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Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells
Experimental Eye Research 93 (2011) 405e412
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Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells Ya-Ling Hsu a, Hsin-Su Yu b, c, Hsien-Chung Lin b, d, Kwou-Yeung Wu d, Rei-Cheng Yang e, Po-Lin Kuo b, f, g, * a
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan c Department of Dermatology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan d Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan e Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan f Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan g Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2011 Accepted in revised form 4 June 2011 Available online 21 June 2011
Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Here, we are the first to investigate the biological effect of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. Our results indicate that heat shock exhibits effective cell proliferation inhibition by inducing apoptosis. Heat shock triggers the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activity. In addition, heat shock triggered the death receptor apoptotic pathway indicated by a change in Fas ligand expression, resulting in caspase-8 activity. Furthermore, we also found that generation of reactive oxygen species (ROS) is a critical mediator in heat shock-induced apoptosis. In addition, the antioxidant vitamin C significantly decreased heat shock-mediated apoptosis. Taken together, these findings suggest a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: heat shock cornea apoptosis ROS mitochondria
1. Introduction Corneal fibroblasts (keratocytes) apoptosis has been observed as an important response to herpes simplex virus infection (Wilson et al., 1997), epithelial injury (Wilson et al., 1996), or epithelial damage associated with surgical procedures such as photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) (Helena et al., 1998). Keratocytes apoptosis after eye surgery, including keratotomy and laser operations, may play a role in the development of post-surgical complications. Subsequent proliferation and generation of myofibroblasts results in corneal haze (Netto et al., 2006). In keratoconus, the most frequent form of corneal dystrophy, keratocytes have been shown to result in apoptosis at some distance from the site of epithelial injury. Excessive keratocytes apoptosis has been hypothesized as a major pathological event in keratoconus (Kim et al.,
* Corresponding author. Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Tel.: þ886 7 312 1101x5528; fax: þ886 7 321 0701. E-mail address: [email protected] (P.-L. Kuo). 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.06.005
1999). These studies have suggested that keratocytes apoptosis plays an important role in influencing corneal function. Previous studies have demonstrated that the disappearance of corneal cells after ultraviolet radiation (UVR)-induced photokeratitis is mediated by apoptosis (Podskochy et al., 2000). FasL is expressed in keratocytes in UVR-exposed rabbit corneas, suggesting the contribution of the Fas/FasL system to apoptosis of corneal cells during photokeratitis after UVR exposure (Podskochy and Fagerholm, 2002). Investigation by Xing et al. revealed that hypoxia preconditioning has a generalized protective effect against UV induced keratocyte apoptosis (Xing et al., 2006). Although many studies have examined the molecular consequences of ultraviolet irradiation, little is known about the effects of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Heat shock produces highly specific stress responses, including the induction of a variety of heat shock proteins which play a protective role against the harmful effects of stressful stimuli (Park et al., 2004; Verbeke et al., 2001; Parcellier et al., 2003). Exposure to elevated ambient temperatures has been proposed to be a risk factor for presbyopia (Kessel et al., 2010; Heys et al., 2007; Miranda, 1980)
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and cataract formation (Kessel et al., 2010; Miranda, 1980; Kojima et al., 2002). With the prospect of increased global temperatures in the future, the prevalence of cataracts may increase and the onset age of presbyopia may decrease if a causal relationship between lens disorders and eye temperature exists (Kessel et al., 2010). In this study, we are the first to investigate the biological effects of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. We show that heat shock exhibits effective cell proliferation inhibition by inducing mitochondrial and death receptor apoptotic pathways. 2. Materials and methods 2.1. Cell culture, treatments and cell proliferation assay Statens Seruminstitut Rabbit Cornea (SIRC), a fibroblastic cell line (keratocytes) (Niederkorn et al., 1990), was obtained from the Bioresource Collection and Research Center (Hsinchu City, Taiwan), and cultured in Dulbecco’s modified Eagle’s minimal essential medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal calf serum (Gibco/BRL, Grand Island, NY, USA) at 37 C in a 5% CO2 atmosphere. SIRC cells were maintained in culture media. Culture dishes were sealed with parafilm and immersed for 30 min in a circulating water bath thermo-regulated at 37 C 0.05 C for the control treatment or at 38 C, 39 C, 40 C, 41 C, 42 C, or 43 C 0.05 C for the heat shock treatments. Culture media were replaced with fresh media and the cells further incubated for the indicated times. Cell proliferation was assessed by Premixed WST-1 Cell Proliferation Reagent (Clontech Laboratories Inc., Mountain View, CA, USA) according to the manufacturer’s instructions (Park et al., 2004). 2.2. Apoptosis assay Apoptotic cells were quantitatively assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling (TUNEL) method, which examines DNAstrand breaks during apoptosis by using BD ApoAlertÔ DNA Fragmentation Assay Kit. Briefly, SIRC cells were maintained in culture media. Culture dishes were sealed with parafilm and immersed for 30 min in a circulating water bath thermo-regulated at 37 C 0.05 C for the control treatment, or at 42 C 0.05 C for the heat shock treatments. Culture media were replaced with fresh media and the cells further incubated for the indicated times. The cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. After washing, the cells were incubated with the reaction mixture for 60 min at 37 C. The stained cells were analyzed with an EPICS flow cytometer and a fluorescence microscope at 20 magnification (Hsu et al., 2010a,b).
