Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czechia.
Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czechia.
Ann Anat. 2022 Jan;239:151781. doi: 10.1016/j.aanat.2021.151781. Epub 2021 Jun 16.
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.
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.
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.
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.
自噬被归类为一种程序性细胞死亡形式。然而，除了诱导死亡的功能外，自噬还能清除受损的细胞器，节省能量，从而促进细胞存活。这尤其适用于更新能力差的细胞，如软骨细胞。自噬受复杂的分子网络调控，包括蛋白酶及其底物。在前足中，已经研究了与自噬相关的蛋白酶在消除指间组织过程中的作用。然而，其表达/激活的诱导死亡效应尚未明确。本研究聚焦于自噬相关蛋白酶（组织蛋白酶、基质金属蛋白酶和胱天蛋白酶）在小鼠前足指骨软骨发育中的作用。
采用 PCR 阵列、实时 PCR 和免疫组织化学技术，在两个发育阶段（胚胎期 E12 与 E14），体内检测与自噬相关的基因表达。然后，采用实时 PCR 研究了雷帕霉素（自噬诱导剂）对体外软骨细胞中与自噬相关的蛋白酶表达的影响，使用微团培养。
体内前软骨细胞向软骨细胞分化过程中，几种蛋白酶的表达水平升高。其中，Ctsb（倍数调控 2.22）、Ctsd（倍数调控 2.37）、Ctss（倍数调控 2.92）、Mmp9（高达 445%）和 Casp8（高达 250%）的表达增加最为显著。这种转变还伴随着关键自噬诱导因子的高表达，如 Atgs。雷帕霉素体外处理软骨细胞，结果显示组织蛋白酶表达显著下调，金属蛋白酶表达轻度上调，胱天蛋白酶表达无变化。
本研究提供了体内软骨形成过程中与自噬相关的蛋白酶的筛选，并明确了其在雷帕霉素体外处理时的表达情况。值得注意的是，所选蛋白酶与骨关节炎有关，因此其调控可能用于临床相关研究。
