Inhibition of Sumoylation Alleviates Oxidative Stress-induced Retinal Pigment Epithelial Cell Senescence and Represses Proinflammatory Gene Expression.

PURPOSE: Advanced age is the largest risk factor for age-related macular degeneration (AMD). Sumoylation is a reversible post-translational modification that conjugates small peptide, small ubiquitin-like modifier (SUMO), to a target protein. Dysregulation of sumoylation is recently found to be critically involved in several age-related disorders. However, the effects of sumoylation during retina senescence and aging remains elusive. This study is aimed to investigate the function and regulation of sumoylation pathway in the aging retina and premature senescent retinal pigment epithelial (RPE) cells. METHODS: 1.5- and 10-month C57/B6 mice were used for comparative aging study. Both ARPE primary cultures and ARPE-19 cells were used for assay systems. The qRT-PCR was used for analysis of mRNA expression. Western blot and immunofluorescence were used to analyze the protein expression. Cell flow cytometry was used for cell cycle progression analysis. RPE barrier function and senescent-associated ß-galactosidase (SA ß-gal) activity were analyzed to measure cellular senescence. RESULTS: We show that the expression of SUMO enzymes and global protein sumoylation were downregulated in the aging mouse retina, and in the oxidative stress (OS) -induced premature senescent RPE cells. Dramatical altered distribution of SUMO E1, E2 and E3 enzymes were observed during RPE senescence. Inhibition of sumoylation alleviated OS-induced cell senescence in RPE cells, as indicated by decreased p21 and p53 expression and decreased percentage of cell cycle arrest at G0/G1 phase. Intriguingly, inhibition of SUMO E1 repressed the expression of proinflammatory cytokine and chemokine in the premature senescent RPE cells. However, inhibition of sumoylation did not prevent DNA damage during the OS-induced RPE senescence process. CONCLUSIONS: Our data indicate sumoylation critically regulates retina and RPE aging and that targeting sumoylation process may provide potential therapeutic strategy for AMD treatment.

Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities.

We have previously demonstrated that the serine/threonine protein phosphatase-1 (PP-1) plays an important role in promoting cell survival. However, the molecular mechanisms by which PP-1 promotes survival remain largely unknown. In the present study, we provide evidence to show that PP-1 can directly dephosphorylate a master regulator of apoptosis, p53, to negatively modulate its transcriptional and apoptotic activities, and thus to promote cell survival. As a transcriptional factor, the function of p53 can be greatly regulated by phosphorylation and dephosphorylation. While the kinases responsible for phosphorylation of the 17 serine/threonine sites have been identified, the dephosphorylation of these sites remains largely unknown. In the present study, we demonstrate that PP-1 can dephosphorylate p53 at Ser-15 and Ser-37 through co-immunoprecipitation, in vitro and in vivo dephosphorylation assays, overexpression and silence of the gene encoding the catalytic subunit for PP-1. We further show that mutations mimicking constitutive dephosphorylation or phosphorylation of p53 at these sites attenuate or enhance its transcriptional activity, respectively. As a result of the changed p53 activity, expression of the downstream apoptosis-related genes such as bcl-2 and bax is accordingly altered and the apoptotic events are either largely abrogated or enhanced. Thus, our results demonstrate that PP-1 directly dephosphorylates p53, and dephosphorylation of p53 has as important impact on its functions as phosphorylation does. In addition, our results reveal that one of the molecular mechanisms by which PP-1 promotes cell survival is to dephosphorylate p53, and thus negatively regulate p53-dependent death pathway.

Roles of SUMOylation in Heart Development and Cardiovascular Diseases.

