Role of Periostin/Periostin-like-factor in BMP-2 induced osteoblastic differentiation

Abstract

1. IntroductionPeriostin is a 90 kDa protein which is predominantly secreted by Mesenchymal stem cells (MSCs) in the bone marrow stroma and by osteoblasts. Periostin-Like-Factor (PLF) is a isoform of Periostin and are highly homologous to Periostin because they are alternatively splices mRNAs from the same gene. Periostin/PLF promotes osteoblastic differentiation in vitro and bone formation in vivo. BMPs are growth factors that are known to induce bone formation and BMP-2 is one of the most potent factor that stimulate osteoblastic differentiation. TNF-α is an inflammatory cytokine that stimulates osteoclastic differentiation while simultaneously inhibiting BMP-2 induced osteoblastic differentiation. The aim of this study is divided into two parts: 1) To investigate whether there is collaboration between BMP-2 and Periostin/PLF during osteoblastic differentiation, 2) To investigate whether there is a correlation between expression level of Periostin/PLF and the inhibitory effect of TNF-α on BMP-2 induced osteoblastic differentiation.2. Materials and MethodsExperiments were done in both C2C12 cells (pluripotent stem cells) and MC3T3-E1 cells (pre-osteoblasts). To clarify the role of Periostin/PLF in BMP-2 induced osteoblastic differentiation, cells were transfected with expression plasmid or siRNA of Periostin/PLF and treated with BMP-2 and/or TNF-α. After treatment, alkaline phosphatase (ALP) was measured as a marker of osteoblastic differentiation. The amount of Periostin/PLF mRNA and protein expressed were determined by real time RT-qPCR and Western blot analysis respectively.3. ResultsPeriostin/PLF protein expression is increased by BMP-2 and decreased by TNF-α in both MC3T3-E1 and C2C12 cells. Transient transfection with periostin/PLF plasmid (over-expression) substantially increased BMP-2 stimulated expression of ALP (p<0.05) and protein level compared to non-transfected cells. Furthermore, transient transfection with Periostin/PLF-siRNA (silencing) substantially reduced BMP-2 stimulated expression of ALP (p<0.05) and protein level compared to non-transfected cells. Finally, Periostin/PLF over-expression or silencing with siRNA did not restore the ALP activity in cells treated with BMP-2 and TNF-α.4. Conclusion1) First of all, BMP-2 increases Periostin/PLF protein expression and secondly Periostin/PLF potentiates the effect of BMP-2 in osteoblastic differentiation. 2) TNF-α decreases Periostin/PLF protein expression and inhibits BMP-2 induced osteoblastic differentiation. In transfected cells, treated with BMP-2 and TNF-α no changes in ALP activity were recorded. Periostin/PLF is not involved in the inhibitory effect of TNF-α on BMP-2 induced osteoblastic differentiation.