Induction of cellular autophagy impairs TGF-β1-mediated extracellular matrix deposition in primary human knee fibroblasts

Abstract

Aims: To evaluate the role of autophagy in primary knee fibroblasts undergoing myofibroblast differentiation as an in vitro model of arthrofibrosis, a complication after total knee arthroplasty characterized by aberrant intra-articular scar tissue formation and limited range of motion.

Methods: We conducted a therapeutic screen of autophagic-modulating therapies in primary human knee fibroblasts undergoing transforming growth factor-beta 1 (TGF-β1)-mediated myofibroblast differentiation. Autophagy was induced pharmacologically with rapamycin or by amino acid deprivation. Picrosirius red staining was performed to quantify collagen deposition. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were conducted to evaluate fibrotic gene expression levels.

Results: Rapamycin, an mTOR complex 1 (mTORC1) inhibitor and autophagy inducer, reduced TGF-β1-mediated collagen deposition. Interestingly, we simultaneously report that myofibrogenic genes, including ACTA2, were highly upregulated following rapamycin-TGF-β1 treatment. When autophagy was induced through amino acid deprivation, we demonstrated suppressed extracellular matrix levels, fibrotic gene expression (e.g. ACTA2), and SMAD2 phosphorylation levels in TGF-β1-stimulated fibroblasts.

Conclusion: Our findings demonstrate that the induction of cellular autophagy suppresses TGF-β1-induced collagen deposition in primary human knee fibroblasts. Taken together, these data suggest that cellular autophagy may be prophylactic against the pathogenesis of arthrofibrosis.

Fig. 1

Impact of autophagy modulators on transforming growth factor-beta 1 (TGF-β1)-mediated extracellular matrix (ECM) deposition. Primary knee fibroblasts derived from patients undergoing primary total knee arthroplasty (TKA) (P1-P2) and revision TKA for arthrofibrosis (A1-A2) were used. a) Schematic illustrating the targets of each autophagy inducer or inhibitor within the autophagy pathway (created using BioRender). On Day 0, fibroblasts were treated with rapamycin (R_L = 10 nM, R_H = 50 nM), bafilomycin A1 (B = 5 nM), or pepstatin A (5 µg/mL) and E64d (5 µg/mL) combination (P/E). On Day 1, fibroblasts were stimulated with TGF-β1 (T) or its vehicle (V) (no media change). b) Picrosirius red (PSR) staining was performed on Day 3 to visualize/quantify collagen deposition (n = 3, mean and SD). Whole-well images (top) and laser scanning microscopy (LSM) scans (bottom). Similarly, cells were treated with rapamycin (Rap., 1 nM) on Day 0 and spiked with TGF-β1 or its vehicle on Day 1. c) PSR staining was performed on Day 3 (n = 4, mean and SD). For significance: a, b, c, d are relative to TGF-β1. a = p ≤ 0.05, b = p ≤ 0.01, c = p ≤ 0.001, d = p ≤ 0.0001. Norm., normalized.

Fig. 2

Gene expression changes induced by rapamycin and transforming growth factor-beta 1 (TGF-β1). Primary knee fibroblasts derived from patients undergoing primary total knee arthroplasty (TKA) (P1-P2) and revision TKA for arthrofibrosis (A1-A2) were used. On Day 0, fibroblasts were treated with rapamycin (Rap.) and spiked with TGF-β1 or its vehicle on Day 1 (no media change). a) On Day 3, messenger RNA (mRNA) transcript levels of various fibrotic genes were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (n = 3 to 4, heatmap displays the mean of log2-transformed values). b) Day 3 ACTA2 protein levels were determined by western blotting (n = 4, mean and SD). For significance: a, b, c, d are relative to TGF-β1. a = p ≤ 0.05, b = p ≤ 0.01, c = p ≤ 0.001, d = p ≤ 0.0001. CTGF, connective tissue growth factor; Exp., expression; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; MMP, matrix metalloproteinase; MTOR, mechanistic target of rapamycin; Norm., normalized; PAI, plasminogen activator inhibitor; TIMP, tissue inhibitor of metalloproteinases.

Fig. 3

Mechanistic analysis of fibrotic gene hyperactivation by rapamycin and transforming growth factor-beta 1 (TGF-β1). Primary knee fibroblasts derived from patients undergoing primary total knee arthroplasty (TKA) (P1) and revision TKA for arthrofibrosis (A2) were used. On Day 0, fibroblasts were treated with rapamycin (Rap.) and spiked with TGF-β1 or its vehicle on Day 1 (no media change). a) On Day 3, messenger RNA (mRNA) levels of the indicated genes were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (n = 3, heatmap displays the mean of log2-transformed values), and b) Day 3 protein levels of ACTA2, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined by western blotting (n = 3, mean and SD). Cells were pretreated with rapamycin (Rap.) for three hours and then stimulated with TGF-β1 (no media change). c) After ten minutes of TGF-β1 exposure, pSMAD2/SMAD2 levels (top bands) were assessed by western blotting (n = 4, mean and SD). For significance: a, b, c, d are relative to TGF-β1. a = p ≤ 0.05, b = p ≤ 0.01, c = p ≤ 0.001, d = p ≤ 0.0001. CTGF, connective tissue growth factor; Exp., expression; MMP, matrix metalloproteinase; MTOR, mechanistic target of rapamycin; Norm., normalized; PAI, plasminogen activator inhibitor; TIMP, tissue inhibitor of metalloproteinases.

Fig. 4

Influence of starvation-induced autophagy on fibrotic processes. Primary knee fibroblasts derived from patients undergoing primary total knee arthroplasty (TKA) (P1) and revision TKA for arthrofibrosis (A2) were used. On Day 0, fibroblasts underwent Earle’s balanced salt solution (EBSS)-induced amino acid deprivation for three hours in the presence/absence of bafilomycin A1 (Baf.). a) At t = 3 hrs, LC3-I/LC3-II protein levels were determined by western blotting. Cells were cultured in EBSS for three hours before receiving transforming growth factor-beta 1 (TGF-β1) or its vehicle (fresh media). On Day 3, b) collagen deposition was assessed by picrosirius red (PSR) staining (n = 3, mean and SD), c) messenger RNA (mRNA) levels by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (n = 3, heatmap displays the mean of log2-transformed values), and d) ACTA2 protein levels by western blotting (n = 3, mean and SD). e) After ten minutes of TGF-β1 exposure, pSMAD2/SMAD2 protein levels (top bands) of previously EBSS-starved cells were determined by western blotting (n = 3, mean and SD). For significance: a, b, c, d are relative to TGF-β1. a = p ≤ 0.05, b = p ≤ 0.01, c = p ≤ 0.001, d = p ≤ 0.0001. CTGF, connective tissue growth factor; Exp., expression; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LC3, light chain 3; MMP, matrix metalloproteinase; MTOR, mechanistic target of rapamycin; Norm., normalized; PAI, plasminogen activator inhibitor; TIMP, tissue inhibitor of metalloproteinases.