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. 2023 Oct;41(10):2163-2174.
doi: 10.1002/jor.25594. Epub 2023 May 13.

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

Rahul G Alenchery  1   2 , Raquel E Ajalik  1   2 , Kyle Jerreld  1   3 , Firaol Midekksa  1   2 , Sylvia Zhong  1   2 , Bashar Alkatib  1 , Hani A Awad  1   2   3
Affiliations

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

Rahul G Alenchery et al. J Orthop Res. 2023 Oct.

Abstract

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 actin 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.

Keywords: PAI-1; TGF-β1; fibrosis; flexor tendon; mTOR.

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Figures

Figure 1.
Figure 1.. Loss of PAI-1 does not alter morphology and proliferation of murine digital flexor tenocytes.
A) Bright field micrographs of primary tenocyte outgrowth from tendon explants and subsequent passaging reveals no qualitative differences in cell morphology. B) Cell proliferation curves for the WT (black) and PAI-1KO (red) tenocytes plotted over 10 days under 1% FBS low serum conditions (Mean±SD for triplicates per genotype). C) ELISA quantification of PAI-1 protein levels in primary tenocytes without or with TGF-β1 (Mean±SD for quadruplicates per genotype).
Figure 2:
Figure 2:. Loss of PAI-1 blunts TGF-β1 activation of α-SMA+ myofibroblasts in murine digital flexor tenocytes.
(A) Representative micrographs of α-SMA-immunostained (WT and PAI-1KO) primary flexor tendon cells treated with TGF-β1 (10 ng/ml) for 24 hours. (B) Quantified α-SMA mean fluorescence normalized to cell count (arbitrary unit). Data represent means and error bars represent standard deviations, n=5–6 replicates. (C) Quantitative real-time RT-PCR analysis of Acta2 gene expression normalized to ActB gen. Data represent means and error bars represent standard deviations, n=6 replicates. Asterisks indicate significant differences inferred from Bonferroni-corrected multiple comparisons following a two-way ANOVA (*=p
Figure 3:
Figure 3:. Loss of PAI-1 modulates ECM synthesis and remodeling and cell cycle genes in murine digital flexor tenocytes.
Quantitative real-time RT-PCR analysis of relative expression of: (A) ECM synthesis genes (Col1a1, Col3a1, and Serpinh1). (B) ECM remodeling genes (Mmp2, Mmp3, and Mmp9). (C) cell cycle genes (Tp53, dkn2a, and Rb). Gene expression was normalized to normalized to β actin gene (Actb) and arbitrarily normalized to untreated WT cells. Data represent means and error bars represent standard deviations, n=3–5 pooled replicates. Asterisks indicate significant differences inferred from Bonferroni-corrected multiple comparisons following a two-way ANOVA (*=p
Figure 4:
Figure 4:. Loss of PAI-1 rescues PTEN activation but does not affect mTORC1 signaling in murine digital flexor tenocytes treated with TGF-β1.
A) schematic representation of the TGF-β1/PAI-1/mTOR signaling axis. B) Representative Western blots of primary mouse flexor tendons (WT & PAI-1KO) stimulated without or with TGF-β1 (10 ng/ml) for 24 hours showing relative abundance and phosphorylation for various nodes in the TGF-β1/PAI-1/mTOR signaling axis. C) Densitometry quantification of phosphorylated proteins normalized to β Actin. Data represent means and error bars represent standard deviations, n=3–5 pooled replicates. Asterisks indicate significant differences inferred from Bonferroni-corrected multiple comparisons following a two-way ANOVA (*=p
Figure 5:
Figure 5:. TGF-β1 and mTOR additive effects on increased myofibroblast activation are dependent on PAI-1.
Flow cytometry histogram of A) myofibroblast differentiation (αSMA), B) DNA damage (γH2AX), and C) proliferation (Ki67) in WT and PAI-1KO tenocytes treated with TGF-β1 (10 ng/ml) for 24 hours. D) Gating strategy to define two mTOR activity populations based on the median fluorescence intensity (MFI) for pAKT and p4EBP1 or pS6. Forward and side scatter gating are used to identify the cells of interest with subsequent doublet exclusion. Tenocytes are then gated for either high and low abundance of pAKT/p4EBP1 or pAKT/pS6 cells. MFI values of E,H) αSMA, F,I) γH2AX, and G,J) Ki67 are graphically represented to illustrate the shifts in cellular protein abundance.
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