Analysis of shared ceRNA networks and related-hub genes in rats with primary and secondary photoreceptor degeneration.

Introduction: Photoreceptor degenerative diseases are characterized by the progressive death of photoreceptor cells, resulting in irreversible visual impairment. However, the role of competing endogenous RNA (ceRNA) in photoreceptor degeneration is unclear. We aimed to explore the shared ceRNA regulation network and potential molecular mechanisms between primary and secondary photoreceptor degenerations. Methods: We established animal models for both types of photoreceptor degenerations and conducted retina RNA sequencing to identify shared differentially expressed long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs). Using ceRNA regulatory principles, we constructed a shared ceRNA network and performed function enrichment and protein-protein interaction (PPI) analyses to identify hub genes and key pathways. Immune cell infiltration and drug-gene interaction analyses were conducted, and hub gene expression was validated by quantitative real-time polymerase chain reaction (qRT-PCR). Results: We identified 37 shared differentially expressed lncRNAs, 34 miRNAs, and 247 mRNAs and constructed a ceRNA network consisting of 3 lncRNAs, 5 miRNAs, and 109 mRNAs. Furthermore, we examined 109 common differentially expressed genes (DEGs) through functional annotation, PPI analysis, and regulatory network analysis. We discovered that these diseases shared the complement and coagulation cascades pathway. Eight hub genes were identified and enriched in the immune system process. Immune infiltration analysis revealed increased T cells and decreased B cells in both photoreceptor degenerations. The expression of hub genes was closely associated with the quantities of immune cell types. Additionally, we identified 7 immune therapeutical drugs that target the hub genes. Discussion: Our findings provide new insights and directions for understanding the common mechanisms underlying the development of photoreceptor degeneration. The hub genes and related ceRNA networks we identified may offer new perspectives for elucidating the mechanisms and hold promise for the development of innovative treatment strategies.

The BET PROTAC inhibitor dBET6 protects against retinal degeneration and inhibits the cGAS-STING in response to light damage.

BACKGROUND: Chronic inflammation significantly contributes to photoreceptor death in blinding retinal diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that act as key proinflammatory factors. We recently found the first-generation BET inhibitor JQ1 alleviated sodium iodate-induced retinal degeneration by suppressing cGAS-STING innate immunity. Here, we investigated the effects and mechanism of dBET6, a proteolysis­targeting chimera (PROTAC) small molecule that selectively degrades BET by the ubiquitin‒proteasome system, in light-induced retinal degeneration. METHODS: Mice were exposed to bright light to induce retinal degeneration, and the activation of cGAS-STING was determined by RNA-sequencing and molecular biology. Retinal function, morphology, photoreceptor viability and retinal inflammation were examined in the presence and absence of dBET6 treatment. RESULTS: Intraperitoneal injection of dBET6 led to the rapid degradation of BET protein in the retina without detectable toxicity. dBET6 improved retinal responsiveness and visual acuity after light damage (LD). dBET6 also repressed LD-induced retinal macrophages/microglia activation, Müller cell gliosis, photoreceptor death and retinal degeneration. Analysis of single-cell RNA-sequencing results revealed cGAS-STING components were expressed in retinal microglia. LD led to dramatic activation of the cGAS-STING pathway, whereas dBET6 suppressed LD-induced STING expression in reactive macrophages/microglia and the related inflammatory response. CONCLUSIONS: This study indicates targeted degradation of BET by dBET6 exerts neuroprotective effects by inhibiting cGAS-STING in reactive retinal macrophages/microglia, and is expected to become a new strategy for treatment of retinal degeneration.

Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration.

