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. HGF increased p70 S6K activity by 300% and KGF by about 200%. Protein kinase C (PKC) activator TPA also induced the phosphorylation of p70 S6K. Both the PI-3K inhibitor wortmannin and PKC inhibitor calphostin C blocked the phosphorylation of p70 S6K mediated by the growth factors. However, the mitogen-activated protein kinase (p42/44 MAPK) cascade inhibitor PD98059 had no effect on p70 S6K activation. Furthermore, HGF and KGF increased the rate of corneal epithelial wound healing in an organ culture model, and wortmannin and rapamycin (the p70 S6K inhibitor) blocked corneal epithelial wound healing promoted by the growth factors. These studies suggest that PI-3K and p70 S6K are important signal transducers in the stimulation of corneal epithelial cells by HGF and KGF. PKC is involved in the PI-3K-dependent activation of p70 S6K but not MAPK. Inhibition of wound closure by PI-3K and p70 S6K inhibitors suggests these enzymes play a significant role in corneal wound repair stimulated by HGF and KGF.

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. High-performance liquid chromatography on an ion exchange column (Partisil-SAX; Whatman, Maidstone, United Kingdom) was used to identify PI-3K reaction products. Cultured bovine lens epithelial cells were stimulated with insulin or IGF-1, and PI-3K activity was determined after immunoprecipitation with antibody against phosphotyrosine. Association of p85 with other proteins after stimulation was determined in anti-p85 immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis using anti-phosphotyrosine antibody. RESULTS: PI-3K activity was found in both lens epithelial cells and fiber cells. The highest specific activity was found in the capsule-epithelium, but there was considerable activity in other fiber cell layers. Insulin and IGF-1 stimulated the PI-3K activity in epithelial cells in culture by more than 100%, and activation of the enzyme resulted in tyrosine phosphorylation of the p85 subunit. After stimulation, the p85 subunit of PI-3K was associated with 100- and 180-kDa tyrosine phosphorylated proteins. CONCLUSIONS: The activation of PI-3K and its association with specific tyrosine-phosphorylated proteins may be important in insulin and IGF-1 signal transduction in lens epithelial cells. The presence of significant PI-3K activity throughout the lens further suggests that this signal transduction enzyme is sustained in fiber cells.

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. METHODS: For the in vivo model, epithelium was collected from rabbit corneas at different stages of wound healing after complete de-epithelialization. For in vitro studies, after 7 mm central scrape wounds were applied, rabbit corneas were maintained in organ culture. Immunoprecipitation and Western blot using anti-p85alpha antibodies were employed to determine PI-3K activity and expression of the p85alpha regulatory subunit of PI-3K. Two specific PI-3K inhibitors, Wortmannin and LY 294002 were used to study the effect of PI-3K activity on corneal epithelial wound healing. RESULTS: Two to four days after in vivo corneal epithelial wound healing, there was a 6-8 fold increase in the expression of the p85alpha subunit of PI-3K. By 8 days, the expression of p85alpha was similar to non-injured tissue. Increased expression of the 85kDa protein was observed mainly in the membrane fraction. Similarly, the expression of PI-3K was increased 24h after injured corneas were maintained in organ culture. Increase of p85alpha was confined to the wound region and surrounding area. No concomitant increase in PI-3K activity was observed in any of the wound models. Forty-eight hours after the central injury, Wortmannin and LY294002 inhibited wound healing by about 50%. CONCLUSIONS: Association of most of the increased p85alpha with the membrane fraction and no detectable increase in PI-3K activity during corneal re-epithelialization indicates that PI-3K activation is transitory. The results also suggest a mechanism of down regulation of the enzyme to avoid uncontrollable growth and cellular hypertrophy after growth factor stimulation during wound healing.

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. The myristoylated protein was not seen in the epithelium of the adult rat. Essentially no protein mass was evident in the 19-20 kDa range when samples of the labeled-soluble protein were fractionated by either HPLC coupled with SDS-PAGE or 2D-electrophoresis. These findings suggest that the myristoylated-soluble protein of 19 kDa in lens (p19L) is a rapidly-turning over minor protein likely associated with lens growth. The absence of any apparent membrane association for a myristoylated protein appears unusual. The trace nature of p19L has frustrated attempts at its identification by MALDI-MS.

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. Cytosolic, membrane and detergent-insoluble fractions from epithelium were analysed by Western blot using monoclonal antibodies against the different PKC isoforms. We have identified PKCalpha, gamma, epsilon, mu and iota. PKCalpha and gamma were expressed only in the cytosolic fraction, with the expression of PKCalpha markedly increasing 4 days after injury. Corneas cultured in the presence of rabbit-specific PKCalpha antisense showed a greater than 50% inhibition of wound closure, compared to controls. The PKCepsilon and mu were expressed in the soluble, as well as in the membrane fraction. Additionally, 12% of PKCmu was found attached to the detergent insoluble fraction. The expression of the membrane-bound PKCepsilon and mu isoforms decreased between 1 and 2 days following injury. Only 10% of the PKCiota expressed in corneal epithelium was membrane bound, but between 4 and 8 days after de-epithelization, the expression in this fraction increased three-fold. Our results suggest that changes in the expression and distribution within the various fractions of selective isoforms of PKC after injury could be involved in events leading to wound healing and that PKCalpha is a key modulator in rabbit corneal wound repair.

