(Received for publication, November 2, 1995; and in revised form, December 18, 1995)
From the
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.
Aldehyde 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) . Aldehyde dehydrogenases
are both constitutively expressed or inducible under a variety of
conditions. Of the three major groups of mammalian ALDHs, ()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) . Class 3 ALDH prefers NAD as coenzyme, but this isoform can use NADP effectively, in vitro(2) . 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, 3) . Both the induced and constitutive forms of Class 3 ALDH are dimers of identical monomers, each 453 amino acids in length(4) . 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) . Southern and Northern analyses indicate that Class 3 ALDH from induced and constitutively expressing liver cell lines is derived from a single gene(6) . Furthermore, current evidence indicates that regulation of ALDH3 occurs at the level of transcription(1) .
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, 7, 8) . 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, 8) . 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, 9) . Xenobiotics are believed to induce Class 3 ALDH activity via increased ALDH3 gene transcription by an Ah-receptor mediated process(10, 11) . 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) 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?
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 HO. 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.
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.
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) . 3`
RACE-amplified DNA was subsequently cloned into the pC(TM)II vector
and the polyadenylation signal sequence was verified by dideoxy
sequencing.
We 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) . Fibroblasts remain trapped within the stromal collagen(20) . Endothelium, which demonstrates limited viability in vitro (21), 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) . Monoclonal antibody, AE5, recognizes the 64-kDa basic cytokeratin, K3(23) . 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, 22) .
Figure 1: Morphological analysis of cultured rat corneal epithelia. A, light microscopic analysis of 7-day rat corneal epithelium cultured from explant. Note characteristic ``cobblestone'' pavement appearance. B, immunofluorescent staining of 10-day rat corneal epithelium cultures using monoclonal antibody AE5 to basic cytokeratin K3. C, transmission electron micrograph of 10-day rat corneal epithelium culture showing tight junction connections between cells (d = desmosomes; n = nucleus; bar = 500 nm). d, low magnification survey (bar = 1 µm) of 10-day rat corneal epithelium showing reduced numbers of mitochondria compared with the distribution of mitochondria in keratinocytes (inset; bar = 250 nm) (n = nucleus; m = mitochondria).
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) , 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.
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, 26, 27) . 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) ; 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.
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, 26, 28) .
This decrease and subsequent resurgence of a protein associated with
differentiated cell function is not uncommon. Frequently, primary
cultures exhibit extensive declines in the levels of noncell cycle
proteins as differentiated cells de-differentiate to enter a highly
proliferative state (G
G
) (29) .
During this time (1-5 days), culture conditions (e.g. epidermal growth factor, insulin, collagen matrix) stimulate
transcription of cell cycle proteins at the expense of differentiated
gene expression. However, as proliferation slows (5-10 days),
transcription resumes for genes expressed in fully differentiated cells (30) .
Figure 4: Aldehyde dehydrogenase activity of cultured corneal epithelium. Assays of cultured corneal extracts were performed at various days in culture with millimolar concentrations of benzaldehyde and NADP (patterns: checkered = tissue standards; diagonal = cultured rat corneal epithelium). Inset, activity of 9-day culture extracts with micromolar concentrations of both propionaldehyde and NAD and benzaldehyde and NADP (patterns: solid = propionaldehyde substrate 20 µM; cross-hatched = benzaldehyde 10 µM). Abbreviations: HTC = rat hepatoma cell line; 3-MC = 3-methycholanthrene-treated rat liver. Error bars denote mean ± S.D.; n = 3.
Determination of ALDH activity with an aliphatic
substrate (propionaldehyde) and NAD yields approximately one-third the
activity as compared with assays with an aromatic substrate and NADP
(data not shown). This 3-fold higher activity from the aromatic
aldehyde-NADP assay is diagnostic of Class 3 ALDH as the major aldehyde
dehydrogenase isoform in a tissue(3) . Additionally, aldehyde
dehydrogenase activity at micromolar aliphatic aldehyde concentrations
is not detectable in cultured corneal epithelium (Fig. 4, inset) or in whole corneal homogenates (data not shown). These
data suggest that mitochondrial Class 2 ALDH, which has a µMK for aliphatic aldehydes(9) , is
present at very low levels in corneal epithelium. This is consistent
with the reduced number of corneal mitochondria noted earlier.
