Table 1.
Clinical Finding in 50 Patients with LDH.
Table 2.
Primer Sequences for PCR.
Fig 1.
The expression of FOXC2 in human nucleus pulposus tissue.
(A) The expression of FOXC2 exhibited significantly higher levels in 4 degenerative nucleus pulposus tissue compared to 4 idiopathic scoliosis nucleus pulposus tissue. (B) The correlation between the expression of FOXC2 and Pfirrmann scores of disc degeneration grade. n = 36,r = 0.881,p<0.05. (C) RT-PCR analysis of FOXC2 in the human nucleus pulposus tissue of 36 patients. Error bars represent SD. As compared with control, ** indicates p<0.01.
Fig 2.
Immunofluorescence characterization and real-time quantitative PCR validation of primary cultured human NP cells.
(A) Fluorescence microscopy images showing collagenase type II was observed in NP cells. Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40×objective. (B) Fluorescence microscopy images showing aggrecan was observed in NP cells. Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40×objective. (C) Real-time RT-PCR analysis of negative NP cell marker gene collagen type I in NP cells, NP and AF. Real-time RT-PCR analysis was performed in triplicate and the expression levels of collagenase type I mRNAs were normalized to GAPDH mRNAs. (D) Real-time RT-PCR analysis of NP cell marker gene collagen type II in NP cells, NP and AF. Real-time RT-PCR analysis was performed in triplicate and the expression levels of collagenase type II mRNAs were normalized to GAPDH mRNAs. Error bars represent SD. As compared with control, ** indicates p<0.01.
Fig 3.
Overexpression of FoxC2 promotes NP cells growth.
(A) Immunohistochemical staining of NP cells against Ki-67. Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40×objective, and had Ki-67-positive percentages in cultured NP cells 48h after transfection with LentiFoxC2 or LentiGFP or no transfection. (B) Real-time RT-PCR analysis of Ki-67 mRNA expression in NP cells after transfection of LentiFoxC2 for 0h, 24h, 48h or 72h. (C) Growth of NP cells was shown after transfection with 50 nmol/L of LentiFoxC2 or LentiGFP or no transfection. The growth index was assessed at 1, 2, 3, 4, and 5 days. Error bars represent SD. As compared with control, * indicates p<0.05, ** indicates p<0.01.
Fig 4.
Immunohistochemical staining of NP cells against FoxC2.
(A) After 1-day serum deprivation, NP cells were treated 50 ng/ml or 100 ng/ml BMP-7 in serum-free medium for 2h. NP cells were Immunohistochemical stained against FoxC2. Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40×objective. FoxC2-positive percentages in cultured NP cells 2h after treated with 50 ng/ml or 100 ng/ml BMP-7 or control was under analysis. (B) NP cells were stimulated by BMP-7 (100 ng/ml) for the indicated periods of time. And then expression level of FoxC2 was measured by real-time RT-PCR. (C) NP cells were stimulated by 10, 50, and 100 ng/ml BMP-7 for 2h or NP cells were stimulated by BMP-7 (100 ng/ml) for the indicated periods of time. Cell lysates were then prepared and analyzed by western blotting with specific anti-FoxC2 antibody. β-actin was used as a control for normalization. Error bars represent SD. As compared with control, * indicates p<0.05, ** indicates p<0.01.
Fig 5.
The interactional promotion effect between FoxC2 and BMP-7 signaling pathway.
(A) FoxC2 mRNA expression levels were examined by real-time PCR in the presence and absence of SiRNA-Smad1 the BMP inhibitor noggin and Scramble. (B) NP cells were treated 100 ng/ml BMP7, 100 ng/ml noggin for 2h and the total RNA was extracted to perform real-time PCR of FoxC2 genes (C) Smad1/5/8 phosphorylation Protein expression in NP cells was transfected with 50 nmol/L of Lenti FoxC2 or Lenti GFP or no transfection. (D) Expression levels of BMP-7 were examined by real-time PCR after transfection of 50 nmol/L of Lenti FoxC2 or Lenti GFP or no transfection. Error bars represent SD. As compared with control, * indicates p<0.05, ** indicates p<0.01.
Fig 6.
FoxC2 enhances the compensatory anabolic gene expression effect by BMP-7.
NP cells were treated 100 ng/ml BMP7, 100 ng/ml noggin or 50 nmol/L of LentiFoxC2 for 2h. After stimulation, cells were harvested and the total RNA was extracted to perform real-time PCR of (A) collagen type-II genes and (B) aggrecan. (C) NP cells were stimulated by 50 nmol/L of Lenti FoxC2, BMP-7 (100 ng/ml), and cocktail of Lenti FoxC2 and BMP-7 for 2h. After stimulation, cells were harvested and the total RNA of Noggin was extracted to perform real-time PCR. As compared with control, ***p<0.01, *p<0.05, and **p<0.01. (D) Cell lysates were then prepared and analyzed by western blotting with specific anti-Noggin antibody. β-actin was used as the control for normalization. Error bars represent SD. As compared with control, * indicates p<0.05, ** indicates p<0.01.
Fig 7.
Schematic diagram of FoxC2-mediated biological effects on the BMP7-SMAD signaling pathways.
BMP/Smad signaling pathway can upregulate FoxC2 expression in the NP cells. FoxC2 inhibits the production of noggin and enhances BMP7-mediated anabolism, what was a positive feedback for maintaining signal activation.