Atrophic ("dry") form of age-related macular degeneration (AMD) is a leading cause of vision loss characterized by macular retinal pigment epithelium (RPE) and the ensuing photoreceptor degeneration. cGAS-STING signaling is a key cytosolic DNA sensor system in innate immunity and have recently been shown promotes RPE degeneration. However, expression regulation and therapeutic potential of cGAS and STING are not explored in retina under dry AMD pathogenic conditions. Our analysis shows upregulated STING RNA and increased chromatin accessibility around cGAS and STING promoters in macular retinas from dry AMD patients. cGAS-STING activation was detected in oxidative stress-induced mouse retina degeneration, accompanied with cytosolic leakage of damaged DNA in photoreceptors. Pharmaceutical or genetic approaches indicates STING promotes retina inflammation and degeneration upon oxidative damage. Drug screening reveals that BRD4 inhibitor JQ1 reduces cGAS-STING activation, inflammation and photoreceptor degeneration in the injured retina. BRD4 inhibition epigenetically suppresses STING transcription, and promotes autophagy-dependent cytosolic DNA clearance. Together, our results show that activation of cGAS-STING in retina may present pivotal innate immunity response in GA pathogenesis, whereas inhibition of cGAS-STING signaling by JQ1 could serve as a potential therapeutic strategy.

Heterochromatin inhibits cGAS and STING during oxidative stress-induced retinal pigment epithelium and retina degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness characterized by degeneration of retina pigment epithelium (RPE) and photoreceptors in the macular region. Activation of the innate immune cGAS-STING signaling has been detected in RPE of dry AMD patients, but the regulatory basis is largely unexplored. Heterochromatin is a highly compact, transcription inert chromatin status. We have recently shown that heterochromatin is required for RPE survival through epigenetically silencing p53-mediated apoptosis signaling. Here, we found that cGAS and STING were dose-dependently upregulated in mouse RPE and retina during oxidative injury, correlated with decreased chromatin compaction in their gene loci. Genetic or pharmaceutical disruption of heterochromatin leads to elevated cGAS and STING expression and enhanced inflammatory response in oxidative stress-induced RPE and retina degeneration. In contrast, application of methotrexate (MTX), a recently identified heterochromatin-promoting drug, inhibits cGAS and STING in both RPE and retina, attenuates RPE/retina degeneration and inflammation. Further, we show that intact heterochromatin is required for MTX to repress cGAS and STING. Together, we demonstrated an unrevealed regulatory function of heterochromatin on cGAS and STING expression and provide potential new therapeutic strategy for AMD treatment.

The E3 Ligase PIAS1 Regulates p53 Sumoylation to Control Stress-Induced Apoptosis of Lens Epithelial Cells Through the Proapoptotic Regulator Bax.

Protein sumoylation is one of the most important post-translational modifications regulating many biological processes (Flotho A & Melchior F. 2013. Ann Rev. Biochem. 82:357-85). Our previous studies have shown that sumoylation plays a fundamental role in regulating lens differentiation (Yan et al., 2010. PNAS, 107(49):21034-9.; Gong et al., 2014. PNAS. 111(15):5574-9). Whether sumoylation is implicated in lens pathogenesis remains elusive. Here, we present evidence to show that the protein inhibitor of activated STAT-1 (PIAS1), a E3 ligase for sumoylation, is implicated in regulating stress-induced lens pathogenesis. During oxidative stress-induced cataractogenesis, expression of PIAS1 is significantly altered at both mRNA and protein levels. Upregulation and overexpression of exogenous PIAS1 significantly enhances stress-induced apoptosis. In contrast, silence of PIAS1 with CRISPR/Cas9 technology attenuates stress-induced apoptosis. Mechanistically, different from other cells, PIAS1 has little effect to activate JNK but upregulates Bax, a major proapoptotic regulator. Moreover, Bax upregulation is derived from the enhanced transcription activity of the upstream transcription factor, p53. As revealed previously in other cells by different laboratories, our data also demonstrate that PIAS1 promotes SUMO1 conjugation of p53 at K386 residue in lens epithelial cells and thus enhances p53 transcription activity to promote Bax upregulation. Silence of Bax expression largely abrogates PIAS1-mediated enhancement of stress-induced apoptosis. Thus, our results demonstrated that PIAS1 promotes oxidative stress-induced apoptosis through positive control of p53, which specifically upregulates expression of the downstream proapoptotic regulator Bax. As a result, PIAS1-promoted apoptosis induced by oxidative stress is implicated in lens pathogenesis.

