April 2004
Volume 45, Issue 4
Free
Cornea  |   April 2004
Regulation of Involucrin Expression in Normal Human Corneal Epithelial Cells: A Role for Activator Protein One
Author Affiliations
  • Gautam Adhikary
    From the Departments of Physiology and Biophysics,
  • James Crish
    From the Departments of Physiology and Biophysics,
  • Jonathan Lass
    Ophthalmology,
  • Richard L. Eckert
    From the Departments of Physiology and Biophysics,
    Oncology,
    Dermatology, and
    Biochemistry, Case School of Medicine, Cleveland, Ohio.
Investigative Ophthalmology & Visual Science April 2004, Vol.45, 1080-1087. doi:10.1167/iovs.03-1180
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      Gautam Adhikary, James Crish, Jonathan Lass, Richard L. Eckert; Regulation of Involucrin Expression in Normal Human Corneal Epithelial Cells: A Role for Activator Protein One. Invest. Ophthalmol. Vis. Sci. 2004;45(4):1080-1087. doi: 10.1167/iovs.03-1180.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Understanding the mechanisms that regulate gene expression in the human cornea is an important goal. In the present study, the involucrin gene was used as a model to study this regulation. Human involucrin (hINV) is a structural protein that is selectively expressed in surface epithelia, including corneal epithelial cells.

methods. Regulation of involucrin gene expression was monitored in cultures of normal human primary corneal epithelial cells.

results. The studies revealed that an activator protein (AP)-1 DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-1, results in a loss of hINV promoter activity. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1, Fra-2, and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased nuclear AP1 factor level and binding to the hINV gene AP1-1 response element. Expression of the endogenous hINV gene is also increased by TPA treatment.

conclusions. These findings point to an important role for AP1 transcription factors in the regulation of human corneal epithelial cell involucrin gene expression.

