TCF3, a novel positive regulator of osteogenesis, plays a crucial role in miR-17 modulating the diverse effect of canonical Wnt signaling in different microenvironments

Abstract

Wnt signaling pathways are a highly conserved pathway, which plays an important role from the embryonic development to bone formation. The effect of Wnt pathway on osteogenesis relies on their cellular environment and the expression of target genes. However, the molecular mechanism of that remains unclear. On the basis of the preliminary results, we observed the contrary effect of canonical Wnt signaling on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the different culture environment. Furthermore, we found that the expression level of miR-17 was also varied with the change in the culture environment. Therefore, we hypothesized that miR-17 and canonical Wnt signaling may have potential interactions, particularly the inner regulation relationship in different microenvironments. In this paper, we observed that canonical Wnt signaling promoted osteogenesis of PDLSCs in the fully culture medium, while inhibited it in the osteogenic differentiation medium. Interestingly, alteration in the expression level of endogenous miR-17 could partially reverse the different effect of canonical Wnt signaling. Furthermore, the role of miR-17 was because of its target gene TCF3 (transcription factor 3), a key transcription factor of canonical Wnt pathway. Overexpression of TCF3 attenuated the effect of miR-17 on modulating canonical Wnt signaling. Finally, we elucidated that TCF3 enhanced osteogenesis both in vitro and in vivo. In brief, the different level of miR-17 was the main cause of the different effect of canonical Wnt signaling, and TCF3 was the crucial node of miR-17-canonial Wnt signaling regulation loop. This understanding of microRNAs regulating signaling pathways in different microenvironments may pave the way for fine-tuning the process of osteogenesis in bone-related disorders.

Figure 1

Canonical Wnt signaling had an opposite effect on osteogenic differentiation of PDLSCs in different culture environment. PDLSCs were cultured in the fully culture medium or osteogenic differentiation medium with 50 ng/ml recombinant Wnt3a for 7 days. (a) ALP staining and activity analyses were performed at day 7. (b) The expression levels of ALP and Runx2 were measured at the indicated time by real-time PCR and western blot. The expression levels of mRNA and protein were normalized to β-actin. The data are shown as mean±S.D. *P<0.05, n=3. Con, control; OD, optical density; OS, osteogenic induction; Wnt3a, 50 ng/ml recombinant human Wnt3a

Figure 2

miR-17 could partially attenuate the effect of canonical Wnt signaling. (a) The expression level of miR-17 was measured by real-time PCR after the PDLSCs were cultured in the fully culture medium or osteogenic differentiation medium with 50 ng/ml Wnt3a for 7 days. (b) PDLSCs were transfected with pre-miR-17, anti-miR-17 and negative controls for 1 day, and then cultured in the osteogenic differentiation medium for an additional 7 days. The expression of ALP and Runx2 was measured by real-time PCR and western blot. (c and d) PDLSCs were transfected with pre-miR-17 and anti-miR-17 for 1 day and then cultured in the fully culture medium or osteogenic differentiation medium with 50 ng/ml Wnt3a for 7 days. ALP staining and activity analyses were performed at day 7 (c) and the expression of ALP and Runx2 was measured by real-time PCR and western blot (d). β-Actin and U6 were used as control for the PCR data. β-Actin was also used as a loading control. The data are shown as mean±S.D. *P<0.05, n=3. Con, control; CM, fully culture medium; miR-Con, siPORT reagent alone; miR-NC, miRNA negative control; OD, optical density; OS, osteogenic induction; Wnt3a, 50 ng/ml recombinant human Wnt3a