Generation of a homozygous CRISPR/Cas9-mediated knockout H9 hESC subline for the MERTK locus.

MERTK mutations are associate with rod-cone dystrophies. To enable investigations into the mechanism of this disease, we generated a cell line resource of H9 human embryonic stem cells harboring large fragment deletion mutation in a homozygous state in exon 19 of the MERTK gene. This subline expressed pluripotent stem cell markers, presented a normal karyotype, and preserved the ability to differentiate into endodermal, mesodermal, and ectodermal lineages.

Design, synthesis and evaluation of cinnamic acid hybrids as multi-target-directed agents for the treatment of Alzheimer's disease.

Herein, combining 1,2,3,4-tetrahydroisoquinoline and benzylpiperidine groups into cinnamic acid derivatives, a series of novel cinnamic acid hybrids was rationally designed, synthesized and evaluated by the multi-target-directed ligands (MTDLs) strategy. Hybrid 4e was the most promising one among these hybrids with a reversible huBuChE inhibitor (IC50 = 2.5 µM) and good MAO-B inhibition activity (IC50 = 1.3 µM) and antioxidant potency (ORAC = 0.4 eq). Moreover, compound 4e significantly inhibited self-mediated Aß1-42 aggregation (65.2% inhibition rate). Compound 4e exhibited remarkable anti-inflammatory propery and neuroprotective effect. Furthermore, compound 4e displayed favourable blood-brain barrier penetration via parallel artificial membrane permeation assay (PAMPA). The obtained results also revealed that compound 4e significantly improved dyskinesia recovery rate and response efficiency on AD model zebrafish. Further, 4e did not show obvious acute toxicity at dose up to 1500 mg/kg in vivo and improved scopolamine-induced memory impairment. Importantly, compound 4e showed good stability in both artificial gastric fluid and artificial intestinal fluid. Therefore, compound 4e presented a promising multi-targeted active molecule for treating AD.

Generation of a homozygous CRISPR/Cas9-mediated knockout H9 hESC subline for the CRB1 locus.

Mutations in the CRB1 gene reportedly cause early-onset autosomal recessive retinitis pigmentosa (RP), which can result in severe loss of vision at an early age. To investigate the mechanism of CRB1-knockout (CRB1-/-) induced RP, we generated a subline of H9 human embryonic stem cells harboring frame shift mutations in a homozygous state in exon 2 of the CRB1 gene. This subline expressed pluripotent stem cell markers, presented a normal karyotype, and preserved the ability to differentiate into endodermal, mesodermal, and ectodermal lineages.

Using inducible lentiviral vectors to generate induced pluripotent stem cell line ZOCi001-A from peripheral blood cells of a patient with CRB1-/- retinitis pigmentosa.

We isolated peripheral blood mononuclear cells from a patient diagnosed with early-onset non-syndromic retinitis pigmentosa caused by compound heterozygous mutations in CRB1 (NM_001257965): c.1369C>T (p.R457X) and c.2027C>T (p.T676M). These cells were transfected with four inducible plasmids encoding human OCT4, SOX2, KLF4 or C-MYC together. Transfected cells were induced to form pluripotent stem cells (iPSCs) expressing the pluripotent stem cell markers SOX2, OCT4, KLF4, SSEA4 and NANOG and presenting a normal karyotype. These cells could be differentiated into three germ layers as well as retinal organoids, and thus provide a valuable cellular model for the study of development of major retina diseases.

Benthic cyanobacterial detritus mats in lacustrine sediment: Characterization and odorant producing potential.

