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Lack of ERK activation and cell migration in FGF-2-deficient endothelial cells¹

GIUSEPPE PINTUCCI^*,, DAVID MOSCATELLI, FIORELLA SAPONARA^*,, PETER R. BIERNACKI^*,, F. GREGORY BAUMANN^*,, COSTAS BIZEKIS, AUBREY C. GALLOWAY^*,, CLAUDIO BASILICO and PAOLO MIGNATTI^*,,2

^* The Seymour Cohn Cardiovascular Surgical Research Laboratory, Division of Cardiothoracic Surgery,
The John H. C. Ranson Basic Science Research Laboratory, Department of Surgery,
Department of Microbiology, and
Department of Cell Biology, New York University School of Medicine, New York, New York, USA

²Correspondence: Department of Cell Biology, NYU School of Medicine, 550 First Ave., New York, NY 10016, USA. E-mail: mignap01{at}med.nyu.edu

SPECIFIC AIMS

We tested the hypothesis that fibroblast growth factor 2 (FGF-2) -induced activation of ERK1/2 is required for endothelial cell migration. We used endothelial cells from mice genetically deficient in FGF-2 to test ERK1/2 activation and cell migration in response to mechanical damage.

PRINCIPAL FINDINGS

1. Lack of ERK1/2 activation and migration in wounded FGF-2 -/- endothelial cells
Exogenous FGF-2 induces a variety of endothelial cell responses through activation of the ERK pathway. To study the role of endogenous FGF-2, we characterized cell migration and ERK-1/2 activation in FGF-2 -/- and wild-type (wt) endothelial cells. By the in vitro wound assay, wt endothelial cells showed high motility; in contrast, wounded FGF-2 -/- endothelial cells migrated very poorly. Neutralizing antibody to FGF-2 greatly reduced wt cell migration; conversely, addition of human recombinant FGF-2 (hrFGF-2) restored normal migration in FGF-2 -/- cells, showing that impaired migration resulted from the genetic deficiency of this growth factor. Analysis of ERK activation showed that FGF-2 -/- endothelial cells had lower levels of active ERK-1/2 than their wt counterpart (Fig. 1 ). Addition of hrFGF-2 resulted in dramatic activation of ERK-1/2 in both cell types. Conversely, wounding of the cell monolayer caused increased ERK-1/2 activation in wt but not FGF-2 -/- cells (Fig. 1) . UO126, a synthetic inhibitor of MEK (MAPK kinase) -1/2, dramatically down-regulated cell migration and abolished ERK1/2 activation in wt and FGF-2 -/- cells; anti-FGF-2 antibody reduced ERK activation only in wt cells. These results show that endogenous FGF-2 controls endothelial cell migration through activation of the ERK pathway.

View larger version (61K): [in this window] [in a new window]   Figure 1. Wounding activates ERK-1/2 in wild-type but not FGF-2 -/- endothelial cells. Western blotting analysis of ERK-1/2 activation in FGF-2 -/- or FGF-2+/+ endothelial cells either wounded (Wound) or treated with 10 ng/ml of hrFGF-2 (+FGF-2) or untreated (Control). The cells were harvested 20 min after growth factor addition or wounding and active ERK was characterized with a phosphospecific antibody. The membrane used for active ERK analysis was stripped and reprobed with anti-ERK-2 antibody to control for equal loading and transfer of cell extract protein. Cell extracts were also analyzed by Western blotting with FGF-2 antibody. High (21.5-22 kDa) and low (18 kDa) molecular mass FGF-2 are present in wt endothelial cells. In contrast, FGF-2 (18 kDa) can be seen only in extracts of FGF-2 -/- cells to which hrFGF-2 was added (+FGF-2). hrFGF-2 (18 kDa) is shown as a control in the rightmost lane. This experiment was repeated 3 times with comparable results.

2. ERK activation occurs at the wound edge
To understand whether ERK is activated in all the cells of a wounded monolayer or only in those cells at the wound edge that participate in the repair response, we characterized ERK activation in situ by immunostaining with antibody to active ERK-1/2. As expected, addition of hrFGF-2 resulted in ERK-1/2 activation in virtually all cells of wt and FGF-2 -/- monolayers. In contrast, wounding induced ERK activation only in wt cells, where active ERK was detected exclusively in cells located at the wound edge. Thus, FGF-2-mediated ERK-1/2 activation after wounding occurs exclusively at the wound edge.

3. FGF-2 induces endothelial cell migration but not proliferation
FGF-2 induces endothelial cell migration and proliferation. To test whether under our wound assay conditions FGF-2 also affected cell proliferation, we characterized FGF-2 -/- endothelial cell migration in the presence of either 0.5% fetal calf serum (FCS) or 10% FCS. With 10% FCS, the number of migrating cells was considerably higher than in the presence of 0.5% FCS. UO126 abolished FGF-2-induced cell migration but had a much lower effect on 10% FCS-stimulated cells. BrdU incorporation experiments showed that 10% FCS induced proliferation among both cells in the monolayer and cells migrated into the wound space. In contrast, hrFGF-2 had no effect on the contact-inhibited cells of the monolayer but induced BrdU incorporation in only a few cells migrated into the wounded area (Fig. 2 ). Migrating wt cells also showed little or no increase in DNA synthesis. Thus, FGF-2 controls endothelial cell migration through ERK activation without affecting proliferation.

