LEDGF activation of PKC gamma and gap junction disassembly in lens epithelial cells

Lens epithelium-derived growth factor (LEDGF) has been shown to enhance survival of lens epithelial cells (LECs) against stress. The objectives of these studies are to determine how LEDGF controls PKC gamma activity in normal LECs: how this control of PKC gamma regulates the phosphorylation of Connexin 43, the inhibition of gap junction activity, and the prevention of assembly of gap junctions in LECs. A rabbit LEC line, N/N1003A, was grown in the absence or presence of LEDGF. PKC gamma protein was translocated from the cytosolic fractions to the membrane fractions upon addition of LEDGF at 10 ng ml(-1). In whole cell extracts of N/N1003A cells, co-immunoprecipitation assays showed a protein-protein interaction between PKC gamma and Connexin 43. In the presence of LEDGF the activation of PKC gamma enhanced the phosphorylation of Connexin 43 by four-fold compared to the absence of LEDGF. The addition of LEDGF for 30 min resulted in a 65% decrease in gap junction Connexin 43 at the cell surface and a 70% decrease in gap junction activity. These results suggest that the activation of PKC gamma by LEDGF plays a major role in gap junction assembly/disassembly, which may enhance survival of LECs against osmolarity-stress induced by high sugar concentration.     

Hypoxia-inducible factor-1 (HIF-1) pathway activation by quercetin in human lens epithelial cells

Quercetin is a dietary bioflavonoid which has been shown to inhibit lens opacification in a number of models of cataract. The objectives of this study were to determine gene expression changes in human lens epithelial cells in response to quercetin and to investigate in detail the mechanisms underlying the responses. FHL-124 cells were treated with quercetin (10 μM) and changes in gene expression were measured by microarray. It was found that 65% of the genes with increased expression were regulated by the hypoxia-inducible factor-1 (HIF-1) pathway. Quercetin (10 and 30 μM) induced a time-dependent increase in HIF-1α protein levels. Quercetin (30 μM) was also responsible for a rapid and long-lasting translocation of HIF-1α from the cytoplasm to the nucleus. Activation of HIF-1 signaling by quercetin was confirmed by qRT–PCR which showed upregulation of the HIF-1 regulated genes EPO, VEGF, PGK1 and BNIP3. Analysis of medium taken from FHL-124 cells showed a sustained dose-dependent increase in VEGF secretion following quercetin treatment. The quercetin-induced increase and nuclear translocation of HIF-1α was reversed by addition of excess iron (100 μM). These results demonstrate that quercetin activates the HIF-1 signaling pathway in human lens epithelial cells.     

Growth factor-induced proliferation in corneal epithelial cells is mediated by 12(S)-HETE

PURPOSE: Previous studies in our laboratory have shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a product of 12-lipoxygenase (12-LOX) activity, is the predominant metabolite formed in rabbit corneas after injury. The present study was undertaken to investigate the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) on 12-LOX expression and activity. We also investigated whether 12(S)-HETE mediated the growth factor-induced proliferation of corneal epithelial cells. METHODS: Rabbit corneas were stimulated with EGF, HGF, and KGF (10 ng ml(-1)) for different times. 12-LOX activity was assayed by incubating corneal microsomal preparations with radiolabeled arachidonic acid (AA) as substrate. For inhibitor studies, the microsomes were pretreated with 12-LOX-specific inhibitors baicalein (BC) or cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC). Lipid extracts were injected onto an Ultramex 5 microm C(18) column and radioactivity was monitored online by a Radiomatic Flo-One Beta detector. Stereochemical analysis of 12-HETE product was determined by chiral-phase HPLC. To evaluate the effects of growth factors on 12-LOX mRNA expression, mRNA was extracted at several time points (12, 24, 36, 48 hr) and subjected to real-time PCR. For 12-LOX protein expression, microsomal preparations from 24- and 48-hr incubations were analyzed by Western blot. In cell-proliferation studies, epithelial cells treated with EGF, HGF, or KGF for 24, 48, and 72 hr were measured with a CyQUANT cell-proliferation assay kit. To determine the role of growth factor-induced 12(S)-HETE synthesis on corneal epithelial cell proliferation, cells were pretreated with 12-LOX-specific inhibitors BC or CDC prior to growth-factor supplementation. RESULTS: Stimulation with EGF, HGF, or KGF for 12 hr induced 12-LOX mRNA expression in rabbit corneal epithelial cells. This gene induction was followed by an increase in protein expression at 24 and 48 hr and a marked increase in 12(S)-HETE synthesis when compared to untreated controls. At 24-hr incubations, KGF showed a greater capacity than did EGF and HGF to stimulate microsomal 12-LOX activity, while at 48 hr 12(S)-HETE synthesis was significantly greater in EGF-treated cells as compared to that of HGF- and KGF-treated cells. Pretreatment with 12-LOX inhibitors blocked the growth factor-induced increase in 12(S)-HETE synthesis. Stimulation with growth factors or 12(S)-HETE for 24, 48, and 72hr produced a significant increase in corneal epithelial proliferation, which was partially inhibited by pretreatment of cells with 12-LOX-specific inhibitors. CONCLUSION: These findings suggest that EGF, HGF, and KGF stimulate 12(S)-HETE production in rabbit corneal epithelial cells through gene induction of 12-LOX. Furthermore, 12(S)-HETE may play a role in regulating epithelial cell proliferation and the rate of corneal re-epithelialization following an injury.     

