagenase solution at 37°C for 30 min in a humidified condition and then the reaction mixtures were added to the collagen-coated wells and incubated for 3 h at 37°C in a humidified condition. The wells were then washed twice with assay buffer and then twice with reagent-grade water. To evaluate the amount of remaining collagen on the microtiter wells, 100 ml of stain solution (0.25% (w/v) Coomasie blue R-250, 10% (v/v) acetic acid, 50% (v/v) methanol) was added in each well and incubated for 25 min at 25°C. The wells were washed three times with reagent grade water and the stained collagen in the wells was quantitated by determining the absorbance at 590 nm with Microplate Reader (Model 3550, Bio-Rad Laboratories, Richmond, CA). The collagenase inhibitory activity in the samples was expressed as either the absorbance at 590 nm or unit that was defined as the amount of inhibitor that inhibited 10 U of collagenase activity by 50% for 3 h at 37°C.

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2.4. Purification of collagenase inhibitors KCMH-1 cells were grown to subconfluence in multilayered cell flasks (Cell factory, Nunc, Roskilde, Denmark) containing RPMI 1640 medium supplemented with 10% FCS, 100 IU/ ml penicillin G and 100 mg/ml streptomycin. After the medium was discarded, the cells were rinsed three times with serum-free RPMI 1640 medium, fed fresh serum-free medium containing the same concentration of penicillin G and streptomycin and incubated for a further 24 h. The cell-free supernatant was then collected, concentrated and dialyzed against Tris buffer (20 mM Tris–HCl, pH 8.0) on Amicon ultrafiltration membrane (Amicon, Beverly, MA). Collagenase inhibitors in the supernatant were purified through four steps of column chromatography, including DEAE-anion exchange column chromatography, gel-chromatography, hydroxyapatite column chromatography and reversed phase-high performance liquid chromatography (RP-HPLC). Purification procedure was as follows: the sample was first applied to DEAE-Sepharose FF column (26 × 450 mm, Pharmacia, Freiburg, Germany) previously equilibrated with Tris buffer. The column was washed with Tris buffer and was eluted with a linear gradient of 0 – 0.5 M NaCl-containing Tris buffer at a flow rate of 100 ml/h. Fractions of 10 ml were collected. Aliquots of each fraction were taken and tested for collagenase inhibitory activity. Fractions containing collagenase inhibitory activity were concentrated on Amicon YM-3 filter to a volume of 3–5 ml and applied to Sephadex G-100 column (26×700 mm, Pharmacia) previously equilibrated with Tris buffer containing 0.1 M NaCl. Chromatography was carried out with same buffer at a flow rate of 25 ml/h. The fractions containing activity were collected, dialyzed against 10 mM phosphate buffer (pH 6.8) containing 0.3 mM CaCl2 and applied to Shim-pack HAC column (7.5 × 55 mm, Shimadzu, Kyoto, Japan) previously equilibrated with 10 mM phosphate buffer (pH 6.8) containing 0.3 mM

CaCl2. The sample was eluted with a linear gradient from 10 mM phosphate buffer (pH 6.8) containing 0.3 mM CaCl2 to 0.3 M phosphate buffer (pH 6.8) containing 0.013 mM CaCl2. The fractions containing activity were collected, concentrated on Amicon YM-3 filter and dialyzed against reagent-grade water containing 0.1% (v/v) trifluoroacetic acid (TFA). This sample was finally purified upon RP-HPLC using YMC-Pack ODS-A column (5 mm, 120 A, , 6× 100 mm, YMC, Kyoto, Japan). Elution buffer A consisted of 0.1% TFA in water and elution buffer B consisted of 0.1% TFA in acetonitrile. The column was equilibrated with a mixture of 70% A and 30% B. After injection of sample, chromatography was carried out with a gradient using buffer A and B at a flow rate of 1 ml/ min. Buffer B was increased from 30 to 50% after 30 min and after that, B was increased to 100% and eluant was fractionated. In some experiments, fractions containing collagenase inhibitory activity after DEAE-Sepharose FF column were collected and applied to Con A-Agarose affinity column (28× 120 mm, Seikagaku, Tokyo, Japan) previously equilibrated with phosphate buffer (0.15 M NaCl, 0.1 M sodium phosphate, pH 7.2). The column was then washed with the same buffer and the sample was eluted with the phosphate buffer containing 0.1 M methyl-a-D-glucoside. The eluant was monitored at 280 nm of UV through all chromatography steps. Each fraction was dialyzed against reagent-grade water and then activity was assayed.

2.5. Polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a Pharmacia Phast System (Pharmacia) using homogenous 20% SDS-gels, according to manufacturer’s instructions. Samples were diluted with the same volume of sample buffer containing 0.5 M Tris–HCl (pH 6.8), 1% SDS and 1% b-mercaptoethanol and heated at 95°C for 5 min prior to loading. After electrophoresis, silver stain of the gels was performed with Silver stain kit (Pharmacia).

