Expression and Role of p53 in the Retina

Abstract

Since it was identified in 1979, p53 has been widely studied for its role in tumor suppression. It is mutated in approximately half of all human cancers, leading to aberrant cell growth. In addition to its role as a tumor suppressor, p53 is activated in response to various cell stress signals, including DNA damage and hypoxia. This activation leads to alterations in target gene expression, giving p53 a regulatory role in diverse cellular functions such as apoptosis, senescence, and cell cycle arrest. Throughout life, the eye is exposed to a multitude of stressors including disease, light-induced damage, and oxidative stress, all of which can lead to debilitating loss of vision. This article examines the role of p53 during ocular development. Finally, the role of p53 is examined in ocular response to intense light exposure, ionizing radiation, oxidative stress, degenerative disorders, and retinoblastoma.

The protein p53 is perhaps best known for its role as a tumor suppressor. However, it was initially thought that TP53, the gene that encodes human p53, was an oncogene, because it is overexpressed in cells transformed with the SV40 DNA tumor virus. ¹ In 1984, Trp53, the gene encoding murine P53, was found to be inactivated in an Abelson murine leukemia virus–transformed cell line, ² providing the first evidence that p53 may be active in tumor suppression. Subsequent research into murine leukemias showed that the Friend erythroleukemia virus also causes Trp53 inactivation. ^3,4 Similarly, in the human leukemia–derived cell line HL-60, much of the coding sequence of TP53 is deleted, resulting in p53 deficiency. ⁵  

Further confirmation that p53 is a tumor suppressor came when researchers realized that the various p53 clones they had been using had different sequences due to various mutations. After the sequence of wild-type TP53 was established, it became clear that mutant p53, but not wild-type p53, can promote transformation. ^6,7 Finally, researchers found that overexpression of wild-type Trp53 can inhibit the transformation of cultured cells by the oncogenes Myc and H-Ras. ^8,9  

In 1990, the tumor-suppressing role of p53 was also demonstrated in experiments with the p53Val135 mutant. This alanine-to-valine substitution generates a temperature-sensitive mutant that behaves like the wild-type at 32.5°C, but is inactivated at 37.5°C. Researchers working with rat embryo fibroblasts co-transformed with the p53Val135 mutant allele and Ras found that oncogene-mediated focus formation occurred at 37.5°C (mutant p53) but was abolished at 32.5°C (wild-type p53). ¹⁰  

The p53 knockout mouse delivered the final, decisive data supporting the role of p53 as a tumor suppressor. This mouse model exhibits significantly increased incidence of multiple types of neoplasms with high penetrance. ¹¹ Similarly, germline mutations in one TP53 allele were found to be responsible for Li-Fraumeni syndrome, ^12,13 in which patients have early onset of a variety of cancers. 

Cellular Functions of p53

Activation of p53

Activation of p53 occurs through multiple mechanisms including (1) increased protein concentration, often caused by decreased p53 degradation, ¹⁴ (2) nuclear translocation, ¹⁵ and (3) covalent and noncovalent protein modifications, ¹⁶ including phosphorylation and acetylation. ¹⁷ These posttranslational modifications can inhibit interaction between p53 and its negative regulators, ¹⁸ but the precise role of the various modifications is complex. For a review of posttranslational modifications to p53, see Boehme and Blattner. ¹⁷  

p53 is synthesized in the cytoplasm and, under normal conditions, is transported between the cytoplasm and the nucleus in a cell-cycle–dependent manner. ¹⁹ It accumulates in the cytoplasm during G₁, localizes to the nucleus during the transition from the G₁ to the S phase, and is shuttled back to the cytoplasm shortly after the start of the S phase. ¹⁵  

p53 may execute some of its functions in the cytoplasm by binding to other components to regulate mitochondrial membrane permeabilization and apoptosis, but it mainly functions in the nucleus. Once in the nucleus, active p53 oligomerizes at its C-terminal domain ²⁰ to form tetramers that bind DNA to affect transcription of target genes. ²¹  

p53 in Cell Cycle Arrest

In response to DNA-damaging γ-irradiation, p53 halts cell growth at G₁ to prevent the synthesis of faulty DNA. ²² This G₁ checkpoint role led to the application to p53 of the name “guardian of the genome.” ²³ Further research revealed that it can also arrest the cell cycle at the S phase completion checkpoint, when there is a depletion of DNA synthesis substrates, ²⁴ and at the G₂/M checkpoint, if there is subsequent DNA damage. ^24,25 p53-mediated cell cycle arrest occurs via transcriptional activation of the p53 target gene Cdkn1a that encodes p21, ²⁶ a cyclin-dependent kinase inhibitor. ²⁷  

p21, also known as WAF1, belongs to the Cip/Kip cyclin-dependent kinase (Cdk) inhibitor family. ²⁸ Its role as a potent Cdk inhibitor is essential for p21 to induce cell cycle arrest at the G₁ and G₂ ²⁹ or the S phase ³⁰ after DNA damage ^{31 –33} (Fig. 1, Cell Cycle Regulation). 

Figure 1.

