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Journal of Peking University(Health Sciences) ›› 2018, Vol. 50 ›› Issue (6): 962-967. doi: 10.19723/j.issn.1671-167X.2018.06.004

• Article • Previous Articles     Next Articles

Effects of integrin metalloproteinases on osteogenic differentiation

Xia LIU1,Ying ni LI2,Xiao li SUN2,Qing lin PENG1,Xin LU1,Guo chun WANG1,△()   

  1. 1. Department of Rheumatology and Immunology, China?Japan Friendship Hospital, Beijing 100029, China
    2. Department of Rheumatology and Immunology, Peking University People’s Hospital, Beijing 100044, China
  • Received:2018-08-31 Online:2018-12-18 Published:2018-12-18
  • Contact: Guo chun WANG E-mail:guochunwang@hotmail.com
  • Supported by:
    Supported by the National Natural Science Foundation of China(81302586)

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Abstract:

Objective: To study the effects of disintegrin and metalloproteinase (ADAM)9, 15 and 17 on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs).Methods: BMMSCs of ADAM9, ADAM15, ADAM17 conditional knockout mice and wild type mice (WT) were induced and cultured. Alkaline phosphatase (ALP) activity was measured by colorimetry, early osteogenic transcription factors Runx and Osterix were detected by Real-time PCR, and mineral formation was analyzed by alizarin red staining. Results:ALP activity was lower in ADAM9 group (8.08±0.34), ADAM15 group (6.46±3.40), ADAM17 group (9.30±2.30) than that in WT group (9.44±2.50), but there was no significant difference (P>0.05). Stimulated with bone morphogenetic protein 2(BMP2),there was significant difference (P<0.05) between ADAM9 group (14.22±3.25), ADAM15 group (10.14±2.40) and WT group (20.89±3.40), and ADAM 17 group (23.56±2.50) was higher than WT group (20.89±3.40), but no significant difference (P>0.05).Similarly, cultured by osteogenic induction medium (OST), compared with WT group(12.97±1.30), ADAM9 group(9.63±1.00) and ADAM15 group(7.75±1.30)were lower,ADAM17 group (20.09±1.68) was higher, and the difference was statistically significant (P<0.05). Using stimulated culture by BMP2 and OST combined , ADAM9 group (15.75±1.30), ADAM 15 group (12.43±1.30) were less than WT group (26.15 ±1.50), while ADAM17 group (29.55±2.10) was higher than WT group were statistically significant (P<0.05). The expression of Runx2 in ADAM9 group (2.02±0.24), ADAM15 group (3.09±0.19), ADAM17 group (3.89±0.91) had no significant difference compared with WT (2.02±0.21) group (P>0.05). ADAM9 group stimulated by BMP2 (7.00±0.23), ADAM15 group (6.04±0.23) were lower than WT group (12.6±0.23), ADAM17 group (18.52±1.39) was higher than WT group (12.6±0.23), and the difference was statistically significant (P<0.05).In non-stimulating culture,there was no significant difference in Osterix expression between ADAM9 group (9.60±3.87) , ADAM17 group (12.40±3.00) and WT group (10.9±1.10, P>0.05),but in ADAM15 group (6.50±1.51) it was slightly lower than that in WT group (P<0.05). After BMP2 stimulation, ADAM9 group (39.20±3.23) and ADAM15 group (20.50±4.80) were less than WT group (60.30±5.93), while ADAM17 group (80.20±3.30) was higher than WT group (P<0.05). Alizarin red staining showed no obvious orange-red mass in the non-induction group. Local calcified nodules could be seen in the BMP2, OST, OST + BMP2 induction culture conditions in all the experimental groups, but there was no significant difference in quantitative analysis (P>0.05). Conclusion: ADAM9, 15, 17 took part in the osteogenic differentiation of BMMSCs, and provided new targets for its regulation.

Key words: A disintegrin and metalloproteinase, Bone marrow mesenchymal stem cells, Osteogenic differentiation

CLC Number: 

  • R593.2

Table 1

ALP activity of BMMSCs after 7 d of cultured"

Items ADAM9 ADAM15 ADAM17 WT F P
Control 8.08±0.34 6.46±3.40 9.30±2.30 9.44±2.50 1.98 0.5
BMP2 (100 μg/L) 14.22±3.25* 10.14±2.40* 23.56±2.50 20.89±3.40 26.44 <0.001
OST 9.63±1.00# 7.75±1.30# 20.09±1.68# 12.97±1.30 93.39 <0.001
OST+BMP2 (100 μg/L) 15.75±1.30△ 12.43±1.30△ 29.55±2.10△ 26.15±1.50 159.86 < 0.001

Figure 1

Expression of Runx2 and Osterix of BMMSCsA,Runx2; B, Osterix; BMMSCs, bone marrow mesenchymal stem cells; BMP2, bone morphogenic protein 2."

Table 2

The Runx2 expression of BMMSCs stimulated by BMP2 for 6 h"

Items ADAM9 ADAM15 ADAM17 WT F P
Control 2.02±0.24 3.09±0.19 3.89±0.91 2.02±0.21 18.56 <0.001
BMP2 (100 μg/L) 7.00±0.23* 6.04±0.23* 18.52±1.39* 12.60±0.23 381.50 <0.001

Table 3

The Osterix expression of BMMSCs stimulated by BMP2 for 6 h"

Items ADAM9 ADAM15 ADAM17 WT F P
Control 9.60±3.87 6.50±1.51* 12.40±3.00 10.90±1.10 5.50 0.006
BMP2 (100 μg/L) 39.20±3.23# 20.50±4.80# 80.20±3.30# 60.30±5.93 201.79 <0.001

Figure 2

Alizarin red staining of BMMSCs cultured for 14 d(×20) BMMSCs, bone marrow mesenchymal stem cells;ADAM, a disintegrin and metalloproteinase; WT, wild type; BMP2, bone morphogenic protein 2; OST, osteogenic induction medium."

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