Fungal keratitis is a major cause of corneal blindness and remains difficult to treat due to poor drug retention, frequent dosing, and inadequate modulation of pathogenic mechanisms. Current therapies largely focus on antifungal efficacy while neglecting inflammation-, fibrosis-, and cell death-related pathways. Here, ferroptosis is identified as a critical pathogenic target, and a synergistic therapeutic strategy is developed using a thermosensitive in situ-forming hydrogel composed of poloxamer and hyaluronic acid, enabling the co-delivery of a ferroptosis inhibitor (ferrostatin-1, Fer-1) and the antifungal agent (voriconazole, VOR), termed PX/HA/Fer-1/VOR hydrogel. Upon administration, the hydrogel exhibits lens-like adhesion to corneal defects, forming a localized drug reservoir that significantly enhances corneal retention and prolongs drug residence time by at least 90 min. In vitro studies demonstrate enhanced antifungal activity, while in vivo fungal keratitis models show superior therapeutic efficacy compared with conventional voriconazole eye drops administered six times daily. Notably, a single daily application effectively suppresses inflammation, reduces corneal fibrosis and scarring, and promotes recovery of visual function. Mechanistically, the composite hydrogel exerts synergistic effects through zinc ion chelation-mediated antifungal activity, inhibition of ferroptosis, and attenuation of fibrosis. This multifunctional hydrogel platform offers a promising therapeutic strategy for fungal keratitis.

Most cancers are currently incurable, partly due to abnormal post-translational modifications (PTMs). In this study, we initially used multiple myeloma (MM) as a working model and found that SUMOylation activating enzyme subunit 1 (SAE1) promotes the malignancy of MM. Through proteome microarray analysis, SAE1 was identified as a potential target for bioactive colcemid or its derivative colchicine. Elevated levels of SAE1 were associated with poor clinical survival and increased MM proliferation in vitro and in vivo. Additionally, SAE1 directly SUMOylated and upregulated the total protein expression of p27, leading to LLPS-mediated nuclear export of p27. Our study also demonstrated the involvement of SAE1 in other types of cancer cells, and provided the first monomer crystal structure of SAE1 and its key binding model with colchicine. Colchicine also showed promising results in the Patient-Derived Tumor Xenograft (PDX) model. Furthermore, a controlled clinical trial with 56 MM patients demonstrated the clinical efficacy of colchicine. Our findings reveal a novel mechanism by which tumor cells evade p27-induced cellular growth arrest through p27 SUMOylation-mediated nuclear export. SAE1 may serve as a promising therapeutic target, and colchicine may be a potential treatment option for multiple types of cancer in clinical settings.

Purpose:Dry eye disease (DED) is a common ocular surface inflammatory disease with a complex pathogenesis. Herein, the role and effect of gasdermin E (GSDME) in DED pathogenesis were explored. Methods:In vitro, flow cytometry, Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assays were used to determine the effects of hyperosmotic stress on pyroptosis, apoptosis, and cell viability in human corneal epithelial cells (HCECs). Quantitative PCR (qPCR) and Western blot assays were used to detect GSDME expression in HCECs and in those transfected with si-GSDMD. In vivo, GSDMD-knockout (KO) mice were used to study the role of GSDME in DED pathogenesis. The qPCR, Western blotting, and immunofluorescence were used to explore the effects of GSDME on HCEC apoptosis, pyroptosis, and the expression of related genes and proteins in GSDMD-KO mice with scopolamine-induced dry eye. Results:Pyroptosis and cell membrane rupture occurred, and caspase-3 and GSDME protein expression increased after HCECs were treated with 312 to 500 mOsm sodium chloride. GSDME gene and protein expression levels were increased in HCECs from both si-GSDMD- and GSDMD-KO mice. Although caspase-3 expression was increased in the dry eye group of GSDMD-KO mice, HCEC apoptosis and the apoptosis-related factors PARP were not detected. The gene and protein expression levels of the pyroptosis-related factors ASC and IL-1β were greater than those in GSDMD-KO mice without dry eye. Conclusions:GSDME is involved in DED pathogenesis by mediating inflammation via the pyroptosis pathway, GSDME inhibition may be a therapeutic target for DED.

