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Impaired repair ability of hsp70.1 KO mouse after UVB irradiation
Journal of Dermatological Science 28 (2002) 144– 151 www.elsevier.com/locate/jdermsci
Impaired repair ability of hsp70.1 KO mouse after UVB irradiation Sun Bang Kwon a, Cui Young a, Dong Seok Kim a, Hyun O. Choi a, Kyu Han Kim a, Jin Ho Chung a, Hee Chul Eun a, Kyoung Chan Park a,*, Chang Kyu Oh b, Jeong Sun Seo b a
Department of Dermatology and Artificial Organ Laboratory of the Clinical Research Institute, Seoul National Uni6ersity College of Medicine, Seoul 110 -744, South Korea b Department of Biochemistry and Ilchun Molecular Medicine Institute MRC, Seoul National Uni6ersity College of Medicine, Seoul, South Korea Received 20 August 2001; received in revised form 1 October 2001; accepted 3 October 2001
Abstract UV light is absorbed in the epidermis and induces sunburn cell formation. It has been reported that HSP70 increases the UVB resistance of cell lines by in vitro experiments using various cell lines. In this study, hsp70.1 − / − KO mouse was used in order to study the role of HSP70 after UVB irradiation. Western blotting showed a decreased level of HSP70 in hsp70.1 − / − KO mouse compared with wild type FVB mouse. Six h after UVB irradiation, there were no significant histologic differences between the hsp70.1 − / − KO mouse and the wild type FVB mouse. A similar degree of nuclear swelling was observed. However, there were significant differences at 12 and 24 h after UVB irradiation. After 12 h, a few apoptotic cells were observed in the wild type mouse, but a large number of cells were apoptotic in the hsp70.1 − / − KO mouse. After 24 h, the epidermis of the wild type FVB mouse was relatively intact, but almost the entire epidermis was necrotic in the hsp70.1 − / − KO mouse. These results showed that epidermal injury of hsp70.1 − / − KO mouse was much more severe than that of wild type mouse although initial changes are similar in both species of mice. These results suggest that susceptibility of hsp70.1 − / − KO mouse to UVB irradiation may originate from a defect in the repair mechanism. This HSP deficient model may be useful in studies of the effects of tissue injury that relate to the impaired tissue repair mechanisms. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: HSP70; UVB; Repair
1. Introduction
* Corresponding author. Tel.: + 82-2-3668-7474; fax: + 822-3675-1187. E-mail address: [email protected] (K.C. Park).
HSPs are a highly conserved class of molecules that were initially characterized in the context of their cellular stress response [1]. Based on molecular mass and sequence homology, HSPs are
0923-1811/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 1 ) 0 0 1 5 6 - 6
S.B. Kwon et al. / Journal of Dermatological Science 28 (2002) 144–151
classified into families, which include ubiquitin, small HSPs of 20 –28 kDa, HSP60, HSP70 and HSP90 [2]. Heat shock protein 70 (HSP70) is the major heat inducible protein [3]. The skin serves as a barrier between a person and injurious environmental influences, such as ultraviolet (UV) light. Most of the damaging UV light is absorbed by the epidermis and leads to an apoptosis of keratinocytes [4]. It has been reported that heat treatment before UVB exposure increases the UVB resistance of the epidermal carcinoma cell line A431 by increasing the expression of the 72 kDa heat shock protein [5]. We also reported that an over-expression of HSP70 can prevent UVB-induced apoptosis of a human melanoma cell line [6]. A heat inducible member of the mouse hsp70 gene family has been characterized [7]. In this study, we analyzed whether the hsp70.1 − / − KO mouse is susceptible to UVB irradiation and we also evaluated the levels of HSP70 after UVB irradiation.
