We have performed a microarray-based comparative global gene expression profiling (Affymetrix) of the two linings covering the ocular surface, the corneal and conjunctival epithelia. Given the embryologic and functionally related nature of these epithelial linings, the identification of genes and biological pathways that are specifically or preferentially expressed in either of these two tissues could selectively contribute to tissue phenotype and/or function. The use of SIs and PS/A calls (definitions for present and absent transcripts described in the Methods section), the LPE statistical approach, and a limitation of the analysis to substantially expressed transcripts led to the identification of 425 and 803 transcripts that were significantly overexpressed in the cornea and conjunctiva, respectively.
The most prominent global feature of the microarray data is the large excess of differentially expressed transcripts in the conjunctival set versus its corneal counterpart, and the high significance of EASE scores for multiple subcategories within the response to stimulus and organismal physiological processes (). This result probably reflects the multiphenotypic nature of the conjunctival epithelium.
Blood-derived intraepithelial antigen-presenting cells are numerous in this tissue () and lymphocytes account for between 1% and 3% of the epithelial mass.18,19
Consistent with this preponderance of intraepithelial leukocytes, the differential analysis identified multiple blood cell– exclusive cell markers (, ). Of the 29 MHC class II transcripts represented in the HG-U133A microarray, 20 were present in the differential conjunctival sets. displays the genes represented by these transcripts. Notably, the expression levels for some of the MHC transcripts match the expression levels of housekeeping genes such as β
-actin and GAPDH. MHC class II genes are expressed only in cells from the hematopoietic lineage. Thus, the results imply that the intraepithelial cells in conjunctiva express class II genes at extraordinary levels. The very intense level of the immunostaining for HLA class II for DP, DQ, and/or DR subtypes () is fully consistent with this conclusion. The presence of lymphocytes is reflected by the prominent location of the lymphocyte-specific protein 1 (LSP1) within the exclusive Cnj gene list (). Likewise, the high concentration of melanocytes within the Cnj epithelial basal layer () is reflected in the substantial representation of TYRP-1 and -2 (, ).
In contrast to conjunctival gene expression, the homogeneous cellular nature of the corneal epithelium generated minimal uncertainty as to the epithelial cell origin of transcripts that showed preferential expression in this tissue. EASE analysis indicated that the preferential expression of transcripts related to genes involved in the control of oxidative damage and heavy metal detoxification were the only biological processes that are more prominent in the corneal than in the conjunctival epithelia at a statistically significant level. The high levels of cornea-preferred genes of two well-known corneal proteins, aldolase dehydrogenase type 3AI and transketolase,20,21
and of an NADH/NADPH reductase (diaphorase), known under the aliases of NQO1,22
diaphorase 4, cytochrome b5 reductase, and dioxin-inducible menadione oxidoreductase, are likely to be the main contributors to the statistical significance of this EASE score (, ). NQO1 has not been shown to be prominent in the cornea. Its function is to catalyze the two-electron reduction of quinones to hydroquinones, thereby preempting one-electron reduction of quinones by other reductases. The latter leads to the formation of detrimental reactive oxygen species. Overall, NQO1 could protect the cornea by directly scavenging free radicals or by allowing the rapid recovery of highly efficient free radical scavengers, such as ascorbate, to their active state.23,24
The biological significance of NQO1 is also indicated by the fact that its absence leads to severe neurologic disability due to deficiencies in lipid and steroid metabolism that cause toxic accumulations in microsomal compartments.25
Consistent with the NQO1 expression differences, our histochemical study of NADPH reductase indicates large differences in total oxiredox activity between corneal and conjunctival epithelia. Aldehyde dehydrogenase type 3A1 has recently been shown to be critical for corneal oxidative protection.26
Considering that the gene expression level for diaphorase/NQO1 is similar to, and the Co/CNJ ratio is larger than, those for ALDH3A1, a detailed study of the role(s) played by this diaphorase in corneal biology appears to be highly warranted.
SIs and Co/Cnj Ratios for Corneal Reductases and Dehydrogenases
In addition to global gene expression analyses, it is also valuable to focus on the significance of individual transcripts (). The highest-expressing cornea-exclusive gene is testican 1. This large secreted multidomain proteoglycan has been shown to have strong inhibitory activity against cathepsin and activation of matrix metalloproteinases (MMPs).27,28
The corneal epithelium expresses multiple MMPs and cathepsins, in particular, cathepsin L/V2,29
which in itself shows a high corneal preference (). Thus, it is intriguing to consider the possibility of a role for testican-1 in locally protecting the corneal epithelial cell surface membrane against the activity of its own proteolytic agents. The third gene in the cornea-exclusive list is a formin isoform. Formins are a conserved family of actin nucleators responsible for the assembly of diverse actin structures such as cytokinetic rings and filopodia. Formin binding to the barbed end of actin filaments increases filament flexibility and has been documented to cause critical modulation of cell adhesion and cell motility. The exclusive presence of a certain formin isoform in the corneal epithelium is likely to play a weighty role in the phenotypic difference of this tissue with that of its ocular surface counterpart. It will be interesting to assess formin’s contribution to the rapid migration in corneal wound healing and response to physical pressure.30
Another intriguing and unexpected corneal gene is cartilage acidic protein 1 (CRTAC1; ), a matrix component with high affinity for integrins. It is profusely expressed in cartilage, a mostly avascular tissue, but it is absent in most other organs and tissues examined, including brain, liver, and muscle.31
Its corneal expression may indicate that its function is somehow related to the avascular environs. Adiponectin, is another cornea-preferred, highly expressed gene. In a fashion similar to CRTAC1, it codes for a protein that until now has been believed to be specific for one cell type, the adipocyte.32
Adiponectin is a secreted protein with multiple systemic functions including growth-promoting activity in epithelial cells.33,34
If indeed, adiponectin is secreted by the corneal epithelium, it may function as part of the proliferation promoter autocrine loop. Other valuable corneal preferred genes to be considered for future investigations are chemokine CXCL14 (BRAK) and Dickkoft 3 (DKK3). CXCL14 plays a central role in monocyte attraction and their in situ conversion to Langerhans cells.35
Thus, it could be important for the ontogeny of these immunosurveillance cells in the limbus. DKK proteins are involved in the regulation of the WNT-β
-catenin cascade, which, in turn may control developmental cell fate36
and the expression of connexin43, another highly expressed corneal preferred transcript (). DKK2, which is not expressed in the mature human cornea (), has recently been found to be a critical component of corneal epithelial development in mouse.37
Thus, it is possible that in humans, this role is performed by DKK3.
