Abstract
purpose. To determine whether polyamines are present in corneal cells, whether corneal cell polyamines can be depleted by blocking the first rate-limiting enzyme in the polyamine synthesis pathway, ornithine decarboxylase (ODC), and whether polyamines are required for proliferation in all three corneal cell types.
methods. Cultured corneal epithelial cells, keratocytes, and endothelial cells were exposed to the specific ODC blocker difluoromethylornithine (DFMO), and ODC activity, intracellular polyamine concentrations, and cell proliferation were measured.
results. DFMO blocked ODC activity in a dose- and time-dependent manner in all three cell types. DFMO treatment completely depleted putrescine and spermidine by 2 days and also significantly depleted spermine. DFMO treatment also inhibited cell growth in all three cell types and this inhibition could be completely reversed by adding exogenous putrescine to the culture medium.
conclusions. Polyamines are present in all cell types of the cornea, their formation is catalyzed at least in part by ODC, and they are an important component of corneal cell proliferation.
Polyamines are polycationic metabolites that have been found in all cell types examined to date. The cellular functions attributed to polyamines are numerous and include important roles in cell migration, proliferation, and differentiation, and to this end, participation in embryogenesis, tumor promotion, and wound healing.
1 In addition, polyamines are important mediators of ion channel rectification, Ca
2+ homeostasis, and protein phosphorylation, and they have been shown to associate with a host of membrane-bound proteins and cytosolic components.
2 To date, only a handful of studies examining polyamine activity and function in the eye have been reported, with only two reports of polyamine activity or function in the cells of the cornea. Forseman et al.
3 examined ornithine decarboxylase (ODC) activity in ocular tissues from developing neonatal rabbits and found low but significant activity in whole corneas at 2 days after birth that decreased with time. Chen et al.
4 determined that exogenously applied polyamines can affect activation of the K3 keratin gene.
After injury, the cells of the cornea migrate to the wound site and organize to perform specialized functions related to the wound-healing process. Corneal epithelial cells, the multilayered cells facing the tear layer and external environment, divide, migrate, and restratify to form a barrier between the external environment and the cornea. In restratifying, they must form an optically correct surface as well as a surface capable of maintaining a proper tear layer. During normal physiological activity, corneal epithelial cell turnover is quite rapid. Corneal endothelial cells, the cells on the inner surface of the cornea responsible for maintaining corneal hydration (and thus transparency), also migrate to cover wounded areas and resume hydration maintenance functions through Na+K+-ATPase–driven membrane transporters. In the rabbit (one of the primary corneal wound-healing animal models) endothelial cells also divide, whereas in the human they typically do not. Corneal keratocytes reside within the corneal stroma. These cells are responsible for the actual healing of the corneal stroma. After wounding, keratocytes activate and migrate to the wound area, where they proliferate and secrete new collagen, metalloproteases, proteoglycans, growth factors, and other compounds needed to repair the corneal wound.
Corneal cell proliferation has been examined in considerable detail, but as mentioned earlier, the influence of polyamines on corneal proliferation has not been addressed. Polyamines probably affect proliferation through their direct interactions with, and stabilization of nucleic acids, stimulating their replication. In addition, polyamines have been shown to activate protein kinases and transcription factors.
5 In the corneal epithelium, it is clear from the literature that epidermal growth factor (EGF) augments proliferation in corneal epithelial cells.
6 7 EGF also stimulates proliferation in corneal endothelial cells and keratocytes.
8 9 10 11 These studies are relevant to the current study in that polyamines have a significant effect on EGF receptor distribution and signaling.
12 Other signaling compounds that affect corneal cell function
10 13 14 15 16 and that also require polyamines for many of their physiological activities
17 18 19 20 21 22 include substance P, basic fibroblast growth factor (bFGF), transforming growth factors-α and -β, integrins, and protein kinase C activators. These obvious connections between corneal cell activities and polyamines have led us to examine their presence and role in corneal cell proliferation.
Ornithine decarboxylase (ODC) is the first rate-limiting enzyme in polyamine synthesis. ODC is a highly selective enzyme that catalyzes the decarboxylation of ornithine to produce putrescine plus CO
2. Putrescine is the precursor of the physiologically relevant polyamines spermidine and spermine
(Fig. 1) . α-Difluoromethylornithine (DFMO) acts as a specific, irreversible inhibitor of ODC and has become an important tool for inhibiting polyamine synthesis and examining the effects of the subsequent depletion of polyamines on specific cell functions. The objectives of this study were to determine whether polyamines are present in the different cell types of the cornea and to examine ODC activity and the role of polyamines in corneal cell proliferation, using DFMO to deplete cellular polyamines.
Cells were grown in 60-mm culture dishes. When the cells reached approximately 60% confluence, they were switched to a serum- and polyamine-free medium that included DMEM plus Ham’s F10 (Gibco). Cells were kept serum-starved for 24 hours to synchronize their growth cycles. To determine ODC sensitivity to DFMO, cells were divided into four groups (each group consisting of three dishes). All groups had 15% dialyzed fetal calf serum (dFCS) added to the medium. Groups 2 to 4 had 0.1, 1, or 5 mM DFMO (provided as a gift from the Merrell Dow Research Institute of Marion Merrell Dow, Cincinnati, OH) added. After 24 hours, the cells were again serum starved (for 48 hours in the presence of DFMO), and then dFCS was again added to all groups for 3 hours (to stimulate ODC activity) in the presence of DFMO (0.0–5.0 mM), at which time the cells were harvested for the ODC assay.
To determine the time course of ODC stimulation by serum, a second series of experiments was performed. Cells were serum starved for 48 hours and divided into multiple groups (each group consisting of three dishes). The control group was collected immediately after 48-hour serum starvation. The remaining groups had dFCS added to the dishes for 1, 3, 8, 24, or 48 hours. As a control, one group had 5 mM DFMO added. As above, experimental groups were identical for each cell type.
ODC assays were performed using a radiometric technique in which the amount of
14CO
2 liberated from
dl-[
l-
14C] ornithine is estimated.
26 Briefly, cells were washed and then harvested in a Tris-EDTA buffer containing pyridoxal 5′-phosphate and dithiothreitol and frozen. Thawed cells were centrifuged at 12,000
g for 10 minutes at 4°C, and the supernatant was assayed for protein content. For the ODC assay, supernatant was added to a buffer containing
14C-ornithine (Dupont-New England Nuclear, Boston, MA).
14CO
2 liberated through ODC activity was trapped on filter paper (Whatman, Clifton, NJ) impregnated with 2 N NaOH, which was then counted on a scintillation counter. Protein concentration was measured using the Bradford method.
27 ODC activity was measured as picomoles per hour per milligram protein. For all these studies, each experiment was performed at least in duplicate to confirm repeatability.