In this study, we have shown that in neonatal retina, apoptosis of RGCs following a hypoxic exposure is associated with hypoxia-mediated nuclear translocation of NF-κB and increased expression of nNOS. It appears that the nuclear translocation of NF-κB had a role in the increased expression of nNOS in hypoxic RGCs. This notion lends its support from the fact that nNOS expression in cultured RGCs exposed to hypoxia was significantly reduced by a NF-κB–specific inhibitor, BAY. Our study further indicated that an increased production of NO through the enhanced nNOS expression in hypoxic RGCs causes the death of the RGCs by activated caspase-3–mediated apoptosis.
In the developing retina, NOS and NO are required for the timely maturation of the inner plexiform layer
12 and for early retinal differentiation,
15 but an excessive induction of NOS isoforms has been implicated in damage to the retina.
17,39 In response to hypoxic insult, in the developing retina, although excessive production of inflammatory mediators
7 and destructive effects of free radicals
8 are implicated in death of RGCs, it appears that increased nNOS expression in RGCs in the hypoxic retina and the subsequent production of NO also may result in their apoptosis via caspase-3 activation. Hypoxia-mediated nNOS expression is well documented
1,39,40 and NO produced from nNOS has been demonstrated as being highly detrimental to RGCs in adult retina,
17,22 yet the mechanisms involved remain to be fully characterized.
It has been speculated that hypoxia-mediated activation of transcription factor, NF-κB,
27,28 may have a critical role in the regulation and activation of genes involved in inflammation, oxidative stress, and apoptosis.
27,41–43 Under physiological conditions, NF-κB is localized in the cytoplasm and its activation is inhibited by IκB.
44 Hypoxic exposure causes degradation of IκB and results in activation and translocation of NF-κB
28,45 to the nucleus, where it regulates the expression of target genes. In the present study, following hypoxic exposure, the expression of NF-κB was increased in the RGCs of developing retina. This was supported further by the finding from cultured RGCs, wherein the concentration of NF-κB was upregulated in the cytoplasmic and nuclear fractions of hypoxic RGCs. However, this increase was abolished when hypoxic cultured RGCs were treated with BAY. Hypoxia-induced NF-κB activation has been reported previously in human retinal progenitor cells,
46 and in RGC-5, an RGC cell line
47,48 and the activation of NF-κB in RGCs has been implicated in the apoptosis of these cells.
48–50 In light of the above and from our results, it appears that the nuclear translocation of NF-κB in RGCs, in response to hypoxia, could lead to the transcription of genes that might result in the death of RGCs.
Additional support for the role of NF-κB in the hypoxia-induced expression of nNOS comes from a previous study, which reported the presence of NF-κB binding site in the promoter of the nNOS gene.
25 The suppression of nNOS expression and NO production in hypoxic RGCs treated with BAY or 7-NINA also supports the view that hypoxia-mediated nuclear translocation of NF-κB is essential for the induction of nNOS and the subsequent production of NO in hypoxic RGCs.
A number of reports claim that an excess production of NO through nNOS expression could mediate RGC death. These include the ability of NO to induce apoptosis in cultured retinal neurons when treated with advanced glycation end products
51 /S-nitroso-N-acetyl-penicillamine (SNAP), a NO donor,
23 and the increased survival of cultured RGCs against NO-mediated neurotoxicity, by the addition of NOS inhibitors, such as L-NAME, to the culture medium.
52 Previously, NO was shown to induce the proapoptotic cascade, in hypoxic neural tissues, by phosphorylating Bcl-2.
53 Once phosphorylated, Bcl-2 loses its antiapoptotic potential and its ability to heterodimerize with the proapoptotic protein Bax, resulting in Bax-mediated activation of caspases and initiation of apoptosis.
54–56 The NO-mediated injury to the RGCs is believed to occur via a caspase-dependent pathway. The addition of caspase inhibitor, Z-VAD-FMK, to SNAP-treated hypoxic RGC-5 cells resulted in partial protection.
23 In the present study, following hypoxia, parallel to the increased NO production there was increased expression of caspase-3 in RGCs in the developing retina. Our in vitro study also depicted the same, wherein there was increased caspase-3 labeling in hypoxic cultured RGCs. This increase in caspase-3-positive RGCs, however, was reduced when treated with 7-NINA or BAY, in vivo and in vitro. The results supported the view that excess NO produced by nNOS in hypoxic RGCs leads to their apoptosis through activation of caspase cascade.