Estrogen and Cyp1b1 Regulate Pparγ in Pulmonary Hypertension Through a Ubiquitin-Dependent Mechanism

Female sex increases risk of Group I pulmonary arterial hypertension by roughly threefold, but the mechanism is unclear. Low expression of Cyp1b1, an enzyme that metabolizes estrogens, is associated with disease penetrance, particularly in women. We previously found that lower Ppar gamma levels in murine PAH models, which may drive disease, are rescued by estrogen blockade. The goal of the current studies was to examine interaction of estrogen, Cyp1b1, and energy metabolism in cell culture and in knockout mice. We found that both estrogen and siRNA to Cyp1b1 resulted in reduction of Ppar gamma at a protein, but not transcript level, in addition to regulating Ppar gamma cofactors. siCyp1b1 reduced both basal and maximal respiration rates in a fatty acid oxidation Seahorse protocol. This Ppar gamma inhibition could be eliminated by blocking ubiquitination. RNA-seq suggested that Cyp1b1 may be having important pulmonary hypertension effects both in concert with and independently of its effect on estrogen. Cyp1b1 knockout mice have lower Ppar gamma levels than WT mice both in normoxia and hypoxia, and develop mild pulmonary hypertension on a high fat diet. RNA-seq on their lungs reflected similar pathways to those altered in endothelial cells alone - lipid metabolism, cytokines, and vasoreactivity-associated genes, among others, but added genes associated with circadian rhythm. These data suggest multiple potential points for intervention in estrogen and Cyp1b1 mediated etiology of PAH, in particular Ppar gamma ubiquitination, but also suggests that both the difference between E2 and 16aOHE and the impact of Cyp1b1 is more complex than simply "degree of estrogenicity".