ARTICLE |
Correspondence to: Mary Ann Stepp, Depts. of Anatomy and Cell Biology and Ophthalmology, the George Washington U. Medical Center, 2300 I St. NW, Washington, DC 20037.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stratified epithelia are exposed to abrasive forces and are required to respond rapidly to injury to minimize fluid loss and the risk for microbial infection. Healing involves a cell migratory phase to reestablish barrier function and cell proliferation to restratify the epithelium. Cell migration during re-epithelialization involves cell sliding, termed sheet movement, during which cells retain their cell-cell junctions while dynamically altering their shape and cell-substrate interactions to permit movement across the exposed wound bed. Proteins of the integrin family of receptor molecules modulate cell shape, cell migration, and signal transduction in many cell types. In epithelial cells, integrins of the ß1 family have been implicated in regulating cell proliferation and differentiation. 9ßb1 is one of the newer members of the integrin ß1 family and has been recently shown to function as a tenascin receptor. Although little is known about its function in vivo, studies in developing mouse cornea and eyelid suggest that it may play a role in epithelial differentiation. Using a debridement wound model in the mouse cornea, we show in this study that (a) in response to small debridement wounds that close without cell proliferation,
9 integrin protein and mRNA are not induced during migration but are induced during restratification, (b) larger debridement wounds that require cell proliferation to generate the cells necessary for sheet movement result in a dramatic induction of
9 protein and its mRNA during both migration and restratification, and (c) tenascin, an
9ß1 ligand, accumulates beneath epithelial cells during restratification but not during cell migration. Therefore,
9 integrin protein production and tenascin accumulation are dynamically regulated in response to corneal epithelial injury. (J Histochem Cytochem 45:189-201, 1997)
Key Words: integrins, cornea, epithelium, differentiation, wound healing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrins are a superfamily of cell adhesion molecules known to be involved in cell migration and development and to be important in mediating a variety of signal transduction events within the cytoplasm (
Recently we reported that among the ß1-family integrins expressed in the epithelial cells of the skin and cornea, only one, the 9 subunit, localized differently in the skin compared to the cornea (
9 integrin forms heterodimers with the ß1 integrin subunit and in adults localizes to the basal cells of the epidermis, skeletal muscle, hepatocytes, to the airway epithelium lining the lungs, and to the basal cells of the corneal limbus. The only well-characterized ligand for
9ß1 integrin is tenascin (
In the epidermis, 9 integrin is localized to the lateral, apical, and basal cell membranes of the epithelial basal cells. In the cornea,
9 is localized to the basal cells within the limbus, a region of the cornea at its periphery that forms its border with the conjunctiva. Stem cells in skin are distributed in clusters and comprise approximately 10% of the cells in the basal cell layer (
In the cornea, the confinement of the 9 integrin subunit to the corneal epithelial limbal basal cells, coupled with studies demonstrating a role for ß1 family integrins in epidermal proliferation and differentiation (Hotchkin et al. 1995;
9 integrin protein and mRNA expression and expression of the
9ß1 extracellular matrix ligand tenascin. The experimental model used is a mouse corneal epithelial debridement wound in which small (30-40% of the epithelial surface) or large (
95% of the epithelial surface) central regions of the corneal epithelium are removed under anesthesia. The manual removal of the epithelium leaves the underlying basement membrane intact.
The biochemical and morphological response of the epithelial cells in the rodent cornea to injury has been studied in detail (
We show here that production of 9 integrin protein and mRNA are enhanced during restratification in smaller epithelial debridement wounds and during both migration and restratification in larger epithelial debridement wounds. We further show that tenascin accumulates beneath the
9-positive epithelial cells during restratification regardless of wound size, but is much more abundant in the larger wounds.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Vivo Wound Healing
All investigations described herein conformed to the regulations of the George Washington University Medical Center Institution Animal Care and Use Committee and are in voluntary compliance with the Statement for the Use of Animals in Ophthalmic and Vision Research established by the Association for Research in Vision and Ophthalmology. Adult male Balb/c mice weighing 18-22 g were anesthetized with 250 µl of a 1:10 dilution of a 1:1 mixture of Ketaset (100 mg/ml) (Aveco; Fort Dodge, IA) and Rompun (20 mg/ml) (Miles; Shawnee Mission, KS). The eyes of anesthetized animals were treated with the topical anesthetic Ophthetic (Allergan America; Hormigueros, PR) several times until the blink response was absent. A 1.5-mm-diameter (smaller wound) or a 3-mm-diameter (larger wound) central corneal area was demarcated with a dulled trephine, and the epithelium within this region was removed by gentle scraping with a dulled scalpel. Immediately after wounding the eyes were treated with erythromycin ophthalmic ointment (E. Fougera; Melville, NY). After 12, 24, and 48 h (for the smaller wound) or 2, 3, 6, and 10 days (for the larger wound), mice were sacrificed by IP injection of 0.5 ml of Fatal-Plus solution containing sodium pentobarbital (Vortech Pharmaceuticals; Dearborn, MI). The corneas were dissected and processed for immunohistochemistry, in situ hybridization, or for in situ cell proliferation as described below.
