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Cystatin A suppresses ultraviolet B-induced apoptosis of keratinocytes
Journal of Dermatological Science (2007) 46, 179—187
www.intl.elsevierhealth.com/journals/jods
Cystatin A suppresses ultraviolet B-induced apoptosis of keratinocytes Hidetoshi Takahashi *, Naritsuna Komatsu, Masaki Ibe, Akemi Ishida-Yamamoto, Yoshio Hashimoto, Hajime Iizuka Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaokaminami, Asahikawa 078-8510, Japan Received 15 October 2006; received in revised form 16 January 2007; accepted 2 February 2007
KEYWORDS Apoptosis; Cystatin A; Keratinocytes; Ultraviolet B
Summary Background: Cystatin A is a cysteine proteinase inhibitor abundantly expressed in keratinocytes. Although cystatin A is one of the cornified cell envelope constituents and expressed in the upper epidermis, its precise function is still unknown. Ultraviolet B irradiation (UVB) induces apoptosis accompanied with the activation of cysteine proteinases, caspases. Objective: We investigated the effect of cystatin A on UVB-induced apoptosis of keratinocytes. Methods: We assessed the caspase activities and apoptotic cell numbers induced by UVB ittadiation in cystatin A gene transfected keratinocytes. Results: UVB-induced pro-caspase 3 cleavage and caspase 3 activation were suppressed in cystatin A expression vector-transfected SV40-transformed human keratinocytes (SVHK). Furthermore, the transfected SVHK cells were resistant to UVB-induced apoptosis. In contrast neither caspase 8 nor caspase 9 activities were affected by UVB irradiation in cystatin A-transfected SVHK cells. The effects were also observed in cystatin A expression adenovirus vector-transfected cultured normal human keratinocytes (NHK). Conversely knockdown of cystatin A by siRNA induced marked apoptosis of NHK cells following UVB irradiation accompanied with increased caspase 3 activity. In order to confirm the antiapoptotic effect of cystatin A in vivo UVB irradiation was performed on cystatin A transgenic mice (cystatin A-tg). The epidermis from cystatin A-tg was resistant to UVB-induced apoptosis compared to control mice epidermis.
Abbreviations: Cystatin A-tg, cystatin A transgenic mouse; NHK, normal cultured human keratinocyte; SVHK, SV40 transformed human keratinocytes; UVB, ultraviolet B * Corresponding author. E-mail address: [email protected] (H. Takahashi). 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.02.003
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H. Takahashi et al. Conclusion: These results indicate that cystatin A suppresses UVB-induced apoptosis of keratinocytes by the inhibition of caspase 3 activation. # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction
in a sub-confluent state by sub-culturing every 4—5 days.
Apoptosis, or programmed cell death, plays an important role on development, cell proliferation, and differentiation of various tissues [1,2]. In skin apoptosis is observed in hair follicles during catagen phase of hair cycle, in lichenoid tissue reaction, and in various skin tumors [3,4]. Apoptosis is also induced by various external stimuli, such as ultraviolet irradiation, hydroxy radical, and anti-cancer agents [5,6]. Ultraviolet B (UVB) is one of the potent inducers of apoptosis forming ‘‘sunburn cells’’. Biochemical analysis revealed that the apoptosis is induced by the activation of caspases, a member of cysteine proteinases [7]. Anti-apoptotic molecule, bcl-2, suppresses UVB-induced apoptosis by inhibiting caspase activity [8]. Cystatin A is a member of cysteine proteinase inhibitors and is a potent inhibitor of cathepsins [9]. In particular, cystatin A efficiently inhibits cathepsin B. Biochemical and immunohistochemical analyses revealed that cystatin A is abundantly expressed in keratinocytes and is among the major constituents of cornified cell envelope of horny cells [10—14]. Although previous study indicated that cystatin a (a rat counterpart of cystatin A)-depleted rat skin is more sensitive to Staphylococcus aureus compared with normal skin [15], other function(s) of this proteinase inhibitor in the skin is still unknown. In the present study, we investigated the effect of cystatin A on UVB-induced apoptosis of keratinocytes both in vitro and in vivo.
Cystatin A cDNA [14] was cloned into a cosmid vector, pAxCALLNLw (donated by Dr. Saito, University of Tokyo, Tokyo, Japan). The procedure was performed as described previously [8]. Infection was performed by adding various titers of recombinant adenovirus. The cells were incubated at 37 8C for 60 min. The medium was changed to fresh KGM medium and the cells were incubated at 37 8C for 24 h before UVB irradiation.
2. Materials and methods
2.4. Si-RNA construction and transfection into NHK cells
2.1. Cell culture SV40-transformed human keratinocytes (SVHK cells) [16] were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum at 37 8C in 5% CO2 atmosphere. Normal human keratinocytes (NHK cells) were obtained from skin during plastic surgery. Informed consent was obtained from each patient. NHK cells were cultured in keratinocytes growth medium (KGM) containing insulin (5 mg/ml), and bovine pituitary extract (50 mg/ml) at 37 8C in air containing 5% CO2. The calcium concentration in the medium was 0.1 mM. NHK cells were maintained
2.2. Establishment of stably cystatin A expressing SVHK cells Cystatin A cDNA was introduced in the EcoRI sites of pSG5 vector (cystain A-pSG5) [14]. Then cystatin A-pSG (5 mg) and pSG-neo (1 mg), which expresses neomycin resistant gene, was transfected into 5 106 SVHK cells by electroporation according to the protocol of Amaxa Biosystems (Koeln, Germany). After the transfection the cells were cultured in the presence of neomycin (1 mg/ml), and neomycin resistant SVHK cells were isolated. Then the cells were examined for the expression of cystatin A by Western blot analysis using anti-cystatin A antibody and the clones, which highly expressed cystatin A, were selected.
2.3. Cystatin A adenovirus vector and its transfection into cultured normal human keratinocytes (NHK cells)
Si-RNA (CUCAAGUUGUUGCUGGAACTT) was designed and constructed by Takara biomedicals (Kyoto, Japan). Ten microgram of cystatin A si-RNA was transfected into 5 106 NHK cells by electroporation according to the protocol of Amaxa Biosystems and the cells were cultured for 48 h. Then transfected NHK cells were collected and various assays were performed.