Eutrophic freshwater lake ecosystems are receiving increasing public attention due to a global increase in large-scale harmful cyanobacterial blooms in surface waters. However, the contribution of phytodetritus accumulation in benthic sediments post-bloom remains unclear. In this study, field investigations were performed using microsensors to evaluate benthic phytodetritus mats by measuring TOC/TN ratios, pigments, biodegradable compounds and odorants as descriptive parameters. Results show that the massive amount of phytodetritus trapped by aquatic plants gradually evolved into benthic cyanobacterial detritus mats, which were characterized as anoxic, reductive and low pH. It was confirmed that the occurrence of odorants is more serious in the detritus mats due to decay and decomposition of the accumulated phytodetritus. The mean odorant content in the vegetated zones was 3-52 times higher than that in the unvegetated zones. The dominant odorants were dimethyl trisulfide (DMTS), ß-ionone and ß-cyclocitral, with mean contents of 52.38 ng·(g·dw)-1, 162.20 ng·(g·dw)-1 and 307.51 ng·(g·dw)-1, respectively, in the sediment. In addition, odorant production appears to be associated with the distribution of biodegradable compounds in the sediment. This is supported by the marked correlation observed between biodegradable compounds and odorants. Multiple regression analysis showed that biodegradable compounds can be used as indicators to predict odorant content in the sediment. It is noteworthy that the odorant trend in the water column and sediment is symmetrical, indicating a risk of diffusion from the sediment to the water column. This study helps to clarifying the contributions of benthic cyanobacterial detritus mats to odorant production in shallow eutrophic lakes. The information provided herein may also be useful for future management of aquatic ecosystems.

Oxidative stress-induced KLF4 activates inflammatory response through IL17RA and its downstream targets in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness worldwide. Oxidative stress (OS), inflammation and genetics are considered the key pathogenic factors contributing to AMD development. Recent evidence shows the pro-inflammatory interleukin 17 (IL17) signaling is activated in AMD patients and promotes disease pathogenesis. However, the interplay between OS and IL17 signaling, and the regulatory mechanism of IL17 pathway are largely unknown. OS-induced retinal pigment epithelial cell (RPE) damage causes both the initial pathogenesis of AMD and secondary degeneration of rods and cones. Healthy RPE is essential for ocular immune privilege, however, damaged RPE cells can activate inflammatory response. In the present study, we identified IL17RA, the principle receptor of IL17 signaling, is one of the most upregulated inflammatory genes in human RPE cells upon OS exposure. The prominent increase of IL17RA was also observed in RPE and retina of an AMD-like mouse model. Knockdown of IL17RA in RPE cells prevented OS-induced RPE cell apoptosis and reduced the inflammatory response in both RPE and macrophages. Furthermore, we found that transcription factor KLF4 directly activates IL17RA expression, therefore, promotes the production of IL1ß and IL8 in an IL17RA-dependent manner. In addition, the mRNA level of KLF4 isoform 2 was positively correlated with that of IL17RA in AMD patients. Together, our study demonstrates an unrevealed relationship between IL17RA and OS, and a new regulatory mechanism of IL17RA by KLF4 in RPE cells. These findings suggest that inhibition of IL17RA as a new potential therapeutic target for AMD through RPE protection and inflammatory suppression upon OS exposure.

Heterochromatin protects retinal pigment epithelium cells from oxidative damage by silencing p53 target genes.

Oxidative stress (OS)-induced retinal pigment epithelium (RPE) cell apoptosis is critically implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Heterochromatin, a compact and transcriptional inert chromatin structure, has been recently shown to be dynamically regulated in response to stress stimuli. The functional mechanism of heterochromatin on OS exposure is unclear, however. Here we show that OS increases heterochromatin formation both in vivo and in vitro, which is essential for protecting RPE cells from oxidative damage. Mechanistically, OS-induced heterochromatin selectively accumulates at p53-regulated proapoptotic target promoters and inhibits their transcription. Furthermore, OS-induced desumoylation of p53 promotes p53-heterochromatin interaction and regulates p53 promoter selection, resulting in the locus-specific recruitment of heterochromatin and transcription repression. Together, our findings demonstrate a protective function of OS-induced heterochromatin formation in which p53 desumoylation-guided promoter selection and subsequent heterochromatin recruitment play a critical role. We propose that targeting heterochromatin provides a plausible therapeutic strategy for the treatment of AMD.