During the corneal epithelial cell life cycle, proliferating cells cease cell division and give rise to cells that populate the multilayered corneal epithelium. Ultimately, these cells are lost from the corneal surface as part of the normal process of cell turnover. 1 This process is associated with marked changes in gene expression. Thus, understanding the mechanisms that regulate corneal epithelial cell gene expression is an important goal. In the present study, we examine the mechanisms that regulate involucrin expression in normal human corneal epithelial cells. 
Involucrin is a 46-kDa, α-helical, rod-shaped structural protein. 2 The amino acid sequence consists of highly homologous 10-amino-acid repeats. 2 3 Each repeat encodes specific amino acids (e.g., glutamine, lysine) that serve as substrates for the formation of covalent interprotein ε-(γ-glutamyl)lysine isopeptide bonds. 4 Involucrin function and expression gave been extensively studied in the epidermis, 2 3 where it is absent from the proliferative epidermal basal cell layer, but is expressed in the nonproliferative cells of the immediate suprabasal layer. It is initially produced as a soluble cytosolic protein. 5 In the upper epidermal cell layers, during the terminal stages in human epidermal keratinocyte differentiation, involucrin is covalently cross-linked at sites in the inner face of the plasma membrane. It then serves as a scaffold protein for assembly of additional protein precursors to complete formation of the cornified envelope. 6 The cornified envelope comprises an array of covalently cross-linked proteins that are assembled to provide a protective surface to resist environmental damage. 7 Type I transglutaminase, 8 an enzyme that catalyzes formation of interprotein ε-(γ-glutamyl)lysine bonds, is responsible for assembly of this structure. Involucrin is a highly efficient transglutaminase substrate. 9 Thus, the presence of involucrin and type I transglutaminase in corneal epithelial cells, suggests that assembly of covalently cross-linked structures is likely to play a role during the corneal epithelial cell life cycle. 10 11 However, a specific function for involucrin in the corneal epithelium has not yet been identified. It is possible that it serves to stabilize the corneal epithelial surface layers. 
In the corneal epithelium, specific information is available regarding the mechanisms that regulate expression of only a few genes (e.g., cytokeratin genes 12 and the gene encoding lactate dehydrogenase 13 ). Moreover, only limited information is available regarding the mechanisms that regulate involucrin (hINV) gene expression in the corneal epithelium, and regulatory mechanisms have not been investigated in primary human corneal epithelial cell cultures. One study examined lens epithelium–derived growth factor regulation of involucrin gene expression in SV40-immortalized human corneal epithelial cells. 14  
In the present experiments, we identified specific DNA sequence elements in the involucrin promoter upstream regulatory region that are necessary for corneal epithelial cell hINV expression. We showed that a specific activator protein one (AP1) transcription-factor–binding site is necessary for involucrin expression in normal human corneal epithelial cells and that specific AP1 factors bind at this site to drive both basal and stimulated involucrin gene expression. We further demonstrated that the AP1 factor binding site is required for an increase in involucrin gene expression observed in response to protein kinase C (PKC) activation. 
Materials and Methods
Chemicals and Reagents
Keratinocyte serum-free medium (KSFM), trypsin, Hanks’ balanced salt solution, and gentamicin were purchased from Invitrogen-Gibco (Grand Island, NY). Phorbol ester (12-O-tetradeconylphorbol-13-acetate; TPA) and dimethyl sulfoxide were obtained from Sigma-Aldrich (St. Louis, MO). The pGL2-Basic plasmid and chemiluminescent luciferase assay system were from Promega (Madison, WI), and chemiluminescence was measured using a luminometer (Berthold, Wildbad, Germany). [γ-32P]ATP was purchased from Perkin Elmer Life Sciences (Boston, MA). The human involucrin-specific polyclonal antibody was generated by injecting rabbits with recombinant human involucrin. This antibody specifically detects human involucrin. 9 Transcription-factor–selective rabbit polyclonal antibodies specifying c-fos (sc-52X), fosB (sc-48X), Fra-1 (sc-605X), Fra-2 (sc-171X), Jun B (sc-46X), c-Jun (sc-1694X), and JunD (sc-74X) were from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody specific for β-actin was purchased from Sigma-Aldrich and used for immunoblot analysis at a dilution of 1:3000. Peroxidase-conjugated goat anti-mouse IgG (sc-2005) and anti-rabbit IgG were obtained from Santa Cruz and used at a dilution of 1:5000. hINV promoter reporter plasmids, containing various lengths of hINV upstream promoter sequence, with or without mutations at specific sites, were constructed in pGL2-basic. 15 16 pRSVα-TAM67 encodes dominant-negative c-Jun. 17 18  
Immunoblot Method
For immunologic analysis, equivalent amounts of protein were electrophoresed on denaturing and reducing 6% polyacrylamide gels and transferred to nitrocellulose membrane. The membrane was blocked by 5% nonfat dry milk and then incubated with the appropriate primary and secondary antibodies. Secondary antibody binding was visualized with chemiluminescent detection technology. 
Human Corneal Epithelial Cell Culture
Human eyes were obtained from the Cleveland Eye Bank, at 5 to 10 hours after death, from patients 50 to 75 years of age. The tissue-procurement protocol was approved by the Case Western Reserve/University Hospitals Institutional Review Board. Eyes were obtained from subjects who had agreed to donate these organs, and all procedures followed the principles articulated in the Declaration of Helsinki. The globes were transferred to the laboratory in KSFM. Adhering conjunctival tissue was removed from the corneal epithelial surface by scraping with a sterile scalpel. The corneal epithelium was then excised by a circumferential incision. Adherent vitreous fluid was removed from the inside corneal surface and the isolated cornea was placed, corneal surface down, in 4 mL sterile Hanks’ balanced salt solution containing 10 mg/mL dispase and 5 μg/mL gentamicin for 15 hours at 4°C. The outer corneal surface was then collected by scraping, treated with 5 mL 0.25% trypsin for 5 minutes at 37°C, gently pipetted to create a single cell suspension, and transferred to stop medium containing (2.5 mL Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 100 U/mL penicillin, and 10 μg/mL streptomycin). The cells were then collected by centrifugation and resuspended in KSFM (Invitrogen-Gibco) containing epidermal growth factor and bovine pituitary extract without antibiotics. The cells from a single corneal epithelium were distributed into two 9.5-cm2 surface area dishes in 4 mL of culture medium. For passage, four near-confluent 9.5-cm2 cultures were harvested with 0.25% trypsin and transferred to one 50-cm2 dish. When confluent, this dish was harvested and transferred to four 50-cm2 dishes. Media were changed every fourth day, and the cultures were confluent after 6 to 8 days. Fifty percent to 70% confluent cultures were used for experimentation. We routinely passage at a split ratio of 1:3 in early cultures and at 1:5 as the cells expand. 
hINV Promoter Activity
For hINV promoter studies, 6 μL transfection reagent (Fugene-6; Roche Diagnostics, Indianapolis, IN) was mixed with 94 μL KSFM and incubated at 25°C for 10 minutes. This mixture was then added to 2 μg hINV promoter reporter plasmid and incubated at 25°C for 20 minutes followed by direct addition to cultures containing 2 mL KSFM. For cotransfection experiments, 1 μg involucrin plasmid and 1 μg TAM67 expression plasmid were used. The final DNA concentration in all groups was maintained constant by addition of empty expression vector. At 24 hours after transfection, 2 mL fresh medium was added containing 0 or 50 ng/mL TPA. After an additional 24 hours, the cells were washed with phosphate-buffered saline (pH 7.5) and scraped into 200 μL cell lysis buffer, 16 and luciferase activity was assayed immediately. All assays were performed in triplicate, and each experiment was repeated a minimum of three times. Luciferase activity was normalized per microgram of protein, as described previously. 16  
