Project Summary/Abstract Lymphangioleiomyomatosis (LAM), characterized by nodular proliferation of abnormal smooth muscle- like cells (LAM cells) and pulmonary cysts, is a significant cause of morbidity and mortality in women with Tuberous Sclerosis Complex (TSC) or with the sporadic form of LAM. LAM is associated with genetic inactivation of TSC1 or TSC2. Critical knowledge gaps include the heterogeneity of cells within LAM nodules, the cellular origin of TSC2-deficient LAM cells, and mechanisms underlying the female predominance of LAM. Early developmental deletion of Tsc2 (E6.5-E14.5) in lung mesenchyme resulted in nodular pulmonary lesions in adulthood, predominantly in female mice. These proliferative nodules are composed of cells with loss of Tsc2 as well as wild-type cells, resembling human LAM nodules. The nodules contain cells expressing smooth muscle and melanocyte lineage proteins, hallmarks of human LAM. Later developmental deletion of Tsc2 (E13.5-P1), sparing the pulmonary vasculature, does not result in pulmonary nodular lesions in adulthood. Metabolic and mitochondrial defects are present in Tsc2-null mesenchymal cells derived from the pulmonary nodules, compared with wild-type controls. Estrogen stimulates proliferation of these Tsc2-null cells, but not wild-type cells, and upregulates mitochondrially-derived reactive oxygen species. These and other preliminary data lead to our central hypothesis: hyperactivation of mTORC1 in subsets of lung mesenchymal cells leads to estrogen-dependent proliferation of nodular-like lesions, associated with metabolic and mitochondrial dysfunction, recruitment of extrapulmonary cells and lung destruction. Two specific aims are proposed: 1) To dissect the molecular and cellular mechanisms of LAM using a novel mouse model with spontaneous LAM-like lung lesions. The role of hyperactivated mTORC1 in the pulmonary LAM-like nodules will be examined using both pharmacologic and genetic approaches. The impact of Tsc2 deficiency on cellular metabolism and mitochondrial function will be addressed for the first time in lung mesenchyme-derived cells as a potential mechanism for the abnormal cell growth. The origin of LAM cells and the mechanisms of recruitment of extrapulmonary mesenchymal cells to LAM-like nodules will be examined using both parabiosis and allograft models. 2) To determine the specific role of gender in contributing to the formation and progression of LAM-like nodules in mice with lung mesenchymal Tsc2 deletion. The roles of estrogen in the pulmonary LAM-like lesions will be investigated in vivo by altering estrogen activity or simultaneous deletion of ERα/Tsc2 or ERβ/Tsc2 and in vitro by metabolic profiling, RNA-seq, ChIP-seq in Tsc2-null vs. wild-type cells. This project will have high scientific and clinical impact by filling knowledge gaps with translational impact for women with LAM and providing a new preclinical therapeutic model.