Regulatory Mechanism Of Osteoblastic Differentiation Of Bone Marrow Derived Mesenchymal Stem Cells In Patients With Sysytemic Lupus Erythematosus

Regulatory mechanism of osteoblastic differentiation of bone marrow derived mesenchymal stem cells in patients with sysytemic lupus erythematosusThe bone remolding in the adult is a balance between bone formation and bone resorption. Up to now, pro-inflammatory cytokines, such as TNF-a, induced osteoclastogenesis either by causing the proliferation of osteoclast precursor cells or by promoting the activation of the differentiated osteoclast are thought to be the main mechanism of osteoporosis (OP) of patients with systemic lupus erythematosus (SLE). However, the study about the osteoblasts of SLE patients is unresolved. Osteoblasts are derived from mesenchymal stem cells (MSCs), which can differentiate into osteoblasts in vitro and in vivo. Roles of TNF-a signaling system in determining osteoblast function and its differentiation remain controversial. Some studies reported TNF-a in vitro could inhibit osteoblastic differentiation of MSCs through activating NF-κB or MAPK pathways, while others found the enhanced effect of TNF-a on them. We previously reported BMMSCs from SLE patients showed significantly decreased bone-forming capacity and impaired reconstruction of bone marrow osteoblastic niche in vivo, however, the cellular mechanism of this deficiency has not yet been elucidated. We postulate here that TNF-a activated NF-κB signaling pathway inhibits bone morphogenetic protein (BMP-2) induced osteoblastic differentiation of BMMSCs from SLE patients by downregulating BMP/Smad signaling pathway.In order to investigate the signaling pathways that regulate osteoblastic differentiation of BMMSCs from SLE patients, we compared the gene expression patterns of bone marrow-derived mesenchymal stem cells (BMMSCs) between 4 healthy controls and 4 lupus individuals. RNAs were reversely transcribed to cDNAs and hybridized to the cDNA microarray. BMP/TGF-P, MAPK and cell cycling'regulation pathways were detected by immunohistochemistry, immunofluoresence and immunoblotting. It was shown that among over 20,000 genes investigated,1905 genes were differentially expressed by BMMSCs derived from SLE patients. And among them,652 genes were up-regulated and 1253 genes were down-regulated. Gene Ontology (GO) analysis showed that the majority of differentially expressed genes were related to cell cycle and protein binding. Pathway analysis showed that differentially regulated signal pathways involved actin cytoskeleton, focal adhesion, tight junction and TGF-(3/BMP signaling pathways. The high level of BMP-5 protein expression and low Idl expression indicated dysregulated BMP/TGF-βsignaling pathway. The protein level of cyclin E decreased in the cell cycling regulation pathway. Moreover, the MAPK signaling pathway was activated in BMMSCs from SLE patients via phosphorylation of ERK1/2 and Junk. Of note, Among the 25 genes detected in BMP/Smad signaling,19 genes were differentially expressed. More importantly, among the 19 differentially expressed genes, all were down-regulated except for BMP-5. The results of cDNA microarray above suggested the BMP/Smad signaling pathway in SE BMMSCs might be suppressed.For further revealing the state of BMP/Smad signaling pathway of SLE BMMSCs, western blotting analysis was used to detect the proteins of this pathway. We found that there was no difference in the expression of BMP receptor MPRIA between normal and SLE BMMSCs, while the phosphorylation of Smad1/5/8 was decreased. The complex of Smad4 with Smad1/5/8 which translocates into nucleus was also found decreased, indicating that BMP/Smad signaling pathway of BMMSCs from SLE patients was depressed.In order to further investigate the osteoblastic differentiation of BMMSCs from lupus patients, SLE BMMSCs were cultured in the osteogenic medium including BMP-2, and the osteogenic markers ALP, osteogenic transcriptor Rux2 and the extracellular mineralization of SLE BMMSCs were evaluated. The results showed that the expressions of ALP, Rux2, the ALP activity and the extracellular mineralization of BMMSCs from SLE patients were decreased as compared with normal BMMSCs at day 2,14 and 21 respectively. These results indicated a low osteogenic capacity of SLE BMMSCs.Next, we investigated the role of TNF-αon the differentiation of BMMSCs. BMMSCs from health controls were induced by BMP-2 in the precence or abcence of TNF-a. We found that TNF-a could induce the activation of NF-κB and inhibited BMP-2 induced activation of BMP/Smad signaling pathway by down-regulating phosphorylation of Smadl/5/8. On the other hand, NF-κB inhibitor PDTC could restor TNF-a induced phosphorylation of Smadl/5/8. In addition, TNF-a could inhibite the expression of ALP and Rux2 at day 2 and the extracellular mineralization of normal BMMSCs at day 21.At last, we found that the pIκB was increased and IκB was decreased in SLE BMMSCs, which indicates that the NF-κB signaling pathway in SLE BMMSCs was inactive. PDTC partially restored the phosphorylation of Smadl/5/8 of SLE BMMSCs. It could also partially restore the BMP-2 induced expression of ALP and Rux2 at day 2 and the extracellular mineralization at day 21 of BMMSCs from SLE patients.In conclusion, we have demonstrated an activated NF-κB and NF-κB inhibited BMP/Smad signaling pathways in BMMSCs from SLE patients. The activated NF-κB pathway inhibits BMP-2-induced osteogenetic differentiation through down-regulating Smad signaling.Our results have uncovered a novel mechanism that TNF-a participates in the pathology of OP in SLE patients and suggested a new approach to increase osteoblastic differentiayion for the treatment of OP in SLE patients.