 

Overview of p53 regulation and function. Under normal conditions, p53 is tightly regulated by Mdm2, Mdm4, Yy1, and p300, leading to the ubiquitination and degradation of p53. However, in response to cell stress, p53 levels are stabilized, and p53 can go on to affect the transcription of a variety of downstream targets involved in cell cycle regulation and apoptosis. Most notably, the p53 target p21 is a Cdk inhibitor that can inhibit cell cycle progression at G₁, S, and G₂. Alternatively, p53 can upregulate the expression of Bax, a regulator of mitochondrial membrane permeabilization, and induce apoptosis by the release of cytochrome c from the mitochondria.

p53/p21-mediated cell-cycle arrest at G₁ is achieved by blocking the Cdk4/cyclin D complex. When active, this complex phosphorylates the retinoblastoma protein (pRb), leading to induction of the transcription factor E2F1, which activates cell cycle progression from G₁ to S. ³⁴  

Cell cycle arrest mediated by p53/p21 can also occur when dysfunctional telomeres are present. Damaged telomeres are recognized as double-stranded DNA breaks, leading to increased p53-driven expression of p21 and senescence. ³⁵ Telomerase reverse transcriptase is the catalytic subunit of telomerase and works to maintain telomere length and avoid senescence, in part by preventing the upregulation of p21 expression in response to telomere damage. ³⁶  

Finally, p53/p21 can induce cell cycle arrest at the S phase by inhibiting proliferating cell nuclear antigen (PCNA), a DNA polymerase δ cofactor that is essential for DNA synthesis and repair. ^{37 –39} In vitro work has shown that p21 can interfere with PCNA interaction with replication factor C, DNA polymerase δ, and flap endonuclease 1, ^{40 –42} resulting in a p21-mediated blockage of DNA replication and repair. ⁴³  

p53 in Apoptosis

The role of p53 in apoptosis was discovered in 1991 when researchers restored Trp53 expression to clone S6 of the M1 murine myeloid leukemia cell line that normally lacks p53. ⁴⁴ The M1 cells were transfected with mRNA from the p53Val135 mutant and observed at both 32.5°C and 37.5°C. Cells incubated at 37.5°C (mutant p53) grew regularly while cells incubated at 32.5°C (wild-type p53) rapidly lost viability. Morphologic studies showed that the cells incubated at 32.5°C were dying from apoptosis, not necrosis, ⁴⁴ suggesting that p53 can promote apoptosis. 

It was also found that when RNA polymerase II–dependent transcription was inhibited, p53 activated the mitochondrial apoptotic pathway ^45,46 by directly inducing transcription of BAX ⁴⁷ and inhibiting the apoptosis regulator protein B-cell lymphoma 2 (bcl-2), ⁴⁶ possibly through repression of bcl-2 transcription or through antagonistic binding of p53 to bcl-2. ⁴⁷  

p53 also plays a role in hypoxia-mediated activation of the mitochondrial apoptotic pathway. Within 1 hour of hypoxic stress, approximately 2% of the total activated p53 localizes to the mitochondria and induces apoptosis. This p53 response occurs upstream of changes in mitochondrial membrane potential, the release of cytochrome c, and the activation of procaspase-3 ⁴⁸ (Fig. 1, Cell Death). Of particular interest, hypoxia also causes a p53-independent increase in p21, ^49,50 indicating that alternative, competing pathways may be at work to arrest the cell cycle in response to hypoxia instead of initiating apoptosis. 

p53 in Stem Cell Homeostasis

More recently, a potential regulatory role for p53 in stem cell homeostasis has been identified. ^51,52 p63, a p53 family member, has been shown to be essential in maintaining the high proliferative potential of epithelial stem cells, ⁵³ and p53 was subsequently linked to stem cell regulation. This link was initially described in a freshwater planarian (flatworm), ⁵¹ but has now been seen in mammary, ⁵² hematopoietic, ⁵⁴ and neural stem cells. ⁵⁵ In these studies, loss of p53 appears to lead to increased self-renewal of the stem cell population and hyperproliferation, which is consistent with p53's established role as a cell cycle regulator. 

Knockdown of Smed-p53, the planarian homolog of p53, via feeding with bacteria-expressing Smed-p53 dsRNA induced a significant increase in cell proliferation between days 3 and 9 after feeding compared with planarians fed with control dsRNA. Cell proliferation decreased steadily in Smed-p53 dsRNA-treated worms, and at 15 days after treatment, histologic markers for stem cells and their progeny showed a collapse of the entire stem cell lineage. ⁵¹ As a result of these findings, p53 was identified as a potential regulator of stem cell homeostasis. 

