Abstract
The Rho family small GTPase Rac1 has been shown to play multiple roles in cell regulation, including actin cytoskeleton organization, transcriptional activation, microtubule dynamics, and endocytosis. Here, we report a novel role of Rac1 in regulating genomic stability and cell senescence. We observed in primary mouse embryonic fibroblasts that deletion of rac1 by gene targeting, as well as expression of the constitutively active Rac1 mutant L61Rac1, led to decreased cell growth that was associated with altered cell cycle progression at both G(1)/S and G(2)/M phases, increased apoptosis, and premature senescence. The senescence induction by either loss or gain of Rac1 activity was due at least in part to an increase in cellular reactive oxygen species (ROS). rac1 gene deletion caused a compensatory up-regulation of a closely related family member, Rac3, in mouse embryonic fibroblasts, the activity of which induced ROS production independently of Rac1. Furthermore, the Rac1-regulated ROS production and senescence correlated with the extent of DNA damage in the Rac1(-/-) and L61Rac1 cells. Treatment of these cells with a ROS inhibitor inhibited phospho-H2AX-positive nuclear focus formation. Finally, phospho-Ser(15) p53 was significantly increased in L61Rac1 and Rac1(-/-) cells, and genetic deletion of p53 from these cells readily reversed the senescence phenotype, indicating that Rac1 is functionally dependent on p53 in regulating cell senescence. Taken together, our results show that Rac1 activity serves as a regulator of cell senescence through modulation of cellular ROS, genomic stability, and p53 activity.
Highlights
Rac GTPases (Rac1, Rac2, and Rac3) are members of the Rho GTPase family that act as molecular switches cycling between an inactive GDP-bound state and an active GTP-bound state in response to a variety of extracellular stimuli [6, 7]
In addition to regulating cell cycle G1/S and G2/M phase transitions and cell survival, appropriately regulated Rac1 activity plays an important role in regulating cell senescence through the maintenance of cellular reactive oxygen species (ROS) levels and genomic stability in primary cells
To achieve a gain of Rac1 activity, WT mouse embryonic fibroblasts (MEFs) were transduced with retrovirus bicistronically expressing the N-terminally HA-tagged constitutively active Rac1 mutant L61Rac1 and enhanced green fluorescent protein (EGFP)
Summary
Rac GTPases (Rac1, Rac2, and Rac3) are members of the Rho GTPase family that act as molecular switches cycling between an inactive GDP-bound state and an active GTP-bound state in response to a variety of extracellular stimuli [6, 7]. Expression of the dominant-negative Rac1 mutant N17Rac1 in WT or Rac1Ϫ/Ϫ MEFs caused significant cell growth inhibition (data not shown) [25].
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