Due to overlapping substrate preferences, it is difficult to assess the levels of specific ALDH isoforms simply by activity assays. Therefore, cell extracts from various stages of corneal cultures were also analyzed by Western analysis using monospecific antibodies to the Class 1, 2, and 3 isoforms. Class 1 ALDH is easily detected in normal rat liver and to a lesser extent appears in 3-methylcholanthrene (3-MC) treated liver and whole, uncultured corneas (Fig. 5A). Class 1 ALDH is not detectable in either cultured corneal epithelium, rat hepatoma cells (HTC), or a normal rat liver epithelial cell line (Clone 9). Class 2 ALDH is present in normal liver, HTC cells, and 3-MC-treated liver (Fig. 5B). Interestingly and consistent with the assay data, Class 2 ALDH protein is absent from whole cornea and cultured corneal epithelia. This again correlates with the apparent lack of mitochondria in corneal epithelium. Compared with HTC cells, 3-MC treated liver and normal liver, Class 3 ALDH is clearly the most abundant ALDH isoform in whole cornea and cultured corneal epithelium (Fig. 5C). Furthermore, the enzyme appears to be the same size, 50 kDa, as authentic Class 3 ALDH from HTC cells. Consistent with the assay data, Western analyses detect the changes in Class 3 ALDH protein levels with increased time in culture. As noted previously, Class 3 ALDH from 3-MC-treated liver appears to be slightly smaller than the enzyme derived from cornea and HTC cells(31) . Whether this represents partial proteolysis or an induction-associated post-translational modification event is yet to be determined.
Figure 5: Western blot analysis of cultured corneal epithelium for aldehyde dehydrogenase isoforms. Aldehyde dehydrogenases detected in cultured rat corneal epithelium using polyclonal, monospecific antibodies to each isoform. A, Class 1 ALDH (1:2000). B, Class 2 ALDH (1:5000). C, Class 3 ALDH (1:7500). All lanes identical for each blot. Lane 1, normal rat liver; lane 2, rat hepatoma cell line, HTC, ALDH3 expressing; lane 3, 3-MC-treated rat liver; lane 4, cultured rat corneal epithelium, 15 days growth; lane 5, cultured rat corneal epithelium, 10 days growth; lane 6, cultured rat corneal epithelium, 5 days growth; lane 7, whole, intact cornea. 10 µg of protein was loaded per lane.
Since the size of the protein, substrate/coenzyme preferences, subcellular localization, and histochemical expression patterns are identical for Class 3 ALDH constitutively expressed in cornea and induced in liver; and since previous evidence indicates regulation of the ALDH3 gene is controlled at the level of transcription (1, 9) , corneal transcripts were compared with liver transcripts. In liver, ALDH3 transcription is from a major start site 45 bp 5` to the initiator codon and produces a 1.7-kb message(5, 6, 9) . Northern analysis indicates ALDH3 transcripts in both whole cornea and cultured corneal epithelium are identical in size to that of ALDH3 transcripts from HTC cells or xenobiotic-treated liver (Fig. 6). Consistent with both assay and Western analysis data, ALDH3 transcript levels decline from the 1st to the 5th day of growth, peak at 10 days, and subsequently decline steadily for the remainder of the culture's viability. This is also consistent with the decrease and subsequent increase in expression of differentiated-function genes in cultured primary cells noted above. Northern hybridization under extremely stringent conditions with a series of cluster probes spanning the ALDH3 exons and untranslated regions indicates corneal transcripts possess all known ALDH3 exons (Fig. 7). Additional or deleted sequences were not evidenced by any obvious differences in transcript size.