2.4. Mitochondrial membrane potential assay We used mitochondrial-specific cationic dye JC-1 (5,50 ,6,60 tetrachloro-1,10,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) (Molecular Probes, Leiden, the Netherlands), which undergoes potential-dependent accumulation in the mitochondria. It is a monomer when the membrane potential (DJm) is lower than 120 mV, and emits a green light (540 nm) following excitation by blue light (490 nm). At higher membrane potentials, JC-1 monomers convert to J-aggregates that emit a red light (590 nm) following excitation by green light (540 nm). Following treatment with heat shock for the indicated times, cells were stained with 25 mM JC-1 for 30 min at 37 C. Fluorescence was monitored with the fluorescence plate reader at wavelengths of 490 nm (excitation)/540 nm (emission) and 540 nm (excitation)/590 nm (emission) pairs. Changes in the ratio between the measurement at test wavelengths of 590 nm (red) and 540 nm (green) fluorescence intensities are indicative of changes in the mitochondrial membrane potential (Hsu et al., 2010a,b). 2.5. Measurements of ROS Levels of intracellular O2 and H2O2 were assessed spectrofluorometrically by oxidation of specific probes: dihydroethidium (DHE, Molecular Probes, Leiden, the Netherlands) and 20 70 dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Leiden, the Netherlands). Cells were plated in 60-mm dishes, allowed to attach overnight, and exposed to vitamin C (100 mM) alone, heat shock (42 C) alone, and vitamin C plus heat shock (42 C) for specified time intervals. The cells were stained with 10 mM H2DCFDA and 20 mM DHE for 10 min at 37 C, and the cells’ fluorescence intensity was determined using flow cytometer (Becton Dickinson and Co., Franklin Lakes, NY) (Kuo et al., 2007). 2.6. Real-time RT-PCR RNA isolation was performed using the TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was prepared using an oligo (dT) primer and reverse transcriptase (Takara, Shiga, Japan) following standard protocols. Real-time PCR was performed using SYBR Green on the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Each PCR reaction mixture contained 200 nM of each primer, 10 mL of 2xSYBR Green PCR Master Mix (Applied Biosystems), and 5 ml cDNA and RNase-free water in
2.3. Assay for caspase activity The assay is based on the ability of the active enzyme to cleave the chromophore from the enzyme substrate LEHD-pNA (for caspase-9), Ac-IETD-pNA (for caspase-8) and Ac-DEVD-pNA (for caspase-3). Cell lysates were prepared and incubated with specific caspase-3 antibodies. Immunocomplexes were incubated with peptide substrate in assay buffer (100 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol and 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), pH 7.4) for 2 h at 37 C. The release of p-nitroaniline was monitored at 405 nm. Results are represented as the percentage of change of the activity in comparison with the untreated control (Hsu et al., 2010a,b).
Fig. 1. The effects of heat shock on cell proliferation inhibition in SIRC cells. Cell proliferation inhibition activity of heat shock was assessed by WST-1. SIRC cells were maintained in culture media for 48 h; the culture dishes were sealed with parafilm and immersed for 30 min in a circulating water bath thermo-regulated at 37 C for the control treatment, or at 38 C, 39 C, 40 C, 41 C, 42 C, or 43 C for the heat shock treatments. Culture media were replaced with fresh media and the cells further incubated for 24 h. Results are expressed as the percentage of cell proliferation relative to the proliferation of control treatment. The data shown are the mean from three independent experiments. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
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Fig. 2. Heat shock-induced apoptosis. Quantitative evaluations of TUNEL assay by flow cytometry, SIRC cells treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. TUNEL-positive cells were examined by flow cytometry, and were examined by fluorescence microscope. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
a total volume of 20 ml. The PCR reaction was carried out with a denaturation step at 95 C for 10 min, and then for 40 cycles at 95 C for 15 s and 60 C for 1 min. All PCRs were performed in triplicate and normalized to internal control glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA. Relative expression was presented using the 2DDCT method (Kuo et al., 2011).
2.7. Immunoblot Cells were lysed on ice for 15 min by M-PER lysis reagent (Pierce, USA). Cell lysate was centrifuged at 14,000 g for 15 min, and the supernatant fraction collected for immunoblot. Equivalent amounts of protein were resolved by SDS-PAGE (8e12%) and transferred to PVDF
Fig. 3. The expression levels of Bcl-2 family members in heat shock-treated SIRC cells. Heat shock increased the expression of Bax mRNA (A) and Bak mRNA (B), and decreased the expression of Bcl-2 mRNA (C) and Mcl-1 mRNA (D), as assessed by real-time PCR. The expression levels of Bcl-2 family proteins were determined by immunoblot analysis (E). SIRC cells treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
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Fig. 4. Heat shock-induced apoptosis through the initiation of the mitochondrial pathway. (A) The loss of DJm, (B) caspase-9 activity and (C) caspase-3 activity in SIRC cells. SIRC cells treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. For (A), the DJm was measured by JC-1 and flow cytometry. For (B), caspase-9 activity was assessed by Caspase-9 Activity Assay kit. For (C), caspase-3 activity was assessed by CaspSELECTTM Caspase-3 Immunoassay Kits. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
membranes. After blocking for 1 h in 5% nonfat dry milk in Trisbuffered saline, the membrane was incubated with the desired primary antibody for 1e16 h, then treated with the appropriate peroxidase-conjugated secondary antibody and the immunoreactive proteins detected using an enhanced chemiluminescence kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. 2.8. Statistical analysis Data were expressed as means SD. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences (p < 0.05) between the means of control and heat shock-treated cells were analyzed by Dunnett’s test.
Fig. 5. Fas ligand/caspase-8 system was involved in heat shock-mediated apoptosis. The expressions of Fas receptor (A) and FasL (B). The activity of caspase-8 (C). SIRC cells treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. For (A) and (B), the levels of Fas and FasL mRNA were assessed by RT-PCR. For (C), caspase-8 activity was assessed by Caspase-8 Activity Assay kit. Each value is the mean S.D. of three determinations. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
shock for 30 min, then further incubated for 24 h resulted in inhibiting the growth of SIRC cells in a temperature-dependent manner.
3. Results 3.1. Heat shock inhibits cell proliferation in SIRC cells
3.2. Heat shock induces apoptotic cell death by triggering the mitochondrial pathway in SIRC cells
To investigate the effect of heat shock on inhibition of SIRC cell proliferation, we first examined the effect of heat shock on proliferation of SIRC cells. As shown in Fig. 1, exposure of SIRC cells to heat
A quantitative evaluation was also made, using TUNEL to detect DNA-strand breaks. Compared to the control, treatment of SIRC cells in a 42 C water bath induced a 27.58% increase of apoptotic cells at 24 h
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Fig. 6. The effect of heat shock on the production of ROS in SIRC cells. The effect of heat shock on the production of O2 (A), the production of H2O2 (B). For (A) and (B), SIRC cells were treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. The amounts of H2O2 and O2 were assayed by H2DCFDA (for H2O2) and DHE (for O2) staining. Results shown are representative of three independent experiments.