2.5. UVB irradiation UVB irradiation source was a Toshiba-Eizai Dermaray instrument (DMR-1, Tokyo, Japan) equipped with
Cystatin A suppresses apoptosis five fluorescence lamps (FL-20-SE-30, Toshiba, Tokyo, Japan). The UVB source emits an energy spectrum with high fluence in the UVB region (290—320 nm) and a peak at 313 nm. In order to block UVC emission a WG-295 long pass filter was used (Schott Glass Technology, Duryea, PA). The emitted dose was routinely evaluated by a UVB radiometer photodetector (IL443 and SEE 240, International Light, Newburyport, MA).
2.6. Cell death assay
181 were clarified by centrifugation and supernatants were used for enzyme assays. Caspase 3 substrate (Z-Asp-Glu-Val-Asp-AFC), caspase 8 substrate (Z-Ile-Glu-Thr-Asp-AFC), and caspase 9 substrate (Z-Leu-Glu-His-Asp-AFC) were purchased from Kamiya Biomedical Co. (Seattle, WA). Caspase assays were carried out using fluorogenic substrates according to the protocol provided by the manufacture (Kamiya Biomedical Co.). The reaction mixtures were incubated at 30 8C for 1 h and fluorescence was measured by a fluorometer with an excitation of 400 nm and emission of 505 nm.
SVHK cells or NHK cells were seeded at 1 104 cells in a 96 well microtiter plate and were washed twice with phosphate-buffered saline (PBS, pH 7.5). After irradiation the cells were stained for 20 min at room temperature with 0.75% crystal violet in 50% ethanol, 0.25% NaCl, and 1.75% formaldehyde, and were washed three times with PBS. Dye uptake was measured by measuring optical density (OD) at 540 nm using an automated Micro-ELISA autoreader [17]. The ratio of viable cells was expressed as percentage of the OD value obtained without UVB treatment.
We excised the cystatin A cDNA from vector pGEM-3cystatin A [11,12] and ligated it into pBSK5 constract [18]. PBSK5-cystatin A was digested with EcoRI, purified, and used to generate transgenic founders on B6 background. Transgenic mice were identified by PCR of genomic DNA with primers specific for the gene encoding human cystatin A; 50 -AATTGGAAGCTGTGCAGTATAAAAC-30 and 50 -TGATGGTTATATTTATCAGCAAGGAT-30 .
2.7. Western blot analysis
2.10. Immunostaining of cystatin A
Cytosolic protein was extracted with cell lysis buffer (50 mM Tris—HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM ethylene glycol-bis[aminoethylether]-tetraacetic acid; 1 mM phenylmethylsulfonyl fluoride; 1 mg/ml each of aprotinin, leupeptin and pepstatin; 1 mM sodium vanadate and 1 mM NaF) and was electrophoresed on a 12.5% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto nylon membrane for 1 h in a buffer containing 25 mM Tris—HCl (pH 8.3), 192 mM glycine and 20% methanol. The blots were blocked with 5% skim milk in Tris-buffered saline (TBS, pH 7.6) for 1 h at room temperature and were then incubated at 4 8C overnight with anti-caspase 3, anti-caspase 8, anti-caspase 9, or anti-cystatin A antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After washing at room temperature with 0.1% Tween-20 in TBS a blotting detection kit for rabbit antibody (Amersham Japan, Tokyo, Japan) was used for immunodetection.
The frozen sections of cystatin A-tg mouse skin were pretreated with 0.3% hydrogen peroxide to block endogeneous peroxidase activity. Following incubation with normal goat serum diluted in PBS for 10 min the sections were incubated with anti-human cystatin A antibody diluted 1:200 in PBS for 1 h at room temperature. The sections were washed with PBS followed by application of Histofine streptavidine-biotin kit (Nichirei, Tokyo, Japan). Biotinylated rabbit anti-mouse antibody was used as secondary antibody and the immune reaction was visualized by avidine/biotin complex with 0.03% hydrogen peroxide and DAB. The sections were then counterstained with hematoxylin.
2.8. Caspase activity assay SVHK cells and NHK cells (1 106 cells) were lysed in a lysis buffer containing 25 mM N-2-hydroxythylpiperazine-N0 -2-ethane-sulfonic acid (pH 7.4), 5 mM ethylenediamine-tetraacetic acid, 2 mM dithiothreitol and 10 mM digitonin. The lysates
2.9. Transgenic mice
2.11. Apoptotic cells counting Biopsy specimens were taken, processed routinely, and stained with hematoxylin and eosin. The number of apoptotic cells in the epidermis was counted and expressed as number of apoptotic cells per cm [19]. Five sections were scored for apoptotic cells as described previously [8].
2.12. Statistics Statistical significance of the data obtained was evaluated by Student’s t-test using unpaired analyses.
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2.13. Materials Dulbecco’s modified Eagle’s medium and KGM medium were purchased from GIBCO (Grand Island, NY). Penicillin and streptomycin were obtained from M.A. Bioproducts (Walkersville, MD). All other chemicals were purchased from Nakarai Chemicals Ltd. (Kyoto, Japan).
3. Results 3.1. Establishment of SVHK cells which stably express cystatin A We transfected cystatin A expression vector into SVHK cells and the keratinocyes were cloned in the presence of G418. Following the selection of seven clones, Western blot analysis was performed to select the clone, which highly expressed cystatin A. Three clones (A3, B2, B3) highly expressed cystatin A with B2 the most potent (Fig. 1). We used B2 clone in the following studies.
3.2. Pro-caspase 3 cleavage is suppressed in UVB-irradiated B2 cells and cystatin Atransfected NHK cells No cleaved caspase 3, caspase 8, or caspase 9 was detected in SVHK cells or in B2 cells without UVB irradiation. UVB irradiation of SVHK cells induced cleaved pro-caspase 3, pro-caspase 8 and pro-caspase 9 to active caspase forms, respectively (Fig. 2, lane 3). In B2 cells cleaved caspase 3 was markedly suppressed following UVB irradiation (Fig. 2, lane 4). The effect was also observed in NHK cells transfected with cystatin A expression-adenovirus vector with the maximal effect at (1—2) 108 pfu/ml (Fig. 3). In contrast cleavage of pro-caspase 8, or pro-caspase 9 was not affected in either UVB-irradiated B2 cells (Fig. 2, lane 4) or adenovirus vectortransfected NHK cells (data not shown).
