leupeptins and Cataract

To investigate the effect of proteasome inhibitor MG132 on the apoptosis of bovine lens epithelial cells (BLECs), the cells were treated with MG132 at different concentrations for 12, 24 and 36 h. The cell viability was analyzed by MTT assay and the effect of MG132 on the apoptosis of BLECs was assessed by flow cytometry (FCM). The results showed that after treatment for the same period, the inhibitory effect of MG132 on BLECs proliferation was enhanced with the increment of the concentration of MG132 (0, 2, 5, 10, mumol/L) (P<0.05). The 50% inhibiting concentration (IC(50)) was 2.03 mumol/L when the BLECs were treated with MG132 for 36 h. MG132 also induced the apoptosis of BLECs obviously. FCM showed that the apoptosis index of the cells treated by MG132 at 2 mumol/L for 12 h was (20.24+/-1.51)%, and that of the control was (0.98+/-0.20)% respectively (P<0.01, n=3). It was concluded that MG132 could lead to apoptosis of BLECs. The decrease of proteasome activity may play an important role in the formation and development of cataract.

Topics: Animals; Apoptosis; Cataract; Cattle; Cells, Cultured; Cysteine Proteinase Inhibitors; Epithelial Cells; Lens, Crystalline; Leupeptins

The durability of the ubiquitin proteasome pathway in the mammalian lens makes this enzyme system a potential contributor to certain cataracts and posterior capsular opacification (PCO). The present study addresses proteasome involvement in TGF-beta induced, cataract-associated gene activation in human lens cells.. HLE B-3 cells were treated with TGF-beta, in combination with the proteasome inhibitors MG-132 or lactacystin. TGF-beta target gene expression was measured by semiquantitative RT-PCR. Annexin-FITC staining and flow cytometry were used to assess apoptosis levels. Western blot analyses were performed with anti-SnoN and anti-Smad2 antibodies.. TGF-beta induced the expression of alpha-smooth muscle actin, fibronectin, and TGF-beta-inducible gene mRNA in HLE B-3 cells and primary cultured human lens cells from donor tissues. TGF-beta also induced a time-dependent decrease in the level of the Smad repressor SnoN. Gamma-glutamyl-cysteine synthetase (gamma-GCS) mRNA levels decreased in the presence of TGF-beta. Proteasome inhibitor cotreatment blocked the induction of alpha-SMA mRNA, the loss of SnoN protein, the decrease in gamma-GCS mRNA, and TGF-beta-induced apoptosis.. The HLE B-3 cell line and primary cultured human lens cells respond similarly to TGF-beta treatments by activating cataract-related gene expression. This response in both of these model systems is blocked by inhibiting the proteasome. This suggests that the proteasome can mediate cataract and PCO-associated changes and therefore is a novel target of medical therapy.

Topics: Acetylcysteine; Actins; Annexins; Blotting, Western; Cataract; Cells, Cultured; Cysteine Proteinase Inhibitors; Epithelial Cells; Fibronectins; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Glutamate-Cysteine Ligase; Humans; Intracellular Signaling Peptides and Proteins; Lens, Crystalline; Leupeptins; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad2 Protein; Transcriptional Activation; Transforming Growth Factor beta

Posterior capsular opacification (PCO) is caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the remaining lens epithelial cells (LECs) after cataract surgery. Studies have shown that proteasome inhibition interferes with EMT and remodeling of the extracellular matrix. This study was conducted to investigate suppression of LEC proliferation by proteasome inhibition and its signaling pathway.. HLE B-3 cells and human lens epithelium explants from 17- to 20-week fetal lenses were cultured and treated with TGF-beta2 (1 or 10 ng/mL), FGF-2 (20 or 50 ng/mL), HGF (10 ng/mL) and 5 or 10 muM MG132. LEC proliferation was determined using both the WST-1 reagent and proliferating cell nuclear antigen (PCNA) expression. Protein expression was observed by Western blot analysis. Transfection with p21/p27 siRNA was performed to evaluate the mechanism of the antiproliferative effect of proteasome inhibition.. TGF-beta2 suppressed proliferation of HLE B-3 cells, whereas FGF-2 and HGF enhanced proliferation. Proliferation suppression by TGF-beta2 was blocked by adding FGF-2 or HGF. Proteasome inhibitor (MG132) treatment strongly inhibited the proliferation of LECs, either alone or in the presence of TGF-beta2, FGF-2, or HGF. These findings were confirmed by observing PCNA expression. Similar results were obtained with primary human LECs. Expression of cell cycle regulatory proteins was determined to evaluate the mechanism of the antiproliferative activity of proteasome inhibition. MG132 caused a significant increase in p21 and p27 protein and decrease in CDK2, but no change in p53, p57, CDK4, or CDK6 protein. The antiproliferative effect of MG132 was significantly reversed in samples transfected with p21 and p27 siRNA, which reduced p21 and p27 protein expression to very low levels that remained below basal control levels, even after treatment with MG132.. Proteasome inhibition decreases the proliferation of LECs in the presence or absence of TGF-beta2, FGF-2, and HGF. This process is mediated in part by an increase in p21 and p27 proteins. These findings suggest that proteasome inhibitors are good candidates for blocking development of PCO.