In vitro work with murine mammary stem cells confirmed the findings of studies on lower organisms and showed that p53 impairment led to aberrant cell growth. ⁵² Each wild-type mammosphere contains one stem cell that typically divides asymmetrically, producing one daughter stem cell and one daughter cell that will differentiate. In contrast, mammospheres from p53-null mice each contain five to six stem cells that divide symmetrically, producing a pool of stem cells with an unlimited potential for self-renewal. ⁵²  

Hematopoietic stem cells from p53-null mice also show increased self-renewal both in vitro and in vivo, leading to an expanded pool of stem cells. ⁵⁴ More recent findings have shown that both short hairpin (sh)RNA–mediated knockdown and genomic deletion of p53 result in aberrant self-renewal of myeloid progenitor cells. ⁵⁶ After ionizing radiation (IR), cells with lower p53 levels or activity proliferated more rapidly than those cells with high p53 levels or activity. ^{57 –59}  

Finally, p53 may have a role in cancer stem cell proliferation. The homeoprotein Nanog is known to be necessary for embryonic stem cell pluripotency ⁶⁰ and is also essential for the cancer stem cell function of glioblastoma multiforme, a type of highly invasive brain tumor. ⁵⁵ p53 is a negative regulator of Nanog, ⁵⁵ suggesting that p53 may be involved in glioblastoma multiforme growth and possibly in other cancer stem cell proliferation. 

Regulators of p53

Mdm2 and Mdm4 Promote p53 Degradation

The murine double minute 2 (Mdm2), a negative regulator of p53, ⁶¹ was identified in 1987 from its amplification in 3T3-DM, a spontaneously transformed BALB/c murine cell line. ⁶² It was later established that Mdm2 overexpression was responsible for the transformation of the 3T3-DM cell line. ⁶³  

Researchers found that Mdm2 is able to bind to oligomerized p53 ^61,64 and act as an E3 ubiquitin ligase, causing p53 degradation. ⁶⁵ This process occurs in the nucleus where ubiquitination of p53 by Mdm2 reveals a p53 nuclear export sequence that causes p53 to localize to the cytoplasm where proteasomal degradation can occur. ^19,66  

Interestingly, when p53 is activated, it initiates transcription of a variety of target genes including Mdm2. ⁶⁷ Thus, p53 and Mdm2 are involved in a negative feedback loop that allows for tightly regulated p53 expression (Fig. 1, Homeostatic Regulation). 

Mdm4, also known as MdmX, was identified as a novel p53 inhibitor in 1996 ⁶⁸ and later as an Mdm2-binding partner in 1999. ⁶⁹ Mdm4 is the closest identified analog of Mdm2 ⁷⁰ with complete conservation of the C-terminal metal-binding domains ⁶⁸ and >53% sequence similarity in the p53-binding domain. ⁷⁰  

Mdm2 and -4 form a stable heterodimer that allows Mdm4 to regulate the ability of Mdm2 to ubiquitinate p53. ⁷⁰ The ratio of Mdm4 to Mdm2 determines the effect on p53 levels. ⁷¹ When Mdm4 levels fall below a 2-to-1 ratio with Mdm2, the stability of p53 is decreased. However, when Mdm4 levels increase above the 2-to-1 ratio, p53 ubiquitination decreases, thus increasing the steady state levels of p53. ⁷¹  

Mdm4 can also regulate p53, independently of Mdm2. Mdm4 is mainly a cytoplasmic protein, and from 10% to 29% of cytoplasmic Mdm4 has been shown to localize to the mitochondria, where it may facilitate binding between p53 and bcl-2 to control the release of cytochrome c and caspase-mediated apoptosis ⁷² (Fig. 1, Cell Death). 

p53 can escape regulation and degradation caused by Mdm2 and -4 through acetylation. Acetylation destabilizes the interaction between p53 and Mdm2 or -4, allowing p53 to respond to cellular stress. Loss of acetylation at the C terminus of p53 and at lysines 120 and 164 abolishes p21 activation and cell cycle arrest. Acetylation at lysine 120 is also necessary for p53-mediated activation of transcription. ⁷³  

Yy1 Negatively Regulates p53 Protein Levels and Activity

The ubiquitously expressed GLI-Krüppel-type zinc finger protein Yin yang 1 (Yy1) was first identified in 1991. ⁷⁴ It is a highly conserved and multifunctional transcription factor that can act as an activator, repressor, or initiator of transcription, depending on its interactions with a variety of regulatory proteins. ⁷⁵ Localization of Yy1 is cell cycle regulated. Although primarily cytoplasmic, Yy1 enters the nucleus during the early and mid-S phase. ⁷⁶  

Three main mechanisms have been identified for Yy1 transcriptional repression ⁷⁵ : Yy1 can competitively bind to promoter elements where transcriptional activators typically bind ⁷⁵ ; it can bind to the transcription initiation region of the promoter ⁷⁴ ; and it can interact with co-repressors such as the RPD3 histone deacetylase. ⁷⁷ In the case of p53, transcriptional activity is repressed by Yy1 through disruption of p53 interaction with p300, ⁷⁸ a transcription factor and acetyltransferase that regulates p53-dependent apoptosis in response to DNA damage. ^79,80  