Fig. 1 Establishment of SVHK cells which highly express cystatin A. Cystatin A expression vector was transfected into SVHK cells and neomycin resistant clones were isolated in the presence of G418. Then Western blot analysis was performed. Three clones, A3, B2, and B3 out of seven, showed high expression of cystatin A with the highest expression in B2.
Fig. 2 Effect of pro-caspase cleavage in UVB-irradiated B2 cells. Following UVB irradiation (50 mJ/cm2) SVHK cells and B2 cells were cultured for 6 h. Then cells were harvested and Western blot analysis was performed. Pro-caspase 3, 8, and 9 and caspase 3, 8, and 9 forms are shown. (1) non-transfected SVHK cells, (2) B2 cells, (3) UVB-irradiated non-transfected SVHK cells, and (4) UVBirradiated B2 cells.
3.3. Caspase 3 activity is suppressed in UVB-irradiated B2 cells and cystatin A transfected NHK cells Following UVB irradiation caspase 3, 8, and 9 activities were markedly stimulated in SVHK cells (Fig. 4A) and in NHK cells (Fig. 4B). Although UVBinduced increase in caspase 3 activity was significantly inhibited in B2 cells and cystatin A adenovirus transfected NHK cells (Fig. 4A and B), suppression of caspase 8 or caspase 9 activities was not observed in these cells (Fig. 4A).
Fig. 3 UVB-induced pro-caspase 3 cleavage in cystatin A expression adenovirus vector-transfected NHK cells. Following cystatin A transfection NHK cells were irradiated with UVB (50 mJ/cm2) and NHK cells were cultured for 6 h. Then cells were collected and Western blot analyses were performed using anti-caspase 3 (upper panel) and anticystatin A (lower panel) antibodies.
Cystatin A suppresses apoptosis
Fig. 4 Suppression of caspase 3 activity in UVB irradiated B2 and cystatin A-transfected NHK cells. (A) Following UVB irradiation (50 mJ/cm2) SVHK cells and B2 cells were cultured for 6 h and caspase assays were performed. Open bars, non-transfected SVHK; closed bars, B2 cells. Caspase 3, 8, and 9 indicate each caspase activity. (B) NHK cells were transfected with various concentration of cystatin A expression adenovirus vectors and were cultured for 24 h. Following UVB irradiation (50 mJ/ cm2) the transfected NHK cells were cultured for 6h. Then the cells were collected and caspase 3 assay was performed. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with non-transfected cells.
3.4. Cystatin A-transfected keratinocytes are resistant to UVB-induced cell death UVB irradiation induced SVHK cell death with the maximal effect at 50 mJ/cm2 showing about 25% of cells survived (Fig. 5A). B2 cells which highly expressed cystatin A were more resistant to UVBinduced cell death and about 50% of B2 cells
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Fig. 5 Cystatin A suppresses UVB-induced apoptosis of keratinocytes. (A) Following UVB irradiation (50 mJ/cm2) SVHK cells and B2 cells were cultured for the indicated time. Viable cells were evaluated by measuring the OD at 540 nm. (*): untransfected SVHK cells; (^): B2 cells. NHK cells were transfected with various concentrations of cystatin A expression-adenovirus vector and were cultured for 24 h. (B) Following UVB irradiation (50 mJ/ cm2) these cells were cultured for another 12 h. Then viable cell assay was performed. Cont. indicates NHK cells without UVB irradiation. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with untransfected cells.
survived (Fig. 5A). The anti-apoptotic effect of cystatin A was also observed in NHK cells and was parallel to cystatin A expression level (Fig. 5B) with the maximal effect at 2 108 pfu/ml.
3.5. Cystatin A si-RNA suppressed cystatin A expression, increased UVB-induced cleavage of pro-caspase 3, and caspase 3 activity in NHK cells In order to confirm the effect of cystatin A on UVBinduced apoptosis the expression of cystatin A was suppressed by si-RNA knockdown. UVB-induced cleavage of pro-caspase 3 was markedly augmented
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Fig. 6 Cystatin A knockdown potentiates UVB-induced cleavage of pro-caspase 3 of keratinocytes. NHK cells were transfected with various concentrations of cystatin A si-RNA and were cultured for 24 h. Following UVB irradiation (50 mJ/cm2) the transfected cells were cultured for another 6 h. Then the cells were collected and Western blot analysis was performed using anti-caspase 3 (upper panel) and anti-cystatin A (lower panel) antibodies.
in si-RNA-transfected NHK cells with the maximal effect at 5—10 mg si-RNA transfection (Fig. 6). The transfection of cystatin A si-RNA increased cleaved caspase 3 (Fig. 6) accompanied with increased caspase 3 activity (Fig. 7) following UVB irradiation. Without UVB irradiation neither cleavage of procaspase 3 nor caspase 3 activation was detected by si-RNA treatment (Fig. 7).
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Fig. 8 Cystatin A knockdown potentiates UVB-induced cell death of keratinocytes. NHK cells were transfected with various concentrations of cystatin A si-RNA and were cultured for 24 h. Following UVB irradiation (50 mJ/cm2) the transfected NHK cells were cultured for 12 h. Then viable cell assay was performed. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with non-transfected cells.
3.6. Cystatin A knockdown increased UVB-induced cell death Cystatin A knockdown by si-RNA potently increased UVB-induced apoptosis compared with control NHK cells (Fig. 8). The effect was dose-dependent with the maximal effect at 10 mg si-RNA. si-RNA treatment without UVB irradiation had no effect on cell viability.
3.7. The epidermis of cystatin A transgenic mice (cystatin A-tg) was more resistant to UVB-induced apoptosis
Fig. 7 Cystatin A knockdown potentiates UVB-induced caspase 3 activation of keratinocytes. NHK cells were transfected with various concentrations of cystatin A siRNA and were cultured for 24 h. Following UVB irradiation (50 mJ/cm2) the transfected NHK cells were cultured for 6 h. Then the cells were collected and caspase 3 activity was measured. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with non-transfected cells.
