Infrared Radiation Affects the Mitochondrial Pathway of Apoptosis in Human Fibroblasts
2004; Elsevier BV; Volume: 123; Issue: 5 Linguagem: Inglês
10.1111/j.0022-202x.2004.23472.x
ISSN1523-1747
AutoresSandra Frank, Lisa Oliver, Corinne Lebreton‐De Coster, Carole Moreau, Marie-Thérèse LeCabellec, Laurence Michel, François M. Vallette, Louis Dubertret, Bernard Coulomb,
Tópico(s)Connexins and lens biology
ResumoWe have previously observed that near-infrared (IR) pre-irradiation protects normal human dermal fibroblasts from ultraviolet (UV) cytotoxicity in vitro. Here, we show that IR pre-irradiation of human fibroblasts inhibited UVB activation of caspase-9 and -3, leading us to study early events in the mitochondrial apoptotic pathway after IR irradiation. IR irradiation led to a partial release of cytochrome c and Smac/Diablo but not apoptosis-inducing factor (AIF). This was accompanied by a slight but transient decrease in the mitochondrial membrane potential (Δψm) and by the insertion of Bax into mitochondrial membrane. Early apoptotic events in the mitochondrial pathway thus occurred after IR irradiation despite a lack of caspase-9 and -3 activation. This could be explained by the induction by IR of the expression of heat shock protein Hsp27, which is known to prevent apoptosome assembly. Furthermore, the balance between pro-apoptotic (i.e., Bax) and anti-apoptotic (i.e., Bcl-2 or Bcl-xL) proteins, which was rather pro-apoptotic after IR exposure, became anti-apoptotic 24 h later, suggesting a protective effect. Together, these actions could also contribute to prepare the cell to resist UVB-triggered apoptosis. Finally, isolated rat liver mitochondria-released cytochrome c in response to IR, demonstrating that mitochondria were a primary target of IR radiation. We have previously observed that near-infrared (IR) pre-irradiation protects normal human dermal fibroblasts from ultraviolet (UV) cytotoxicity in vitro. Here, we show that IR pre-irradiation of human fibroblasts inhibited UVB activation of caspase-9 and -3, leading us to study early events in the mitochondrial apoptotic pathway after IR irradiation. IR irradiation led to a partial release of cytochrome c and Smac/Diablo but not apoptosis-inducing factor (AIF). This was accompanied by a slight but transient decrease in the mitochondrial membrane potential (Δψm) and by the insertion of Bax into mitochondrial membrane. Early apoptotic events in the mitochondrial pathway thus occurred after IR irradiation despite a lack of caspase-9 and -3 activation. This could be explained by the induction by IR of the expression of heat shock protein Hsp27, which is known to prevent apoptosome assembly. Furthermore, the balance between pro-apoptotic (i.e., Bax) and anti-apoptotic (i.e., Bcl-2 or Bcl-xL) proteins, which was rather pro-apoptotic after IR exposure, became anti-apoptotic 24 h later, suggesting a protective effect. Together, these actions could also contribute to prepare the cell to resist UVB-triggered apoptosis. Finally, isolated rat liver mitochondria-released cytochrome c in response to IR, demonstrating that mitochondria were a primary target of IR radiation. apoptosis-inducing factor heat shock protein infrared mitochondrial transmembrane potential Infrared (IR) irradiation is used for therapeutic purposes including surgery (Kaufmann et al., 1994Kaufmann R. Hartmann A. Hibst R. Cutting and skin-ablative properties of pulsed mid-infrared laser surgery.J Dermatol Surg Oncol. 1994; 20: 112-118Crossref PubMed Scopus (150) Google Scholar), or promotion of wound healing (Horwitz et al., 1999Horwitz L.R. Burke T.J. Carnegie D. Augmentation of wound healing using monochromatic infrared energy. Exploration of a new technology for wound management.Adv Wound Care. 1999; 12: 35-40PubMed Google Scholar;Danno et al., 2001Danno K. Mori N. Toda K. Kobayashi T. Utani A. Near-infrared irradiation stimulates cutaneous wound repair: Laboratory experiments on possible mechanisms.Photodermatol Photoimmunol Photomed. 2001; 17: 261-265https://doi.org/10.1034/j.1600-0781.2001.170603.xCrossref PubMed Scopus (70) Google Scholar;Toyokawa et al., 2003Toyokawa H. Matsui Y. Uhara J. et al.Promotive effects of far-infrared ray on full-thickness skin wound healing in rats.Exp Biol Med (Maywood). 