Figure 3

TCF3 was a direct target of miR-17. (a) The PDLSCs, transfected with pre-miR-17, anti-miR-17 or negative control, were harvested after 24 h for real-time PCR or after 48 h for western blot. (b) A schematic of the miR-17 putative target sites in the human TCF3 3′-UTR and the alignment of miR-17 with the WT, MUT1 and MUT2 3′-UTR regions of TCF3. Two mutated nucleotides were shown in red. (c) The pMIR vector control, pMIR-TCF3-WT, pMIR-TCF3-MUT1 and pMIR-TCF3-MUT2 luciferase constructs were cotransfected with pre-miR-17, anti-miR-17 or negative control. Luciferase activity was measured after 48 h of transfection and normalized with β-galactosidase activity. (d) The expression pattern of TCF3 during osteogenic differentiation at the indicated time points was measured by western blot. (e) The endogenous expression of miR-17 was determined by real-time PCR at the same time points during osteogenic differentiation. The expression levels of mRNA and protein were normalized to β-actin. The data are shown as mean±S.D. *P<0.05, n=3. Con, control; miR-Con, siPORT reagent alone; miR-NC, miRNA negative control; OS, osteogenic differentiation; Vector, pMIR vector; WT, wild type; MUT, mutated type

Figure 4

Overexpression of TCF3 attenuated the effect of pre-miR-17 on inhibiting osteogenic differentiation. PDLSCs were transfected with pre-miR-17 after stably overexpressing TCF3 by lentiviral construct and then cultured in osteoblastic differentiation medium for an additional 7 or 14 days. (a) Osteoblastic differentiation was determined by ALP staining at day 7 and alizarin red staining at day 14. (b) ALP activity and calcium level analyses were performed at days 7 and 14. (c) The expressions of ALP and Runx2 were measured by real-time PCR and western blot at day 7. The expression levels of mRNA and protein were normalized to β-actin. The data are shown as mean±S.D. *P<0.05, n=3. miR-Con, siPORT reagent alone; OD, optical density; OS, osteoblastic induction; plenti-TCF3, lentiviral construct for upregulating TCF3

Figure 5

miR-17 modulated the contrary effect of canonical Wnt signaling mainly through targeting TCF3. (a) The expression level of TCF3 was measured by real-time PCR and western blot after the PDLSCs were cultured in the fully culture medium or osteogenic differentiation medium with 50 ng/ml Wnt3a for 7 days. (b and c) PDLSCs were transfected with pre-miR-17 after stably overexpressing TCF3 by lentiviral construct and then cultured in the fully culture medium with 50 ng/ml Wnt3a for an additional 7 days. ALP staining and activity analyses were performed at day 7 (b) and the expression of TCF3 and ALP was measured by real-time PCR and western blot (c). The expression levels of mRNA and protein were normalized to β-actin. The data are shown as mean±S.D. *P<0.05, n=3. CM, fully culture medium; OD, optical density; OS, osteogenic induction; plenti-TCF3, lentiviral construct for upregulating TCF3; Wnt3a, 50 ng/ml recombinant human Wnt3a

Figure 6

TCF3 promoted osteogenic differentiation of PDLSCs in vitro and ectopic bone formation in vivo. To confirm the effect of TCF3 on osteogenesis, PDLSCs were transduced with lentiviral constructs for 3 days and then cultured in osteogenic differentiation medium for an additional 7 or 14 days. (a) Osteogenic differentiation was determined by ALP staining at day 7 and alizarin red staining at day 14. (b) ALP activity and calcium level analyses were performed at days 7 and 14. (c) The expressions of ALP and Runx2 were confirmed by real-time PCR and western blot analyses at day 7. The expression levels of mRNA and protein were normalized to β-actin. *P<0.05, n=3. (d) PDLSCs were transduced with sh-Con or sh-TCF3 for 3 days and then implanted into non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. H&E and Masson's Trichrome staining was performed after 8 weeks of implantation. Osteoid formation was quantified as total osteoid volume per total volume by Masson's staining. Scale bar: 50 μm. Six implants per treatment were engrafted into the mice, and three sections of each implant were quantified to minimize variations within the implants. Data are shown as the means±S.D. *P<0.05, n=6. OD, optical density; OS, osteogenic induction; sh-Con, virus produced from the two packaging vectors; sh-NC, lentivirus negative control; sh-TCF3, lentiviral construct for downregulating TCF3

Figure 7

Working model of miR-17 in the modulating contrary effect of canonical Wnt signaling in different microenvironments