A redox signaling mechanism for density-dependent inhibition of cell growth

Reactive oxygen species (ROS) have recently drawn significant attention as putative mitogenic mediators downstream of activated growth factor receptor s and oncogenic Ras; however, the possibility that a redox-related mechanis m also operates in the negative control of cell proliferation by inhibitory signals has not been investigated thus far. Here we show that the arrest o f growth induced by cell confluence ("contact inhibition") is due, at least in part, to a decrease in the steady-state levels of intracellular ROS and the consequent impairment of mitogenic redox signaling. In confluent fibro blast cultures, the decrease in the concentration of oxygen species was ass ociated with diminished activity of the small GTPase Rac-1, a signal transd ucer directly involved in the ligand dependent generation of oxygen-derived molecules, and was effectively mimicked by exposure of sparse cultures to dithiothreitol (DTT) and inhibitors of enzymes (phospholipase A2 and Lipoxy genase) acting in the arachidonic acid cascade downstream of growth factor receptors and Rac-1, Sparse fibroblasts treated with nontoxic amounts of DT T underwent growth arrest, whereas a low concentration of hydrogen peroxide significantly increased thymidine incorporation in confluent cultures, dem onstrating a causal link between redox changes and growth control by cell d ensity. Removal of oxygen species from sparse cultures was accompanied by a drastic decrease of protein tyrosine phosphorylation after epidermal growt h factor stimulation, which, at a biochemical level, reproduced the signali ng hallmarks of contact inhibition, Moreover, the cytosolic tyrosine phosph atase SHP-2 was identified as a putative target for redox signaling by cell density because the enzyme itself and the associated substrates appear mar kedly dephosphorylated in both confluent and reductant-treated cells after exposure to epidermal growth factor, and SHP-2 enzymatic activity is strong ly activated by DTT in vitro. Taken together, these data support a model in which impaired generation of ROS and increased protein tyrosine phosphatas e activity impede mitogenic signaling in contact-inhibited cells.