Heart is an extremely important organ, and cardiovascular disorders emerge as primary life-threatening disease in human life. Aberrant post-translational modifications (PTMs) on cardiac proteins are closely correlated with pathological abnormalities in heart. SUMOylation, one of the most prevalent PTMs with thousands of substrates throughout the cell including critical subcellular organelles, has been shown to precisely finetune the cell survival and proliferation during heart development, and delicately control the function of mitochondrion and sarcoplasmic reticulum in physiological heart functioning. The silver lining is pathologically cardiacspecific SUMOylation being considered as target for cardiovascular disease intervention and treatment. Here, we summarize the recent progress in heart-specific functions of the SUMOylation pathway. In particular, we discuss the biological significance of SUMO conjugation/deconjugation during heart development, and in physiological cardiovascular health involving cardiac mitochondrial function, cardiac contractility, stress adaption and protein homeostasis. We also discuss the crosstalks between sumoylation and other post-translational modifications such as acetylation and ubiquitination. These crosstalks not only shed light to our understanding of the regulatory mechanisms on cardiovascular disorders but also contributes to their future therapy.

Sumoylation Pathway as Potential Therapeutic Targets in Cancer.

Sumoylation is a covalent protein posttranslational modification that conjugates the small ubiquitin-like peptide SUMO to substrate. Sumoylation is critically implicated in multiple biological processes, including cell proliferation, differentiation, senescence and apoptosis, etc. Therefore, it is not surprising that dysregulation of sumoylation has been implicated in tumorigenesis and different types of cancer were found to be addicted to functional sumoylation pathway. The potential role for sumoylation as a therapeutic target in caner is emerging. In this review, we summarize current knowledge regarding the involvement of sumoylation in genome stability and DNA damage response. We will further discuss the therapeutic potential of sumoylation as synthetic lethal partner and as a key signaling pathway in cancer stem cells.

Sumoylation in Cellular Senescence and Aging.

Sumoylation is a reversible post-translational modification that conjugates small peptide SUMO (small ubiquitin-like modifier) to a target protein. Global protein sumoylation and expression of components in sumoylation pathway were recently found to be altered in the process of organismal aging. In addition, key factors controlling cellular senescence are known to be sumoylated. This review will summarize current information on the function of sumoylation in cellular senescence and aging.

Effects of Crosstalks Between Sumoylation and Phosphorylation in Normal Cellular Physiology and Human Diseases.

Post-translational modifications (PTMs) such as phosphorylation, acetylation, methylation, ubiquitylation, sumoylation are important mechanisms to regulate functions of different proteins. Among various PTMs, phosphorylation, discovered about 60 years ago, is probably the most common modification. In contrast, sumoylation, identified about two decades ago is emerging as a key regulatory mechanism modulating protein functions. Although studies on protein phosphorylation and sumoylation have been extensively reviewed, much less attention has been paid to their cross-talk and their co-regulation of the same protein target. Here we summarize various examples of the cross-talks between protein phosphorylation and sumoylation, and discuss their functions in regulating normal physiology and pathogenesis.

SUMOylation in Neurological Diseases.

Since the discovery of SUMOs (small ubiquitin-like modifiers) over 20 years ago, sumoylation has recently emerged as an important posttranslational modification involved in almost all aspects of cellular physiology. In neurons, sumoylation dynamically modulates protein function and consequently plays an important role in neuronal maturation, synapse formation and plasticity. Thus, the dysfunction of sumoylation pathway is associated with many different neurological disorders. Hundreds of different proteins implicated in the pathogenesis of neurological disorders are SUMO-modified, indicating the importance of sumoylation involved in the neurological diseases. In this review, we summarize the growing findings on protein sumoylation in neuronal function and dysfunction. It is essential to have a thorough understanding on the mechanism how sumoylation contributes to neurological diseases in developing efficient therapy for these diseases.

Regulation of CREB Functions by Phosphorylation and Sumoylation in Nervous and Visual Systems.

