TGF-β1 modulated 440 genes in all three experiments: of these, 198 (45%) were co-modulated by EP for at least one phenotype in at least two experiments, and 42 (9.5%) were co-modulated by EP for at least one phenotype in all three experiments. For the 1961 genes which responded to TGF-β1 in at least two experiments, equivalent co-modulation values were 755 (39%) and 187 (9.5%) genes, respectively.
Table 5 shows 20 genes from the 135-member hepatic fibrosis/hepatic stellate cell activation canonical pathway that were EP-modulated, with suppression of extracellular matrix (ECM) components (
COL1A1,
COL1A2,
COL3A1, and
FN1) and of some autocrine signaling contributors (
TGF-B2,
TGF-B3, and
TGF-BR1). Osteoprotegerin (
TNFRSF11B) is known to increase in the corneal stroma after epithelial scrape injury and to contribute to monocyte infiltration of the cornea,
45 and so its downregulation is a therapeutic advantage. However, other EP-evoked changes would not be advantageous (i.e., upregulation of
IL6R and
IL8 suggests increased inflammatory activity, whereas increased expression of
FAS is proapoptotic
46 ). Elevated levels of
TGF-B1 partially repletes the autocrine signaling, and upregulation of
LAMA1 suggests increased basement membrane formation. The ambiguous nature of these canonical pathway changes led us to examine the larger group of 42 co-modulated genes referred to earlier. These appear in
Table 6, together with genes (bold entries) that showed at least two EP changes in each phenotype (i.e., at least four changes of six).
Table 6 is divided into four groups, according to the effects of TGF-β1 and EP. In group 1, six known transcripts (and three additional panels) were consistently decreased by TGF-β and this decrease was inhibited by EP. In this group
ALDH3A1 and
NR0B1 are of particular interest because they are more highly expressed in keratocytes than in either myofibroblasts or fibroblasts.
47 In our hands,
ALDH3A1 was the sixth most highly expressed gene in the keratocytes and the only gene expressed at this level that was EP-modulated.
ALDH3A1 is a corneal crystallin (structural component) with enzymatic activity that protects against oxidative damage.
48 It may also play a role in suppressing keratocyte proliferation.
49 The protein encoded by
NR0B1 (DAX-1) is a powerful transcriptional repressor,
50 which may also contribute to the keratocytes' quiescent state. In group 2, 28 genes were upregulated by TGF-β, an increase attenuated by EP. This group contained a robust subset (
COL3A1,
CSRP2,
LOX,
MXRA5,
SPARC,
ST6GAL2,
TNC, and
VCAN) that confirms the conclusion drawn from
Table 5 that EP modifies the myofibroblast's ability to synthesize ECM. Groups 3 and 4 contain genes that were modulated in the same direction by both EP and TGF-β. In group 3, the transcription factor
BNC1 (basonuclin 1) is of interest because it is diagnostically low in keratocytes relative to either myofibroblasts or (not shown) fibroblasts. Although the EP-induced upregulation of
NR0B1 may preserve the keratocytes' quiescent phenotype, an EP-induced upregulation of
BNC1 apparently acts in the opposite direction. Another gene of interest in group 3 is
HBEGF, a candidate for stromal-to-epithelial signaling during wound healing.
51,52 EP increased
HBEGF expression in keratocytes but markedly inhibited its upregulation by TGF-β1. Our group 4 exemplar is
EDNRA. We have reported in another study, without enumeration, that the expression of
EDNRA is lower in cultured corneal stromal myofibroblasts than in the equivalent fibroblasts (
Table 3 in Ref.
22). The related transcript
EDNRB was also downregulated by TGF-β1, with respective responses of 0.14- and 0.20-fold (not shown). The present data yield similar values, with a 1/8.82 = 0.11-fold change for
EDNRA from
Table 6, and a mean change of 0.21-fold for
EDNRB. These transcripts encode, respectively, ET
A (endothelin-1 specific) and ET
B (endothelin-1 and -3 binding) receptors, which mediate the angiogenic effects of endothelin in the cornea.
53 Although both were downregulated by TGF-β1,
EDNRA is further downregulated by EP (
Table 6), whereas EP upregulated
EDNRB in myofibroblasts (
Table 5). The physiological significance of these different responses to EP merit further investigation.