Loss of bone mass and aggravation of micro-fractures increase the bone fragility and susceptibility to fractures in patients of osteoporosis [39]. However, there is currently a lack of enough safe and effective treatment strategy for dealing with this gap [40, 41]. Many studies have confirmed that the pathogenesis of osteoporosis is highly correlated with the tendency of increased adipocytes and decreased osteoblasts [42–44],and our analysis found the number of the DEGs in the AD was more than that of OS corresponding to previous studies (Fig. 1–2). Therefore, clarifying the detailed mechanisms about the differentiation of MSCs into OS and AD would be contributed to develop a new strategy and treatment for osteoporosis. As the differentiation of MSCs into a specific lineage was determined at the early stage of differentiation [45, 46], we selected early OS and AD profiles to explore the early differentiation mechanisms that determined lineage fates of BMSCs.
After a series of integration analyses, we identified FoxO3, IL-6 and CAT as key genes for osteogenic differentiation, and these genes were all enriched in the FoxO signaling pathway (Fig. 4 and S1). FoxO, an intracellular signaling factor belongs to transcriptional regulators family of forhead box O, consisting of FoxO1, FoxO3, FoxO4 and FoxO6, plays a critical regulatory role in the multiple biological processes, including cell cycle, anti-apoptosis and anti-oxidation [47, 48]. Previous studies had discovered that FoxO3 could reduce ROS and promote OS differentiation of MSCs by activating autophagy [49–51],which demonstrated that the up-regulation of FoxO had an essential role on the activity of the OS in MSCs. Wen Sun had verified that Sirt1 overexpression promoted FoxO3a deacetylation and inhibited oxidative stress and resisted the apoptosis to increase the osteogenesis and partially restoring the defects of the skeletal system in osteoporosis [52]. IL-6, a pleiotropic cytokine with multiple physiological functions including immune regulation, hematopoiesis and tissue regeneration, plays a paramount role in the tissue regeneration engineer, especially in the bone metabolism [53, 54]. It could stimulate the hepatocytes to return to the cell cycle progression through triggering the initiative signal of liver regeneration [55]. Research showed that HIF1A-AS2 could erase the antagonistic effect of IL-6 exerted by miR-665, and then promote osteogenic differentiation of the BMSCs [56]. Zhongyu Xie also confirmed that IL-6 had an important role in the osteogenic differentiation of BMSCs [57]. As for CAT, Mao Li had illuminated that it could promote osteogenic differentiation through enhancing resistance to oxidative stress [58]. Next, we extracted the expression value of FOXO3, IL-6 and CAT (Fig. 8A), suggesting FOXO3 and CAT were positively correlated with osteogenic differentiation, while IL-6 was negative. Based on the previous studies, we surmised that FOXO3, IL-6 and CAT were key genes in the OS and FoxO was a core signaling pathway, especially in the early stage of OS.
In the AD, we identified hub-genes of VEGFA and FGF2 in the Rap1 signaling pathway was the essential mechanisms involved in the early stage of adipogenic differentiation (Fig. 5 and S2). Frank Yeung had indicated that Rap1, a mammalian telomere-binding protein, played a key role in the AD through its additional non-telomeric functions, which was known as co-factor of transcriptional cascade and regulator of NF-kB pathway[59]. VEGFA, a key angiogenic factor, was initially considered as an important molecular in the angiogenesis, and latterly was identified that it had multiple biological functions including bone cellular survival [60]. Wen Zhang pointed out that miR-128 promoted adipogenic differentiation of hMSCs by the suppression of the VEGF pathway [61]. As a heparin-binding growth factor stored in the extracellular matrix, FGF2 had been identified to be an important modulator in the early differentiation and development of cells, owning to its multiple biological functions including chemotaxis, angiogenesis and mitotic activity [62]. Kim, S had found that BMSCs with deficiency of FGF2 showed a strong capacity of adipogenic differentiation, which indicated that down-regulated FGF2 played an important role in the AD[63]. Thus, VEGFA and FGF2 were both negative factors during adipogenic differentiation, which was consistent with our analysis (Figure.8B). In the AD, FGF2 might increase the conduction of extracellular signals through molecular adhesion, and then regulate the Rap1 signaling pathway to promote the differentiation of BMSCs.
In the trigger phase of BMSCs differentiation, we speculated PI3K-Akt signaling pathway played a key role in triggering the differentiation of stem cells into various progenitor lines, at least in the OS and AD, based on the previous studies and our surveys (Fig. 1,2D-F and Figure.3A-C). As shown in our analysis, the PI3K-Akt signaling pathway enriched the most DEGs during the whole process of the early stage in OS and AD (Fig. 1D-F and 2D-F), which meant a paramount role in the differentiation of BMSCs. Activation of PI3K-Akt signaling pathway had been demonstrated not only to promote the OS and AD, but stimulate the chondrogenic differentiation of BMSCs, while it’s antagonist could lead to inhibition of BMSCs differentiation [64–66]. Through the PI3K-Akt signaling pathway,BMSCs can also differentiate into other progenitor cells, such as endothelial cells (ECs), Vascular smooth muscle cells [67, 68]. Researches had confirmed that stimulation of PI3K-Akt signaling pathway had the ability to activate its downstream target of rapamycin-p70S6 kinase, and promote BMSCs differentiate into coulvascular smooth muscle. Proangiogenic bioscaffold composited of porous β-CaSiO(3)/PDLGA advanced the ECs differentiation of BMSCs via activating PI3K-Akt signaling pathway, which in turn promoted phosphorylation of endothelial nitric oxide synthase (eNOS), production of nitric oxide (NO) and increased secretion of vascular endothelial growth factor (VEGF). Therefore, we surmised activation of PI3K-Akt signaling pathway in the trigger phase of BMSCs differentiation might subsequently activate the FoxO and Rap1 signaling pathways, respectively, thereby promoting OS and AD. And expression level of the key gene PIK3R1 from PI3K-Akt signaling pathway was up-regulated both in the OS and AD, which are in line with our speculation (Fig. 8A and B).
Based on the GO enrichment analysis of OS, protein binding, transcription factor binding, nucleus, membrane, cytosol and Cytoplasm were clustered in the FoxO3 and IL-6. These aggregations were similar in the adipogenesis (Fig. 6D-F and 7D-F). From these results, it seemed that osteogenic and adipogenic differentiations were consistent in biological processes, both through protein-protein binding and transmission of signaling molecules inside and outside the membrane.