Biomedical subjects

V Trinkaus-Randall

Publications and source records attributed to V Trinkaus-Randall.

At least 19 recordsLinked to original sources

PubMed2004

Cellular injury induces activation of MAPK via P2Y receptors.

Wound healing is a complex process that involves cell communication, migration, proliferation, and changes in gene expression. One of the first events after injury is the rapid release of Ca(2+) that propagates as a wave to neighboring cells (Klepeis et al. [2001]: J. Cell. Sci. 114:4185-4195). Our goal was to examine the signaling events induced by cellular injury and identify extracellular molecules that induce the activation of extracellular signal responsive kinase (ERK) (p42/44). In this study we demonstrated that injury induced ERK1/2 activation occurred within 2 min and was negligible by 15 min. Treatment of unwounded cells with wound media caused activation of ERK that could be inhibited by apyrase III. Stimulation with epidermal growth factor (EGF) did not mimic the injury response and it was not detected in the wound media. To identify the active component, size fractionation was performed and factor(s) less than 3 kDa that induced the release of Ca(2+) and activation of ERK1/2 were identified. Activity was not altered by heat denaturation, incubation with proteinase K but it was lost by treatment with apyrase. Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) promoted activation by 2 min with similar profiles as that generated by injury. Preincubation with phospholipase C inhibitor, U73122, inhibited activation that was induced by injury and/or nucleotides. Lack of activation by alpha-beta-methylATP (alpha, beta-MeATP) and beta-gamma-methylATP (beta, gamma-MeATP) to purinergic (P)2X receptors further indicated that activation occurs via P2Y and not P2X purinergic receptors. These results indicate that injury-induced activation of ERK1/2 is mediated by a P2Y signaling pathway.

Adenosine Diphosphate↗

PubMed2003

Role of epidermal growth factor and epidermal growth factor receptor on hemidesmosome complex formation and integrin subunit beta4.

Epidermal growth factor (EGF) stimulates integrin beta4 expression and synthesis in corneal epithelium through ligand binding to the EGF receptor, receptor dimerization and activation of the intracellular domain. We hypothesized that inhibition of EGF receptor messenger RNA (mRNA) would block integrin beta4 expression, which is induced by EGF. We also tested the hypothesis that EGF would cause the degradation of hemidesmosomes in control and injured corneal organ cultures. Primary rabbit corneal epithelial cell cultures or corneas were cultured in keratinocyte medium in the presence or absence of an antisense 20-mer phosphorothioate oligonucleotide complementary to the initiation codon region of EGF receptor mRNA. Cells were also cultured in the presence or absence of EGF. Sense and scrambled oligonucleotides similarly modified were used as controls. The concentration of EGF receptor mRNA was semiquantitatively determined by reverse transcriptase/polymerase chain reaction (RT-PCR). We found that transfection did inhibit EGFR expression and migration of epithelial cells and also demonstrated that EGFR mediated expression of integrin beta4 mRNA. Injury induced a decrease in hemidesmosomes that was enhanced with EGF but was not caused by the presence of growth factor in unwounded tissue. These results indicate that injury causes the activation of EGFR but that EGF alone is not responsible for the degradation of hemidesmosomes and that other growth factors play a role in the complex repair of wounds in an avascular tissue.

Animals↗

PubMed2002

Extraction and purification of decorin from corneal stroma retain structure and biological activity.

We developed a method to purify decorin core protein from tissue with the goal of preserving its native structure and biological function. Currently, most procedures rely on the use of denaturing reagents potentially altering the biological activity. Decorin was purified from corneal stromas without the use of detergents or chaotropic reagents. Proteoglycans isolated using anion exchange chromatography on Q-Sepharose were treated with chondroitinase ABC. Decorin was isolated by a second Q-Sepharose chromatography with affinity chromatographies on heparin-Sepharose and concanavalin A-Sepharose. SDS-PAGE revealed a 98.4% pure 44kDa protein identified as decorin with a yield of 35mg per 100 bovine corneas. Identification was confirmed by NanoESI and MALDI qTOF. The novel inclusion of 20% propylene glycol in extraction and column buffers resulted in recoveries of proteoglycans comparable with those observed with detergents and urea. Purified decorin did alter the rate of fibrillogenesis of type I collagen and inhibited the lateral fusion of collagen fibrils. It also bound to [125I]TGF-beta1 with an apparent K(d) of 40nM. Circular dichroism spectroscopy of decorin displayed the spectra of alpha-helices and beta-pleated sheets consistent with those obtained from recombinant decorin. Urea-induced unfolding was cooperative and reversible while thermal denaturation caused irreversible unfolding. Native decorin can be purified from tissue in quantity and quality for biophysical, biochemical, and biological assays.

