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• W2008018303 abstract "Mammalian Class 3 aldehyde dehydrogenase (ALDH) is normally associated with neoplastic transformation or xenobiotic induction by aromatic hydrocarbons in liver. However, Class 3 ALDH is constitutively expressed at it's highest specific activity in corneal epithelium. Tissue-specific, differential gene expression is often controlled by alternative, independent molecular pathways. We report here the development of an in vitro corneal epithelium culture system that retains constitutive high expression of the ALDH3 gene. This model system was used to establish, by enzymatic assays, Western and Northern analyses, histochemical and immunocytochemical staining, and 5′3′ RACE methodologies that constitutive and xenobiotic induction of Class 3 ALDHs occurs from a single gene. Our results also provide a plausible explanation for the very high Class 3 ALDH activity in mammalian cornea, as the primary mechanism of oxidation of lipid peroxidation-derived aldehydes. Further studies with corneal epithelium suggest the presence of additional mechanisms, other than Ah-receptor-mediated, by which the ALDH3 gene can be differentially regulated in a tissue-specific manner. Mammalian Class 3 aldehyde dehydrogenase (ALDH) is normally associated with neoplastic transformation or xenobiotic induction by aromatic hydrocarbons in liver. However, Class 3 ALDH is constitutively expressed at it's highest specific activity in corneal epithelium. Tissue-specific, differential gene expression is often controlled by alternative, independent molecular pathways. We report here the development of an in vitro corneal epithelium culture system that retains constitutive high expression of the ALDH3 gene. This model system was used to establish, by enzymatic assays, Western and Northern analyses, histochemical and immunocytochemical staining, and 5′3′ RACE methodologies that constitutive and xenobiotic induction of Class 3 ALDHs occurs from a single gene. Our results also provide a plausible explanation for the very high Class 3 ALDH activity in mammalian cornea, as the primary mechanism of oxidation of lipid peroxidation-derived aldehydes. Further studies with corneal epithelium suggest the presence of additional mechanisms, other than Ah-receptor-mediated, by which the ALDH3 gene can be differentially regulated in a tissue-specific manner. INTRODUCTIONAldehyde dehydrogenases are a family of NAD-dependent enzymes that catalyze the oxidation of cellular aldehydes to carboxylic acids. Physiological substrates include ethanolderived acetaldehyde, aldehydes from membrane lipid peroxidation and aldehydes from neurotransmitter, drug, and xenobiotic metabolism (1.Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar). Aldehyde dehydrogenases are both constitutively expressed or inducible under a variety of conditions. Of the three major groups of mammalian ALDHs, ( 1The abbreviations used are: ALDHaldehyde dehydrogenasekbkilobase pair(s)PBSphosphate-buffered saline3-MC3-methylcholanthrenebpbase pair(s)UTRuntranslated regionPCRpolymerase chain reaction.) Class 1 enzymes are cytosolic and either constitutive or drug-inducible. Class 1 isoforms are NAD-specific and prefer aliphatic aldehydes as substrate. Class 2 ALDH is localized to the mitochondria. This isoform appears to be primarily responsible for oxidizing acetaldehyde, as well as several aldehydes generated by lipid peroxidation. Like Class 1 ALDHs, Class 2 ALDH uses NAD and preferentially functions at micromolar concentrations of small aliphatic aldehydes. Both Class 1 and Class 2 ALDHs are tetramers of identical subunits, the monomers being approximately 500 amino acids long with molecular masses of 55 kDa.Class 3 aldehyde dehydrogenase is cytosolic and appears to be either constitutively produced or inducible, depending on the tissue(1.Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar). Class 3 ALDH prefers NAD as coenzyme, but this isoform can use NADP effectively, in vitro(2.Lindahl R. Feinstein R. Biochim. Biophys. Acta. 1976; 452: 345-355Crossref PubMed Scopus (54) Google Scholar). Class 3 ALDH preferentially catalyzes the oxidation of aromatic aldehydes, such as benzaldehyde, and medium chain length aliphatic aldehydes, such as hexanal. Consequently, in vitro assays using benzaldehyde and NADP serve as a marker for Class 3 ALDH activity(1.Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar, 3.Marselos M. Lindahl R. Toxicol. Applied Pharmacol. 1988; 95: 339-345Crossref PubMed Scopus (26) Google Scholar). Both the induced and constitutive forms of Class 3 ALDH are dimers of identical monomers, each 453 amino acids in length(4.Lindahl R. Evces S. J. Biol. Chem. 1984; 259: 11991-11996Abstract Full Text PDF PubMed Google Scholar). The subunit molecular weight is approximately 50 kDa.Cloning and characterization of rat ALDH3 indicates the gene spans 9 kb and has 11 exons, the first of which is noncoding(5.Asman D. Takimoto K. Pitot H. Dunn T. Lindahl R. J. Biol. Chem. 1993; 268: 12530-12536Abstract Full Text PDF PubMed Google Scholar). Southern and Northern analyses indicate that Class 3 ALDH from induced and constitutively expressing liver cell lines is derived from a single gene(6.Lin K. Brennan M. Lindahl R. Cancer Res. 1988; 48: 7009-7012PubMed Google Scholar). Furthermore, current evidence indicates that regulation of ALDH3 occurs at the level of transcription(1.Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar).Among the aldehyde dehydrogenases, Class 3 ALDH is most clearly expressed in a tissue-specific manner. It is found at its highest constitutive levels in mammalian cornea(1.Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar, 7.Evces S. Lindahl R. Arch. Biochem. Biophys. 1989; 274: 518-524Crossref PubMed Scopus (40) Google Scholar, 8.Abedinia M. Pain T. Algar E. Holmes R. Exp. Eye Res. 1990; 51: 419-425Crossref PubMed Scopus (121) Google Scholar). Class 3 ALDH is believed to play a protective role in corneal tissue as either an NAD binding protein with UV absorption capabilities or as a catalytic enzyme involved in oxidizing lipid aldehydes generated during UV assault(7.Evces S. Lindahl R. Arch. Biochem. Biophys. 1989; 274: 518-524Crossref PubMed Scopus (40) Google Scholar, 8.Abedinia M. Pain T. Algar E. Holmes R. Exp. Eye Res. 1990; 51: 419-425Crossref PubMed Scopus (121) Google Scholar). Given the enzyme's constitutive abundance in the cornea, it has also been suggested that Class 3 ALDH functions as a structural protein. While not detectable in normal liver, Class 3 ALDH is expressed in liver following exposure to certain xenobiotics or during neoplastic transformation(1.Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar, 9.Takimoto K. Lindahl R. Pitot H. Arch. Biochem. Biophys. 1992; 298: 492-497Crossref Scopus (21) Google Scholar). Xenobiotics are believed to induce Class 3 ALDH activity via increased ALDH3 gene transcription by an Ah-receptor mediated process(10.Nebert D. Gonzalez F.J. Annu. Rev. Biochem. 1987; 56: 945-993Crossref PubMed Scopus (1405) Google Scholar, 11.Korkalainen M. Torronen A. Karenlamp S. Chem. Biol. Interact. 1995; 94: 121-134Crossref PubMed Scopus (14) Google Scholar). Constitutive expression in cornea and stomach, however, suggests that an independent mechanism may underlie tissue-specific expression.We are interested in the mechanisms controlling high constitutive, tissue-specific ALDH3 expression. Because of its extremely high Class 3 ALDH activity, we have chosen the rat cornea as our model. This required the development of an in vitro corneal culture system that expresses near in vivo levels of Class 3 ALDH for extended periods. Here we report a modified method (12.Zieska J. Exp. Cell Res. 1994; 214: 621-633Crossref PubMed Scopus (166) Google Scholar) for successfully culturing rat corneal epithelium that maintains its differentiated properties, including high Class 3 ALDH activity. This in vitro system is then used to begin examining constitutive expression of the ALDH3 gene. In doing so we address the following questions. 1) Does rat corneal epithelium produce Class 3 ALDH constitutively in culture? Does expression continue at near in vivo levels for an extended period? 2) How is corneal Class 3 ALDH distributed intracellularly? 