(Fig. 2). To determine whether heat shock induces apoptosis by triggering the mitochondrial apoptotic pathway, we measured changes in mitochondrial membrane potential (DJm), caspase activity, and expression of the Bcl-2 family members. Real-time PCR analysis and immunoblot showed that treatment of SIRC cells with heat shock increased Bax and Bak mRNA, together with protein levels (Fig. 3A, B and E). In contrast, heat shock decreased Bcl-2 and Mcl-1 levels, which led to a time-dependent increase in the proapoptotic/anti-apoptotic ratio of Bcl-2 family members as early as 3 h after treatment (Fig. 3CeE). We also investigated mitochondrial dysfunction by measuring DJm in heat shock-treated SIRC cells. As shown in Fig. 4A, heat shock treatment resulted in the diminishment of mitochondrial membrane potential in a time-dependent manner. The apoptotic process includes activation of cysteine proteases, which represent both initiators and executors of cell death. Furthermore, initiator caspase-9 activities increased significantly, as indicated by the observation that heat shock treatment increased caspase-9 activity in SIRC cells based on the results of statistical analysis (Fig. 4B). Heat shock also increased the activity of caspase-3 in SIRC cells (Fig. 4C). The time relationship between the expression of proapoptotic/anti-apoptotic ratio of Bcl-2 family members at 3 h of treatment, and the occurrence of DNA fragmentation at 12 h of treatment, supports the hypothesis that the mitochondrial pathway may mediate heat shock-induced apoptosis of SIRC cells. 3.3. Fas ligand/caspase-8 is also involved in heat shock-mediated apoptotic cell death RT-PCR data shows that heat shock increased expression of Fas ligand (FasL) in SIRC cells as early as 3 h after treatment in time-
dependent manner (Fig. 5B). However, heat shock did not affect the expression of Fas receptor (Fig. 5A). In addition, heat shock also increased caspase-8 activity after 3 h of treatment, and reached maximum induction after heat shock (42 C) treatment (Fig. 5C). The caspase-8 activity (at 3 h) was before the production of DNA fragmentation (at 12 h) showing caspase-8 activity was required in heat shock-induced apoptosis. 3.4. Heat shock increases the generation of ROS in SIRC cells Because ROS generation plays an important role in physiological and nonphysiological environmental stressor such as ultraviolet radiation and hypoxiaereoxygenation (Shimmura et al., 1996, 1998, 1999), we tested the possibility that heat shock-induced apoptosis triggers ROS accumulation. DHE-based FACS detection revealed that intracellular O2 levels in SIRC cells increased following treatment with heat shock. ROS was detected as early as 1 h after SIRC cells were treated at 42 C, and peaked at 2 h (Fig. 6A). Similar results were obtained from H2DCFDA-based FACS detection data, which revealed that intracellular H2O2 levels increased in examined cells after heat shock (42 C) treatment (Fig. 6B). 3.5. The role of ROS on heat shock-induced apoptosis To investigate whether ROS generation is directly associated with heat shock-induced proliferation inhibition, caspase-9 activity and apoptosis, we assessed these events in SIRC cells which had been pretreated with vitamin C (100 mM) for 1 h then treated with heat shock (42 C). As shown in Fig. 7, the antioxidant vitamin C significantly decreased heat shock-mediated proliferation inhibition,
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Fig. 7. The role of ROS on heat shock-mediated proliferation inhibition, caspase-9 activity and apoptosis. Antioxidant agents inhibited heat shock-mediated proliferation inhibition (A), apoptosis (B), caspase-9 (C) and caspase-8 activity (D). SIRC cells were pretreated with vitamin C (100 mM) for 1 h, then treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for 24 h. Cell proliferation inhibition activity was assessed by WST-1. Caspase-9 and Caspase-8 activity was assessed by Caspase-9 and Caspase-8 Activity Assay kits. The induction of apoptosis was determined by TUNEL analysis. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between two test groups, as analyzed by Dunnett’s test (p < 0.05). The asterisk indicates a significant difference between control and test groups, *p < 0.05.
caspase-9/-8 activity, and apoptosis. These data confirm that heat shock treatment of SIRC cells activates the mitochondrial and death receptor apoptotic pathway via release of ROS. 4. Discussion Apoptosis within the corneal epithelium has been inferred from the discovery of the dark cells during the regeneration stage of the rat corneal epithelium after chemical abrasion (Glasø et al., 1993). However, dark cells in the corneal epithelium resulting from damage caused by UVR have also been described (Ringvold, 1983; Clarke et al., 1990). Using TEM, Ringvold and Davanger found keratocytes with nuclear fragments of high electron density and abnormal inclusions in the rabbit corneal stroma after UV light exposure (Ringvold and Davanger, 1984). 310-nm UVR caused more extensive damage to the corneal stroma and endothelium than did 280-nm UVR (Podskochy et al., 2000). Apoptosis appears to be a mechanism of corneal cell death upon exposure to UVR. Little is known about the biological effects of IR on human eyes, however. Heat is likely to be generated as a consequence of IR irradiation, leading to an increase in skin temperature after exposure to the sun, and heat shock is widely considered a major extracellular stimulus (Park et al., 2004). A recent study has reported that in the face of global warming, the question of a link between eye disease and ambient temperature is more relevant than ever. It suggested that the effect of global warming on the prevalence of eye disease will be more severe for those living in cold climates getting warmer than for those in hot climates getting hotter (Kessel et al., 2010). This study, the first to investigate the biological effects of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells, shows that heat shock induces apoptosis through reactive oxygen species/ mitochondrial pathways in cornea cells.