2003; 228: 724-729PubMed Google Scholar). This laser medicine is based on the mechanism of photoenergy conversion in heating, but has also been suggested to activate photoacceptors such as cytochrome c oxidase, pointing to a particular role for mitochondria (Karu, 1999Karu T. Primary and secondary mechanisms of action of visible to near-IR radiation on cells.J Photochem Photobiol. 1999; 49: 1-17https://doi.org/10.1016/S1011-1344(98)00219-XCrossref PubMed Scopus (937) Google Scholar). In addition, despite the fact that IR (700–4000 nm) accounts for 40% of the solar radiation reaching the earth's surface, very little is known about its biological effects. Nevertheless, several mechanisms of action of non-coherent IR radiation have been proposed. Mitogen-activated protein kinases (MAPK) have been shown to be involved in IR induction of matrix metalloproteinase 1, or collagenase (Schieke et al., 2002Schieke S.M. Stege H. Kürten V. Grether-Beck S. Sies H. Krutmann J. Infrared-A radiation-induced matrix metalloproteinase 1 expression is mediated through extracellular signal-regulated kinase 1/2 activation in human dermal fibroblasts.J Invest Dermatol. 2002; 119: 1323-1329https://doi.org/10.1046/j.1523-1747.2002.19630.xCrossref PubMed Scopus (97) Google Scholar). Ferritin has also been proposed as a possible cell defense system induced by IR (Applegate et al., 2000Applegate L.A. Scaletta C. Panizzon R. Frenk E. Hohlfeld P. Schwarzkopf S. Induction of the putative protective protein ferritin by infrared radiation: Implications in skin repair.Int J Mol Med. 2000; 5: 247-251PubMed Google Scholar). Furthermore, most of our knowledge on solar radiation is based on monochromatic ultraviolet (UV) and interactions between the different solar wavelengths have been overlooked. Nevertheless, we have previously shown that prior treatment with naturally occurring doses of IR protects normal human dermal fibroblasts from UV toxicity, in vitro (Menezes et al., 1998Menezes S. Coulomb B. Lebreton – De Coster C. Dubertret L. Non-coherent near infra-red radiation protects normal human dermal fibroblasts from solar ultraviolet toxicity.J Invest Dermatol. 1998; 111: 629-633https://doi.org/10.1046/j.1523-1747.1998.00338.xCrossref PubMed Scopus (81) Google Scholar). This effect was observed under temperature-controlled conditions, independently of heat shock protein induction (Hsp72–70) (Menezes et al., 1998Menezes S. Coulomb B. Lebreton – De Coster C. Dubertret L. Non-coherent near infra-red radiation protects normal human dermal fibroblasts from solar ultraviolet toxicity.J Invest Dermatol. 1998; 111: 629-633https://doi.org/10.1046/j.1523-1747.1998.00338.xCrossref PubMed Scopus (81) Google Scholar;Schieke et al., 2002Schieke S.M. Stege H. Kürten V. Grether-Beck S. Sies H. Krutmann J. Infrared-A radiation-induced matrix metalloproteinase 1 expression is mediated through extracellular signal-regulated kinase 1/2 activation in human dermal fibroblasts.J Invest Dermatol. 2002; 119: 1323-1329https://doi.org/10.1046/j.1523-1747.2002.19630.xCrossref PubMed Scopus (97) Google Scholar), and was independent of cell division (Menezes et al., 1998Menezes S. Coulomb B. Lebreton – De Coster C. Dubertret L. Non-coherent near infra-red radiation protects normal human dermal fibroblasts from solar ultraviolet toxicity.J Invest Dermatol. 1998; 111: 629-633https://doi.org/10.1046/j.1523-1747.1998.00338.xCrossref PubMed Scopus (81) Google Scholar). In order to better understand how IR prevents UV cytotoxicity (Menezes et al., 1998Menezes S. Coulomb B. Lebreton – De Coster C. Dubertret L. Non-coherent near infra-red radiation protects normal human dermal fibroblasts from solar ultraviolet toxicity.J Invest Dermatol. 1998; 111: 629-633https://doi.org/10.1046/j.1523-1747.1998.00338.xCrossref PubMed Scopus (81) Google Scholar), we evaluated the effect of IR radiation on UVB-induced apoptosis and investigated more particularly the influence of IR on early apoptotic events involving mitochondria, which contain IR chromophores such as cytochrome c oxidase as previously suggested (Karu, 1999Karu T. Primary and secondary mechanisms of action of visible to near-IR radiation on cells.J Photochem Photobiol. 