3) What characteristics are similar between corneal Class 3 ALDH and xenobiotic-induced or transformed liver Class 3 ALDH? 4) Are the constitutively expressed and xenobiotic-activated Class 3 ALDHs expressed from a single gene? 5) What mechanisms may be involved in constitutive ALDH3 expression?MATERIALS AND METHODSReagentsUnless otherwise specified, all tissue culture media, supplements, growth factors, assay reagents, buffers, developing reagents, and collagens were from Sigma. Tissue culture dishes came from Corning (Catalog number 25000-35). In our hands, only Corning dishes supported corneal epithelial growth and differentiation. The following reagents were purchased from the supplier indicated: nitrocellulose (Bio-Rad), goat anti-rabbit IgG horseradish peroxidase (Sigma), [α-32P]dCTP (Amersham Corp.), DNA Random Priming Kit (U. S. Biochemical Corp.), Geneclean DNA purification (Intermountain Scientific), UltraPure agarose (Life Technologies, Inc.), Hybond-N membrane (Amersham), 5′ RACE kit (Life Technologies, Inc.), Sequenase sequencing kit (U. S. Biochemical Corp.), 35S-dATP (Amersham), SequaGel acrylamide system (National Diagnostics), Nunc push columns (Stratagene), T∗A cloning vector (InVitrogen), Taq polymerase (Perkin-Elmer), Sprague-Dawley rats (Sasco), AE3 and AE5 antibodies to cytokeratins (ICN), polyvinylidene difluoride 0.2-μm membrane (Bio-Rad), Spurr's resin (EM Sciences), Unicryl (Goldmark Biologicals), colloidal-gold goat anti-rabbit IgG antibody (British BioCell). A 1.5-kb cDNA fragment from the ALDH5 gene was a generous gift from Dr. David Crabb (Indiana University School of Medicine, Indiana).Collagen Coating DishesThirty-five millimeter Corning culture dishes were aseptically coated with 0.1% rat tail collagen type I in 0.1 N acetic acid at 37°C for 24 h prior to explant attachment. Collagen coating solution was removed following incubation, and the plates were rinsed once with sterile phosphate-buffered saline (PBS), pH 7.5. Dishes were thoroughly aseptically air-dried prior to use.Cornea ExplantationCorneas were removed from whole, proptosed rat eyes by circumcision of the tissue just internal to the limbus. Following debridement, corneas were sectioned into 1-mm2 explants. A single explant was transferred, epithelial side up, to the center of a 35-mm2, collagen-coated Petri dish. Explants were allowed to anchor to the dish for 5 min before media was added. One milliliter of complete media (Dulbecco's modified Eagle's medium/F-12 with high glucose; 10% fetal calf serum, dimethyl sulfoxide, 0.005 ml/ml; cholera toxin, 0.1 μg/ml; epidermal growth factor, 10 ng/ml; insulin, 5 μg/ml; gentamicin, 40 μg/ml; glutamine, 20 mM) was added, and the explants were incubated in a 37°C, 5% CO2 environment, in continuous dark. Medium was changed daily. At 4 days post-explantation, tissue explants were removed. Cultures were confluent at 13 days and viable for an additional 11-15 days. For certain studies, some cultures were exposed to a 6-h cycling light regimen using a Phillips 14-watt, cool white lamp.Ultrastructure AnalysisMedia was removed from the culture dishes, and the cells were rinsed with 0.01 M PBS, pH 7.4. The cells were digested with 1.0% trypsin/PBS for 10 min at room temperature. The cell suspension was collected into a microcentrifuge tube and fixed in 3.0% paraformaldehyde, 0.5% gluteraldehyde for 6 h at 20°C. The cells were dehydrated through graded alcohols and infiltrated overnight in Spurr's resin. The cells were then embedded in fresh Spurr's resin. Ultrathin sections were cut on a Reichert-Jung Ultracut E (Leica, Chicago, IL). The grids were viewed using a JOEL-JEM-1210 electron microscope.In Situ Immunofluorescence of CytokeratinsCultures at various stages of growth were fixed for 2 min in ice-cold methanol and rinsed three times with PBS. Cultures were blocked for 1 h in 2% goat serum at 37°C. Following three rinsed with PBS, cultures were incubated with mouse IgG monoclonal antibody to cytokeratin K3 (AE5) (diluted 1:50 in PBS) for 4 h at 37°C. The second antibody was fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Incubation with the second antibody was at 37°C for 2 h. Fluorescent epithelia were visualized with 480/512-nm light under a Wild Leitz GMBH microscope.Cytokeratin AnalysisAfter 10 days of growth, corneal cultures were rinsed with PBS and scraped from their dishes with a rubber policeman. Cultures from ten dishes were pooled and pelleted at 12,000 × g. Cytokeratins were extracted from the cell pellet in 9 M urea, 10 mM Tris, pH 8.4, 1% protease inhibitor mixture, by subjecting the suspension to two freeze/thaw cycles. Similar protein extracts were prepared from whole cornea. Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and the membranes incubated with mouse monoclonal anti-keratin antibodies AE3 and AE5 (1:1000 in PBS). Membranes were then incubated with rabbit anti-mouse IgG alkaline phosphatase (1:1000) and developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.ALDH Histochemistry and ImmunocytochemistryEpithelial cultures at 4 and 9 days growth were rinsed three times with phosphate-buffered saline, pH 7.2. A histochemical staining solution (13.Lindahl R. Clark R. Evces S. Cancer Res. 1983; 43: 5972-5977PubMed Google Scholar) containing benzaldehyde (4 mM), NADP (1.3 mM), phenazine methosulfate (0.13 mM), and nitro blue tetrazolium (0.48 mM) in PBS was applied at 2 ml/dish. The reaction was allowed to proceed in the dark for 15 min. The explants were then rinsed with PBS. Cultures were examined and photographed by phase contrast microscopy.Cells were fixed as described for ultrastructure analysis. Following dehydration, cells were infiltrated and embedded in Unicryl and sectioned. The grids were rinsed in 1.0% ovalbumin/PBS (O/PBS) for 10 min. Grids were incubated for 4 h at room temperature in a humid chamber with rabbit polyclonal anti-Class 3 ALDH antibody diluted 1:100 in O/PBS. After incubation, the grids were rinsed in six washes of O/PBS. The tissues were then transferred to 0.05 M Tris-buffered saline for 10 min. Grids were then incubated with colloidal gold-labeled secondary goat anti-rabbit IgG for 1 h at room temperature in a humid chamber. Grids were rinsed in a series of washes with Tris-buffered saline followed by distilled, deionized H2O. Excess water was removed, and the grids were counterstained with 2.0% aqueous uranyl acetate for 4 min. The grids were allowed to air-dry before viewing on JOEL-JEM-1210 transmission electron microscope.Determination of ALDH ActivityAt various days of growth, five dishes of cultured epithelium were rinsed once with cold PBS. Anchored cells were scraped with a rubber policeman, pooled, and pelleted at 10,000 rpm for 5 min in the cold. The pellets were resuspended in 200 μl of ice-cold PBS. Triton X-100 was added to 1.0% total volume. Each cell suspension was transferred to a glass microhomogenizer and dounced 10 times on ice. Following 30 min on ice, the slurry was centrifuged at 14,000 rpm for 15 min in the cold. Supernatants were assayed for ALDH activity as described previously (14.Lindahl R. Evces S. Biochem. Pharmacol. 1984; 33: 3383-3389Crossref PubMed Scopus (68) Google Scholar). Briefly, 25 μl of cell extract was added to an assay mixture containing either 1 mM benzaldehyde and 1 mM NADP (for Class 3 ALDH activity) or 20 mM propionaldehyde and 1 mM NAD (for Class 1 and Class 2 activity) in assay buffer (60 mM phosphate, 1 mM EDTA, 1 mM β-mercaptoethanol, pH 8.5). The increase in absorbance due to NADP(H) or NAD(H) production during oxidation of the respective substrate was monitored at 340 nm. Protein determinations were by the method of Bradford(15.Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213154) Google Scholar). Specific activities are reported as mIU/mg protein.Western Blot AnalysisTen micrograms of whole epithelial protein extract prepared for enzymatic assays (see above) were denatured with SDS, separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with rabbit polyclonal antibodies to Class 1 ALDH (1:3500), Class 2 ALDH (1:5000), or Class 3 ALDH (1:7500)(7.Evces S. Lindahl R. Arch. Biochem. Biophys. 1989; 274: 518-524Crossref PubMed Scopus (40) Google Scholar, 16.Lindahl R. Biochim. Biophys. Acta. 1978; 525: 9-17Crossref PubMed Scopus (10) Google Scholar), respectively. Membranes were then incubated with goat anti-rabbit IgG whole molecule conjugated to horseradish peroxidase (1:1000) and developed with 9-aminoethylcarbazole and hydrogen peroxide as described(7.Evces S. Lindahl R. Arch. Biochem. Biophys. 