At present, the main treatment method for wet AMD is single anti-VEGF therapy, which can require multiple injections, is costly and may have poor efficacy. Studies and clinical experiments have shown that the oral Chinese medicine Xueshuantong combined with anti-VEGF therapy is more effective, and this study aims to explore the molecular mechanism. The TCMSP database was used to identify the main Xueshuantong components. The PubChem database and SWISS Target Prediction data were used to find the SMILES molecular formulas of compounds and corresponding target genes and disease-related genes were searched using the GEO, DisGeNET, and GeneCards databases. Venny was used to identify the intersecting wet AMD-related genes and Xueshuantong targets and Cytoscape software was used to construct direct links between the drug components and disease targets. Then, PPI networks were constructed using the STRING website. R software was used for GO and KEGG enrichment analyses. Cytoscape software was used for topological analyses, and AutoDock Vina v.1.1.2 software was used for molecular docking. 64 compounds corresponding to four drugs were found by the TCMSP database, 1001 total drug targets were found by the PubChem database, 607 wet AMD target genes were found by the GEO, DisGeNET, and GeneCards databases, and 87 Xueshuantong target genes for wet AMD were obtained. Then, by constructing the drug component and disease target network and PPI network, we found that the components closely interacted with VEGF, TNF, caspase 3, CXCL8, and AKT1, which suggested that the therapeutic effects might be related to the inhibition of neovascularization, inflammation, and AKT pathway. Then, GO enrichment analysis showed that the biological processes response to hypoxia, positive regulation of angiogenesis, and inflammatory response were enriched. KEGG enrichment results showed that the HIF-1 and pi3k-akt pathways may mediate the inhibition of wet AMD by Xueshuantong. Topological analysis results identified 10 key proteins, including VEGF, TNF, AKT1, and TLR4. The results of molecular docking also confirmed their strong binding to their respective compounds. In this study, it was confirmed that Xueshuantong could inhibit wet AMD by targeting VEGF, TNF, TLR4, and AKT1, multichannel HIF-1, and the PI3K-AKT pathway, which further proved the therapeutic effects of Xueshuantong combined with single anti-VEGF therapy on wet AMD and provided new insights into the study of novel molecular drug targets for the treatment of wet AMD.

Dry age-related macular degeneration (AMD) is one of the main diseases that causes blindness in humans, and the number of cases is increasing yearly. However, effective treatments are unavailable, and arbutin (ARB) has been reported to have antioxidant, anti-inflammatory, and anti-aging effects in other age-related diseases. However, whether ARB can be used to treat dry AMD remains unknown. To explore the therapeutic potential and molecular mechanism of arbutin in the treatment of dry AMD. MTT assays, reactive oxygen species (ROS) production assays, flow cytometry assays, qPCR and western blotting were used to assess the impact of ARB on human RPECs induced by H2O2. A transcriptome sequencing assay was used to further explore how ARB acts on human RPECs treated with H2O2. Hematoxylin and eosin (H&E) staining and total antioxidant capacity (T-AOC) assays were used to observe the impact of ARB on mouse retina induced by sodium iodate. ARB counteracted the H2O2-induced reduction in human RPECs viability, ARB reversed H2O2-induced cellular ROS production by increasing the expression of antioxidant-related genes and proteins, ARB also reversed H2O2-induced cell apoptosis by altering the expression of apoptosis-related genes and proteins. Transcriptome sequencing and western blotting showed that ARB reduced ERK1/2 and P-38 phosphorylation to prevent H2O2-induced oxidation damage. The in vivo experiments demonstrated that ARB protected against retinal morphology injury in mice, increased serum T-AOC levels and increased antioxidant oxidase gene expression levels in the mouse retina induced by sodium iodate. We concluded that ARB reversed the H2O2-induced decrease in human RPECs viability through the inhibition of ROS production and apoptosis. The ERK1/2 and P38 MAPK signaling pathways may mediate this process. ARB maintained retinal morphology, increased serum T-AOC level and improved the expression of antioxidant oxidase genes in mice.