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. The organization of alpha-crystallin at the membrane surface was proved by the examination of various chemical reactivities and a functional property of the membrane bound protein. The carboxyl-terminal domain of membrane bound alpha-crystallin appeared to be as readily cleaved by mild trypsinolysis as that of the soluble protein and the cleaved protein remained bound to the membrane. The immobilized protein was more extensively crosslinked by a bifunctional primary amine-reactive agent than the soluble protein. No evidence for crosslinking to membrane intrinsic protein was obtained. Like soluble alpha-crystallin, the membrane bound protein displayed chaperone-like activity, a property dependent upon quaternary structure. These findings were interpreted to indicate that alpha-crystallin binds to lens membrane as an aggregate with only a fraction of each aggregate in direct contact with the membrane's hydrophobic surface. It is suggested that the nonsaturable binding reflects low affinity association of soluble alpha-crystallin with a layer of membrane-immobilised protein.

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. Essentially all of the water-insoluble protein from young to aged to cataractous human lens appeared membrane associated. In young lens (20-37 years old), most of the membrane banded at the 25/45% sucrose interface fraction. This fraction contained relatively little urea-soluble protein and likely represents fiber-cell plasma membrane with its physiologically associated extrinsic and intrinsic proteins. With aging (62-80 years old), about one-third of the membrane, as judged by the distribution of cholesterol, banded at a much higher density (50/58% sucrose fraction). The higher density was due to a great increase in the membrane's relative protein content (protein/cholesterol). Although this extra protein was composed of both urea-insoluble and -soluble fractions, the urea-soluble protein predominated in all lenses. Cataractous lens differed from aged-clear lens in that much more of the total membrane (70-75%) had shifted to the high density and participated in this massive binding of cytosolic proteins. Although alpha-crystallin was the principal extrinsic-membrane protein in young lens, high molecular weight aggregate of modified (acidic) crystallins accounted for the increased extrinsic protein in aging. The extrinsic proteins bound to both clear-aged and cataractous lens membrane were aggregated. In conclusion, examination of human lens native membrane fractions revealed that the association of crystallins with membrane in both aging and cataracts was much greater than previously recognized and most of this increased protein was non-covalently bound to the membrane. Much more of the lens total membrane from cataractous than clear-aged lens was involved in this massive protein association and the protein bound to cataract membrane appeared more highly aggregated.

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. By contrast in gamma IIIa, gamma IVa and gamma Vb, all tryptophans were readily accessible to acrylamide and 70% of the fluorescence could be quenched with KI. Spectral analysis, before and after quenching, time-resolved spectroscopy and simulations of the quenching curves suggested that gamma IIIa, gamma IVa and gamma IVb contain two classes of tryptophan residues. One class (tau 0 = 0.52 ns, fa = 0.3, lambda max = 324 nm) which was completely inaccessible to KI and relatively inaccessible to acrylamide (Ksv = 0.25 M-1), was assigned to the topologically equivalent residues in positions 42 and 131. The other class (tau 0 = 2.1-3.4 ns, fa = 0.7, lambda max = 330 nm) was accessible to both quenchers (Ksv = 5.00-5.15 M-1 and 2.47-2.60 M-1, for acrylamide and KI, respectively) and corresponded to the tryptophan residues in positions 68 and 157. The same classes may be present in the other low molecular weight proteins (tau 0 = 0.47-0.55 and 1.55-1.74) but the lower emission and low accessibilities to quenchers prevented their distinction and suggested that these proteins had more compact structures.

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. During this period polypeptides beta B1a and beta A3 almost completely disappeared and several new components of 23-26 kDa in beta-crystallin region appeared. Extensive modification of alpha A resulted in appearance of new components of less than 20 kDa. Most of the gamma-crystallins disappeared from the water soluble proteins in advanced cataract lenses of 18 day old rats presumably by leaking out of the lens. The water insoluble proteins which accumulated in the cataract were very similar to modified crystallins which appeared in the water soluble fraction. In vitro incubation of normal lens water soluble proteins with calcium duplicated most of the protein changes seen during cataract progression. Immunoblotting studies with antisera to rat alpha- and beta-crystallins revealed the identity of most of the modified water soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

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. No apparent differences were seen between high capacity binding of carboxyl terminal-truncated alpha A (by trypsin) and intact alpha A, although each crystalline could antagonize binding of the other. However, once membrane bound, neither crystallin appeared to grossly displace the other. Using the carboxyl terminal-truncated alpha crystallin as a model substrate, native membrane was seen to have a higher capacity to bind the truncated alpha crystallin than urea-extracted membrane and binding was better correlated with the preexisting alpha A content of the native membrane than its MP26 content. An artificial native membrane was prepared by prebinding the truncated alpha A to urea-extracted membrane. This preparation bound more intact alpha A than urea-extracted membrane bearing no prebound crystallin. We conclude that lens native membrane possesses a high capacity to bind alpha crystallins and that this binding could be mediated through protein-protein interactions with alpha crystallin bound in situ to the membrane as extrinsic protein.

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 +/- 3.90 units (mean +/- SD) in cataract compared to 1.46 +/- 0.57 in normals; antitryptic, 0.54 +/- 0.38 and 0.12 +/- 0.04; antichymotryptic, 1.03 +/- 0.61 and 0.297 +/- 0.055). Antielastase activity was distinctly higher in adult normal human lenses compared to infant lenses (0.159 +/- 0.068). Elastase- and trypsin-like activities were detected at low levels in all mammalian lenses. Chymotrypsin-like activity could not be observed in the lenses. The cataractous lenses had lower trypsin- and elastase-like activities compared to normal human lenses (elastase 1.20 +/- 0.643 in normal compared to 0.062 +/- 0.035 in cataract; trypsin, 0.367 +/- 0.154 and 0.069 +/- 0.038). The role of protease: inhibitor complexes in the expression of the individual activities and their role in cataractogenesis are discussed.