Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125 I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.

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 small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways. ß

Stimulatory autoantibodies against PDGFR appear to be a specific hallmark of scleroderma. Their biologic activity on fibroblasts strongly suggests that they have a causal role in the pathogenesis of the disease.

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