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Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell
Natalie M Walker1, Elizabeth A Belloli1, Linda Stuckey2
1From the Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine.
Abstract:
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.
Insights
Fibrotic diseases involve mesenchymal cell activation and collagen deposition. Targeting mTORC1 and mTORC2 signaling pathways, particularly the mTORC1-4E-BP1 axis, may offer new therapeutic strategies for fibrotic conditions.
Area of Science:
- Cell Biology
- Molecular Biology
- Pathology
Background:
- Fibrotic diseases are characterized by mesenchymal cell (MC) activation and excessive matrix protein deposition, notably collagen.
- Understanding the molecular mechanisms driving MC activation and collagen production is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the roles of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) in regulating collagen expression in mesenchymal cells.
- To elucidate the specific signaling pathways involved in mTOR-mediated collagen production in fibrotic conditions.
Main Methods:
- Comparison of signaling pathways in mesenchymal cells from fibrotic lung allografts (Fib-MCs) versus normal mesenchymal cells (non-Fib MCs).
- Assessment of phosphoinositide-3kinase (PI3K) dependent activation of mTORC1 and mTORC2 via substrate phosphorylation (S6K1, 4E-BP1, AKT).
- Pharmacological inhibition using dual TORC1/2 inhibitor (AZD8055) and mTORC1-specific inhibitor (rapamycin).
- Genetic manipulation including raptor gene silencing, dominant-inhibitory 4E-BP1 overexpression, and rictor knockdown via siRNA.
Main Results:
- Fib-MCs exhibited increased PI3K-dependent activation of both mTORC1 and mTORC2 compared to non-Fib MCs.
- The dual inhibitor AZD8055, unlike rapamycin, significantly reduced 4E-BP1 phosphorylation and collagen I expression in Fib-MCs.
- mTORC1 signaling positively regulated collagen I expression in non-Fib MCs.
- The mTORC1/4E-BP1 pathway was identified as a key driver of collagen I expression in Fib-MCs.
- mTORC2 signaling, through rictor, was necessary for full mTORC1 activation and collagen expression in Fib-MCs.
Conclusions:
- Fibrotic activation of MCs enhances PI3K-dependent mTORC1 and mTORC2 signaling.
- Collagen I expression in fibrotic MCs is driven by the mTORC1-dependent 4-E-binding protein 1/eukaryotic initiation factor 4E pathway.
- These findings suggest targeting specific mTOR pathway components as a therapeutic strategy for fibrotic diseases.
- Further research is needed to fully understand mTORC2's role in activated cell states during fibrosis.
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