Nuclear Extract Preparation and Gel Mobility Shift Assay
Sixty percent confluent human corneal epithelial cells, growing in KSFM, were incubated with 0 or 50 ng/mL TPA for 24 hours. The cells were then washed with phosphate-buffered saline, and total cell extract was prepared, in the presence of proteinase inhibitors, as previously described. 16 Nuclear extracts were prepared according to the method of Schreiber et al. 19 in the presence of 5 μg/mL leupeptin, 5 μg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein content was measured with a protein assay reagent (DC; Bio-Rad, Hercules, CA). Identification of transcription factors binding to the hINV promoter AP1-1 site was detected by electrophoretic mobility shift assay. 15 Three micrograms of nuclear extract was incubated for 25 minutes at room temperature in a total volume of 20 μL containing 20 mM HEPES (pH 7.5), 10% glycerol, 50 mM KCl, 2 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol (DTT), 1 μg/mL poly(dI-dC), 0.1 mg/mL bovine serum albumin, and 50,000 cpm radioactive, double-stranded, 32P-labeled AP1-1 site oligonucleotide (5′-CCTGTGGTGAGTCAGGAAGGG). The hINV AP1-1 binding site is indicted in bold. The mutated AP1-1 site is indicated in bold with the altered nucleotide underscored. The Sp1c oligonucleotide encodes a Sp1 consensus site (5′-ATTCGATCGGGGCGGGGCGAGC). The Sp1 site is indicated in bold. For competition studies, radioinert competitor oligonucleotide was added to the DNA-binding reaction. For the gel mobility supershift assay, AP1-specific antibodies (2 μg) were added to the reaction mixture and incubated at 4°C for 45 minutes. The 32P-labeled probe was then added, and the incubation was continued for an additional 20 minutes at room temperature. Protein-DNA complexes were then resolved in nondenaturing 6% polyacrylamide gels, and the position of the complex was determined by autoradiography. 
Results
DNA Sequence Elements Required for hINV Expression in Corneal Epithelium
We began by examining the effects of TPA, an agent that activates differentiation-associated gene responses, on endogenous hINV gene expression in corneal epithelial cells. Involucrin is a marker of corneal epithelial cell differentiation. 10 As shown in Figure 1 , TPA treatment increased the level of both hINV mRNA and protein. 
A major goal of this study was to gain new insight regarding the mechanism(s) that maintain basal hINV expression and mediate the TPA-stimulated increase in hINV gene expression shown in Figure 1 . We began by monitoring activity of the hINV promoter constructs shown in Figure 2A in primary cultures of human corneal epithelial cells. The full-length hINV promoter construct pINV-2473 drove substantial basal activity (Fig. 2B) . Moreover, TPA treatment increased activity by threefold. Truncation to nucleotide −241 resulted in reduced overall activity, but the TPA-dependent response was retained. In contrast, pINV-110, pINV-97, and pINV-41 were inactive and did not respond to TPA. Two AP1 sites, AP1-1 and AP1-5 (Fig. 2A) , were removed by truncation to nucleotide −110, suggesting that these AP1 sites may be necessary for activity. To assess the importance of these sites, constructs containing inactivating mutations, in one or both sites (Fig. 3A) , were tested for activity. Figure 3 confirms that pINV-2473 was active and that activity was increased by TPA treatment. Mutation of the AP1-5 site resulted in a slight reduction in activity, whereas mutation of the AP1-1 site or simultaneous mutation of both the AP1-1 and AP1-5 sites resulted in a substantial loss of activity and elimination of the TPA-dependent increase. As a control, we tested a construct containing only the basal promoter, pINV-41. As expected, this construct, which contains only the hINV TATA box, was not active and did not respond to TPA. 
These studies suggest that AP1 factors may be necessary for expression of hINV in cultured human corneal epithelial cells. To test this hypothesis, corneal epithelial cells were transfected with pINV-2473, with or without TAM67. TAM67, a mutant form of c-Jun, is a dominant-negative inhibitor of AP1 factor function. 17 18 As shown in Figure 4 , TPA-dependent hINV expression was markedly reduced in TAM67-positive cells, suggesting a role for AP1 factors in mediating the response to TPA. TAM67, in contrast, did not suppress basal promoter (pINV-41) activity. 
AP1 Factor Binding to the hINV Promoter AP1 Sites
Our studies confirm that the AP1-1 site is necessary for optimal corneal epithelial cell hINV gene expression. To gain insights regarding the mechanism of regulation and the transcription factors involved, we performed gel mobility shift experiments. An oligonucleotide, 32P-AP1-1, encoding the hINV promoter AP1-1 site and surrounding sequence (Fig. 5A) , was incubated with nuclear extract prepared from TPA treated and nontreated corneal epithelial cells. As shown in Figure 5B , a single slow mobility band indicated by the arrow (AP1) was observed when nuclear extracts were incubated with 32P-AP1-1 before nondenaturing gel electrophoresis. As is shown in Figure 5B and was confirmed by gel scanning (not shown), the level of binding increased 2.8-fold after TPA treatment. Moreover, this binding was specific, as a marked reduction in band intensity was observed when the reaction mixture included a 10- or 100-fold molar excess of radioinert AP1-1 oligonucleotide. In addition, as a further proof of specificity, neither AP1-1m (Fig. 5A) , an oligonucleotide in which the AP1-1 site is mutated so that it does not bind AP1 factors, 22 nor Sp1c, which encodes a consensus site for Sp1 transcription factors (Fig. 5A) , 23 competed with 32P-AP1-1 for this binding (Fig. 5B)
Regulation of AP1 Factor Levels
The TPA-dependent increase in intensity of the AP1 band in the gel mobility shift assay suggests increased binding of AP1 factors at the site. We hypothesized that this may be due to an increase in AP1 factor level. To test this hypothesis, primary human corneal epithelial cells were treated for 24 hours with TPA, followed by preparation of nuclear extracts. The level of each individual AP1 factor was then assessed by immunoblot. Figure 6 shows that TPA treatment resulted in an increase in nuclear Fra-1, Fra-2, JunD, and JunB levels and a coordinate decrease in the c-Jun level. 
Transcription Factor Interaction with the AP1-5 Site
Because AP1 factors comprise a large family of transcriptional regulators that frequently function in combination, it is important to evaluate which family members interact with the hINV AP1-1 site in corneal epithelial cell nuclear extracts. We therefore used gel mobility supershift assays to identify potential AP1 proteins that interact at the AP1-1 site. Nuclear extract, prepared from TPA-treated cells, was incubated with 32P-AP1-1 in the presence of antibodies that detect specific AP1 factors and DNA–protein interaction was monitored by nondenaturing gel electrophoresis. As shown in Figure 7 , in the absence of nuclear extract, migration of the AP1-1-P32 oligonucleotide was not retarded. Addition of nuclear extract resulted in the appearance of the slow-mobility AP1 band. Addition of anti-pan-fos, an antibody that reacts with all fos family members, resulted in a strong supershift of the slow-mobility band, indicating fos factor binding at the AP1-1 site. Treatment with anti-Fra-1 also resulted in a supershift, indicating the presence of Fra-1. These supershifted bands are indicated by a single asterisk. Anti-Fra-2 also produced a supershift (double asterisk). In contrast, no supershift was observed when the mixture was incubated with anti-c-fos. A similar analysis of binding of Jun family members, revealed a strong reduction in binding when extract is incubated with anti-JunB, indicating the presence of JunB in the complex (Fig. 7) . However, only a slight change in band intensity was observed after treatment with anti-c-Jun or anti-JunD. An unrelated antibody (anti-IgG), as expected, did not produce a supershift. 
Discussion
Corneal epithelial cells transiently proliferate giving rise to daughter cells that then migrate from the limbus toward the central cornea. 24 25 26 27 28 29 These cells ultimately cease proliferation and undergo terminal differentiation to form the multilayered tissue. This process proceeds at a steady rate during the lifetime of the tissue and requires that specific gene products be turned on and off in a tightly controlled spatial and temporal manner. Several gene products, including cytokeratins K3 12 13 30 and K12, 27 31 involucrin, 10 and type I transglutaminase, 32 are selectively expressed in the corneal epithelium, and this expression is controlled by specific transcription factors. However, despite this knowledge, information is limited regarding the mechanisms that regulate gene expression in this tissue. 