Immunohistochemical analysis revealed that cystatin A was potently expressed in the epidermis of cystatin A-tg compared with control mice (Fig. 9A). Western blot analysis confirmed the high expression of cystatin A (Fig. 9B). UVB-irradiation was performed on cystatin A-tg. The induction of apoptosis was significantly decreased in cystatin A-tg epidermis compared with control mice epidermis (Fig. 10A and B). The suppressive effect was observed by 12 h, more marked at 24 h, and remained until 48 h (Fig. 10).
4. Discussion Our results clearly demonstrated that cystatin A protects keratinocytes from UVB-induced apoptosis both in vitro and in vivo. The suppressive effect was closely associated with the expression level of
Cystatin A suppresses apoptosis
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Fig. 9 Expression of cystatin A in transgenic mouse skin. Cystatin A-tg and control mice skin was excised and immunohistochemical (A) and Western blot (B) analyses for cystatin A were performed. Scale bar, 100 mm.
Fig. 10 UVB-induced apoptosis in cystatin A transgenic mice. Cystatin A-tg or control mouse skin was irradiated with UVB (100 mJ/cm2). Mouse skin samples were excised at 24 h (A) and at the indicated time (B) and histochemical analysis was performed. (A) Hematoxylin and eosin staining of the excised skin at 24 h following UVB irradiation. Arrows indicate apoptotic cells (sunburn cells). Scale bar: 100 mm. (B) Apoptotic cells (sunburn cells) were counted at the indicated time. Open bars indicate control mouse. Shaded bars indicate cystatin A-tg mouse. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with control mouse.
186 cystatin A. To the best of our knowledge this is the first report showing antiapoptotic effect of cystatin A in keratinocytes. Previous studies showed that UVB irradiation induces apoptosis of keratinocytes with increased caspase 8, 9, and 3 activities [7,20]. In the present study, cystatin A significantly suppressed UVB-induced caspase 3 activity without affecting caspase 8 or caspase 9 activities. This is consistent with that caspase 3 locates downstream of caspase cascade with more direct involvement in UVB-induced apoptosis. While specific inhibitors of caspases markedly suppressed UVB-induced apoptosis [20], the suppressive effect of cystatin A was less potent. However, the knockdown of cystatin A significantly augmented UVB-induced apoptosis suggesting a critical role of this proteinase inhibitor against apoptotic process of keratinocytes. Jones et al. reported that cystatin A reduced bile saltinduced apoptosis of rat hepatoma cell line [21], which was apparently independent of the inhibition of caspase 3 activation. They also showed that the inhibition of caspase 3 blocked the activation of cathepsin B suggesting that cathepsin B activation follows caspase 3 activation. However, careful analysis of their results suggests that cystatin A considerably suppressed caspase 3 activity [21], which is consistent with the direct inhibition of caspase 3 by cystatin A. Our results might indicate cystatin Adependent suppressive pathway of caspase 3, which has apparently been ignored in the literature. Nevertheless, the overall anti-apoptotic effect was up to 10—20% in cystatin A-transgenic mice. This might be due to the interaction of other antiapoptotic molecules, such as bcl-2, and Akt, in keratinocytes. UVB irradiation induces p53 protein in normal human keratinocytes [22]. It is known that UVB irradiation activates p53 through a conformational change and induces apoptosis following the inhibition of cell cycle progression [23]. Cystatin A efficiently inhibits cathepsin B with an inhibition constant (Ki) of 8.2 nM [24] and previous report indicates that the inhibition of cathepsin B blocks p53-induced apoptosis [25]. Since cathepsin B is widely expressed including keratinocytes [26], the mechanism of the suppressive effect of cystatin A on UVB-induced apoptosis might be related to cathepsin B. Roberts et al. [27] reported that cathepsin B is activated and translocates from cytoplasm to nucleus during bile salt-induced apoptosis of hepatocytes. In their report the effect of cathepsin B was suppressed by cystatin A showing the important role of cystatin A on the regulation of apoptotic phenomena. Cystatin A is highly expressed in epidermal keratinocytes and the concentration is up to
H. Takahashi et al. 10,000-fold higher than most other cells. Polymorphonuclear leukocytes also express abundant cystatin A [28]. Immunohistochemical and biological analysis revealed that cystatin A is more markedly expressed in the psoriatic epidermis. Our previous study revealed that psoriatic hyperproliferative epidermis highly expresses anti-apoptotic molecules, such as Bcl-xL and inhibitor of caspase-activated DNase, suggesting anti-apoptotic condition in this inflammatory skin disorder [29]. The increased expression of cystatin A indicates another antiapoptotic condition in the psoriatic epidermis, which is also characterized by infiltration of cystatin A-rich polymorphonuclear leukocytes. Psoriasis also shows an aberrant expression of differentiation-related molecules. This includes increased expression of involucrin and cystatin A, and decreased expression of loricrin [30]. Long-term observation of cystatin A-tg mice, however, did not show any features of psoriatic skin, such as scaly erythematous plaques or nail deformity. Thus, the increased expression of cystatin A appears to be not the cause, but among the features of psoriatic hyperproliferative epidermis. In conclusion, our results indicate that cystatin A suppresses UVB-induced apoptosis of keratinocytes by inhibiting caspase 3 activity. The results indicate a novel function of cystatin A as an antiapoptotic molecule. This should be appreciated among the functions of cystatin A, a cysteine proteinase inhibitor, and a cornified cell envelope precursor of epidermal keratinocytes.