Mitochondria are thought to be another pathway for apoptosis, and mitochondrial function is regulated through Bcl-2 family members, comprised of both anti-apoptotic (Bcl-2, Mcl-1) and proapoptotic members (Bax, Bak) (Hengartner, 2000; Shroff et al., 2007). A previous study has reported the Bcl-2 family plays a predominant role in the regulation of early corneal wound healing, such as keratocyte depletion, endothelial changes, and the inflammatory responses (Li et al., 2000). Our results showed that heat shock results in a significant increase of Bax and Bak mRNA and protein expression, and a decrease of Bcl-2 and Mcl-1, suggesting that changes in the ratio of proapoptotic and anti-apoptotic Bcl-2 family members might contribute to the apoptosis-promotion activity of heat shock. In addition, our data also revealed the degree of caspase-9 and caspase-3 activity after SIRC cells were treated with heat shock. Because the proapoptotic/anti-apoptotic ratio of Bcl-2 family members was found to be significantly changed by treating breast cancer cell lines with heat shock, and the changes of Bax and Bak (at 3 h) occurred earlier than the caspase activity (6 h for caspase-9 and caspase-3), this suggests that heat shock increases caspase activity through the regulation of Bcl-2 family members. This result is consist with several studies reported that Bcl-2 family proteins regulate the activation of caspase-9 and caspase-3 during apoptotic cell death (Weng et al., 2009; Hung et al., 2010). These mitochondrial apoptotic events play an important role in heat shock-mediated apoptosis. Fas/FasL system is a key signaling transduction pathway of apoptosis in cells and tissues (Mahmood and Shukla, 2010). Ligation of Fas by agonistic antibody or its mature ligand induces receptor oligomerization and formation of death-inducing signaling complex (DISC), followed by activation of caspase-8, then further activating a series caspase cascades resulting in cell apoptotic death (Mahmood and Shukla, 2010). Investigation by Podskochy and Fagerholm
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revealed that may play an important role in apoptosis of corneal cells after ultraviolet radiation (Podskochy and Fagerholm, 2002). In addition, some insults, such as HSV-1 and Pseudomonas aeruginosa infection, have been reported to induce apoptosis by Fas signaling pathway in cornea cells (Qian and Atherton, 2003; Zhou et al., 2010). Our study indicated that FasL, increased in heat shock-treated SIRC cells. Moreover, the levels of FasL and the activity of caspase-8 were simultaneously enhanced in SIRC cells following by apoptotic cell death (DNA fragmentation). Thus, these findings are the first to show that the Fas ligand/caspase-8 system plays an important role in heat shock-mediated SIRC cellular apoptosis. All aerobic organisms are subjected to physiological oxidant stress as a consequence of aerobic metabolism. Superoxide anion (O2), hydrogen peroxide (H2O2), hydroxyl radical (OH) and hypochlorous acid are included among the reactive oxygen species (ROS) that are natural by-products of normal cell metabolism (Rao et al., 1987; Halliwell, 1990). The redox status of all aerobic cells is balanced by enzyme and nonenzyme systems (Halliwell, 1990; Kornas et al., 2010). Oxidative stress occurs when there is an imbalance between cellular oxidant species production and antioxidant capability (Kondo et al., 2006; Matés et al., 2008). Previous studies demonstrated that physiological and nonphysiological environmental stressor such as ultraviolet radiation and hypoxiaereoxygenation can cause transient formation of reactive oxygen species (ROS) in corneal tissue (Shimmura et al., 1996, 1998). The response of perturbed tissue against ROS formation ranges from modification of cytokine regulation, to organelle dysfunction in lysosomes and mitochondria, to cell death (Aragane et al., 1997; Cullen, 1980; Shimmura and Tsubota, 1997; Ren and Wilson, 1994; Shimmura et al., 1999). Previous studies have demonstrated reactive oxygen species (ROS) induces apoptosis through mitochondrial pathway (Azad et al., 2010; Nakamura and Sakamoto, 2001; Nie et al., 2008; Hsu et al., 2010a,b; Kuo et al., 2007). Another recent study has also suggested increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts (Chwa et al., 2006). Our study has shown that heat shock-mediated oxidative stress primarily by increasing the production of O2 and H2O2. We further observed that heat shock generates ROS, resulting in caspase-9 and caspase-8 activation. Furthermore, vitamin C, an agent which can regulate ROS detoxification, blocked proliferation inhibition, caspase-9 and caspase-8 activity and apoptosis induced by heat shock. These data suggest that ROS accumulation contributes to heat shock-induced cell death in cornea cells. In conclusion, our data indicate that Statens Seruminstitut Rabbit Cornea (SIRC) cells are highly sensitive to growth inhibition and apoptosis induction by heat shock. Heat shock-induced apoptosis is associated with mitochondrial and death receptor pathways, which are in turn mediated by ROS generation. Our study demonstrates a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells.
Acknowledgments This work is supported by grants from the National Science Council of Taiwan (NSC 99-2632-B-037-001-MY3) and the Center of Excellence for Environmental Medicine, Kaohsiung Medical University (KMU-EM-100-4).
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The role of apoptosis in the early corneal wound healing after excimer laser keratectomy in the rat. Graefes Arch. Clin. Exp. Ophthalmol. 238, 853e860. Mahmood, Z., Shukla, Y., 2010. Death receptors: targets for cancer therapy. Exp. Cell Res. 316, 887e899. Matés, J.M., Segura, J.A., Alonso, F.J., Márquez, J., 2008. Intracellular redox status and oxidative stress: implications for cell proliferation, apoptosis, and carcinogenesis. Arch. Toxicol. 82, 273e299. Miranda, M.N., 1980. Environmental temperature and senile cataract. Trans. Am. Ophthalmol. Soc. 78, 255e264. Nakamura, T., Sakamoto, K., 2001. Reactive oxygen species up-regulates cyclooxygenase-2, p53, and Bax mRNA expression in bovine luteal cells. Biochem. Biophys. Res. Commun. 284, 203e210. Netto, M.V., Mohan, R.R., Sinha, S., Sharma, A., Dupps, W., Wilson, S.E., 2006. Stromal haze, myofibroblasts, and surface irregularity after PRK.Exp. Eye Res. 82, 788e797. Nie, C., Tian, C., Zhao, L., Petit, P.X., Mehrpour, M., Chen, Q., 2008. Cysteine 62 of Bax is critical for its conformational activation and its proapoptotic activity in response to H2O2-induced apoptosis. J. Biol. Chem. 283, 15359e15369. Niederkorn, J.Y., Meyer, D.R., Ubelaker, J.E., Martin, J.H., 1990. Ultrastructural and immunohistological characterization of the SIRC corneal cell line. In Vitro Cell Dev. Biol. 1990 (26), 923e930. Parcellier, A., Gurbuxani, S., Schmitt, E., Solary, E., Garrido, C., 2003. Heat shock proteins, cellular chaperones that modulate mitochondrial cell death pathways. Biochem. Biophys. Res. Commun. 304, 505e512. Park, C.H., Lee, M.J., Ahn, J., Kim, S., Kim, H.H., Kim, K.H., Eun, H.C., Chung, J.H., 2004. Heat shock-induced matrix metalloproteinase (MMP)-1 and MMP-3 are mediated through ERK and JNK activation and via an autocrine interleukin-6 loop. J. Invest. Dermatol. 123, 1012e1019. Podskochy, A., Fagerholm, P., 2002. The expression of Fas ligand protein in ultraviolet-exposed rabbit corneas. Cornea 21, 91e94.
