Substance P (SP) is a neuropeptide, predominantly released from sensory nerve fibers, with a potentially protective role in diabetic corneal epithelial wound healing. However, the molecular mechanism remains unclear. We investigated the protective mechanism of SP against hyperglycemia-induced corneal epithelial wound healing defects, using type 1 diabetic mice and high glucose–treated corneal epithelial cells. Hyperglycemia induced delayed corneal epithelial wound healing, accompanied by attenuated corneal sensation, mitochondrial dysfunction, and impairments of Akt, epidermal growth factor receptor (EGFR), and Sirt1 activation, as well as decreased reactive oxygen species (ROS) scavenging capacity. However, SP application promoted epithelial wound healing, recovery of corneal sensation, improvement of mitochondrial function, and reactivation of Akt, EGFR, and Sirt1, as well as increased ROS scavenging capacity, in both diabetic mouse corneal epithelium and high glucose–treated corneal epithelial cells. The promotion of SP on diabetic corneal epithelial healing was completely abolished by a neurokinin-1 (NK-1) receptor antagonist. Moreover, the subconjunctival injection of NK-1 receptor antagonist also caused diabetic corneal pathological changes in normal mice. In conclusion, the results suggest that SP-NK-1 receptor signaling plays a critical role in the maintenance of corneal epithelium homeostasis, and that SP signaling through the NK-1 receptor contributes to the promotion of diabetic corneal epithelial wound healing by rescued activation of Akt, EGFR, and Sirt1, improvement of mitochondrial function, and increased ROS scavenging capacity.

The early events that occur rapidly after injury trigger signal cascades that are essential for proper wound closure of corneal epithelial cells. We hypothesize that injury releases ATP, which stimulates purinergic receptors and elicits the phosphorylation of epidermal growth factor receptor (EGFR) tyrosine residues and subsequent cell migration by a MMP and HB-EGF dependent pathway. We demonstrated that the inhibition of purinergic receptors with the antagonist, Reactive Blue 2, abrogated the phosphorylation of EGFR and ERK. Preincubation of cells with the EGFR kinase inhibitor, AG1478, and subsequent stimulation by injury or ATP resulted in a decrease in phosphorylation of EGFR and migration. Furthermore, downregulation of EGFR by siRNA, inhibited the EGF induced intracellular Ca 2+ wave. However, the response to injury and ATP was retained indicating the presence of 2 signaling pathways. Inhibition with either CRM197 or TIMP-3 decreased injury and nucleotide induced phosphorylation of both EGFR and ERK. Incubation in the presence of a functional blocking antibody to HB-EGF also resulted in a decrease in the phosphorylation of EGFR. In addition, cell migration was inhibited by CRM197 and rescued when cells were incubated with HB-EGF. We showed that injury induced phosphorylation of specific tyrosine residues and found that a similar pattern of phosphorylation was induced by trinucleotides. These studies indicate that injury induced purinergic receptor activation leads to phosphorylation of EGFR, ERK and migration.

Wound healing is a complex process that involves cell communication, migration, proliferation, and changes in gene expression. One of the first events after injury is the rapid release of Ca(2+) that propagates as a wave to neighboring cells (Klepeis et al. [2001]: J. Cell. Sci. 114:4185-4195). Our goal was to examine the signaling events induced by cellular injury and identify extracellular molecules that induce the activation of extracellular signal responsive kinase (ERK) (p42/44). In this study we demonstrated that injury induced ERK1/2 activation occurred within 2 min and was negligible by 15 min. Treatment of unwounded cells with wound media caused activation of ERK that could be inhibited by apyrase III. Stimulation with epidermal growth factor (EGF) did not mimic the injury response and it was not detected in the wound media. To identify the active component, size fractionation was performed and factor(s) less than 3 kDa that induced the release of Ca(2+) and activation of ERK1/2 were identified. Activity was not altered by heat denaturation, incubation with proteinase K but it was lost by treatment with apyrase. Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) promoted activation by 2 min with similar profiles as that generated by injury. Preincubation with phospholipase C inhibitor, U73122, inhibited activation that was induced by injury and/or nucleotides. Lack of activation by alpha-beta-methylATP (alpha, beta-MeATP) and beta-gamma-methylATP (beta, gamma-MeATP) to purinergic (P)2X receptors further indicated that activation occurs via P2Y and not P2X purinergic receptors. These results indicate that injury-induced activation of ERK1/2 is mediated by a P2Y signaling pathway.

PURPOSE. To investigate how Sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1) promotes high glucose-attenuated corneal epithelial wound healing.METHODS. The effects of high glucose on SIRT1 expression were assessed in primary human corneal epithelial cells (CECs) in treatment of 5 mM D-glucose (normal glucose [NG]) and 25 mM D-glucose (high glucose [HG]) and corneas from Ins2 Akita/þ mice by Western blotting. The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. Pifithrin-a (PFT-a) was used to inhibit the expression of p53 and an adenovirus was used for overexpression of SIRT1 in vivo and in vitro. How overexpression of SIRT1 promotes HG-attenuated corneal epithelial wound healing via p53 regulation of the IGFBP3 (insulin-like growth factor binding protein-3)/IGF-1 (insulin-like growth factor-1)/AKT pathway was investigated in CECs and Ins2 Akita/þ mice. RESULTS. HG induced the downregulation of SIRT1 and the upregulation of p53 acetylation in primary human CECs and corneas from Ins2Akita/þ mice. The results of cell migration assay and corneal wound healing from Ins2Akita/þ mice demonstrated that SIRT1 overexpression strongly promoted wound healing in the presence of HG levels via the downregulation of the IGFBP3 protein. The levels of total p53 expression and acetylated p53 decreased dramatically in the presence of PFT-a, whereas the IGF-1R/AKT pathway was activated in CECs. The results of cell migration assay suggested this posttranslational modification of p53 was involved in the response to cell injury under HG conditions in CECs.CONCLUSIONS. The molecular mechanism by which SIRT1 promotes corneal epithelial wound healing was involved in an enhancement of the IGFBP3/IGF-1/AKT pathway through the deacetylation of p53. This study also suggests that SIRT1 has a protective role in the pathogenesis of diabetic keratopathy.