Background The aim of this study was to explore the preventive and therapeutic potential of salvianolic acid B in inhibiting senile cataracts through network pharmacology and experimental validation. Methods Disease-related genes were obtained from the DisGeNET and GeneCards databases. Drug targets were identified from the Swiss Target Prediction and PharmMapper databases, shared genes were identified via the Venny website, links between genes were identified via a protein–protein interaction (PPI) network, and GO and KEGG analyses were subsequently performed via R software. The key genes were identified via Cytoscape software, and their binding with Sal-B was demonstrated by molecular docking. Then, the results were verified by cell experiments. A CCK-8 assay was used to assess the activity of human LECs with or without H2O2 or Sal-B treatment, and the cell apoptosis rate of each group was determined by flow cytometry. The gene expression levels of caspase 3, TNF-α and MMP-9 in human LECs treated with or without H2O2 or Sal-B were determined by qPCR. Results A total of 705 and 152 cataract-related genes and salvianolic acid B-related genes, respectively, were identified, with 37 shared genes. The PPI results showed that MMP9, IL-2, JUN, TNF-α and caspase 3 were the core genes. GO data analysis revealed that the biological process, cell component and molecular function terms were most enriched in the categories “apoptosis progress”, “cytosol” and “protein binding”. Through KEGG enrichment analysis, we found that the core genes were related to the IL-17 and TNF signalling pathways. Cytoscape results showed that MMP9, TNF-α and caspase 3 were the key genes, and molecular docking showed that the drugs interacted well with the target molecules. The experimental results showed that salvianolic acid B inhibited H2O2-induced decreases in LEC activity and apoptosis and inhibited the gene expression of MMP9, TNF-α and caspase 3. Conclusion Network pharmacology and molecular docking results showed that salvianolic acid B has the potential to prevent and treat senile cataracts. The experimental results verified the finding that salvianolic acid B can inhibit the decrease in LEC activity and apoptosis induced by H2O2 and verified the expression of the key molecules MMP9, TNF-α and caspase 3.

Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90β (HSP90β) plays a fundamental role in suppressing cataractogenesis. HSP90β is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90β is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90β silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90β can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90β silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90β and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90β is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.

Background To investigate different visual evoked potential (VEP) patterns in neuromyelitis optica spectrum disorder-related optic neuritis (NMOSD-ON) and idiopathic demyelinating optic neuritis (IDON). Methods This was a longitudinal, prospective, case-control study. Eighty-four Chinese patients with acute optic neuritis were enrolled, including 26 NMOSD-ON patients and 58 IDON patients. All the patients underwent best-corrected visual acuity (BCVA) and full-field pattern reversal VEP recordings at the onset, 1 month, 3 months, and 6 months. Results Within 15′ checks, the NMOSD-ON patients had more severe VEP amplitude reduction at 6 months (2.39 ± 4.63 μV vs. 6.96 ± 8.88 μV, P = 0.034). However, the IDON patients showed more frequently normal VEP response at 3 months (24.0% vs. 4.5%, P = 0.017), and only prolonged P100 peak latency with normal amplitude (L) at 6 months (30.0% vs. 57.8%, P = 0.048). Within 60′ checks, no significant difference in VEP parameters between the two groups was found at each follow-up ( P > 0.05). Conclusions The NMOSD-ON patients showed more severe axonal damage and worse axonal recovery than the IDON patients. VEP elicited by smaller check size was more sensitive to visual pathway abnormality in NMOSD-ON.

BACKGROUND:Acute kidney injury is a clinical syndrome with both high morbidity and mortality. However, the underlying molecular mechanism of AKI is still largely unknown. The role of SENP1 in AKI is unclear, while one of its substrates, HIF-1α possesses nephroprotective effect in AKI. Herein, this study aimed to reveal the role of SENP1/HIF-1α axis in AKI by using both cell and animal models. METHODS:We investigated the effects of AKI on SENP1 expression using clinical samples, and cisplatin-induced AKI model based on mice or HK-2 cells. The influence of SENP1 knockdown or over-expression on cisplatin-induced AKI was studied in vitro and in vivo. Following the exploration of the change in HIF-1α expression brought by AKI, the synergistic effects of SENP1 knockdown and HIF-1α over-expression on AKI were examined. RESULTS:The results showed the up-regulation of SENP1 in clinical specimens, as well as cell and animal models. The knockdown or over-expression of SENP1 in HK-2 cells could promote or inhibit AKI through regulating cell apoptosis, respectively. Moreover, SENP1+/- mice suffered from much more serious AKI compared with mice in wild type group. Furthermore, we found that HIF-1α over-expression could attenuate the promoted cell apoptosis as well as AKI induced by SENP1 knockdown. CONCLUSIONS:we showed that SENP1 provided protection for kidney in AKI via regulating cell apoptosis and through the regulation of HIF-1α. This study could benefit for the understanding of the pathogenesis of AKI and provide potential therapeutic target for AKI treatment.