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Podskochy, A., Gan, L., Fagerholm, P., 2000. Apoptosis in UV-exposed rabbit corneas. Cornea 19, 99e103. Qian, H., Atherton, S., 2003. Apoptosis and increased expression of Fas ligand after uniocular anterior chamber (AC) inoculation of HSV-1. Curr. Eye Res. 26, 195e203. Rao, N.A., Romero, J.L., Fernandez, M.A., Sevanian, A., Marak Jr., G.E., 1987. Role of free radicals in uveitis. Surv. Ophthalmol. 32, 209e213. Ren, H., Wilson, G., 1994. The effect of ultraviolet-B irradiation on the cell shedding rate of the corneal epithelium. Acta Ophthalmol. (Copenh) 72, 447e452. Ringvold, A., Davanger, M., 1984. Changes in the rabbit corneal stroma caused by UV-radiation. Acta Ophthalmol. (Copenh) 63, 601e606. Ringvold, A., 1983. Damage of the cornea epithelium caused by ultraviolet radiation. A scanning electron microscopic study in rabbit. Acta Ophthalmol. (Copenh) 61, 898e907. Shimmura, S., Tsubota, K., 1997. Ultraviolet B-induced mitochondrial dysfunction is associated with decreased cell detachment of corneal epithelial cells in vitro. Invest. Ophthalmol. Vis. Sci., 620e626. Shimmura, S., Suematsu, M., Shimoyama, M., Tsubota, K., Oguchi, Y., Ishimura, Y., 1996. Subthreshold UV radiation-induced peroxide formation in cultured corneal epithelial cells: the protective effects of lactoferrin. Exp. Eye Res. 63, 519e526. Shimmura, S., Shimoyama, M., Hojo, M., Urayama, K., Tsubota, K., 1998. Reoxygenation injury in a cultured corneal epithelial cell line protected by the uptake of lactoferrin. Invest. Ophthalmol. Vis. Sci. 39, 1346e1351.
Shimmura, S., Masumizu, T., Nakai, Y., Urayama, K., Shimazaki, J., Bissen-Miyajima, H., Kohno, M., Tsubota, K., 1999. Excimer laser-induced hydroxyl radical formation and keratocyte death in vitro. Invest. Ophthalmol. Vis. Sci. 40, 1245e1249. Shroff, E.H., Snyder, C., Chandel, N.S., 2007. Bcl-2 family members regulate anoxiainduced cell death. Antioxid. Redox Signal. 9, 1405e1409. Verbeke, P., Fonager, J., Clark, B.F., Rattan, S.I., 2001. Heat shock response and ageing: mechanisms and applications. Cell Biol. Int. 25, 845e857. Weng, C.J., Yang, Y.T., Ho, C.T., Yen, G.C., 2009. Mechanisms of apoptotic effects induced by resveratrol, dibenzoylmethane, and their analogues on human lung carcinoma cells. J. Agric. Food Chem. 57, 5235e5243. Wilson, S.E., He, Y.G., Weng, J., Li, Q., McDowall, A.W., Vital, M., Chwang, E.L., 1996. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp. Eye Res. 62, 325e327. Wilson, S.E., Pedroza, L., Beuerman, R., Hill, J.M., 1997. Herpes simplex virus type-1 infection of corneal epithelial cells induces apoptosis of the underlying keratocytes. Exp. Eye Res. 64, 775e779. Xing, D., Sun, X., Li, J., Cui, M., Tan-Allen, K., Bonanno, J.A., 2006. Hypoxia preconditioning protects corneal stromal cells against induced apoptosis. Exp. Eye Res. 82, 780e787. Zhou, Z., Wu, M., Barrett, R.P., McClellan, S.A., Zhang, Y., Hazlett, L.D., 2010. Role of the Fas pathway in Pseudomonas aeruginosa keratitis. Invest. Ophthalmol. Vis. Sci. 51, 2537e2547.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells Ya-Ling Hsu a, Hsin-Su Yu b, c, Hsien-Chung Lin b, d, Kwou-Yeung Wu d, Rei-Cheng Yang e, Po-Lin Kuo b, f, g, * a
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan c Department of Dermatology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan d Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan e Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan f Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan g Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2011 Accepted in revised form 4 June 2011 Available online 21 June 2011
Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Here, we are the first to investigate the biological effect of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. Our results indicate that heat shock exhibits effective cell proliferation inhibition by inducing apoptosis. Heat shock triggers the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activity. In addition, heat shock triggered the death receptor apoptotic pathway indicated by a change in Fas ligand expression, resulting in caspase-8 activity. Furthermore, we also found that generation of reactive oxygen species (ROS) is a critical mediator in heat shock-induced apoptosis. In addition, the antioxidant vitamin C significantly decreased heat shock-mediated apoptosis. Taken together, these findings suggest a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: heat shock cornea apoptosis ROS mitochondria
1. Introduction Corneal fibroblasts (keratocytes) apoptosis has been observed as an important response to herpes simplex virus infection (Wilson et al., 1997), epithelial injury (Wilson et al., 1996), or epithelial damage associated with surgical procedures such as photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) (Helena et al., 1998). Keratocytes apoptosis after eye surgery, including keratotomy and laser operations, may play a role in the development of post-surgical complications. Subsequent proliferation and generation of myofibroblasts results in corneal haze (Netto et al., 2006). In keratoconus, the most frequent form of corneal dystrophy, keratocytes have been shown to result in apoptosis at some distance from the site of epithelial injury. Excessive keratocytes apoptosis has been hypothesized as a major pathological event in keratoconus (Kim et al.,
* Corresponding author. Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Tel.: þ886 7 312 1101x5528; fax: þ886 7 321 0701. E-mail address: [email protected] (P.-L. Kuo). 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.06.005
1999). These studies have suggested that keratocytes apoptosis plays an important role in influencing corneal function. Previous studies have demonstrated that the disappearance of corneal cells after ultraviolet radiation (UVR)-induced photokeratitis is mediated by apoptosis (Podskochy et al., 2000). FasL is expressed in keratocytes in UVR-exposed rabbit corneas, suggesting the contribution of the Fas/FasL system to apoptosis of corneal cells during photokeratitis after UVR exposure (Podskochy and Fagerholm, 2002). Investigation by Xing et al. revealed that hypoxia preconditioning has a generalized protective effect against UV induced keratocyte apoptosis (Xing et al., 2006). Although many studies have examined the molecular consequences of ultraviolet irradiation, little is known about the effects of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Heat shock produces highly specific stress responses, including the induction of a variety of heat shock proteins which play a protective role against the harmful effects of stressful stimuli (Park et al., 2004; Verbeke et al., 2001; Parcellier et al., 2003). Exposure to elevated ambient temperatures has been proposed to be a risk factor for presbyopia (Kessel et al., 2010; Heys et al., 2007; Miranda, 1980)
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and cataract formation (Kessel et al., 2010; Miranda, 1980; Kojima et al., 2002). With the prospect of increased global temperatures in the future, the prevalence of cataracts may increase and the onset age of presbyopia may decrease if a causal relationship between lens disorders and eye temperature exists (Kessel et al., 2010). In this study, we are the first to investigate the biological effects of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. We show that heat shock exhibits effective cell proliferation inhibition by inducing mitochondrial and death receptor apoptotic pathways. 2. Materials and methods 2.1. Cell culture, treatments and cell proliferation assay Statens Seruminstitut Rabbit Cornea (SIRC), a fibroblastic cell line (keratocytes) (Niederkorn et al., 1990), was obtained from the Bioresource Collection and Research Center (Hsinchu City, Taiwan), and cultured in Dulbecco’s modified Eagle’s minimal essential medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal calf serum (Gibco/BRL, Grand Island, NY, USA) at 37 C in a 5% CO2 atmosphere. SIRC cells were maintained in culture media. Culture dishes were sealed with parafilm and immersed for 30 min in a circulating water bath thermo-regulated at 37 C 0.05 C for the control treatment or at 38 C, 39 C, 40 C, 41 C, 42 C, or 43 C 0.05 C for the heat shock treatments. Culture media were replaced with fresh media and the cells further incubated for the indicated times. Cell proliferation was assessed by Premixed WST-1 Cell Proliferation Reagent (Clontech Laboratories Inc., Mountain View, CA, USA) according to the manufacturer’s instructions (Park et al., 2004). 2.2. Apoptosis assay Apoptotic cells were quantitatively assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling (TUNEL) method, which examines DNAstrand breaks during apoptosis by using BD ApoAlertÔ DNA Fragmentation Assay Kit. Briefly, SIRC cells were maintained in culture media. Culture dishes were sealed with parafilm and immersed for 30 min in a circulating water bath thermo-regulated at 37 C 0.05 C for the control treatment, or at 42 C 0.05 C for the heat shock treatments. Culture media were replaced with fresh media and the cells further incubated for the indicated times. The cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. After washing, the cells were incubated with the reaction mixture for 60 min at 37 C. The stained cells were analyzed with an EPICS flow cytometer and a fluorescence microscope at 20 magnification (Hsu et al., 2010a,b).