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Fig. 1. Expression of mRNA for TIMPs. mRNA expression for TIMP-1, TIMP-2 and TIMP-3 were analyzed in (A) cultured human keratinocytes and (B) mouse KCMH-1 cells by RT-PCR. Lane 1: 100-bp DNA ladder; lane 2: mRNA for TIMP-1; lane 3: mRNA for TIMP-2; lane 4: mRNA for TIMP-3. The predicted sizes of the PCR products were 552-bp for human TIMP-1, 583-bp for human TIMP-2, 563-bp for human TIMP-3, 543-bp for mouse TIMP-1, 582-bp for mouse TIMP-2 and 564-bp for mouse TIMP-3.

2.6. Amino acid sequencing N-terminal amino acid sequencing of the samples was performed with automated amino acid sequencer (PSQ-2, Shimadzu).

2.7. Protein assay Protein concentration of samples was determined by the method of Bradford using Protein assay kit 2 (Bio-Rad).

3. Results

3.1. Detection of mRNA for TIMPs With RT-PCR technique, we examined the expression of mRNA for TIMPs in cultured human keratinocytes and mouse transformed keratinocyte cell line, KCMH-1. Based on the previ-

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ously reported nucleotide sequences of the human TIMP-1 [15], TIMP-2 [16] and TIMP-3 [17], as well as mouse TIMP-1 [18], TIMP-2 [19] and TIMP-3 [8], we designed specific primers for amplifying TIMP cDNAs (Table 1). The expression of mRNA for TIMPs in cultured human epidermal keratinocytes as well as KCMH-1 cells is presented in Fig. 1. In cultured human keratinocytes DNA bands, with size of approximately 550-bp and 580-bp were detected for human TIMP-1 and TIMP-2, respectively. In KCMH-1 cells DNA bands were also detected with the primers for mouse TIMP-1 and TIMP-2. These amplified cDNAs were confirmed to be the cDNAs for TIMP-1 and TIMP-2 by digesting with several restriction enzymes (MspI, BamHI, HindIII and PstI) (data not shown), although a minor non-specific 240-bp band was seen in KCMH-1 cells with mouse TIMP-1 primers. However, only faint band was seen with primers for TIMP-3 in cultured human epidermal keratinocytes.

3.2. Purification of TIMPs from the conditioned medium obtained from mouse keratinocyte cell line cells We used KCMH-1 to further analyze collagenase inhibitor production, because KCMH-1 is considered to be a pure keratinocyte-derived cell line [12]. Therefore, we excluded the effects of contaminating other epidermal cells such as fibroblasts, Langerhans cells, or melanocytes. The medium conditioned from KCMH-1 cells was tested for collagenase inhibitory activity using human PMN collagenase preparation. The collagenase activity was inhibited by adding the KCMH-1 conditioned medium in a dose-dependent manner (Fig. 2). When bacterial collagenase was used, no inhibition was seen. This indicates that the KCMH-1 conditioned medium contains specific inhibitor(s) for mammalian collagenase. In order to characterize the inhibitor(s) found in KCMH-1 conditioned medium, we then analyzed the conditioned medium on several column chromatography steps. Serum-free KCMH-1 conditioned medium was collected, concentrated on Amicon YM-3 membrane and dialyzed against

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Tris buffer. When the concentrated KCMH-1 conditioned medium was chromatographed upon DEAE-Sepharose FF anion exchange-column, two peaks of the collagenase inhibitory activity were detected (Fig. 3). The first inhibitory activity (tentatively designated as inhibitor 1) was seen in the fractions passed through the DEAE-column and the second peak (tentatively designated as inhibitor 2) was detected in the fractions where eluted with approximately 0.15 M NaCl. These

Fig. 4. Con A-Agarose affinity chromatography of inhibitor 1. Protein content was measured at OD 280 nm () and collagenase inhibitory activity was expressed as OD 595 nm ( ).

Fig. 2. Collagenase inhibitory activity in KCMH-1 conditioned medium. PMN collagenase () and Clostridium histolycum collagenase ( ) were incubated with serially diluted KCMH-1 conditioned medium and remained collagenase activity was measured by using a spectrophotometrical assay technique.

