At this point, the cellular population affected by these network alterations remains uncertain. This lack of hypothesis led us to perform, in a first approach, a full-thickness corneal study. The recovered amounts of mRNAs were largely in favor of epithelial cells, and this could have biased the results and consequently their interpretation. Nevertheless, one can imagine that both epithelial cells and keratocytes are key players. Keratocytes are specialized fibroblasts, derived from neural crest mesenchyme in common with the cartilaginous stromal layer behind the eyeball,
71 that produce collagen fibers and proteoglycans constituting the corneal stroma. Throughout life, most keratocytes are in a quiescent state.
72 By the end of eye development, a keratocyte network, interconnected through dendritic processes, is established. Keratocyte apoptosis, either of quiescent or actively dividing cells, is a process of great interest for corneal growth and remodeling. Previous reports have speculated that keratocytes may be involved in KC development through increased catabolism, either misregulation of metalloprotease activity
67 or a modification of collagen subtype composition.
73 We suggest here the novel hypothesis that keratocyte implication would be mediated by a tissue-specific misregulation of apoptosis that may be due to less redundant antiapoptotic pathways in the cornea. In a healthy cornea, programmed cell death is a rare occurrence, but immediately after an injury, keratocytes directly below the injury site undergo apoptosis when the basal membrane is broken. In the following steps of the healing and scarring process, this cell loss is counteracted by mitoses among the remaining adjacent keratocytes.
62 In KC corneas, impairment of apoptotic signaling pathways on minor corneal injuries sustained through life may affect appropriate keratocyte proliferation, itself necessary for the recovery of the initial normal cell density. Keratocyte-restricted deficiencies in the regulation of apoptosis may lead to the gene expression differences we have observed in this study. This could provide an explanation for the loss of cell density and corneal thinning observed in KC. Interestingly, a recent study underlined the role of
TWIST2, a bHLH transcription factor, in keratocyte proliferation in mouse leading to a corneal thinning.
74 The epithelium, with cells that are continuously renewed, may also be affected by the antiproliferative and hyperapoptotic phenotypes. The 18 overexpressed genes we observed to be significantly differentially expressed included
KRT78,
ROR2,
S100A6,
MUC4, and
KRT78 is a structural protein of epithelial cells whose cross-linking helps withstand mechanical and chemical stresses.
75 ROR2 encodes a nuclear orphan receptor in the noncanonical Wnt pathway that appears to trigger, among other effects, the maintenance and proliferation of stem cells.
76 S100A6 (calcyclin) is involved in the fibroblast cell cycle
77 and overexpressed during wound healing after corneal injury. In addition, sialomucin (
MUC4) is expressed by the corneal epithelium, which raises the question of how epithelial cells may also be involved in the pathogenesis of KC. We found increased expression of the whole transcript population of
MUC4 in KC corneas. The apical location of mucins could suggest their general involvement in mechanisms of response to epithelial damage.
78 In addition, among mucins,
MUC4 displays tissue-specific expression patterns—notably, a conjunctival-type expression pattern is observed on the corneal surface in limbal stem cell deficiency.
79 Both mucin-4 (the product of the
MUC4 gene) and calcyclin are implicated in corneal protection and wound healing by the constitution of the lacrimal film.
80,81 Mutations in other mucin genes are already implicated in human pathology, notably the dry eye syndromes
82 (
Supplementary Table S6). Alternatively sialomucin is also an activator partner of the epidermal growth factor ErbB2 receptor, potentially implicating its overexpression in growth factor signaling pathways leading to either differentiation or increased cell proliferation.
78 Each of these overexpressed transcripts therefore could be either pathogenic or simply deregulated as a consequence of other causal molecular mechanisms. Overexpression of
MUC4 may be a protective response or could be causative in KC, and a corneal-specific mouse overexpression model would be welcome to resolve this question.