The p53/miR-17/Smurf1 pathway mediates skeletal deformities in an age-related model via inhibiting the function of mesenchymal stem cells

Abstract

Osteoporosis is an age-related progressive bone disease. Trp53 (p53) is not only a famous senescence marker but also a transcription regulator which played a critical role in osteogenesis. However, how p53 contributes to the bone mass loss in age-related osteoporosis is still unclear. Here, we found that bone mass and osteogenic differentiation capacity of mesenchymal stem cells (MSCs) is significantly reduced with advancing age. Serum levels of TNF-α and INF-γ and senescence-associated β-galactosidase, p16, p21 and p53 are significantly increased in elder mice, but antipodally, osteogenic marker expression of Runx2, ALP and osterix are reduced. Overexpression p53 by lentivirus inhibits osteogenesis in young MSCs in culture and upon implantation in NOD/SCID mice through inhibiting the transcription of miR-17-92 cluster, which is decreased in old mice. In addition, miR-17 mimics could partially rescue the osteogenesis of old MSCs both in vitro an in vivo. More importantly, Smurf1 as a direct target gene of miR-17, plays an important role in the p53/miR-17 cascade acting on osteogenesis. Our findings reveal that p53 inhibits osteogenesis via affecting the function of MSCs through miRNA signaling pathways and provide a new potential target for treatment in future.

Keywords: aging; mesenchymal stem cells; miR-17; osteogenesis; p53.

Figure 1

The osteogenic capacity of old mice is significantly reduced both in vivo and in vitro. Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A) Micro-CT analysis of trabecular bone mass in the tibiae of 4 (young) and 16 month-old (old) mice. Quantitative analyses were performed via volumetric bone mineral density (BMD) and trabecular bone volume fraction (BV/TV) measurements. (B) HE stainings of histological sections from femur derived from young and old mice for detection of the number of bone trabeculae. (C-D) Representative images of the CFU-F assay for determination of proliferation capacity and of the CFU-Ob assay for osteogenic differentiation ability of BMMSCs obtained from young and old mice and stained with crystal violet and alizarin red, respectively. CFU efficiency was determined by the number of colonies relative to the total number of seeded cells in each plate. (E) Serum levels of TNF-α and INF-γ in young and old mice determined via ELISA. Results are expressed as pg/ml.

Figure 2

BMMSCs from old mice express higher levels of senescence markers and lower osteoblast markers compared to young ones. Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A) In vitro staining of the senescence-related marker ß-galactosidase in BMMSCs cultures derived from young and old mice. Quantitative analysis of the total number of positively stained cells. (B-C) Real-time PCR analyses on whole bone tissue extracts (B) and on BMMSCs (C) for the senescence-related genes p16, p21 and p53. Normalization to ß-actin. (D) The western blot showed that the protein level changed as the mRNA. (E) Alizarin red staining of BMMSCs from young and old mice osteogenically induced for 14 d. Cont = Control, OS = osteogenically induced. (F-G) Real-time PCR and western blot analyses on BMMSCs for the osteogenic markers Runx2, ALP, osterix. Normalization to ß-actin.

Figure 3

Overexpression of p53 changed the phenotype of young BMMSCs into old BMMSCs. BMMSCs from young mice were lentivirally transduced to upregulate the expression level of p53 (= pLenti-p53) or were transduced as lentiviral control (= pLenti-Cont). Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A) Alizarin red staining of pLenti-p53 and of pLenti-Cont after osteogenic inducing for 14 days. Cont = Control, OS = osteogenically induced. The values show the mean ± SD (n=10). * p < 0.05. (B-C) Real-time PCR and western blot analyses on BMMSCs with lentiviral transduction (pLenti-p53 and pLenti-Cont) and with/without osteogenic induction for the osteogenic markers Runx2, ALP, osterix. Normalization to ß-actin. (D-E) Histological analyses and corresponding statistical analysis of tissue sections from subcutaneous pockets on the backs of 6-week-old NOD/SCID mice with implanted HA/TCP ceramic particles mixed with BMMSCs from young mice with lentiviral transduction of p53 and control.

