Background

Autophagy is classified as a form of programmed cell death. Nevertheless, besides the death-inducing function, autophagy enables removal of damaged organelles, energy savings, and thus cell survival. This applies in particular to cells with poor renewal capabilities, such as chondroblasts. Autophagy is regulated by a complex molecular network, including proteases and their substrates. In autopodium, autophagy-related proteases have been examined particularly within the context of the elimination of the interdigital tissue. However, the death-inducing effects of their expression/activation have not been specified yet. This work focuses on autophagy-associated proteases (cathepsins, matrix metalloproteinases, and caspases) in development of phalangeal cartilage of the mouse autopodium.

Methods

PCR Array, Real-time PCR, and immunohistochemistry were used to follow the expression of autophagy-associated genes in vivo at two developmental stages prenatal/embryonic (E)12 vs. E14. Real-time PCR was then applied to investigate the influence of rapamycin (an inducer of autophagy) on the expression of autophagy-associated proteases in chondroblasts in vitro using micromass culture.

Results

Several proteases showed increased expression levels during the transition of pre-chondrogenic cells into chondroblasts in vivo. The most significant increases were observed for Ctsb (fold regulation 2.22), Ctsd (fold regulation 2.37), Ctss (fold regulation 2.92), Mmp9 (up to 445%), and Casp8 (up to 250%). The transition was associated also with the high expression of crucial autophagic inducers, such as Atgs. The in vitro treatment of chondroblasts by rapamycin showed significantly decreased expression of cathepsins, a mild increase in expression of metalloproteinases, and no effect in caspase expression.

Conclusions

The present data provide a screening of autophagy-associated proteases accompanying the formation of cartilage in vivo and specify their expression under rapamycin treatment in vitro. Notably, the selected proteases are assigned to osteoarthritis, therefore their regulation might be used in clinically oriented studies.

中文翻译：

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自噬相关蛋白酶伴随着前成软骨细胞向成软骨细胞的转变

背景

自噬被归类为程序性细胞死亡的一种形式。然而，除了诱导死亡的功能外，自噬还可以去除受损的细胞器，节省能量，从而使细胞存活。这尤其适用于更新能力较差的细胞，例如成软骨细胞。自噬受复杂的分子网络调控，包括蛋白酶及其底物。在 autopodium 中，自噬相关的蛋白酶已被检查，特别是在消除指间组织的背景下。然而，它们的表达/激活的死亡诱导作用尚未明确。这项工作的重点是自噬相关蛋白酶（组织蛋白酶、基质金属蛋白酶和半胱天冬酶）在小鼠自体趾骨软骨发育中的作用。

方法

PCR 阵列、实时 PCR 和免疫组织化学用于跟踪自噬相关基因在产前/胚胎 (E)12和12 两个发育阶段的体内表达。E14. 然后应用实时 PCR 来研究雷帕霉素（自噬诱导剂）对体外使用微团培养的成软骨细胞中自噬相关蛋白酶表达的影响。

结果

几种蛋白酶在体内前成软骨细胞转变为成软骨细胞的过程中表现出增加的表达水平。观察到Ctsb（调节倍数 2.22）、Ctsd（调节倍数 2.37）、Ctss（调节倍数 2.92）、Mmp9（高达 445%）和Casp8（高达 250%）的最显着增加。这种转变也与关键的自噬诱导剂（如 Atgs）的高表达有关。在体外治疗雷帕霉素显示出软骨细胞的显著降低组织蛋白酶的表达，在金属蛋白酶的表达的轻度增加，并且在半胱天冬表达没有影响。

结论

目前的数据提供了对伴随体内软骨形成的自噬相关蛋白酶的筛选，并在体外雷帕霉素治疗下指定了它们的表达。值得注意的是，选定的蛋白酶被指定为骨关节炎，因此它们的调节可能用于临床导向的研究。