Figure 6:
Northern analysis of ALDH3 transcript from
cultured rat corneal epithelium. Total RNA from corneal epithelium at
various stages of culture, probed with
[-
P]dCTP-labeled EcoRI/BglII fragment of ALDH3 cDNA. The
1.7-kb transcript is indicated by an arrow. Lane 1,
whole intact cornea; lane 2, cultured corneal epithelium at
five days growth; lane 3, cultured corneal epithelium at ten
days growth; lane 4, cultured corneal epithelium at 15 days
growth; lane 5, cultured corneal epithelium at 25 days growth; lane 6, rat hepatoma cell line, 7777, non-ALDH3 expressing; lane 7, normal liver epithelial cell line,
Clone 9, non-ALDH3 expressing; lane 8, rat hepatoma
cell line, HTC, ALDH3 expressing. 5 µg of total RNA was
loaded per lane as verified by nondenaturing electrophroesis of sample
RNAs with equivalent optical density concentrations (see
``Materials and Methods'').
Figure 7: ALDH3 exon verification in cultured rat corneal transcripts. 1.7-kb ALDH3 transcript from cultured rat corneal epithelium verified by cluster hybridization. Lane 1, 10 day cultured rat corneal epithelium; lane 2, 15-day cultured corneal epithelium; lane 3, whole intact rat cornea; lane 4, normal rat liver epithelial cell line, Clone 9 (ALDH3 nonexpressing); lane 5, whole normal rat liver; lane 6, rat hepatoma cell line, HTC (ALDH3 expressing). A, RNAs probed with EcoRI/RsaI cDNA fragment to detect exons 1, 2, and 3 and 5`0UTR. B, RNAs probed with RsaI/RsaI cDNA fragment to detect exons 5, 6, 7, and 8. Remaining exons and 3`-UTR produced identical results (data not shown).
RACE methodologies were employed to identify the transcriptional start site and polyadenylation signal sequence for corneal ALDH3 transcripts. First pass 5` RACE generated an anticipated 600-bp PCR fragment from cultured corneal mRNA (Fig. 8). Nested amplification of this fragment then yielded the expected 350-bp piece. If the transcriptional start site for the ALDH3 gene in cornea was dramatically different from that of ALDH3 in liver, then more or less 5`-UTR sequence would first be evident by the 5` RACE product size. Dideoxy sequencing of the cloned corneal 5`-UTR indicated 100% sequence identity through the first 150 bp of cornea and liver ALDH3 transcripts. This confirms that identical transcriptional start sites are used in both cornea and liver. For both tissues, the transcriptional start site defines the same first noncoding exon, 45 bp upstream of the translational initiation codon, which itself resides in exon two. 3` RACE also produced appropriately sized amplification products from cultured corneal mRNA, relative to HTC cell ALDH3 transcripts(18) , 700 and 300 bp, respectively (Fig. 9). Dideoxy sequencing likewise confirmed the use of the same polyadenylation signal sequence for both liver and corneal transcripts. Finally, termination codons for the constitutively expressed and inducible ALDH3 transcripts are in the same nucleotide sequence and are located at the same distance from the polyadenylation signal.
Figure 8: RACE amplification of the ALDH3 5`-UTR from rat corneal epithelium in culture. Lane 1, standard size markers (in base pairs); lane 2, 350-bp PCR fragment from nested amplification of first pass 5` RACE product containing 5`-UTR sequence, transcriptional start site, and initiator codon; lane 3, 600 bp first pass 5` RACE product generated from rat corneal ALDH3 cDNA.
Figure 9: RACE amplification of the ALDH3 3`-UTR from rat corneal epithelium in culture. Lane 1, standard size markers (in base pairs); lane 2, 700-bp first pass 3` RACE product generated from rat corneal ALDH3 cDNA; lane 3, 300-bp nested amplification of first pass 3` RACE product containing 3`-UTR sequence, polyadenylation signal, and terminator codon.
