Regulation of GelB During Corneal Resurfacing

Abstract

Abstract: : Purpose:Matrix metalloproteinases (MMPs) are key regulators of tissue remodeling in cornea and other organs. MMPs such as MMP–1 and –3 are expressed only on demand, typically stimulated by autocrine or paracrine cytokines. In contrast, we have found that expression of gelatinase B (GelB; MMP–9), a MMP expressed by corneal epithelial cells (CECs), is relatively refractory to cytokine stimulation. Nevertheless, reports from other labs suggest that there may be physiologically relevant conditions under which cytokines could act as significant stimulators of MMP–9 expression by CECs. The purpose of this study was to investigate some of these possibilities. Methods: CECs were harvested from rabbits, plated in 24 well culture dishes, and allowed to adhere overnight, when cells were thoroughly washed to abrogate all receptor–mediated signaling. Many labs use cholera toxin in their CEC cultures because it stimulates cell growth by up–regulating intracellular cAMP levels. We hypothesized that this provides a secondary signal needed for cytokine induction of GelB. Cultures were treated with IL–1ß, TNF–α, TGF–ß, EGF, HB–EGF, or FGF–2 at 10ng/mL, in the presence or absence of 30ng/mL of cholera toxin. PMA (1µM) was used as a positive control. Conditioned media was then collected to quantify GelB secretion by zymography. To ensure a CE phenotype, immunocytochemistry was performed using antibodies against keratin 12. Results: GelB expression is induced at the migrating front of the CE in healing wounds. GelB is also expressed constitutively by CECs in culture, with highest expression levels at low cell density, mimicking the conditions of low cell contact at the migrating wound front. GelB can also be induced to high levels by agents such as PMA that activate signal transduction pathways downstream of cytokine receptors. None of the cytokines, alone or with cholera toxin, stimulated GelB expression above constitutive levels. As EGF is endogenously produced by migrating CECs, we treated with AG1478, an EGFR antagonist, to inhibit endogenous EGFR activity; this did not reduce constitutive expression. Additionally, EGF combined with IL–1beta was unable to elicit upregulation of GelB expression, with or without cholera toxin. Experiments were performed multiple times at different plating densities (5*10⁴, 1*10⁵, 2*10⁵, 4*10⁵, and 8*10⁵ cells/well) with unvarying results. In contrast, GelB expression was stimulated to high levels by the positive control PMA. Conclusions: The results presented here provide further support for the idea that GelB expression is not under significant paracrine or autocrine regulation, but is instead controlled by cell–cell contact or physical forces at the migrating wound edge.

Keywords: cornea: epithelium • enzymes/enzyme inhibitors • cytokines/chemokines