2. Materials and methods
2.1. Construction of hsp70.1 targeting 6ector and generation of knockout mice A murine genomic clone of the hsp70.1 locus was cloned from a l FixII phage library prepared from 129/Sv embryonic stem (ES) cells using a human hsp70 cDNA probe and was characterized by Southern blot analysis and DNA sequencing. The targeting vector contained a 7.5 kb NotI – XhoI fragment from the 5% promoter and the regulatory region of the hsp70.1 gene as the long arm, a 1.8 kb neomycin-resistant gene, a 0.8 kb NotI– SmaI fragment derived from hsp70.1 exon as the short arm, and a 3.4 kb fragment containing two copies of the herpes simplex virus thymidine kinase gene. Some of coding sequences of promotor were deleted and replaced by a PGK-neo expression cassette. Ten mg of NotI-linearized targeting vector was electroporated into E14/ BK4 ES cells and correct targeted clones were
145
selected with G418 (0.2 mg/ml, Life Technologies, Gaithersburg, MD) and FIAU (200 mM, Syntex, Palo Alto, CA) in DMEM medium. Southern blot analysis using a 520 bp 3% untranslated region (UTR) as a probe recognized a 6 kb BamHI homologous recombinant and a 10 kb wild-type BamHI one. Three independent homologous recombinant hsp70.1 ES cell clones were injected into FVB blastocysts. Heterozygous mutant mice were generated from one line. The mutant mice used in all experiments have been backcrossed onto the FVB strain for four generations. Genotypes were determined by a four-primer PCR approach and confirmed by Southern blotting of genomic DNA isolated from tail biopsies. Mice were genotyped by isolating tail DNA and digesting it with BamHI then Southern blotting analysis. For PCR genotyping, the following two primer sets were used: forward primer A (5%-AGGAGCTGACCCTTAACAGC-3%) and reverse primer B (5%-GTCGTTGGCGATGATCTC-3%) annealing to deleted part of genomic sequences; a forward primer C (5%-CGAGATCAGCAGCCTCTGTTCC-3%) located within the PGK promoter in the neomycin-resistance cassette; and a reverse primer D (5%-CCA AGCAGCTATCAAGTGTTCC-3%) which anneals to the genomic sequences in the 3% arm homologous region. A 500 bp PCR fragment was generated from the wild-type allele with primers A and B, whereas a 1250 bp fragment was generated from the targeted allele with primers C and D. All animal works was conducted in accordance with institutional guidelines.
2.2. UVB irradiation Mice were 12–16 weeks old when they were used in the experiments. Groups of mice (n= 3) were irradiated with UVB. The source of UVB was BLE-1T158 (Spectronics Corp, Westbury, NY). A Kodacel filter (TA401/407. Kodak, Rochester) was used to block wavelengths of less than 290 nm (ultraviolet C). The energy was measured with a Waldmann UV meter (model No. 585100; Waldmann Co, VS-Schwenningen, Germany).
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2.3. Verhoeff iron hematoxylin staining Specimens were fixed in 10% formalin, and embedded in paraffin. Then samples were sectioned at 6 mm thickness, mounted onto slides, and stained by Verhoeff iron hematoxylin staining methods [8].
2.4. Electron microscopic examination Skin specimens were fixed in 2.5% paraformaldehyde glutaraldehyde in 0.1 M cacodylate buffer(pH 7.4) and postfixed in 1% osmium tetroxide. Then, samples were dehydrated in graded acetone and embedded in epon 812. Ultrathin sections were double stained with 4% uranyl acetate and lead citrate solutions. They were observed in a transmission electron microscope (TEM, JEM-1200EX, JEOL, Japan).
mouse. Furthermore, levels of HSP70 increased more dramatically in the wild type FVB mouse after UVB irradiation in a time dependent manner (Fig. 1).
3.2. Loose collagen bundles in hsp70.1 − / − KO mouse Light microscopic examination showed that the distribution of collagen in dermis of hsp70.1 − / − KO mouse was loose compared with wild type FVB mouse (Fig. 2, arrow). Electron microscopic examination also showed loose distribution by both longitudinal and cross sectional view in hsp70.1 − / − KO mouse compared with wild type FVB mouse (Fig. 3, arrow).
3.3. UVB irradiation induced se6ere epidermal injury in the hsp70.1 − / − KO mouse but not in the wild type FVB mouse
2.5. Western blot analysis Collected samples were lyzed in cell lysis buffer (0.0625M Tris–HCl (pH 6.8), 5% b-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 2% SDS, 10 mM EDTA). About 5 – 10 mg of protein per lane was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and was blotted onto a nitrocellulose paper. Blots were incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using an enhanced chemiluminiscence plus kit (Amersham International, Little Chalfont, UK). Antibodies used in this experiment were as follows: antiHSP70 (sc-24, Santa Cruz Biotech Inc, Santa Cruz, CA), and anti-actin (sc-1615, Santa Cruz Biotech Inc, Santa Cruz, CA).
