The levels of Ras proteins in human primary fibroblasts are regulated by PDGF (platelet-derived growth factor). PDGF induced post-transcriptionally Ha-Ras by stimulating reactive oxygen species (ROS) and ERK1/2. Activation of ERK1/2 and high ROS levels stabilize Ha-Ras protein, by inhibiting proteasomal degradation. We found a remarkable example in vivo of amplification of this circuitry in fibroblasts derived from systemic sclerosis (scleroderma) lesions, producing vast excess of ROS and undergoing rapid senescence. High ROS, Ha-Ras, and active ERK1/2 stimulated collagen synthesis, DNA damage, and accelerated senescence. Conversely ROS or Ras inhibition interrupted the signaling cascade and restored the normal phenotype. We conclude that in primary fibroblasts stabilization of Ras protein by ROS and ERK1/2 amplifies the response of the cells to growth factors and in systemic sclerosis represents a critical factor in the onset and progression of the disease.Although the detailed molecular nature of the link between oncogenesis and senescence remains obscure, they appear to be two sides of the same coin. Ras and reactive oxygen species (ROS) 3 are two important players that underlie both phenotypes (transformation and senescence), but their effects are somewhat enigmatic. For example, in mammalian cells, expression in fibroblasts of the oncogenic allele of ras (v-Ha-Ras) triggers rapid senescence (1). Also, ROS mediate apoptosis, DNA damage (2), RNA synthesis (3), as well as growth inhibition (4).ROS and Ras signaling are linked. In the yeast Saccharomyces cerevisiae, cAMP-PKA signals are located downstream of Ras. However, constitutively active Ras2 Val19 affects endogenous ROS production and oxygen consumption in a PKA-independent way (5). Ras isoforms in higher eukaryotes are uncoupled from cAMP-PKA signaling, and control many aspects of redox metabolism. We and others have presented data showing that Ha-Ras induced production of superoxide by stimulating the membrane NADPH oxidase complex via ERK1/2 (6 -8). On the other hand, we have found that Ki-Ras-stimulated mitochondrial MnSOD via ERK1/2 and reduced cellular ROS levels (7). Different anchors may dictate different membrane compartments, localizing Ha and Ki-Ras in proximity of specific substrates (9, 10). We note, also, an important difference between the oncogenic activated form and the wild-type version of ras genes. This is illustrated by the opposing effects of these forms on life span of S. cerevisiae: deletion of ras2 or expression of the active RAS Val19 allele decreased life span; overexpression of yeast wild-type RAS2 extended life span (11).In this work, we present a novel level of regulation of Ras proteins, dependent on ERK1/2 signaling. Specifically, we have found that PDGF and ROS induce Ha-Ras in primary fibroblasts. This has revealed a novel and hitherto unknown pathway, which links ROS to Ras protein levels through ERK1/2. We find a remarkable example of this circuitry in vivo in cells derived from patients affected by systemic sclerosi...

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 (H 2 O 2 ). 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 H 2 O 2 , 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 H 2 O 2 . 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 H 2 O 2 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

These findings suggest that dilatation of cerebral arterioles by receptor-mediated activation of adenylate cyclase and by direct activation of guanylate cyclase in the rat is mediated in large part by activation of Ca(2+)-dependent K+ channels.

The cytotoxicity of dental monomers has been widely investigated, but the underlying mechanisms have not been elucidated. We studied the molecular mechanisms involved in cell death induced by HEMA. In human primary fibroblasts, HEMA induced a dose-dependent apoptosis that was confirmed by the activation of caspases-8, -9, and -3. We found an increase of reactive oxygen species (ROS) and NF-kappaB activation after HEMA exposure. Blocking of ROS production by anti-oxidants had no direct influence on apoptosis caused by HEMA, but inhibition of NF-kappaB increased the fraction of apoptotic cells. Accordingly, mouse embryonic fibroblasts (MEF) from p65-/- mice were more susceptible to HEMA-induced apoptosis than were wild-type controls. Our results indicate that exposure to HEMA triggers apoptosis and that this mechanism is not directly dependent upon redox signaling. Nevertheless, ROS induction by HEMA activates NF-kappaB, which exerts a protective role in counteracting apoptosis.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.