Preparation of Probes
The murine 9 cDNA clone (209 base pairs), generously provided by D. Sheppard (Lung Biology Center; UCSF, San Fransciso, CA), was linearized with BamH1 and Xho1. Anti-sense and sense probes were transcribed from linearized plasmids using an in vitro RNA transcription kit (Ambion MAXI Script; Austin, TX) in the presence of [35S]-UTP (DuPont NEN; Boston, MA). The transcription reaction was carried out with T7 (anti-sense) and T3 (sense) polymerases for 30 min at 37C. Approximately 60-80% of the [35S]-UTP was incorporated, to yield a final specific activity of 1 x 108 cpm/µg RNA.
In Situ Hybridization
In situ hybridization with radiolabeled RNA probes was performed as described previously (
Immunohistochemistry
The polyclonal antiserum against the cytoplasmic domain of 9 integrin was also provided by D. Sheppard. The monoclonal anti-mouse tenascin antibody (T3413) was obtained from Sigma Immunochemicals (St Louis, MO).
Indirect immunofluorescence was performed as previously described (
Confocal microscopy was performed in the Center for Microscopy and Image Analysis at GWU Medical Center as previously described (
In Situ Cell Proliferation
The in situ cell proliferation kit AP (Boehringer Mannheim; Indianapolis, IN) was used to identify epithelial cells that were engaged in DNA synthesis at the time of sacrifice. Briefly, after in vivo wound healing, dissected corneas were placed in dishes containing media (DMEM; Gibco LTI, Bethesda, MD) supplemented with bromodeoxyuridine (BrdU) labeling solution at a final concentration of 10 µm BrdU for 30 min at 37C in a humidified atmosphere (5% CO2). After incubation, corneas were frozen in tissue embedding medium and 6-µm sections obtained. BrdU was visualized with an anti-BrdU serum conjugated with alkaline phosphatase, as suggested by the manufacturer. After color development, slides were briefly stained with Toluidine blue and were viewed and photographed in brightfield. For quantitation, the number of labeled cells per unit area of the basal membrane surface of the basal cells was determined by morphometry. No fewer than five visual fields were counted per time point. At least two corneas from two different animals and two slides for each time point studied were used for these studies.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Localization and Expression of 9 Protein and mRNA Are Altered in Corneal Epithelium During Restratification After Small (1.5-mm) Debridement
Shown in Figure 1 are data from a representative experiment involving control mice or mice at 12, 24, or 48 hr after small (1.5-mm) debridement. As we have reported previously (9 primarily in the basal cells of the limbus (Figure 1A and Figure 1B). At 12 hr after injury there was no detectable
9 in the cells at the leading edge, nor was there a change in the localization of
9 within the limbus (Figure 1C and Figure 1D). In this model, re-epithelialization is complete at 19-22 hr. At 24 hr, there was upregulation of
9 protein in some of the basal cells of the central corneal epithelium (Figure 1E and Figure 1F). By 48 hr after injury, there was less
9 in the basal cells of the central cornea (Figure 1G and Figure 1H). At each time point studied, the distribution of
9 within the limbal basal cells remained unaltered.