Animals↗

PubMed2002

TGF-beta1 regulates TGF-beta1 and FGF-2 mRNA expression during fibroblast wound healing.

AIMS: To evaluate the expression of transforming growth factor beta1 (TGF-beta1) and fibroblast growth factor 2 (FGF-2) mRNA in stromal cells in response to injury in the presence of either TGF-beta1 or FGF-2. It has been shown previously that heparan sulfate proteoglycans and FGF-2 are present transiently during wound repair in vivo and that an increase in TGF-beta1 mRNA is detected rapidly after injury. METHODS: Primary corneal fibroblasts were cultured to confluency, serum starved, and linear wound(s) were made in medium containing TGF-beta1 or FGF-2. TGF-beta1 and FGF-2 mRNA expression were evaluated using both northern blot analysis and in situ hybridisation. Both dose dependent and time course experiments were performed. Whole eye organ culture experiments were also carried out and growth factor expression was assessed. RESULTS: Injury and exogenous TGF-beta1 increased TGF-beta1 mRNA values. The increase in expression of FGF-2 mRNA was not detected until wound closure. In contrast, FGF-2 inhibited the expression of TGF-beta1. TGF-beta1 increased TGF-beta1 mRNA stability but did not alter that of FGF-2. Migration assay data demonstrated that unstimulated stromal cells could be activated to migrate to specific growth factors. CONCLUSIONS: TGF-beta1 specifically enhances cellular responsiveness, as shown by increased stability after injury and the acquisition of a migratory phenotype. These data suggest that there is an integral relation during wound repair between TGF-beta1 and FGF-2.

Blotting, Northern↗

PubMed2001

cDNA and genomic cloning of lacritin, a novel secretion enhancing factor from the human lacrimal gland.

Multiple extracellular factors are hypothesized to promote the differentiation of unstimulated and/or stimulated secretory pathways in exocrine secretory cells, but the identity of differentiation factors, particularly those organ-specific, remain largely unknown. Here, we report on the identification of a novel secreted glycoprotein, lacritin, that enhances exocrine secretion in overnight cultures of lacrimal acinar cells which otherwise display loss of secretory function. Lacritin mRNA and protein are highly expressed in human lacrimal gland, moderately in major and minor salivary glands and slightly in thyroid. No lacritin message or protein is detected elsewhere among more than 50 human tissues examined. Lacritin displays partial similarity to the glycosaminoglycan-binding region of brain-specific neuroglycan C (32 % identity over 102 amino acid residues) and to the possibly mucin-like amino globular region of fibulin-2 (30 % identity over 81 amino acid residues), and localizes primarily to secretory granules and secretory fluid. The lacritin gene consists of five exons, displays no alternative splicing and maps to 12q13. Recombinant lacritin augments unstimulated but not stimulated acinar cell secretion, promotes ductal cell proliferation, and stimulates signaling through tyrosine phosphorylation and release of calcium. It binds collagen IV, laminin-1, entactin/nidogen-1, fibronectin and vitronectin, but not collagen I, heparin or EGF. As an autocrine/paracrine enhancer of the lacrimal constitutive secretory pathway, ductal cell mitogen and stimulator of corneal epithelial cells, lacritin may play a key role in the function of the lacrimal gland-corneal axis.

Amino Acid Sequence↗

PubMed2001

Injury and EGF mediate the expression of alpha6beta4 integrin subunits in corneal epithelium.