The small ubiquitin-like modifier (SUMO) system serves an important role in the regulation of protein stability and function. SUMOylation sustains the homeostatic equilibrium of protein function in normal tissues and numerous types of tumor. Accumulating evidence has revealed that SUMO enzymes participate in carcinogenesis via a series of complex cellular or extracellular processes. The present review outlines the physiological characteristics of the SUMOylation pathway and provides examples of SUMOylation participation in different cancer types, including in hematological malignancies (leukemia, lymphoma and myeloma). It has been indicated that the SUMO pathway may influence chromosomal instability, cell cycle progression, apoptosis and chemical drug resistance. The present review also discussed the possible relationship between SUMOylation and carcinogenic mechanisms, and evaluated their potential as biomarkers and therapeutic targets in the diagnosis and treatment of hematological malignancies. Developing and investigating inhibitors of SUMO conjugation in the future may offer promising potential as novel therapeutic strategies.

Background: To investigate the remedial efficacy and safety of intravenous cyclophosphamide (CP) in the acute phase in patients with neuromyelitis optica spectrum disorder-related optic neuritis (NMOSD-ON) who are refractory to intravenous methylprednisolone (MP) treatment. Design: This study was a single-center, retrospective, observational case-control cohort study. Methods: Thirty-six patients who had acute NMOSD-ON attacks and were refractory to MP treatment were included. Patents were divided into two groups: the remedial CP group, and the MP group. The best-corrected visual acuity (BCVA), mean deviation (MD) of the visual field (VF), visual evoked potential amplitude (VEP-A), visual evoked potential latency (VEP-T), and average thickness of the retinal nerve fiber layer (RNFL) at onset, 1 month (m), 3 m, and 6 m after the attack were analyzed. Routine blood test results, liver and kidney function, routine urinalysis results and general condition were analyzed for safety issues at each follow-up. Fisher's exact test, the Mann-Whitney U test, the Kruskal-Wallis test and the Wilcoxon rank-sum test were used for statistical analysis. Results: The remedial CP group showed significant improvement over 6 m with regard to BCVA and MD ( P < 0.05),whereas MP group only showed significant improvement in MD ( P < 0.05). Regarding remedial CP intervention time window, the CP ≤ 30 days group showed significant improvement over 6 m with regard to BCVA ( P = 0.002), MD ( P = 0.003), and VEP-A ( P = 0.036), while those CP > 30 days group did not. Both two subgroups showed significantly RNFL thickness reduction, however, BCVA, MD, VEP-A, VEP-T, and RNFL thickness showed no significant differences between the two subgroups at any follow-up point ( P > 0.05). Conclusion: CP within 30 days of attack onset is safe and might have a beneficial degree of therapeutic efficacy for acute-phase treatment of NMOSD-ON that is refractory to MP treatment alone.

Posterior capsule opacification (PCO) is a common ocular fibrosis disease related to the epithelial-mesenchymal transition (EMT) of human lens epithelial cells (HLECs). However, safe and effective drugs that prevent or treat PCO are lacking. Metformin (Mtf) has been used to treat fibrosis-related diseases affecting many organs and tissues, but its effect on ocular fibrosis-related diseases is unclear. We investigated whether Mtf can inhibit EMT and fibrosis in HLECs to prevent and treat PCO and elucidated the potential molecular mechanism. Here, we established an HLEC model of TGF-8-induced EMT and found that 400 mu M Mtf inhibited vertical and lateral migration and EMT-related gene and protein expression in HLECs. Smad2/3 are downstream molecules of TGF-8 that enter the nucleus to regulate EMT-related gene expression during the occurrence and development of PCO. We revealed that Mtf suppressed TGF-8-induced Smad2/3 phosphorylation and nuclear translocation. Mtf induces AMP-activated protein kinase (AMPK) phosphorylation. In this study, we found that Mtf induced the activation of AMPK phosphorylation in HLECs. To further explore the mechanism of Mtf, we pretreated HLECs with Compound C (an AMPK inhibitor) to repeat the above experiments and found that Compound C abolished the inhibitory effect of Mtf on HLEC EMT and the TGF-8/Smad2/3 signalling pathway. Thus, Mtf targets AMPK phosphorylation to inhibit the TGF-8/Smad2/3 signalling pathway and prevent HLEC EMT. Notably, we first illustrated the AMPK/TGF-8/Smad2/3 signalling pathway in HLECs, which may provide a new therapeutic strategy for PCO.