|
To determine whether the patchy localization of 9 protein observed in the central corneal epithelial basal cells at 24 and 48 hr was due to alterations in the steady-state levels of
9 mRNA, in situ hybridization analyses were performed using mouse
9 35S-labeled anti-sense RNA probes. Frozen sections from control mice and mice at 12, 24, or 48 hr after debridement were used in these procedures. The data presented in Figure 2 are brightfield photomicrographs taken of sections after slides were dipped in emulsion, exposed in the dark, developed, and stained with Toluidine blue. In control unwounded mouse cornea,
9 mRNA was detected in both the limbus and the central cornea (Figure 2A and Figure 2B; sense control inset in 2B). In fact, there was no restriction of the
9 mRNA to the limbus, as might be expected from the localization of the protein to the limbus (compare Figure 2A and Figure 2B with Figure 1A and Figure 1B). During migration, there was no apparent change in the localization of
9 mRNA at the leading edge (Figure 2C and Figure 2D). At 24 and 48 hr after injury (Figure 2E-H), there appeared to be more
9 mRNA expressed in the basal cells of both the limbus and central cornea especially at 48 hr after wounding (compare Figure 2G and Figure 2H, with Figure 2A and Figure 2B).
|
The data presented in Figure 1 and Figure 2 show that migration per se does not alter expression and localization of 9 protein and mRNA. However, the events that occur after migration is complete do result in the accumulation of
9 protein within some of the basal cells of the central corneal epithelium and appear to indicate an increase in expression of
9 mRNA in the limbus and in the central corneal epithelium.
To more directly correlate 9 expression with cell proliferation, experiments were conducted using bromodeoxyuridine (BrdU) labeling. BrdU becomes incorporated into the DNA synthesized by cells that are in S-phase. Labeled nuclei are shown in Figure 3A-D; data are quantitated in Figure 3E. Incorporation of BrdU into DNA is visible within the nuclei of a few scattered epithelial basal cells in control unwounded cornea (Figure 3A and Figure 3E). No change was observed in the numbers of labeled nuclei at 12 hr after 1.5-mm wounding, while the epithelium is actively migrating (Figure 3B and Figure 3E). However, at 24 hr, when migration is complete, a significant increase in the incorporation of BrdU was observed (Figure 3C and Figure 3E). At 48 hr, there were fewer nuclei labeled relative to 24 hr (Figure 3D and Figure 3E). However, there were still more cells labeled than in the control unwounded cornea. These data support the assumption that migration in response to small 1.5-mm debridement wounds occurs in the absence of any increase in the rate of cell proliferation and that as soon as migration is complete, cells begin to proliferate to restratify the epithelium.
|
Removal of Most of the Corneal Epithelial Cells Results in Dramatic Upregulation of 9 Protein and mRNA in the Epithelial Cells During Migration and Restratification
The data presented above showed that after a 1.5-mm debridement injury to the cornea, 9 integrin protein accumulated in the central corneal epithelial basal cells after epithelial cell migration was complete and during the time when cell division was occurring to re-populate the central corneal to its normal thickness. When larger areas of the corneal epithelium are removed, cell proliferation must occur during migration to generate new cells to cover the exposed wound bed. In the cornea, these cells are generated by the proliferation of a small population of cells located in the basal cell layer of the limbus. This is the same population of cells that express
9 in the unwounded control cornea. To determine whether cells that are required both to migrate and to proliferate alter their localization and expression of
9 integrin, 3-mm debridement wounds were made which involved removal of most of the epithelial surface. Because the Balb/c adult mouse cornea is only slightly larger than 3 mm in diameter, the 3-mm wound removes
95% of the corneal epithelial surface; care was taken to avoid injuring the limbus. These debridement wounds took 2.5-3 days to close; they healed without corneal scarring and neovascularization. Figure 4 shows the results obtained when these tissues were analyzed for both
9 integrin protein by indirect immunofluorescence (Figure 4A, Figure 4C, Figure 4E, and Figure 4G) and
9 integrin mRNA by in situ hybridization (Figure 4B, Figure 4D, Figure 4F, and Figure 4H). Compared with control unwounded tissues, the expression of
9 protein was elevated both during migration and during restratification. As a result, there was no longer evidence of differential accumulation of
9 in the basal cells of the limbus, and only the central cornea is shown in Figure 4. Expression of
9 protein in cells at the leading edge at 2 days during active migration was observed (Figure 4A). At 3 days, migration was complete and
9 was detected in all of the cells of the central cornea (Figure 4C). By 6 days, only the basal cells of the central cornea expressed
9 (Figure 4E). At the last time point presented, Day 10, expression of
9 in the cells of the central cornea was almost absent (Figure 4G).