1989; 274: 518-524Crossref PubMed Scopus (40) Google Scholar, 16.Lindahl R. Biochim. Biophys. Acta. 1978; 525: 9-17Crossref PubMed Scopus (10) Google Scholar).Northern AnalysisTotal RNA from tissue samples (e.g. whole cornea, cultured cells, normal liver, normal testes, 3-methylcholanthrene (3-MC) treated liver etc.) was prepared by the guanidinium isothiocyanate/lithium chloride method(17.Carthala G. Savouret J. Mendez B. West B. Karin M. Martial J. Baxter J. DNA (N. Y.). 1983; 2: 329-335PubMed Google Scholar). RNA concentrations were calculated from A620 determinations. Equivalent concentrations of each RNA were electrophoresed under nondenaturing conditions and stained with ethidium bromide to assess RNA integrity and loading amounts for subsequent Northern analysis (based on 18 and 28 S ribosomal RNA banding). Comparable levels of 18 and 28 S ribosomal RNA were detected in all samples. Five micrograms of each RNA were then denatured in formaldehyde and separated by formaldehyde-agarose electrophoresis. RNA was transferred to Hybond-N membrane and UV-cross-linked. Membranes were prehybridized, then hybridized with [α-32P]dCTP-labeled cDNA probes for 24 h at 42°C. Membranes were washed under progressively more stringent conditions and autoradiographed for 3 days at −70°C.cDNA Probe PreparationAn ALDH3 cDNA (18.Jones D. Brennan M. Hempel J. Lindahl R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1782-1786Crossref PubMed Scopus (70) Google Scholar) derived from HTC rat hepatoma cells was digested with EcoRI/BglII to release a 1180-bp fragment containing the 5′-untranslated region and the coding sequence for the first 8 exons. This fragment was purified from Geneclean glassmilk suspension and random primed using [α-32P]dCTP. Total counts for hybridization approximated 2 × 107 cpm.Clustered exon probes were prepared by digesting the ALDH3 cDNA with the appropriate restriction enzymes and labeled as above. A cDNA fragment containing the 5′-UTR and exons 1 and 2 was isolated from an EcoRI/RsaI digest. A 284-bp RsaI fragment contained exons 3 and 4. Exon 5 was retrieved from a separate RsaI fragment. An RsaI/BglII digest released cDNA sequences corresponding to exons 6, 7, and 8. Finally, sequences for exons 9, 10, 11, and the 3′-UTR were obtained from a BglII/XhoI digest.Human ALDH5 cDNA was digested with XhoI and ClaI to release a 240-bp fragment containing sequences common to both ALDH5 and ALDH2, and unique for ALDH5, based on amino acid alignments. Following purification from glassmilk (Geneclean), the isolated cDNA piece was radioactively labeled as described above.Determining the Transcriptional Start Site and Polyadenylation Signal SequenceA 5′ RACE methodology was used to determine the transcriptional start site for rat corneal ALDH3 transcripts. Specific instructions were provided by the manufacturer. Briefly, first strand cDNA synthesis was accomplished by reverse-transcribing 1 μg of total RNA from cultured rat corneal epithelium. The synthetic oligonucleotide sequence ←3′-GAGTAGGGAGTCATATACCTG-5′ from within exon 4 of the ALDH3 cDNA served as the gene-specific primer (GSP#1) for reverse transcription(5.Asman D. Takimoto K. Pitot H. Dunn T. Lindahl R. J. Biol. Chem. 1993; 268: 12530-12536Abstract Full Text PDF PubMed Google Scholar). Following degradation of the mRNA template with RNase H, terminal D-transferase was used to tail the 3′ end of the cDNA with poly(dCTP). Nested PCR amplification was then performed with 2.5 pmol of both the anchor primer 5′-GGGIIGGGIIGGGII-3′→ and a second gene-specific oligonucleotide sequence, ←3′-GGACTAACCCGACTCCTACTC-5′, corresponding to a region of ALDH3 exon 3, approximately 225 bp upstream of the first gene-specific primer(5.Asman D. Takimoto K. Pitot H. Dunn T. Lindahl R. J. Biol. Chem. 1993; 268: 12530-12536Abstract Full Text PDF PubMed Google Scholar). The resulting 400-bp 5′ RACE PCR fragment was then inserted into a modified EcoRI site of the pCR(™)II vector by T∗A cloning. The transcriptional start site for corneal ALDH3 was then confirmed by dideoxy sequencing of the 5′ RACE fragment cloned into pCR(™)II.The polyadenylation signal for corneal ALDH3 transcript was determined by a similar technique. Starting with 1 μg of total RNA from cultured corneal epithelium, a DNA copy was reverse-transcribed from the poly(A) tail using 2.5 pmol of poly(dTTP) primer. The resulting cDNA was released from its mRNA template by RNase H digestion. Nested PCR amplification was then performed with a gene-specific oligonucleotide primer 5′-TTGAATGAAGAAGCTCACAAG-3′→ from within exon 10 of the liver ALDH3 cDNA and the poly(dTTP) primer(5.Asman D. Takimoto K. Pitot H. Dunn T. Lindahl R. J. Biol. Chem. 1993; 268: 12530-12536Abstract Full Text PDF PubMed Google Scholar). 