To answer the question whether HSP70 deficient mouse is susceptible to UVB irradiation, 500 mJ/cm2 of UVB were irradiated to both the wild type FVB and hsp70.1 − / − KO mouse. Five min after UVB irradiation, electron microscopic examination did not show evidences of cell membrane damage in either type of mice. But, condensation of the nucleus and disruption of organelles were observed. Six hours after UVB irradiation, there
3. Results
3.1. Decreased le6el of HSP expression in skin of hsp70.1 KO mouse Western blotting with anti-HSP70 antibody disclosed decreased levels of HSP 70 in hsp70.1 − / − KO mouse compared with the wild type FVB
Fig. 1. Western blot analysis of HSP70 in of hsp70.1 − / − KO mouse and wild type FVB mouse. Total proteins were isolated and separated on 12% SDS-polyacrylamide gels. After transfer of the protein, the membrane was hybridized with monoclonal anti-HSP70 antibody. Samples were taken before, and 6, 12, 24 h after 500 mJ/cm2 of UVB irradiation.
S.B. Kwon et al. / Journal of Dermatological Science 28 (2002) 144–151
147
Fig. 2. Verhoeff iron Hematoxylin staining of of hsp70.1 − / − KO mouse and FVB control mouse. Biopsy samples were fixed and embedded in paraffin and sections were stained. Samples were taken before (C), and 5 min, 6, 12, 24 h after 500 mJ/cm2 of UVB irradiation, ( ×200 in the original magnification, arrow: cleft in the dermis).
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Fig. 3. Electron microscopic findings of hsp70.1 − / − KO mouse and wild type FVB mouse. Biopsy specimens were fixed and embedded in epon 812. Samples were taken before (C), and 5 min, 6, 12, 24 h after 500 mJ/cm2 UVB irradiation, ( × 5000 in the original magnification, D: dermis, small arrows: dermo-epidermal junction, big arrow: apoptotic cell at basal layer in KO mouse).
S.B. Kwon et al. / Journal of Dermatological Science 28 (2002) 144–151
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Fig. 4. Electron microscopic findings of collagen fibers of hsp70.1 − / − KO mouse and wild type FVB mouse Biopsy specimens were fixed and embedded in epon 812. Collagen bundels were examined (arrow: compact fiber in wild type mouse and loose fiber in KO mouse).
were no significant histologic differences between wild type and hsp70.1 − / − KO mouse. Electron microscopically, basal epidermal cells showed a similar degree of nuclear swelling in both types of mice. Twelve hours after UVB irradiation, only a few damaged cells were observed and the nucleus had the similar granular appearance as it had before the UVB irradiation in wild type mouse. However, a number of cells became apoptotic in the hsp70.1 − / − KO mouse. Twenty-four hours after UVB irradiation, relatively intact epidermis was observed in wild type FVB mouse. However, almost the entire epidermis was injured in hsp70.1 − / − KO mouse. Compared with the wild type FVB mice, marked basophilic degeneration of the upper dermis and a moderate infiltration of inflammatory cells was observed in hsp70.1 − / − KO mouse. Electron microscopically, basal cells with normal morphology were observed in wild type FVB mice. However, almost all basal cells were apoptotic in hsp70.1 − / − KO mouse (Fig. 4).