|
The expression of 9 mRNA was also upregulated at all time points studied. Figure 4B shows expression of
9 mRNA at Day 2. Inset i in Figure 4B shows the sense probe control for this experiment. Inset ii shows the results of anti-sense
9 RNA probe hybridization to control unwounded corneal tissue in this experiment. Inset ii in Figure 4B and Figure 2B show
9 mRNA expression in control tissues. Comparison of these two photomicrographs shows that approximately the same number of silver grains are bound to control tissues in both experiments. The in situ hybridization results presented in Figure 4 were generated by exposure of slides to emulsion for the same length of time as those presented in Figure 2. At all time points evaluated after the larger debridement wounds, there was intense accumulation of silver grains, indicating a dramatic increase in
9 mRNA within these tissues. Interestingly, at 6 and 10 days (Figure 4F and Figure 4H) there is evidence of enhanced accumulation of
9 mRNA in the basal cells. Protein and mRNA expression at earlier time points was also studied.
9 integrin protein and mRNA expression were elevated also at 24 hr, the earliest time point at which epithelial cells could be visualized moving over the corneal surface away from the limbus (data not shown). Therefore, the immunohistochemical and the in situ hybridization data both indicate that a significant increase in expression of
9 integrin occurs in the cells that are migrating over the wound surface and during restratification in response to the 3-mm epithelial debridement wound.
Tenascin Accumulates Beneath Epithelial Cells Expressing 9 Integrin
To determine the localization of tenascin, the 9 ligand, relative to that of the
9 protein in the cornea after wounding, double labeling immunofluorescence microscopy was performed on corneal tissue sections and viewed by confocal microscopy. The results are presented in Figure 5. In control tissues (Figure 5A), note the presence of tenascin in the stroma underlying the region anterior to the
9-positive limbal basal cells in the control cornea. The central region of the cornea is negative for both
9 protein and tenascin in the epithelium, at the basement membrane junction, and in the stroma (Figure 5A, inset). At 24 hr after the smaller (1.5-mm) wounds, a few
9-positive epithelial basal cells overlie patchy regions of tenascin-positive matrix. The
9-positive epithelial cells themselves are negative for tenascin protein (Figure 5B). Tenascin also begins to accumulate in the posterior stroma in the smaller wounds at 24 hr. In the larger (3-mm) wounds,
9 expression in the epithelial cells is not correlated with expression of tenascin by the epithelial cells or at the basement membrane zone during migration at Day 2. There is, however, accumulation of tenascin in the stroma (Figure 5C). By 3 days in the larger wounds, patchy areas of tenascin-positive extracellular matrix appear beneath the epithelial cells (Figure 5D) and by 6 days an intense band of tenascin-positive extracellular matrix material has accumulated beneath the epithelial cells (Figure 5E). At later time points, the expression of both
9 integrin in the epithelial basal cells and tenascin at the basement membrane zone beneath the epithelial cells begins to decrease, as shown in a representative micrograph from a cornea 10 days after injury (Figure 5F).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The data presented above show that 9 integrin protein and mRNA expression are enhanced when cell proliferation is induced by debridement. The in situ hybridization studies indicate that
9 integrin mRNA levels increase along with protein levels during cell proliferation in response to injury. This suggests that the changes observed in
9 integrin production are regulated at the level of transcription. Studies are under way to quantitate mRNA levels, using RNAse protection analyses, to enable us to quantify the steady state expression of integrin mRNAs in the cornea in response to injury. It is also interesting that
9 mRNA can be observed in cells that do not express detectable levels of the
9 protein. This is easily observed in the central region of the normal cornea and in the failure of the mRNA to be restricted to the basal cells of the limbus (Figure 2A and Figure 2B). We lack sufficient data to enable us to determine whether there is
9 protein expressed and rapidly degraded in these cells or whether the mRNA is being stored and not used for translation of protein. In the normal cornea, therefore,
9 protein accumulation is regulated at both transcriptional and non-transcriptional levels; in the injured cornea during cell proliferation, however, there appears to be increased expression of
9 mRNA suggesting transcriptional regulation.
By comparing the integrin profile after smaller wounds, which close without the requirement for cell mitosis, vs larger wounds whose closure requires both mitosis and migration, we have shown that 9 protein and mRNA expression are induced coordinately with cell proliferation. In addition, tenascin accumulated under the corneal epithelial cells expressing
9 integrin during restratification, and was especially apparent 6 days after the larger wound. This intense localization of tenascin observed after the larger debridement appears to correlate with active remodeling of the basement membrane and re-insertion of hemidesmosomes, as suggested by studies showing co-distribution of tenascin with ß4 integrin at this time point (data not shown).
