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(Investigative Ophthalmology and Visual Science. 2003;44:2512-2517.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.02-0889
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Polyamines in Cultured Rabbit Corneal Cells

Haiming Du, Mary Jane Viar, Leonard R. Johnson, and Mitchell A. Watsky

From the Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
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, Ca2+ 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 function10 13 14 15 16 and that also require polyamines for many of their physiological activities17 18 19 20 21 22 include substance P, basic fibroblast growth factor (bFGF), transforming growth factors-{alpha} and -ß, 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 CO2. Putrescine is the precursor of the physiologically relevant polyamines spermidine and spermine (Fig. 1) . {alpha}-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.



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  		FIGURE 1. Flow diagram showing the major intracellular synthesis pathway of polyamines from ornithine. DFMO blocks the first rate-limiting step in the pathway by inhibiting ODC activity.

 

   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Cell Culture
Corneal epithelial, endothelial, and stromal cells were isolated from the corneas of 1 kg New Zealand White rabbits, as previously described.23 24 25 Cells were grown using DMEM (Cellgro; Mediatech, Inc., Herndon, VA) and Ham’s F12 (Gibco, Rockville, MD) plus 15% fetal calf serum (FCS). Cells were passaged at approximately 70% confluence. Cells of passages 2 to 4 were used in all experiments. Rabbits were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

ODC Assay
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 14CO2 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 12,000g 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). 14CO2 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.

Polyamine Assay
Polyamine concentrations were examined using high-performance liquid chromatography (HPLC).28 Rabbit cells (epithelium, endothelium, and keratocytes) were cultured as described earlier in polyamine-free medium, with several concentrations of DFMO added at several time points (1, 5, and 10 mM DFMO added for both 2 and 4 days). Each group was examined in triplicate, unless noted otherwise (some groups had lower samples due to technical difficulties with the HPLC. Results were not compromised). Briefly, cells were lysed and stabilized in perchloric acid, harvested by scraping, and centrifuged at 12,000g for 10 minutes at 4°C. The pellet was assayed for protein content, whereas the supernatant was neutralized and dansylated for the reversed-phase HPLC assay. Dansylation was accomplished by adding of an equal volume of dansyl chloride (10 mg/mL in acetone) to each sample or standard. The mixture was heated for 30 minutes at 70°C in a dry bath. The resultant dansylated polyamines were detected by means of a fluorescence detector (420-AC; Waters Chromatography, Milford, MA). The area under recorded peaks was then compared with similar peaks from polyamine standards (Sigma, St. Louis, MO).

Proliferation Assay
Experiments were performed to examine the influence of polyamines on corneal cell proliferation. These experiments were designed specifically to determine whether depletion of polyamines with DFMO affects proliferation and whether addition of putrescine to the cultures would reverse the effect. Cells were transferred to 100-mm culture dishes (5 x 104 cells/dish) and separated into four groups (three dishes per group per time point). Group 1 was cultured in standard polyamine-free medium (including 15% dFCS). Group 2 had 0.1 mM putrescine added, group 3 had 1.0 mM DFMO added, and group 4 had both 0.1 mM putrescine and 1.0 mM DFMO added. Cells in each culture dish were counted (model Zf; Coulter Electronics Inc., Hialeah, FL) at 24-hour intervals from 72 to 120 hours after plating. All proliferation assays were performed at least twice to confirm repeatability.

Statistics
Day-2 versus day-4 polyamine concentrations were compared by Student’s t-test. All other comparisons were made by ANOVA with the Tukey-Kramer multiple comparison test.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
ODC Assay
Representative results for ODC assays in all three cell types are shown in Figures 2 and 3 . Figure 2 shows the effect of serum and different concentrations of DFMO on ODC activity. For all cell types, serum significantly stimulated ODC activity, whereas DFMO significantly inhibited this serum-stimulated ODC activity in a dose-dependent manner. In addition, DFMO significantly inhibited serum-stimulated ODC activity to levels below those of the serum-free groups in keratocytes and endothelial cells, with a similar trend in epithelial cells.



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  		FIGURE 2. Concentration-dependent DFMO block of serum-stimulated ODC activity in cultured rabbit corneal epithelial cells, keratocytes, and endothelial cells. Both the culture medium and the serum (FCS) were free of exogenous polyamines. All values are mean ± SE. *P < 0.01 versus 15% FCS control; {Delta}P < 0.05 versus serum-free; {dagger}P < 0.01 versus serum-free.

