Differential effects of hepatocyte growth factor and keratinocyte growth factor on corneal epithelial cell cycle protein expression, cell survival, and growth.

Purpose: Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are secreted in the cornea in response to injury. In this study, we investigated the HGF- and KGF-mediated effect on the expression of cell cycle and apoptosis controlling proteins, cell survival, and growth in the corneal epithelium to better understand the possible role of their signaling mechanisms in repairing epithelial injuries.

Methods: The cell survival capability of HGF and KGF in epithelial primary cultures was evaluated by using a staurosporine-induced apoptosis model.

Regulation of Cdc42 expression and signaling is critical for promoting corneal epithelial wound healing.

Purpose: Cdc42, a member of Rho GTPases (guanosine triphosphatases), participates in cytokine- and growth factor-controlled biological functions in mammalian tissues. Here, we examined Cdc42 role in corneal epithelial wound healing and the influence of hepatocyte, keratinocyte, and epidermal growth factor (HGF, KGF, and EGF)-mediated signaling on Cdc42.

Methods: Epithelial wounds were created on the corneas of live rabbits by complete debridement and in rabbit corneal epithelial primary cultures through scratch injury.

Honokiol, a chemopreventive agent against skin cancer, induces cell cycle arrest and apoptosis in human epidermoid A431 cells.

Honokiol is a plant lignan isolated from bark and seed cones of Magnolia officinalis. Recent studies from our laboratory indicated that honokiol pretreatment decreased ultraviolet B-induced skin cancer development in SKH-1 mice. The aim of the present investigation was to study the effects of honokiol on human epidermoid squamous carcinoma A431 cells and to elucidate possible mechanisms involved in preventing skin cancer.

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells.

Background: alpha-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of alpha-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of alpha-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.

Phosphatidylinositol 3-kinase (PI-3K)/Akt but not PI-3K/p70 S6 kinase signaling mediates IGF-1-promoted lens epithelial cell survival.

Purpose: To investigate the ability of insulin-like growth factor (IGF)-1 to prevent apoptosis in lens epithelial cells and the involvement of phosphatidylinositol 3-kinase (PI-3K)/Akt and PI-3K/p70 S6 kinase (p70 S6K) signaling in the cell-survival process.

Methods: Apoptosis in rabbit lens epithelial cell cultures was induced by staurosporine (10 ng/mL). Cellular apoptosis was detected by identifying the characteristic ladder-like fragmentation of genomic DNA in agarose gels and the intense blue fluorescence exhibited by apoptotic nuclei of cells in live cultures in the presence of Hoechst 33,258 dye.

HGF protects corneal epithelial cells from apoptosis by the PI-3K/Akt-1/Bad- but not the ERK1/2-mediated signaling pathway.

Purpose: Cell survival is critical during corneal epithelial regeneration after injury, and growth factors could be fundamental in cytoprotection. The goal of this study was to investigate the involvement of the paracrine hepatocyte growth factor (HGF) in the prevention of corneal epithelial cell apoptosis and to identify signal transducers in this process.

Methods: Apoptosis in human and rabbit corneal epithelial (HCE and RCE) cells was induced with a nutrient-deprived exhausted medium (ExM) or by treatment with staurosporine (20-100 ng/mL) or the calcium ionophore A23187 (0.

Alterations in lens protein tyrosine phosphorylation and phosphatidylinositol 3-kinase signaling during selenite cataract formation.

Purpose: Protein tyrosine phosphorylation is an important event in the cell signal transduction process. Phosphatidylinositol-3 kinase (PI-3K) is an intracellular signal mediator and plays a key role in many cellular functions. In this study we have examined the changes in lens protein tyrosine phosphorylation and its impact on phosphatidylinositol 3-kinase (PI-3K) signaling during selenite cataract development.

Differential activation of phosphatidylinositol 3-kinase signaling during proliferation and differentiation of lens epithelial cells.

Purpose: To investigate whether phosphatidylinositol 3-kinase (PI-3K) signaling is involved in lens epithelial cell proliferation and differentiation promoted by growth factors.

Methods: Proliferation of rabbit lens epithelial cells grown in culture was measured with a DNA-binding fluorescent dye in a proliferation assay. Primary cultures of embryonic chicken lens epithelial cells that develop lentoids were used for differentiation-related studies, and delta-crystallin synthesis in these cultures was determined by metabolic labeling with [(35)S]methionine.

Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor.

Purpose: To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing.

Methods: A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF.

HGF- and KGF-induced activation of PI-3K/p70 s6 kinase pathway in corneal epithelial cells: its relevance in wound healing.

In this study we have investigated the involvement of PI-3K and its downstream target p70 S6K in the signaling response of corneal epithelial cells after HGF and KGF stimulation. HGF induced three- to five-fold increase in PI-3K activity in 5-10 min, whereas KGF stimulation resulted in two- to three-fold increase in activity in 2-10 min. Both growth factors also caused the phosphorylation of p70 S6K and stimulation of its activity.

Phosphatidylinositol 3-kinase in bovine lens and its stimulation by insulin and IGF-1.