Under normal cellular conditions, p300 forms a complex with Mdm2 and p53 that helps to mediate the homeostatic turnover of p53. ⁸¹ However, after UV-induced DNA damage, p300 acetylates lysines in the C terminus of nuclear p53 to stabilize p53 levels, a requirement for apoptosis. ⁷⁹ Researchers found that Yy1 and p53 interact with overlapping p300 domains, allowing Yy1 to competitively bind to p300 and repress p53 levels and activity. ⁷⁸ Yy1 can also suppress levels of p53 by regulating its interaction with Mdm2 ⁷⁸ and Hdm2 (the human ortholog of Mdm2), ⁸² to promote Mdm2-mediated ubiquitination and degradation of p53. ⁷⁸  

Relationship between p53, the Retinoblastoma Protein, and E2F

Retinoblastoma (Rb) is a childhood cancer in which a malignant tumor develops in retinal progenitor cells. Patients with Rb have mutations in both copies of the RB1 tumor suppressor gene through somatic loss of heterozygosity, leading to the growth of death-resistant tumor cells. ^83,84  

The protein product of the RB1 gene, pRb, along with its family members p107 and p130, belongs to the pocket protein family. Like the p53 family, members of the pocket protein family act as tumor suppressors, in part by inhibiting cell cycle progression through interaction with the E2F family of transcription factors. ⁸⁵ The phosphorylation state of pRb and its related family members ultimately decides whether a progenitor cell will continue to divide or instead will differentiate and become postmitotic. ⁸⁶  

The E2F family of proteins includes both the transcriptional activators E2F1 and E2F3 and the transcriptional repressors E2F4 and E2F5. E2F proteins play important roles in the regulation of the cell cycle. In plants, the E2F/retinoblastoma-related pathway has been linked to stem cell maintenance and the regulation of differentiation and gametogenesis. ^87,88 In humans, E2F family members can transactivate a variety of downstream effectors or repress transcription by recruiting chromatin modifiers and remodeling factors. ⁸⁹ Expression of E2F1 leads quiescent cells to transition from the G₁ to the S phase. ⁹⁰  

Wild-type TP53 is expressed in Rb tumor cells, leading researchers to assume initially that Rb develops despite the presence of nonmutated TP53 alleles, possibly in cells that are inherently resistant to p53-mediated cell death. ⁸⁴ However, in vitro experiments have indicated that RB1-deficient retinoblasts undergo p53-mediated apoptosis. This apparent contradiction was resolved by more recent studies showing that during retinal development, the loss of RB1 induces overexpression of Mdm4 along with consequent increased degradation of p53 ^84,91 and tumor promotion. In addition, Mdm4 has been shown to interact with proteins such as E2F, Numb, and p21 to promote Rb tumor progression. ^84,91  

These findings support the view that the p53 pathway may be inactivated in Rb as a result of increased p53 degradation, suggesting that these tumors do not develop from cells that are inherently resistant to death. It is, therefore, clear that even in tumors that are initiated by mutations in other genes such as RB1, p53 and other members of its pathway play a critical role in tumor development. 

p53 in Development

The p53 Family in General Development

Cell cycle regulation and apoptosis are critical features of normal embryogenesis and development, and the role of p53 in some of these processes has been explored. Zebrafish embryos injected with P53 antisense morpholinos develop normally, ⁹² but show decreased apoptosis in response to DNA damage from both UV radiation and camptothecin, a topoisomerase inhibitor. 

In addition, transgenic zebrafish that express P53 with the missense mutation M214K in the DNA-binding domain are unable to activate transcription of p21. ⁹³ Although they develop normally in comparison to wild-type littermates, the M214K fish exhibit decreased fertility and decreased apoptotic responses to irradiation, which is comparable to that of p53 morphants. ⁹³  

Similarly, most P53 knockout mice exhibit normal morphologic and histologic development. ¹¹ However, some homozygous null embryos, especially females, show exencephaly and neural tube closure defects. ^94,95  

These results suggest that, in general, p53 is not necessary for normal embryonic development in zebrafish and mice. However, the p53 family members p63 and p73 and their isoforms play integral roles in normal development. ^{96 –101}  

p63 is essential for ectoderm-derived tissues to develop normally. ^96,97 Knockout mice that do not express the amino terminal transactivation domain isoform (Tap63) or the amino terminally truncated isoform (ΔNp63) do not develop limbs or stratified squamous epithelia. ^96,97 Zebrafish embryos injected with ΔNp63 morpholinos have skin lesions and do not develop pectoral fins. ^98,99  

During mouse development, P73 is expressed in the nasal epithelium, hippocampus, and hypothalamus. ¹⁰⁰ When all isoforms of P73 are deficient, embryos experience hydrocephalus, chronic inflammation, and defects in neuronal development and pheromone sensing. ¹⁰⁰ Because the P73 isoform Tap73α is expressed in the olfactory system, telencephalon, pharyngeal endoderm, pronephric ducts, and slow muscle cells in developing zebrafish, ^{100, 101} injection of P73 morpholinos leads to developmental defects in the olfactory system, telencephalon, and craniofacial cartilage. ¹⁰¹  