9 integrin expression, cell proliferation, and tenascin accumulation at the epithelial basement membrane are correlated.
9 integrin is one of the most recently characterized of the integrin molecules (
9 is regulated developmentally in the mouse (
9 is expressed transiently (
9 protein expression is upregulated in the developing cornea at the same time that the corneal epithelium undergoes a period of rapid cell proliferation and becomes fully stratified. Therefore, in the corneal epithelium, expression of
9 integrin is regulated both developmentally and during proliferation in response to injury.
9 integrin forms heterodimers with the ß1 integrin subunit and in adults localizes to the basal cells of the epidermis, skeletal muscle, hepatocytes, to the airway epithelium lining the lungs, and to the basal cells of the corneal limbus. The only well-characterized ligand for
9ß1 integrin is tenascin (
9ß1 in tissues that lack tenascin, as well as the failure of
9 to co-localize with tenascin in some cells that express both proteins (
9 integrin advances. Because
9ß1 is present on lateral, apical, and basal membranes of epidermal cells, it cannot be ruled out that
9ß1 functions in cell-cell adhesion.
2ß1 and
3ß1 are two of the most abundant integrins expressed in epithelial tissues and have been suggested to play a role in cell-cell adhesion, possibly via homophilic integrin-integrin interaction (
Tenascin is a large extracellular matrix protein believed to play a role in epithelial-mesenchymal interactions both during development and in adults (9ß1 and another ß1 family integrin heterodimer,
8ß1 (
8ß1 and
9ß1,
vß6 is believed to interact with tenascin (
A role for tenascin in wound healing in the skin and in cornea has been suggested by the dramatic increase in tenascin in the healing dermis (
Several studies have shown that production of specific integrin and ß chains appears to be increased in the skin and cornea after injury (
v are increased at the leading edge of migrating epithelial sheets (
vß6 is upregulated in human epidermal wound healing (
In the cornea, we recently showed that the hemidesmosomal integrin 6ß4 was upregulated in the epithelial cell early (3-8 hr after injury) during migration in response to debridement wounding (
9 is not altered early in response to small injuries but later during the restratification stages of wound healing, the corneal epithelial cells clearly regulate ß4 and
9 integrins via distinct mechanisms. In our previous studies we have been unable to document any significant changes in the expression of ß1 or
v integrin in the cornea during wound healing (
9 forms heterodimers with ß1, much more ß1 is present in the epithelial cells forming heterodimers with
2 and
3. Therefore, it is not surprising that the modest increase in expression of
9 we observed in these studies in the smaller wounds at 24 and 48 hr (Figure 1E-H) occurred in the absence of changes in the abundant ß1 integrins. The increased expression of
vß6 in skin during healing was demonstrated using a complex specific monoclonal antibody against human
vß6 (
v subunit biochemically in the corneal epithelium but did not look at expression of ß5 and ß6.
Evidence suggests that integrin expression and function in the epidermis are involved in mediating epithelial cell proliferation and differentiation (Hotchkin et al. 1995; 2ß1 and
3ß1. Cells expressing high levels of these two integrin heterodimers have the proliferative properties expected of epidermal stem cells, with cells expressing low levels having low proliferative potential. The role of
9 in epidermal proliferation has yet to be evaluated.
Although progress has been achieved that implicates integrins in cell proliferation and migration, less progress has been made in the analysis of the extracellular ligands for integrins. The relative lack of progress in this area is due primarily to the large number of distinct extracellular matrix proteins present in the epithelial basement membrane. Several different laminin isoforms are present whose molecular identity has only recently been solved (ß heterodimers have been found to bind to more than a single extracellular matrix molecule. For example,
2ß1 can mediate attachment both to various collagen proteins and to many laminin isoforms. The variety of matrix molecules present, coupled with the ability of many integrins to interact with multiple distinct matrix proteins, has resulted in slower progress in the identification of the extracellular matrix molecules involved in integrin-mediated cell attachment during wound healing.