4. Discussion It has been shown that heat shock modulates UVB-induced cell death in human epidermal keratinocytes [9]. Heat shock increases the levels of HSPs, and 72 kDa HSP is reported to be a mediator of resistance to ultraviolet B light [5,10]. Over-expression of HSP70 is known to protect murine fibroblasts against UV-light [11]. HSP70 is also known to prevent the activation of stress kinases such as JNK and p38 kinase. These effects are thought to be a novel pathway of cellular thermotolerance [12]. Recently, it has also been demonstrated that keratinocytes injected with a mAb to HSP72 died rapidly following UV irradiation [13]. We also demonstrated that hsp70.1 transfected melanoma cells were resistant to UVB irradiation [6]. These results suggest that HSP70 may play an essential role in modulating UVB injury to cells. HSP70 is heat-inducible by expression of two genes, hsp70.1 and hsp70.3, which is located in the MHC class III region [7]. However,
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differential regulation of these two genes has not been studied yet. In this study, we explore the in vivo role of HSP70 in UVB induced damage using hsp70.1 − / − KO mouse. Our results show that the level of HSP70 was down-regulated in hsp70.1 − / − KO mouse. The effects of low levels of HSP70 on susceptibility to UVB irradiation were studied by light microscopic and electron microscopic examination. Light microscopically, relatively intact epidermis was observed in wild type FVB mouse 24 h after 500 mJ/cm2 of UVB irradiation, but most of cells in the epidermis were necrotic in the hsp70.1 − / − KO mouse. Electron microscopically, a similar degree of nuclear swelling was observed in both hsp70.1 − / − KO mouse and wild type FVB mouse at 6 h after UVB irradiation. These results suggest that similar degree of damage is induced in both hsp70.1 − / − KO mouse and wild type FVB mouse. However, there are significant differences between hsp70.1 − / − KO mouse and wild type FVB mouse at 12 and 24 h after UVB irradiation. There are plenty of abnormal cells in hsp70.1 − / − KO mouse compare with wild type FVB mouse. Most of these cells have condenced chromatin which is a characteristic of apoptotic cell. Some apoptotic cells were engulfed by neighboring cells (Fig. 4, arrow). These findings suggest that increased apoptotic cells are induced by UVB irradiation in hsp70.1 − / − KO mouse even though similar degree of damages are observed 6 h after UVB irradiation. These results suggest that hsp70.1 − / − KO mouse is quite susceptible to UVB irradiation because of an impaired repair after UVB-induced apoptotic injuries. In addition to the chaperoning function for folding, transport, and assembly of newly synthesized polypeptides, HSP70 protects cells from a number of apoptoic stimuli, including heat shock, tumor necrosis factor, growth factor withdrawal, oxidative stress, chemotheapeutic agents, ceramides, and radiation [10,14– 16]. In this study, we also observed increased fragility and loose arrangement of collagen fiber in hsp70.1 − / − KO mouse. These findings need further study to clarify the relationship between HSP70 and collagen synthesis. In conclusion, our results suggest that hsp70.1 − / − KO mouse is quite susceptible to
UVB irradiation as a result of an impaired repair ability. These findings show that our model might be useful to study the effects of depletion of HSP70 from the aspect of tissue injury in its relation to tissue repair failure.
Acknowledgements This study was supported by the Korea Science and Engineering Foundation (98-0403-1901-5). The authors also wish to acknowledge the financial support of the Pacific Co.
References [1] Welch WJ, Suhan JP. Cellular biochemical events in mammalian cells during and after recovery from physiological stress. J Cell Biol 1986;103:2035 – 52. [2] Lindquist S, Craig EA. The heat shock proteins. Ann Rev Genet 1988;22:631 – 77. [3] Welch WJ, Feramisco JR. Nuclear and nucleolar localization of the 72 000 dalton heat shock protein in heat shocked mammalian cells. J Biol Chem 1984;259:4501 – 13. [4] Leverkus M, Yaar M, Gilchrest BA. Fas/Fas ligand interaction contributes to UV-induced apoptosis in human keratinocytes. Exp Cell Res 1997;232:255 – 62. [5] Trautinger F, Kindas-Mugge I, Barlan B, Neuner P, Knobler RM. 72 kDa heat shock protein is a mediator of resistance to ultraviolet B light. J Invest Dermatol 1995;105:160 – 2. [6] Park KC, Kim DS, Choi HO, Kim KH, Chung JH, Eun HC, Lee JS, Seo JS. Overexpression of HSP70 prevents ultraviolet B-induced apoptosis of a human melanoma cell line. Arch Dermatol Res 2000;292:482 – 7. [7] Ito Y, Ando A, Ando H, Ando J, Saijoh Y, Inoko H, Fujimoto H. Genomic structure of the spermatid-specific hsp70 homolog gene located in the class III region of the major histocompatibility complex of mouse and man. J Biochem (Tokyo) 1998;124:347 –53. [8] Augsburger HR. Elastic fibre system of the female canine urethra. Histochemical identification of elastic, elaunin and oxytalan fibres. Anat Histol Embryol 1997;26(4):297 – 302. [9] Maytin EV, Wimberly JM, Kane KS. Heat shock modulates UVB-induced cell death in human epidermal keratinocytes: evidence for a hyperthermia-inducible protective response. J Invest Dermatol 1994;103:547 – 53. [10] Samali A, Cotter TG. Heat shock proteins increase resistance to apoptosis. Exp Cell Res 1996;223:163 – 70. [11] Simon MM, Reikerstorfer A, Schwarz A, Krone C, Luger TA, Jaattela M, Schwarz T. Heat shock protein 70 over-
S.B. Kwon et al. / Journal of Dermatological Science 28 (2002) 144–151 expression affects the response to ultraviolet light in murine fibroblasts. J Clin Invest 1995;95:926 –33. [12] Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI, Sherman MY. Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 1997;272:18033 –7. [13] Bayerl C, Ung EG. Microinjection of an antibody against HSP72 in keratinocytes to study acute UV injury. Exp Dermatol 1999;8:247 –53. [14] Sasahira M, Lowry T, Simon RP, Greenberg DA.
