MAB21L1 promotes survival of lens epithelial cells through control of αB-crystallin and ATR/CHK1/p53 pathway.

The male abnormal gene family 21 (mab21), was initially identified in C. elegans. Since its identification, studies from different groups have shown that it regulates development of ocular tissues, brain, heart and liver. However, its functional mechanism remains largely unknown. Here, we demonstrate that Mab21L1 promotes survival of lens epithelial cells. Mechanistically, Mab21L1 upregulates expression of αB-crystallin. Moreover, our results show that αB-crystallin prevents stress-induced phosphorylation of p53 at S-20 and S-37 through abrogating the activation of the upstream kinases, ATR and CHK1. As a result of suppressing p53 activity by αB-crystallin, Mab21L1 downregulates expression of Bak but upregulates Mcl-1 during stress insult. Taken together, our results demonstrate that Mab21L1 promotes survival of lens epithelial cells through upregulation of αB-crystallin to suppress ATR/CHK1/p53 pathway.

MYPT1/PP1-Mediated EZH2 Dephosphorylation at S21 Promotes Epithelial-Mesenchymal Transition in Fibrosis through Control of Multiple Families of Genes.

The methyltransferase EZH2 plays an important role in regulating chromatin conformation and gene transcription. Phosphorylation of EZH2 at S21 by AKT kinase suppresses its function. However, protein phosphatases responsible for the dephosphorylation of EZH2-S21 remain elusive. Here, it is demonstrated that EZH2 is highly expressed in the ocular lens, and AKT-EZH2 axis is important in TGFß-induced epithelial-mesenchymal transition (EMT). More importantly, it is identified that MYPT1/PP1 dephosphorylates EZH2-S21 and thus modulates its functions. MYPT1 knockout accelerates EMT, but expression of the EZH2-S21A mutant suppresses EMT through control of multiple families of genes. Furthermore, the phosphorylation status and gene expression modulation of EZH2 are implicated in control of anterior subcapsular cataracts (ASC) in human and mouse eyes. Together, the results identify the specific phosphatase for EZH2-S21 and reveal EZH2 dephosphorylation control of several families of genes implicated in lens EMT and ASC pathogenesis. These results provide important novel information in EZH2 function and regulation.

PP-1ß and PP-2Aα modulate cAMP response element-binding protein (CREB) functions in aging control and stress response through de-regulation of αB-crystallin gene and p300-p53 signaling axis.

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-1ß 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-1ß 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.

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.

Aging-dependent loss of GAP junction proteins Cx46 and Cx50 in the fiber cells of human and mouse lenses accounts for the diminished coupling conductance.

The homeostasis of the ocular lens is maintained by a microcirculation system propagated through gap junction channels. It is well established that the intercellular communications of the lens become deteriorative during aging. However, the molecular basis for this change in human lenses has not been well defined. Here, we present evidence to show that over 90% of Cx46 and Cx50 are lost in the fiber cells of normal human lenses aged 50 and above. From transparent to cataractous lenses, while Cx43 was upregulated, both Cx46 and Cx50 were significantly down-regulated in the lens epithelia. During aging of mouse lenses, Cx43 remained unchanged, but both Cx46 and Cx50 were significantly downregulated. Under oxidative stress treatment, mouse lenses develop in vitro cataractogenesis. Associated with this process, Cx43 was significantly upregulated, in contrast, Cx46 and Cx50 were sharply downregulated. Together, our results for the first time reveal that downregulation in Cx46 and Cx50 levels appears to be the major reason for the diminished coupling conductance, and the aging-dependent loss of Cx46 and Cx50 promotes senile cataractogenesis.

The transcription factor CREB acts as an important regulator mediating oxidative stress-induced apoptosis by suppressing αB-crystallin expression.

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 αB-crystallin gene to sensitize CREB-expressing cells undergoing oxidative stress-induced apoptosis. CREB knockdown via CRISPR/CAS9 technology led to upregulation of αB-crystallin and enhanced resistance against oxidative stress-induced apoptosis. Moreover, overexpression of exogenous human αB-crystallin can restore the resistance against oxidative stress-induced apoptosis. Finally, we provided first evidence that CREB directly regulates αB-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 αB-crystallin expression.

Glucose Oxidase- and UVA-Induced Changes in the Expression Patterns of Seven De-sumoylation Enzymes (SENPs) Are Associated with Cataract Development.

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.

Differential Expression of Sumoylation Enzymes SAE1, U BA2, UBC9, PIAS1 and RanBP2 in Major Ocular Tissues of Mouse Eye.

Background: It is now well established that protein sumoylation is an important mechanism to regulate multiple cellular processes including gene transcription, chromatin structure, cell proliferation and differentiation, as well as pathogenesis. Objective: In the vertebrate eye, we and others have previously shown that sumoylation can regulate differentiation of major ocular tissues including retina and lens. However, the expression patterns of the three types of sumoylation enzymes, the activating enzymes SAE1 and UBA2, the conjugating enzyme UBC9, and the ligating enzymes such as RanBP2 and PIAS1 have not been well studied in the ocular tissues. Conclusion: In the present study, using QRT-PCR and western blot analysis, we have determined the differentiatial expression patterns of the above three types of enzymes, and the obtained results lay down a foundation for further exploration of sumoylation functions in vertebrate eye.