Our goal was to evaluate the role of epidermal growth factor and injury on the expression of integrin subunits alpha6(alpha6) and beta4(beta4). An in vitro wound model was used to evaluate corneal wound repair and cellular migration. Primary rabbit corneal epithelial cell cultures were serum-starved and injured in the presence or absence of EGF or tyrphostin AG1478, an inhibitor of EGF receptor kinase activity. Repair was monitored morphologically and expression was analyzed using in situ hybridization and immunohistochemistry accompanied by confocal microscopy. The addition of EGF to cell cultures induced a dose-dependent increase in beta4 mRNA expression but the constitutive expression of alpha6 was several fold greater. In the wounded cultures there was a rapid change in expression at the edge of the wound that was enhanced with EGF. In our model there was an increase in beta4 and alpha6 protein in migrating cells. Changes in integrin expression were accompanied by a transient increase in activation of the EGF receptor. The addition of tyrphostin inhibited migration of cells and wound repair, the activation of the EGF receptor and phosphorylation of beta4 in the cytoplasm. These data indicate that the activation of the EGF receptor plays a critical role in the regulation of integrin receptors and the mediation of cellular migration.

Animals↗

PubMed2001

Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells.

This paper characterizes the early responses of epithelial cells to injury. Ca2+ is an important early messenger that transiently increases in the cytoplasm of cells in response to external stimuli. Its elevation leads to the regulation of signaling pathways responsible for the downstream events important for wound repair, such as cell migration and proliferation. Live cell imaging in combination with confocal laser scanning microscopy of fluo-3 AM loaded cells was performed. We found that mechanical injury in a confluent region of cells creates an elevation in Ca2+ that is immediately initiated at the wound edge and travels as a wave to neighboring cells, with [Ca2+]i returning to background levels within two minutes. Addition of epidermal growth factor (EGF), but not platelet-derived growth factor-BB, resulted in increased [Ca2+]i, and EGF specifically enhanced the amplitude and duration of the injury-induced Ca2+ wave. Propagation of the Ca2+ wave was dependent on intracellular Ca2+ stores, as was demonstrated using both thapsigargin and Ca2+ chelators (EGTA and BAPTA/AM). Injury-induced Ca2+ waves were not mediated via gap junctions, as the gap-junction inhibitors 1-heptanol and 18alpha-glycyrrhetinic acid did not alter wave propagation, nor did the cells recover in photobleaching experiments. Additional studies also demonstrated that the wave could propagate across an acellular region. The propagation of the injury-induced Ca2+ wave occurs via diffusion of an extracellular mediator, most probably via a nucleotide such as ATP or UTP, that is released upon cell damage.

Animals↗

PubMed2001

Regulation of heparan sulfate proteoglycan nuclear localization by fibronectin.

Heparan sulfate proteoglycans (HSPG) regulate multiple cellular processes and mediate the cellular uptake of numerous molecules. While heparan sulphate glycosaminoglycan chains are known to modulate receptor binding of several heparin-binding proteins, here we show that distinct extracellular matrices direct HSPG to the nucleus. We analyzed HSPG localization in primary corneal fibroblasts, cultured on fibronectin or collagen type I matrices, using confocal laser scanning microscopy and cell fractionation. Image analysis revealed that the nuclear localization of HSPG core proteins was greater when cells were cultured on fibronectin versus collagen. Matrices containing the heparin-binding domain of fibronectin, but not the integrin-activating domain, demonstrated increased nuclear staining of core proteins. Furthermore, activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited nuclear targeting of HSPG in cells on fibronectin, whereas inhibition of protein kinase C with Ro-31-8220 greatly enhanced nuclear localization of HSPG in cells on both collagen and fibronectin. We propose a matrix-dependent mechanism for nuclear localization of cell surface HSPG involving protein kinase C-mediated signaling. Nuclear localization of HSPG might play important roles in regulating nuclear function.

Animals↗

PubMed2001

[Keratoprosthesis design and biological response to a synthetic cornea].

OBJECTIVE: To evaluate the biological response to three different designs of synthetic cornea in vivo. METHODS: All devices consist of a transparent center and a porous blown periphery. Thirty-five devices were implanted into 35 rabbits' corneas and followed up to more than 6 months. Rabbit corneal stromal cells were inoculated onto the anterior surface of the synthetic cornea and cultured in vitro for one week before the operation. The anterior surface of the optic center hydrogel was modified with radio frequency (rf)-argon plasma treatment. RESULTS: Fibroplasia occurred 2 weeks after surgery and collagen was detected by 28 days. The anterior chamber was normal without detectable leakage of aqueous humor. Basic fibroblast growth factor (bFGF) was detected at the interface between the device and the tissue by 42 days. CONCLUSIONS: It is demonstrated that rabbit limbal epithelial cells can migrate onto the synthetic cornea in vivo.

