Study Of The Mechanism That TNF-α Inhibits Osteogenic Differentiation Of Bone Marrow-derived Mesenchymal Stem Cells Through MicroRNAs During Osteoporosis

Background Postmenopausal osteoporosis(PMOP) is one of the most common types of osteoporosis. The pathological characteristics of PMOP includes low bone mass, damage of micro-structure and decrease of mechanical property, resulting in high incidence of non-stress fracture. PMOP is becoming the major degenerative disease threatening the healthy and life of aged female. The fundamental mechanism of osteoporosis is the imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Previous studies focused on the terminal differentiated functioning cells. However, bone marrow-derived mesenchymal stem cells(BMSCs), the origin of osteoblast progenitors, have been recently found to play crucial role in bone development, remodeling and regeneration. Osteogenic differentiation capacity of BMSCs is decreased during osteoporosis, resulting in defect of bone formation. However, the mechanism of abnormal BMSC osteogenic differentiation remains elusive.Recent studies have shown that estrogen indirectly function in BMSCs. Elevated inflammatory factors secreted by T cells after estrogen-deficiency are important etiological factors of osteoporosis. Tumor necrosis factor-alpha(TNF-α) is a major inflammatory factor during osteoporosis. Inhibition of TNF-α by gene knock-out or specific antibody efficiently prevents estrogen-deficiency-associated osteoporosis. Moreover, TNF-α has been shown to inhibit osteogenic differentiation of BMSCs. Since the function and downstream signaling of TNF-α is complicated, the molecular mechanism of TNF-α to inhibit BMSC differentiation remains unknown. As important regulators of gene expression, micro RNAs(mi RNAs) are recently found to play important role in differentiation of BMSCs. A number of mi RNAs control osteogenic differentiation of BMSCs by targeting master transcription factors and signaling molecules. Moreover, differential expression of mi RNAs has been observed in numbers of age-related degenerative diseases including tumor, cardiovascular diseases and nerve degenerative diseases. Specific mi RNAs might take part in the pathogenesis and development of diseases. But whether mi RNA expression in BMSCs is affected during osteoporosis and whether the change of mi RNA expression is related with osteoporosis are needed to be answered.Purpose Our study aims to take advantage of technologies of mi RNA micro-array, molecular biology and cell biology to explore the molecular mechanism of TNF-α to inhibit BMSC osteogenic differentiation and confirm the function of specific mi RNAs. This study would provide experimental evidence to improve our understanding of etiology of PMOP and explore novel targets of osteoporosis treatment.Methods 1. PMOP mice model was established by ovariectomy(OVX), and micro CT and histology assay were used to determine the change of bone mass. 2. Based on in vitro osteogenic differentiation assay, osteogenic differentiation capacity of OVX BMSCs was determined by alizarin red staining and realtime RT-PCR analysis of osteogenesis makers. 3. TNF-α levels of OVX mice were determined by ELISA analysis. 4. Downstream signaling pathways activated by TNF-α were determined by Western blot assay. 5. si RNA was used to knockdown specific signaling molecules in loss-of-function assays. 6. mi RNA micro-array was used to screen differentially expressed mi RNAs between BMSCs derived from OVX and Sham surgery(SHAM) mice. 7. Gain- and loss-of-function analysis based on mi RNA mimics and inhibitor were performed to determine the function of mi R-3077-5p and mi R-705 in BMSCs. 8. Bioinformatics analysis was performed to find the candidate target gene of mi RNA. 9. Western blot assay and luciferase reporter assay were used to determine whether a mi RNA directly binds target m RNAs. 10. In vivo estradiol injection and in vitro estrogen treatment were used to explore whether mi R-3077-5p and mi R-705 are directly regulated by estrogen. 11. In vivo injection of recombinant TNF-α or TNF-α antibody was used to determine the function of TNF-α on mi R-3077-5p and mi R-705 expression and the downstream signaling pathway. 12. TNF-α in vitro treatment assay was used to investigate molecular signaling pathway of TNF-α to regulate mi R-3077-5p and mi R-705. 13. ROS in BMSCs was labeled by DCFH-DA, and detected with fluorescence microscope and flow cytometry. 14. Antioxidant NAC was used to determine the effect of ROS on BMSC osteogenic differentiation. 15. Immunochemistry was performed to determine the levels of Forkhead box-containing protein, O1 sub-family(FOXO1) in osteoporotic bone. 16. si RNA and overexpression lenti-virus were used to determine the function of FOXO1 in antioxidant defense of BMSCs. 