p53 in Neuronal Development

Evidence of the involvement of p53 in the normal differentiation and apoptosis of neurons that are destined to become postmitotic was first reported in primary cultures of rat oligodendrocytes ¹⁰² and in the neuronal PC12 pheochromocytoma cell line. ¹⁰³ It has been shown that these cells constitutively express p53, which changes its subcellular localization at a critical point in the maturation of these cells in vitro. During differentiation, p53 is mainly nuclear, but in mature differentiated neuronal cells, it is localized to the cytoplasm, which correlates with changes in levels of immunoprecipitated p53. Cells infected with p53 DD, a recombinant retrovirus encoding a C-terminal p53 miniprotein that acts as a dominant negative inhibitor of endogenous wild-type p53 activity, showed inhibited differentiation of oligodendrocytes and PC12 cells and protected neurons from spontaneous apoptotic death. These findings indicate that p53, on receiving the appropriate signals, is recruited into the nucleus, where it plays a regulatory role in directing primary neurons, oligodendrocytes, and PC12 cells toward either differentiation or apoptosis. ¹⁰³ This suggests that p53 plays an important role in neural development. 

The p53 Family in Ocular Development

The role of p53 in murine ocular development has been shown to vary with the genetic background of the mice being studied. p53-null mice on the C57BL x CBA background ¹¹ and the 129/Sv x C57BL/6 background ¹⁰⁴ have no reported ocular developmental defects and otherwise develop normally, except for a small percentage of embryos that develop exencephaly. ⁹⁵  

However, p53-null mice on the BALB/c OlaHsd background and the pure C57BL/6 background exhibit some ocular developmental abnormalities. Specifically, p53-null mice on the BALB/c OlaHsd background exhibit hyaloid vasculature that persists past the age at which these vessels undergo apoptosis in wild-type mice and have an increased frequency of cataract formation. ¹⁰⁵ Similarly, p53-null mice on the C57BL/6 background also exhibit abnormal hyaloid vasculature in addition to fibrous retrolental tissue, vitreal opacities, retinal folding, and hypoplastic optic nerves. ¹⁰⁶ This nerve fiber loss suggests that p53 affects retinal ganglion cells (RGCs); however, no further studies assessing this question have been undertaken. The researchers hypothesized that because the vitreal vascularization, proliferation of fibrous tissue, and optic nerve hypoplasia were observed in p53-null mice on the C57BL/6 background and not in those on the 129/Sv background, 129/Sv mice may have alleles that are protective and compensate for P53 loss. ¹⁰⁶  

To better understand the role of p53 and its regulators, Mdm2, Mdm4, and Yy1, and its family members, p63 and p73, in retinal development, their mRNA and protein expression levels were examined in the retinas of an in-house inbred line of mice ¹⁰⁷ at various developmental time points. 

qRT-PCR was used to measure Trp53 transcripts in murine retinal samples between embryonic day (E)15 and postnatal day (P)30. Trp53 transcripts were detected at E15 but were markedly higher by E17 followed by a gradual decrease until P15, when they reached the lowest levels (Vuong L et al., manuscript submitted). Similarly, by E18 there was an abundant amount of P53 protein detected in the developing retina (Vuong L et al., manuscript submitted). However, by P3, ocular P53 protein levels were barely detectable on immunoblots. This decrease in P53 levels coincides with the time that retinal cells exit the cell cycle, differentiate, and become postmitotic and also with the end of developmental apoptotic cell death. ¹⁰⁸  

In contrast to Trp53 transcript levels, which peaked at E17 and E18, Mdm2 transcript levels were at their highest between P1 and P7. Of note, retinal Mdm2 protein levels remained constant at all time points tested and did not reflect the fluctuation observed on the transcript level (Vuong L et al., manuscript submitted). This may suggest that retina cells maintain a reservoir of Mdm2 message for specific biological functions or that there is an increased rate of Mdm2 protein degradation at certain developmental time points. 

The expression profile of Mdm4 is different from that of both p53 and Mdm2. Mdm4 transcript levels peak at E15 and are reduced by P1 to the relatively low levels seen in adult animals, whereas protein levels remain constant at all retinal developmental stages tested (Vuong L et al., manuscript submitted). 

Immunohistologic analysis of P53 and Mdm2 in developing retinas was consistent with the Western blot analysis. Both P53 and Mdm2 are abundantly expressed at E18 with P53 localized to the nucleus and Mdm2 present in the cytoplasm. During early postnatal development, expression of both P53 and Mdm2 is dramatically reduced (Vuong L et al., manuscript submitted), probably owing to retinal cells exiting the cell cycle and differentiating. ¹⁰⁹ Finally, during late postnatal retinal development, P53 is almost undetectable in any retinal layer, whereas Mdm2 is observed in both the outer plexiform layer and photoreceptor inner segments (Vuong L et al., manuscript submitted). 

Because P53 levels are low during early postnatal retinal development, other members of the p53 family may play a critical role in the developmental apoptosis that occurs in the murine retina during the first three weeks of life. ¹¹⁰ The Trp63 transcript is expressed at relatively high levels at E15 and rapidly drops by E17, and P63 protein levels follow message levels. Although levels of Trp73 message fluctuate with the highest levels detected at early postnatal stages, P73 protein levels are maintained at very low levels throughout retinal development (Vuong L et al., manuscript submitted), suggesting that it has no role in the development or maintenance of the retina. 

