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on January 28, 2002

A more recent version of this article appeared on February 26, 2002

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Submitted on July 31, 2001
Revised on December 10, 2001
Accepted on December 21, 2001

Protection of Human Endothelial Cells From Oxidative Stress. Role of Ras-ERK1/2 Signaling

Giovanni Cuda MD, Roberto Paternò MD, Roberto Ceravolo MD, Mafalda Candigliota MD, Nicola Perrotti MD, Francesco Perticone MD, Maria Concetta Faniello PhD, Filippo Schepis MD, Antonio Ruocco MD, Evelina Mele PhD, S. Cassano PhD, Maurizio Bifulco MD, Mariarosaria Santillo PhD, and Enrico V. Avvedimento MD^*

From the Dipartimento di Medicina Sperimentale e Clinica "G. Salvatore" (G.C., R.C., M.C., N.P., F.P., M.C.F., F.S., E.M., M.B, E.V.A.), Università di Catanzaro "Magna Graecia," and Dipartimento di Biologia e Patologia Cellulare e Molecolare (E.V.A.), Centro di Endocrinologia ed Oncologia Sperimentale del CNR (E.V.A., S.C.), Dipartimento di Medicina Clinica e Sperimentale (R.P., A.R.), and Dipartimento di Neuroscienze e Scienze del Comportamento Sezione di Fisiologia (M.S.), Università Federico II, Napoli, Italy.

^* To whom correspondence should be addressed. E-mail: avvedim{at}unina.it.

Background—Reactive oxygen species play a critical role in inducing apoptosis. The small GTPase p21 Ras and the ERK1/2 MAPK have been proposed as key regulators of the signaling cascade triggered by oxidative stress (H₂O₂). Harvey-Ras (Ha-Ras) and Kirsten-Ras (Ki-Ras) isoforms are so far functionally indistinguishable, because they activate the same downstream effectors, including ERK1/2. Moreover, ERK1/2 signaling has been involved in both protection and induction of apoptosis.

Methods and Results—Human umbilical vein endothelial cells (HUVECs) were subjected to H₂O₂, and apoptosis was detected by fluorescence-activated cell sorting analysis, fluorescence microscopy, and caspase-3 activation. Transfection of Ha-Ras and Ki-Ras genes in HUVECs was performed to evaluate the response to H₂O₂. We have found that, whereas Ha-Ras decreases tolerance to oxidative stress, Ki-Ras has a potent antiapoptotic activity. Both effects are mediated by ERK1/2. Tolerance to H₂O₂ is encoded by a unique stretch of lysines at the COOH terminus of the Ki-Ras, lacking in Ha-Ras, and it is relatively independent of the farnesylated anchor. Inhibition of p21 Ras signaling by farnesylation inhibitors increased the resistance to apoptosis in Ha-Ras--expressing cells.

Conclusions—These findings explain the opposite effects of ERK1/2 stimulation on apoptosis found in different cell types and suggest that local activation of ERK1/2 signaling may account for the opposing response to oxidative stress by Ha-Ras or Ki-Ras--expressing cells. Modulation of cell reactivity to oxidative stress by p21 Ras points to the specific and predictive effects of Ras inhibitors in vivo as potential therapeutic drugs in disorders produced by increase of reactive oxygen species inside the cells.

Key words: endothelium • apoptosis • stress • free radicals • hypoxia

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