CREB is an ubiquitous transcription factor regulating diverse cellular responses. Its phosphorylation at S133 is an essential event for its activation in both nervous and visual systems. The activated CREB is implicated in the regulation of development, protection, learning, memory and plasticity in the nerve system. Moreover, sumoylation, an important post-translational modification of protein, plays a key role in sustaining CREB activation in the rat hippocampus in order to enhance the long-term memory and other aspects. In the visual system, although the CREB activation by phosphorylation at S133 is similar to that as observed in the nervous system, the role of CREB sumoylation remains to be explored. This review will discuss the aspects of CREB functions and their regulation by phosphorylation and sumoylation in both systems.

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.

Sumoylation in Lens Differentiation and Pathogenesis.

Sumoylation, a post-translational modification discovered over a decade ago, turns out to be a very important regulatory mechanism mediating multiple cellular processes. Recent studies from our laboratory and others also revealed that it plays a crucial role in regulating both differentiation and pathogenesis of the ocular lens. This review will summarize these progresses.

Molecular Cloning and Developmental Expression Patterns of the Striatin Gene Encoding A Member of the Regulatory Subunits for the Protein Serine/Threonine Phosphatase-2A in Fish.

PURPOSE: The protein phosphatase-2A (PP-2A) is one of the most important serine/threonine phosphatases in eukaryotes. The holoenzyme of PP-2A consists of three subunits: a scaffold A subunit, a catalytic C subunit and a regulatory B subunit. While both A and C subunits are coded by two different genes, the B subunits exist in 26 or more isoforms which are encoded by at least 15 different genes. Previous studies have shown that besides regulating specific PP-2A activity, various B subunits may have other functions. To explore the possible roles of the regulatory subunits of PP-2A in vertebrate development, we have cloned the gene encoding goldfish striatin, a member of the B'" family regulatory subunits for PP-2A, and determined their tissue-specific and temporal expression patterns. METHODS: The cDNA cloning was conducted with RT-PCR-based RACE. The mRNA expression levels for the goldfish striatin were analyzed with RT-PCR. The expression levels of the striatin protein from goldfish were determined with Western blot analysis. The semi-quantitation of the mRNA and protein expression levels was conducted with the software of U-scanning. RESULTS: Our study revealed that the full length cDNA for striatin consists of 2965 bp coding for a deduced protein of 769 amino acids, which bears a very high level of amino acid sequence identity with the homolog protein from other species. The striatin mRNA is highly expressed in the kidney, to a less degree in brain, fin, muscle, liver, ovary and gill, and the lowest in testis and heart. Similar pattern of protein expression is detected in the above 9 tissues. During the development of goldfish, the striatin mRNA maintains a relatively high level at the 2-cell, multiple cell and blastula stages. Then, it drops down substantially at gastrula stage and fluctuates around this level in the next 8 different stages. At the protein level, the striatin maintained higher level from 2-cell to gastrula stages, then decreased at neurula and optic vesicle stages, and gradually increased again to peak at eye pigmentation stage, then slightly decreased in the next few stages of development. CONCLUSIONS: Our results suggest that the striatin may play an important role in regulating goldfish development and adult tissue homeostasis. While the former function may or may not occur through PP- 2A functions, the later function appears to occur via PP-2A activity.

SUMOylation Regulation of Retina Development and Functions.

The structure and developmental mechanisms of vertebrate retina are highly conserved. One of the most distinctive events during retinogenesis is the temporally and spatially generation of seven types of retinal cells from the multipotent retinal progenitor cells. The importance and prevalence of SUMOylation in regulation of this process through modulation of gene expression and protein function diversity have been increasingly appreciated. Here, we review the biological significance of SUMOylation in retina development, examine how SUMOylation balances the proliferation and cell cycle exit of retinal progenitor cells, and finally discuss the molecular mechanisms mediating the specification of different retina neurons and photoreceptors through modulation of various transcription factors. The potential role of SUMOylation in normal retina function is illustrated by the abundant expression of key components of SUMOylation machinery in mouse retina, and is also exemplified by the highly conserved SUMOylation site on neurotransmission receptors in ganglion cells.

The Male Abnormal Gene Family 21 (Mab21) Members Regulate Eye Development.