Regulation of potassium transport in human lens epithelial cells

The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1 mM) and bumetanide (5 microM) discriminated between the Na/K pump ( approximately 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) ( approximately 50%). Cl-replacement with nitrate or sulfamate revealed <10% residual [ouabain+bumetanide]-insensitive K-Cl cotransport (KCC). At 0.3-0.5 mM, NEM stimulated the Na/K pump by 2-fold independent of external Na, KCC between 2 and 4-fold, and abolished approximately 90% of NKCC. Calyculin-A, a serine/threonine protein phosphatase-1 inhibitor, did not affect NKCC but inhibited KCC, whereas 10 microM staurosporine, a serine/threonine kinase inhibitor, abolished NKCC, and stimulated KCC only when followed by NEM treatment. The tyrosine-kinase inhibitor genistein, at concentrations >100 microM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria.     

Temporal and regional production of 12(S)hydroxyeicosatetraenoic acid [12(S)-HETE] in rat lens.

Enzymatic production of 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] from exogenous arachidonic acid (AA) was studied in the 9000 g supernatant fraction of either whole rat lens or the capsule-epithelium and cortex-nucleus regions with age (4-180 days). Whole rat lens 12(S)-HETE synthetic capacity measured either by RIA or HPLC was significantly decreased with lens growth. 12(S)-HETE production was highest in the 4-day-old rat lens, the earliest time point measured, and declined to background levels by 6 months of age. Regional studies demonstrated that the capsule-epithelium possessed a higher specific activity in comparison to the cortex-nucleus. However, lipoxygenase activity decreased significantly faster in the capsule-epithelium compared to the cortex-nucleus. The bulk of the 12-lipoxygenase activity was located in the cortex-nucleus due to the higher amount of tissue in the region. The elevated lipoxygenase activity in the neonate and its rapid decline with growth suggests that the enzyme may contribute in transforming the epithelial cells to fiber cells.     

Transcriptional activation in lens epithelial cells following an ocular blunt trauma

PURPOSE: To determine whether an ocular blunt trauma activates anterior ocular segment (cornea and lens) by examining the expression patterns of c-fos and c-jun mRNAs in these tissues of an eye of adult rat following a blunt trauma. SETTING: Department of Ophthalmology, Wakayama Medical University School of Medicine, Kimiidera, Wakayama, Japan. METHODS: Adult Wistar rats (n=36) were generally anesthetized by ether inhalation. One eye was hit with an iron sphere (30 gram) that fell to the eye from 1 m. After the procedure, the animals were killed and the affected eye was enucleated at 15, 30, 60, 120, and 180 minutes. In situ hybridization using radiolabeled oligoprobes was used to detect mRNAs of c-fos and c-jun in tissue. RESULTS: The c-fos and c-jun mRNAs were not detected in the epithelium of uninjured cornea and lens by in situ hybridization. The mRNAs for c-fos and c-jun were then detected in corneal epithelium from 15 to 60 minutes posttreatment, and were no longer observed thereafter. In lens epithelium, mRNA for c-fos or c-jun were transiently detected from 15 to 60 minutes or 30 minutes posttreatment, respectively. CONCLUSION: The c-fos and c-jun mRNAs were transiently expressed in corneal and lens epithelial cells after blunt trauma. Ocular blunt trauma activates corneal and lens epithelial cells without apparent corneal ablation or direct injury in the lens epithelium. Such activation in lens epithelium might be involved in cataractogenesis.     