Determination of Expression Patterns of Seven De-sumoylation Enzymes in Major Ocular Cell Lines.

PURPOSE: Accumulated evidence have well established that protein sumoylation plays multiple roles in various cellular processes. In the vertebrate eye, we and others have demonstrated that sumoylation displays indispensable roles in regulating eye development. Various ocular cell lines including human embryonic cell line (FHL124), the SV40-large T-transformed human lens epithelial cell line (HLE), the SV40-large T-transformed mouse lens epithelial cell line (αTN4-1), the rabbit lens epithelial cell line (N/N1003A) and the human retina pigment epithelial cell line (ARPE-19) have been extensively used for studying various cellular functions and disease processes including sumoylation functions, and mechanisms for cataract and age-related macular degeneration (AMD). However, the sumoylation enzyme systems have not been well established. METHODS: FHL124, HLE, αTN4-1, N/N1003A and ARPE-19 were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% FBS and 1% penicillin & streptomycin. The expression levels of seven SENP mRNAs were analyzed with qRT-PCR, and the expression levels of seven SENP proteins were detected with Western blot analysis. RESULTS: Using both qRT-PCR and Western blot analysis, we have obtained the followings: 1). The 3 human ocular cell lines, FHL124, HLE and ARPE-19 express all types of SENP mRNA and proteins. 2). In mouse lens epithelial cell line αTN4-1, and rabbit lens epithelial cells line N/N1003A, however, only the mRNAs for SENP1, 2, 3, 6 and 7 are expressed. At the protein level, SENP8 was absent in both αTN4-1 and N/N1003A cells; 3). Each cell line has different dominant SENP enzymes. For FHL124, SENP3, 5, 7 and 8 proteins are relatively dominant. SENP3, 5 and 6 are the major de-sumoylation enzymes in HLE cells. Different from human lens epithelial cells, FHL124 and HLE, human retina pigment epithelial cells (ARPE-19) have SENP3, 7, and 8 as the dominant forms of de-sumoylation enzymes. For mouse lens epithelial cells, SENP1, 3 and 7 are the major de-sumoylation enzymes. On the other hand, the rabbit lens epithelial cells have SENP1, 2 and 7 as the major isoforms. CONCLUSION: Our results for the first time defined the differential expression patterns of the seven types of de-sumoylation enzymes (SENPs) in 5 major ocular cell lines. These results help to establish the basis for the future study of sumoylation functions and the related mechanisms in vertebrate eye.

The Sumoylation Modulated Tumor Suppressor p53 Regulates Cell Cycle Checking Genes to Mediate Lens Differentiation.

PURPOSE: 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 and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Furthermore, we have recently demonstrated that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. In addition, we have also shown that p53 controls both transcription factors, C-Maf and Prox-1 as well as lens crystallin genes, αA, ß- and γ-crystallins. Here, we have examined whether p53 also regulates other known target genes during its modulation of lens differentiation. The human and mouse lens epithelial cells, FHL124 and αTN4-1 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. METHODS: Mice used in this study were handled in compliance with the "Protocol for the Care and Use of Laboratory Animals" (Sun Yat-sen University). Adult mice were used for the collection of lens cells. These samples were used for extraction of total proteins. A total of 32 embryonic mice {8 at 14.5 ED, 8 at 17.5 ED and 8 newborns for wild type} were used for immunohistochemistry, which were used for co-localization study. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: Immunohistochemistry revealed that both the cell cycle checking genes, p21 and Gadd45α and the apoptotic genes, Bcl-2 and PUMA, display developmental changes associated with p53 during mouse lens development. Knockdown of p53 in the mouse lens epithelial cells caused inhibition of lens differentiation. Associated with this inhibition, the cell cycle genes displayed significant downreglation, the apoptotic genes was also attenuated but to a much less degree. In addition, we found that bFGF can induce dose-dependent upregulation of the upstream kinases, CHK1/2 and ERK1/2, both known to phosphorylate p53 and activate the later. Furthermore, We showed that in both developing lens and human lens epithelial cells, p53 can be co-localized with the catalytic subunit of the protein phoshphatase-1 (PP-1), suggesting that PP-1 regulates p53 phosphorylation status both in vivo and in vitro. CONCLUSION: Taken together, our results suggest that during mouse lens development, p53 activity is regulated by ERK and CHK kinases-mediated activation, and by PP-1-mediated inactivation. p53 can regulate multiple groups of genes to mediate lens differentiation.