View larger version (120K): [in this window] [in a new window]   Figure 2. FGF-2 stimulates cell migration without affecting proliferation. BrdU incorporation in wounded FGF-2 -/- endothelial cells. Wounded cells were incubated with either hrFGF-2 (10 ng/ml) or FCS (10%) or no addition (Control) for 24 h. BrdU uptake was analyzed by immunocytochemistry. Two magnifications of the same fields are shown (left panels, ob 10x; right panels, ob 20x). This experiment was repeated twice with comparable results.

CONCLUSIONS AND SIGNIFICANCE

Endothelial cell repair of mechanical damage is fundamental for the reconstitution of vessel integrity. FGF-2 controls a variety of endothelial cell functions, and its expression levels in vascular endothelial cells correlate with the efficiency of wound repair in vitro. Here we show that 1) mechanical damage of FGF-2 -/- endothelial cells does not result in ERK activation; 2) wound-induced cell migration is strongly decreased in FGF-2-/- endothelial cells; 3) upon mechanical damage, endogenous FGF-2 induces ERK activation only in the cells lining the wound edge; 4) FGF-2-induced ERK activation controls endothelial cell migration without a significant effect on proliferation. Because FGF-2 -/- cells respond to exogenous FGF-2, these findings unequivocally demonstrate a link between FGF-2-induced ERK activation and cell migration (Fig. 3 ).

View larger version (23K): [in this window] [in a new window]   Figure 3. FGF-2-induced ERK activation controls cell migration without affecting proliferation. A) FGF-2-expressing endothelial cells respond to wounding with increased ERK activation, which prompts them to migrate. In contrast, the absence of endogenous FGF-2 results in lack of ERK activation and migration. B) Exogenous FGF-2 restores ERK activation and migration in wounded FGF-2-deficient endothelial cells. C) FGF-2 addition to wounded FGF-2-deficient endothelial cells induces migration via ERK activation without affecting cell proliferation. Serum factors induce activation of a variety of signaling pathways, including ERK activation, with an increase in cell proliferation.

In light of the number of growth factors produced by cultured endothelial cells and the redundancy of FGFs, it is remarkable that FGF-2 alone regulates endothelial wound repair. ERK activation in wounded wt endothelial cells is localized at the wound edge. This effect may be mediated by FGF-2 derived from extracellular matrix or released from damaged cells at the wound edge. ERK activation induced by this growth factor in an autocrine or paracrine manner mediates the intracellular signaling for cell migration.

FGF-2 -/- mice are viable, fertile, and have no gross abnormalities. However, they show neuronal defects in certain brain areas and a delayed repair of excisional skin wounds. No other FGFs appear to compensate for this defect. Because mice deficient in FGF-7, a potent keratinocyte growth factor, have normal excisional skin wound repair, the delayed wound healing observed in FGF-2 -/- mice suggests the involvement of cells other than epidermal keratinocytes. Endothelial cells are key candidates, as new vessel formation is required for the repair of deep skin wounds. Our results indicate a major mechanism to explain the wound repair defect of FGF-2-/- mice.

Most growth factors exert their biological effects mostly by activating the ERK and the phosphoinositide 3 kinase (PI-3K) signaling pathways. These pathways have been implicated in cell proliferation, migration, differentiation, and survival in a variety of cell types. Our results show that endothelial cell migration in response to injury is mediated by FGF-2-induced activation of the ERK pathway. FGF-mediated control of endothelial cell migration but not proliferation represents an efficient mechanism to achieve a rapid angiogenic response. A separate control of proliferation and migration has been described in several systems, including the control of C. elegans myoblast migration by egl-17, a member of the FGF family. Similarly, inhibition of the ERK pathway decreases PDGF-BB-induced migration of vascular smooth muscle cells without inhibition of focal adhesion kinase phosphorylation. However, endothelial cell proliferation requires activation of the ras/ERK and PI-3K/Akt pathways. In our experimental model, FGF-2 strongly activated ERK-1/2, but its effect on Akt activation was much lower than that of serum (Fig. 3) . This mechanism could explain the differential effect of FGF-2 on cell migration and proliferation we observed.

In conclusion, our data establish a link between FGF-2 expression, activation of the ERK pathway, and endothelial cell migration and may provide indications for the development of tools aimed to either block or promote endothelial cell migration in a variety of pathophysiological settings that involve new vessel formation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0815fje; to cite this article, use FASEB J. (February 25, 2002) 10.1096/fj.01-0815fje

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