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...

The sensing by T cells of metabolic and energetic changes in the microenvironment can determine the differentiation, maturation, and activation of these cells. Although it is known that mammalian target of rapamycin (mTOR) gauges nutritonal and energetic signals in the extracellular milieu, it is not known how mTOR and metabolism influence CD4+CD25−FOXP3− effector T cell (Teff) responses. In this article, we show that leptin-induced activation of mTOR, which, in turn, controls leptin production and signaling, causes a defined cellular, biochemical, and transcriptional signature that determine the outcome of Teff responses, both in vitro and in vivo. The blockade of leptin/leptin receptor signaling, induced by genetic means or by starvation, leads to impaired mTOR activity that inhibits the proliferation of Teffs in vivo. Notably, the transcriptional signature of Teffs in the presence of leptin blockade appears similar to that observed in rapamycin-treated Teffs. These results identify a novel link between nutritional status and Teff responses through the leptin–mTOR axis and define a potential target for Teff modulation in normal and pathologic conditions.

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

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.

For decades, obesity has been considered to be the result of the complex interaction between genes and the environment and its pathogenesis is still unresolved. The discovery of hormones and neural mediators responsible for the control of food intake and metabolism at the hypothalamic level has provided fundamental insights into the complicated pathways that control food intake. However, the molecular basis for the association between obesity and low-degree chronic inflammation is still unknown. More recently, the discovery of leptin, one of the most abundant adipocyte-derived hormones, has suggested that nutritional status, through leptin secretion, can control immune self-tolerance modulating Treg suppressive function and responsiveness. Furthermore, recent experimental evidence has shown the presence of an abundant adipose tissue-resident Treg population responsible for the control of metabolic parameters and glucose homeostasis. Better knowledge of the intricate network of interactions among leptin-related energy regulation, Treg activities and obesity could lead to valuable strategies for therapeutic intervention in obesity and obesity-associated insulin resistance.