Cyclooxygenase-2 negatively regulates osteogenic differentiation in murine bone marrow mesenchymal stem cells via the FOXO3a/p27kip1 pathway

Abstract

Aims: Cyclooxygenase-2 (COX-2) is an enzyme that synthesizes prostaglandins from arachidonic acid. Previous reports have indicated that COX-2 is constitutively expressed in osteogenic cells instead of being expressed only after pathogenic induction, and that it facilitates osteoblast proliferation via PTEN/Akt/p27^kip1 signalling. However, the role of COX-2 in osteogenic differentiation of murine bone marrow mesenchymal stromal cells (BMSCs) remains controversial. In this study, we investigated the function of COX-2 in the osteogenic differentiation of BMSCs.

Methods: COX-2 inhibitor, COX-2 overexpression vector, and p27^kip1 small interfering RNA (siRNA) were used to evaluate the role of COX-2 in osteogenic differentiation and related signalling pathways in BMSCs.

Results: We found that the messenger RNA (mRNA) and protein levels of COX-2 decreased gradually during osteogenic differentiation. Inhibition of COX-2 activity promoted FOXO3a and p27^kip1 expression and simultaneously enhanced osteogenesis, as indicated by increased osteogenic gene expression and mineralization in BMSCs. Furthermore, when p27^kip1 was silenced, the suppressive effects of COX-2 on osteogenesis were reversed. It demonstrated that the negative regulatory effect of COX-2 on osteogenesis was mediated by p27^kip1. In addition, our results showed that overexpression of COX-2 reduced the mRNA and protein levels of FOXO3a and p27^kip1, and thus attenuated osteogenic gene expression. These results indicate that COX-2 negatively regulates osteogenic differentiation by reducing the expression of osteogenic genes via the FOXO3a/p27^kip1 signalling pathway.

Conclusion: Together with the findings from previous and current studies, these results indicate that COX-2 has a different role in proliferation versus differentiation during osteogenesis via FOXO3a/p27^kip1 signalling in osteoblasts or BMSCs.

Fig. 1

Gene expression and protein levels of cyclooxygenase-2 (COX-2) during osteogenesis. a) COX-2 gene expression decreased from 100% on day 0 (subconfluence) to approximately 40% on day 2 (confluence) and decreased gradually until day 5. b) The protein level of COX-2 also decreased from day 0 to day 3 and was maintained until day 7 (**p < 0.01; n = 4; one-way analysis of variance and Scheffe’s method). mRNA, messenger RNA.

Fig. 2

Cyclooxygenase-2 (COX-2) inhibitor NS398 promotes osteogenic gene expression and mineralization. a) The gene expression levels of Runx-2, osteocalcin (OC), and alkaline phosphatase (ALP) increased in osteoinduction medium (OIM) from day 1 to day 3 and then decreased on day 5 (**p < 0.01 compared to the day 0 control group). After 10 µM and 20 µM NS398 treatment, the gene expression levels of Runx-2, OC, and ALP were significantly greater on days 1 to 3 than those in the control group on the same day (#p < 0.05, ##p < 0.01 compared to the control group on the same day). b) Mineralization was increased by 5 to 25 µM NS398 on days 4, 6, and 7 (*p < 0.05, **p < 0.01 compared to the day 0 control group; #p < 0.05, ##p < 0.01 compared to the control group on the same days). All p-values calculated using two-way analysis of variance and Scheffe’s method.

Fig. 3

Cyclooxygenase-2 (COX-2) inhibitor promoted p27^kip1 and FOXO3a messenger RNA (mRNA) and protein expression. a) p27^kip1 gene expression was increased during osteogenic differentiation from day 1 to day 7 (**p < 0.01 compared to the day 1 control group). After treatment with 20 μM NS398, the gene expression of p27^kip1 increased from day 1 to day 7 (##p < 0.01 compared to the control group on the same day). b) The protein levels of FOXO3a and p27^kip1 increased from day 1 to day 3, and then decreased from day 5 to day 7 during osteogenesis (**p < 0.01 compared to the day 0 control group). Compared with those in the control group, the FOXO3a and p27^kip1 protein levels in the NS398 treatment group were also increased on day 1 or day 3 (##p < 0.01, compared with the control group on the same day). All p-values calculated using two-way analysis of variance and Scheffe’s method.