3′ RACE-amplified DNA was subsequently cloned into the pC(™)II vector and the polyadenylation signal sequence was verified by dideoxy sequencing.RESULTS AND DISCUSSIONWe have successfully cultured rat corneal epithelium by explanting corneal tissue plugs onto collagen-coated plastic dishes. The culture conditions have consistently produced confluent, near-homogenous populations of rat corneal epithelium virtually devoid of stromal fibroblasts and endothelium. Outgrowth of epithelia is encouraged by supplementing with growth factors that selectively stimulate epithelial mitosis (e.g. epidermal growth factor)(19.Hsu L. Chang W.C. J. Biol. Chem. 1991; 266: 12257-12265Abstract Full Text PDF PubMed Google Scholar). Fibroblasts remain trapped within the stromal collagen(20.McDevitt D. Cell Biology of the Eye. Academic Press, New York1982Google Scholar). Endothelium, which demonstrates limited viability in vitro (21.Kruse F. Tseng S. Invest. Ophthalmol. & Visual Sci. 1993; 34: 163-176Google Scholar), is suppressed by anchoring the explant endothelial side down.The characteristic “pavement” morphology of epithelium growing in sheets down off of the top of the explant is apparent (Fig. 1A). That the cultures are predominantly epithelial was confirmed by in situ immunofluorescence and Western analysis of cell type-specific cytokeratins. Cytokeratins are cellular matrix proteins that serve as markers for both cell type classification and differentiation state. The basic/acidic cytokeratin pair (K3/K12) is specifically expressed in differentiated, corneal-like epithelia(22.Freshney R. Culture of Animal Cells. 3rd Edition. Wiley Press, New York1994Google Scholar). Monoclonal antibody, AE5, recognizes the 64-kDa basic cytokeratin, K3(23.Kruse F. Chen J. Tsai R. Tseng S. Invest. Ophthalmol. & Visual Sci. 1990; 31: 1903-1913PubMed Google Scholar). In situ immunofluorescence of 10-day cultured rat corneal epithelium using monoclonal antibody AE5 discerns intracellular keratin fibers throughout the cultured cell population (Fig. 1B). At the ultrastructural level, the appearance of desmosomes confirms that the cultured cells are epithelial (Fig. 1C). Compared with other normal epithelial cells (Fig. 1, inset), few mitochondria or other organelles (e.g. Golgi, endoplasmic reticulum, lysosomes) are observed in cultured rat corneal epithelium (Fig. 1D). We estimate the number of mitochondria in corneal epithelium to be approximately 10% that of keratinocytes. These results are consistent with those observed for rabbit corneal epithelium(20.McDevitt D. Cell Biology of the Eye. Academic Press, New York1982Google Scholar, 22.Freshney R. Culture of Animal Cells. 3rd Edition. Wiley Press, New York1994Google Scholar).Western analysis of protein extracts from cultured corneal epithelium and whole cornea, using monoclonal antibody AE3, which detects a range of Type II basic cytokeratins(24.Cooper D. Schermer A. Sun T. Lab. Invest. 1985; 52: 243-256PubMed Google Scholar), indicates the presence of a variety of cytoskeletal proteins (Fig. 2A). However, when identical blots were incubated with the AE5 antibody (specific to corneal cytokeratin K3), a single 64-kDa polypeptide band was detected (Fig. 2B), indicating the cells in culture are differentiated and of epithelial origin. Lower molecular weight bands are believed to be degradation products of K3, as removing the protease inhibitor mixture significantly increased both the number and intensity of cross-reacting polypeptides at the expense of the 64-kDa protein (data not shown). The ability to maintain confluent primary rat corneal epithelium in culture for 24-28 days now allows for further investigation of constitutive ALDH3 gene expression.Figure 2:Western blot analysis of cytokeratins from rat corneal epithelium. A, basic cytokeratins detected with monoclonal antibody AE3; lane 1, cytokeratin markers K1 (molecular mass = 68 kDa) and K5 (molecular mass = 58 kDa); lane 2, 10-day rat corneal epithelial culture protein extracts; lane 3, whole cornea protein extracts. B, specific 64-kDa cytokeratin K3 detection using monoclonal antibody AE5; lane 4, ten day rat corneal epithelial culture protein extracts; lane 5, whole cornea protein extracts. 