Pattern of p53 Expression in the Eye

Transgenic mice expressing a bacterial chloramphenicol acetyl transferase (CAT) reporter gene under the control of the human p53 promoter were established, and an initial survey of tissue distribution of p53 was conducted. ¹¹¹ In adult mice aged 4 to 6 months, p53 promoter activity was observed predominantly in the testes, cerebellum, and whole eye. Within the eye, activity in the cornea accounted for more than 70% of total p53 promoter activity. The p53 promoter activity in the cornea was 4.1 times higher than in the lens and eight times higher than in the retina and sclera. Immunohistochemical analysis showed p53 promoter-driven CAT expression in the photoreceptor layer, but the strongest expression was in the corneal epithelium. ¹¹¹  

In another study, ¹¹² expression of Trp53 mRNA in ocular tissues of adult Sprague-Dawley rats was mapped to the smooth posterior surface of the iris, simple epithelial cells covering the ciliary processes, and the retina, specifically the ganglion cell layer, inner nuclear layer, and external limiting membrane. The same cells also expressed mRNA for bcl-2, suggesting that p53 and bcl-2 are involved in normal ocular cell survival and death. 

p53 and Retinal Stressors

p53 in Retinal Response to Light Exposure

Intense light can damage the retina ¹¹³ and cause photoreceptor ¹¹⁴ and retinal pigment epithelial (RPE) cell ¹¹⁵ apoptosis, an outcome characteristic of many retinal dystrophies. Although it is logical to expect the involvement of p53 in this process, the role of p53 in light-induced retinal apoptosis may be mixed. ^{115 –117}  

Both wild-type and p53-null mice bred from heterozygous matings on a C57BL/6 background have normal retinal morphology and demonstrate normal electroretinogram (ERG) responses before prolonged exposure to bright light. After 2 hours of exposure of wild-type and p53-null mice to light at 15,000 lux followed by 12 hours in darkness, the ERG responses of the mice were significantly reduced compared with the responses of wild-type and p53 null controls that were not exposed to bright light. However, there was no significant difference between the ERG responses of the light-exposed wild-type and light-exposed p53-null mice. ¹¹⁷  

Structural and morphologic indicators of retinal damage were also similar between light-exposed wild-type and light-exposed p53-null mice. Histologic examination after 2 hours of light exposure at 8500 lux followed by 12 hours in darkness showed condensed photoreceptor nuclei in the outer nuclear layer (ONL) and deteriorated rod outer segments. In mice left to recover for 36 hours, internucleosomal fragmentation of retinal DNA was observed. However, in all cases, damage was equivalent in wild-type and p53-null mice. ¹¹⁶ These results suggest that light-induced apoptosis of photoreceptors is independent of p53. 

In contrast, evidence suggests that p53 may be involved in the response of the RPE to bright light. The A2E fluorophore is a major component of RPE lipofuscin and has been shown to mediate damage to RPE cells caused by high-energy visible (HEV) light. When the human RPE cell line ARPE-19 was incubated with A2E and then exposed to HEV light, p53 was upregulated. When cells were transfected with siRNA specific to TP53 before exposure to HEV light, the amount of apoptosis was reduced. ¹¹⁵  

These results suggest that although light-induced photoreceptor cell death is p53-independent, p53 may play a role in the response of RPE cells to light. Of interest, age-related macular degeneration (AMD), a leading cause of legal blindness in the elderly, ¹¹⁸ is often accompanied by lipofuscin accumulation in the RPE and by RPE cell death. ^115,119,120 Given the role that p53 plays in lipofuscin-associated cell death in vitro and that inhibition of Mdm2, one of the main negative regulators of p53, has been shown to sensitize human RPE cells to apoptosis, ¹²¹ it is reasonable to hypothesize that RPE cell death in AMD involves the p53 pathway. 

Retinal Cell Death Due to Ionizing Radiation Is p53 Dependent

In response to DNA damage by IR, ^122,123 ataxia telangiectasia mutated kinase (ATMK) phosphorylates downstream effectors, including p53, and induces cell cycle arrest or apoptosis. ¹²³ After ATMK-mediated phosphorylation at serine 18, p53 activates expression of the growth arrest and DNA damage (gadd45) genes that mediate G₁ arrest ¹²³ and induce apoptosis. ¹²²  

Postmitotic cells and non-S-phase proliferating cells in the neonatal retina died 4 to 6 hours after exposure to 2 Gy of IR, and the death of proliferating cells followed at 24 hours after irradiation. ¹²⁴ In mice carrying one Trp53 allele, there was a significant reduction in IR-induced apoptosis in the retina, and retinal IR-induced apoptosis was abolished in p53-null mice. Induction of p21 in the retina was also observed in response to IR, but the knockout mouse for the p21 gene Cdkn1a showed no changes in IR-induced apoptosis, implying that p53-regulated apoptosis is independent of p21. ¹²² Further experiments using mature mice may help to further clarify the role of p53 in the overall retinal response to IR. 

