Mammalian Target of Rapamycin Positively Regulates Collagen Type I Production via a Phosphatidylinositol 3-Kinase-independent Pathway

Abstract

The mammalian target of rapamycin (mTOR) is a multifunctional protein involved in the regulation of cell growth, proliferation, and differentiation. The goal of this study was to determine the role of mTOR in type I collagen regulation. The pharmacological inhibitor of phosphatidylinositol (PI) 3-kinase, LY294002, significantly inhibited collagen type I protein and mRNA levels. The effects of LY294002 were more pronounced on the collagen ␣1(I) chain, which was inhibited at the transcriptional and mRNA stability levels versus collagen ␣2(I) chain, which was inhibited through a decrease in mRNA stability. In contrast, addition of the PI 3-kinase inhibitor, wortmannin, did not alter type I collagen steady-state mRNA levels. This observation and further experiments using an inactive LY294002 analogue suggested that collagen mRNA levels are inhibited independent of PI 3-kinase. Additional experiments have established that mTOR positively regulates collagen type I synthesis in human fibroblasts. These conclusions are based on results demonstrating that inhibition of mTOR activity using a specific inhibitor, rapamycin, reduced collagen mRNA levels. Furthermore, decreasing mTOR expression by about 50% by using small interfering RNA resulted in a significant decrease of collagen mRNA (75% COL1A1 decrease and 28% COL1A2 decrease) and protein levels. Thus, mTOR plays an essential role in regulating basal expression of collagen type I gene in dermal fibroblasts. Together, our data suggest that the classical PI 3-kinase pathway, which places mTOR downstream of PI 3-kinase, is not involved in mTOR-dependent regulation of type I collagen synthesis in dermal fibroblasts. Because collagen overproduction is a main feature of fibrosis, identification of mTOR as a critical mediator of its regulation may provide a suitable target for drug or gene therapy.A common characteristic of all fibrotic diseases, including scleroderma, is an abnormal accumulation of extracellular matrix proteins. Fibrotic lesions disrupt normal tissue architecture and contribute to organ failure. Type I collagen, the primary component of fibrotic lesions, is a triple helix composed of two ␣1 chains and one ␣2 chain. These chains, although coordinately expressed, are not regulated via the same mechanisms. It is well established that collagen protein degradation, mRNA stability, and transcription are tightly regulated during collagen biosynthesis. Signals from external stimuli such as cytokines (1-3), nutrients (4), and cell interactions (5, 6) modulate these processes via several pathways, including TGF-␤ 1 / p38 (7, 8), PKC (9), and stress-activated protein kinase/c-Jun N-terminal kinase (10, 11). Despite these advances, the pathways controlling collagen biosynthesis are not fully characterized, prompting us to further examine potential signaling molecules involved in type I collagen regulation.Prior studies suggested the involvement of phosphatidylinositol 3-kinase (PI 3-kinase), a ubiquitous lipid kinase, in collagen regulation. For example, Ivarsson et al....

Rapamycin increases CCN2 expression of lung fibroblasts via phosphoinositide 3-kinase

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Differential modulation of the cytokine-induced MMP-9/TIMP-1 protease–antiprotease system by the mTOR inhibitor rapamycin

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