Fig. 4

p27^kip1 silencing reduces cyclooxygenase-2 (COX-2) inhibition-promoted osteogenesis. a) The gene expression of p27^kip1 was significantly reduced after D1 cells were transfected with 10 or 20 nM p27^kip1 small interfering RNA (siRNA) from day 1 to day 5 (**p < 0.01, compared to the mock control group day 1; ##p < 0.01, compared to the mock control group day 5). b) The gene expression levels of Runx-2, osteocalcin (OC), and alkaline phosphatase (ALP) were increased after 20 µM NS398 treatment, but the expression levels of these genes were decreased after combined p27^kip1 siRNA and NS398 treatment (**p < 0.01, NS398 group compared to the mock siRNA group; ##p < 0.01, the mock control with NS398 group compared with the siRNA p27^kip1 with NS398 group). c) Quantification of the increase in mineralization after treatment with 10 and 20 NS398 and mock siRNA (**p < 0.01, compared to the 0 μM NS control group). However, after treatment with p27^kip1 siRNA, NS398-induced mineralization was attenuated (##p < 0.01, compared to the mock siRNA group at the same concentration in the NS398 group). All p-values calculated using two-way analysis of variance and Scheffe’s method. mRNA, messenger RNA.

Fig. 5

Overexpression of cyclooxygenase-2 (COX-2) reduced osteogenic gene expression. a) In the control group, COX-2 gene expression was reduced from day 1 to day 3 during osteogenic differentiation (##p < 0.01 compared with the day 0 vehicle control group). COX-2 gene expression was significantly increased by 200-fold three days after COX-2 overexpression (**p < 0.01, compared with the day 0 COX-2 overexpression group). b) to e) The gene expression levels of Runx-2, bone morphogenetic protein 2 (BMP-2), osteocalcin (OC), and alkaline phosphatase (ALP) were significantly increased during osteogenic differentiation, but the expression of these genes decreased after overexpression of the COX-2 gene on days 2 and 3 ($$p < 0.01, compared with the vehicle control group). All p-values calculated using two-way analysis of variance and Scheffe’s method.

Fig. 6

Cyclooxygenase-2 (COX-2) overexpression reduced FOXO3a and p27^kip1 protein levels. a) The western blotting showed the protein levels of COX-2, FOXO3a, p27^kip1, and Runx-2. The protein levels of actin were included as the internal control. b) The protein levels of COX-2 decreased in a time-dependent manner in the vehicle control group and in the COX-2 overexpression group (##p < 0.01 compared with the day 0 vehicle control group; **p < 0.01 compared with the day 0 COX-2 overexpression group). COX-2 protein levels were significantly increased after transfection with the COX-2 overexpression vector in day 0 and day 1 ($$p < 0.01 compared with the control group on the same day). c) Western blotting revealed that the FOXO3a protein level increased with time in the vehicle control group, but decreased with time after COX-2 was overexpressed that compared with the control group on the same day (##p < 0.01 compared with the day 0 vehicle control group; $$p < 0.01 compared with the vehicle control group on the same day). d) The protein level of p27^kip1 increased in the control group in a time-dependent manner, but decreased with time after COX-2 overexpression (##p < 0.01 compared with the day 0 vehicle control group; $$p < 0.01 compared with the vehicle control group on the same day). e) Runx-2 protein levels were also reduced after COX-2 was overexpressed compared with the control group on the same day (##p < 0.01 compared with the day 0 vehicle control group; $$p < 0.01 compared with the vehicle control group on the same day). All p-values calculated using two-way analysis of variance and Scheffe’s method.

Fig. 7

Diagram illustrating the molecular mechanism by which cyclooxygenase-2 (COX-2) negatively regulates osteogenic differentiation via the FOXO3a/p27^kip1 pathway. ALP, alkaline phosphatase; BMSC, bone marrow mesenchymal stromal cell; CDK, cyclin-dependent kinase; OC, osteocalcin; Runx2, Runt-related transcription factor 2.