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Cystatin A suppresses apoptosis [8] Takahashi H, Honma M, Ishida-Yamamoto A, Namikawa K, Miwa A, Okado H, et al. In vitro and in vivo transfer of bcl-2 gene into keratinocytes suppresses UVB-induced apoptosis. Photochem Photobiol 2001;74:579—86. [9] Dubin G. Proteinaceous cystaine proteinase. Cell Mol Life Sci 2005;62:653—69. [10] Zettergren JG, Peterson LL, Wuepper KD. Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue. Proc Natl Acad Sci 1984;81: 238—42. [11] Takahashi H, Kinouchi M, Wuepper KD, Iizuka H. Cloning of human keratolinin cDNA: keratolinin is identical with a cysteine proteinase inhibitor, cystatin A, and is regulated by Ca2+ TPA. J Invest Dermatol 1997;108:843—7. [12] Takahashi H, Asano K, Kinouchi M, Ishida-Yamamoto A, Wuepper KD, Iizuka H. Structute and transcriptional regulation of the human cystatin A gene. The 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive element-2 site (272 to 278) on cystatin A gene is critical for TPA-dependent regulation. J Biol Chem 1998;273:17375—80. [13] Takahashi H, Honma M, Ishida-Yamanoto A, Namikawa K, Kiyama H, Iizuka H. Expression of human cystatin A by keratinocytes is positively regulated via Ra/MEKK1/MKK7/ ERK pathway but negatively regulated via the Ra/Raf-1/ MEK/ERK pathway. J Biol Chem 2001;276:36632—8. [14] Steinert PM, Marekov LN. 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;270:117702—11. [15] Takahashi M, Tezuka T, Katunuma N. Inhibition of growth and cysteine proteinase activity of Staphylococcus aureus V8 by phosphorylated cystatin a in skin cornified envelope. FEBS Lett 1994;355:275—8. [16] Steinberg ML, Defendi V. Transformation and immortalization of human keratinocytes by SV40. J Invest Dematol 1983;81:131S—6S. [17] Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991;66:233—43. [18] Tarutani M, Itami S, Okabe M, Ikawa M, Tezuka T, Yoshikawa K, et al. Tissue-specific knockout of the mouse Pig-a gene
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Cystatin A suppresses ultraviolet B-induced apoptosis of keratinocytes Hidetoshi Takahashi *, Naritsuna Komatsu, Masaki Ibe, Akemi Ishida-Yamamoto, Yoshio Hashimoto, Hajime Iizuka Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaokaminami, Asahikawa 078-8510, Japan Received 15 October 2006; received in revised form 16 January 2007; accepted 2 February 2007
KEYWORDS Apoptosis; Cystatin A; Keratinocytes; Ultraviolet B
Summary Background: Cystatin A is a cysteine proteinase inhibitor abundantly expressed in keratinocytes. Although cystatin A is one of the cornified cell envelope constituents and expressed in the upper epidermis, its precise function is still unknown. Ultraviolet B irradiation (UVB) induces apoptosis accompanied with the activation of cysteine proteinases, caspases. Objective: We investigated the effect of cystatin A on UVB-induced apoptosis of keratinocytes. Methods: We assessed the caspase activities and apoptotic cell numbers induced by UVB ittadiation in cystatin A gene transfected keratinocytes. Results: UVB-induced pro-caspase 3 cleavage and caspase 3 activation were suppressed in cystatin A expression vector-transfected SV40-transformed human keratinocytes (SVHK). Furthermore, the transfected SVHK cells were resistant to UVB-induced apoptosis. In contrast neither caspase 8 nor caspase 9 activities were affected by UVB irradiation in cystatin A-transfected SVHK cells. The effects were also observed in cystatin A expression adenovirus vector-transfected cultured normal human keratinocytes (NHK). Conversely knockdown of cystatin A by siRNA induced marked apoptosis of NHK cells following UVB irradiation accompanied with increased caspase 3 activity. In order to confirm the antiapoptotic effect of cystatin A in vivo UVB irradiation was performed on cystatin A transgenic mice (cystatin A-tg). The epidermis from cystatin A-tg was resistant to UVB-induced apoptosis compared to control mice epidermis.
Abbreviations: Cystatin A-tg, cystatin A transgenic mouse; NHK, normal cultured human keratinocyte; SVHK, SV40 transformed human keratinocytes; UVB, ultraviolet B * Corresponding author. E-mail address: [email protected] (H. Takahashi). 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.02.003
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H. Takahashi et al. Conclusion: These results indicate that cystatin A suppresses UVB-induced apoptosis of keratinocytes by the inhibition of caspase 3 activation. # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction
in a sub-confluent state by sub-culturing every 4—5 days.
Apoptosis, or programmed cell death, plays an important role on development, cell proliferation, and differentiation of various tissues [1,2]. In skin apoptosis is observed in hair follicles during catagen phase of hair cycle, in lichenoid tissue reaction, and in various skin tumors [3,4]. Apoptosis is also induced by various external stimuli, such as ultraviolet irradiation, hydroxy radical, and anti-cancer agents [5,6]. Ultraviolet B (UVB) is one of the potent inducers of apoptosis forming ‘‘sunburn cells’’. Biochemical analysis revealed that the apoptosis is induced by the activation of caspases, a member of cysteine proteinases [7]. Anti-apoptotic molecule, bcl-2, suppresses UVB-induced apoptosis by inhibiting caspase activity [8]. Cystatin A is a member of cysteine proteinase inhibitors and is a potent inhibitor of cathepsins [9]. In particular, cystatin A efficiently inhibits cathepsin B. Biochemical and immunohistochemical analyses revealed that cystatin A is abundantly expressed in keratinocytes and is among the major constituents of cornified cell envelope of horny cells [10—14]. Although previous study indicated that cystatin a (a rat counterpart of cystatin A)-depleted rat skin is more sensitive to Staphylococcus aureus compared with normal skin [15], other function(s) of this proteinase inhibitor in the skin is still unknown. In the present study, we investigated the effect of cystatin A on UVB-induced apoptosis of keratinocytes both in vitro and in vivo.
Cystatin A cDNA [14] was cloned into a cosmid vector, pAxCALLNLw (donated by Dr. Saito, University of Tokyo, Tokyo, Japan). The procedure was performed as described previously [8]. Infection was performed by adding various titers of recombinant adenovirus. The cells were incubated at 37 8C for 60 min. The medium was changed to fresh KGM medium and the cells were incubated at 37 8C for 24 h before UVB irradiation.
2. Materials and methods
2.4. Si-RNA construction and transfection into NHK cells
2.1. Cell culture SV40-transformed human keratinocytes (SVHK cells) [16] were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum at 37 8C in 5% CO2 atmosphere. Normal human keratinocytes (NHK cells) were obtained from skin during plastic surgery. Informed consent was obtained from each patient. NHK cells were cultured in keratinocytes growth medium (KGM) containing insulin (5 mg/ml), and bovine pituitary extract (50 mg/ml) at 37 8C in air containing 5% CO2. The calcium concentration in the medium was 0.1 mM. NHK cells were maintained
2.2. Establishment of stably cystatin A expressing SVHK cells Cystatin A cDNA was introduced in the EcoRI sites of pSG5 vector (cystain A-pSG5) [14]. Then cystatin A-pSG (5 mg) and pSG-neo (1 mg), which expresses neomycin resistant gene, was transfected into 5 106 SVHK cells by electroporation according to the protocol of Amaxa Biosystems (Koeln, Germany). After the transfection the cells were cultured in the presence of neomycin (1 mg/ml), and neomycin resistant SVHK cells were isolated. Then the cells were examined for the expression of cystatin A by Western blot analysis using anti-cystatin A antibody and the clones, which highly expressed cystatin A, were selected.
