Somatic mutations in the tuberous sclerosis complex-2 (TSC2) gene are associated with pulmonary lymphangioleiomyomatosis (LAM), a disorder characterized by benign lesions of smooth muscle and/or smooth muscle-like cells in the lung. However, the cellular mechanisms underlying LAM disease are largely unknown. Given that the TSC2 gene product tuberin is involved in the regulation of cell growth and proliferation, the present study was designed to investigate the potential roles of TSC2 in regulation of the cell cycle. We studied cell cycle profiles of pulmonary vascular smooth muscle cells (SMCs) derived from Eker rats (Tsc2(+/EK)), a genetic model carrying a germline insertional deletion in one copy of the Tsc2 gene, and the wild-type rats (Tsc2(+/+)), a noncarrier counterpart. We found that Tsc2(+/EK), but not Tsc2(+/+), SMCs displayed increases in cells with > or =4N DNA content (> or =4N cells) and in the bromodeoxyuridine (BrdU) incorporation of > or =4N cells. Centrosome number was also increased in Tsc2(+/EK) SMCs, but the mitotic index was comparable between Tsc2(+/+) and Tsc2(+/EK) SMCs. Furthermore, Tsc2(+/EK) SMCs showed elevated phosphorylation of p70S6K and increased expression of cell cycle regulatory proteins Cdk1, cyclin B, Cdk2, and cyclin E. Inhibition of the mammalian target of rapamycin (mTOR) pathway by rapamycin not only inhibited the phosphorylation of p70(S6K) and the expression of cell cycle regulatory proteins but also reduced accumulation of > or =4N cells and BrdU incorporation of >4N cells. Therefore, our results demonstrate that Tsc2(+/EK) SMCs are predisposed to undergo tetraploidization, involving activation of the mTOR pathway. These findings suggest an important role of Tsc2 in regulation of the cell cycle.