The function of the transcription factor, cAMP response element-binding protein (CREB), is activated through S133 phosphorylation by PKA and others. Regarding its inactivation, it is not well defined. cAMP response element-binding protein plays an essential role in promoting cell proliferation, neuronal survival and the synaptic plasticity associated with long-term memory. Our recent studies have shown that CREB is an important player in mediating stress response. Here, we have demonstrated that CREB regulates aging process through suppression of αB-crystallin and activation of the p300-p53-Bak/Bax signaling axis. First, we determined that two specific protein phosphatases, PP-1β and PP-2Aα, can inactivate CREB through S133 dephosphorylation. Subsequently, we demonstrated that cells expressing the S133A-CREB, a mutant mimicking constant dephosphorylation at S133, suppress CREB functions in aging control and stress response. Mechanistically, S133A-CREB not only significantly suppresses CREB control of αB-crystallin gene, but also represses CREB-mediated activation of p53 acetylation and downstream Bak/Bax genes. cAMP response element-binding protein suppression of αB-crystallin and its activation of p53 acetylation are major molecular events observed in human cataractous lenses of different age groups. Together, our results demonstrate that PP-1β and PP-2Aα modulate CREB functions in aging control and stress response through de-regulation of αB-crystallin gene and p300-p53-Bax/Bak signaling axis, which regulates human cataractogenesis in the aging lens.

Posterior capsule opacification (PCO) is a common ocular fibrosis disease related to the epithelial-mesenchymal transition (EMT) of human lens epithelial cells (HLECs). However, safe and effective drugs that prevent or treat PCO are lacking. Metformin (Mtf) has been used to treat fibrosis-related diseases affecting many organs and tissues, but its effect on ocular fibrosis-related diseases is unclear. We investigated whether Mtf can inhibit EMT and fibrosis in HLECs to prevent and treat PCO and elucidated the potential molecular mechanism. Here, we established an HLEC model of TGF-β-induced EMT and found that 400 μM Mtf inhibited vertical and lateral migration and EMT-related gene and protein expression in HLECs. Smad2/3 are downstream molecules of TGF-β that enter the nucleus to regulate EMT-related gene expression during the occurrence and development of PCO. We revealed that Mtf suppressed TGF-β-induced Smad2/3 phosphorylation and nuclear translocation. Mtf induces AMP-activated protein kinase (AMPK) phosphorylation. In this study, we found that Mtf induced the activation of AMPK phosphorylation in HLECs. To further explore the mechanism of Mtf, we pretreated HLECs with Compound C (an AMPK inhibitor) to repeat the above experiments and found that Compound C abolished the inhibitory effect of Mtf on HLEC EMT and the TGF-β/Smad2/3 signalling pathway. Thus, Mtf targets AMPK phosphorylation to inhibit the TGF-β/Smad2/3 signalling pathway and prevent HLEC EMT. Notably, we first illustrated the AMPK/TGF-β/Smad2/3 signalling pathway in HLECs, which may provide a new therapeutic strategy for PCO.

Esophageal squamous cell carcinoma (ESCC) is a common malignant cancer worldwide. Despite recent improvements in surgical techniques and adjuvant therapies, the prognosis of patients with advanced ESCC remains poor. Resistance to chemoradiotherapy (CRT) remains a major cause of treatment failure for advanced ESCC patients. Here, we report that NRIP3 (nuclear receptor interacting protein 3) promotes ESCC tumor cell growth and resistance to CRT in ESCC cells by increasing and binding to DDI1 (DNA-damage inducible 1 homolog 1) and RTF2 (homologous to Schizosaccharomyces pombe Rtf2), and accelerating the removal of RTF2, which is a key determinant for the ability of cells to manage replication stress. In addition, we found that NRIP3 could increase DDI1 expression via PPARα. The NRIP3-PPARα-DDI1-RTF2 axis represents a protective molecular pathway in ESCC cells that mediates resistance to replication stress signals induced by chemoradiotherapy. In addition, elevated NRIP3 is associated with the poor clinical outcome of ESCC patients receiving radiotherapy and/or cisplatin-based chemotherapy. Our study therefore reveals that NRIP3 is a prognostic factor in ESCC and could have some predictive value to select patients who benefit from CRT treatment. A common mechanism that protects ESCC tumor cells from DNA damage induced by CRT is also revealed in this study.