Fig. 3. DEAE-Sepharose FF chromatography of KCMH-1 conditioned medium. Protein content was measured at OD 280 nm () and collagenase inhibitory activity was expressed as OD 595 nm ( ).

two inhibitors were separately collected. When these two inhibitors were applied to Con AAgarose affinity column, inhibitor 1 bound to the column and eluted with methyl-a-D- glucoside (Fig. 4), however, inhibitor 2 passed through the column (data not shown). Upon gel-chromatography with Sephadex G-100 column, molecular weight of inhibitor 1 and inhibitor 2 were estimated to be 25–45 kDa and 10–50 kDa, respectively (Fig. 5). Sephadex G-100 column fractions with inhibitory activity were pooled and loaded on a hydroxyapatite-column. Inhibitor 1 eluted with approximately 0.25 M phosphate buffer and inhibitor 2 eluted with approximately 0.28 M phosphate buffer (data not shown). Final purification of the inhibitors was obtained by a RPHPLC using ODS column. This step resulted in purification as single peaks of these two inhibitors (Fig. 6). The purity of the last preparation was then verified by SDS-PAGE (Fig. 7). Single protein bands were identified with a Mr 29 kDa for inhibitor 1 and with a Mr 22 kDa for inhibitor 2, respectively. The purification achieved with 80 l of KCMH-1 conditioned medium was summarized in Table 2. Upon DEAE-Sepharose FF column chromatography, two inhibitors, inhibitors 1 and 2, were separately detected and after this step the activity of inhibitor 1 and inhibitor 2 were determined as 55 200 U and 86 000 U, respectively (Table 2). The final preparation of inhibitor 1 had a specific

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activity of 56 000 U/mg and that of inhibitor 2 had a specific activity of 26 200 U/mg. The purification procedures resulted in approximately 8% recovery for inhibitor 1 and 33% for inhibitor 2. The purity was achieved 157-fold for inhibitor 1 and 73-fold for inhibitor 2.

3.3. Determination of N-terminal amino acid sequence To further characterize these two inhibitors, their N-terminal amino acid sequence were analyzed. The first 25 N-terminal sequence of inhibitor 1 was identical with that of mouse TIMP-1 previously reported [18], except for three unidentified amino acids (Fig. 8). The N-terminal sequence of inhibitor 2 was consistent with that of mouse TIMP-2 previously reported [19], although

Fig. 6. RP-HPLC profile of (A) inhibitor 1 and (B) inhibitor 2. Protein content was monitored at OD 280 nm. Fractions were collected peakwise and collagenase inhibitory activity was expressed at OD 595 nm (shaded area).

in this sequence six amino acids were unidentified. These results indicate that inhibitor 1 with a Mr 29 kDa is TIMP-1 and inhibitor 2 with a Mr 22 kDa is TIMP-2.

4. Discussion

Fig. 5. Sephadex G-100 gel-chromatography of (A) inhibitor 1 and (B) inhibitor 2. Protein content was measured at OD 280 nm () and collagenase inhibitory activity was expressed as OD 595 nm ( ).

By RT-PCR analysis for mRNA expression in cultured human keratinocytes and mouse transformed keratinocytes, we demonstrated that these keratinocytes express mRNA for TIMP-2 in addition to TIMP-1. We successfully purified these two collagenase inhibitors, TIMP-1 and TIMP-2, from culture medium conditioned from mouse transformed keratinocytes, KCMH-1. These results indicate that keratinocytes have essentially an ability to produce both TIMP-1 and TIMP-2. Although TIMP-3 mRNA was detected in cultured human keratinocytes by RT-PCR, the band of TIMP-3 cDNA was far less intense compared with those of TIMP-1 and TIMP-2 (Fig. 1). In

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Fig. 8. Comparison of the N-terminal amino acid sequences of mouse TIMP-1, mouse TIMP-2, purified inhibitor 1 and purified inhibitor 2. Asterisks indicate unidentified amino acids.

Fig. 7. SDS-PAGE of purified inhibitor 1 and inhibitor 2. Samples were electrophoresed on a homogeneous polyacrylamide gel (20%) and silver-stained. Lane 1: molecular weight marker; lane 2: purified inhibitor 2; lane 3: purified inhibitor 1.

addition, TIMP-3 was not isolated from KCMH1 conditioned medium. Therefore, it would appear that keratinocytes secrete predominantly TIMP-1 and TIMP-2, but the secretion of TIMP3 is minimum. More recently, cDNA for TIMP-4 has been identified from human heart cDNA library [20]. However, TIMP-4 expression is quite restricted.