Understanding of epithelial cell-cell and cell-substrate adhesion at the molecular level is just beginning to be achieved. Data point to integrins as mediators of epithelial adhesion, migration, proliferation, and differentiation. Research on epithelial cell proliferation and migration in response to injury has clinical relevance for improving our understanding of the basis of treatment for patients with defects in healing. In ophthalmology, for example, recurrent epithelial erosions and related corneal dystrophies are significant corneal diseases. Minor trauma causes the debridement of the epithelium in these patients. A popular treatment for this condition involves debriding the epithelial tissue around the site of the erosion, followed by bandaging the eye (
Taken together, our results and those of others studying skin and corneal wound healing point to roles for specific integrin ß heterodimers in the proliferation, migration, and differentiation of epithelial cells.
9 integrin is abundant in the corneal limbus in cells that are believed to be the corneal epithelial stem cells. In response to corneal debridement, we have shown here that enhanced
9 integrin expression is correlated with induction of cell proliferation. We have also shown that accumulation of the
9ß1 ligand tenascin is altered during epithelial cell proliferation. Further studies must be performed to determine whether these changes are essential for appropriate healing of epithelia.
![]() |
Acknowledgments |
---|
Supported by NIH grant EYO8512-08 (MAS).
We thank James Kendrick and the photography staff in Biomedical Communications at the George Washington University Medical Center for help with photography, Fred Lightfoot in the Center for Microscopy and Image Analysis for help with the confocal microscopy, and Dr Abdo Romano Jurjus, Visiting Associate Professor in the Department of Anatomy and Cell Biology at the George Washington University Medical Center and Associate Professor, Department of Human Morphology, Faculty of Medicine, American University of Beirut, Lebanon, for comments on the manuscript.
Received for publication July 23, 1996; accepted October 3, 1996.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aukhil I, Sahlberg C, Thesleff I (1996) Basal layer of epithelium expressed tenascin mRNA during healing of incision skin wounds. J Periodont Res 31:105-112[Medline]
Betz P, Nerlich A, Tubel J, Penning R, Eisenmenger W (1993) Localization of tenascin in human skin woundsan immunohistochemical study. Int J Leg Med 105:325-328[Medline]
Burgeson RE, Chiquet M, Deutzmann R, Ekbloom P, Engel J, Kleinman H, Martin GR, Meneguzzi G, Paulsson M, Sanes J, Timpl R, Tryggvason K, Yamada Y, Yurchenco PD (1994) A new nomenclature for thelaminins. Matrix Biol 14:209-211[Medline]
Buxton JN, Constad WH (1987) Superficial epithelial keratectomy in the treatment of epithelial basement membrane dystrophy. Cornea 6:292-297[Medline]
Carter WG, Kaur P, Gil SG, Gahr PJ, Wayner EA (1990) Distinct functions for integrins 3ß1 in focal adhesions and
6ß4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes. J Cell Biol 111:1817-1823
Cavani A, Zambruno G, Marconi A, Manca V, Marchetti M, Giannetti A (1993) Distinctive integrin expression in the newly forming epidermis during wound healing in humans. J Invest Dermatol 101:600-604[Abstract]
Cheresh DA (1992) Structural and biologic properties of integrin-mediated cell adhesion. Clin Lab Med 12:217-236[Medline]
Chiquet-Ehrismann R, Tannheimer M, Koch M, Brunner A, Spring J, Martin D, Baumgartner S, Chiquet M (1994) Tenascin-C expression by fibroblasts is elevated in stressed collagen gels. J Cell Biol 127:2093-2101[Abstract]
Chung E-H, DeGregorio PG, Wasson M, Zieske JD (1995) Epithelial regeneration after limbus-to-limbus debridement: expression of -enolase in stem and transient amplifying cells. Invest Ophthalmol Vis Sci 36:1336-1343[Abstract]
Clark EA, Brugghe JS (1995) Integrins and signal transduction pathways: the road taken. Science 268:233-239[Medline]
Clark EA, Hynes RO (1996) Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization. J Biol Chem 271:14814-14818
