Venkat N. Reddy

Ubiquitin-dependent lysosomal degradation of the HNE-modified proteins in lens epithelial cells

4‐Hydroxynonenal (HNE), a highly reactive lipid peroxidation product, may adversely modify proteins. Accumulation of HNE‐modified proteins may be responsible for pathological lesions associated with oxidative stress. The objective of this work was to determine how HNE‐modified proteins are removed from cells. The data showed that αB‐crystallin modified by HNE was ubiquitinated at a faster rate than that of native αB‐crystallin in a cell‐free system. However, its susceptibility to proteasome‐dependent degradation in the cell‐free system did not increase. When delivered into cultured lens epithelial cells, HNE‐modified αB‐crystallin was degraded at a faster rate than that of unmodified αB‐crystallin. Inhibition of the lysosomal activity stabilized HNE‐modified αB‐crystallin, but inhibition of the proteasome activity alone had little effect. To determine if other HNE‐modified proteins are also degraded in a ubiquitin‐dependent lysosomal pathway, lens epithelial cells were treated with HNE and the removal of HNE‐modified proteins in the cells was monitored. The levels of HNE‐modified proteins in the cell decreased rapidly upon removal of HNE from the medium. Depletion of ATP or the presence of MG132, a proteasome/lysosome inhibitor, resulted in stabilization of HNE‐modified proteins. However, proteasome‐specific inhibitors, lactacystin‐β‐lactone and epoxomicin, could not stabilize HNE‐modified proteins in the cells. In contrast, chloroquine, a lysosome inhibitor, stabilized HNE‐modified proteins. The enrichment of HNE‐modified proteins in the fraction of ubiquitin conjugates suggests that HNE‐modified proteins are preferentially ubiquitinated. Taken together, these findings show that HNE‐modified proteins are degraded via a novel ubiquitin and lysosomal‐dependent but proteasome‐independent pathway.

Cellular distribution of lens epithelium-derived growth factor (LEDGF) in the rat eye: loss of LEDGF from nuclei of differentiating cells

Lens epithelium-derived growth factor (LEDGF) enhances the survival and growth of cells. To understand LEDGFs spatial localization and its putative function(s) during proliferation and differentiation, we localized LEDGF during terminal differentiation in whole rat lenses, lens epithelial cell (LEC) explants stimulated with FGF-2, and insulin, iris, human LECs with lentoids. In addition, intracellular localization of LEDGF was performed in other ocular tissues: ciliary body, retina, and cornea. We found the immunopositivity of nuclear LEDGF decreased in LECs of the equatorial region. In contrast, immunopositivity of LEDGF was detected in the cytoplasm of LECs and superficial fiber cells. After treating LEC explants with FGF-2 and insulin, which are known to be differentiating factors for LECs, the nuclei of these cells showed no LEDGF immunopositivity, but explants did express p57kip2, a differentiation marker protein. Also, immunopositive LEDGF was not detected in the nuclei of differentiated cells, lentoid body, and corneal epithelial cells. This demonstrated that the loss of LEDGF from the nucleus may be associated with the process of terminal differentiation that might be in some way common with the biochemical mechanisms of apoptosis. The spatial and temporal distribution of LEDGF in the present study also provides a vision for further investigation as to how this protein is involved in cell fate determination.

Inhibitory Effects of Arg-Gly-Asp (RGD) Peptide on Cell Attachment and Migration in a Human Lens Epithelial Cell Line

Posterior capsule opacification (PCO) after cataract surgery is caused by growth of residual human lens epithelial (HLE) cells on the posterior capsule. We have shown that extracellular matrix (ECM) is an essential factor for HLE cell attachment and migration. The purpose of this study was to examine the inhibitory effects of Arg-Gly-Asp (RGD) peptide on cell attachment and migration in an HLE cell line. HLE cell line cells (SRA 01/04) that were obtained by transfection of large T antigen of SV40 were cultured in the absence of serum. The culture dishes were coated with type IV collagen, laminin or fibronectin, and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) RGD peptide (0.1, 0.3, 1.0, 2.0 mg/ml) was added to the medium. The number of attached cells was counted after 90 min of incubation, and the inhibitory effects of GRGDSP RGD peptide on cell attachment were calculated. Cell attachment on the fibronectin-coated dishes was inhibited by GRGDSP RGD peptide at concentrations higher than 0.3 mg/ml; the inhibitory rate was 80% at a concentration of 2.0 mg/ml. The inhibition of cell attachment by GRGDSP RGD peptide on laminin-coated dishes appeared only at a concentration of 2.0 mg/ml, whereas no effects were observed on the type IV collagen-coated dishes. The inhibitory effects of GRGDSP RGD peptide on cell migration were measured in medium containing 2.0 mg/ml of GRGDSP RGD peptide after 1, 3, 5 and 7 days of culture. Cell migration was inhibited by GRGDSP RGD peptide from 1 day of culture on the fibronectin-coated dishes and from 5 days of culture on the laminin-coated dishes, whereas no effects were observed on the type IV collagen-coated dishes. GRGDSP RGD peptide inhibited cell attachment and migration on laminin and fibronectin that have RGD sequences. These data suggested that RGD peptide may have the potential to prevent PCO.

Functional analysis of the promoter and chromosomal localization for human LEP503, a novel lens epithelium gene.

LEP503 is a novel gene product isolated from lens epithelial cells by a subtractive cDNA cloning strategy. It is highly conserved in different vertebrate species and developmentally regulated in postnatal rat lens, suggesting that LEP503 may be an important lens epithelium gene involved in the processes of lens epithelial cell differentiation. The expression of LEP503 is highly restricted to lens epithelial cells in vivo. To investigate the molecular mechanisms regulating the promoter of the human LEP503, we cloned and characterized the promoter of the human LEP503 gene. The transcription start site was localized to a nucleotide C 22 base pairs (bp) 5 of the initiation methionine codon. By reporter gene transfection experiments, we found that approximately 2.5-kb of LEP503 5-flanking sequence directed high level luciferase activity in human lens epithelial cells; further deletion analysis revealed positive regulatory element between bp -401 and +22. Mutation analysis in each of the seven potential binding sites for transcription factors within the region between -401 and +22 showed that the AP-1 element at -131 and the Sp1 element at -48 are the most important sites for the tissue-specific expression of LEP503. Consistent with lens epithelial cell-restricted expression of LEP503 mRNA, we found that the approximately 2.5-kb 5-flanking sequence directed high-level promoter activity in lens epithelial cells but not in other cell types. Understanding the LEP503 promoter will allow us to investigate lens epithelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to lens epithelial cells. The LEP503 gene is mapped to human chromosome 1q22, the same location to which zonular pulverulent cataract was previously mapped.