X-ray induced DNA strand break induction and rejoining in cultured bovine lens epithelial cells.

DNA strand break induction and rejoining in cultured bovine lens epithelial cells were measured by the alkaline unwinding technique followed by hydroxyapatite chromatography. DNA damage is described in terms of dose effect curves for unwinding immediately after irradiation (0 min) and for unwinding after a recovery period (30 min). The dose effect curves obtained are purely exponential with D0 values of 29.3 Gy (0 min) and 138 Gy (30 min), respectively. Rejoining data are given in terms of rejoining enhancement curves with half-times for overall rejoining being in the range from 4 to 10 min, independent of the dose. Kinetics of strand breaks are also presented in terms of decay curves, which are best described by the sum of three exponential components. The half-times of these components have been determined as tau(I) approximately 2 min, tau(II) approximately 15 min and tau(III) approximately 500 min. These components of damage decay curves are discussed in relation to cell killing.     

Biosynthesis of proteoglycans by lens epithelial cells of cataractous mouse (Nakano strain)

The capsules (with epithelial cells attached) of lenses from normal and cataractous mice (Nakano strain) were biosynthetically labeled in vitro with radioactive precursors. The labeled macromolecules were chromatographed on a Sepharose CL-4B column and analyzed by specific enzyme digestion. The incorporation of [3H]-proline and [3H]-glucosamine into macromolecules was comparable in the cataract and normal capsules, while that of [35S]-sulfate was reduced by 60% in the cataract capsules, indicating that the proteoglycan synthesis was specifically decreased in the cataract lens. Glycosaminoglycan analyses showed an increased synthesis of hyaluronic acid and decreased synthesis of heparan sulfate in the cataract capsules. It is possible that the alterations in the synthetic level and glycosaminoglycan components of proteoglycan affect the permeabilities of macromolecules to lens capsule and lead to cataract in Nakano mouse lens.     

Identification of 12(S)-hydroxyeicosatetraenoic acid in the young rat lens

Evidence is presented indicating that young rat lens has the capacity to synthesize 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) from exogenous arachidonic acid (AA). A 9,000 xg supernatant prepared from 15 day old rat lenses, when incubated with calcium and U-[14C]-AA, generated a radiolabelled product with a retention time identical to authentic unlabelled 12-HETE in two different HPLC solvent systems. Mass spectral analysis provided evidence that the metabolite was 12-HETE while chiral studies demonstrated the exclusive presence of the 12(S) isomer. Radiolabelled 12-HETE synthesis was inhibited by preincubating the 15 day old rat lens supernatant with 0.2 Mm curcumin, a mixed cyclooxygenase/lipoxygenase inhibitor. Radiolabelled 12(S)-HETE synthetic capacity was highest in the 4 day old rat lens, the earliest time period measured, and rapidly declined with age reaching negligible levels at about 4 months. These results suggest that the young rat lens possess the biosynthetic capacity to generate radiolabelled 12(S)-HETE from U-[14]C-AA. The profile of 12-HETE synthetic activity suggests a possible regulatory function of the hydroxylated AA metabolite in the developing lens.     

Iron uptake by cultured lens epithelial cells

Background: Transferrin and Fe concentrations increase in the intraocular fluids in pathological conditions and the lens accumulates Fe during ocular inflammation. Tissues take up Fe from transferrin by two mechanisms, receptor-medicated endocytosis of diferric transferrin and a process occurring at the cell membrane which may be mediated by an oxido-reductase. However, Fe metabolism, transport and storage have not been previously investigated in the lens. This study was designed to characterize the uptake of Fe from transferrin by lens epithelial cells in culture. Methods: Primary, secondary and tertiary cultures of canine lens epithelial cells and cultures obtained from cataractous lenses were studied. Uptake of ⁵⁹Fe from transferrin by these cultured cells was measured. Transferrin receptor populations were determined in receptor-binding assays. Results: There was a distinct relationship between the amount of Fe-transferrin added and the amount of Fe taken up, which was linear for the primary cultures but significantly reduced for the secondary, tertiary and cataract cultures (252±21, 169±14, 153±14 and 96±2 ng Fe/mg protein, respectively).Transferring receptor expression in lens cell cultures was reduced 10-fold within 2 days of addition of serum to cells grown in low-Fe, serum-free medium for 1 week. Conclusions: The reduction of Fe uptake by the subcultured and cataract cell lines probably reflects a decrease in transferrin receptor expression and in the activity of an alternative pathway for Fe transferrin uptake occurring over time. This reduced Fe uptake may result from long-term exposure to relatively high Fe concentration in the media. A reduction in the expression of the transferrin receptor after incubation with high concentrations of Fe supports this conclusion.     