Involucrin is produced as a soluble cytosolic protein and subsequently assembled through the action of transglutaminase to form part of the protective surface epithelial structure. 5 This structure, called the cornified envelope, is assembled on the inner surface of the plasma membrane during the terminal stages of cell differentiation. At steady state, involucrin exists in two compartments: one is a soluble pool of involucrin monomer present in the cell cytosol, and the second pool consists of involucrin that is covalently cross-linked to the inner surface of the plasma membrane. 5 33 Involucrin protein and mRNA are expressed in the corneal epithelium. 10 11 Moreover, immunohistological studies (not shown) indicate that the level of expression is low in the limbal region and high in the central cornea, suggesting that the expression may be differentiation dependent. In the present study, we examine the regulation of hINV gene expression in primary cultures of human corneal epithelial cells. Our studies indicate that involucrin mRNA and protein are expressed in primary cultures of human corneal epithelial cells. The goal of the present study was to identify DNA regulatory elements that are required for expression. Two AP1 transcription factor binding sites, AP1-1 and AP1-5, have been shown to be necessary for involucrin gene expression in cultured human epidermal keratinocytes and transgenic mice. 15 22 34 Our studies showed that the AP1 transcription factor binding site, AP1-1, located within the proximal regulatory region (nucleotides −241/−1), 15 is necessary for optimal involucrin expression in corneal epithelial cells. In contrast, mutation of the AP1-5 site, located in the distal regulatory region (nucleotides −2473/−2088) had a minimal impact on corneal epithelial cell expression. This is in contrast to the situation in normal human keratinocytes where the AP1-1 and AP1-5 sites each drove one half of promoter activity. 15 These findings suggest that the relative role of these sites differs in the two cell types and that the regulation is subtly different. 
Role of AP1 Transcription-Factor–Binding Sites in Corneal Epithelial hINV Expression
To investigate further the mechanism responsible for these differences, we examined AP1 transcription factor function. The AP1 family of transcription factors includes c-Jun, JunB, JunD, c-Fos, Fra-1, and Fra-2. 35 36 37 These factors regulate a variety of processes in different tissues and have a prominent role in regulating gene expression in surface epithelia. 22 38 AP1 factors are known to be expressed in the corneal epithelium, 39 40 41 and some have been shown to be expressed in specific locations. For example, in rat corneal epithelium, JunB is reported to be constitutively expressed in the superficial layers. 39 Our studies suggest a central role for AP1 factors in regulating hINV expression in the corneal epithelial cells. First, promoter truncation experiments showed that removal of the AP1-1 transcription factor binding site resulted in a loss of TPA-dependent promoter activity. Basal promoter activity was also somewhat reduced. Second, mutation of this site resulted in a loss of activity. Taken together, these results argue that the AP1-1 site is necessary for expression. Third, treatment of cells with a dominant-negative form of c-Jun, a c-Jun mutant that functions to inactivate all Jun/Fos factors, 17 resulted in a complete loss of promoter activity. Thus, leaving the promoter intact, but interfering with AP1 factor function, also inactivates transcription. 
The present study shows that cultured corneal epithelial cells express Fra-1, Fra-2, JunD, JunB, and c-Jun and that the complex that forms at the hINV AP1-1 site includes only a subset of these factors—specifically, Fra-1, Fra-2, and JunB. This binding is specific, as it is competed by the homologous oligonucleotide, but not by oligonucleotides that encode binding sites for other transcription factors. The finding that only selected members of the AP1 factor family directly participate in regulation of hINV gene expression is consistent with previous reports indicating selectivity in this process. For example, JunB, JunD, and Fra-1 selectively interact at the AP1-1 site in extracts prepared from cultured human epidermal keratinocytes. These factors are necessary for appropriate involucrin expression in epidermis. 15 42 Thus, the subset of AP1 factors that interact at the AP1-5 site varies slightly in extracts from epidermal cells compared with corneal cell extracts. This is likely to be important, as the mix of AP1 factors that interact at a particular AP1 site can control the pattern, timing, and level of expression of the corresponding gene. 38 43 44 45 This may explain, for example, why immunohistology revealed that involucrin is not expressed in basal layer keratinocytes in epidermis, but is expressed in basal layer cells in the central cornea (not shown). It is interesting that JunB has been detected in the superficial layers in rat cornea, 39 a location suggesting a possible role in regulating gene expression. Moreover, Fra-1 is expressed in human corneal epithelium, whereas c-fos, Fra-2, and FosB are not detected. 46 Thus, Fra-1 and JunB, implicated in the present study, are viable in vivo candidates as regulators of hINV gene expression. 
Regulation of AP1 Factor Level by TPA
Previous studies indicate that TPA stimulates hINV gene expression in normal human epidermal keratinocytes. 47 48 Promoter activity studies suggest that the hINV AP1-1 transcription factor binding site is necessary for TPA-stimulated hINV transcription. Moreover, as measured by gel mobility shift analysis, the TPA-dependent increase in gene expression is associated with increased complex formation at the AP1 sites. 15 This increased binding is probably a mass-action effect caused by an increase in intranuclear AP1 factor level. Indeed, our studies in corneal cells showed that TPA treatment increased the nuclear AP1 factor concentration. Based on these findings, we propose that TPA treatment leads to increased nuclear Fra-1 and JunB levels and that this leads to increased DNA binding at the AP1-1 site and increased transcription. This model is similar to that observed in normal epidermal keratinocytes where TPA treatment increases AP1 factor levels and leads to increased involucrin expression. 16 47 48 The TPA-dependent increase in hINV level suggests a role for PKC, as TPA is a specific activator of PKC-dependent pathways. Indeed, in human epidermal keratinocytes, activation of hINV gene expression proceeds through a mechanism that involves activation of novel PKC which, in turn, activates a p38 MAPK cascade that increases AP1 transcription factor levels. 16 47 48 49 That TPA increases hINV expression in corneal cells suggests that PKC and p38 MAPK may also drive expression in corneal cells. Additional studies are needed to assess this possibility. 
AP1: Another Factor Regulating Corneal Epithelial Gene Expression
Several families of transcription factors have been shown to regulate gene expression in the corneal epithelium. Chen et al. 12 reported that Sp1 and AP2 regulate cytokeratin K3 expression in cultured rabbit corneal epithelial cells. 12 Lactate dehydrogenase, which is increased during corneal epithelial cell differentiation, is also regulated by Sp1 and AP2 factors, suggesting a shared mode of regulation with cytokeratin K3. 13 Sp1 has also been implicated by cotransfection studies as a suppressor of α1-proteinase inhibitor expression in corneal epithelial cells, 50 and is involved in regulation of other corneal epithelial genes, including the α5-integrin subunit 51 and cytokeratin 4. 52 In addition, the ESE-1 transcription factor has been reported to be selectively expressed in differentiated cells and thus may have a role in differentiation. 53 These earlier studies provide evidence that a host of transcription factors (e.g., AP2, Sp1) regulate corneal epithelial function. Our present studies add AP1 factors to this list of regulators. 
 