2.3. Cystatin A adenovirus vector and its transfection into cultured normal human keratinocytes (NHK cells)
Si-RNA (CUCAAGUUGUUGCUGGAACTT) was designed and constructed by Takara biomedicals (Kyoto, Japan). Ten microgram of cystatin A si-RNA was transfected into 5 106 NHK cells by electroporation according to the protocol of Amaxa Biosystems and the cells were cultured for 48 h. Then transfected NHK cells were collected and various assays were performed.
2.5. UVB irradiation UVB irradiation source was a Toshiba-Eizai Dermaray instrument (DMR-1, Tokyo, Japan) equipped with
Cystatin A suppresses apoptosis five fluorescence lamps (FL-20-SE-30, Toshiba, Tokyo, Japan). The UVB source emits an energy spectrum with high fluence in the UVB region (290—320 nm) and a peak at 313 nm. In order to block UVC emission a WG-295 long pass filter was used (Schott Glass Technology, Duryea, PA). The emitted dose was routinely evaluated by a UVB radiometer photodetector (IL443 and SEE 240, International Light, Newburyport, MA).
2.6. Cell death assay
181 were clarified by centrifugation and supernatants were used for enzyme assays. Caspase 3 substrate (Z-Asp-Glu-Val-Asp-AFC), caspase 8 substrate (Z-Ile-Glu-Thr-Asp-AFC), and caspase 9 substrate (Z-Leu-Glu-His-Asp-AFC) were purchased from Kamiya Biomedical Co. (Seattle, WA). Caspase assays were carried out using fluorogenic substrates according to the protocol provided by the manufacture (Kamiya Biomedical Co.). The reaction mixtures were incubated at 30 8C for 1 h and fluorescence was measured by a fluorometer with an excitation of 400 nm and emission of 505 nm.
SVHK cells or NHK cells were seeded at 1 104 cells in a 96 well microtiter plate and were washed twice with phosphate-buffered saline (PBS, pH 7.5). After irradiation the cells were stained for 20 min at room temperature with 0.75% crystal violet in 50% ethanol, 0.25% NaCl, and 1.75% formaldehyde, and were washed three times with PBS. Dye uptake was measured by measuring optical density (OD) at 540 nm using an automated Micro-ELISA autoreader [17]. The ratio of viable cells was expressed as percentage of the OD value obtained without UVB treatment.
We excised the cystatin A cDNA from vector pGEM-3cystatin A [11,12] and ligated it into pBSK5 constract [18]. PBSK5-cystatin A was digested with EcoRI, purified, and used to generate transgenic founders on B6 background. Transgenic mice were identified by PCR of genomic DNA with primers specific for the gene encoding human cystatin A; 50 -AATTGGAAGCTGTGCAGTATAAAAC-30 and 50 -TGATGGTTATATTTATCAGCAAGGAT-30 .
2.7. Western blot analysis
2.10. Immunostaining of cystatin A
Cytosolic protein was extracted with cell lysis buffer (50 mM Tris—HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM ethylene glycol-bis[aminoethylether]-tetraacetic acid; 1 mM phenylmethylsulfonyl fluoride; 1 mg/ml each of aprotinin, leupeptin and pepstatin; 1 mM sodium vanadate and 1 mM NaF) and was electrophoresed on a 12.5% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto nylon membrane for 1 h in a buffer containing 25 mM Tris—HCl (pH 8.3), 192 mM glycine and 20% methanol. The blots were blocked with 5% skim milk in Tris-buffered saline (TBS, pH 7.6) for 1 h at room temperature and were then incubated at 4 8C overnight with anti-caspase 3, anti-caspase 8, anti-caspase 9, or anti-cystatin A antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After washing at room temperature with 0.1% Tween-20 in TBS a blotting detection kit for rabbit antibody (Amersham Japan, Tokyo, Japan) was used for immunodetection.
The frozen sections of cystatin A-tg mouse skin were pretreated with 0.3% hydrogen peroxide to block endogeneous peroxidase activity. Following incubation with normal goat serum diluted in PBS for 10 min the sections were incubated with anti-human cystatin A antibody diluted 1:200 in PBS for 1 h at room temperature. The sections were washed with PBS followed by application of Histofine streptavidine-biotin kit (Nichirei, Tokyo, Japan). Biotinylated rabbit anti-mouse antibody was used as secondary antibody and the immune reaction was visualized by avidine/biotin complex with 0.03% hydrogen peroxide and DAB. The sections were then counterstained with hematoxylin.
2.8. Caspase activity assay SVHK cells and NHK cells (1 106 cells) were lysed in a lysis buffer containing 25 mM N-2-hydroxythylpiperazine-N0 -2-ethane-sulfonic acid (pH 7.4), 5 mM ethylenediamine-tetraacetic acid, 2 mM dithiothreitol and 10 mM digitonin. The lysates
2.9. Transgenic mice
2.11. Apoptotic cells counting Biopsy specimens were taken, processed routinely, and stained with hematoxylin and eosin. The number of apoptotic cells in the epidermis was counted and expressed as number of apoptotic cells per cm [19]. Five sections were scored for apoptotic cells as described previously [8].
2.12. Statistics Statistical significance of the data obtained was evaluated by Student’s t-test using unpaired analyses.
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2.13. Materials Dulbecco’s modified Eagle’s medium and KGM medium were purchased from GIBCO (Grand Island, NY). Penicillin and streptomycin were obtained from M.A. Bioproducts (Walkersville, MD). All other chemicals were purchased from Nakarai Chemicals Ltd. (Kyoto, Japan).
3. Results 3.1. Establishment of SVHK cells which stably express cystatin A We transfected cystatin A expression vector into SVHK cells and the keratinocyes were cloned in the presence of G418. Following the selection of seven clones, Western blot analysis was performed to select the clone, which highly expressed cystatin A. Three clones (A3, B2, B3) highly expressed cystatin A with B2 the most potent (Fig. 1). We used B2 clone in the following studies.