Altered Expression Patterns of the Sumoylation Enzymes E1, E2 and E3 Are Associated with Glucose Oxidase- and UVA-Induced Cataractogenesis.

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.

Analysis of Non-Sumoylated and Sumoylated Isoforms of Pax-6, the Master Regulator for Eye and Brain Development in Ocular Cell Lines.

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.

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 Analysis of Seven De-sumoylation Enzymes (SENPs) in Ocular Cell Lines.

PURPOSE: It is now well established that protein sumoylation acts as an important regulatory mechanism modulating functions over three thousand proteins. In the vision system, protein conjugation with SUMO peptides can regulate differentiation of multiple ocular tissues. Such regulation is often explored through analysis of biochemical and physiological changes with various cell lines in vitro. We have recently analyzed the expression levels of both mRNAs and proteins for seven de-sumoylation enzymes (SENPs) in five major ocular cell lines. In continuing the previous study, here we have determined their cellular localization of the seven de-sumoylation enzymes (SENP1, 2, 3, 5, 6, 7 and 8) in the above 5 major ocular cell lines using immunocytochemistry. 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 7 major de-sumoylation enzymes (SENPs) in the 5 major ocular cell lines were determined with immunohistochemistry. The images were captured with a Zeiss LSM 880 confocal microscope. RESULTS: 1) The SENP1 was localized in both cytoplasm and nucleus of 3 human ocular cell lines, FHL124, HLE and ARPE-19; In N/N1003A and αTN4-1, SENP 1 was more concentrated in the cytoplasm. SENP1 appears in patches; 2) SENP2 was distributed in both cytoplasm and nucleus of all ocular cell lines in patches. In HLE and ARPE-19 cells, SENP2 level was higher in nucleus than in cytoplasm; 3) SENP3 was almost exclusively concentrated in the nuclei in all ocular cells except for N/N1003A cells. In the later cells, a substantial amount of SENP3 was also detected in the cytoplasm although nuclear SENP3 level was higher than the cytoplasmic SENP3 level. SENP3 appeared in obvious patches in the nuclei; 4) SENP5 was dominantly localized in the cytoplasm (cellular organelles) near nuclear membrane or cytoplasmic membrane ; 5) SENP6 was largely concentrated in the nuclei of all cell lines except for αTN4-1 cells. In the later cells, a substantial amount of SENP6 was also detected in the cytoplasm although nuclear SENP6 level was higher than the cytoplasmic SENP6 level. 6) SENP7 has an opposite localization pattern between human and animal cell lines. In human cell lines, a majority of SENP7 was localized in nuclei whereas in mouse and rabbit lens epithelial cells, most SENP7 was distributed in the cytoplasm. SENP8 was found present in human cell lines. The 3 human ocular cell lines had relatively similar distribution pattern. In FHL124 and ARPE-19 cells, SENP8 was detected only in the cytoplasm, but in HLE cells, patches of SENP8 in small amount was also detected in the nuclei. CONCLUSIONS: Our results for the first time defined the differential distribution patterns of seven desumoylation enzymes (SENPs) in 5 major ocular cell lines. These results help to understand the different functions of various SENPs in maintaining the homeostasis of protein sumoylation patterns during their functioning processes.

Analysis of the Differential Expression Patterns of Sumoylation Enzymes E1, E2 and E3 in Ocular Cell Lines.

PURPOSE: Protein sumoylation is a well established regulatory mechanism to control many cellular processes such as chromatin structure dynamics, transcriptional regulation of gene expression, cell proliferation and differentiation, cell transformation and carcinogenesis, autophagy and senescence. In the vertebrate vision system, we and others have revealed that sumoylation plays important roles in regulating differentiation of several ocular tissues during eye development. To further elucidate the functional mechanisms of sumoylation, in vitro assay systems are needed. Currently, the five major cell lines including αTN4-1, FHL124, HLE, N/N1003A and ARPE-19 have been extensively used to test the biochemical and molecular aspects of normal vision physiology and various disease processes. Thus, we conducted the study on the expression patterns of the three types of sumoylation enzymes, the activating enzymes SAE1 and UBA2, the conjugating enzyme UBC9, and the ligating enzymes such as RanBP2 and PIAS1 in these 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 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) For the mRNAs encoding E1 SAE1 and UBA2, E2 UBC9 and E3 PIAS1, the highest level of expression was observed in αTN4-1 cells; For the mRNA encoding RanBP2, the highest level of expression was detected in N/N1003A cells; 2) In contrast to the mRNA expression patterns, a similar level of the SAE1 protein was observed in the all five cell lines, and so is true with UBA2 protein in all cells except for N/N1003A where over fourfold of enrichment in UBA2 protein was observed compared with other cell lines; 3) A similar level of UBC9 protein was also detected in all cells except for N/N1003A where more than one-fold of decrease in UBC9 level was found compared with other cell lines; 4) For E3 ligases, we did not identify the regular PIAS1 band in N/N1003A cells, the remaining cells have a level of PIAS1 with difference of less than 0.6-fold; all cells except for FHL124 cells have a similar level of RanBP2, and a 70% drop in RanBP2 was observed in FHL124 cell. CONCLUSIONS: Our determination of the differential expression patterns of the three types of sumoylation enzymes in the 5 ocular cell lines help to understand sumoylation 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.

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 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.