p53 Regulates Response to Oxidative Stress in Retinal Cells

The retina is particularly sensitive to oxidative stress because of its oxygen and lipid-rich environment. ^{125 –127} Damage owing to oxidative stress is thought to contribute to AMD, cataracts, primary open-angle glaucoma, and other eye diseases. ^{128 –130}  

Oxidative stress can be induced in vitro by exposure to CI-1010 ((R)-α-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide), a bioreductive nitroimidazole that induces selective, irreversible photoreceptor apoptosis. ^131,132 After 24 hours of CI-1010 exposure, cone photoreceptor–derived 661W cells begin to undergo apoptosis, and after 48 hours of treatment, the cells enter its final stages. ¹³³ To further understand this process, researchers studied the cellular signaling that precedes cell death in the stressed 661W cells. After 12 hours of CI-1010 exposure, caspase-3 activity increased by approximately 12-fold, and pretreatment with a broad or specific caspase inhibitor led to a ∼50% protection from apoptosis. 

Total p53 levels were increased at 12 hours after exposure but returned to baseline 24 hours after exposure. In contrast, levels of activated p53 phosphorylated at serines 6 and 15 remained elevated from 12 to 24 hours after exposure, and expression of p53 phosphorylated at serine 20 was elevated at 18 and 24 hours after exposure. Flow cytometry showed that 19.21% ± 0.16% of the cells were arrested in G₂ after 48 hours of exposure to CI-1010. These results led the authors to conclude that CI-1010 exposure causes irreparable DNA damage to 661W cells, leading to activation of p53-mediated apoptosis. ¹³³  

p53 is also thought to play a role in oxidative-stress–mediated cell death in RGCs. When levels of p53 were downregulated in the ganglion cell line RGC-5 by transfection with an anti-p53 siRNA, there was a 70% decrease in catalase, an enzyme that breaks down hydrogen peroxide into oxygen and water. Viability assays showed that the p53-deficient RGC-5 cells were 15% more susceptible to oxidative-signaling–induced apoptosis than RGC-5 cells with normal p53 expression. ¹³⁴ This observation suggests that, although p53 mediates apoptosis in 661W cells in response to oxidative stress, p53 may prevent oxidative-stress–induced apoptosis in RGC-5 cells. 

p53 may also regulate apoptosis in response to retinal ischemia. ^135,136 Hypoxic conditions may arise in the retina from ischemia, ¹³⁷ which involves a lack of blood flow to the eye. ¹³⁸ Both p53 gene ¹³⁵ and protein ¹³⁶ expression have been shown to be upregulated in response to retinal ischemia, implying that the apoptosis that occurs in response to ischemia is p53 mediated. 

Cell Death in Retinitis Pigmentosa Is p53 Independent

Retinitis pigmentosa (RP) is a genetically heterogeneous group of eye conditions that exhibits similar clinical features. ¹³⁹ Patients with RP typically experience night blindness followed by loss of peripheral vision that eventually results in tunnel vision. ^140,141 With a few exceptions, apoptosis of photoreceptors and RPE cells is a hallmark of RP and leads to thinning of the ONL and development of lesions and pigment deposits in the fundus. ¹⁴¹ Because of the overall role of p53 in apoptosis, it is reasonable to hypothesize that p53 is involved in the retinal cell death associated with RP. However, results identifying the role of p53 in RP have been mixed. 

One commonly used model is the retinal dystrophic (rd) mutant mouse, which has a recessive null mutation in the β-subunit of the rod photoreceptor cGMP phosphodiesterase and is regarded as a good model of early-onset RP. ¹⁴² At P12, retinas from wild-type, rd, and p53 null rd mice showed similar ONL morphology with only a slight increase in TUNEL labeling in the rd mutant retinas. However, almost all rods were lost in the rd mutant retinas by P16, regardless of p53 expression. The authors observed a slight decrease in TUNEL labeling in the central retina of p53-null rd mice at P14 and a slight increase in TUNEL labeling in the peripheral retina at P16 compared with that in rd mice. This minor difference led researchers to conclude that p53-null rd mice may show a slight delay in apoptosis but that the retinal degeneration in the rd model is largely p53-independent. ¹⁴³  

These findings were independently confirmed by another group. TUNEL staining of retinal sections from rd mice with normal p53 expression showed rod photoreceptor and inner nuclear layer apoptosis kinetics similar to that in sections from rd mice that were p53 null. Furthermore, cone photoreceptor survival, measured by the total number of peanut agglutinin–positive cells in each eye, was also unaffected by the absence of p53. ¹⁴⁴  

The disease process was similarly p53-independent in the N-methyl-N-nitrosourea (MNU)-induced murine model of retinal degeneration. MNU is an alkylating agent that causes photoreceptor apoptosis in mice. When eyes from age-matched p53 wild-type, heterozygous, and null mice injected with MNU were examined histologically and morphologically, no differences in photoreceptor cell loss were seen between genotypes in either the central or peripheral retina. ¹⁴⁵  

