Wenfeng Hu

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.

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 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.

Protein serine/threonine Phosphotase-2A is differentially expressed and regulates eye development in vertebrates

Protein serine/threonine phosphatase-2A (PP-2A) is one of the key enzymes responsible for dephosphorylation in vertebrates. PP-2A-mediated dephosphorylation participates in many different biological processes including cell proliferation, differentiation, transformation, apoptosis, autophage and senescence. However, whether PP-2A directly controls animal development remains to be explored. Here, we present direct evidence to show that PP-2A displays important functions in regulating eye development of vertebrates. Using goldfish as a model system, we have demonstrated the following novel information. First, inhibition of PP-2A activity leads to significant death of the treated embryos, which is derived from blastomere apoptosis associated with enhanced phosphorylation of Bcl-XL at Ser-62, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of PP-2A with morpholino oligomers leads to significant death of the injected embryos. The survived embryos from PP-2A knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each catalytic subunit of PP-2A also causes death of majority of the injected embryos and leads to absence of goldfish eye lens or severely disturbed differentiation. Together, our results provide direct evidence that protein phosphatase-2A is important for normal eye development in goldfish.

Contrast functions of αA- and αB-crystallins in cancer development

α-Crystallins, initially identified as the structural proteins of the ocular lens, belong to the small heat shock protein family. They play significant roles in maintaining the lens transparency and preventing protein aggregation. α-Crystallins exist in two isoforms: αA and αB, and they display differential tissue distribution. Their mutations are implicated in several human diseases including cardiac myopathies, neurodegenerative diseases, cataracts and various types of cancers. Increased αB expression was detected in retinoblastoma, breast cancer, glioblastoma, prostate and renal cell carcinomas, indicating its role in promoting tumor growth. A complex picture emerges for αA. Although earlier studies suggest that αA may promote cancer development, recent studies from our laboratory demonstrate that αA can act as a tumor suppressor inhibiting cell transformation and retarding cell migration through modulating MAP kinase activity. In this review, we summarize the recent progress about the functions of αA and αB in cancer development.

The small heat shock protein αA-crystallin negatively regulates pancreatic tumorigenesis.

Our recent study has shown that αA-crystallin appears to act as a tumor suppressor in pancreas. Here, we analyzed expression patterns of αA-crystallin in the pancreatic tumor tissue and the neighbor normal tissue from 74 pancreatic cancer patients and also pancreatic cancer cell lines. Immunocytochemistry revealed that αA-crystallin was highly expressed in the normal tissue from 56 patients, but barely detectable in the pancreatic tumor tissue. Moreover, a low level of αA-crystallin predicts poor prognosis for patients with pancreatic duct adenocarcinoma (PDAC). In the 12 pancreatic cell lines analyzed, except for Capan-1 and Miapaca-2 where the level of αA-crystallin was about 80% and 65% of that in the control cell line, HPNE, the remaining pancreatic cancer cells have much lower αA-crystallin levels. Overexpression of αA-crystallin in MiaPaca-1 cells lacking endogenous αA-crystallin significantly decreased its tumorigenicity ability as shown in the colony formation and wound healing assays. In contrast, knockdown of αA-crystallin in the Capan-1 cells significantly increased its tumorigenicity ability as demonstrated in the above assays. Together, our results further demonstrate that αA-crystallin negatively regulates pancreatic tumorigenesis and appears to be a prognosis biomarker for PDAC.

p53 Regulates Developmental Apoptosis and Gene Expression to Modulate Lens Differentiation

The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle progress and cell differentiation. It mainly acts as a transcription factor. In addition, it can also directly interact with apoptosis regulators in mitochondria to control apoptosis. Recent studies from our laboratory and others have shown that p53 plays an active role in regulating lens differentiation. It does so by modulating developmental apoptosis and also controlling expression of lens differentiation-specific genes. In this chapter, we summary the current progresses in this field.