1999; 49: 1-17https://doi.org/10.1016/S1011-1344(98)00219-XCrossref PubMed Scopus (937) Google Scholar). In fact, mitochondria play a key role in type II apoptosis, by releasing pro-apoptotic factors, such as cytochrome c, Smac/Diablo and AIF (apoptosis-inducing factor), from the intermembrane space into the cytoplasm. Cytochrome c, when associated with Apaf-1 (apoptosis protease activating factor 1), activates caspase-9 (Li et al., 1997Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (5991) Google Scholar), which in turn activates caspase-3 (Li et al., 1997Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. 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Zamzami N. et al.Molecular characterization of mitochondrial apoptosis-inducing factor.Nature. 1999; 397: 441-446https://doi.org/10.1038/17135Crossref PubMed Scopus (3343) Google Scholar). In addition, a reduction in mitochondrial transmembrane potential (Δψm) is also detected during the early stages of the apoptotic process (Zamzami et al., 1996Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. Mitochondrial control of nuclear apoptosis.J Exp Med. 1996; 183: 1533-1544https://doi.org/10.1084/jem.183.4.1533Crossref PubMed Scopus (1238) Google Scholar), when cells are not irreversibly committed to death (Vayssiere et al., 1994Vayssiere J.L. Petit P.X. Risler Y. Mignotte B. Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40.Proc Natl Acad Sci USA. 1994; 91: 11752-11756Crossref PubMed Scopus (302) Google Scholar). 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In the present work, we show that IR inhibited UVB-induced caspase-9 and -3 activation, and focus on the effects of IR on mitochondria, by analyzing the release of the pro-apoptotic molecules such as cytochrome c, Smac/Diablo, or AIF, and the changes in Δψm. The importance of the role of mitochondria in the action of IR is confirmed by studying cytochrome c release by isolated rat liver mitochondria. Finally, to determine how IR impaired caspase activation, we studied the effects of IR on the balance of pro- (Bax) and anti- (Bcl-2, Bcl-xL, Hsp27) apoptotic molecules modulating the Δψm and the release of mitochondrial proteins. Caspase activation was studied using the most efficient IR protocol found to prevent UV-induced cytotoxicity (Menezes et al., 1998Menezes S. Coulomb B. Lebreton – De Coster C. Dubertret L. Non-coherent near infra-red radiation protects normal human dermal fibroblasts from solar ultraviolet toxicity.J Invest Dermatol. 1998; 111: 629-633https://doi.org/10.1046/j.1523-1747.1998.00338.xCrossref PubMed Scopus (81) Google Scholar), i.e., three 30 min sessions (3 × 810 kJ per m2) followed 24 h later, by UVB irradiation. Caspase activation was analyzed 4 d later. A significant increase in both caspase-9 and -3 activity was found in UVB-irradiated cells (1500 J per m2). IR pre-irradiation significantly reduced UVB-induced caspase activation. No activation of either caspase-9 or 3, however, was detected after IR irradiation only (Figure 1). These results showed that IR pre-irradiation caused an inhibition of UVB-induced caspase activation. Early apoptotic events involving the mitochondrial pathway were studied after a single 60 min IR irradiation (1620 kJ per m2). The cellular localization of cytochrome c was examined using laser confocal microscopy. In non-irradiated cells, cytochrome c staining showed a punctuate cytoplasmic pattern in keeping with its mitochondrial localization. In contrast, IR-irradiated cells exhibited a diffuse cytochrome c staining, suggesting a translocation from mitochondria to the cytoplasm (Figure 2). A time-course study of cytochrome c release showed the presence of a cytosolic fraction of cytochrome c as early as 3 h after IR irradiation, a further increase was observed at 6 h (Figure 2), which persisted until 24 h. The pattern of cytochrome c staining resembled control cells 72 h after IR treatment (not shown). Staining with an anti-cytochrome oxidase subunit IV antibody (a marker of the inner mitochondrial membrane) showed the particular mitochondrial pattern (Figure 2). Similar results were obtained when a Schott (Schott France, Clichy, France) RG 715 filter was used, indicating that cytochrome c