Sodium Iodate-Induced Mouse Model of Age-Related Macular Degeneration Displayed Altered Expression Patterns of Sumoylation Enzymes E1, E2 and E3.

PURPOSE: Protein sumoylation is a highly dynamic and reversible post-translational modification, involving covalently conjugation of the small ubiquitin-like modifier (SUMO) to the lysine residue of the target protein. Similar to ubiquitination, sumoylation is catalyzed by E1, E2 and several E3 ligases. However, sumoylation usually does not cause protein degradation but alter the target function through diverse mechanisms. Increasing evidences have shown that sumoylation plays pivotal roles in the pathogenesis of human diseases, including neuron degeneration, cancer and heart disease, etc. We and others have shown that sumoylation is critically implicated in mouse eye development. However, the expression of sumoylation machinery has not been characterized in normal and pathogenic retina. Worldwide, age-related macular degeneration (AMD) is the leading cause of irreversible blindness in aged person. In the present study, we investigated the expression of the major sumoylation enzymes in normal mice and sodium iodateinduced AMD mouse model. METHODS: Four-week-old C57BL/6J mice were used in our experiment. A sterile 1% NaIO3 solution was freshly prepared in PBS from solid NaIO3. Experimental mice were injected with 70 mg/kg NaIO3, and similar volumes of PBS as control. Eyes were enucleated and immersion in FAA fixation overnight and processed for eye cross-sections. After fixation, cross sections eyes were dehydrated, embedded in paraffin, and 6 mm transverse sections were cut using the rotary microtome. Then paraffin sections were stained with hematoxylin and eosin (H&E), and mouse retinal thickness was observed to assess the histopathologic changes. RESULTS: Significantly declined RNA levels of E1, E2 and E3 ligase PIAS1 in NaIO3-injected mouse RPE one day-post treatment. Consistently, the protein level of PIAS1 was also decreased at this time point. At the late stage of treatment (three days post-injection), significantly reduced expression of E1 enzyme SAE1/UBA2 was detected in NaIO3-injected mouse retinas. In the contrary, dramatically increased E3 ligase RanBP2 was found in the injected-retinas. CONCLUSION: Together, our results demonstrated for the first time the dynamic expression of sumoylation pathway enzymes during the progression of retina degeneration induced by oxidative stress. Dynamic expression of E1, E2 and E3 enzymes were found during the time course of RPE and retina degeneration, which revealed the potential regulatory roles of sumoylation in AMD pathogenesis.

Differential Expression of Seven De-sumoylation Enzymes (SENPs) in Major Ocular Tissues of Mouse Eye.

PURPOSE: Protein Sumoylation is one of the most important and prevalent posttranscriptional modification. Increasing evidence have shown that the SENPs (sentrin/SUMOspecific proteases) are critical for steady-state levels of SUMO modification of target proteins, and protein de-sumoylation modulates a great diversity of biological processes including transcription, development, differentiation, neuroprotection, as well as pathogenesis. In the vertebrate eye, we and others have previously shown that sumoylation participated in the differentiation of major ocular tissues including retina and lens. However, the biological significance of seven SENP enzymes: SENP1 to 3 and SENP5 to 8 have not be fully investigated in the ocular tissues. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: At the mRNA level, all SENPs were highly expressed in retina, and much reduced expression patterns in cornea, lens epithelium and lens fiber. At the protein level, SENP1 to -3, and SENP6 were highly abundant in cornea, while SENP5, SENP7 and SENP8 were enriched in retina, and these SENPs were relatively less abundant in lens tissues. CONCLUSION: Our results for the first time established the differentiation expression patterns of the 7 de-sumoylation enzymes (SENPs), which provides a basis for further investigation of protein desumoylation functions in vertebrate eye.

