Abstract
purpose. Migration of retinal pigment epithelial (RPE) cells can be triggered by disruption of the RPE monolayer or injury to the neural retina. Migrating cells may re-establish a confluent monolayer, or they may invade the neural retina and disrupt visual function. The purpose of this study was to examine the role of endogenous polyamines in mechanisms of RPE migration.
methods. Endogenous polyamine levels were determined in an immortalized RPE cell line, D407, using HPLC. Activities of the two rate-limiting enzymes for polyamine synthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (SAMdc), were measured by liberation of (14CO2). Migration was assessed in confluent cultures by determining the number of cells migrating into a mechanically denuded area. All measurements were obtained both in control cultures and in cultures treated with synthesis inhibitors that deplete endogenous polyamines. Subcellular localization of endogenous polyamines was determined using a polyamine antibody.
results. The polyamines, spermidine and spermine, as well as their precursor, putrescine, were normal constituents of RPE cells. The two rate-limiting synthetic enzymes were also present, and their activities were stimulated dramatically by addition of serum to the culture medium. Cell migration was similarly stimulated by serum exposure. When endogenous polyamines were depleted, migration was blocked. When polyamines were replenished through uptake, migration was restored. Polyamine immunoreactivity was limited to membrane patches in quiescent cells. In actively migrating and dividing cells, immunoreactivity was enhanced throughout the cytoplasm.
conclusions. Polyamines are essential for RPE migration. Pharmacologic manipulation of the polyamine pathway could provide a therapeutic strategy for regulating anomalous migration.
Retinal pigment epithelium (RPE) normally exists as a mitotically quiescent monolayer of stationary cells that provides structural and metabolic support for visual function of the neural retina. Migration of RPE cells may be triggered by release of chemotactic agents or growth factors. Release of these factors from blood or vitreous is triggered in response to trauma, such as retinal detachment,
1 2 or certain pathologic conditions, such as subretinal neovascularization.
3 4 5 6 In some cases, migratory RPE cells may play a beneficial role in the healing process. They may aggregate along injured capillaries to slow leakage and thus impede further neovascular growth.
7 In contrast, exuberant migration and proliferation can also lead to accumulation of ectopic RPE cells in the form of epiretinal membranes that cause severe vision loss.
8 Thus, it is of considerable interest to examine the migratory behavior of RPE and to understand ways in which migration can be controlled clinically.
To achieve movement, specific cellular mechanisms interact to produce coordinated cycles of four basic steps: protrusion of the leading edge, formation of attachment sites (focal adhesions), generation of contractile forces, and detachment of the trailing edge.
9 Cytoskeletal elements provide the intracellular framework necessary for each of these steps. Migration is initiated at cellular protrusion sites by polymerization of an actin network along the cell’s leading edge. Focal adhesions are composed of complexes of integrin receptors, actin filaments, and associated proteins that provide anchor points for actin stress fibers. Movement is initiated and maintained by contraction of myosin in association with stress fibers.
9
A number of factors appear to regulate these cytoskeletal elements. In RPE cells,
10 as well as other epithelial cell types,
11 protein kinase C stimulates migration, perhaps through phosphorylation of integrin.
12 Mitogen-activated protein kinase has also been shown to be necessary for RPE migration.
13 Other regulatory factors have been identified in several epithelial cell types but have not been studied in RPE. Among these are members of the Rho family of small GTPases, Rho, Cdc42, and Rac, which regulate new protrusions and adhesions through their effects on polymerization and reorganization of actin.
14 Through regulation of p21 activated kinase, they also promote phosphorylation of myosin light chain and hence dimerization and interactions of myosin with actin to drive contraction.
15
Additional regulatory factors recently identified include Src family kinases,
16 focal adhesion kinase (FAK),
17 and calpain.
18 FAK is activated by integrins and targeted to the focal adhesion complex. There, it undergoes additional phosphorylation to provide binding sites for other proteins, such as paxillin, within the focal adhesion complex. Paxillin, in turn, recruits actin to the complex and stimulates formation of stress fibers.
19
Additional potent regulators of migration, the polyamines, have been described in intestinal epithelial cells.
20 21 22 The effect of polyamines on RPE migration has not been studied. Polyamines are composed of flexible carbon chains with amino groups that are positively charged at physiologic pH, and their synthesis is tightly regulated through the inducible enzyme, ornithine decarboxylase (ODC; for review, see Ref.
23 ). Although the exact mechanism of action of polyamines has not been determined, blockage of polyamine synthesis has been shown to cause reorganization of F-actin and tropomyosin and to disrupt the association of F-actin with activated epidermal growth factor (EGF) receptors.
24 25 In addition, polyamine depletion decreases phosphorylation of FAK, recruitment of paxillin, and focal adhesion complex formation.
26
Endogenous polyamines are present in primary cultures of bovine RPE cells, and they are essential for proliferation.
27 Given the role of polyamines in intestinal epithelial cell migration, we wanted to determine whether polyamines also play a role in RPE cell migration. Using a human RPE cell line (D407), we found that polyamine deficiency disrupted migration and that the disruption was prevented by addition of exogenous polyamines. Biochemical analysis coupled with immunocytochemical localization studies suggest that polyamine content increases dramatically, and its subcellular localization changes in cells that migrate in response to disruption of the RPE monolayer.