Animals↗

PubMed2000

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell-layer associated pepsin-resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils.

Animals↗

PubMed2000

Transforming growth factor-beta1 expression in cultured corneal fibroblasts in response to injury.

The mechanisms underlying TGF-beta regulation in response to injury are not fully understood. We have developed an in vitro wound model to evaluate the expression and localization of transforming growth factor-beta1 in rabbit corneal fibroblasts in response to injury. Experiments were conducted in the presence or absence of serum so that the effect of the injury could be distinguished from exogenous wound mediators. Cultures were wounded and evaluations conducted over a number of time points. Expression of TGF-beta1 RNA was determined using Northern blot analysis and in situ hybridization, while the TGF-beta receptors were identified by affinity cross-linking. Injury increased the expression of TGF-beta1 mRNA in cells at the wound edge after 30 min; this response was amplified by the addition of serum. TGF-beta1 mRNA expression was observed in a number of cells distal from the wound. After wound closure, TGF-beta1 mRNA was negligible and resembled unwounded cultures. The half-life of TGF-beta1 mRNA was two times greater in the wounded cultures, indicating that the injury itself maintained the expression, while cell migration was present. Analogous to these findings, we found that binding of TGF-beta to its receptors was maximal at the wound edge, decreasing with time and distance from the wound. These results indicate that injury increases the level of expression of TGF-beta1 mRNA and maintains a higher level of receptor binding during events in wound repair and that these might facilitate the migratory and synthetic response of stromal fibroblasts.

Animals↗

PubMed2000

Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi.

Our goal was to evaluate early signaling events that occur as epithelial cells make initial contact with a substrate and to correlate them with phosphorylation. The corneal epithelium was chosen to study signaling events that occur with adhesion because it represents a simple system in which the tissue adheres to a basal lamina, is avascular, and is bathed by a tear film in which changes in the local environment are hypothesized to alter signaling. To perform these experiments we developed a novel adhesion assay to capture the changes in intracellular Ca(2+) and pH that occur as a cell makes its initial contact with a substrate. The first transient cytosolic Ca(2+) peak was detected only as the cell made contact with the substrate and was demonstrated using fluorimetric assays combined with live cell imaging. We demonstrated that this transient Ca(2+) peak always preceded a cytoplasmic alkalization. When the intracellular environment was modified, the initial response was altered. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N, N'N'-tetraacetic acid (BAPTA), an intracellular chelator, inhibited Ca(2+) mobilization, whereas benzamil altered the duration of the oscillations. Thapsigargin caused an initial Ca(2+) release followed by a long attenuated response. An inositol triphosphate analog induced a large initial response, whereas heparin inhibited Ca(2+) oscillations. Inhibitors of tyrosine phosphorylation did not alter the initial mobilization of cytosolic Ca(2) but clearance of cytosolic Ca(2+) was inhibited. Exposing corneal epithelial cells to BAPTA, benzamil, or thapsigargin also attenuated the phosphorylation of the focal adhesion protein paxillin. However, although heparin inhibited Ca(2+) oscillations, it did not alter phosphorylation of paxillin. These studies demonstrate that the initial contact that a cell makes with a substrate modulates the intracellular environment, and that changes in Ca(2+) mobilization can alter later signaling events such as the phosphorylation of specific adhesion proteins. These findings may have implications for wound repair and development.

Animals↗

PubMed2000

Tyrosine phosphorylation: a critical component in the formation of hemidesmosomes.