Figure 1.
 
Regulation of endogenous involucrin gene expression. Near-confluent corneal epithelial cell cultures were incubated without or without 50 ng/mL TPA. After 24 hours, cell extracts were prepared for detection of hINV protein immunoblot (top). The β-Actin level was measured as a normalizing control. Also shown (bottom) is an RNA blot using RNA prepared from cells treated as above. The cells were harvested, and involucrin mRNA level was measured by hybridization blot. 20 GAPDH mRNA level was monitored in parallel to provide a loading control. 21 Twenty micrograms of total RNA was electrophoresed in each lane of a denaturing formaldehyde gel, transferred to hybridization membrane, and incubated with random-primed, 32P-CTP-labeled hINV cDNA. The hybridized membrane was then washed and exposed on x-ray film.
Figure 1.
 
Regulation of endogenous involucrin gene expression. Near-confluent corneal epithelial cell cultures were incubated without or without 50 ng/mL TPA. After 24 hours, cell extracts were prepared for detection of hINV protein immunoblot (top). The β-Actin level was measured as a normalizing control. Also shown (bottom) is an RNA blot using RNA prepared from cells treated as above. The cells were harvested, and involucrin mRNA level was measured by hybridization blot. 20 GAPDH mRNA level was monitored in parallel to provide a loading control. 21 Twenty micrograms of total RNA was electrophoresed in each lane of a denaturing formaldehyde gel, transferred to hybridization membrane, and incubated with random-primed, 32P-CTP-labeled hINV cDNA. The hybridized membrane was then washed and exposed on x-ray film.
Figure 2.
 
Regulation of hINV promoter activity in human corneal epithelial cells. (A) Structure of the hINV upstream regulatory region luciferase reporter constructs. Filled boxes and arrows: the luciferase gene and the start site and direction of transcription, respectively. The negative numbers indicate the nucleotide position upstream of the transcription start site (+1). Each plasmid is named based on the length of the upstream regulatory sequence present (i.e, pINV-2473 contains 2473 nucleotides of upstream regulatory region sequence). The AP1-1, AP1-5, Sp1, and C/EBP sites are indicated. 15 (B) Near-confluent cultures of normal human corneal epithelial cells were transfected with 2 μg of the indicated hINV promoter construct and then treated for 24 hours with TPA at 50 ng/mL. At 24 hours, the cells were harvested, and luciferase activity was monitored using a fluorometer. The error bars represent the mean ± SEM of four separate experiments.
Figure 2.
 
Regulation of hINV promoter activity in human corneal epithelial cells. (A) Structure of the hINV upstream regulatory region luciferase reporter constructs. Filled boxes and arrows: the luciferase gene and the start site and direction of transcription, respectively. The negative numbers indicate the nucleotide position upstream of the transcription start site (+1). Each plasmid is named based on the length of the upstream regulatory sequence present (i.e, pINV-2473 contains 2473 nucleotides of upstream regulatory region sequence). The AP1-1, AP1-5, Sp1, and C/EBP sites are indicated. 15 (B) Near-confluent cultures of normal human corneal epithelial cells were transfected with 2 μg of the indicated hINV promoter construct and then treated for 24 hours with TPA at 50 ng/mL. At 24 hours, the cells were harvested, and luciferase activity was monitored using a fluorometer. The error bars represent the mean ± SEM of four separate experiments.
Figure 3.
 
