Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-κB-dependent pathway

The small GTP-binding protein Rac1, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide production. Cyclin D1 abundance is rate-limiting in normal G₁ phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Ras and Rac1. Nuclear factor-κB (NF-κB) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the IκB family of inhibitor proteins. In the current studies, activating mutants of Rac1 (Rac(Leu-61), Rac(Val-12)) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rac1 required both an NF-κB and an ATF-2 binding site. Inhibiting NF-κB by overexpression of an NF-κB trans-dominant inhibitor (nonphosphorylatable IκBα) reduced cyclin D1 promoter activation by the Rac1 mutants, placing NF-κB in a pathway of Rac1 activation of cyclin D1. Specific amino acid mutations in the amino- terminal effector domain of Rac(Leu-61) had comparable effects on NF-κB transcriptional activity and activation of the cyclin D1 promoter. The NF-κB factors Rel A (p65) and NF-κB₁ (p50) induced the cyclin D1 promoter, requiring both the NF-κB binding site and the ATF-2 site. Stable overexpression of Rac(Leu-61) increased binding of Rel A and NF-κB₁ to the cyclin D1 promoter NF-κB site. Activation of Rac1 in NIH 3T3 cells induces both NF-κB binding and activity and enhances expression of cyclin D1 through an NF-κB and ATF-2 site in the proximal promoter, suggesting a critical role for NF-κB in cell cycle regulation through cyclin D1 and Rac1.