In the case of the conjunctival epithelium, the presence of substantial levels of infiltrated leukocytes and resident melanocytes implies that for most transcripts, confirmation of a bona fide epithelial origin will require specific spatial techniques such as in situ hybridization and/or immunohistochemistry. In the present study, we performed such a test for sPLA2
-IIA, a natural antimicrobial protein in tears38,39
and a systemic proinflammatory mediator40
that occupies the top position in the list of conjunctiva-expressed transcripts not expressed in the cornea (). Immunohistology showed that the protein is abundantly expressed in the conjunctival Goblet cells. The stain displays spatial patterns and features suggestive of a secretory process (). To our knowledge, this is the first report of a nonmucinous secretion by mucin-rich cells anywhere in the body. Thus, the potential role(s) of this sPLA2
n in Goblet cell biology and secretory activity warrant further study. The more tenuous expression in other differentiated cells is consistent with the identification of high levels of gene and polypeptide expression of sPLA2
class II, in a human conjunctival epithelial cell line that does not form mature Goblet cells.41
Other very prominent conjunctival novel genes within the conjunctival lists that deserve future attention include lipocalin 2, an iron sequester protein that is part of tissue innate immunity42
; insulin-like growth factor binding protein-3 (IGFBP3), a multifunctional soluble protein that regulates the potent mitogenic and antiapoptotic effects of IGF-I and -II and has been recently shown to have decreased expression in pterygium43
; RARRES1, a retinoid receptor; trefoil factor 1, an estrogen-inducible protein; ceruloplasmin (), and the Na-dependent phosphate transporter isoform NaPi-IIb (). In the kidney proximal tubule and intestine, this transporter is localized at apical membranes, where it determines reabsorption or uptake of phosphate toward the circulation.44,45
A similar role in the conjunctiva implies vigorous phosphate removal from the tears, which may be important in corneal protection; recent studies have shown that phosphate catalyzes corneal calcification.46,47
For some proteins (e.g., IGFBPs48
) the two tissues displayed isoform expression differences. These secreted, high-affinity, IGF-binding proteins act as modulators of IGF bioavailability. Differential patterns of spatial and temporal expression in dynamic processes such a development and cellular transformation, indicate subtle differences in function. The corneal epithelium expresses high levels of the isoforms 6 and 7 (). These isoforms are poorly expressed in the conjunctival epithelium which, conversely, shows a selectivity for isoform 3 ().
Finally, in relation to keratins, the fundamental markers of epithelial phenotype, the microarray measurements yielded, as expected, high expression levels of the two cornea-specific keratins K3 and K1249
(). In addition, consistent with its cornea-specific classification, K3 was absent from the conjunctiva. K12, however, was expressed in this tissue at substantial levels. This result reflects the fact that patches of K12-expressing cells are intercalated within the conjunctival epithelium. As proposed by Kawasaki et al.,50
the simplest interpretation of the patch pattern is the existence of a misdirected cell migration of corneal epithelial precursors from the limbus toward the conjunctiva. However, examination of other genes highly expressed in the cornea does not fit such a model. For instance, K3, testican-1, and ECM-1 transcripts well expressed in the cornea, are not found at all within the conjunctival set (). In addition, the conjunctiva/cornea SI ratios for certain transcripts showing abundant corneal expression (e.g., cathepsin L2/V and NQO1 in ) are much lower than those for K12. Thus, none of those genes are substantially expressed within the K12-positive cells in the conjunctival cells. Therefore, the possibility that the cells in the K12-positive patches are not corneal cells but rather K12-expressing conjunctival cells requires attention. Other keratins also provide interesting distribution patterns. The simple epithelial keratin type 18 was moderately expressed by both tissues whereas K4, K13, K15, and K19 expression occurred at very high levels in the conjunctival domain only. These gene expression patterns in humans are generally coincident with reports of keratin protein expression in the ocular surface of rodents.51
Finally, K19 has been mentioned occasionally as a potential limbal stem cell marker.52
Given the conjunctival levels observed (), it is unlikely that it plays such a function in the conjunctiva.
Keratin Gene Expression Levels in Human Cornea and Conjunctiva
In summary, our microarray analysis of the embryologically related, yet phenotypically disparate corneal and conjunctival epithelia, allowed the identification of a large number of novel conjunctiva- and cornea-preferred transcripts for proteins that can have important roles in the ocular epithelial physiology of each of these tissues.