Figure 4

p53 contribute to impaired osteogenesis of BMMSCs via inhibiting the transcription of miR-17-92 cluster. BMMSCs were lentivirally transduced to upregulate the expression level of p53 (= pLenti-p53) or were transduced as lentiviral control (= pLenti-Cont). Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A-C) Real-time PCR analyses for the expression of miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a in bone (A), bone marrow (B) and BMMSCs (C) of young and old mice. Normalization to ß-actin. (D-F) Real-time PCR and western blot analysis of p53 (D, E) and real-time PCR of miR-17 (F) expression in BMMSCs derived from young and old mice after osteogenic differentiation for 7 d. Normalization to ß-actin and U6. (G) Pri-miR-17 transcript analysis by Taqman-based qPCR. Normalization to GAPDH. (H) Real-time PCR analysis of the mature miR-17-92 cluster after upregulating P53 for 48 h. Normalization to U6.

Figure 5

Up-regulation of miR-17 by miR-17 mimics reversed the effect of p53 on inhibiting osteogenic differentiation in old BMMSCs. miR-17 was stable upregulated in BMMSCs by miR-17 mimics (= miR-17 mimics). miRNA control (= miR Cont). Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A) Osteogenic induction for 14 d of BMMSCs derived from old mice and subsequent alizarin red staining resulted in a heightened osteogenic differentiation of BMMSCs with previously upregulated miR-17 expression. (B-C) Real-time PCR and western blot analyses on old BMMSCs with miR-17 mimic treatment or miRNA control and with/without osteogenic induction for the osteogenic markers Runx2, ALP, osterix. Normalization to ß-actin. (D-E) Histological analysis (D) and corresponding statistical analysis (E) on osteoid formation of tissue sections from subcutaneous pockets on the backs of 6-week-old NOD/SCID mice with implanted HA/TCP ceramic particles mixed with BMMSCs from 16-month-old mice with/without miR-17 upregulation.

Figure 6

Smurf1 plays an important role in miR-17-mediated osteogenic differentiation of BMMSCs. BMMSCs were lentivirally transduced to upregulate the expression level of P53 (= pLenti-P53) or were transduced as lentiviral control (= pLenti-Cont). miR-17 was stable upregulated in BMMSCs by miR-17 mimics (= miR-17 mimics). miRNA control (= miR Cont). 16 Mon Cont = control BMMSCs, Smurf1 siRNA = downregulated Smurf1 level via si-RNA, miR-17 inhibitor = transfection with anti-miR-17. Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A) Real-time PCR and western blot analysis on the expression of Smurf1 and TCF3 after upregulation of p53 (pLenti-p53) in BMMSCs derived from young mice. Normalization to ß-actin. (B) Western blot analysis on the expression of Smurf1. Transfection of miR-17 mimics in stable upregulated p53 BMMSCs derived from young mice. Normalization to ß-actin. (C-D) Real-time PCR and western blot analysis of Smurf1 expression in osteogenically differentiated BMMSCs from young and old mice. Normalization to ß-actin. (E) Alizarin red staining after osteogenic induction for 14 d of BMMSCs derived from old mice with/without siRNA-downregulated Smurf1 level and with/without transfection with miR-17 inhibitor. (F) Western blot analysis on old BMMSCs with/without siRNA-down-regulated Smurf1 level and with/without transfection with anti-miR-17 for the osteogenic markers Runx2, ALP, osterix. Normalization to ß-actin.

Figure 7

Schematic diagram of p53/miR-17/Smurf1 cascade. (A, B) p53 regulates the osteogenic differentiation of BMMSCs through inhibiting transcription of miR-17-92 cluster and subsequent modulating Smurf1, a direct target gene of miR-17, aslo acts as a negative regulator for osteogenic differentiation of mesenchymal stem cells.