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Epileptic tolerance: prior seizures protect against seizure-induced neuronal injury. Neurosci Lett 1995;185:95 – 8. [15] Mosser DD, Martin LH. Induced thermotolerance to apoptosis in a human T lymphocyte cell line. J Cell Physiol 1992;151:561 – 70. [16] Li CY, Lee JS, Ko YG, Kim JI, Seo JS. Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upsream of caspase-3 activation. J Biol Chem 2000;275:25665 – 71.
Impaired repair ability of hsp70.1 KO mouse after UVB irradiation Sun Bang Kwon a, Cui Young a, Dong Seok Kim a, Hyun O. Choi a, Kyu Han Kim a, Jin Ho Chung a, Hee Chul Eun a, Kyoung Chan Park a,*, Chang Kyu Oh b, Jeong Sun Seo b a
Department of Dermatology and Artificial Organ Laboratory of the Clinical Research Institute, Seoul National Uni6ersity College of Medicine, Seoul 110 -744, South Korea b Department of Biochemistry and Ilchun Molecular Medicine Institute MRC, Seoul National Uni6ersity College of Medicine, Seoul, South Korea Received 20 August 2001; received in revised form 1 October 2001; accepted 3 October 2001
Abstract UV light is absorbed in the epidermis and induces sunburn cell formation. It has been reported that HSP70 increases the UVB resistance of cell lines by in vitro experiments using various cell lines. In this study, hsp70.1 − / − KO mouse was used in order to study the role of HSP70 after UVB irradiation. Western blotting showed a decreased level of HSP70 in hsp70.1 − / − KO mouse compared with wild type FVB mouse. Six h after UVB irradiation, there were no significant histologic differences between the hsp70.1 − / − KO mouse and the wild type FVB mouse. A similar degree of nuclear swelling was observed. However, there were significant differences at 12 and 24 h after UVB irradiation. After 12 h, a few apoptotic cells were observed in the wild type mouse, but a large number of cells were apoptotic in the hsp70.1 − / − KO mouse. After 24 h, the epidermis of the wild type FVB mouse was relatively intact, but almost the entire epidermis was necrotic in the hsp70.1 − / − KO mouse. These results showed that epidermal injury of hsp70.1 − / − KO mouse was much more severe than that of wild type mouse although initial changes are similar in both species of mice. These results suggest that susceptibility of hsp70.1 − / − KO mouse to UVB irradiation may originate from a defect in the repair mechanism. This HSP deficient model may be useful in studies of the effects of tissue injury that relate to the impaired tissue repair mechanisms. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: HSP70; UVB; Repair
1. Introduction
* Corresponding author. Tel.: + 82-2-3668-7474; fax: + 822-3675-1187. E-mail address: [email protected] (K.C. Park).
