Platelet-derived Growth Factor and Reactive Oxygen Species (ROS) Regulate Ras Protein Levels in Primary Human Fibroblasts via ERK1/2

Abstract

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...

Integrin α1β1 Controls Reactive Oxygen Species Synthesis by Negatively Regulating Epidermal Growth Factor Receptor-Mediated Rac Activation

“…Other cell types, including fibroblasts, can increase collagen in a ROS-dependent manner (50). In these cells, ROS contribute to the stabilization of H-Ras protein with subsequent ERK1/2-mediated collagen stimulation (50). Thus, as in fibroblasts, the ROS-dependent increased collagen production by ␣1KO mesangial cells may be due to activation of the Rasmitogen-activated protein kinase pathway.…”

“…Other cell types, including fibroblasts, can increase collagen in a ROS-dependent manner (50). In these cells, ROS contribute to the stabilization of H-Ras protein with subsequent ERK1/2-mediated collagen stimulation (50).…”

Smad1 pathway is activated in systemic sclerosis fibroblasts and is targeted by imatinib mesylate

“…Constitutive activation of ERK-1/2 signaling is also observed in cultured SSc fibroblasts (22,23). Since imatinib mesylate blocks fibrosis through a mechanism that is also independent of Smad2/3, we wished to determine whether imatinib mesylate affects activation of the Smad1 and/or ERK-1/2 pathways.…”

Epitope Specificity Determines Pathogenicity and Detectability of Anti–Platelet‐Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis

“…To quantify collagen gene transcription, total RNA was extracted from the primary human fibroblasts using the Aurum total RNA Mini kit (Bio-Rad) at baseline and after 1 The ROS bioassay was performed as described previously (3,16), using a fluorometric assay to determine the amount of intracellular ROS generated by adherent fibroblasts.…”

“…Finally, direct and competitive ELISAs were performed with serum samples from 29 consecutive patients with SLE (SLE Disease Activity Index range [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The distribution of PDGFRa binding in SLE serum was comparable to that in healthy control serum, as determined by direct ELISA with PDGFRa-His.…”