The Role of αA-Crystallin in Experimental Autoimmune Uveitis.

Uveitis refers to a group of ocular inflammatory diseases that can lead to blindness. For years, researchers have been trying to decipher the underlying mechanisms and develop therapeutic strategies using the model of experimental autoimmune uveitis (EAU). Recently, αA-crystallin has been found to be upregulated in EAU and can even ameliorate its severity through different mechanisms, suggesting its use as a potent therapeutic factor against uveitis. Here we review the protective role of αA-crystallin and discuss its functional mechanisms in EAU.

The tumor suppressor, p53 regulates the γA-crystallin gene during mouse lens development.

The tumor suppressor, p53 regulates a large number of target genes to control cell proliferation and apoptosis. In addition, it is also implicated in the regulation of cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates several genes including c-Maf and Prox1, two important transcription factors for lens differentiation, and αA and ßA3/A1, the lens differentiation markers. In the present study, we present evidence to show that the γA-crystallin gene distal promoter and the first intron also contain p53 binding sites and are capable of mediating p53 control during mouse lens development. First, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from human lens epithelial cells (HLE) directly binds to the p53 binding sites present in the γA-crystallin gene. Second, the exogenous wild type p53 induces the dose-dependent expression of the luciferase reporter gene driven by the basic promoter containing the γA-crystallin gene p53 binding site. In contrast, the exogenous dominant negative mutant p53 causes a dose-dependent inhibition of the same promoter. Third, ChIP assays revealed that p53 binds to the γA-crystallin gene promoter in vivo. Finally, in the p53 knockout mouse lenses, the expression level of the γAcrystallin gene was found attenuated in comparison with that in the wild type mouse lenses. Together, our results reveal that p53 regulates γA-crystallin gene expression during mouse lens development. Thus, p53 directly regulates all 3 types of crystallin genes to control lens differentiation.

Protein serine/threonine phosphotase-1 is essential in governing normal development of vertebrate eye.

Protein serine/threonine phosphatase-1 (PP-1) is one of the key enzymes responsible for dephosphorylation in vertebrates. Protein dephosphorylation via PP-1 is implicated in many different biological processes including gene expression, cell cycle control, transformation, neuronal transmission, apoptosis, autophage and senescence. However, whether PP-1 directly controls animal development remains to be investigated. Here, we present direct evidence to show that PP-1 plays an essential role in regulating eye development of vertebrates. Using goldfish as a model system, we have shown the following novel results. First, inhibition of PP-1 activity leads to death of a majority of the treated embryos, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of each catalytic subunit of PP-1 with morpholino oligomers leads to partial (PP-lα knockdown) or complete (PP-lß or PP-lγ knockdown) death of the injected embryos. The survived embryos from PP-1α knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each subunit of PP-1 also causes death of majority of the injected embryos and leads to abnormal development of goldfish eye. Mechanistically, Pax-6 is one of the major downstream targets mediating the effects of PP-1 function since the eye phenotype in Pax-6 knockdown fish is similar to that derived from overexpression of PP-1. Together, our results for the first time provide direct evidence that protein phosphatase-1 plays a key role in governing normal eye formation during goldfish development.

The PP2A-Aß gene is regulated by multiple transcriptional factors including Ets-1, SP1/SP3, and RXRα/ß.

Protein phosphatase-2A (PP-2A) is a major serine/threonine phosphatase abundantly expressed in eukaryotes. PP-2A is a heterotrimer that contains a 65 kD scaffold A subunit, a 36 kD catalytic C subunit, and a regulatory B subunit of variable isoforms ranging from 54-130 kDs. The scaffold subunits, PP2A-Aα/ß, act as platforms for both the C and B subunits to bind, and thus are key structural components for PP-2A activity. Mutations in both genes encoding PP2A-Aα and PP2A-Aß lead to carcinogenesis and likely other human diseases. Our previous work showed that the gene coding for PP2A-Aα is positively regulated by multiple transcription factors including Ets-1, CREB, and AP-2α but negatively regulated by SP-1/SP-3. In the present study, we have functionally dissected the promoter of the mouse PP2A-Aß gene. Our results demonstrate that three major cis-elements, including the binding sites for Ets-1, SP1/SP3, and RXRα/ß, are present in the proximal promoter of the mouse PP2A-Aß gene. Gel mobility shifting assays reveal that Ets-1, SP1/SP3, and RXRα/ß all bind to PP2A-Aß gene promoter. In vitro mutagenesis and reporter gene activity assays demonstrate that while Ets-1 displays negative regulation, SP1/SP3 and RXRα/ß positively regulate the promoter of the PP2A-Aß gene. Co-expression of the cDNAs encoding Ets-1, SP1/SP3, or RXRα/ß and the luciferase reporter gene driven by PP2A-Aß promoter further confirm their control over the PP2A-Aß promoter. Finally, ChIP assays demonstrate that Ets-1, SP1/SP3, and RXRα/ß can all bind to the PP2A-Aß gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aß gene. Moreover, our results provide important information explaining why PP2A-Aα and PP2A-Aß display distinct expression levels.

The tumor suppressor p53 regulates c-Maf and Prox-1 to control lens differentiation.

The tumor suppressor p53 plays a key role in regulating apoptosis and cell cycle progression. In addition, p53 is implicated in control of cell differentiation in muscle, the circulatory system, ocular lens and various carcinoma tissues. However, the mechanisms by which p53 controls cell differentiation are not fully understood. Here we present evidence that p53 directly regulates c-Maf and Prox1, two important transcription factors controlling differentiation in the ocular lens. First, human and murine c-Maf and Prox1 gene promoters contain authentic p53 DNA binding sites. Second, p53 directly binds to the p53 binding sites found in the promoter regions. Third, exogenous p53 induces dose-dependent expression of the luciferase report gene driven by both c-Maf and Prox1 promoters, and p53 binds to both promoters in the ChIP assays. Fourth, in the in vitro differentiation model, knockdown of p53 significantly inhibits lens differentiation which is associated with downregulated expression of c-Maf and Prox1. Finally, in p53 knockout mice, the expression of c-Maf and Prox1 are significantly altered. Together, our results reveal that p53 regulates lens differentiation through modulation of two important transcription factors, c-Maf and Prox1, and through them p53 thus controls expression of various differentiation-related downstream crystallin genes.

A 5-year follow up of mildly dyskaryotic smears, comparing colposcopy with expectant management.

The purpose of this study was to see if the National Health Service Cervical Screening Programme's guidelines were appropriate when they recommended that mildly dyskaryotic smears were repeated before referral for colposcopy. We identified all those with a mildly dyskaryotic smear, and followed them over 5 years, to compare those that had colposcopy and those that did not. In total, 269 women were followed and 35% of these only had the one mild smear. Of those whose smear returned to normal after the initial mild smear, 84% remained normal over the 5-year period. Of those whose smear did not return to normal, i.e. those that required a colposcopy, 74% returned to normal after the colposcopy.