Osteoporosis is a disease that causes progressive bone loss, increasing the susceptibility of affected patients to bone fractures. The ability of hMSCs to differentiate into osteoblasts, osteocytes, and adipocytes ultimately controls to development of bone and fat tissues [16, 17]. Impairment of hMSC osteogenesis can impair bone formation, and such impairment is a common hallmark of osteoporosis [6, 18]. It is therefore essential that the molecular mechanisms regulating hMSC hallmark be better understood in order to guide the treatment of osteoporosis and bone fractures.
Herein, we found that IGFBP5 expression in hMSCs increased over time during osteoblastic differentiation, with maximum expression levels being reached on day 7 of this process (Fig. 1A). This showed that IGFBP5 may be a key regulator of the early phases of the osteogenic differentiation process in these cells. To test this possibility, we generated hMSCs in which IGFBP5 was stably overexpressed or knocked down using lentiviral constructs (Fig. 1B-D).
We found that cells overexpressing IGFBP5 exhibited more robust ALP staining and activity, whereas the opposite was true in cells in which this gene was knocked down (Fig. 2A,C; Fig. 2B,D). In line with these findings, osteogenic marker gene expression was markedly increased during hMSC osteogenesis in cells overexpressing IGFBP5 (Fig. 2E,G), while the expression of these marker genes was suppressed following IGFBP5 knockdown (Fig. 2F,H). These data suggest that IGFBP5 functions as a positive regulator of early hMSC osteoblastogenesis.
Of the six known IGFBP family members, IGFBP5 is the most broadly bioactive and is expressed in many different cells and tissues [13, 19, 20]. The relationship between IGFBP5 and osteogenic differentiation, however, remains to be fully clarified. There is some evidence that IGFBP5 can enhance the osteogenic differentiation of umbilical cord stem cells and periodontal ligament stem cells (PDLSCs) [21], and recombinant human IGFBP5 (rhIGFBP5) can promote PDLSC migration, chemotaxis, and osteo/dentinogenic differentiation [22]. However, IGFBP5 overexpression has also been shown to decrease in vitro osteoblastogenesis [12], and there is some evidence that this protein can also restrain skeletal growth [23]. As such, IGFBP5 may play cell- and tissue-specific roles in regulating physiological activities. As such, in the present study, we specifically evaluated the impact of IGFBP5 on hMSC osteogenesis.
The mechanistic basis by which IGFBP5 controls osteogenesis has yet to be clarified. The differentiation of hMSCs into osteoblasts is controlled by the coordinated simultaneous activation of many signaling pathways, making it essential to understand which of these pathways function downstream of IGFBP5. In prior research, IGFBP5 was shown to modulate dental pulp stem cell dentinogenesis by controlling the ERK signaling pathway [21]. Consistent with such activity, IGFBP5 also impacts the growth of pancreatic cancer cells by modulating ERK1/2 signaling [24]. As such, we hypothesized that IGFBP5 may also control osteogenesis via the ERK1/2 signaling pathway. Consistent with this model, we determined that overexpressing or knocking down IGFBP5 was sufficient to alter ERK1/2 phosphorylation levels during hMSCs osteogenesis (Fig. 3A and B). The treatment of these cells with ERK1/2 inhibitors was also sufficient to reverse the impact of IGFBP5 overexpression, on hMSC osteogenic differentiation (Fig. 3C and Fig. 4). These data therefore confirmed that IGFBP5 signals via ERK1/2 in order to control the early osteogenic differentiation of hMSCs.
Together, our data indicate that IGFBP5 serves as a key regulator of early-stage osteoblastogenesis in hMSCs. As a novel positive regulator of this important differentiation process, IGFBP5 may thus be a viable target for future studies of the treatment of osteoporosis and other bone-related diseases.