HSPs are a highly conserved class of molecules that were initially characterized in the context of their cellular stress response [1]. Based on molecular mass and sequence homology, HSPs are
0923-1811/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 1 ) 0 0 1 5 6 - 6
S.B. Kwon et al. / Journal of Dermatological Science 28 (2002) 144–151
classified into families, which include ubiquitin, small HSPs of 20 –28 kDa, HSP60, HSP70 and HSP90 [2]. Heat shock protein 70 (HSP70) is the major heat inducible protein [3]. The skin serves as a barrier between a person and injurious environmental influences, such as ultraviolet (UV) light. Most of the damaging UV light is absorbed by the epidermis and leads to an apoptosis of keratinocytes [4]. It has been reported that heat treatment before UVB exposure increases the UVB resistance of the epidermal carcinoma cell line A431 by increasing the expression of the 72 kDa heat shock protein [5]. We also reported that an over-expression of HSP70 can prevent UVB-induced apoptosis of a human melanoma cell line [6]. A heat inducible member of the mouse hsp70 gene family has been characterized [7]. In this study, we analyzed whether the hsp70.1 − / − KO mouse is susceptible to UVB irradiation and we also evaluated the levels of HSP70 after UVB irradiation.
2. Materials and methods
2.1. Construction of hsp70.1 targeting 6ector and generation of knockout mice A murine genomic clone of the hsp70.1 locus was cloned from a l FixII phage library prepared from 129/Sv embryonic stem (ES) cells using a human hsp70 cDNA probe and was characterized by Southern blot analysis and DNA sequencing. The targeting vector contained a 7.5 kb NotI – XhoI fragment from the 5% promoter and the regulatory region of the hsp70.1 gene as the long arm, a 1.8 kb neomycin-resistant gene, a 0.8 kb NotI– SmaI fragment derived from hsp70.1 exon as the short arm, and a 3.4 kb fragment containing two copies of the herpes simplex virus thymidine kinase gene. Some of coding sequences of promotor were deleted and replaced by a PGK-neo expression cassette. Ten mg of NotI-linearized targeting vector was electroporated into E14/ BK4 ES cells and correct targeted clones were
145
selected with G418 (0.2 mg/ml, Life Technologies, Gaithersburg, MD) and FIAU (200 mM, Syntex, Palo Alto, CA) in DMEM medium. Southern blot analysis using a 520 bp 3% untranslated region (UTR) as a probe recognized a 6 kb BamHI homologous recombinant and a 10 kb wild-type BamHI one. Three independent homologous recombinant hsp70.1 ES cell clones were injected into FVB blastocysts. Heterozygous mutant mice were generated from one line. The mutant mice used in all experiments have been backcrossed onto the FVB strain for four generations. Genotypes were determined by a four-primer PCR approach and confirmed by Southern blotting of genomic DNA isolated from tail biopsies. Mice were genotyped by isolating tail DNA and digesting it with BamHI then Southern blotting analysis. For PCR genotyping, the following two primer sets were used: forward primer A (5%-AGGAGCTGACCCTTAACAGC-3%) and reverse primer B (5%-GTCGTTGGCGATGATCTC-3%) annealing to deleted part of genomic sequences; a forward primer C (5%-CGAGATCAGCAGCCTCTGTTCC-3%) located within the PGK promoter in the neomycin-resistance cassette; and a reverse primer D (5%-CCA AGCAGCTATCAAGTGTTCC-3%) which anneals to the genomic sequences in the 3% arm homologous region. A 500 bp PCR fragment was generated from the wild-type allele with primers A and B, whereas a 1250 bp fragment was generated from the targeted allele with primers C and D. All animal works was conducted in accordance with institutional guidelines.
2.2. UVB irradiation Mice were 12–16 weeks old when they were used in the experiments. Groups of mice (n= 3) were irradiated with UVB. The source of UVB was BLE-1T158 (Spectronics Corp, Westbury, NY). A Kodacel filter (TA401/407. Kodak, Rochester) was used to block wavelengths of less than 290 nm (ultraviolet C). The energy was measured with a Waldmann UV meter (model No. 585100; Waldmann Co, VS-Schwenningen, Germany).
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S.B. Kwon et al. / Journal of Dermatological Science 28 (2002) 144–151
2.3. Verhoeff iron hematoxylin staining Specimens were fixed in 10% formalin, and embedded in paraffin. Then samples were sectioned at 6 mm thickness, mounted onto slides, and stained by Verhoeff iron hematoxylin staining methods [8].
