Laboratory of Odontogenesis and Osteogenesis, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Brno, Czech Republic.
Department of Physiology, Faculty of Veterinary Medicine, Veterinary University, Brno, Czech Republic.
Histochem Cell Biol. 2022 Apr;157(4):403-413. doi: 10.1007/s00418-021-02067-9. Epub 2022 Jan 9.
Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation.
除了细胞死亡，半胱天冬酶-9 还参与非凋亡事件，包括细胞分化。为了评估其对软骨/成骨因子表达的可能影响，在体外测试了一种半胱天冬酶-9 抑制剂。为此，使用了最常见的体外软骨形成模型——鼠前肢衍生的微团培养物。基于聚合酶链反应（PCR）阵列和实时 PCR 进行了以下分析。结果表明，一些与软骨发生相关的基因表达发生改变，其中一些可能影响软骨分化（Bmp4、Bmp7、Sp7、Gli1）、矿物质沉积（Alp、Itgam）或与软骨内骨化相关的细胞外基质重塑（Col1a2、Mmp9）。在表达改变的基因簇中，Mmp9 的表达下降最为显著，超过 50 倍。此外，我们还确定了半胱天冬酶-9 下调对其他 Mmp 基因表达的可能影响。观察到 Mmp14 的表达略有增加，但其他研究的 Mmp 基因（Mmp1、Mmp3、Mmp8、Mmp10、Mmp12、Mmp13）的表达没有变化。有趣的是，在微团中抑制 Mmp9 导致与半胱天冬酶-9 相关的一些软骨形成标志物表达降低。这些样本也显示半胱天冬酶-9 本身的表达降低，表明这两种酶存在双向调节。这些结果表明半胱天冬酶-9 抑制剂对半胱天冬酶-9 表达的特异性影响。在体内发育过程中，静止、增殖和预肥大软骨细胞中这两种酶的定位重叠，支持它们的多种功能，无论是凋亡还是非凋亡。值得注意的是，在 Pik3cg 中鉴定出了一种可能的 Mmp9 调节候选物，其表达模式与这两种酶重叠。
