PAI-1 mediates TGF-β1-induced myofibroblast activation in tenocytes via mTOR signaling.

Rahul G Alenchery, Raquel E Ajalik, Kyle Jerreld, Firaol Midekksa, Sylvia Zhong, Bashar Alkatib, Hani A Awad

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2023 Oct

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Transforming growth factor-beta (TGF-β1) induces plasminogen activator inhibitor 1 (PAI-1) to effect fibrotic pathologies in several organs including tendon. Recent data implicated PAI-1 with inhibition of phosphatase and tensin homolog (PTEN) suggesting that PAI-1-induced adhesions involves phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (mTOR) signaling. Ergo, we investigated effects of TGF-β1, PAI-1, and mTOR signaling crosstalk on myofibroblast activation, senescence, and proliferation in primary flexor tenocytes from wild-type (WT) and PAI-1 knockout (KO) mice. PAI-1 deletion blunted TGF-β1-induced myofibroblast activation in murine flexor tenocytes and increased the gene expression of Mmp-2 to confer protective effects against fibrosis. While TGF-β1 significantly reduced phosphorylation of PTEN in WT cells, PAI-1 deletion rescued the activation of PTEN. Despite that, there were no differences in TGF-β1-induced activation of mTOR signaling (AKT, 4EBP1, and P70S6K) in WT or KO tenocytes. Phenotypic changes in distinct populations of WT or KO tenocytes exhibiting high or low mTOR activity were then examined. TGF-β1 increased alpha-smooth muscle acti n abundance in WT cells exhibiting high mTOR activity, but this increase was blunted in KO cells exhibiting high 4EBP1 activity but not in cells exhibiting high S6 activity. DNA damage (γH2AX) was increased with TGF-β1 treatment in WT tenocytes but was blunted in KO cells exhibiting high mTOR activity. Increased mTOR activity enhanced proliferation (Ki67) in both WT and KO tenocytes. These findings point to a complex nexus of TGF-β1, PAI-1, and mTOR signaling in regulating proliferation, myofibroblast differentiation, and senescence in tenocytes, which could define therapeutic targets for chronic tendon adhesions and other fibrotic pathologies. © 2023 Orthopaedic Research Society.

Citation

Rahul G Alenchery, Raquel E Ajalik, Kyle Jerreld, Firaol Midekksa, Sylvia Zhong, Bashar Alkatib, Hani A Awad. PAI-1 mediates TGF-β1-induced myofibroblast activation in tenocytes via mTOR signaling. Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 2023 Oct;41(10):2163-2174