Our goal was to evaluate the role of tyrosine phosphorylation in the complete formation of hemidesmosomes that occurs during development or during remodeling after injury. A corneal organ culture system was used to study hemidesmosome formation as it would occur in an intact tissue. Phosphorylation of the integrin subunit beta 4 and bullous pemphigoid antigen-1 (BPAG-1) was examined, as these proteins are known to play a role in linking the electron-dense plaques along the basal surface with the intermediate filaments to complete the formation of hemidesmosomes. Corneal epithelial sheets were placed on substrata that contained an intact basal lamina or basal laminae that had been either modified or removed. These constructs were incubated for up to 18 h, and hemidesmosome formation was evaluated by using transmission electron microscopy. When epithelial sheets were placed on intact basal laminae and incubated in the presence of the tyrosine kinase inhibitor genistein (200 microM), hemidesmosome formation was impaired. The formation of electron-dense regions was delayed, and no association of intermediate filaments was detected. Results were confirmed by biochemical studies. When the epithelium and underlying proteins were extracted and immunoprecipitated with beta 4 or BPAG-1, tyrosine phosphorylation decreased in the presence of genistein. In addition, the phosphorylation of beta 4 decreased when epithelial sheets were incubated on substrata from which the basal lamina had been removed or altered. Thus, a reduction in phosphorylation of tyrosine residues impairs the formation of mature hemidesmosomes, and substrata that fail to support hemidesmosome formation also demonstrate decreased phosphorylation of tyrosine residues.

Alkalies↗

PubMed1999

Regulation of basic fibroblast growth factor binding and activity by cell density and heparan sulfate.

The role of cell density in modulating basic fibroblast growth factor binding and activity was investigated. A primary corneal stromal fibroblast cell culture system was used, since these cells do not constitutively express heparan sulfate proteoglycans in vivo except after injury. A 3-5-fold reduction in bFGF binding per cell was observed as cell density increased from 1000 to 35,000 cells/cm2. The cell density-dependent change in bFGF binding was not the result of altered FGFR expression as determined by equilibrium binding experiments and by immunoblot analysis. However, bFGF-cell surface receptor binding affinities were measured to be 10-20-fold higher at low cell densities than at intermediate and high cell density. bFGF-induced cell proliferation was also cell density-dependent, with maximal stimulation of proliferation 190-280% greater at intermediate densities (15,000 cells/cm2) than at other cell densities. This effect was specific to bFGF as serum, epidermal growth factor, and transforming growth factor-beta did not exhibit the same density-dependent profile. Further, heparan sulfate proteoglycans and, specifically, syndecan-4 were implicated as the modulator of bFGF binding and activity. Pretreatment of cell cultures with heparinase resulted in reduced bFGF binding to the cells and abrogated bFGF induced proliferation. These data suggest a mechanism by which cell density regulates heparan sulfate proteoglycan expression and modulates the cellular response to bFGF. Modulation of heparan sulfate proteoglycan expression might be an important aspect of the regulation of stromal cell migration and proliferation during wound healing.

Cell Division↗

PubMed1999

Characterization of proteoglycans synthesized by cultured corneal fibroblasts in response to transforming growth factor beta and fetal calf serum.

A culture system was developed to analyze the relationship between proteoglycans and growth factors during corneal injury. Specifically, the effects of transforming growth factor beta-1 (TGF-beta1) and fetal calf serum on proteoglycan synthesis in corneal fibroblasts were examined. Glycosaminoglycan synthesis and sulfation were determined using selective polysaccharidases. Proteoglycan core proteins were analyzed using gel electrophoresis and Western blotting. Cells cultured in 10% dialyzed fetal calf serum exhibited decreased synthesis of more highly sulfated chondroitin sulfate and heparan sulfate compared with cells cultured in 1% dialyzed fetal calf serum. The amount and sulfation of the glycosaminoglycans was not significantly influenced by TGF-beta1. The major proteoglycan species secreted into the media were decorin and perlecan. Decorin was glycanated with chondroitin sulfate. Perlecan was linked to either chondroitin sulfate, heparan sulfate, or both chondroitin sulfate and heparan sulfate. Decorin synthesis was reduced by either TGF-beta1 or serum. At early time points, both TGF-beta1 and serum induced substantial increases in perlecan bearing chondroitin sulfate and/or heparan sulfate chains. In contrast, after extended periods in culture, the amount of perlecan bearing heparan sulfate chains was unaffected by TGF-beta1 and decreased by serum. The levels of perlecan bearing chondroitin sulfate chains were elevated with TGF-beta1 treatment and were decreased with serum. Because both decorin and perlecan bind growth factors and are proposed to modulate their activity, changes in the expression of either of these proteoglycans could substantially affect the cellular response to injury.

Animals↗

PubMed1998

Biological response to a synthetic cornea.

