C-Jun N-terminal kinase (JNK)–mediated induction of mSin1 expression and mTORC2 activation in mesenchymal cells during fibrosis

Natalie M Walker,Serina M Mazzoni,Ragini Vittal,Diane C Fingar,Vibha N Lama

doi:10.1074/jbc.ra118.003926

Natalie M Walker, Serina M Mazzoni + Show 3 more

Open Access

https://doi.org/10.1074/jbc.ra118.003926

Copy DOI

Abstract

Mammalian target of rapamycin complex 2 (mTORC2) has been shown to regulate mTORC1/4E-BP1/eIF4E signaling and collagen I expression in mesenchymal cells (MCs) during fibrotic activation. Here we investigated the regulation of the mTORC2 binding partner mammalian stress-activated protein kinase-interacting protein 1 (mSin1) in MCs derived from human lung allografts and identified a novel role for mSin1 during fibrosis. mSin1 was identified as a common downstream target of key fibrotic pathways, and its expression was increased in MCs in response to pro-fibrotic mediators: lysophosphatidic acid (LPA), transforming growth factor β, and interleukin 13. Fibrotic MCs had higher mSin1 protein levels than nonfibrotic MCs, and siRNA-mediated silencing of mSIN1 inhibited collagen I expression and mTORC1/2 activity in these cells. Autocrine LPA signaling contributed to constitutive up-regulation of mSin1 in fibrotic MCs, and mSin1 was decreased because of LPA receptor 1 siRNA treatment. We identified c-Jun N-terminal kinase (JNK) as a key intermediary in mSin1 up-regulation by the pro-fibrotic mediators, as pharmacological and siRNA-mediated inhibition of JNK prevented the LPA-induced mSin1 increase. Proteasomal inhibition rescued mSin1 levels after JNK inhibition in LPA-treated MCs, and the decrease in mSin1 ubiquitination in response to LPA was counteracted by JNK inhibitors. Constitutive JNK1 overexpression induced mSin1 expression and could drive mTORC2 and mTORC1 activation and collagen I expression in nonfibrotic MCs, effects that were reversed by siRNA-mediated mSIN1 silencing. These results indicate that LPA stabilizes mSin1 protein expression via JNK signaling by blocking its proteasomal degradation and identify the LPA/JNK/mSin1/mTORC/collagen I pathway as critical for fibrotic activation of MCs.

Highlights

Mammalian target of rapamycin complex 2 has been shown to regulate mTORC1/4E-BP1/eIF4E signaling and collagen I expression in mesenchymal cells (MCs) during fibrotic activation
We investigated the regulation of the Mammalian target of rapamycin complex 2 (mTORC2) binding partner mammalian stress-activated protein kinase–interacting protein 1 in MCs derived from human lung allografts and identified a novel role for mSin1 during fibrosis. mSin1 was identified as a common downstream target of key fibrotic pathways, and its expression was increased in MCs in response to pro-fibrotic mediators: lysophosphatidic acid (LPA), transforming growth factor ␤, and interleukin 13
To determine whether mSin1 regulation in lung MCs is exclusive to LPA signaling or a common phenomenon in response to pro-fibrotic mediators, nonfib MCs were treated for 16 h with TGF-␤ or interleukin 13 (IL-13), and the level of mSin1 protein expression was assessed by Western blotting (Fig. 1D). mSin1 levels were significantly increased in response to both TGF-␤ and IL-13, demonstrating a common signaling mechanism by which levels of the mTORC2 component-mSin1 are increased, potentially priming the cell to ramp up mTORC2 assembly and, total cellular translation

Summary

Results

Pro-fibrotic stimulation up-regulates the mTORC2 binding partner mSin in lung mesenchymal cells. To determine whether mSin regulation in lung MCs is exclusive to LPA signaling or a common phenomenon in response to pro-fibrotic mediators, nonfib MCs were treated for 16 h with TGF-␤ or IL-13, and the level of mSin protein expression was assessed by Western blotting (Fig. 1D). SiRNA-mediated silencing of mSIN1 in fibrotic MCs significantly blunted collagen I expression and, as expected, phosphorylation of AKT (Ser-473) (Fig. 2F). MSIN1-silenced fib MCs demonstrated decreased phosphorylation of the direct AKT substrates TSC2 (Thr-1462) and FOXO3a (Ser-253) compared with the scrambled siRNA control (Fig. 2F). Inhibition of mTORC1 and 2 activities and reduced mSin and collagen I protein expression were noted in fibrotic MCs transfected with LPAR1 siRNA (Fig. 2G), further reiterating a potential role of LPA in regulating mSin expression

Collagen I

Discussion

Total mTOR rictor

Isolation of mesenchymal cells from human lung allografts

Statistical analysis