The AP1-1 site is necessary for optimal transcription. (A) hINV upstream regulatory region reporter plasmids. The top construct encodes the intact full-length hINV promoter upstream regulatory region linked to luciferase. Filled boxes and arrows: the luciferase gene and the start site and direction of transcription, respectively. The additional transgenes have mutated AP1-1, AP1-5, and AP1-1/AP1-5 sites. (B) Cultured human corneal epithelial cells were transfected with 2 μg of each plasmid. After 24 hours, the cells were incubated with or without 50 ng/mL TPA for 24 hours. The cells were then harvested and extracts were assayed for luciferase activity. pINV-41, which encodes the minimal hINV promoter, was included as a control. The error bars represent the mean ± SEM of results in three experiments.
Figure 3.
 
The AP1-1 site is necessary for optimal transcription. (A) hINV upstream regulatory region reporter plasmids. The top construct encodes the intact full-length hINV promoter upstream regulatory region linked to luciferase. Filled boxes and arrows: the luciferase gene and the start site and direction of transcription, respectively. The additional transgenes have mutated AP1-1, AP1-5, and AP1-1/AP1-5 sites. (B) Cultured human corneal epithelial cells were transfected with 2 μg of each plasmid. After 24 hours, the cells were incubated with or without 50 ng/mL TPA for 24 hours. The cells were then harvested and extracts were assayed for luciferase activity. pINV-41, which encodes the minimal hINV promoter, was included as a control. The error bars represent the mean ± SEM of results in three experiments.
Figure 4.
 
TAM67 (dominant-negative c-Jun) inhibited promoter activity. Corneal epithelial cells were transfected with 1 μg of the indicated hINV luciferase reporter construct in presence of 1 μg empty expression vector (pRSV2, control) or 1 μg TAM67-encoding expression vector and incubated with or without 50 ng/mL TPA for 24 hours. Cells extracts were then prepared and assayed for promoter activity. The data represent the mean ± SEM of results in five experiments.
Figure 4.
 
TAM67 (dominant-negative c-Jun) inhibited promoter activity. Corneal epithelial cells were transfected with 1 μg of the indicated hINV luciferase reporter construct in presence of 1 μg empty expression vector (pRSV2, control) or 1 μg TAM67-encoding expression vector and incubated with or without 50 ng/mL TPA for 24 hours. Cells extracts were then prepared and assayed for promoter activity. The data represent the mean ± SEM of results in five experiments.
Figure 5.
 
Complex formation at the hINV promoter AP1-1 site. (A) The oligonucleotides used for the gel mobility shift assay included AP1-1, AP1-1m, and Sp1c. The native and mutated AP1-1 sites are shown in bold in the AP1-1 and AP1-1m constructs. The mutated nucleotides are underscored in the AP1-1m oligonucleotide. Sp1c encodes a consensus Sp1 transcription-factor–binding site. (B) Nuclear extract (NE) was prepared from human corneal epithelial cells treated in the presence and absence of TPA (50 ng/mL) for 24 hours. Nuclear extract, prepared from each group of cells, was incubated with double-stranded 32P-end-labeled AP1-1-P32 for 25 minutes at room temperature. Some reactions were supplemented with a 10- or 100-fold molar excess of radioinert AP1-1, AP1-1m, or Sp1c. The reactions were then fractionated on nondenaturing gels, and bands were visualized by autoradiography. Arrows: migration of the AP1/DNA complex (AP1) and migration of noncomplexed free probe (FP). (C) Complex formation is not inhibited by competition with AP1-1m or Sp1c. Migration of the complex was visualized by autoradiography.
Figure 5.
 
Complex formation at the hINV promoter AP1-1 site. (A) The oligonucleotides used for the gel mobility shift assay included AP1-1, AP1-1m, and Sp1c. The native and mutated AP1-1 sites are shown in bold in the AP1-1 and AP1-1m constructs. The mutated nucleotides are underscored in the AP1-1m oligonucleotide. Sp1c encodes a consensus Sp1 transcription-factor–binding site. (B) Nuclear extract (NE) was prepared from human corneal epithelial cells treated in the presence and absence of TPA (50 ng/mL) for 24 hours. Nuclear extract, prepared from each group of cells, was incubated with double-stranded 32P-end-labeled AP1-1-P32 for 25 minutes at room temperature. Some reactions were supplemented with a 10- or 100-fold molar excess of radioinert AP1-1, AP1-1m, or Sp1c. The reactions were then fractionated on nondenaturing gels, and bands were visualized by autoradiography. Arrows: migration of the AP1/DNA complex (AP1) and migration of noncomplexed free probe (FP). (C) Complex formation is not inhibited by competition with AP1-1m or Sp1c. Migration of the complex was visualized by autoradiography.
Figure 6.
 
TPA treatment increased the AP1 transcription factor level. Primary cultured human corneal epithelial cells were incubated with or without 50 ng TPA/mL for 24 hours before nuclear extract preparation. Equivalent amounts of nuclear extract (based on protein content) from TPA treated and control cells was electrophoresed on a 10% denaturing gel and transferred to nitrocellulose for immunodetection with antibodies specific for each Jun/Fos protein. The bands were visualized by incubation with peroxidase-conjugated secondary antibody followed by visualization with chemiluminescent detection reagents.
Figure 6.
 
TPA treatment increased the AP1 transcription factor level. Primary cultured human corneal epithelial cells were incubated with or without 50 ng TPA/mL for 24 hours before nuclear extract preparation. Equivalent amounts of nuclear extract (based on protein content) from TPA treated and control cells was electrophoresed on a 10% denaturing gel and transferred to nitrocellulose for immunodetection with antibodies specific for each Jun/Fos protein. The bands were visualized by incubation with peroxidase-conjugated secondary antibody followed by visualization with chemiluminescent detection reagents.
Figure 7.
 
