FGFR2 accommodates osteogenic cell fate determination in human mesenchymal stem cells

Stem cell-based therapies offer significant potential for treating disease or injury through the regeneration of damaged tissues. Among the various stem cells available, mesenchymal stem cells (MSCs) are garnering considerable interest because of their availability, ease of isolation, and multipotency. Importantly, under appropriate culture conditions, MSCs can be selectively induced to differentiate into various lineages, such as osteoblasts, adipocytes, chondrocytes, and myoblasts (Caplan, 1991, Prockop, 1997). This makes them popular for studies of factors that control cell fate. Understanding the mechanism governing commitment and progression through these lineages is crucial for the development of effective strategies that lead to tissue regeneration.

The commitment of MSCs to the osteoblast lineage is characterized by the initial expression of Runt-related transcription factor 2 (RUNX2), followed by type 1 collagen (COL1A, encoded by COL1A1 and COL1A2 genes) and alkaline phosphatase (ALP, encoded by the ALPL gene), and finally mineralization of the extracellular matrix (Lian et al., 2004). Osteogenic lineage commitment of MSCs can be induced by in vitro supplementation of growth media with dexamethasone, ascorbic acid and β-glycerol phosphate (Jaiswal et al., 1997). In addition, growth factors and other signaling proteins, such as bone morphogenetic proteins (BMPs) (Chen et al., 2012, Smith et al., 2018) and WNT proteins (Ling et al., 2009), regulate osteogenic lineage commitment and differentiation.

Fibroblast growth factors (FGFs) are essential for early osteogenesis and bone homeostasis, although FGF signaling itself does not directly induce osteoblast differentiation (Charoenlarp et al., 2017). Nonetheless, FGFs could modulate osteogenesis by regulating the expression of multiple genes involved in bone formation. In particular, FGF is important for the proliferation of osteoprogenitor cells and their subsequent maturation into bone-forming cells (Nakamura et al., 1998, Zellin and Linde, 2000, Lisignoli et al., 2001). Loss of FGF2 expression in mice caused a reduction in bone volume, mineral deposition, and bone formation efficiency (Montero et al., 2000). FGFs function through interaction with FGF receptors (FGFRs), a group of cell surface receptor tyrosine kinases. There are five FGFRs. In the early phase of bone formation, both FGFR1 and FGFR2 are expressed in developing bone tissue, and in the perichondrium and periosteum (Ornitz and Marie, 2002). In contrast, FGFR3 is mainly expressed by chondrocytes (Wang et al., 2001) and has been shown to inhibit bone formation by promoting chondrocyte proliferation (Deng et al., 1996).

Among all the FGFRs, FGFR2 has been shown to positively regulate osteogenesis (Ornitz and Marie, 2002, Wilkie, 2002, Wilkie et al., 2002, Jackson et al., 2006). Frequently, murine cell lines or in vivo models are used to evaluate the role of FGFR2 during osteogenesis. However, conflicting results have been observed. Notably, increased FGFR2 transcripts have been reported during osteogenesis in immortalized murine MSCs (Kahkonen et al., 2018), whereas the opposite result (decreased FGFR2) has been observed in hMSCs (Simann et al., 2017). Such conflicting observations warrant further investigation into the role of FGFR2 during osteogenesis, particularly in hMSCs.

Osteogenesis of MSCs is a complex process involving cross-talk between many epigenetic and transcriptional factors (Perez-Campo and Riancho, 2015). Histones in the promoter region of RUNX2 and PPARγ, which are involved in lineage commitment, undergo modification during MSC differentiation (Meyer et al., 2016). Additional epigenetic switches involving Enhancer of zeste 2 (EZH2), a component of the polycomb repressor complex 2 (PRC2) that enhances the methylation of H3K2, govern MSC differentiation (Hemming et al., 2014, Dudakovic et al., 2015, Samsonraj et al., 2018, Galvan et al., 2021).

In this context, EZH2 regulates the methylation status of H3K27 on the promoter of RUNX2, PPARγ and CEBPA. Notably, downregulation of EZH2 expression promotes osteogenesis (Hemming et al., 2014), while overexpression downregulates the expression of RUNX2 and OC (Osteocalcin) (Hemming et al., 2016).

In the current study, we employed siRNA to knockdown FGFRs in hMSCs to determine their functions in mediating osteogenesis. Combined with a focused PCR gene array study, we evaluated how FGFR2 could modulate the osteo-adipogenic lineage commitment of hMSCs. Moreover, we sought to establish a link between FGFR2 and EZH2 in regulating MSC osteogenesis by examining their expression under osteogenic induction. The data show that under osteogenic stimuli, EZH2 levels decrease while FGFR2 and ALP increase resulting in the deposition of a mineralized matrix. Under the same osteogenic conditions, FGFR2 knockdown resulted in a greater reduction of EZH2 levels and decreased ALP and matrix mineralization. These results highlight a possible FGFR2-EZH2 osteogenic axis and that epigenetic changes regulated by EZH2 may contribute to the reduced osteogenic differentiation observed upon FGFR2 knockdown in MSCs. Furthermore, the current study suggests FGFR2 is critical for the control of osteogenic fate , and thus constitutes a potential target for strategies that enhance bone regeneration.

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