By Northern blot analysis, only the adult heart showed abundant TIMP-4 transcripts and very low levels of the transcripts were detected in the kidney, placenta, colon and testes. By contrast, no transcripts were detected in the liver, brain, lung, thymus and spleen [20]. Keratinocytes are unlikely to produce this fourth inhibitor, since no additional inhibitor for collagenase was purified from the KCMH-1 conditioned medium except TIMP1 and TIMP-2. Our results presented here suggest that in order to understand the role of keratinocytes in tissue-remodelling, expression of TIMP-2 in addition to TIMP-1 should be taken into consideration. Both TIMP-1 and TIMP-2 showed similar inhibitory activity for leukocyte-collagenase with the specific activity of 56 000 and 26 200 U/mg, respectively, however, chromatographic behavior of these two inhibitors was different. Upon DEAE-anion exchange column chromatography TIMP-1 and TIMP-2 were clearly separated. TIMP-1 passed through the DEAE-Sepharose,

Table 2 Purification of collagenase inhibitors from KCMH-1 conditioned medium Purification step

Total protein (mg)

Total activity (U)a

Yield (%)

Conditioned medium

927

329 600

(100)

Inhibitor 1 DEAE Sephadex G-100 HAC RP-HPLC

115.2 0.252 0.224 0.075

55 200 2940 4320 4200

16.7 0.9 1.3 1.3

479 11 700 19 300 56 000

Inhibitor 2 DEAE Sephadex G-100 HAC RP-HPLC

84.5 28.8 9.0 1.1

86 000 44 800 23 400 28 800

26.1 13.6 7.1 8.7

1018 1560 2600 26 200

a

Specific activity (U/mg) 356

Purity (fold) 1 1.3 32.9 54.2 157 2.86 4.38 7.3 73.6

One unit is defined as the amount of inhibitor that inhibits 10 U of PMN collagenase by 50% for 3 h at 37°C.

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whereas TIMP-2 bound to the column. This indicates that TIMP-1 has an isoelectric point at neutral or basic area and TIMP-2 has an anionic isoelectric point. The chromatographic behavior of these two TIMPs on Con A-Agarose column were also different. TIMP-1 bound to the Con A column and TIMP-2 passed through the Con A column. This behavior was consistent with previously reported results that TIMP-1 is glycosylated protein and TIMP-2 is not glycosylated [21]. A principal role of TIMPs is to regulate the degradation of the basement membrane and extracellular matrix by metalloproteinases by binding to metalloproteinases with a stoichiometry of 1:1 on a molar basis. Petersen et al. reported that human keratinocytes in culture produce procollagenase [9] indicating that keratinocytes are able to degrade ECM. Two TIMPs, TIMP-1 and TIMP2, produced from keratinocytes are likely to regulate this collagenolytic enzyme. Therefore, remodelling of ECM is considered to be an important function of keratinocytes. The physiological significance of multiple TIMPs production by keratinocytes remains to be further investigated. The demonstration of significant amount of TIMP-2 production comparable to that of TIMP-1 in keratinocytes suggests the possibility that TIMP-1 and TIMP-2 regulate different metalloproteinases. In fact, although TIMP-1 has been characterized as binding only to the activated form of interstitial collagenase, TIMP-2 is capable of binding to both the latent and activated forms of type IV collagenase [22]. So far, functional difference between TIMP-1 and TIMP-2 has not been reported. Leco et al. have studied mRNA expression pattern by Northern blotting for three TIMPs (TIMP-1, TIMP-2 and TIMP-3) in mouse fibroblasts and revealed that expression of these TIMPs are regulated by different fashion [8]. In unstimulated fibroblasts, only TIMP-2 transcripts were detectable. TIMP-1 and TIMP-3 transcripts accumulate in response to phorbol 12-myristate 13-acetate and transforming growth factor-b. In contrast, only a slight increase of TIMP-2 expression was found in stimulated cells. Although we have not studied the regulation of TIMP expression in keratinocytes, it is conceivable that expres-

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sion of TIMPs in keratinocytes are also up-regulated by some stimulative agents. Human PMN are known to release several proteolytic enzymes. Among these enzymes, elastase is another proteinase to degrade a number of ECM proteins, type III and type IV collagen and proteoglycans as well as elastin. An elastase inhibitor, Elafin, which was originally purified from psoriatic scales, is reported to be produced from keratinocytes [23]. This inhibitor is highly up-regulated in the lesional epidermis of psoriatic patients [24] and is considered to play a protective role against leukocyte-elastase released in the lesion [23]. As two TIMPs produced from keratinocytes inhibit leukocyte-collagenase, TIMPs secreted from keratinocytes may play a protective role for epidermis from collagenase digestion by PMN infiltrated into the skin. Interestingly, TIMP-1 was also characterized as a factor with erythroid-potentiating activity [25]. Gasson et al. demonstrated that human TIMP-1 stimulates colony formation of mature erythroid precursors in human peripheral blood [15]. Subsequently TIMP-2 was found to possess erythroidpotentiating activity [26]. Moreover, by using primary cultured human keratinocytes, human TIMP-1 was found to stimulate the growth of normal human keratinocytes [27]. These results suggest that through TIMP production, keratinocytes show wide physiological function in regulating not only extracellular matrix degradation by metalloproteinases but also the growth of several cell lineages including itself.

Acknowledgements This work was carried out with the kind cooperation of the Research Center for Molecular Medicine, Faculty of Medicine, Hiroshima University, Japan.

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