AP1 transcription factor binding at the AP1-1 site. Nuclear extract, prepared from TPA-treated corneal cells, was incubated with the indicated antibody (anti-Fra-1) for 45 minutes at 4°C. Trace amounts of AP1-1-P32 were then added to each reaction. After an additional 20 minutes at room temperature, the reactions were electrophoresed and bands were visualized by autoradiography. Asterisks: supershifted complexes. The gel was electrophoresed for a long period to facilitate separation of the antibody-shifted bands from the top of the gel. The free probe is not visible, because it had migrated off the bottom of the gel. NE, nuclear extract.
Figure 7.
 
AP1 transcription factor binding at the AP1-1 site. Nuclear extract, prepared from TPA-treated corneal cells, was incubated with the indicated antibody (anti-Fra-1) for 45 minutes at 4°C. Trace amounts of AP1-1-P32 were then added to each reaction. After an additional 20 minutes at room temperature, the reactions were electrophoresed and bands were visualized by autoradiography. Asterisks: supershifted complexes. The gel was electrophoresed for a long period to facilitate separation of the antibody-shifted bands from the top of the gel. The free probe is not visible, because it had migrated off the bottom of the gel. NE, nuclear extract.
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Figure 1.
 
Regulation of endogenous involucrin gene expression. Near-confluent corneal epithelial cell cultures were incubated without or without 50 ng/mL TPA. After 24 hours, cell extracts were prepared for detection of hINV protein immunoblot (top). The β-Actin level was measured as a normalizing control. Also shown (bottom) is an RNA blot using RNA prepared from cells treated as above. The cells were harvested, and involucrin mRNA level was measured by hybridization blot. 20 GAPDH mRNA level was monitored in parallel to provide a loading control. 21 Twenty micrograms of total RNA was electrophoresed in each lane of a denaturing formaldehyde gel, transferred to hybridization membrane, and incubated with random-primed, 32P-CTP-labeled hINV cDNA. The hybridized membrane was then washed and exposed on x-ray film.
Figure 1.
 
Regulation of endogenous involucrin gene expression. Near-confluent corneal epithelial cell cultures were incubated without or without 50 ng/mL TPA. After 24 hours, cell extracts were prepared for detection of hINV protein immunoblot (top). The β-Actin level was measured as a normalizing control. Also shown (bottom) is an RNA blot using RNA prepared from cells treated as above. The cells were harvested, and involucrin mRNA level was measured by hybridization blot. 20 GAPDH mRNA level was monitored in parallel to provide a loading control. 21 Twenty micrograms of total RNA was electrophoresed in each lane of a denaturing formaldehyde gel, transferred to hybridization membrane, and incubated with random-primed, 32P-CTP-labeled hINV cDNA. The hybridized membrane was then washed and exposed on x-ray film.
Figure 2.
 
Regulation of hINV promoter activity in human corneal epithelial cells. (A) Structure of the hINV upstream regulatory region luciferase reporter constructs. Filled boxes and arrows: the luciferase gene and the start site and direction of transcription, respectively. The negative numbers indicate the nucleotide position upstream of the transcription start site (+1). Each plasmid is named based on the length of the upstream regulatory sequence present (i.e, pINV-2473 contains 2473 nucleotides of upstream regulatory region sequence). The AP1-1, AP1-5, Sp1, and C/EBP sites are indicated. 15 (B) Near-confluent cultures of normal human corneal epithelial cells were transfected with 2 μg of the indicated hINV promoter construct and then treated for 24 hours with TPA at 50 ng/mL. At 24 hours, the cells were harvested, and luciferase activity was monitored using a fluorometer. The error bars represent the mean ± SEM of four separate experiments.
Figure 2.
 
Regulation of hINV promoter activity in human corneal epithelial cells. (A) Structure of the hINV upstream regulatory region luciferase reporter constructs. Filled boxes and arrows: the luciferase gene and the start site and direction of transcription, respectively. The negative numbers indicate the nucleotide position upstream of the transcription start site (+1). Each plasmid is named based on the length of the upstream regulatory sequence present (i.e, pINV-2473 contains 2473 nucleotides of upstream regulatory region sequence). The AP1-1, AP1-5, Sp1, and C/EBP sites are indicated. 15 (B) Near-confluent cultures of normal human corneal epithelial cells were transfected with 2 μg of the indicated hINV promoter construct and then treated for 24 hours with TPA at 50 ng/mL. At 24 hours, the cells were harvested, and luciferase activity was monitored using a fluorometer. The error bars represent the mean ± SEM of four separate experiments.
Figure 3.
 
The AP1-1 site is necessary for optimal transcription. (A) hINV upstream regulatory region reporter plasmids. The top construct encodes the intact full-length hINV promoter upstream regulatory region linked to luciferase. Filled boxes and arrows: the luciferase gene and the start site and direction of transcription, respectively. The additional transgenes have mutated AP1-1, AP1-5, and AP1-1/AP1-5 sites. (B) Cultured human corneal epithelial cells were transfected with 2 μg of each plasmid. After 24 hours, the cells were incubated with or without 50 ng/mL TPA for 24 hours. The cells were then harvested and extracts were assayed for luciferase activity. pINV-41, which encodes the minimal hINV promoter, was included as a control. The error bars represent the mean ± SEM of results in three experiments.
Figure 3.
 
The AP1-1 site is necessary for optimal transcription. (A) hINV upstream regulatory region reporter plasmids. The top construct encodes the intact full-length hINV promoter upstream regulatory region linked to luciferase. Filled boxes and arrows: the luciferase gene and the start site and direction of transcription, respectively. The additional transgenes have mutated AP1-1, AP1-5, and AP1-1/AP1-5 sites. (B) Cultured human corneal epithelial cells were transfected with 2 μg of each plasmid. After 24 hours, the cells were incubated with or without 50 ng/mL TPA for 24 hours. The cells were then harvested and extracts were assayed for luciferase activity. pINV-41, which encodes the minimal hINV promoter, was included as a control. The error bars represent the mean ± SEM of results in three experiments.
Figure 4.
 