release was induced at wavelengths between 700 and 2000 nm (Figure 2). An evaluation of the release of cytochrome c was determined by analyzing the degree of co-localization of F1-ATPase (inner mitochondrial membrane) and cytochrome c using Metamorph 4.6 software (Universal Imaging, Roper Scientific, Evry, France) after laser confocal microscopy analysis. At 6 h after IR treatment, an extra-mitochondrial fraction of cytochrome c was clearly detectable in cells after IR irradiation, whereas a marked co-localization was found in control fibroblasts. IR was estimated to induce the release of 30% of mitochondrial cytochrome c (Figure 3). To confirm this cytochrome c release, fibroblast subcellular fractionation was performed 6 and 18 h after IR irradiation. The localization of cytochrome c was analyzed by immunoblotting of the mitochondrial and cytosolic fractions. As shown in Figure 4, a band corresponding to cytochrome c was clearly visible in the cytosolic fraction of irradiated cells. Cytochrome c was still present in the mitochondrial fraction of both irradiated and control cells, confirming the partial release suggested by confocal analysis. A release of Smac/Diablo was also observed in IR-irradiated cells. AIF, another pro-apoptotic protein, however, was not detected in the cytosolic fraction at either 6 or 18 h after IR-irradiation (Figure 4). As a control, we examined the distribution of porin (Figure 4), a mitochondrial transmembrane protein, also known as VDAC (voltage-dependent anion channel) and Hsp60 (Fig S1), a soluble mitochondrial matrix protein. As expected, those proteins were only found in the mitochondrial fraction of both irradiated and control fibroblasts showing that the mitochondria were not fragmented during cell fractionation. Finally, IR irradiation did not reduce the amount of procaspase-3 present in the cells nor induce the proteolytic activation of procaspase-3 (Figure 1 and Figure 4). To determine whether cytochrome c release was due to a direct action of IR on mitochondria, isolated rat liver mitochondria were resuspended in mitochondrial isolation buffer (MIB), then IR irradiated and the presence of cytochrome c was determined in the mitochondrial pellet and in the supernatant. Cytochrome c was detected in the supernatant immediately after IR irradiation (1620 kJ per m2), and also 30 min later (Figure 5). The absence of porin in the buffer indicated that the mitochondria were still intact and that the release of cytochrome c was not due to a disruption of the mitochondrial outer membrane (Figure 5). The use of a Schott RG 715 filter did not affect the results underlining the fact that the efficient wavelengths were between 700 and 2000 nm (Figure 5). Thus, IR-irradiated mitochondria release cytochrome c, even in the absence of cytosolic components, demonstrating a direct effect of IR on this organelle. A decrease in Δψm is another early event in apoptosis and is often associated with cytochrome c release. We thus analyzed DiOC6 fluorescence by flow cytometry. No change in Δψm was detected immediately after a 60 min IR irradiation (1620 kJ per m2) as compared to control cultures. In contrast, depending on the experiment and on the strain of fibroblast, a 10%–30% decrease was always detected between 2 and 3 h (Figure 6). This decrease sometimes persisted 5 h after irradiation; however, the Δψm always returned to the control level after 18 h. Propidium iodide nuclear staining of IR-irradiated fibroblasts was always negative in keeping with the absence of cell death. This decrease in Δψm further supports the notion that mitochondria are targets of IR. In addition, the recovery of Δψm is compatible with the absence of apoptosis after IR irradiation. In healthy cells, Bax is predominantly localized to the cytosol in a inactive form. After apoptotic stimuli, however, Bax is translocated into mitochondria (Wolter et al., 1997Wolter K.G. Hsu Y.T. Smith C.L. Nechushtan A. Xi X.G. Youle R.J. Movement of Bax from the cytosol to mitochondria during apoptosis.J Cell Biol. 1997; 139: 1281-1292https://doi.org/10.1083/jcb.139.5.1281Crossref PubMed Scopus (1539) Google Scholar). By immunohistochemistry, using an antibody that recognizes active Bax, we showed a punctuated