SUMOylation and deacetylation affect NF-κB p65 activity induced by high glucose in human lens epithelial cells.

AIM: To explore the effects of IκBα SUMOylation and NF-κB p65 deacetylation on NF-κB p65 activity induced by high glucose in cultured human lens epithelial cells (HLECs). METHODS: HLECs (SRA01/04) were cultured with 5.5, 25, and 50 mmol/L glucose media for 24h, and with 50 mmol/L glucose media for 0, 12, and 24h respectively. SUMO1 and SIRT1 expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot (WB). IκBα and NF-κB p65 expressions were detected by WB. With NAC, DTT, MG132 or Resveratrol (RSV) treatment, SUMO1 and SIRT1 expressions were detected by WB. Protein expression localizations were examined by immunofluorescence and co-immunofluorescence. The effects of SUMO1 or SIRT1 overexpression, as well as MG132 and RSV, on the nuclear expression and activity of IκBα and NF-κB p65 were analyzed by immunoblot and dual luciferase reporter gene assay. RESULTS: SUMO1 and SIRT1 expressions were influenced by high glucose in mRNA and protein levels, which could be blocked by NAC or DTT. SUMO1 was down-regulated by using MG132, and SIRT1 was up-regulated under RSV treatment. IκBα nuclear expression was attenuated and NF-κB p65 was opposite under high glucose, while IκBα and NF-κB p65 location was transferred to the nucleus. SUMO1 or SIRT1 overexpression and MG132 or RSV treatment affected the nuclear expression and activity of IκBα and NF-κB p65 under high glucose condition. CONCLUSION: IκBα SUMOylation and NF-κB p65 deacetylation affect NF-κB p65 activity in cultured HLECs under high glucose, and presumably play a significant role in controlling diabetic cataract.

HIF-1α SUMOylation affects the stability and transcriptional activity of HIF-1α in human lens epithelial cells.

PURPOSE: High blood glucose can induce oxidative damage and result in diabetic cataract. Oxidative stress induces various signal pathways including HIF-1α transcriptional signal to attenuate the damage of lenses. Whether HIF-1α SUMOylation can increase the activation of HIF-1α or if high glucose can affect the SUMOylation of HIF-1α in cultured human lens epithelial cells (HLECs) is still unknown, as well as the function of HIF-1α SUMOylation in oxidative damage induced by high glucose in HLECs. In the present study, we examined SUMO and SUMO E3 (Cbx4 and PIASy) expression induced by high glucose, and investigated SUMO or SUMO E3 overexpression that enhanced HIF-1α SUMOylation in HLECs. METHODS: SRA01/04 cells, one kind of human lens epithelial cell line, were addressed in media with 5.5 mmol/l (normal control group), 25 mmol/l (high glucose1 group) and 50 mmol/l (high glucose2 group) final glucose respectively. Expression of SUMO1 ~ 4, Cbx4, PIASy, HIF-1α, GLUT1, and VEGFA were detected in the mRNA and protein levels by RT-PCR and Western blot analysis. Protein expression localization and co-localization were examined by immunofluorescence and co-immunofluorescence. The effects of SUMO overexpression, SUMO E3 overexpression, and Proteasome inhibitor MG132 respectively on the stability and transcriptional activity of HIF-1α were analyzed by immunoblot. RESULTS: High glucose treatment induced SUMO1-4 expression and enhanced the expression of Cbx4 and PIASy. It also increased the expression of HIF-1α, GLUT1, and VEGFA. The co-localization of HIF-1α and SUMO was mainly in the nucleus induced by high glucose. Further studies showed that SUMO overexpression or SUMO E3 overexpression could enhance HIF-1α stability and transcriptional activity in HLECs. Proteasome inhibitor MG132 protected the stability and transcriptional activity of HIF-1α in the SRA01/04 cells. CONCLUSIONS: HIF-1α SUMOylation affected the stability and transcriptional activity of HIF-1α in cultured human lens epithelial cells; SUMO overexpression or SUMO E3 overexpression enhanced the expression of HIF-1α, which is involved in inhibiting cell apoptosis and protecting lens opacification, and presumably plays a key role in protecting lenses from diabetic cataract.

Lactulose biosynthesis by ß-galactosidase from a newly isolated Arthrobacter sp.

Lactulose, a ketose disaccharide, is used in both pharmaceutical and food industries. This study was undertaken to screen and isolate potent ß-galactosidase-producing bacteria and to evaluate their enzymatic production of lactulose. Soil samples from fruit gardens were collected. One isolate designated LAS was identified whose cell extract could convert lactose and fructose into lactulose. The 16S rDNA gene analysis of LAS revealed its phylogenetic relatedness to Arthrobacter sp. The ß-galactosidase produced by LAS was purified 15.7-fold by ammonium sulfate precipitation and subsequent Phenyl-Sepharose hydrophobic chromatography. The optimum pH and temperature for lactulose synthesis by this ß-galactosidase were 6.0 and 20 °C, respectively. The low optimum temperature of this enzyme compared to the currently used ones for lactulose production has the advantage of reducing the nonenzymatic browning in biotransformations. The results indicated that Arthrobacter could be used as a novel bacterial ß-galactosidase source for lactulose production.