Clark RA (1990) Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin. J Invest Dermatol 94:128s-134s[Medline]
Cotsarelis G, Cheng SZ, Dong G, Sun TT (1989) Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 57:201-209[Medline]
Damsky C, Werb Z (1992) Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information. Curr Cell Res 195:315-322
Erickson HP (1993) Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions. Curr Opin Cell Biol 5:869-876[Medline]
Gailit J, Welch MP, Clark RAF (1994) TGF-ß1 stimulates expression of keratinocyte integrins during re-epithelialization of cutaneous wounds. J Invest Dermatol 103:321-327
Gipson IK, Kiorpes TC (1982) Epithelial sheet movement: protein and glycoprotein synthesis. Dev Biol 92:259-262[Medline]
Gipson IK, Kiorpes TC, Brennan SJ (1984) Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis. Dev Biol 101:212-220[Medline]
Guo M, Kim LT, Akiyama SK, Gralnick HR, Yamada KM, Grinnell J (1991) Altered processing of integrin receptors during keratinocyte activation. Exp Cell Res 195:315-322[Medline]
Haapasalmi K, Zhang K, Tonnesen M, Olerud J, Sheppard D, Salo T, Kramer R, Clark RAF, Uitto V-J, Larjava H (1996) Keratinocytes in human wounds express v
6 integrin. J Invest Dermatol 106:42-48[Abstract]
Haas TA, Plow EF (1994) Integrin-ligand interactions: a year in review. Curr Opin Cell Biol 6:656-662[Medline]
Hanna C (1966) Proliferation and migration of epithelial cells during corneal wound repair in the rabbit and rat. Am J Ophthalmol 61:55-63[Medline]
Hertle MD, Kubler MD, Leigh IM, Watt FM (1992) Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis. J Clin Invest 89:1892-1901[Medline]
Hotchkin NA, Gandarillas Watt FM (1995) Regulation of cell surface ß1 integrin levels during keratinocyte terminal differentiation. J Cell Biol 128:1209-1219[Abstract]
Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11-25[Medline]
Jensen PJ, Wheelock MJ (1995) ß1 integrins do not have a major role in keratinocyte intercellular adhesion. Exp Cell Res 219:322-331[Medline]
Jones PH, Harper S, Watt FM (1995) Stem cell patterning and fate in human epidermis. Cell 80:83-93[Medline]
Jones PH, Watt FM (1993) Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73:713-724[Medline]
Juhasz I, Murphy GF, Yan H-C, Herlyn M, Albelda SM (1993) Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo. Am J Pathol 143:1458-1469[Abstract]
Juliano RL, Haskill S (1993) Signal transduction from the extracellular matrix. J Cell Biol 120:577-585[Medline]
Larjava H, Peltonen J, Akiyama SK, Yamada SS (1990) Novel function for ß1 integrins in keratinocyte cell-cell interactions. J Cell Biol 110:803-815[Abstract]
Larjava H, Salo T, Haapasalmi K, Kramer RH, Heino J (1993) Expression of integrins and basement membrane. J Clin Invest 92:1425-1435[Medline]
Latvala T, Tervo K, Mustonen R, Tervo T (1995) Expression of cellular fibronectin and tenascin in the rabbit cornea after photorefractive keratectomy: a 12 month study. Br J Ophthalmol 79:65-69[Abstract]
Lightner VA (1994) Tenascin: does it play a role in epidermal morphogenesis and homeostasis. J Invest Dermatol 102:273-277[Abstract]
Ljubimov AV, Burgeson RE, Butkowski RJ, Michael AF, Sun TT, Kenney MC (1995) Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms. Lab Invest 72:461-473[Medline]
Lo SH, Chen LB (1994) Focal adhesion as a signal transduction organelle. Cancer Metast Rev 13:9-24[Medline]
Mackie EJ, Halfter W, Liverani D (1988) Induction of tenascin in healing wounds. J Cell Biol 107:2757-2767[Abstract]
Marinkovich MP, Lunstrum GP, Burgeson RE (1992a) The anchoring filamentprotein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem 267:17900-17906
Marinkovich MP, Lunstrum GP, Keene DR, Burgeson RE (1992b) The dermal-epidermal junction of human skin contains a novel laminin variant. J Cell Biol 119:695-703[Abstract]
Onda H, Poulin ML, Tassava RA, Chiu I (1991) Characterization of a newt tenascin cDNA and localization of tenascin mRNA during newt limb regeneration by in situ hybridization. Dev Biol 115:1127-1136
