Roles Of FOXC2 And BMP-7 In Disc Degeneration Disease And Mechanisms Involved

Degenerative disc disease(DDD) is thought to be a major cause of lower back pain which interferes with normal activities and impacts the ability to work. Although the process of DDD remains unclear, progressive breakdown of the extracellular matrix(ECM) is thought to be one of the most important causative factors. Changes in the structure of the ECM are associated with adverse genetic, biomechanical and nutritional factors. In the course of DDD, the breakdown of the ECM from age changes and adverse biomechanical loading can be enhanced by a genetically inferior matrix. Bone morphogenetic protein-7(BMP-7) is a growth factor that plays a major role in mediating anabolism and anti-catabolism of the intervertebral disc matrix and cell homeostasis. It is known that activation of the transcription factor Smad through the BMP/Smad signaling pathways is essential for enhance ECM repair and regeneration in NP cells.Forkhead box protein C2(Fox C2) is a member of the forkhead box(FOX) family of transcription factors. Fox C2 is reported to serve as a key regulator in diverse biological and pathological processes, including cell proliferation, differentiation and apoptosis. Following treatment of skeletal precursors with BMPs, Foxc2 may play a role in the early stage of chondrogenic differentiation, because increased expression of Foxc2 is observed in relation to SOX9, noggin and aggrecan. In osteoblasts, Fox C2 is a downstream target of BMPs and promotes cell proliferation and differentiation. A similar study has found that forced expression of FOXC2 in C2C12 cells results in upregulation of BMP-4. Overall, these results suggest that Foxc2 is a vital element of ECM secretion via facilitation of BMP signaling pathways and failure of proper Foxc2 transcript regulation may result in DDD. BMPs play an important role in regulating anabolic processes of the NP cells in which Fox C2 may have significant effect in DDD pathogenesis. However, the role Fox C2 may play in degenerative human intervertebral disc tissue and the relationship between Fox C2 and BMP-7 in nucleus pulposus(NP) cells remain to be elucidated. Therefore to investigate the the relationship between Fox C2 and degenerative human intervertebral disc, as well as whether Fox C2 play a functional role on the proliferation and anabolism of primary cultured rat NP cells. Specifically, understand the molecular mechanism between Fox C2 and BMP signaling pathways。It has a great significance in understand the disc degeneration disease and discover new treatment.Objective:FOXC2 is a member of the FOX family who has the winged helix transcription factor with a DNA-binding domain. Recent studies imply that BMP signaling is required for Foxc2 expression and that FOXC2 acts as a modulator of the pathway. Therefore, we studied the expression of FOXC2 in human degeneration disc tissue, and its relationship with NP cell proliferation. Also this study aims to investigate the presence and signaling mechanisms of Fox C2 in rat NP cells, in order to investigate the role of FOXC2 in the disc degeneration disease and discover new treatment.Method:1. The expression of FOXC2 pattern by real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR) analyses study on in human disc tissue, analysis of the correlation between FOXC2 expression and disc degeneration grade.2. Primary culture and phenotypic identification of rat NP cells. Immunofluorescence and real-time PCR were performed to investigate the collagen I, collagen II expression and aggrecan.3. The proliferation of NP cells was detected by CCK8 method after being plated and treated in different times of over expression of Fox C2 in NP cells. The expression of Ki-67,a mark of cell proliferation, were detected by real-time q RT-PCR and immunofluorescence to realize the influence of Fox C2 in rat NP cells.4. Transfections were carried out to measure the effect of Fox C2 on BMP-7-mediated extracellular matrix upregulation. Adenoviral knock-down of Smad1 was performed to investigate the mechanism of BMP-7-induced Fox C2 expression. Real-time q RT-PCR analyses were used to measure the relationship between Fox C2 and noggin. Also, we performed to detect the changes of m RNA expression level of collagen type I and collagen type II in the condition of BMP-7, Fox C2 and noggin.Results:1. Of the collected 36 DDD patients, 19 females and 17 males, the average age of these patients was range 33–57 years. FOXC2 expression in the degenerative NP tissue was significantly higher compared to the idiopathic scoliosis NP tissue as determined by real-time PCR. In human degenerative NP tissue, Fox C2 was markedly upregulated and positively correlated with increased disc degeneration. no significant difference was observed between samples from different herniation types or genders. However, expression of FOXC2 was positively correlated with the disc degeneration grade.2. Rat NP cells cultured in monolayer were round or polygonal with a granular cytoplasm. Collagen II and aggrecan expression in these cells were observed by immunofluorescence. Collagen type I had significantly lower expression levels in NP cells and NP tissue than in AF tissue, whilst Collagen II was found to have significantly higher expression levels in NP cells and NP tissue than in AF tissue.3. Induction of NP cell proliferation was confirmed by using cell counting kit-8 assay, immunocytochemistry and real-time q RT-PCR for Ki67. We transfected Lenti-Fox C2 in NP cells and transfected Lenti-GFP as the control. Transfecting Lenti-Fox C2 in NP cells significantly increased the expression of Ki-67. Also, transfecting Lenti-Fox C2 in NP cells induced the up-regulation of Ki-67 mRNA level as a time-dependent manner, and the Ki-67 m RNA level was significantly increased after 48 h and 72 h when compared to control. We used CCK-8 proliferation assay to compare the effects of Proliferation between Lenti Fox C2 and Lenti-GFP transfected to NP cells. There was a significant increase of cell proliferation in the Lenti-Fox C2 transfected NP cells compared with the control.4. We investigated whether BMP-7 could induce Fox C2 expression in rat NP cells. Immunofluorescence analysis confirmed this result, showing that BMP-7 treatment(50?100 ng/ml, 2 h) promoted the expression of Fox C2 protein, and higher concentrations of BMP-7 had a more significant effect compared with lower concentrations and the control. Fox C2 protein was induced by adding BMP-7 to the culture medium in a dose-dependent and time-dependent manner. Western blots over the 60-min time frame showed that Fox C2 was significantly up-regulated at the optimal concentration of 100ng/ml BMP-7. Furthermore, we found that Fox C2 overexpression induced the phosphorylation of Smad/1/5/8 obviously, which were known to be activated by BMPs. BMP-7 was positively upregulated whilst noggin was suppressed by Fox C2 overexpression.5. The real-time q PCR results demonstrated BMP-7 stimulated the expression of ECM genes. Specifically, Fox C2 had complementary effects on Collagen II and aggrecan expression increased when compared with controls. Combination of Fox C2 with BMP-7 further increased Collagen II and aggrecan gene expression above levels induced by BMP-7 alone. Concordant with our hypothesis, when the BMP signaling pathway was inhibited by Si RNA-smad1, this led to a significant downregulation of the anabolism effect that Fox C2 increased in NP cells, compared to in the presence of only in the presence of BMP-7.Conclusion:1. We identified that the transcription factor Fox C2 was over expressed in human degenerative discs. The m RNA expression level of Fox C2 was positively correlated with disc degeneration grade.2. We demonstrated that overexpression of Fox C2 increased the proliferation of NP cells.3. Importantly, BMP-7 induced Fox C2 expression, which was a positive feedback for maintaining signal activation.4. Upregulation of Fox C2 results in increased anabolic effects. Future studies are warranted to elucidate the relationship between Fox C2 and other signaling pathways and crosstalk between them. Combination therapy using BMP-7 and Fox C2 may be beneficial to the treatment of intervertebral disc degeneration.