17. Antagomir was injected in vivo to knockdown mi R-705 expression and determine the function and target gene of mi R-705 in vivo. 18. CHIP assay was used to determine whether NF-κB signaling pathway directly regulates transcription of mi R-705.Results 1. TNF-α inhibits osteogenic differentiation of BMSCs during osteoporosis. Three month after OVX, osteogenic differentiation capacity of BMSCs was decreased compared with Sham surgery group. TNF-α levels in serum were significantly increased after OVX. In vitro assay confirmed that redundant TNF-α inhibited BMSCs osteogenic differentiation. Further signaling pathway analysis confirmed that TNF-α did not trigger apoptosis pathway, while activate NF-κB pathway. Inhibition of NF-κB by Ikka si RNA effectively rescued osteogenic differentiation of OVX BMSCs, and blocked the inhibitory function of TNF-α on BMSC differentiation. 2. TNF-α directly inhibits osteogenic differentiation of BMSCs by up-regulating mi R-3077-5p. Mi RNA micro-array screening found that 10 mi RNAs were differentially expressed between SHAM and OVX BMSCs. Among them, the difference of mi R-3077-5p expression was the most significant. Further analysis found that mi R-3077-5p expression was increased in OVX BMSCs and bone tissue, which could be recovered by estradiol in vivo injection. But estradiol in vitro treatment promoted the expression of mi R-3077-5p. TNF-α robustly enhanced mi R-3077-5p expression, suggesting that estrogen indirectly regulates mi R-3077-5p expression through TNF-α. Moreover, mi R-3077-5p was highly expressed in bone tissue and significantly decreased during BMSC osteogenic differentiation. Functional analysis showed that mi R-3077-5p inhibited osteogenic differentiation of BMSCs by directly bind the m RNA 3’ untranslated region(UTR) of Runx2, a master transcription factor of osteogenesis. Knockdown of mi R-3077-5p efficiently improved the osteogenic differentiation defect of OVX BMSCs, while partly prevented the effect of TNF-α on BMSC differentiation. 3. TNF-α indirectly inhibits osteogenic differentiation of BMSCs through enhancing oxidative stress. In vitro and in vivo analysis confirmed that elevated TNF-α during osteoporosis enhanced ROS levels in BMSCs, leading to inhibition of BMSC osteogenic differentiation. Oxidative stress was resulted from defect of antioxidant defense mediated by SOD2 and CAT. FOXO1, a key regulator of antioxidant defense, was decreased in osteoporotic bone and BMSCs. Gain- and loss-of-function assay confirmed that FOXO1 promoted expression of Sod2 and Cat to suppress oxidative stress. TNF-α decreased the protein levels of FOXO1, leading to oxidative damage in OVX BMSCs. Overexpression of FOXO1 significantly alleviated oxidative damage caused by OVX and TNF-α, and recovered osteogenic differentiation capacity of BMSCs. 4. TNF-α leads to persistent oxidative damage by increasing mi R-705 expression TNF-α did not directly regulate transcription of Fox O1 m RNA. Based on bioinformatics analysis, gain-and-loss-of function assay and luciferase reporter system, mi R-705 was proved to directly target 3’UTR of Fox O1 m RNA to suppression its expression. In vivo and in vitro experiments confirmed that TNF-α increased mi R-705 expression. Knockdown of mi R-705 increased FOXO1 protein levels, Sod2 and Cat m RNA levels and suppressed oxidative damage. Signaling pathway analysis showed that TNF-α activated NF-κB pathway to promote nuclear translocation of P65 to activate mi R-705 transcription. Moreover, ROS also activated NF-κB to promote mi R-705 expression. Long-term TNF-α treatment activated a feed-forward stem-loop of NF-κB-mi R-705-FOXO1-ROS, resulting in persistent ROS damage and osteogenic differentiation defect of BMSCs.Conclusion 1. TNF-α plays important role in the defect of bone formation mediated by BMSCs during PMOP. TNF-α levels are elevated after estrogen deficiency. On the one hand, redundant TNF-α directly inhibits osteogenic differentiation of BMSCs by suppression the expression of RUNX2, a master transcription factor of osteogenesis. One the other hand, TNF-α indirectly inhibits osteogenic differentiation of BMSCs by suppressing FOXO1-mediated antioxidant defense. 2. A number of mi RNA are differentially expressed in BMSCs derived from OVX mice, and are important factors of osteogenic defect of BMSCs. TNF-α inhibits osteogenic differentiation of BMSCs by regulating the expression of specific mi RNAs. Suppression of mi R-3077-5p and mi R-705 efficiently blocks the inhibitory effect of TNF-α on BMSCs differentiation, leading to recovery of osteogenesis of osteoporotic BMSCs, suggesting abnormal post-transcriptional regulation mediated by mi RNAs plays crucial role in etiology and development of PMOP. 3. TNF-α activates NF-κB signaling pathway to enhance mi R-3077-5p expression. Redundant mi R-3077-5p binds the 3’UTR of Runx2 m RNA to decrease RUNX2 protein accumulation at post-transcriptional level, resulting in direct inhibition of osteogenic differentiation of BMSCs. 4. TNF-α activates mi R-705 transcription through NF-κB signaling pathway. Redundant mi R-705 targets 3’UTR of Fox O1 m RNA to inhibit FOXO1, a key regulator of SOD2 and CAT-mediated antioxidant defense, resulting in oxidative damage. Moreover, long-term TNF-α stimulation activates a feedforward stem-loop of NF-κB-mi R-705-FOXO1-ROS to persistently cause oxidative damage of BMSC-mediated bone formation.