Palmer EL, Ruegg C, Gerrando R, Pytela R, Sheppard D (1993) Sequence and tissue distribution of the integrin 9 subunit, a novel partner of ß1 that is widely distributed in epithelia and muscle. J Cell Biol 123:1289-1297[Abstract]
Potten CS, Morris RJ (1988) Epithelial stem cells in vivo. J Cell Sci (suppl) 10:45-62[Medline]
Prieto AL, Edelman GM, Crossin KL (1993) Multiple integrins mediate cell attachment to cytotactin/tenascin. Proc Natl Acad Sci USA 90:10154-10158[Abstract]
Sastry SK, Horwitz AF (1995) Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intra-cellular at transmembrane signaling. Curr Opin Cell Biol 5:819-831
Schermer A, Galvin S, Sun TT (1986) Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 103:49-62[Abstract]
Schnapp LM, Hatch N, Ramos DM, Klimanskaya IV, Sheppard D, Pytela R (1995) The human integrin 8ß1 functions as a receptor for tenascin, fibronectin, and vitronectin. J Biol Chem 270:23196-23202
Schwartz MA (1993) Signaling by integrins: implications for tumorigenesis. Cancer Res 53:1503-1506[Medline]
Steindler DA, Settles D, Erickson HP, Laywell ED, Yoshiki A, Falssner A, Kusakabe M (1995) Tenascin knockout mice: barrels, boundary molecules, and glial scars. J Neurosci 15:1971-1983[Abstract]
Stepp MA, Spurr-Michaud S, Gipson IK (1993) Integrins in the wounded and unwounded stratified squamous epithelium of the cornea. Invest Ophthalmol Vis Sci 34:1829-1844[Abstract]
Stepp MA, Urry LA, Hynes RO (1994) Expression of 4 integrin mRNA and protein and fibronectin in the early chicken embryo. Cell Adhes Commun 2:359-375[Medline]
Stepp MA, Zhu L, Cranfill RL (1996) Changes in ß4 integrin expression and localization in vivo in response to corneal epithelial injury. Invest Ophthalmol Vis Sci 37:1593-1601[Abstract]
Stepp MA, Zhu L, Sheppard D, Cranfill RL (1995) Localized distribution of 9 integrin in the cornea and changes in expression during corneal epithelial cell differentiation. J Histochem Cytochem 43:529-537
Symington BE, Takada Y, Carter WG (1993) Interaction of integrins 3ß1 and
2ß1: potential role in keratinocyte intercellular adhesion. J Cell Biol 120:523-535[Abstract]
Tenchini M, Adams JC, Gilbery C, Steel J, Hudson DL, Malcovati M, Watt FM (1993) Evidence against a major role for integrins in calcium-dependent intercellular adhesion of epidermal keratinocytes. Cell Adhes Commun 1:55-66[Medline]
Tervo K, Tervo T, van Setten G-B, Virtanen I (1991) Integrins in human corneal epithelium. Cornea 10:461-465[Medline]
Tucker RP (1991) The distribution of J1/tenascin and its transcript during the development of the avian cornea. Differentiation 48:59-66[Medline]
van Setten G-B, Koch JW, Tervo K, Lang GK, Tervo T, Naumann G, Kolkmeier J, Virtanen I, Tarkkanen A (1992) Expression of tenascin and fibronectin in the rabbit cornea after excimer laser surgery. Graefes Arch Clin Exp Ophthalmol 230:178-183[Medline]
Wang A, Patrone L, McDonald JA, Sheppard D (1995) Expression of integrin subunit 9 in the murine embryo. Dev Dyn 204:421-431[Medline]
Watt FM, Kubler M-D, Hotchkin NA, Nocholson LJ, Adams JC (1993) Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions. J Cell Sci 175ndash182
Weller A, Beck S, Ekblom P (1991) Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis. J Cell Biol 112:355-362[Abstract]
Wood TO, Griffith ME (1988) Surgery for corneal epithelial basement membrane dystrophy. Ophthalmic Surg 19:20-24[Medline]
Yokosaki Y, Palmer EL, Prieto AL, Crossin KL, Bourdon MA, Pytela R, Sheppard D (1994) The integrin 9ß1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin. J Biol Chem 296:26691-26696
Zieske JD, Bukusoglu G, Gipson IK (1989) Enhancement of vinculin synthesis by migrating stratified squamous epithelium. J Cell Biol 109:571-576[Abstract]
Zieske JD, Bukusoglu G, Yankauckas MS (1992) Characterization of a potential marker of corneal epithelial stem cells. Invest Ophthalmol Vis Sci 33:143-152[Abstract]
Zieske JD, Gipson IK (1986) Protein synthesis during corneal epithelial wound healing. Invest Ophthalmol Vis Sci 27:1-7[Abstract]
Zieske JD, Mason VS, Wasson ME Meunier SF (1994) Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction. Exp Cell Res 214:621-633[Medline]