 


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  		FIGURE 3. Time-dependent serum responsiveness of ODC activity in cultured rabbit corneal epithelial cells, keratocytes, and endothelial cells. Both the culture medium and the serum were free of exogenous polyamines. The x-axis represents the duration of serum stimulation. Data are the mean ± SE. *P < 0.01 versus serum-free; {dagger}P < 0.001 versus 8 hours FLS; {Delta}P < 0.001 versus 24 hours with serum.

 
Figure 3 shows the time course of ODC activation by serum. For all three cell types, serum stimulation of ODC activity was time dependent and, as in Figure 2 , was inhibited by DFMO. For epithelial cells, ODC activity became significantly elevated 3 hours after serum was added to the medium, with a peak in activity at 8 hours. Stromal cells showed a similar time course of ODC activity stimulation, whereas endothelial cell ODC activity peaked 24 hours after serum was added to the medium.

Polyamine Assay
Figure 4 shows control and DFMO depleted polyamine concentrations for epithelium, keratocytes, and endothelium, respectively. Each panel in Figure 4 shows putrescine, spermidine, and spermine concentrations in cells cultured for 2 and 4 days in polyamine-free medium and in polyamine-free medium plus different DFMO concentrations. Control putrescine concentrations declined during the 4-day culture period in all three cell types (although not significant in endothelial cells), whereas all concentrations of DFMO completely depleted putrescine at both time points and at all DFMO concentrations examined. Spermidine concentrations varied between cell types during the 4-day culture period in the control group, but as with putrescine, DFMO, at all concentrations examined, completely depleted spermidine in all cell types examined. Spermine, which is the final metabolic byproduct within the polyamine synthetic pathway, was decreased by DFMO at day 2 only in the keratocyte group. Compared with the day-4 control group, spermine concentrations in cells exposed to DFMO for 4 days were significantly reduced at all DFMO concentrations in keratocytes and epithelial cells. In addition, when compared with the 2-day groups, spermine levels in the 4-day groups were significantly lower in all but three groups (1 mM and 5 mM DFMO epithelium and 10 mM DFMO keratocytes), although nowhere near as low as putrescine or spermidine.



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  		FIGURE 4. Polyamine concentrations in cultured corneal epithelial cells, keratocytes, and endothelial cells after 2 and 4 days in culture and after treatment with several concentrations of DFMO. Putrescine and spermidine were undetectable in DFMO-treated cells. Data are the mean ± SE. *P < 0.01 versus matched polyamine control (same day); {Delta}P < 0.05 versus matched polyamine control (same day); {dagger}P < 0.05 versus matched polyamine group (4-day versus 2-day).

 
Proliferation Assay
Figure 5 shows the effects of polyamines and polyamine depletion on the proliferation of corneal epithelium, keratocytes, and endothelium. These results demonstrate that for all cell types, inhibition of putrescine synthesis by DFMO and its subsequent depletion of polyamines almost completely inhibited cell proliferation. Addition of putrescine to the medium prevented the inhibition of proliferation in all cell types. Addition of putrescine alone had no effect on cell proliferation.



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  		FIGURE 5. Growth curves (cell counts per dish) of cultured corneal epithelial cells, keratocytes, and endothelial cells grown in polyamine-free medium after addition of putrescine (100 µM), DFMO (1 mM), or DFMO+putrescine. Addition of putrescine to the DFMO group reversed the effects of DFMO. Statistical comparisons were made between different groups at each time point. All values are mean ± SE. *P < 0.001 versus DFMO group.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
This work demonstrates that polyamines are present in all cell types of the cornea, that their formation is catalyzed at least in part by ODC, and that they are an important component of corneal cell proliferation. As has been reported in other cell types,29 30 31 32 ODC activity, the first rate-limiting catalyst of polyamine synthesis (Fig. 1) , was stimulated by FCS in all three corneal cell types, with DFMO inhibiting this activity in a dose-dependent manner. Serum stimulation of ODC activity was time dependent, with significant elevation by 3 hours in epithelial cells and by 8 hours in keratocytes and endothelial cells. Maximum stimulation of ODC activity occurred by 8 hours in epithelial cells and keratocytes and by 24 hours in endothelial cells. This time-dependent increase in ODC activity may be related to stimulation of the cell cycle, given that reduction of ODC activity by DFMO inhibited serum-stimulated cell proliferation in all three corneal cell types (Fig. 5) . The decline in ODC activity over time, despite the continuous presence of serum, is probably related to ODC’s having one of the shortest half-lives of any known mammalian enzyme, in addition to the fact that its activity is inhibited through a negative-feedback pathway linked to increases in polyamine concentrations.33 34