Topics: Blotting, Western; Cataract; Cell Proliferation; Cells, Cultured; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Proteinase Inhibitors; Epithelial Cells; Fibroblast Growth Factor 2; Hepatocyte Growth Factor; Humans; Intracellular Signaling Peptides and Proteins; Lens Capsule, Crystalline; Lens, Crystalline; Leupeptins; Proliferating Cell Nuclear Antigen; Proteasome Inhibitors; RNA, Small Interfering; Tetrazolium Salts; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta2

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, is an important mechanism for protection against carcinogenesis. The transcription factor Nrf2, which binds to the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes, is essential for the induction of these enzymes. We have investigated the effect of the potent enzyme inducer and anticarcinogen 3H-1,2-dithiole-3-thione (D3T) on the fate of Nrf2 in murine keratinocytes. Both total and nuclear Nrf2 levels increased rapidly and persistently after treatment with D3T but could be blocked by cotreatment with cycloheximide. Nrf2 mRNA levels increased approximately 2-fold 6 h after D3T treatment. To examine the transcriptional activation of Nrf2 by D3T, the proximal region (1 kb) of the nrf2 promoter was isolated. Deletion and mutagenesis analyses demonstrated that nrf2 promoter-luciferase reporter activity was enhanced by treatment with D3T and that ARE-like sequences were required for this activation. Gel shift assays with nuclear extracts from PE cells indicated that common factors bind to typical AREs and the ARE-like sequences of the nrf2 promoter. Direct binding of Nrf2 to its own promoter was demonstrated by chromatin immunoprecipitation assay. Overexpression of Nrf2 increased the activity of the nrf2 promoter-luciferase reporter, while expression of mutant Nrf2 protein repressed activity. Thus, Nrf2 appears to autoregulate its own expression through an ARE-like element located in the proximal region of its promoter, leading to persistent nuclear accumulation of Nrf2 and protracted induction of phase 2 genes in response to chemopreventive agents.

Topics: Animals; Anticarcinogenic Agents; Antioxidants; Cataract; Cell Line; Cell Nucleus; Cysteine Endopeptidases; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Genes, Reporter; Leupeptins; Mice; Multienzyme Complexes; Mutagenesis, Site-Directed; NF-E2-Related Factor 2; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Response Elements; RNA, Messenger; Thiones; Thiophenes; Trans-Activators; Transcription, Genetic

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.

Topics: Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Culture Techniques; Cysteine Proteinase Inhibitors; Dipeptides; Drug Interactions; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Lens, Crystalline; Leucine; Leupeptins; Molecular Sequence Data; Rats; Rats, Sprague-Dawley

Lens-protein-degrading activity in human aqueous humor was determined biochemically. Proteolytic activity was found in the pellet of aqueous humor from patients with Morgagnian cataract, traumatic cataract, after cataract, traumatic lens luxation, and phacolytic glaucoma. The activity was found at pH 3.8 and it was inhibited partly by pepstatin or leupeptin. Possibly, cathepsin D and lysosomal thiol proteinases in the macrophages play a major role in the degradation of escaping lens protein under pathologic conditions.

Topics: Animals; Aqueous Humor; Cataract; Cattle; Crystallins; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Leupeptins; Pepstatins; Protease Inhibitors

Incubation of lens in Ca2+-containing media, considered by several investigators to be a useful model of cataract formation, gave rise to significant alterations in the covalent structures of various proteins. In rabbit lens, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used after reduction of disulfides in urea, the most readily observable changes were (i) disappearance of 210K, 95K, and 60K proteins, (ii) modifications of alpha crystallin subunits, (iii) alterations of beta H crystallins, and (iv) de novo production of 55K and higher molecular weight polymers. The addition of leupeptin inhibited the disappearances of 210K, 95K, and 60K proteins and the alteration of alpha crystallins, suggesting that all these were caused by a Ca2+-activated protease. The proteolytically sensitive 60K species was identified as vimentin, a component of intermediate filaments. Formation of the 55K material and of higher molecular weight polymers during Ca2+ treatment of the lens could be prevented by histamine, a compound known to inhibit the transglutaminase-mediated cross-linking of proteins by epsilon-(gamma-glutamyl)lysine peptide bonds in other biological systems. It could also be shown by immunoblotting that an antibody raised against the 55K material reacted selectively with beta crystallins of normal lens. This indicates that the 55K product is in all likelihood an essential intermediate toward higher polymers and that the 55K product is a cross-linked dimer of certain polypeptides of beta crystallin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Cataract; Cross Reactions; Crystallins; Electrophoresis, Polyacrylamide Gel; Lens, Crystalline; Leupeptins; Macromolecular Substances; Molecular Weight; Rabbits; Vimentin