However, lack of p53 expression has been shown to delay, but not prevent, cell death in the rds mouse. These mice have a null mutation in the peripherin/rds gene, which encodes a protein essential for the structural stability of photoreceptor outer segments. In this model, photoreceptor apoptosis is observed starting at P14, with 50% thinning of the ONL by P60. At P58, p53-null rds mice had the same number of photoreceptor nuclei as the rds mice with wild-type p53 expression had. When TUNEL staining was performed at earlier time points, the researchers found that the peak of photoreceptor loss in p53-null rds mice was at P19, in contrast to rds mice with P53 who experienced peak apoptosis at P16. This delay in degenerative apoptosis in the absence of p53 increased ONL thickness in p53-null rds mice compared with rds mice from P16 through P26 ¹⁴⁶ and suggests that in some degenerative RP models, the p53 pathway is involved in apoptosis. 

Perspectives

Research on p53 has focused mainly on its role in cancer. To understand the role of p53 in normal retinal function, it is important to further study its role in retinal development and in retinal responses to extracellular stressors and disease. 

There is no clearly defined single role for p53 in the stressed or diseased retina (Table 1). p53 has been shown to be dispensable for apoptosis in response to light-induced damage, as there is no difference in morphology or function between wild-type and p53-null mice exposed to intense light. In addition, MNU-exposed and p53-null rd mice show no differences in retinal morphology compared with wild-type p53 controls, implying that photoreceptor cell death in RP is p53-independent. 

Table 1.

 

Identified Functions of p53 in the Retina

Condition in the Retina	Involvement of p53
Age-related macular degeneration	p53 is upregulated in RPE cells in response to high-energy light exposure that leads to cellular apoptosis. AMD is generally believed to start with RPE cell death. 115,119,120
Development	p53 is generally not required in development of zebrafish and mice. 11,92,104
Light-induced damage	Photoreceptor apoptosis in response to intense light exposure is p53-independent. 116,117
Irradiation	Phosphorylation of p53 is required for apoptosis in neonatal retinas after exposure to up to 2 Gy of radiation. 122
Oxidative stress	p53 has been shown to regulate the response of cone-photoreceptor-derived cells exposed to CI-1010, a bioreductive nitroimidazole, 131,132 and rat RGCs to hydrogen peroxide exposure. 134
Retinitis pigmentosa	The rd mouse model of early-onset RP 143,144 and mice with MNU-induced photoreceptor cell apoptosis 145 show no differences in retinal structure between mice expressing P53 and those that are p53 null. However, rds mice that are p53 null experience a delay in photoreceptor cell apoptosis. 146
Retinoblastoma	p53 typically remains wild-type in this type of cancer. However, Mdm4 is overexpressed in response to loss of RB1, leading to the degradation of p53. 84,91

However, p53 plays many roles in maintaining cellular/tissue homeostasis, which is evident from the extensive posttranslational modifications that it undergoes. ¹⁷ These roles seem to hold true for p53 in the retina as well. For example, evidence suggests that p53 may be differentially involved in retinal apoptosis in specific cell types. One such example is that of the mild protective role of p53 in RGC-5 cells compared with 661W cells under oxidative stress. Another example is the delayed apoptosis in the rds retina in absence of P53. This delay may reflect a protective effect for p53, further supporting a direct role for p53 in photoreceptor apoptosis. However, in the absence of any potential interactions between p53 and the RDS protein, the delay may result from the utilization of an alternative death pathway. Besides a potential cell-type–specific role, the role of p53 in the retina may also be stressor specific. p53 has been linked to retinal responses to irradiation, oxidative stress, and Rb. 

During retinal development, levels of Trp53 are highest at E17 and E18 and drop to very low levels afterward. Therefore, it is not clear what role p53 plays in retinal development past E18, at the peak of differentiation of retinal cells. ¹⁰⁹ Because the peak of rod photoreceptor birth is immediately before birth, ¹⁴⁷ it is possible that p53 plays a role in rod photoreceptor differentiation and/or apoptosis. However, rod photoreceptor differentiation and apoptosis proceed unabated in the p53-null mouse, suggesting potential functional redundancy. 

Although p73 is necessary for embryonic neuronal development, the levels of P73 remain very low throughout murine retinal development, suggesting that it plays little or no role in the process. On the other hand, P63 protein levels are elevated, at times coinciding with developmental retinal apoptosis. However, significant further work is needed to prescribe a specific role for p63 in these developmental processes. 

More intriguing is the pattern of expression of the p53 regulators Mdm2, Mdm4, and Yy1. Although P53 protein levels are drastically reduced after E18, protein levels of Mdm2, the major regulator of p53, remain constant throughout retinal development. The same is true of Mdm4 and Yy1, which are constantly expressed at low protein levels from E15 through P30. 

In conclusion, further research is needed to fully elucidate the functions and mechanisms of p53 in the retina. It is critical to determine whether p63 and p73 play any role in retinal function in the absence of p53. 

Footnotes

 Disclosure: L. Vuong, None; S.M. Conley, None; M.R. Al-Ubaidi, None