Sumoylation is one of the key regulatory mechanisms in eukaryotes. Our previous studies reveal that sumoylation plays indispensable roles during lens differentiation (Yan et al. 2010. Proc Natl Acad Sci USA. 107:21034-21039; Gong et al. 2014. Proc Natl Acad Sci USA. 111:5574-5579). Whether sumoylation is implicated in cataractogenesis, a disease largely derived from aging, remains elusive. In the present study, we have examined the changing patterns of the sumoylation ligases and de-sumoylation enzymes (SENPs) and their substrates including Pax6 and other proteins in cataractous lenses of different age groups from 50 to 90 years old. It is found that compared with normal lenses, sumoylation ligases 1 and 3, de-sumoylation enzymes SENP3/7/8, and p46 Pax6 are clearly increased. In contrast, Ubc9 is significantly decreased. Among different cataract patients from 50s to 70s, male patients express more sumoylation enzymes and p46 Pax6. Ubc9 and SENP6 display age-dependent increase. The p46 Pax6 displays age-dependent decrease in normal lens, remains relatively stable in senile cataracts but becomes di-sumoylated in complicated cataracts. In contrast, sumoylation of p32 Pax6 is observed in senile cataracts and increases its stability. Treatment of rat lenses with oxidative stress increases Pax6 expression without sumoylation but promotes apoptosis. Thus, our results show that the changing patterns in Ubc9, SENP6, and Pax6 levels can act as molecular markers for senile cataract and the di-sumoylated p46 Pax6 for complicated cataract. Together, our results reveal the presence of molecular signature for both senile and complicated cataracts. Moreover, our study indicates that sumoylation is implicated in control of aging and cataractogenesis.

The general transcription factor, CREB has been shown to play an essential role in promoting cell proliferation, neuronal survival and synaptic plasticity in the nervous system. However, its function in stress response remains to be elusive. In the present study, we demonstrated that CREB plays a major role in mediating stress response. In both rat lens organ culture and mouse lens epithelial cells (MLECs), CREB promotes oxidative stress-induced apoptosis. To confirm that CREB is a major player mediating the above stress response, we established stable lines of MLECs stably expressing CREB and found that they are also very sensitive to oxidative stress-induced apoptosis. To define the underlying mechanism, RNAseq analysis was conducted. It was found that CREB significantly suppressed expression of the alpha B-crystallin gene to sensitize CREB-expressing cells undergoing oxidative stress-induced apoptosis. CREB knockdown via CRISPR/CAS9 technology led to upregulation of alpha B-crystallin and enhanced resistance against oxidative stress-induced apoptosis. Moreover, overexpression of exogenous human alpha B-crystallin can restore the resistance against oxidative stress-induced apoptosis. Finally, we provided first evidence that CREB directly regulates alpha B-crystallin gene. Together, our results demonstrate that CREB is an important transcription factor mediating stress response, and it promotes oxidative stress-induced apoptosis by suppressing alpha B-crystallin expression.