2.4. Mitochondrial membrane potential assay We used mitochondrial-specific cationic dye JC-1 (5,50 ,6,60 tetrachloro-1,10,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) (Molecular Probes, Leiden, the Netherlands), which undergoes potential-dependent accumulation in the mitochondria. It is a monomer when the membrane potential (DJm) is lower than 120 mV, and emits a green light (540 nm) following excitation by blue light (490 nm). At higher membrane potentials, JC-1 monomers convert to J-aggregates that emit a red light (590 nm) following excitation by green light (540 nm). Following treatment with heat shock for the indicated times, cells were stained with 25 mM JC-1 for 30 min at 37 C. Fluorescence was monitored with the fluorescence plate reader at wavelengths of 490 nm (excitation)/540 nm (emission) and 540 nm (excitation)/590 nm (emission) pairs. Changes in the ratio between the measurement at test wavelengths of 590 nm (red) and 540 nm (green) fluorescence intensities are indicative of changes in the mitochondrial membrane potential (Hsu et al., 2010a,b). 2.5. Measurements of ROS Levels of intracellular O2 and H2O2 were assessed spectrofluorometrically by oxidation of specific probes: dihydroethidium (DHE, Molecular Probes, Leiden, the Netherlands) and 20 70 dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Leiden, the Netherlands). Cells were plated in 60-mm dishes, allowed to attach overnight, and exposed to vitamin C (100 mM) alone, heat shock (42 C) alone, and vitamin C plus heat shock (42 C) for specified time intervals. The cells were stained with 10 mM H2DCFDA and 20 mM DHE for 10 min at 37 C, and the cells’ fluorescence intensity was determined using flow cytometer (Becton Dickinson and Co., Franklin Lakes, NY) (Kuo et al., 2007). 2.6. Real-time RT-PCR RNA isolation was performed using the TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was prepared using an oligo (dT) primer and reverse transcriptase (Takara, Shiga, Japan) following standard protocols. Real-time PCR was performed using SYBR Green on the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Each PCR reaction mixture contained 200 nM of each primer, 10 mL of 2xSYBR Green PCR Master Mix (Applied Biosystems), and 5 ml cDNA and RNase-free water in
2.3. Assay for caspase activity The assay is based on the ability of the active enzyme to cleave the chromophore from the enzyme substrate LEHD-pNA (for caspase-9), Ac-IETD-pNA (for caspase-8) and Ac-DEVD-pNA (for caspase-3). Cell lysates were prepared and incubated with specific caspase-3 antibodies. Immunocomplexes were incubated with peptide substrate in assay buffer (100 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol and 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), pH 7.4) for 2 h at 37 C. The release of p-nitroaniline was monitored at 405 nm. Results are represented as the percentage of change of the activity in comparison with the untreated control (Hsu et al., 2010a,b).
Fig. 1. The effects of heat shock on cell proliferation inhibition in SIRC cells. Cell proliferation inhibition activity of heat shock was assessed by WST-1. SIRC cells were maintained in culture media for 48 h; the culture dishes were sealed with parafilm and immersed for 30 min in a circulating water bath thermo-regulated at 37 C for the control treatment, or at 38 C, 39 C, 40 C, 41 C, 42 C, or 43 C for the heat shock treatments. Culture media were replaced with fresh media and the cells further incubated for 24 h. Results are expressed as the percentage of cell proliferation relative to the proliferation of control treatment. The data shown are the mean from three independent experiments. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
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Fig. 2. Heat shock-induced apoptosis. Quantitative evaluations of TUNEL assay by flow cytometry, SIRC cells treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. TUNEL-positive cells were examined by flow cytometry, and were examined by fluorescence microscope. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
a total volume of 20 ml. The PCR reaction was carried out with a denaturation step at 95 C for 10 min, and then for 40 cycles at 95 C for 15 s and 60 C for 1 min. All PCRs were performed in triplicate and normalized to internal control glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA. Relative expression was presented using the 2DDCT method (Kuo et al., 2011).
2.7. Immunoblot Cells were lysed on ice for 15 min by M-PER lysis reagent (Pierce, USA). Cell lysate was centrifuged at 14,000 g for 15 min, and the supernatant fraction collected for immunoblot. Equivalent amounts of protein were resolved by SDS-PAGE (8e12%) and transferred to PVDF
Fig. 3. The expression levels of Bcl-2 family members in heat shock-treated SIRC cells. Heat shock increased the expression of Bax mRNA (A) and Bak mRNA (B), and decreased the expression of Bcl-2 mRNA (C) and Mcl-1 mRNA (D), as assessed by real-time PCR. The expression levels of Bcl-2 family proteins were determined by immunoblot analysis (E). SIRC cells treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
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Fig. 4. Heat shock-induced apoptosis through the initiation of the mitochondrial pathway. (A) The loss of DJm, (B) caspase-9 activity and (C) caspase-3 activity in SIRC cells. SIRC cells treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. For (A), the DJm was measured by JC-1 and flow cytometry. For (B), caspase-9 activity was assessed by Caspase-9 Activity Assay kit. For (C), caspase-3 activity was assessed by CaspSELECTTM Caspase-3 Immunoassay Kits. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
membranes. After blocking for 1 h in 5% nonfat dry milk in Trisbuffered saline, the membrane was incubated with the desired primary antibody for 1e16 h, then treated with the appropriate peroxidase-conjugated secondary antibody and the immunoreactive proteins detected using an enhanced chemiluminescence kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. 2.8. Statistical analysis Data were expressed as means SD. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences (p < 0.05) between the means of control and heat shock-treated cells were analyzed by Dunnett’s test.