3.2. Pro-caspase 3 cleavage is suppressed in UVB-irradiated B2 cells and cystatin Atransfected NHK cells No cleaved caspase 3, caspase 8, or caspase 9 was detected in SVHK cells or in B2 cells without UVB irradiation. UVB irradiation of SVHK cells induced cleaved pro-caspase 3, pro-caspase 8 and pro-caspase 9 to active caspase forms, respectively (Fig. 2, lane 3). In B2 cells cleaved caspase 3 was markedly suppressed following UVB irradiation (Fig. 2, lane 4). The effect was also observed in NHK cells transfected with cystatin A expression-adenovirus vector with the maximal effect at (1—2) 108 pfu/ml (Fig. 3). In contrast cleavage of pro-caspase 8, or pro-caspase 9 was not affected in either UVB-irradiated B2 cells (Fig. 2, lane 4) or adenovirus vectortransfected NHK cells (data not shown).
Fig. 1 Establishment of SVHK cells which highly express cystatin A. Cystatin A expression vector was transfected into SVHK cells and neomycin resistant clones were isolated in the presence of G418. Then Western blot analysis was performed. Three clones, A3, B2, and B3 out of seven, showed high expression of cystatin A with the highest expression in B2.
Fig. 2 Effect of pro-caspase cleavage in UVB-irradiated B2 cells. Following UVB irradiation (50 mJ/cm2) SVHK cells and B2 cells were cultured for 6 h. Then cells were harvested and Western blot analysis was performed. Pro-caspase 3, 8, and 9 and caspase 3, 8, and 9 forms are shown. (1) non-transfected SVHK cells, (2) B2 cells, (3) UVB-irradiated non-transfected SVHK cells, and (4) UVBirradiated B2 cells.
3.3. Caspase 3 activity is suppressed in UVB-irradiated B2 cells and cystatin A transfected NHK cells Following UVB irradiation caspase 3, 8, and 9 activities were markedly stimulated in SVHK cells (Fig. 4A) and in NHK cells (Fig. 4B). Although UVBinduced increase in caspase 3 activity was significantly inhibited in B2 cells and cystatin A adenovirus transfected NHK cells (Fig. 4A and B), suppression of caspase 8 or caspase 9 activities was not observed in these cells (Fig. 4A).
Fig. 3 UVB-induced pro-caspase 3 cleavage in cystatin A expression adenovirus vector-transfected NHK cells. Following cystatin A transfection NHK cells were irradiated with UVB (50 mJ/cm2) and NHK cells were cultured for 6 h. Then cells were collected and Western blot analyses were performed using anti-caspase 3 (upper panel) and anticystatin A (lower panel) antibodies.
Cystatin A suppresses apoptosis
Fig. 4 Suppression of caspase 3 activity in UVB irradiated B2 and cystatin A-transfected NHK cells. (A) Following UVB irradiation (50 mJ/cm2) SVHK cells and B2 cells were cultured for 6 h and caspase assays were performed. Open bars, non-transfected SVHK; closed bars, B2 cells. Caspase 3, 8, and 9 indicate each caspase activity. (B) NHK cells were transfected with various concentration of cystatin A expression adenovirus vectors and were cultured for 24 h. Following UVB irradiation (50 mJ/ cm2) the transfected NHK cells were cultured for 6h. Then the cells were collected and caspase 3 assay was performed. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with non-transfected cells.
3.4. Cystatin A-transfected keratinocytes are resistant to UVB-induced cell death UVB irradiation induced SVHK cell death with the maximal effect at 50 mJ/cm2 showing about 25% of cells survived (Fig. 5A). B2 cells which highly expressed cystatin A were more resistant to UVBinduced cell death and about 50% of B2 cells
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Fig. 5 Cystatin A suppresses UVB-induced apoptosis of keratinocytes. (A) Following UVB irradiation (50 mJ/cm2) SVHK cells and B2 cells were cultured for the indicated time. Viable cells were evaluated by measuring the OD at 540 nm. (*): untransfected SVHK cells; (^): B2 cells. NHK cells were transfected with various concentrations of cystatin A expression-adenovirus vector and were cultured for 24 h. (B) Following UVB irradiation (50 mJ/ cm2) these cells were cultured for another 12 h. Then viable cell assay was performed. Cont. indicates NHK cells without UVB irradiation. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with untransfected cells.
survived (Fig. 5A). The anti-apoptotic effect of cystatin A was also observed in NHK cells and was parallel to cystatin A expression level (Fig. 5B) with the maximal effect at 2 108 pfu/ml.
3.5. Cystatin A si-RNA suppressed cystatin A expression, increased UVB-induced cleavage of pro-caspase 3, and caspase 3 activity in NHK cells In order to confirm the effect of cystatin A on UVBinduced apoptosis the expression of cystatin A was suppressed by si-RNA knockdown. UVB-induced cleavage of pro-caspase 3 was markedly augmented
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Fig. 6 Cystatin A knockdown potentiates UVB-induced cleavage of pro-caspase 3 of keratinocytes. NHK cells were transfected with various concentrations of cystatin A si-RNA and were cultured for 24 h. Following UVB irradiation (50 mJ/cm2) the transfected cells were cultured for another 6 h. Then the cells were collected and Western blot analysis was performed using anti-caspase 3 (upper panel) and anti-cystatin A (lower panel) antibodies.
in si-RNA-transfected NHK cells with the maximal effect at 5—10 mg si-RNA transfection (Fig. 6). The transfection of cystatin A si-RNA increased cleaved caspase 3 (Fig. 6) accompanied with increased caspase 3 activity (Fig. 7) following UVB irradiation. Without UVB irradiation neither cleavage of procaspase 3 nor caspase 3 activation was detected by si-RNA treatment (Fig. 7).
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Fig. 8 Cystatin A knockdown potentiates UVB-induced cell death of keratinocytes. NHK cells were transfected with various concentrations of cystatin A si-RNA and were cultured for 24 h. Following UVB irradiation (50 mJ/cm2) the transfected NHK cells were cultured for 12 h. Then viable cell assay was performed. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with non-transfected cells.