Purpose: To identify and characterize phosphatidylinositol 3-kinase (PI-3K) in the lens and to study its involvement as a signal mediator in lens epithelial cells exposed to insulin and insulin-like growth factor (IGF)-1, which are known to induce lens epithelial cell proliferation and differentiation into fiber cells.

Methods: Concentric fiber cell layers from single bovine lens were prepared by dissolution in buffer. PI-3K activity in capsule-epithelium and fiber cell layers was determined after immunoprecipitation with antibodies against p85, the regulatory subunit of PI-3K.

Corneal epithelial wound healing increases the expression but not long lasting activation of the p85alpha subunit of phosphatidylinositol-3 kinase.

Purpose: Corneal epithelial wound healing is a complex process involving several growth factors whose interaction with tyrosine kinase receptors (RTK) leads to the recruitment of enzymes coupled to second messengers that propagate and amplify growth factor-induced signals inside the cells. Phosphatidylinositol-3 kinase (PI-3K) is one such enzyme. Here we have investigated changes in PI-3K activity and expression during re-epithelialization after in vivo and in vitro corneal injury.

Intense myristoylation of a single protein in the ocular lens.

A single protein of the ocular lens was intensely myristoylated following short term incubation of cultured bovine lens epithelial cells and intact rat lenses with 3H-myristic acid. It was acidic (pI <5), about 19 kDa and present exclusively in the cytosol of both cultured epithelial cells and the epithelium of the young rat lens. Fiber cell proteins were not labeled.

Selective changes in protein kinase C (PKC) isoform expression in rabbit corneal epithelium during wound healing. Inhibition of corneal epithelial repair by PKCalpha antisense.

Protein kinase C (PKC) isoforms display different sensitivities to modulators, tissue specificities and subcellular localizations. PKCalpha increases during rabbit corneal epithelial wound healing. Here we report differential expression of PKC isoforms in the cornea of rabbits at 1, 2, 4 and 8 days during re-epithelization.

Properties of alpha-crystallin bound to lens membrane: probing organization at the membrane surface.

Alpha crystallin, one of the three major soluble proteins of the eye lens, appears to be a natural extrinsic protein of lens plasma membrane. Membrane-immobilized alpha-crystallin could provide a template for the increased association of protein with lens membrane seen in aging and cataracts. Alpha-crystallin binds to lens membrane through both a high-affinity saturable and low-affinity nonsaturable process.

Protein associated with human lens 'native' membrane during aging and cataract formation.

Plasma membrane contains extrinsic as well as intrinsic proteins. Changes in the extrinsic proteins of lens membrane during human aging and cataract formation have not been investigated in detail. Unlike previous studies which examined lens membrane after being stripped of extrinsic proteins by treatment with chaotropic agents, we have isolated whole or 'native' lens membrane on a sucrose gradient by ultracentrifugation of the total water-insoluble protein.

Probing the microenvironments of tryptophan residues in the monomeric crystallins of the bovine lens.

Tryptophan microenvironments have been examined in bovine beta s-, gamma II-, gamma IIIa-, gamma IIIb-, gamma IVa- and gamma IVb-crystallins by fluorescence methods. The proteins could be divided into two groups on the basis of the accessibilities of their tryptophan residues. The first group, comprising beta s, gamma II and gamma IIIb, appeared to have a compact structure with none of the tryptophans accessible to KI and only moderately so to acrylamide.

Calcium activated proteolysis and protein modification in the U18666A cataract.

Proteolytic modifications of specific water soluble lens crystallins during U18666A cataract formation in young rats were identified by two dimensional gel electrophoresis and contrasted with those produced by incubating control lens homogenates with calcium. Protein changes which began in clear precataractous lenses at 12 days age included a decrease in 31 and 27 kDa (likely to be beta B1a and beta A3, respectively) crystallin polypeptides, increase in 25 kDa basic polypeptide, appearance of new polypeptide at 30 kDa and modification of alpha A-crystallin. Further modification of both alpha- and beta-crystallins occurred as cataracts formed; they progressed from early to advanced stage within a span of 4 days.

High capacity binding of alpha crystallins to various bovine lens membrane preparations.

This study examines the high capacity binding of intact and carboxyl-terminal-truncated alpha A(alpha A) crystallin to two types of lens membrane preparations; membrane stripped of extrinsic protein and some lipid by extraction with urea and alkali and unextracted membrane isolated by centrifugation of total water insoluble protein on a sucrose gradient (native membrane). High capacity binding of alpha A crystallin to the urea-treated membrane was seen once the alpha A substrate concentration reached about 1 mg/ml of media. The membrane bound up to one mg of alpha A per mg of intrinsic protein (MP26) at a concentration of 5 mg alpha A/ml media, binding 5 to 10 times greater than that seen by others at saturation of the high affinity but low capacity binding sites.

Proteases and protease inhibitory activities in normal mammalian lenses and human cataractous lenses.

Trypsin inhibition (reduction in benzoyl arginine p-nitroanilide hydrolysis), elastase inhibition (reduction in succinyl trialanyl p-nitroanilide hydrolysis), and chymotrypsin inhibition (reduction in acetyl tyrosine ethyl ester hydrolysis) by neutral extracts of mammalian lenses were estimated. The activities were found to be markedly elevated in human cortical cataract lenses compared to normal adult lenses (antielastase 7.21 +/- 3.