Fig. 5. Fas ligand/caspase-8 system was involved in heat shock-mediated apoptosis. The expressions of Fas receptor (A) and FasL (B). The activity of caspase-8 (C). SIRC cells treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. For (A) and (B), the levels of Fas and FasL mRNA were assessed by RT-PCR. For (C), caspase-8 activity was assessed by Caspase-8 Activity Assay kit. Each value is the mean S.D. of three determinations. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p < 0.05.
shock for 30 min, then further incubated for 24 h resulted in inhibiting the growth of SIRC cells in a temperature-dependent manner.
3. Results 3.1. Heat shock inhibits cell proliferation in SIRC cells
3.2. Heat shock induces apoptotic cell death by triggering the mitochondrial pathway in SIRC cells
To investigate the effect of heat shock on inhibition of SIRC cell proliferation, we first examined the effect of heat shock on proliferation of SIRC cells. As shown in Fig. 1, exposure of SIRC cells to heat
A quantitative evaluation was also made, using TUNEL to detect DNA-strand breaks. Compared to the control, treatment of SIRC cells in a 42 C water bath induced a 27.58% increase of apoptotic cells at 24 h
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Fig. 6. The effect of heat shock on the production of ROS in SIRC cells. The effect of heat shock on the production of O2 (A), the production of H2O2 (B). For (A) and (B), SIRC cells were treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for the indicated times. The amounts of H2O2 and O2 were assayed by H2DCFDA (for H2O2) and DHE (for O2) staining. Results shown are representative of three independent experiments.
(Fig. 2). To determine whether heat shock induces apoptosis by triggering the mitochondrial apoptotic pathway, we measured changes in mitochondrial membrane potential (DJm), caspase activity, and expression of the Bcl-2 family members. Real-time PCR analysis and immunoblot showed that treatment of SIRC cells with heat shock increased Bax and Bak mRNA, together with protein levels (Fig. 3A, B and E). In contrast, heat shock decreased Bcl-2 and Mcl-1 levels, which led to a time-dependent increase in the proapoptotic/anti-apoptotic ratio of Bcl-2 family members as early as 3 h after treatment (Fig. 3CeE). We also investigated mitochondrial dysfunction by measuring DJm in heat shock-treated SIRC cells. As shown in Fig. 4A, heat shock treatment resulted in the diminishment of mitochondrial membrane potential in a time-dependent manner. The apoptotic process includes activation of cysteine proteases, which represent both initiators and executors of cell death. Furthermore, initiator caspase-9 activities increased significantly, as indicated by the observation that heat shock treatment increased caspase-9 activity in SIRC cells based on the results of statistical analysis (Fig. 4B). Heat shock also increased the activity of caspase-3 in SIRC cells (Fig. 4C). The time relationship between the expression of proapoptotic/anti-apoptotic ratio of Bcl-2 family members at 3 h of treatment, and the occurrence of DNA fragmentation at 12 h of treatment, supports the hypothesis that the mitochondrial pathway may mediate heat shock-induced apoptosis of SIRC cells. 3.3. Fas ligand/caspase-8 is also involved in heat shock-mediated apoptotic cell death RT-PCR data shows that heat shock increased expression of Fas ligand (FasL) in SIRC cells as early as 3 h after treatment in time-
dependent manner (Fig. 5B). However, heat shock did not affect the expression of Fas receptor (Fig. 5A). In addition, heat shock also increased caspase-8 activity after 3 h of treatment, and reached maximum induction after heat shock (42 C) treatment (Fig. 5C). The caspase-8 activity (at 3 h) was before the production of DNA fragmentation (at 12 h) showing caspase-8 activity was required in heat shock-induced apoptosis. 3.4. Heat shock increases the generation of ROS in SIRC cells Because ROS generation plays an important role in physiological and nonphysiological environmental stressor such as ultraviolet radiation and hypoxiaereoxygenation (Shimmura et al., 1996, 1998, 1999), we tested the possibility that heat shock-induced apoptosis triggers ROS accumulation. DHE-based FACS detection revealed that intracellular O2 levels in SIRC cells increased following treatment with heat shock. ROS was detected as early as 1 h after SIRC cells were treated at 42 C, and peaked at 2 h (Fig. 6A). Similar results were obtained from H2DCFDA-based FACS detection data, which revealed that intracellular H2O2 levels increased in examined cells after heat shock (42 C) treatment (Fig. 6B). 3.5. The role of ROS on heat shock-induced apoptosis To investigate whether ROS generation is directly associated with heat shock-induced proliferation inhibition, caspase-9 activity and apoptosis, we assessed these events in SIRC cells which had been pretreated with vitamin C (100 mM) for 1 h then treated with heat shock (42 C). As shown in Fig. 7, the antioxidant vitamin C significantly decreased heat shock-mediated proliferation inhibition,
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Fig. 7. The role of ROS on heat shock-mediated proliferation inhibition, caspase-9 activity and apoptosis. Antioxidant agents inhibited heat shock-mediated proliferation inhibition (A), apoptosis (B), caspase-9 (C) and caspase-8 activity (D). SIRC cells were pretreated with vitamin C (100 mM) for 1 h, then treated for 30 min in a water bath at 37 C or 42 C. Culture media were replaced with fresh media and the cells further incubated for 24 h. Cell proliferation inhibition activity was assessed by WST-1. Caspase-9 and Caspase-8 activity was assessed by Caspase-9 and Caspase-8 Activity Assay kits. The induction of apoptosis was determined by TUNEL analysis. Each value is the mean SD of three determinations. The asterisk indicates a significant difference between two test groups, as analyzed by Dunnett’s test (p < 0.05). The asterisk indicates a significant difference between control and test groups, *p < 0.05.