Age-related changes of human lens epithelial cells in vivo.

We examined the density and morphology of lens epithelial cells (LECs) in vivo in a group of normal volunteers and cataract patients by using a newly developed noncontact specular microscope. There was a statistically significant decrease in the cell density of LECs in a group of cataract patients over the age of 80 years. The coefficient of variation of the cell area and the number of large black spots that were observed in the enhanced specular images were not related to aging or cataract formation. Our data indicate that the cell density of LECs decreases after reaching the age of 80, but cataract formation does not affect the cell density or the coefficient of variation of the cell area until the age of 80.     

Matrix metalloproteinase secretion is stimulated by TGF-beta in cultured lens epithelial cells.

PURPOSE. To determine if TGF-beta regulates the expression of metalloproteinases in chick lens annular pad cells. METHODS. The activity of secreted matrix metalloproteinases was examined with gelatin zymography in primary cultures exposed to TGF-beta. RESULTS. Metalloproteinases with electrophoretic mobilities corresponding to MMP2 and MMP9 were tentatively identified. Activated, processed forms of the two metalloproteinases were also observed. Plasminogen activators potentially capable of initiating metalloproteinase cascades were concomitantly elicited. Metalloproteinase secretion was shown to be specific for TGF-beta stimulation and independent of substrate composition. CONCLUSIONS. These results indicate that TGF-beta-mediated processes could be responsible for localized lens capsular heterogeneity, establishing a substrate suitable for cell migration or the release of matrix-bound factors which influence the terminal differentiation of lens cells.     

地塞米松诱导晶状体上皮细胞凋亡的实验研究

目的 探讨地塞米松(dexamethasone,Dex)诱发体外培养的牛晶状体上皮细胞(lens epithelial cells,LEC)凋亡情况.方法 取健康1 a龄内牛眼,提取LEC,传代培养;分为2组:对照组和Dex组.透射电镜观察Dex诱导LEC凋亡的超微结构改变,流式细胞术探讨Dex对LEC核内DNA含量的影响.结果 透射电镜下可观察到Dex组LEC细胞核内染色质凝集、固缩、边集、核碎裂等细胞凋亡的形态学改变,核内DNA含量下降,出现典型的细胞凋亡峰,凋亡百分率从第1天的(1.522±0.593)%提高到第7天的(25.318±3.432)%,与对照组相比有统计学意义(P<0.01),并呈时间依赖关系.结论 Dex诱导LEC凋亡可能是激素性白内障发生发展的细胞学和分子生物学机制之一.     

Human lens cells in culture. I. Isolation of adult lens epithelial cells from lens capsule preparations and reactivation of nucleus-containing fiber cells

BACKGROUND: Bovine lens epithelial cells in culture revealed a high sensitivity against micromolar concentrations of linoleic acid. To prove the assumption that unsaturated free fatty acids are risk factors for cataractogenesis, human lens cell lines are needed. Furthermore, the reactivation of nucleus-containing fiber cells to mitotic growth may hint at their role in after cataract genesis. MATERIAL AND METHODS: Epithelium-capsule-preparations obtained by capsulorhexis were cultured in serum containing medium. Subculturing of these adult human lens epithelial cells was done by trypsinization. Fiber cell bundles from the equator region of a fetal human lens were transferred into culture medium. Aggregates of nucleus containing fiber cells were isolated from floating fiber cell bundles by trypsinization. Subculturing and cryoconservation of suitable cell lines. RESULTS: Primary culture of epithelium-capsule-preparations results in flattening, migration and proliferation of adult human lens epithelial cells. Nucleus containing fiber cells were reactivated to mitotic growth after adhesion to a suitable substratum. Established cell lines were received from adult human lens epithelial cells and fetal human fiber cells after repeated subculturing. CONCLUSIONS: Lens-capsule-preparations available from cataract surgery are well suited for the isolation of human lens cell lines, which were needed for testing cytotoxicity of drugs and for tracing of cataractogenic risk factors. The finding that nucleus containing fiber cells from the equator of human lenses can be reactivated to proliferating cells let us suppose, that these cells, which can not be removed easily from the posterior lens capsule, contribute to the after cataract formation.