Localization Patterns of Sumoylation Enzymes E1, E2 and E3 in Ocular Cell Lines Predict Their Functional Importance.

PURPOSE: It is well established now that protein sumoylation acts as an important regulatory mechanism mediating control of ocular development through regulation of multiple transcription factors. Yet the functional mechanisms of each factor modulated remain to be further explored using the available in vitro systems. In this regard, various ocular cell lines including HLE, FHL124, αTN4-1, N/N1003A and ARPE-19 have been demonstrated to be useful for biochemical and molecular analyses of normal physiology and pathogenesis. We have recently examined that these cell lines express a full set of sumoylation enzymes E1, E2 and E3. Following this study, here we have examined the localization of these enzymes and determined their differential localization patterns in these major ocular cell lines. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The localization of the 3 major sumoylation enzymes in the 5 major ocular cell lines were determined with immunohistochemistry. The images were captured with a Zeiss LSM 880 confocal microscope. RESULTS: we have obtained the following results: 1) The sumoylation enzymes SAE1, UBC9 and PIAS1 are distributed in both nucleus and cytoplasm, with a much higher level concentrated in the nucleus and the neighboring cellular organelle zone in all cell lines; 2) The sumoylation enzyme UBA2 was highly concentrated in both cytoplasm membrane, cytoskeleton and nucleus of all cell lines; 3) The ligase E3, RanBP2 was exclusively localized in the nucleus with homogeneous distribution. CONCLUSIONS: Our results for the first time established the differential localization patterns of the three types of sumoylation enzymes in 5 major ocular cell lines. Our establishment of the differential localization patterns of the three types of sumoylation enzymes in these cell lines help to predict their functional importance of sumoylation in the vision system. Together, our results demonstrate that these cell lines can be used for assay systems to explore the functional mechanisms of sumoylation mediating ocular development and pathogenesis.

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.

Sumoylation differentially regulates Sp1 to control cell differentiation.

The mammalian small ubiquitin-like modifiers (SUMOs) are actively involved in regulating differentiation of different cell types. However, the functional differences between SUMO isoforms and their mechanisms of action remain largely unknown. Using the ocular lens as a model system, we demonstrate that different SUMOs display distinct functions in regulating differentiation of epithelial cells into fiber cells. During lens differentiation, SUMO1 and SUMO2/3 displayed different expression, localization, and targets, suggesting differential functions. Indeed, overexpression of SUMO2/3, but not SUMO1, inhibited basic (b) FGF-induced cell differentiation. In contrast, knockdown of SUMO1, but not SUMO2/3, also inhibited bFGF action. Mechanistically, specificity protein 1 (Sp1), a major transcription factor that controls expression of lens-specific genes such as ß-crystallins, was positively regulated by SUMO1 but negatively regulated by SUMO2. SUMO2 was found to inhibit Sp1 functions through several mechanisms: sumoylating it at K683 to attenuate DNA binding, and at K16 to increase its turnover. SUMO2 also interfered with the interaction between Sp1 and the coactivator, p300, and recruited a repressor, Sp3 to ß-crystallin gene promoters, to negatively regulate their expression. Thus, stable SUMO1, but diminishing SUMO2/3, during lens development is necessary for normal lens differentiation. In support of this conclusion, SUMO1 and Sp1 formed complexes during early and later stages of lens development. In contrast, an interaction between SUMO2/3 and Sp1 was detected only during the initial lens vesicle stage. Together, our results establish distinct roles of different SUMO isoforms and demonstrate for the first time, to our knowledge, that Sp1 acts as a major transcription factor target for SUMO control of cell differentiation.