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A3825 - Caveolin1 Regulates Mitochondrial Homeostasis in the Alveolar Epithelium
Author Block: H. Shaghaghi1, D. Schriner1, X. Hong2, M. I. Ramirez1, F. Romero1, R. S. Summer1; 1Center for Translational Medicine and The Jane and Leonard Korman Institute, Thomas Jefferson University, Philadelphia, PA, United States, 2Medical Research Center, North China University of Science and Technology, Thangshan City, China.
Rationale: Caveolin1 (Cav1) is a multifunctional protein whose deficiency in mice leads to a complex phenotype characterized by spontaneous lung fibrosis and widespread systemic metabolic derangements. Although Cav1 has recently been identified in type II alveolar epithelial cells (AE2), which are one of, if not the most, metabolically active cells in the lung, its role in regulating metabolic homeostasis has not been investigated. Objectives: To determine whether Cav1 regulates metabolic homeostasis in AE2 cells. Methods: Primary AE2 cells were isolated from the lungs of B6129 control mice and Cav1 knockout mice (Cav1-/- mice). Alveolar epithelial A549 cells were transfected with Cav1 shRNA lentivirus in order to evaluate the effects of Cav1 deficiency in culture. Cellular O2 consumption (OCR), acid generation, and mitochondrial complex activity were assessed using a Seahorse Bioscience XFp instrument. Western blot analysis was performed for various proteins including total and phosphorylated AMPK, several lipid synthesis genes and the mitochondrial-associated proteins PGC1-α and TOM20. ROS levels were measured by a commercially available kit. Transcript levels for TGFβ1 were measured by RT-qPCR. DNA damage and cellular senescence markers were assessed by measuring proteins levels of γ-H2AX, p21, p-p53 and the SAPS proteins MMP2 and MMP9. Results: Cav1 deficiency markedly impaired mitochondrial function in AE2 cells, as demonstrated by a decrease in OCR, a decrease in ATP production and an increase in mitochondrial membrane potential. Mitochondrial mass was also found to be significantly reduced in primary and immortalized Cav1 deficient cells. Further, we found mitochondrial dysfunction was associated with an increase in AMPK activation, an increase in mitochondrial ROS production and an accumulation of DNA damage, as judged by an increase in nuclear γ-H2AX levels. Hallmark features of senescent cells were also observed in Cav1 deficient cells including a decrease in LMNB1 expression, an increase in p21 and p-p53 levels as well as an increase in production of several SAPS proteins including MMP2, MMP9, and TGFβ1. Interestingly, pyruvate supplementation reversed most, if not all, of the effects of Cav1 deficiency on alveolar epithelial cells in culture, including restoring mitochondrial function, reducing DNA damage and decreasing cellular senescence. Conclusions: Our data provide evidence that Cav1 regulates mitochondrial function in the alveolar epithelium and suggest that mitochondrial dysfunction induced by Cav1 deficiency could promote lung fibrosis by inducing cellular senescence.