Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

• Keywords
• ABHD2, Caspase 9, diaPASEF, migration, osteoblasts, proteomics,
• MeSH
• Cell Line MeSH
• Gene Knockout Techniques MeSH
• Caspase 9 * metabolism   genetics   MeSH
• Mice MeSH
• Osteoblasts * metabolism   cytology   MeSH
• Cell Movement * MeSH
• Proteomics * methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Casp9 protein, mouse MeSH Browser
• Caspase 9 * MeSH

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

• Keywords
• Animal model, Apoptotic, Caspases, Deficiency, Mouse, Non-apoptoic,
• MeSH
• Apoptosis MeSH
• Caspases * metabolism   genetics   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Mice, Knockout * MeSH
• Mice MeSH
• Inflammation enzymology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Caspases * MeSH

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.

• Keywords
• Caspase-9, Chondrogenic differentiation, Micromasses, Mmp-9, Non-apoptotic functions,
• MeSH
• Cell Differentiation MeSH
• Chondrocytes * MeSH
• Chondrogenesis * physiology   MeSH
• Caspase Inhibitors metabolism   pharmacology   MeSH
• Caspase 9 genetics   metabolism   MeSH
• Cells, Cultured MeSH
• Mice MeSH
• Osteogenesis MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Caspase Inhibitors MeSH
• Caspase 9 MeSH