Objective

The forkhead box O1 (FOXO1) transcription factor is a key regulator of autophagy. In chondrocytes, reduced FOXO1 expression with aging causes osteoarthritis due to dysfunction of autophagy, but the mechanisms underlying regulation of FOXO1 expression and the reduction in expression with aging remain unclear. We investigated the mechanism by which transforming growth factor β1 (TGFβ1) signaling regulates the FOXO1–autophagy axis.

Methods

Expression of FOXO1 was measured in chondrocytes after TGFβ1 treatment. Immunohistochemistry was performed to estimate the levels of activin receptor-like kinase 5 (ALK5) and FOXO1 in the knee joints of young, middle-aged and old mice. The effects of the ALK5 inhibitor and SMAD3 or SMAD2 knockdown on FOXO1 expression were evaluated. The role of TGFβ1 in autophagy after hydrogen peroxide (H₂O₂) treatment was analyzed. The protective effect of TGFβ1 against H₂O₂ treatment was assessed by cell viability assay and TUNEL assay.

Results

TGFβ1 promoted the expression of FOXO1 mRNA and protein. Both ALK5 and FOXO1 expression decreased with aging. ALK5 inhibition and SMAD3 knockdown suppressed induction of FOXO1 expression by TGFβ1, whereas SMAD2 knockdown increased it. TGFβ1 promoted the expression of microtubule-associated proteins 1A/1B light chain 3B (LC3)-I protein via the SMAD3–FOXO1 pathway. Furthermore, under H₂O₂ treatment, TGFβ1 promoted expression of LC3-II. TGFβ1 pretreatment suppressed cell death of chondrocytes following H₂O₂ treatment, but this protective effect was abolished by FOXO1 knockdown.

Conclusions

TGFβ1 protects chondrocytes against oxidative stress via the FOXO1–autophagy axis, and a reduction in ALK5 expression might cause reduced FOXO1 expression with aging.

中文翻译：

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TGFβ1信号通过FOXO1-自噬轴保护软骨细胞免受氧化应激

客观的

叉头盒 O1 (FOXO1) 转录因子是自噬的关键调节因子。在软骨细胞中，随着年龄增长，FOXO1 表达降低会导致自噬功能障碍导致骨关节炎，但 FOXO1 表达调控和衰老表达降低的潜在机制仍不清楚。我们研究了转化生长因子β1（TGFβ1）信号调节FOXO1-自噬轴的机制。

方法

在TGFβ1处理后测量软骨细胞中FOXO1的表达。进行免疫组织化学以估计年轻、中年和老年小鼠膝关节中激活素受体样激酶 5 (ALK5) 和 FOXO1 的水平。评估了 ALK5 抑制剂和 SMAD3 或 SMAD2 敲低对 FOXO1 表达的影响。分析了过氧化氢（H ₂ O ₂）处理后TGFβ1在自噬中的作用。通过细胞活力测定和TUNEL测定评估TGFβ1对H ₂ O _{2处理的保护作用。}

结果

TGFβ1促进FOXO1 mRNA和蛋白的表达。ALK5 和 FOXO1 的表达都随着年龄的增长而下降。ALK5 抑制和 SMAD3 敲低抑制了 TGFβ1 对 FOXO1 表达的诱导，而 SMAD2 敲低增加了它。TGFβ1 通过 SMAD3-FOXO1 通路促进微管相关蛋白 1A/1B 轻链 3B (LC3)-I 蛋白的表达。此外，在H ₂ O ₂处理下，TGFβ1促进了LC3-II的表达。_{TGFβ1预处理抑制了H 2} O ₂处理后软骨细胞的细胞死亡，但这种保护作用被FOXO1敲低消除了。

结论

TGFβ1 通过 FOXO1-自噬轴保护软骨细胞免受氧化应激，ALK5 表达的降低可能导致 FOXO1 的表达随着衰老而降低。