2.4. Electron microscopic examination Skin specimens were fixed in 2.5% paraformaldehyde glutaraldehyde in 0.1 M cacodylate buffer(pH 7.4) and postfixed in 1% osmium tetroxide. Then, samples were dehydrated in graded acetone and embedded in epon 812. Ultrathin sections were double stained with 4% uranyl acetate and lead citrate solutions. They were observed in a transmission electron microscope (TEM, JEM-1200EX, JEOL, Japan).
mouse. Furthermore, levels of HSP70 increased more dramatically in the wild type FVB mouse after UVB irradiation in a time dependent manner (Fig. 1).
3.2. Loose collagen bundles in hsp70.1 − / − KO mouse Light microscopic examination showed that the distribution of collagen in dermis of hsp70.1 − / − KO mouse was loose compared with wild type FVB mouse (Fig. 2, arrow). Electron microscopic examination also showed loose distribution by both longitudinal and cross sectional view in hsp70.1 − / − KO mouse compared with wild type FVB mouse (Fig. 3, arrow).
3.3. UVB irradiation induced se6ere epidermal injury in the hsp70.1 − / − KO mouse but not in the wild type FVB mouse
2.5. Western blot analysis Collected samples were lyzed in cell lysis buffer (0.0625M Tris–HCl (pH 6.8), 5% b-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 2% SDS, 10 mM EDTA). About 5 – 10 mg of protein per lane was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and was blotted onto a nitrocellulose paper. Blots were incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using an enhanced chemiluminiscence plus kit (Amersham International, Little Chalfont, UK). Antibodies used in this experiment were as follows: antiHSP70 (sc-24, Santa Cruz Biotech Inc, Santa Cruz, CA), and anti-actin (sc-1615, Santa Cruz Biotech Inc, Santa Cruz, CA).
To answer the question whether HSP70 deficient mouse is susceptible to UVB irradiation, 500 mJ/cm2 of UVB were irradiated to both the wild type FVB and hsp70.1 − / − KO mouse. Five min after UVB irradiation, electron microscopic examination did not show evidences of cell membrane damage in either type of mice. But, condensation of the nucleus and disruption of organelles were observed. Six hours after UVB irradiation, there
3. Results
3.1. Decreased le6el of HSP expression in skin of hsp70.1 KO mouse Western blotting with anti-HSP70 antibody disclosed decreased levels of HSP 70 in hsp70.1 − / − KO mouse compared with the wild type FVB
Fig. 1. Western blot analysis of HSP70 in of hsp70.1 − / − KO mouse and wild type FVB mouse. Total proteins were isolated and separated on 12% SDS-polyacrylamide gels. After transfer of the protein, the membrane was hybridized with monoclonal anti-HSP70 antibody. Samples were taken before, and 6, 12, 24 h after 500 mJ/cm2 of UVB irradiation.
S.B. Kwon et al. / Journal of Dermatological Science 28 (2002) 144–151
147
Fig. 2. Verhoeff iron Hematoxylin staining of of hsp70.1 − / − KO mouse and FVB control mouse. Biopsy samples were fixed and embedded in paraffin and sections were stained. Samples were taken before (C), and 5 min, 6, 12, 24 h after 500 mJ/cm2 of UVB irradiation, ( ×200 in the original magnification, arrow: cleft in the dermis).
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Fig. 3. Electron microscopic findings of hsp70.1 − / − KO mouse and wild type FVB mouse. Biopsy specimens were fixed and embedded in epon 812. Samples were taken before (C), and 5 min, 6, 12, 24 h after 500 mJ/cm2 UVB irradiation, ( × 5000 in the original magnification, D: dermis, small arrows: dermo-epidermal junction, big arrow: apoptotic cell at basal layer in KO mouse).