DFMO inhibition of ODC activity led to significantly reduced polyamine concentrations. By day 2, both putrescine and spermidine were undetectable in all three cell types. Spermine was reduced only in keratocytes at day 2. After cells were exposed to DFMO for 4 days, spermine concentrations were significantly lower than in their day-2 counterparts in almost every group. Administration of DFMO has been shown to lead to a similar pattern of reduced polyamine concentrations in IEC-6 cells, a cell line derived from rat intestinal crypt cells.35 36 The relative resistance of spermine to DFMO treatment is probably related to its being the most highly charged of the group and thus the most tightly bound. It is likely that only the free and less tightly bound polyamines are depleted by DFMO treatment and that these are responsible for most of the physiological effects of the compounds.

Cell proliferation is an important event for all three cell types during wound healing in the rabbit cornea. In our study, depletion of polyamines resulted in a concomitant inhibition of cell proliferation in all three corneal cell types, and addition of exogenous putrescine to the culture medium prevented the inhibition of growth. A similar DFMO-induced inhibition of growth and prevention of inhibition by addition of exogenous polyamines has been demonstrated previously.32 The prevention of inhibition is due to transport of the externally added polyamine into the cells and metabolism of that polyamine downstream of ODC. Once in the cells, the polycationic polyamines bind to RNA,37 affecting the synthesis of proteins involved in the cell cycle. These proteins include, but are not limited to, p21Waf1/Cip1, p27Kip1, and p53.38 39 In addition, polyamines interact with K+ channels, and this may also play a role in cell growth. Addition of exogenous putrescine to the control cells (cultured in the presence of dFCS) had no effect on cell proliferation, probably because of serum stimulation of ODC activity in the cells, leading to increased polyamine synthesis in the cells and stimulation of proliferation, which was not affected any further by addition of exogenous putrescine.

A number of growth factors and signaling pathways leading to cell proliferation have been described in corneal cells, including the pathways involving EGF and TGFß.6 7 8 9 10 11 EGF generally stimulates proliferation, whereas TGFß inhibits it. IEC-6 cells showed similar responses to EGF and TGFß. In these cells, polyamine depletion with DFMO disrupted the cytoskeletal architecture and prevented the prompt association of EGF-bound EGF receptor (EGFR) with actin. In addition, EGFR protein tyrosine phosphorylation and kinase activity were reduced by 50%.12 In contrast, polyamine depletion increased the expression of the TGFß type 1 receptor, leading to an increased sensitivity to TGFß–induced inhibition of growth.40 Both the EGF and TGF effects induced by DFMO were overcome by addition of exogenous polyamines to the growth media. It is not known whether these same events occur in corneal cells.

This is the first comprehensive examination of polyamines and ODC activity in corneal cells. We have determined that the three major cell types of the cornea—epithelial cells, keratocytes, and endothelial cells—all have similar, significant DFMO-inhibitable ODC activity, polyamine concentrations, and dependence on polyamines for cell growth. Future studies will examine polyamine transport into corneal cells in more detail and will continue to examine the role of polyamines in the physiological functions of these cells.


   Footnotes
 
Supported by National Eye Institute Grants EY12821 (MAW), DK16505 (LRJ), and a research grant from the University of Tennessee Medical Group.

Submitted for publication September 30, 2002; revised November 25, 2002; accepted January 9, 2003.

Disclosure: H. Du, None; M.J. Viar, None; L.R. Johnson, Merrell Dow (F); M.A. Watsky, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Mitchell A. Watsky, Department of Physiology, The University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163; mwatsky{at}physio1.utmem.edu.


   References
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 Abstract
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 Discussion
 References
 

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