Our results demonstrate that the polyamines, spermidine and spermine, as well as their precursor, putrescine, are normal constituents of RPE cells. The observation that the activities of both rate-limiting enzymes for polyamine synthesis, ODC and SAMdc, appeared to be highly regulated is important. Low enzyme activities were observed in serum-free medium, whereas the presence of serum transiently stimulated both activities many fold within 2 hours. This suggests that pools of newly synthesized polyamines in RPE can fluctuate rapidly in response to changes in the extracellular environment.
Blockage of polyamine synthesis by DFMO led to a complete depletion of putrescine and spermidine, while spermine levels remained near control levels. This suggests that putrescine and spermidine turn over within 8 hours or less. We were unable to establish the turnover rate for spermine from these data. It is possible that spermine, because it is the most highly charged of the polyamines, remains tightly bound and thus protected from catabolism. Alternatively, if all the putrescine and spermidine lost after DFMO treatment is converted to a stable pool of spermine that does not turn over, the total levels of spermine after DFMO treatment should be several fold higher than control values. The fact that spermine levels were lower than this predicted value suggests that at least some portion, perhaps the major portion, of the spermine pool turns over within 8 hours.
Based on studies in other epithelial cell types,
20 21 22 24 25 changes in newly synthesized polyamine pools may be tied to a number of important functions in RPE cells. Our studies showed for the first time that serum-stimulated migration of RPE cells is dependent on synthesis of polyamines. Cells normally migrate when contact inhibition is lost. This can occur with trauma (e.g., retinal detachment or various surgical procedures), or retinal disease (e.g., epiretinal membranes), or as a natural consequence of aging when random RPE cells die. In our experimental model, contact inhibition was removed by physically scraping away portions of a confluent culture and counting the number of cells that migrate into the denuded area. Blockage of polyamine synthesis by DFMO was found to inhibit the migration that is normally seen 6 hours after serum stimulation. Thus, the migratory response occurs within the time frame we observed for the serum stimulated increase in polyamine synthesis in control cultures. When exogenous polyamines were added to replenish endogenous pools, DFMO had no effect on migration. We thus conclude that RPE migration is dependent on the availability of polyamines that are either endogenously synthesized or taken up from endogenous sources. This also demonstrates that the effects of DFMO are caused by the absence of polyamines and not by a direct action of DFMO itself.
It is interesting to note that the addition of exogenous spermine (or other polyamines) to DFMO treated cells was needed to restore migration, even though the endogenous level of spermine was found to be near normal under these conditions. This suggests that spermine could exist in two pools: one that is sequestered and not readily available for support of migration and a second that is provided by stimulated synthesis (or uptake) and is essential for migration.
Our immunocytochemical data provide the first description of the subcellular localization of polyamines in RPE cells, as well as changes in distribution that occur when cells are actively dividing and migrating. In quiescent cells, polyamine immunoreactivity is limited to discrete membrane patches at apparent points of attachment among adjacent cells. Recent reports have shown that polyamines regulate the structure of the cellular cytoskeleton, particularly the distribution of actin filaments and stress fibers.
22 We speculate that the polyamine-stained regions we observe could represent junctional complexes that serve to anchor the actin cytoskeleton to the membrane and, through integrin, to elements of the extracellular matrix and adjacent cells. If our conjecture is correct, binding of polyamines to junctional complexes could represent one of the sites at which this regulation is effected.
Polyamine immunoreactivity was greatly enhanced in random groups of D407 cells that did not appear contact inhibited and that accumulated on the outer surface of an underlying, confluent monolayer of cells. A similar staining pattern was observed in cells within the monolayer that had lost contact inhibition after adjacent cells had been experimentally removed. Under both of these conditions, dark staining was observed throughout the cytoplasm. Light staining was observed in the nucleus. Numerous unstained vesicles were seen throughout the cytoplasm. We interpret this staining pattern to be a reflection of the stimulated synthesis of polyamines that would be expected to occur in cells undergoing cell division and/or migration.
The discrete subcellular localization of polyamines at presumptive sites of attachment for actin filaments in quiescent cells suggests that polyamines may also serve a housekeeping function in maintaining the organization of the cytoskeleton. DFMO has been shown to cause disruption of actin stress fibers in intestinal epithelial cells in culture and thus to alter cell shape.
22 It is reasonable to suggest that there may be a similar interaction between polyamines and actin filaments during migration, when both polyamine levels and the number of stress fibers increase.
In summary, polyamine function has been well studied in a number of epithelial cell types; however, little is known about the specific roles polyamines may play in RPE. In our findings polyamines were essential for RPE cell migration. Migration is required to establish a confluent monolayer during development and to maintain the monolayer during random loss of cells during aging. Of specific clinical importance is the abnormal RPE cell migration that leads to the formation of epiretinal membranes and vision loss after various forms of retinal trauma or disease. Our results provide a useful experimental model for further examination of RPE cell migration. One future goal will be to develop potential strategies for the pharmacologic regulation of migration that could be suitable for clinical use. This experimental model will also be useful in further defining the basic mechanisms of cell migration and how they are influenced by signaling molecules, such as polyamines.
Supported by Research to Prevent Blindness, National Eye Institute Grants EYO–1655 and EY-13080 and the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52784, a Fight for Sight Student’s Fellowship, and the Thomas A. Gerwin Endowment.
Submitted for publication July 13, 2001; revised November 19, 2001; accepted November 30, 2001.
Commercial relationships policy: N.
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: Dianna A. Johnson, Department of Ophthalmology, University of Tennessee Health Science Center, 855 Monroe Avenue, Memphis TN 38163;
djohnson@mail.eye.utmem.edu.
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