Idiopathic pulmonary fibrosis is a fatal interstitial lung disease characterized by the TGF-β (transforming growth factor-β)–dependent differentiation of lung fibroblasts into myofibroblasts, which leads to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by myofibroblasts requires de novo synthesis of glycine, the most abundant amino acid found in collagen protein. TGF-β upregulates the expression of the enzymes of the de novo serine–glycine synthesis pathway in lung fibroblasts; however, the transcriptional and signaling regulators of this pathway remain incompletely understood. Here, we demonstrate that TGF-β promotes accumulation of ATF4 (activating transcription factor 4), which is required for increased expression of the serine–glycine synthesis pathway enzymes in response to TGF-β. We found that induction of the integrated stress response (ISR) contributes to TGF-β–induced ATF4 activity; however, the primary driver of ATF4 downstream of TGF-β is activation of mTORC1 (mTOR Complex 1). TGF-β activates the PI3K-Akt-mTOR pathway, and inhibition of PI3K prevents activation of downstream signaling and induction of ATF4. Using a panel of mTOR inhibitors, we found that ATF4 activation is dependent on mTORC1, independent of mTORC2. Rapamycin, which incompletely and allosterically inhibits mTORC1, had no effect on TGF-β–mediated induction of ATF4; however, Rapalink-1, which specifically targets the kinase domain of mTORC1, completely inhibited ATF4 induction and metabolic reprogramming downstream of TGF-β. Our results provide insight into the mechanisms of metabolic reprogramming in myofibroblasts and clarify contradictory published findings on the role of mTOR inhibition in myofibroblast differentiation.

特发性肺纤维化是一种致命的间质性肺病，其特征是 TGF-β（转化生长因子-β）依赖性肺成纤维细胞分化为肌成纤维细胞，导致胶原蛋白过度沉积和进行性瘢痕形成。我们之前已经表明，肌成纤维细胞合成胶原蛋白需要从头合成甘氨酸，这是在胶原蛋白中发现的最丰富的氨基酸。TGF-β 上调de novo酶的表达肺成纤维细胞中的丝氨酸-甘氨酸合成途径；然而，该途径的转录和信号调节剂仍未完全了解。在这里，我们证明 TGF-β 促进 ATF4（激活转录因子 4）的积累，这是响应 TGF-β 增加丝氨酸 - 甘氨酸合成途径酶表达所必需的。我们发现综合应激反应 (ISR) 的诱导有助于 TGF-β 诱导的 ATF4 活性；然而，TGF-β 下游 ATF4 的主要驱动因素是 mTORC1（mTOR Complex 1）的激活。TGF-β 激活 PI3K-Akt-mTOR 通路，抑制 PI3K 可阻止下游信号的激活和 ATF4 的诱导。使用一组 mTOR 抑制剂，我们发现 ATF4 激活依赖于 mTORC1，独立于 mTORC2。雷帕霉素，不完全和变构地抑制 mTORC1，对 TGF-β 介导的 ATF4 诱导没有影响；然而，专门针对 mTORC1 激酶域的 Rapalink-1 完全抑制了 TGF-β 下游的 ATF4 诱导和代谢重编程。我们的研究结果提供了对肌成纤维细胞代谢重编程机制的深入了解，并澄清了关于 mTOR 抑制在肌成纤维细胞分化中的作用的已发表的相互矛盾的发现。