TAM67 (dominant-negative c-Jun) inhibited promoter activity. Corneal epithelial cells were transfected with 1 μg of the indicated hINV luciferase reporter construct in presence of 1 μg empty expression vector (pRSV2, control) or 1 μg TAM67-encoding expression vector and incubated with or without 50 ng/mL TPA for 24 hours. Cells extracts were then prepared and assayed for promoter activity. The data represent the mean ± SEM of results in five experiments.
Figure 4.
 
TAM67 (dominant-negative c-Jun) inhibited promoter activity. Corneal epithelial cells were transfected with 1 μg of the indicated hINV luciferase reporter construct in presence of 1 μg empty expression vector (pRSV2, control) or 1 μg TAM67-encoding expression vector and incubated with or without 50 ng/mL TPA for 24 hours. Cells extracts were then prepared and assayed for promoter activity. The data represent the mean ± SEM of results in five experiments.
Figure 5.
 
Complex formation at the hINV promoter AP1-1 site. (A) The oligonucleotides used for the gel mobility shift assay included AP1-1, AP1-1m, and Sp1c. The native and mutated AP1-1 sites are shown in bold in the AP1-1 and AP1-1m constructs. The mutated nucleotides are underscored in the AP1-1m oligonucleotide. Sp1c encodes a consensus Sp1 transcription-factor–binding site. (B) Nuclear extract (NE) was prepared from human corneal epithelial cells treated in the presence and absence of TPA (50 ng/mL) for 24 hours. Nuclear extract, prepared from each group of cells, was incubated with double-stranded 32P-end-labeled AP1-1-P32 for 25 minutes at room temperature. Some reactions were supplemented with a 10- or 100-fold molar excess of radioinert AP1-1, AP1-1m, or Sp1c. The reactions were then fractionated on nondenaturing gels, and bands were visualized by autoradiography. Arrows: migration of the AP1/DNA complex (AP1) and migration of noncomplexed free probe (FP). (C) Complex formation is not inhibited by competition with AP1-1m or Sp1c. Migration of the complex was visualized by autoradiography.
Figure 5.
 
Complex formation at the hINV promoter AP1-1 site. (A) The oligonucleotides used for the gel mobility shift assay included AP1-1, AP1-1m, and Sp1c. The native and mutated AP1-1 sites are shown in bold in the AP1-1 and AP1-1m constructs. The mutated nucleotides are underscored in the AP1-1m oligonucleotide. Sp1c encodes a consensus Sp1 transcription-factor–binding site. (B) Nuclear extract (NE) was prepared from human corneal epithelial cells treated in the presence and absence of TPA (50 ng/mL) for 24 hours. Nuclear extract, prepared from each group of cells, was incubated with double-stranded 32P-end-labeled AP1-1-P32 for 25 minutes at room temperature. Some reactions were supplemented with a 10- or 100-fold molar excess of radioinert AP1-1, AP1-1m, or Sp1c. The reactions were then fractionated on nondenaturing gels, and bands were visualized by autoradiography. Arrows: migration of the AP1/DNA complex (AP1) and migration of noncomplexed free probe (FP). (C) Complex formation is not inhibited by competition with AP1-1m or Sp1c. Migration of the complex was visualized by autoradiography.
Figure 6.
 
TPA treatment increased the AP1 transcription factor level. Primary cultured human corneal epithelial cells were incubated with or without 50 ng TPA/mL for 24 hours before nuclear extract preparation. Equivalent amounts of nuclear extract (based on protein content) from TPA treated and control cells was electrophoresed on a 10% denaturing gel and transferred to nitrocellulose for immunodetection with antibodies specific for each Jun/Fos protein. The bands were visualized by incubation with peroxidase-conjugated secondary antibody followed by visualization with chemiluminescent detection reagents.
Figure 6.
 
TPA treatment increased the AP1 transcription factor level. Primary cultured human corneal epithelial cells were incubated with or without 50 ng TPA/mL for 24 hours before nuclear extract preparation. Equivalent amounts of nuclear extract (based on protein content) from TPA treated and control cells was electrophoresed on a 10% denaturing gel and transferred to nitrocellulose for immunodetection with antibodies specific for each Jun/Fos protein. The bands were visualized by incubation with peroxidase-conjugated secondary antibody followed by visualization with chemiluminescent detection reagents.
Figure 7.
 
AP1 transcription factor binding at the AP1-1 site. Nuclear extract, prepared from TPA-treated corneal cells, was incubated with the indicated antibody (anti-Fra-1) for 45 minutes at 4°C. Trace amounts of AP1-1-P32 were then added to each reaction. After an additional 20 minutes at room temperature, the reactions were electrophoresed and bands were visualized by autoradiography. Asterisks: supershifted complexes. The gel was electrophoresed for a long period to facilitate separation of the antibody-shifted bands from the top of the gel. The free probe is not visible, because it had migrated off the bottom of the gel. NE, nuclear extract.
Figure 7.
 
AP1 transcription factor binding at the AP1-1 site. Nuclear extract, prepared from TPA-treated corneal cells, was incubated with the indicated antibody (anti-Fra-1) for 45 minutes at 4°C. Trace amounts of AP1-1-P32 were then added to each reaction. After an additional 20 minutes at room temperature, the reactions were electrophoresed and bands were visualized by autoradiography. Asterisks: supershifted complexes. The gel was electrophoresed for a long period to facilitate separation of the antibody-shifted bands from the top of the gel. The free probe is not visible, because it had migrated off the bottom of the gel. NE, nuclear extract.
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