The bone marrow stromal contains multipotent cells that are capable of differentiating into osteoblasts, chondrocytes, adipocytes, and myoblasts. BMSCs have the characteristics of self-proliferation and multi-differentiation[7]. During osteogenesis, different cellular phenotypes were tentatively defined as osteoprogenitors, preosteoblasts, mature osteoblasts and mineralizing osteoblasts, each of them characterized by an overlapping set of marker genes[8]. Classical cytochemical markers of osteoblast differentiation are alkaline phosphatase and mineralized bone nodules. This study showed that simvastatin, a HMG-CoA reductase inhibitor, promoted osteoblastic differentiation in rat BMSCs. Simvastatin stimulated the ALP activity, which is an early osteoblastic differentiation marker and promoted the mineralization of the matrix by osteoblasts. These results are consistent with previous observations showing that the statins enhanced osteogenesis in MC3T3-E1 cells (a clonal pre-osteoblastic cell line derived from mouse calvaria) and aging rat marrow stromal cells[9-10].
Previously, the effect of simvastatin on osteoblast differentiation was examined mostly at cellular or single gene level. Many external regulating factors, critical molecular steps in osteoblast differentiation and bone formation are largely unknown. The rat oligonucleotide array used in the present study has enabled us to specifically examine a large number of genes involved in the process of osteoblast differentiation. In our study, we examined the expression profiles of 22575 genes of BMSCs, 103 genes provided significant signals (Ratio Cy3/Cy5 ≥2 or ≤0.5). Among them more than 10 genes are related to ostoeblastic lineage differentiation.
The upregulated genes involved in transcription factor are C/EBPδ, Cited2. CCAAT-enhancer binding proteins (C/EBP) are a family of transcription factors that regulate cell differentiation[11]. C/EBPs are expressed in multiple cell types including osteoblasts and adipocytes, and play critical roles in adipocyte differentiation. C/EBPδ regulates IGF I transcription, and activates osteocalcin transcription by interacting with runt related protein (Runx)-2[12-13]. The Runx2 transcriptional activators are critical factors for the development of hematopoietic and skeletal tissues. Early during the process of differentiation, Cbp/p300-interacting transactivator 2 (Cited2) is upregulated. Cited2 is a transcriptional co-activator that physically sociates with Cbp/p300 enhancing its ability to transactivate a variety of genes[14]. Cbp/p300 activates gene expression by acting as a bridge between activators and the general transcriptional machinery and may regulate transcription by enhancing chromatin remodeling.
We found that a number of genes involved in some well known osteogenesis related signaling pathways such as TGFβ/BMP pathway[15-16], mitogen-activated protein kinase (MAPK) pathway[17-19], WNT/β-catenin pathway were up-regulated or down-regulated[20-23]. For example, two of the critical enzymes that participate in MAPK signal pathway (Ptpn16, Map3k8) were differentially expressed. Xiao et al[24] found that the MAPK pathway provides a plausible link between cell surface integrin activation by ECM and subsequent stimulation of Cbfa1-dependent transcription, suggesting that this pathway has an important role in the control of osteoblast-specific gene expression. Some proteins involved in WNT/β-catenin pathway (Wisp2) were also down-regulated. Clearly, how these regulatory signals affect osteoblast activity are needed to further study.
Cytokines, such as Cxcl10, Il10, Il1a, showed differential expression after simvastatin stimulation for 4 days. Lisignoli et al[25] demonstrated that stimulation of OBs with CXCL10 significantly induces both b-N-acetylhexosaminidase (Hex) release and ALP activity. Hex is an enzyme used to evaluate the standard response of leukocytes after chemokine stimulation, but it is also known to play a role in endochondral ossification and bone remodeling[26-27], since it degrades glycosaminoglycans. As reported by other authors, CXCL10 is highly effective in the inhibition of primary human microvascular endothelial cell proliferation and in stimulating human mesangial cells[28-29]. These different effects of CXCL10 on the proliferation of various cell types may be due to the phenotypical characteristics of cells or to the level of cell differentiation. In another study, they also found that CXCL10 and CXCL13 induced a dose-dependent increase of cell proliferation in OB from young donors[30].
In conclusion, in this study we have identified the multiple biological events as reflected by gene expression prolifes in the early period of ostoeblastic lineage differentiation of bone marrow stromal cells treated with simvastatin, as well as the promoting effects of simvastatin on BMSCs differentiation and minerlization indicating by ALP staining and Von Kossa Staining. Simvastatin is able to modulate a broad range of biological processes. The precise interaction between these genes is not clear. Clearly, more studies are needed. First, primary cell culture may contain heterogenous cell population, often containing contaminated cells of different types and cells in variable differentiation states. This may have leaded to a less precise demonstration of the effect of simvastatin on BMSCs. However, we believe that the present report provides new data on the genetic effect of simvastatin on bone marrow stromal cells.
