As a continuous, rigorous, and dynamic process, bone remodeling plays a critical role in maintaining bone mass and the integrity of the skeletal structure (Mizoguchi and Ono, 2021, Shi et al., 2020). The osteogenic differentiation of mesenchymal stem cells (MSCs) is required for bone remodeling. The decline in MSCs may cause a decrease in bone volume and deterioration of bone microarchitecture, both of which are important for bone strength (Kiernan et al., 2016). Mouse embryonic fibroblasts (MEFs), which are multipotent progenitor cells, possess similar properties to MSCs and are widely used for stem cell biology research, such as osteogenesis (Pi et al., 2016, Tang et al., 2009). Various pathways are involved in the osteoinduction of MEFs, such as the bone morphogenetic protein (BMP) pathway. In addition, some pathways can synergistically promote osteogenic differentiation even if they cannot initiate this process alone, such as the Wnt/β-catenin, fibroblast growth factor, Notch, insulin-like growth factor, and retinoic acid pathways (Jiang et al., 2019, Kang et al., 2009, Mostafa et al., 2019). Even though osteogenic differentiation is a well-orchestrated and complex process, cellular osteogenic potential and quality can be evaluated by detecting transcriptional regulators of the osteogenic process. Essential transcription factors of early osteoblast differentiation include runt-related transcription factor 2 (Runx2), osterix, and DLX-5. Alkaline phosphatase (ALP) is an early marker of osteoblast differentiation; osteopontin (OPN) is expressed in the intermediate stage of osteogenesis; and osteocalcin (OCN) is expressed in the late stage, concomitantly with mineralization (Kang, Song, 2009).

BMPs are members of the transforming growth factor beta superfamily. More than 20 BMP members have been identified since its discovery, and BMP2 and BMP7 have been approved for clinical use in the treatment of bone diseases (Begam et al., 2017, Jiang, Xia, 2019). BMPs play multiple roles in skeletal development, such as homeostasis and regeneration, through Smad (canonical) or non-Smad (non-canonical) signaling pathways. The effect of BMPs on promoting cell differentiation in MSCs are regulated by many extracellular or intracellular proteins associated with BMP signaling (Gomez-Puerto et al., 2019, Lowery and Rosen, 2018, Wu et al., 2016). BMP9 is a potent osteoinduction factor. To induce the osteoblastic differentiation of MSCs, BMP9 binds to and activates its receptor kinase, which eventually phosphorylates Smad1/5/8 and promotes heterodimerization with Smad4. The complex then translocates to the nucleus to regulate the transcription of downstream genes (Li et al., 2020, Mostafa, Pakvasa, 2019). In addition to osteoinduction, BMP9 can also induce and maintain the development of cholinergic neurons, induce hepcidin in hepatocytes, and promote or inhibit tumorigenesis (Mostafa, Pakvasa, 2019).

The Wnt/β-catenin pathway has an important role in skeletal development and osteogenic differentiation. The activity of Wnt/β-catenin pathway can be enhanced by BMP9, and BMP9-induced mineralization and ectopic bone formation are inhibited by β-catenin knockdown (Tang, Song, 2009, Tu et al., 2015). Moreover, osteogenic differentiation generally occurs concurrently with vascularization. The expression of hypoxia inducible factor 1 alpha (HIF-1α) can be upregulated by BMP9, and the osteogenic potential of BMP9 can be enhanced by HIF-1α in MSCs (Hu et al., 2013, Majmundar et al., 2010, Wan et al., 2010). However, the detailed mechanism by which BMP9 regulates Wnt/β-catenin and/or HIF-1α remain unclear.

The development, differentiation, and self-renewal of MSCs are closely related to extracellular matrix (ECM) remodeling (Arai et al., 2021). Collagenase-3 (MMP13), a member of the ECM-degrading endopeptidase family, has a binding site characterized by a zinc-binding motif and plays an important role in ECM remodeling (Leeman et al., 2002). MMP13 is capable of degrading collagen such as collagen type II, which is found primarily in bones and joints, and activating the MMP cascade. MMP13-deficient mice exhibit abnormal cartilage growth plates. In addition, mutation of MMP13 may result in human epiphyseal dysplasia, and dysregulated MMP13 expression is associated with osteoarthritis (Lausch et al., 2009, Mazur et al., 2019, S et al., 2018). Therefore, MMP13 may be involved in regulating skeletal system development. However, it is unclear whether MMP13 has effects on the osteogenic differentiation induced by BMP9 in MEFs.

In this study, we determined whether MMP13 is associated with the osteoinduction potential of BMP9, and investigated the underlying mechanism.