If the gene is the same and the protein is the same, but expression is tissue-dependent, then perhaps the reason for differential gene expression is the occurrence of multiple regulatory mechanisms. Previous studies have indicated that activation of the ALDH3 gene in xenobiotic-treated liver is Ah-receptor mediated and controlled at the level of transcription(32, 33, 34) . Our observations from corneal epithelium exposed to light suggest an additional pathway of ALDH3 regulation also exists. The data presented earlier on ALDH3 activity in cultured corneal epithelium was obtained from cultures grown in continuous dark. Corneal epithelium cultured under cycling light conditions consistently maintains higher levels of Class 3 ALDH, earlier in culture, and without as severe a decline and subsequent rebound in enzyme activity (Fig. 10). Although there is some variability in specific activity which reflects the difficulties of biochemical analysis of organ cultured cells, the results from several light versus dark grown corneal cultures are qualitatively identical. Steady-state levels of ALDH3 mRNA similarly mirror continued expression of the protein in 5-15-day lighted cultures (Fig. 10, inset). These observations are consistent with reports of increased Class 3 ALDH activity correlated with eye opening in newborn mice(35) . Until the eyes are open and exposed to light, Class 3 ALDH activity is barely detectable in whole cornea. While Class 3 ALDH detection correlates to eye opening, this observation does not imply a cause-effect relationship. Presumably, however, if constitutive corneal ALDH3 gene expression is light-influenced, then Class 3 ALDH levels would expectedly decrease in epithelial cultures grown in the dark. Conversely, cultures grown under light conditions could anticipate a steady-state production of Class 3 ALDH. How dark grown cultures re-establish high Class 3 ALDH activity, and why light-exposed corneal cultures do not exhibit higher steady-state specific activities, remains under investigation. However, it is tempting to speculate that light maintains cultured corneal epithelia in a more highly differentiated state and that maintenance of relatively high levels of Class 3 ALDH reflect this.
Figure 10: ALDH activity in rat corneal epithelium cultured under lighted conditions. Class 3 aldehyde dehydrogenase activity (benzaldehyde and NADP) of rat corneal epithelium grown under light and dark conditions. Patterns: solid lines, closed circles = continuous dark cultured epithelium; broken lines, open circles = cycled light cultured epithelium; dash/dot lines, solid triangles = growth curve for both light and dark conditions. Inset, ALDH3 mRNA detected in 0, 5, 10, 15, and 20 day epithelial cultures grown under cycling light conditions. Compared with ALDH3 mRNAs from dark grown cultures (Fig. 6), note steady-state production of transcript from 5-15 days (in the light), which is consistent with enzyme activity levels for the same period. Error bars denote mean ± S.D.; n = 3.
These observations suggest an activation or maintenance pathway in ALDH3 constitutively expressing tissue that is independent from the Ah-receptor mediated pathway utilized during xenobiotic induction. Spectral analysis of the light source (a cool white fluorescent bulb) documents that 95% of energy emissions are above 400 nm(36) . While the major energy output would suggest a cellular response to visible light, we cannot discount UV induction of corneal ALDH3. This is of particular interest given the location of two putative UV-responsive-element consensus sequences within the first 1 kb of ALDH3 5`-flanking region(5) . Studies are underway to determine the molecular basis for possible light-regulated ALDH3 expression in corneal epithelium.
Finally, the work of Holmes and colleagues suggests that a novel ALDH, ALDH5, detectable at the level of ALDH5 transcripts in human and bovine cornea, may also oxidize aldehydes in corneal tissue(35, 37) . ALDH5 transcript, abundant in liver and testes, is 65% homologous to Class 2 ALDH and 25% homologous to Class 3 ALDH(19) . Like Class 2 ALDH, ALDH5 transcripts encode a putative 17 amino acid leader peptide which may function in mitochondrial targeting of Class 5 ALDH(19) . However, ALDH5 transcripts are considerably longer than ALDH2 transcripts (3.0 versus 2.0 kb). To date, neither ALDH5 transcript nor Class 5 ALDH protein has been identified in any rat tissue. Northern analysis of rat liver, testes, and corneal transcripts, using human ALDH5 cDNA probes spanning sequences common to ALDH5 and ALDH2, and unique to ALDH5, did not detected the 3.0-kb message for ALDH5 (Fig. 11). The cDNA probe to common regions of ALDH5/ALDH2 does detect Class 2 transcripts from liver and testes, but not from cornea. The lack of detectable authentic (ALDH2) or putative (ALDH5) mitochondrial transcripts by Northern analysis in corneal epithelium is again consistent with the greatly reduced number of mitochondria observed in these cells.