S.B. Kwon et al. / Journal of Dermatological Science 28 (2002) 144–151
149
Fig. 4. Electron microscopic findings of collagen fibers of hsp70.1 − / − KO mouse and wild type FVB mouse Biopsy specimens were fixed and embedded in epon 812. Collagen bundels were examined (arrow: compact fiber in wild type mouse and loose fiber in KO mouse).
were no significant histologic differences between wild type and hsp70.1 − / − KO mouse. Electron microscopically, basal epidermal cells showed a similar degree of nuclear swelling in both types of mice. Twelve hours after UVB irradiation, only a few damaged cells were observed and the nucleus had the similar granular appearance as it had before the UVB irradiation in wild type mouse. However, a number of cells became apoptotic in the hsp70.1 − / − KO mouse. Twenty-four hours after UVB irradiation, relatively intact epidermis was observed in wild type FVB mouse. However, almost the entire epidermis was injured in hsp70.1 − / − KO mouse. Compared with the wild type FVB mice, marked basophilic degeneration of the upper dermis and a moderate infiltration of inflammatory cells was observed in hsp70.1 − / − KO mouse. Electron microscopically, basal cells with normal morphology were observed in wild type FVB mice. However, almost all basal cells were apoptotic in hsp70.1 − / − KO mouse (Fig. 4).
4. Discussion It has been shown that heat shock modulates UVB-induced cell death in human epidermal keratinocytes [9]. Heat shock increases the levels of HSPs, and 72 kDa HSP is reported to be a mediator of resistance to ultraviolet B light [5,10]. Over-expression of HSP70 is known to protect murine fibroblasts against UV-light [11]. HSP70 is also known to prevent the activation of stress kinases such as JNK and p38 kinase. These effects are thought to be a novel pathway of cellular thermotolerance [12]. Recently, it has also been demonstrated that keratinocytes injected with a mAb to HSP72 died rapidly following UV irradiation [13]. We also demonstrated that hsp70.1 transfected melanoma cells were resistant to UVB irradiation [6]. These results suggest that HSP70 may play an essential role in modulating UVB injury to cells. HSP70 is heat-inducible by expression of two genes, hsp70.1 and hsp70.3, which is located in the MHC class III region [7]. However,
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differential regulation of these two genes has not been studied yet. In this study, we explore the in vivo role of HSP70 in UVB induced damage using hsp70.1 − / − KO mouse. Our results show that the level of HSP70 was down-regulated in hsp70.1 − / − KO mouse. The effects of low levels of HSP70 on susceptibility to UVB irradiation were studied by light microscopic and electron microscopic examination. Light microscopically, relatively intact epidermis was observed in wild type FVB mouse 24 h after 500 mJ/cm2 of UVB irradiation, but most of cells in the epidermis were necrotic in the hsp70.1 − / − KO mouse. Electron microscopically, a similar degree of nuclear swelling was observed in both hsp70.1 − / − KO mouse and wild type FVB mouse at 6 h after UVB irradiation. These results suggest that similar degree of damage is induced in both hsp70.1 − / − KO mouse and wild type FVB mouse. However, there are significant differences between hsp70.1 − / − KO mouse and wild type FVB mouse at 12 and 24 h after UVB irradiation. There are plenty of abnormal cells in hsp70.1 − / − KO mouse compare with wild type FVB mouse. Most of these cells have condenced chromatin which is a characteristic of apoptotic cell. Some apoptotic cells were engulfed by neighboring cells (Fig. 4, arrow). These findings suggest that increased apoptotic cells are induced by UVB irradiation in hsp70.1 − / − KO mouse even though similar degree of damages are observed 6 h after UVB irradiation. These results suggest that hsp70.1 − / − KO mouse is quite susceptible to UVB irradiation because of an impaired repair after UVB-induced apoptotic injuries. In addition to the chaperoning function for folding, transport, and assembly of newly synthesized polypeptides, HSP70 protects cells from a number of apoptoic stimuli, including heat shock, tumor necrosis factor, growth factor withdrawal, oxidative stress, chemotheapeutic agents, ceramides, and radiation [10,14– 16]. In this study, we also observed increased fragility and loose arrangement of collagen fiber in hsp70.1 − / − KO mouse. These findings need further study to clarify the relationship between HSP70 and collagen synthesis. In conclusion, our results suggest that hsp70.1 − / − KO mouse is quite susceptible to
UVB irradiation as a result of an impaired repair ability. These findings show that our model might be useful to study the effects of depletion of HSP70 from the aspect of tissue injury in its relation to tissue repair failure.
Acknowledgements This study was supported by the Korea Science and Engineering Foundation (98-0403-1901-5). The authors also wish to acknowledge the financial support of the Pacific Co.
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