Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell Activation

• Published in: The Journal of biological chemistry 2016-03, Vol.291 (12), p.6262-6271
• Main Authors: Walker, Natalie M., Belloli, Elizabeth A., Stuckey, Linda, Chan, Kevin M., Lin, Jules, Lynch, William, Chang, Andrew, Mazzoni, Serina M., Fingar, Diane C., Lama, Vibha N.
• Format: Article
• Language: English
• Subjects: Bronchiolitis obliterans syndrome; Cell Biology; Cells, Cultured; Chronic allograft rejection; collagen; Collagen Type I - metabolism; extracellular matrix protein; fibrosis; Humans; Lung - pathology; Lung Transplantation; mammalian target of rapamycin (mTOR); Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; mesenchymal cell; Morpholines - pharmacology; Multiprotein Complexes - antagonists & inhibitors; Multiprotein Complexes - metabolism; pulmonary fibrosis; Pulmonary Fibrosis - metabolism; Pulmonary Fibrosis - pathology; Signal Transduction; Sirolimus - pharmacology; TOR Serine-Threonine Kinases - antagonists & inhibitors; TOR Serine-Threonine Kinases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.672170

Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states.