3.6. Cystatin A knockdown increased UVB-induced cell death Cystatin A knockdown by si-RNA potently increased UVB-induced apoptosis compared with control NHK cells (Fig. 8). The effect was dose-dependent with the maximal effect at 10 mg si-RNA. si-RNA treatment without UVB irradiation had no effect on cell viability.
3.7. The epidermis of cystatin A transgenic mice (cystatin A-tg) was more resistant to UVB-induced apoptosis
Fig. 7 Cystatin A knockdown potentiates UVB-induced caspase 3 activation of keratinocytes. NHK cells were transfected with various concentrations of cystatin A siRNA and were cultured for 24 h. Following UVB irradiation (50 mJ/cm2) the transfected NHK cells were cultured for 6 h. Then the cells were collected and caspase 3 activity was measured. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with non-transfected cells.
Immunohistochemical analysis revealed that cystatin A was potently expressed in the epidermis of cystatin A-tg compared with control mice (Fig. 9A). Western blot analysis confirmed the high expression of cystatin A (Fig. 9B). UVB-irradiation was performed on cystatin A-tg. The induction of apoptosis was significantly decreased in cystatin A-tg epidermis compared with control mice epidermis (Fig. 10A and B). The suppressive effect was observed by 12 h, more marked at 24 h, and remained until 48 h (Fig. 10).
4. Discussion Our results clearly demonstrated that cystatin A protects keratinocytes from UVB-induced apoptosis both in vitro and in vivo. The suppressive effect was closely associated with the expression level of
Cystatin A suppresses apoptosis
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Fig. 9 Expression of cystatin A in transgenic mouse skin. Cystatin A-tg and control mice skin was excised and immunohistochemical (A) and Western blot (B) analyses for cystatin A were performed. Scale bar, 100 mm.
Fig. 10 UVB-induced apoptosis in cystatin A transgenic mice. Cystatin A-tg or control mouse skin was irradiated with UVB (100 mJ/cm2). Mouse skin samples were excised at 24 h (A) and at the indicated time (B) and histochemical analysis was performed. (A) Hematoxylin and eosin staining of the excised skin at 24 h following UVB irradiation. Arrows indicate apoptotic cells (sunburn cells). Scale bar: 100 mm. (B) Apoptotic cells (sunburn cells) were counted at the indicated time. Open bars indicate control mouse. Shaded bars indicate cystatin A-tg mouse. Data are means S.E.M. of four independent experiments. *P < 0.01 compared with control mouse.
186 cystatin A. To the best of our knowledge this is the first report showing antiapoptotic effect of cystatin A in keratinocytes. Previous studies showed that UVB irradiation induces apoptosis of keratinocytes with increased caspase 8, 9, and 3 activities [7,20]. In the present study, cystatin A significantly suppressed UVB-induced caspase 3 activity without affecting caspase 8 or caspase 9 activities. This is consistent with that caspase 3 locates downstream of caspase cascade with more direct involvement in UVB-induced apoptosis. While specific inhibitors of caspases markedly suppressed UVB-induced apoptosis [20], the suppressive effect of cystatin A was less potent. However, the knockdown of cystatin A significantly augmented UVB-induced apoptosis suggesting a critical role of this proteinase inhibitor against apoptotic process of keratinocytes. Jones et al. reported that cystatin A reduced bile saltinduced apoptosis of rat hepatoma cell line [21], which was apparently independent of the inhibition of caspase 3 activation. They also showed that the inhibition of caspase 3 blocked the activation of cathepsin B suggesting that cathepsin B activation follows caspase 3 activation. However, careful analysis of their results suggests that cystatin A considerably suppressed caspase 3 activity [21], which is consistent with the direct inhibition of caspase 3 by cystatin A. Our results might indicate cystatin Adependent suppressive pathway of caspase 3, which has apparently been ignored in the literature. Nevertheless, the overall anti-apoptotic effect was up to 10—20% in cystatin A-transgenic mice. This might be due to the interaction of other antiapoptotic molecules, such as bcl-2, and Akt, in keratinocytes. UVB irradiation induces p53 protein in normal human keratinocytes [22]. It is known that UVB irradiation activates p53 through a conformational change and induces apoptosis following the inhibition of cell cycle progression [23]. Cystatin A efficiently inhibits cathepsin B with an inhibition constant (Ki) of 8.2 nM [24] and previous report indicates that the inhibition of cathepsin B blocks p53-induced apoptosis [25]. Since cathepsin B is widely expressed including keratinocytes [26], the mechanism of the suppressive effect of cystatin A on UVB-induced apoptosis might be related to cathepsin B. Roberts et al. [27] reported that cathepsin B is activated and translocates from cytoplasm to nucleus during bile salt-induced apoptosis of hepatocytes. In their report the effect of cathepsin B was suppressed by cystatin A showing the important role of cystatin A on the regulation of apoptotic phenomena. Cystatin A is highly expressed in epidermal keratinocytes and the concentration is up to
H. Takahashi et al. 10,000-fold higher than most other cells. Polymorphonuclear leukocytes also express abundant cystatin A [28]. Immunohistochemical and biological analysis revealed that cystatin A is more markedly expressed in the psoriatic epidermis. Our previous study revealed that psoriatic hyperproliferative epidermis highly expresses anti-apoptotic molecules, such as Bcl-xL and inhibitor of caspase-activated DNase, suggesting anti-apoptotic condition in this inflammatory skin disorder [29]. The increased expression of cystatin A indicates another antiapoptotic condition in the psoriatic epidermis, which is also characterized by infiltration of cystatin A-rich polymorphonuclear leukocytes. Psoriasis also shows an aberrant expression of differentiation-related molecules. This includes increased expression of involucrin and cystatin A, and decreased expression of loricrin [30]. Long-term observation of cystatin A-tg mice, however, did not show any features of psoriatic skin, such as scaly erythematous plaques or nail deformity. Thus, the increased expression of cystatin A appears to be not the cause, but among the features of psoriatic hyperproliferative epidermis. In conclusion, our results indicate that cystatin A suppresses UVB-induced apoptosis of keratinocytes by inhibiting caspase 3 activity. The results indicate a novel function of cystatin A as an antiapoptotic molecule. This should be appreciated among the functions of cystatin A, a cysteine proteinase inhibitor, and a cornified cell envelope precursor of epidermal keratinocytes.
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