T-614 Promotes Osteoblastic Cell Differentiation by Increasing Dlx5 Expression and Regulating the Activation of p38 and NF- κ B

Abstract

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by bone loss. Degree of inflammation has been identified as an important initiator of skeletal damage in RA. Iguratimod (T-614) is an anti-inflammatory agent which has been reported to show the inhibitory effect of bone destruction in RA. However, the role of T-614 in osteoblast differentiation is still not clear. In this study, we intended to find the effect of T-614 on the osteogenesis process. We detected osteogenesis markers and transcription factors associated with osteoblastic lineage and bone formation in the culture of mesenchymal stem cells which differentiate osteoblast. The contents and activity of alkaline phosphatase, levels of collagen type I and bone gla protein, and calcium nodule formation were increased significantly after T-614 treated. Meanwhile, the mRNAs expressions of Osterix and Dlx5 were also found to be increased significantly by real-time PCR. The changes of levels of phosphorylation of p38 and NF-κB were also detected by Western blot. The results showed that T-614 promotes osteoblastic differentiation by increasing the expression of Osterix and Dlx5 and increasing the activation of P38. T-614 could advance the ectopic expression of NF-κB to suppress inflammation, which indirectly inhibits the damage of the osteoblasts.

Figure 1

ALP staining and ALP activity in MSCs. MSCs were treated with rhBMP-2 and/or T-614 for 3 days. The cells were tested for ALP content by using an ALP staining kit; ALP activity was calculated as the total activity units/total protein concentration. (a) The stainings are representative of 3 separate experiments with similar results. (b) Statistical analysis of ALP activity. The data are expressed as the means +/− SD (n = 3). ^∗P < 0.05, ^∗∗P < 0.01, compared with the vehicle control; ^#P < 0.05, compared with the BMP2 group.

Figure 2

Calcium nodule formation in MSCs. MSCs were cultured in α-MEM supplemented with 10% FBS and 10 mM b-glycerophosphate in the absence or presence of 50 ng/ml rhBMP-2 and 10 μg/ml T-614 for 14 days. Then, the calcium content of the mineralized nodules in the cells was stained with Alizarin Red S at pH 4.3. (a) The data are representative of 3 separate experiments with similar results. (b) Statistical analysis of absorbance of Alizarin Red S staining. The data are expressed as the means +/− SD (n = 3). ^∗∗P < 0.01, compared with the vehicle control; ^##P < 0.01, compared with the BMP2 group.

Figure 3

Osteogenesis markers and the related transcription factor expressions in MSCs. The mRNA levels of the osteogenesis markers Alp, Col1, and Bgp in MSCs cultured in the indicated condition (a) and the mRNA levels of the related transcription factors (Osterix and Dlx5) (n = 3) (b) were measured by real-time PCR. The β-actin gene was used as a reference to normalize the differences in total RNA in each sample (n = 3). Statistical analysis is expressed as the means +/− SD. The data are representative of five experiments with similar results. ^∗∗P < 0.01, compared with the vehicle control; ^#P < 0.05, ^##P < 0.01, compared with the BMP2 group.

Figure 4

p38 signaling pathway in osteoblast differentiation in MSCs. MSCs were cultured in α-MEM supplemented with 10% FBS and 50 ng/ml rhBMP-2 in the absence or presence of 10 μg/ml T-614. Then, the cells were harvested at 1.5, 2.5, 3, 4, and 6 h to detect the expressions of phospho-p38 by Western blot. The data are representative of three experiments with similar results.

Figure 5

The effect of T-614 on the NF-κB signal pathway. MSCs were cultured in α-MEM supplemented with 10% FBS and 20 ng/ml TNF-α in the absence or presence of 10 μg/ml T-614. Then, the cells were harvested at 0.75, 1, 1.5, 2, and 3 h to detect the expressions of NF-κB and p-NF-κB by Western blot. The data are representative of three experiments with similar results.