PURPOSE Protein sumoylation is a well established regulatory mechanism that regulates chromatin structure and dynamics, cell proliferation and differentiation, stress response and cell apoptosis. In the vertebrate eye, we and others have shown that sumoylation plays an indispensable role in regulating eye development. During stress induction and aging process, the ocular tissues gradually loss their normality and develop major ocular diseases such as cataract and aging-related macular degeneration. We have recently demonstrated that sumoylation actively regulates differentiation of lens cells, whether this process is implicated in lens pathogenesis remains to be investigated. In this study, we have demonstrated that transparent mouse lenses treated with glucose oxidase and UVA irradiation undergo in vitro cataract formation, and associated with this process, the expression patterns of the 3 sumoylation enzymes have been found significantly altered. METHODS Four-week-old C57BL/6J mice were used in our experiment. Lenses were carefully excised from eyes and cultured in M199 medium (Sigma 3769) for at least 12 hours. Transparent lenses (without surgical damage) were selected for experimentation. The lenses were exposed to UVA for 60 min or treated with 30 mU/mL glucose oxidase (GO, MP Biomedicals, 1673) to induce cataract formation. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS we have obtained the following results: 1) Both GO treatment and UVA irradiation can induce cataract formation in the in vitro cultured mouse lenses; 2) With GO treatment, the mRNAs and proteins for the 5 sumoylation enzymes were all significantly downregulated; 3) With UVA irradiation, the changes in the expression patterns of the mRNAs and proteins for the SAE1, UBA2 , UBC9 and PIAS1 were opposite, while the mRNAs were upregulated either significantly (for SAE1, UBA2 and UBC9) or slightly (PIAS1), the proteins for all 4 sumoylation enzymes were downregulated; For RanBP2, the UVA induced changes in both mRNA and protein are consist with the GO treatment. CONCLUSION Under GO and UVA irradiation conditions, the expression levels of both mRNA and protein for the three major sumoylation enzymes were significantly changed. Our results suggest that altered expression patterns of the sumoylation enzymes are associated with oxidative stressinduced cataractogenesis.

Objective: It has been well established that sumoylation acts as an important regulatory mechanism that controls many different cellular processes. We and others have shown that sumoylation plays an indispensable role during mouse eye development. Whether sumoylation is implicated in ocular pathogenesis remains to be further studied. In the present study, we have examined the expression patterns of the de-sumoylation enzymes (SENPs) in the in vitro cataract models induced by glucose oxidase and UVA irradiation. Methods: Four-week-old C57BL/6J mice were used in our experiments. Lenses were carefully dissected out from mouse eyes and cultured in M199 medium for 12 hours. Transparent lenses (without surgical damage) were selected for experimentation. The lenses were exposed to UVA for 60 min or treated with 20 mU/mL glucose oxidase (GO) to induce cataract formation. The mRNA levels were analyzed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. Results: GO treatment and UVA irradiation can induce cataract formation in lens cultured in vitro. GO treatment significantly down-regulated the mRNA levels for SENPs from 50% to 85%; on the other hand, expression of seven SENP proteins under GO treatment appeared in 3 situations: upregulation for SENP1, 2 and 6; downregulation for SENP 5 and 8; and unchanged for SENP3 and 7. UVA irradiation upregulates the mRNAs for all seven SENPs; In contrast to the mRNA levels for 7 SENPs, the expression levels for 6 SENPs (SENP1-3, 5-6 and 8) appeared down-regulated from 10% to 50%, and only SENP7 was slightly upregulated. Conclusion: Our results for the first time established the differentiation expression patterns of 7 de-sumoylation enzymes (SENPs) under treatment by GO or UVA, which provide preliminary data to link sumoylation to stress-induced cataractogenesis.

PURPOSE Pax-6 is a master regulator for eye and brain development. Previous studies including ours have shown that Pax-6 exists in 4 major isoforms. According to their sizes, they are named p48, p46, p43 and p32 with the corresponding molecular weight of 48, 46, 43 and 32 kd, respectively. While p48 and p46 is derived from alternative splicing, p32 Pax-6 is generated through an internal translation initiation site. As for 43 kd Pax-6, two resources have been reported. In bird, it was found that an alternative splicing can generate a p43 Pax-6. In human and mouse, we reported that the p43 kd Pax-6 is derived from sumoylation: addition of a 11 kd polypeptide SUMO1 into the p32 Pax-6 at the K91 residue. Whether other Pax-6 isoforms can be sumoylated or not remains to be explored. 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 Both non-sumoylated and sumoylated isoforms of Pax-6 exist in 6 major types of ocular cells among which five are lens epithelial cells, and one is retinal pigment epithelial cell. Our results revealed that the most abundant isoforms of Pax-6 are the p32 and p46 Pax-6. These two major isoforms can be sumoylated to generate p43 (mono-sumoylated p32 Pax-6), p57 and p68 Pax-6 (mono- and di-sumoylated p46 Pax-6). In addition, the splicing-generated p48 Pax-6 is also readily detected. CONCLUSION Our results for the first time, have determined the relative isoform abundance and also the sumoylation patterns of pax-6 in 6 major ocular cell lines.