Figure 11: Northern analysis of ALDH transcripts in rat cornea, testes, and liver. 10 µg total RNA from rat cornea (lanes 1, 4, and 7), testes (lanes 2, 5, and 8), and liver (lanes 3, 6, and 9) probed with radioactively labeled cDNA fragments of ALDH3 (A), ALDH2 (B), and ALDH5 (C), see ``Materials and Methods'' for cDNA probe preparation. ALDH3 transcript (1.7 kb) is expressed only in cornea. ALDH2 message (2.0 kb) is found primarily in normal liver and to a lesser extent in testes. The 3.0-kb transcript for ALDH5 is not detectable in any rat tissue RNAs tested. The 2.0-kb band in lanes 8 and 9 (testes and liver, respectively) reflects cross-hybridization of ALDH5 cDNA to homologous regions of mitochondrial ALDH2. Corneas were processed from adult rats subjected to normal 12-h cycles of light/dark. Prior to Northern analysis, adult rat corneas were demonstrated to have high levels of Class 3 ALDH activity as determined by enzymatic assays (data not shown).
Physiologically, high expression ALDH3 in cornea may be
explained in terms of enzyme availability and enzyme efficiency
relative to other ALDHs, particularly Class 2 ALDH. Cornea is exposed
to very high levels of ultraviolet light that can subsequently induce
oxidative stress via reactive oxygen species. Cytotoxic aldehydes from
reactive oxygen speciesinduced lipid peroxidation, including
malondialdehyde and 4-hydroxynonenal, are normally oxidized by Class 1
ALDH (K = 160 µM) (1) and mitochondrial Class 2 ALDH (38, 39) (K
= 17
µM), respectively. To maintain transparency, however,
corneal epithelium has limited cellular organelles, including
mitochondria(25) . Consequently, mitochondrial Class 2 ALDH is
not available at sufficient levels to function effectively in lipid
aldehyde oxidation. Recent studies also indicate that Class 2 ALDH is
inactivated by suicide adduct formation when exposed to physiologically
relevant (nM to µM) concentrations of cytotoxic
lipid aldehyde substrates(38, 39) . Furthermore,
Western analysis of corneal epithelium indicates only minimal levels of
Class 1, compared with Class 3 ALDH. Given the absence of Class 2 ALDH
and limited amounts of Class 1 ALDH in corneal epithelium, high levels
of Class 3 ALDH obtain. Although somewhat less efficient than other
ALDH's in oxidizing some lipid aldehydes (40) (e.g. malondialdehyde, 4-hydroxynonenal), Class 3 ALDH is far less
sensitive to adduct inactivation by substrate aldehydes(39) .
Moreover, Class 3 ALDH preferentially oxidizes medium chain length
aliphatic aldehydes (C
to C
) produced by lipid
peroxidation (K
values = 1-20
µM)(40) . Finally, Class 3 ALDH may function as a
UV-sink by simply binding NAD and absorbing ultraviolet
light(41) . In either capacity, Class 3 ALDH appears to perform
a major role in protecting the cornea from ultraviolet damage.
Summarily, constitutive corneal Class 3 ALDH is identical to xenobiotic-induced liver Class 3 ALDH in subcellular localization, culture expression patterns, substrate preferences, polypeptide size, and antibody cross-reactivity. Furthermore, identical transcriptional start sites, polyadenylation signals, termination codons, and transcript lengths indicate that a single ALDH3 gene is indeed differentially expressed in a tissue-specific manner. However, constitutive expression of the ALDH3 gene in corneal epithelium may be controlled by a light inducible-light maintenance pathway, separate from the Ah-receptor mediated process found in liver. Given the reduced numbers of mitochondria in corneal epithelium and the resultant low levels of Class 2 and putative Class 5 ALDHs, Class 3 aldehyde dehydrogenase may be a key enzyme in protecting the cornea from the cytotoxic effects of UV radiation.