We have designed a synthetic cornea that has a transparent hydrogel optic and a porous skirt. The device has been implanted in rabbit corneas. We have shown that keratocytes migrate into the device and deposit a complex extracellular matrix. The immediate response is detected in the surrounding stroma, and the secondary response is seen after cells have deposited a matrix in the disc. After implantation, a decrease in keratan sulfate accompanied by an increase in dermatan sulfate was detected in the surrounding tissue compared to the unwounded corneal stroma. The glycosaminoglycans in the disc resemble that of an injured stroma. The appearance of heparan sulfate and growth factors, bFGF and TGFbeta, was not detected until 6 weeks after implantation. The growth factors were detected at the interface between the device and the tissue and become more diffuse over time. Methods of controlled release in vivo were used to enhance the rate of fibroplasia and wound repair. While these were successful in the cornea itself, when combined with the synthetic cornea the response was magnified and the initial attempts yielded neovascularization and edema. Currently, efforts are being directed at controlling the release within the porous haptic so that fibroplasia is enhanced while minimizing an inflammatory response.

Animals↗

PubMed1998

Elastogenesis in the developing chick lung is transcriptionally regulated.

The overall goals of this study were to establish the level at which elastin gene expression is regulated during chick lung embryogenesis and to identify the temporal and spatial relationships among elastogenesis, smooth muscle cell differentiation, and cell proliferation. A comparison of lung elastin mRNA and transcriptional levels during embryogenesis shows that elastin expression is developmentally regulated at the transcriptional level. The increase in elastogenic activity occurs during the late stages of lung embryogenesis and coincides with terminal maturation of the tertiary bronchi. In situ hybridization analysis demonstrates that the increase in elastin mRNA expression is confined to the tertiary bronchial respiratory subunits, connective tissue septa, and supporting vasculature of the lung parenchyma. Immunohistochemical localization of smooth muscle cell alpha-actin and tropoelastin suggests that alpha-actin-immunoreactive cells of the lung parenchyma are a major contributor to the increase in elastin expression during embryogenesis. This observation is also reflected by Northern blot analysis, which demonstrates a temporal coincidence in the increase of both alpha-actin and elastin mRNA levels. Histone mRNA expression, which was used as an index of cellular proliferation, reveals a level and spatial pattern inversely related to that of the elastin transcript. Tissue transfections of chick lungs isolated from 18-day embryos with various elastin gene deletion/reporter constructs illustrate that the elastin promoter is not promiscuous within a tissue environment and that sequences spanning the -500 to +2 region are capable of directing promoter activity spatially comparable to the endogenous elastin gene.

Actins↗

PubMed1998

Injury of stromal fibroblasts induces phosphorylation of focal adhesion proteins.

PURPOSE: The extracellular matrix serves as a structural support for the corneal stroma and mediates signaling events that regulate the intracellular environment of stromal keratocytes. We hypothesize that adhesion and injury mediate signal transduction events causing the phosphorylation of tyrosine residues of specific adhesion proteins and that phosphorylation is required for cellular adhesion and migration. METHODS: For the adhesion experiments; primary rabbit stromal fibroblasts were seeded and phosphorylation of tyrosine residues was followed from 1 min to 24 h. For the injury experiments, confluent primary cultures were rendered quiescent, wounded, and tyrosine phosphorylation was followed from 30 s to 6 h. The antibody (py-20) was used to detect proteins phosphorylated on tyrosine residues. We examined changes in the phosphorylation of focal adhesion kinase (FAK), paxillin and cortactin, using immunoprecipitation and Western blot analysis. RESULTS: In the adhesion experiments, the phosphorylation of a 68-kDa protein was detected after 1 min, and the phosphorylation of a 125-kDa protein was not detected until 15 min. These proteins were identified in re-probed blots as paxillin and FAK. In the injury experiments, FAK phosphorylation was detected within 30 s and remained elevated for 6 h when cells were cultured on fibronectin. Both FAK and paxillin phosphorylation were prominent after injury, but unlike FAK phosphorylation, paxillin phosphorylation decreased over time. Phosphorylation was prominent at the wound margin. After wound closure, it returned to background levels. Tyrosine kinase inhibitors, genistein and herbimycin, decreased the number of adherent cells and altered the rate of cell migration after injury, compared to control (DMSO alone). CONCLUSION: The results indicate that injury and cell-matrix interaction mediate the phosphorylation of specific adhesion proteins and that phosphorylation is required for wound repair.

Animals↗