20 μg of total protein was loaded per lane.View Large Image Figure ViewerDownload (PPT)By a variety of methods, our cultured rat corneal epithelial cells have been demonstrated to maintain Class 3 ALDH activity for extended periods. Histochemical staining of epithelial cultures indicates strong aldehyde dehydrogenase activity (Fig. 3A). Like hepatoma cell lines, Class 3 ALDH in corneal epithelial cultures is expressed in discrete regions, rather than homogenously(25.Hogan M. Alvarado J. Weddell J. Histology of the Human Eye. W. B. Saunders, Philadelphia1971Google Scholar, 26.Schermer A. Galvin S. Sun T. J. Cell Biol. 1986; 103: 49-62Crossref PubMed Scopus (1202) Google Scholar, 27.Lin K. Leach M. Winters A. Lindahl R. In Vitro Cell. & Dev. Biol. 1986; 22: 263-272Crossref PubMed Google Scholar). Interestingly, the most homogenous staining occurs just internal to the periphery of the culture's leading edge where cells are most actively dividing. By immunogold cytochemistry, corneal epithelial cells in regions of high Class 3 ALDH activity show an exclusively cytosolic distribution of the enzyme (Fig. 3C). This is the first, direct cytochemical confirmation of the cytosolic localization of Class 3 ALDH (1.Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar); for cornea this distribution is consistent with the paucity of subcellular organelles.Figure 3:Subcellular Localization of aldehyde dehydrogenase. A, phase contrast image of control histochemical staining of 9-day cultured corneal epithelium in the absence of benzaldehyde substrate. Although cell structures are readily identifiable, no dark purple formazan deposition was detected. B, histochemical staining of 9-day cultured epithelium with benzaldehyde/NADP. Note nonhomogenous distribution of aldehyde dehydrogenase activity (white cross-bars = positive ALDH expressing regions; black dashes = negative regions. C, immunogold localization of Class 3 ALDH in 9-day cultured corneal epithelium. Gold deposition, indicated by arrows, denotes detection of Class 3 ALDH protein. Distribution appears random and cytosolic (bar = 100 nm). No apparent accumulation associated with any organelle membrane.View Large Image Figure ViewerDownload (PPT)Corneal epithelium Class 3 ALDH activity was determined with benzaldehyde and NADP at various times from 24 h to 25 days post-explantation, for cultures maintained in continuous dark (Fig. 4). Initially, the level of Class 3 ALDH in epithelial cultures is comparable with that of intact cornea. Activity in corneal cultures decreases over the following 4 days, despite active cell division. By the 5th day in culture, Class 3 ALDH activity has declined to approximately 25% of the original activity. Interestingly, Class 3 ALDH activity then begins to rise, peaking again at 10 days growth. By 10 days, activity in dark-maintained cultures is greater than 70% of intact cornea. From 11-25 days, Class 3 ALDH activity in cultured corneal epithelia steadily declines to a level approximating that of the moderately Class 3 ALDH expressing hepatoma cell line, HTC (Fig. 4) (25.Hogan M. Alvarado J. Weddell J. Histology of the Human Eye. W. B. Saunders, Philadelphia1971Google Scholar, 26.Schermer A. Galvin S. Sun T. J. Cell Biol. 1986; 103: 49-62Crossref PubMed Scopus (1202) Google Scholar, 28.Lin K. Winters A. Lindahl R. Cancer Res. 1984; 44: 5219-5226PubMed Google Scholar)" @default.
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• W2008018303 title "Constitutive Expression of Class 3 Aldehyde Dehydrogenase in Cultured Rat Corneal Epithelium" @default.
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