The male abnormal gene family contains 3 members, named mab21l1, mab21l2 and mab21l3. Since their first discovery in C. elegans, homologues of mab21l1 and mab21l2 have been found in Drosophila, Zebrafish, Xenopus, chicken, mouse and human. A number of studies have revealed that mab21 gene family members, mab21l1 and mab21l2, play important roles in regulating eye development. Here, we review the functions of the mab genes in regulating ocular development.

Human alphaA- and alphaB-crystallins bind to Bax and Bcl-X(S) to sequester their translocation during staurosporine-induced apoptosis.

AlphaA- and alphaB-crystallins are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have recently demonstrated that alphaB-crystallin interacts with the procaspase-3 and partially processed procaspase-3 to repress caspase-3 activation. Here, we demonstrate that human alphaA- and alphaB-crystallins prevent staurosporine-induced apoptosis through interactions with members of the Bcl-2 family. Using GST pulldown assays and coimmunoprecipitations, we demonstrated that alpha-crystallins bind to Bax and Bcl-X(S) both in vitro and in vivo. Human alphaA- and alphaB-crystallins display similar affinity to both proapoptotic regulators, and so are true with their antiapoptotic ability tested in human lens epithelial cells, human retina pigment epithelial cells (ARPE-19) and rat embryonic myocardium cells (H9c2) under treatment of staurosporine, etoposide or sorbitol. Two prominent mutants, R116C in alphaA-crystallin and R120G, in alphaB-crystallin display much weaker affinity to Bax and Bcl-X(S). Through the interaction, alpha-crystallins prevent the translocation of Bax and Bcl-X(S) from cytosol into mitochondria during staurosporine-induced apoptosis. As a result, alpha-crystallins preserve the integrity of mitochondria, restrict release of cytochrome c, repress activation of caspase-3 and block degradation of PARP. Thus, our results demonstrate a novel antiapoptotic mechanism for alpha-crystallins.

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.

Regulation of Eye Development by Protein Serine/Threonine Phosphatases-1 and -2A.

The protein serine/threonine phosphatases-1 and -2A are major cellular phosphatases, playing a fundamental role in organisms from prokaryotes to eukaryotes. They contribute to 90% dephosphorylation in eukaryote proteins. In the eye, both phosphatases are highly expressed and display important functions in regulating normal eye development. Moreover, they are implicated in pathogenesis through modulation of stress-induced apoptosis. Here we review the recent progresses on these aspects.

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.

ERK signaling pathway regulates embryonic survival and eye development in goldfish, Carassius auratus.

The extracellular signal-regulated kinase (ERK) is one of the three major types of mitogen-activated protein kinases. Previous studies showed that ERKs mediate various signaling pathways for cell proliferation, differentiation, survival and transformation in mammals. In the present study, we use goldfish as a model system and demonstrate that ERK kinases play important roles in promoting embryonic survival and regulate development of eye and trunk in vertebrates. ERKs are highly expressed in multiple tissues including lens epithelial cells, lens fiber cells, retina, brain, muscle and heart of adult goldfish. Injection of the dominant negative ERK mutant (DNM-ERK) into the fertilized eggs of goldfish significantly inhibited ERK activity at blastula stage, and completely blocked ERK activity at gastrula and later stages. As a result, the blastula cells were induced into apoptosis, and majority of the injected embryos were lethal at embryonic stages. At the molecular level, inhibition of ERK activity by DNM-ERKs suppressed phosphorylation of Bad at Ser-112 to promote apoptosis. Similar results were observed when MEK activity was inhibited by U0126 treatment. The survived embryos display significant abnormality in the phenotypes of both eye and trunk. Associated with the abnormality in the eye development, phosphorylation in Pax-6 and expression of HSF4 were significantly decreased and expression of the ß-crystallin gene was also downregulated. These results provide novel information regarding the roles of ERKs in regulating vertebrate development.

p53 directly regulates αA- and ßA3/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 ßA3/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.

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.