caspase-9/-8 activity, and apoptosis. These data confirm that heat shock treatment of SIRC cells activates the mitochondrial and death receptor apoptotic pathway via release of ROS. 4. Discussion Apoptosis within the corneal epithelium has been inferred from the discovery of the dark cells during the regeneration stage of the rat corneal epithelium after chemical abrasion (Glasø et al., 1993). However, dark cells in the corneal epithelium resulting from damage caused by UVR have also been described (Ringvold, 1983; Clarke et al., 1990). Using TEM, Ringvold and Davanger found keratocytes with nuclear fragments of high electron density and abnormal inclusions in the rabbit corneal stroma after UV light exposure (Ringvold and Davanger, 1984). 310-nm UVR caused more extensive damage to the corneal stroma and endothelium than did 280-nm UVR (Podskochy et al., 2000). Apoptosis appears to be a mechanism of corneal cell death upon exposure to UVR. Little is known about the biological effects of IR on human eyes, however. Heat is likely to be generated as a consequence of IR irradiation, leading to an increase in skin temperature after exposure to the sun, and heat shock is widely considered a major extracellular stimulus (Park et al., 2004). A recent study has reported that in the face of global warming, the question of a link between eye disease and ambient temperature is more relevant than ever. It suggested that the effect of global warming on the prevalence of eye disease will be more severe for those living in cold climates getting warmer than for those in hot climates getting hotter (Kessel et al., 2010). This study, the first to investigate the biological effects of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells, shows that heat shock induces apoptosis through reactive oxygen species/ mitochondrial pathways in cornea cells.
Mitochondria are thought to be another pathway for apoptosis, and mitochondrial function is regulated through Bcl-2 family members, comprised of both anti-apoptotic (Bcl-2, Mcl-1) and proapoptotic members (Bax, Bak) (Hengartner, 2000; Shroff et al., 2007). A previous study has reported the Bcl-2 family plays a predominant role in the regulation of early corneal wound healing, such as keratocyte depletion, endothelial changes, and the inflammatory responses (Li et al., 2000). Our results showed that heat shock results in a significant increase of Bax and Bak mRNA and protein expression, and a decrease of Bcl-2 and Mcl-1, suggesting that changes in the ratio of proapoptotic and anti-apoptotic Bcl-2 family members might contribute to the apoptosis-promotion activity of heat shock. In addition, our data also revealed the degree of caspase-9 and caspase-3 activity after SIRC cells were treated with heat shock. Because the proapoptotic/anti-apoptotic ratio of Bcl-2 family members was found to be significantly changed by treating breast cancer cell lines with heat shock, and the changes of Bax and Bak (at 3 h) occurred earlier than the caspase activity (6 h for caspase-9 and caspase-3), this suggests that heat shock increases caspase activity through the regulation of Bcl-2 family members. This result is consist with several studies reported that Bcl-2 family proteins regulate the activation of caspase-9 and caspase-3 during apoptotic cell death (Weng et al., 2009; Hung et al., 2010). These mitochondrial apoptotic events play an important role in heat shock-mediated apoptosis. Fas/FasL system is a key signaling transduction pathway of apoptosis in cells and tissues (Mahmood and Shukla, 2010). Ligation of Fas by agonistic antibody or its mature ligand induces receptor oligomerization and formation of death-inducing signaling complex (DISC), followed by activation of caspase-8, then further activating a series caspase cascades resulting in cell apoptotic death (Mahmood and Shukla, 2010). Investigation by Podskochy and Fagerholm
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revealed that may play an important role in apoptosis of corneal cells after ultraviolet radiation (Podskochy and Fagerholm, 2002). In addition, some insults, such as HSV-1 and Pseudomonas aeruginosa infection, have been reported to induce apoptosis by Fas signaling pathway in cornea cells (Qian and Atherton, 2003; Zhou et al., 2010). Our study indicated that FasL, increased in heat shock-treated SIRC cells. Moreover, the levels of FasL and the activity of caspase-8 were simultaneously enhanced in SIRC cells following by apoptotic cell death (DNA fragmentation). Thus, these findings are the first to show that the Fas ligand/caspase-8 system plays an important role in heat shock-mediated SIRC cellular apoptosis. All aerobic organisms are subjected to physiological oxidant stress as a consequence of aerobic metabolism. Superoxide anion (O2), hydrogen peroxide (H2O2), hydroxyl radical (OH) and hypochlorous acid are included among the reactive oxygen species (ROS) that are natural by-products of normal cell metabolism (Rao et al., 1987; Halliwell, 1990). The redox status of all aerobic cells is balanced by enzyme and nonenzyme systems (Halliwell, 1990; Kornas et al., 2010). Oxidative stress occurs when there is an imbalance between cellular oxidant species production and antioxidant capability (Kondo et al., 2006; Matés et al., 2008). Previous studies demonstrated that physiological and nonphysiological environmental stressor such as ultraviolet radiation and hypoxiaereoxygenation can cause transient formation of reactive oxygen species (ROS) in corneal tissue (Shimmura et al., 1996, 1998). The response of perturbed tissue against ROS formation ranges from modification of cytokine regulation, to organelle dysfunction in lysosomes and mitochondria, to cell death (Aragane et al., 1997; Cullen, 1980; Shimmura and Tsubota, 1997; Ren and Wilson, 1994; Shimmura et al., 1999). Previous studies have demonstrated reactive oxygen species (ROS) induces apoptosis through mitochondrial pathway (Azad et al., 2010; Nakamura and Sakamoto, 2001; Nie et al., 2008; Hsu et al., 2010a,b; Kuo et al., 2007). Another recent study has also suggested increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts (Chwa et al., 2006). Our study has shown that heat shock-mediated oxidative stress primarily by increasing the production of O2 and H2O2. We further observed that heat shock generates ROS, resulting in caspase-9 and caspase-8 activation. Furthermore, vitamin C, an agent which can regulate ROS detoxification, blocked proliferation inhibition, caspase-9 and caspase-8 activity and apoptosis induced by heat shock. These data suggest that ROS accumulation contributes to heat shock-induced cell death in cornea cells. In conclusion, our data indicate that Statens Seruminstitut Rabbit Cornea (SIRC) cells are highly sensitive to growth inhibition and apoptosis induction by heat shock. Heat shock-induced apoptosis is associated with mitochondrial and death receptor pathways, which are in turn mediated by ROS generation. Our study demonstrates a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells.
Acknowledgments This work is supported by grants from the National Science Council of Taiwan (NSC 99-2632-B-037-001-MY3) and the Center of Excellence for Environmental Medicine, Kaohsiung Medical University (KMU-EM-100-4).
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