Apoptotic cell death occurs in both light-induced
10 29 30 and inherited retinal degeneration.
9 31 32 33 34 Previous studies have demonstrated the presence of two distinct pathways in light-induced apoptosis in mice.
35 Bright light is thought to induce apoptosis that is independent of transducin, which is accompanied by induction of transcriptional factor AP-1. By contrast, low-light-induced apoptosis has been shown to require transduction. In the present study, AP-1 activation was clearly seen in bright-light-induced retinal degeneration, but was inhibited by pretreatment with PBN. In lipopolysaccharide (LPS)-treated rats and in LPS-stimulated cells, preadministration of PBN has been shown to inhibit significantly the activation of LPS-mediated NF-κB and AP-1,
27 36 both of which are considered to be involved in the LPS effect.
A single administration of PBN before exposure to light was protective, suggesting that PBN is prophylactic rather than therapeutic in this model. Therefore, it is speculated that for PBN-mediated protection, its presence is necessary during the early signaling events that occur immediately after exposure to light. To explore early events in PBN protection from light damage, we investigated changes in AP-1, which mainly consists of c-fos/junD heterodimer.
11 AP-1 is often involved in cellular responses caused by various external stimuli and regulates various gene expressions. In retinal light damage models, AP-1 DNA binding activity has been shown to increase during exposure to light.
4 DNA binding of c-fos is essential for a specific apoptotic pathway induced by light, but not for the execution of apoptosis induced by other stimuli such as
N-methyl-
N-nitrosourea.
11 In the present study, even in retinas exposed to light for 6 hours, AP-1 activation was detected despite the absence of TUNEL staining and histologic changes, which confirms that AP-1 activation is an early event for light-induced photoreceptor degeneration. To our knowledge, this is the first report of AP-1 activation induced by light in rat photoreceptors. Western blot analysis showed a significant increase in c-fos level in retinal nuclear proteins obtained from light-exposed rats, which is consistent with DNA AP-1 binding activity. PBN inhibited the increase of both c-fos protein level and AP-1 activation. The presence of PBN in the EMSA gel did not affect AP-1 DNA binding activity (data not shown). In addition to the fact that PBN inhibits LPS-induced cytokine gene upregulation and AP-1 activation in rats,
27 Ahmed et al.
37 have shown that PBN suppresses IL-1-stimulated expression of matrix metalloproteinase-13 in human osteoarthritis chondrocytes, through the inhibition of JNK and AP-1. Inhibition of AP-1 activation has also been demonstrated in the protection from retinal light damage mediated by dexamethasone-induced glucocorticoid receptor activation.
38 Thus, AP-1 inhibition may be a critical target in drug-mediated protection of retina from damaging light.
Caspase-dependent apoptotic pathways are suggested to play a major role in photoreceptor degenerations.
39 40 41 42 In the present experiments, there was upregulation of caspase-3 gene expression after exposure to light, and this upregulation was significantly inhibited by preadministration of PBN. Other genes that were upregulated by exposure to light were unaffected by PBN. However, there was no significant change in caspase-3 amounts or catalytic activities in any animal
(Fig. 7) , suggesting that there were discrepancies between mRNA and protein levels. Others have noted increased transcription in the absence of an increase in translation.
43 44 45 46 47 48 Under oxidative stress conditions, it has been reported that reactive oxygen inhibits the synthesis of proteins.
49 Wu et al.
50 exposed rats to 6 hours of intense blue light and then dark adapted them for various times up to 24 hours and measured caspase-3 activity in retinal homogenates. No enzyme activity was seen immediately after the animals were removed from the exposure to light, but significant activity was found after 8 and 16 hours of dark adaptation. Their Western blots showed an increase in a 32-kDa protein at 8 and 16 hours, which was not found in our Western blots performed on retinas removed immediately after exposure to bright light. Taken together, these results suggest that activation of caspase-3 occurs in the dark and may not occur as long as the animals remain in the light. Because we found apoptotic nuclei in the absence of increased caspase-3 activity, we suggest that this enzyme is not involved in apoptotic cell death in light-induced retinal degeneration.
Caspase-1 has been shown to be a major player in light-induced photoreceptor degeneration in mice.
39 41 51 52 In our present study, probes in an RPA include Fas, Bcl-xl/S, FasL, caspase-1, caspase-2, caspase-3, Bax, Bcl-2, L32, and GAPDH. We did not detect any caspase-1 mRNA expression. The RPA is less sensitive than RT-PCR methods, and so we attempted to maximize its sensitivity by using radiolabeled probes that can detect mRNA of 5 × 10
5 molecules. We cannot exclude the possibility that the RPA’s sensitivity was too low to allow detection of caspase-1 in our animals. The other possibility is that there are differences in the apoptotic process of light-induced retinal degeneration between rats and mice. Further studies are needed to clarify this point.
Because photoreceptor cell death in light damage and hereditary retinal degenerations is by apoptosis, the light damage model has been used extensively
14 38 53 54 55 to screen putative neuroprotective drugs that ultimately were tested in mutant models of human retinal disease. Although some cytokines
47 56 57 58 59 60 61 62 63 and other drugs
8 were effective in both models, some were not.
15 64 We found that PBN protected against light-induced apoptosis in transgenic rats with a P23H or S334ter rhodopsin mutation, but not against photoreceptor degeneration due to expression of the mutant proteins,
15 suggesting that different apoptotic pathways may be involved in photoreceptor degeneration mediated by light damage and heredity. This lack of effect in the mutant rats is understandable in light of our current finding that PBN reduced c-fos expression in light-stressed rats. Hafezi et al.
13 showed that mice lacking c-fos were not damaged by light, but that reducing c-fos in mice with inherited retinal degeneration (c-
fos −/− Rpe65L) did not prevent the loss of photoreceptor cells.
65 Thus, although the final common pathway in light-induced and hereditary retinal degenerations may be apoptosis, there are fundamental differences in the upstream events that ultimately lead to cell death.
The authors thank Mark Dittmar of the Dean McGee Eye Institute for maintaining the animal colonies used in this study and Charles Stewart of the Oklahoma Medical Research Foundation for help in developing the RPA.