Weike Ji

αA- and αB-Crystallins Interact with Caspase-3 and Bax to Guard Mouse Lens Development

The small heat shock protein, α-crystallin, exists in two isoforms, αA and αB, and displays strong ability against stress-induced apoptosis. Regarding their functional mechanisms, we and others have demonstrated that they are able to regulate members in both caspase and Bcl-2 families. In addition, we have also shown that αA and αB may display differential anti-apoptotic mechanisms under certain stress conditions. While αA-crystallin regulates activation of the AKT signaling pathway, αB negatively regulates the MAPK pathway to suppress apoptosis induced by UV and oxidative stress. Although previous studies revealed that αA and αB could regulate members in both caspase and Bcl-2 families, the molecular mechanism, especially the in vivo regulation still waits to be elucidated. In the present communication, we present both in vitro and in vivo evidence to further demonstrate the regulation of caspase-3 and Bax by αA and αB. First, Surface Plasmon Resonance (SPR) and yeast two-hybrid selection analysis demonstrate that αA and αB directly bind to caspase-3 and Bax with differential affinities. Second, immunohistochemistry reveals that αA and αB regulate caspase-3 and Bax at different developmental stages of mouse embryo. Third, coimmunoprecipitation shows that αA and αB form in vivo interacting complexes with caspase-3 and Bax. Together, our results further confirm that αA and αB regulate caspase-3 and Bax in vitro and in vivo to regulate lens differentiation.

Apoptosis: its functions and control in the ocular lens.

The ocular lens is a non-vascular and non-innervated transparent organ that plays an important role in vision processing. This unique organ is derived from the embryonic ectoderm of the brain region through a complicated differentiation process in which apoptosis plays a key role. First, when the committed ectoderm becomes thickened and invaginated, the defined number of cells required to form the lens vesicle is partially determined by apoptosis. Second, separation of lens vesicle from the above corneal ectoderm is executed through apoptosis of the lens stalk cells. Finally, differentiation of the lens epithelial cells is controlled by the regulators, most of which are involved in control of apoptosis at multiple signaling steps. The lens is also characterized by continuous growth and differentiation in the adulthood. Through the different stages of growth and differentiation in the adult lens, various stress conditions can induce apoptosis of the lens epithelial cells, leading to eventual non-congenital cataractogenesis. The present review summarizes the current knowledge on the functions and regulators of apoptosis in the ocular lens.

Alpha-Crystallins and Tumorigenesis

αA- and αB-crystallins, the major lens structure proteins and members of the small heat-shock proteins (sHSPs) family, play essential roles in maintaining normal cellular structure and physiology of both ocular and some non-ocular tissues. Mutations and abnormal expression of these sHSPs are associated with various human diseases such as cataract, neural disorders, and cardiovascular diseases. In addition, recent studies have revealed that the abnormal expressions and functions of both α-crystallins are associated with several types of tumors. In this regard, αA- and αB-crystallins seem to function differentially or even oppositely during tumorigenesis, and diverse molecular mechanisms have been proposed to explain their roles in cell apoptosis, cell proliferation and tumor metastasis. In this review, we have summarized the current status regarding the expression patterns and functions of αA- and αB-crystallins implicated in tumorigenesis, and discussed the possible mechanisms underlying their functions.

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.

p53 Directly Regulates αA- and βA3/A1-Crystallin Genes to Modulate Lens Differentiation

It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating 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 c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the αA and βA3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.

The p53-Bak apoptotic signaling axis plays an essential role in regulating differentiation of the ocular lens.

The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells (Qin et al. 2008. Cancer Research. 68(11):4150) and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Here, we demonstrate that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. First, during mouse lens development, p53 is expressed and differentially phosphorylated at different residues. Associated with p53 expression, Bak is also significantly expressed during mouse lens development. Second, human p53 directly regulates Bak promoter and Bak expression in p53 knockout mice (p53-/-) was significantly downregulated. Third, during in vitro bFGF-induced lens cell differentiation, knockdown of p53 or Bak leads to significant inhibition of lens cell differentiation. Fourth, besides the major distribution of Bak in cytoplasm, it is also localized in the nucleus in normal lens or bFGF-induced differentiating lens cells. Finally, p53 and Bak are co-localized in both cytoplasm and nucleus, and their interaction regulates the stability of p53. Together, these results demonstrate for the first time that the p53-Bak apoptotic signaling axis plays an essential role in regulating 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-lβ 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-Aβ gene is regulated by multiple transcriptional factors including Ets-1, SP1/SP3, and RXRα/β

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/β, 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-Aβ 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-Aβ gene. Our results demonstrate that three major cis-elements, including the binding sites for Ets-1, SP1/SP3, and RXRα/β, are present in the proximal promoter of the mouse PP2A-Aβ gene. Gel mobility shifting assays reveal that Ets-1, SP1/SP3, and RXRα/β all bind to PP2A-Aβ gene promoter. In vitro mutagenesis and reporter gene activity assays demonstrate that while Ets-1 displays negative regulation, SP1/SP3 and RXRα/β positively regulate the promoter of the PP2A-Aβ gene. Co-expression of the cDNAs encoding Ets-1, SP1/SP3, or RXRα/β and the luciferase reporter gene driven by PP2A-Aβ promoter further confirm their control over the PP2A-Aβ promoter. Finally, ChIP assays demonstrate that Ets-1, SP1/SP3, and RXRα/β can all bind to the PP2A-Aβ gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aβ gene. Moreover, our results provide important information explaining why PP2A-Aα and PP2A-Aβ display distinct expression levels.

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 βA3/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.

GEFT, A Rho Family Guanine Nucleotide Exchange Factor, Regulates Lens Differentiation through a Rac1-Mediated Mechanism

The Rho-family of small GTPase specific guanine nucleotide exchange factor, GEFT, is expressed at high levels in adult human excitable tissues including the brain, heart, and skeletal muscle. Previously, we demonstrated that GEFT is specifically expressed in the adult mouse hippocampus and cerebellum, and that overexpression of this protein can result in neurite and dendrite remodeling. This finding prompted us to explore the expression of GEFT in other tissues, which share common developmental ancestry to the nervous system, specifically the ocular system. Using immunohistochemical analysis specific for GEFT protein expression, we observed the highest ocular expression of GEFT occurring in the neuroblastic layer and differentiating lens fibers of the late-stage mouse embryo, and in the postnatal corneal epithelium, lens epithelium, and throughout the retina. Exogenous expression of GEFT in N/N1003A rabbit lens epithelial cells induced lens fiber differentiation as reflected by cell elongation and lentoid formation, as well as a strong increase in β-crystallin and filensin expression. Moreover, transfection of lens epithelial cells with GEFT resulted in a Rac-1 mediated up-regulation of αA-, αB-, βB-, γC-, or γF-crystallin promoter activities that is in part dependent on the nuclear localization of Rac1. Furthermore, pharmacological inhibition of Rac1 blocked GEFT-induced N/N1003A lens fiber differentiation and βB-crystallin expression in ex vivo mouse lens explants. These results demonstrate for the first time a role for GEFT in lens cell differentiation and mouse eye development. Moreover, GEFT regulation of lens differentiation and eye development occurs through a Rac1-dependent mechanism.