distribution of Bax 6 and 24 h after IR irradiation (1620 kJ per m2), which was consistent with mitochondrial localization, whereas in non-irradiated cells, the staining was absent (Figure 7). Bax translocation after IR irradiation could thus contribute to the permeability of the outer mitochondrial membrane observed. To identify the mechanisms leading to the inhibition of caspase activation despite cytochrome c and Smac/Diablo release, we analyzed by Western blot the expression of Hsp27, a protein known to inhibit cytochrome c-induced caspase-9 activation. The data presented in Figure 8 show that Hsp27 was induced 48h after IR irradiation (1620 kJ per m2) and this induction persisted 72 h after IR irradiation. Whatever the conditions, Hsp27 was always induced after 48h by IR irradiation; however, depending on the fibroblast strain, this induction could be detected earlier. Similar results were obtained with or without a Schott RG 715 filter. The expression of Bcl-2, Bcl-xL (anti-apoptotic proteins), and Bax (pro-apoptotic protein) was determined by western blots. Bax expression increased immediately after a 60 min IR irradiation (1620 kJ per m2). Sometime this induction persisted for 24 h after IR irradiation, then Bax expression decreased gradually until 72 h (Figure 9). Depending on the fibroblast strain, Bcl-2 and Bcl-xL could be detected immediately after IR exposure and continued to accumulate over the following 72 h. Similar results were obtained with and without a Schott RG 715 filter. Thus, the balance between Bax, Bcl-2, and Bcl-xL was pro-apoptotic during the first 24 h after IR exposure, before becoming anti-apoptotic. This study demonstrates that IR induces mitochondrial release of cytochrome c and Smac/Diablo, showing that early events in the mitochondrial apoptotic pathway occur despite the lack of caspase activation after IR exposure, and of course despite the absence of apoptotic cells. Thus, the apoptotic process initiated by IR is rapidly stopped after the release of pro-apoptotic molecules from mitochondria. Once in the cytoplasm, cytochrome c interacts with its adapter molecule Apaf-1 and contributes to the processing and activation of procaspase-9 in the presence of dATP (Li et al., 1997Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (5991) Google Scholar). In turn, caspase-9 cleaves and activates procaspase-3, thereby initiating the final phase of apoptosis (Li et al., 1997Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (5991) Google Scholar). These steps do not occur after IR exposure. Smac/Diablo normally potentiates caspase activation by binding to inhibitors of apoptotic proteins (IAP) and blocking their inhibitory activity (Du et al., 2000Du C. Fang M. Li Y. Li L. Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition.Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2736) Google Scholar). After IR exposure, however, we found that neither Smac/Diablo nor cytochrome c generated active caspase-9 or -3. We also found that IR did not induce the release of AIF, known to lead to DNA fragmentation (Susin et al., 1999Susin S.A. Lorenzo H.K. Zamzami N. et al.Molecular characterization of mitochondrial apoptosis-inducing factor.Nature. 1999; 397: 441-446https://doi.org/10.1038/17135Crossref PubMed Scopus (3343) Google Scholar). The lack of AIF release from the mitochondrial intermembrane space to the cytoplasm agrees with the normal DAPI nuclear staining observed 24 and 72 h after IR exposure, reflecting a lack of DNA fragmentation (data not shown). We thus demonstrate that IR induces selective release of pro-apoptotic factors from mitochondria, leading to the release of caspase-dependent cell death proteins such as cytochrome c and Smac/Diablo but not of caspase-independent cell death proteins such as AIF. These findings are consistent with results fromArnoult et al., 2002Arnoult D. Parone P. Martinou J.C. Antonsson B. Estaquier J. Ameisen J.C. Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli.J Cell Biol. 2002; 159: 923-929https://doi.org/10.1083/jcb.200207071Crossref PubMed Scopus (274) Google Scholar,Arnoult et al., 2003Arnoult D. Gaume B. Karbowski M. Sharpe J.C. Cecconi F. Youle R.J. 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