Leukaemia inhibitory factor (LIF) has a wide variety of biological activities. While recent studies have focused on the role of LIF in osteoblast differentiation, the exact role of LIFR during the early stage of osteogenic differentiation remains unclear. We observed that LIFR expression gradually decreased during the early stage of osteogenic differentiation of hMSCs. To evaluate how LIFR
regulates osteogenic differentiation in greater depth, we transfected hMSCs with LIFR overexpression and siRNA lentiviral plasmids. Cells were divided into four groups:a negative overexpression control group, a LIFR overexpression group, a negative siRNA control group, and a LIFR siRNA group. On different days (0, 3, and 6) of the osteogenic differentiation of hMSCs, alkaline phosphatase (ALP) activity was assayed with an ALP staining and activity assay kit. Cells were harvested to assess the mRNA and protein expression of LIF, LIFR, and osteogenesis-related factors (ALP; RUNX2; osteonectin) by qRT-PCR and western blot analyses, respectively. In addition, culture supernatants were tested for the LIF content by ELISA. Our results showed that overexpression of LIFR significantly suppressed the osteoblast differentiation of hMSCs. In contrast, LIFR siRNA markedly improved this osteoblast differentiation as determined by ALP staining and activity measurements. Moreover, RUNX2, ALP, and ONN expre-sion was also significantly changed by altering LIFR expression. We further analysed the expression of LIF and LIFR, revealing consistent LIF and LIFR trends during the osteogenic differentiation of hMSCs. Together, these results suggested that LIFR may be a novel negative regulator during the early stage of hMSC osteogenic differentiation. and Corresponding authors: Tao Wang, Meirong Zheng, Weidong Li
Insulin-like growth factor binding protein 5 (IGFBP5) is broadly bioactive, but its role in osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) remains to be clarified. Here, we demonstrated that IGFBP5 expression was markedly increased during the early osteogenic differentiation of hMSCs. We then over-expressed and knocked down this gene in hMSCs and evaluated the impact of manipulation of IGFBP5 expression on osteogenic differentiation based upon functional assays, ALP staining, and expression of osteogenic markers. Together, these analyses revealed that IGFBP5 over-expression enhanced early osteogenic differentiation, as evidenced by increased ALP staining and osteogenic marker induction, whereas knocking down this gene impaired the osteogenic process. Over-expression of IGFBP5 also markedly bolstered the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation level, while IGFBP5 knockdown suppressed this signalling activity. We additionally compared the impact of simultaneous IGFBP5 overexpression and ERK1/2 inhibitor treatment to the effect of IGFBP5 over-expression alone in these hMSCs, revealing that small molecule-mediated EKR1/2 inhibition was sufficient to impair osteogenic differentiation in the context of elevated IGFBP5 levels. These findings indicated that IGFBP5 drives the early osteogenic differentiation of hMSCs via the ERK1/2 signalling pathway. Our results offer value as a foundation for future efforts to study and treat serious bone-related diseases including osteoporosis.