Figure 1.
Effect of the PKC activator PMA on AP-1, c-fos or c-jun transcription in IVD cells.
(A) AP-1 was transfected with the pGL4.74 vector into NP cells, and the cells were stimulated with PMA (200 nM) for 24 h. NC = negative control, (= 1.0 fold); PC = positive control. (B) Effect of activation or inhibition of the PKC pathway on AP-1 promoter activity in NP cells. (C,D) c-fos (C) or c-jun (D) was transfected with the pGL4.74 vector into NP cells, AF cells, and AP-1 reporter stable cells, and the cells were stimulated with PMA (10, 100 or 200 nM) for 24 h. *P < 0.05 indicates significant differences between groups. Error bars represent the standard deviation (SD). n.s. = not significant.
Figure 2.
PMA enhanced c-fos expression in NP cells.
(A) c-fos was transfected with the pGL4.74 vector into NP cells, and the cells were stimulated with PMA (200 nM) for 6–24 h (left panel). In other experiments, c-fos was transfected with the pGL4.74 vector into NP cells, and the cells were stimulated with various concentrations of PMA (0, 10, 100, and 200 nM) for 12–24 h (right panel). (B) Effect of activation or inhibition of the PKC pathway on c-fos promoter activity in NP cells. *P < 0.05 between groups. Error bars represent SD. n.s. = not significant. (C) Detection of c-fos protein expression by immunofluorescence microscopy. After 1-day of serum deprivation, NP cells were incubated in serum-free medium containing PMA. NP cells were cultured with or without 200 nM PMA for 24 h, fixed, and stained with an antibody against c-fos. Left: cells stained with antibody to c-fos. Middle: cells stained with DAPI to identify healthy nuclei, (blue). Right: cells stained with antibody to c-fos and with DAPI. Scale bar= 50 μm (original magnification 20×). (D) Detection of c-fos (left) and c-jun (right) protein by western blot analysis after treatment with PMA (0–200 nM) for 24 h. Densitometry analyses were performed to quantify the levels of c-fos and c-jun protein 24 h after PMA treatment, after normalization to the level of β-actin. *P < 0.05 indicates significant differences between groups.
Figure 3.
The expression of c-fos in IVD.
(A) Sagittal sections from 3-week-old (a-d), 11-week-old (e-h) rats and a mouse embryo (day 14.5 of gestation). Detection of c-fos expression by immunofluorescence microscopy. b and f: AF, c- d and g- h: NP, i- k: notchordal cell of embryonic mouse (Left: The cells stained with antibody to c-fos. Middle: The cells were stained with DAPI to identify healthy nuclei, shown in blue. Right: The cells were stained with an antibody to c-fos and with DAPI). Scale bar= 20 μm-100 μm (original magnification 4×- 40×). (B) Real-time RT-PCR analysis of c-fos mRNA levels in NP and AF tissue from 11-week-old rats (left panel); c-fos mRNA levels in NP tissue from 3-week-old rats and 11-week-old rats (right panel). (C) Basal activities of the c-fos promoter in NP cells and AF cells were determined by Dual-Luciferase assays. (D) Western blot analysis of c-fos expression in AF and NP cells. *P < 0.05 indicates significant differences between groups.
Figure 4.
Effect of PMA on MAPK regulation of c-fos expression in NP cells.
(A) ERK1, ERK2, p38, and JNK mRNA expression after exposure of NP cells to PMA (0-200 nM) for 24 h assessed by real-time RT-PCR. (B) Western blot analysis of ERK and p38 activation following treatment of NP cells with PMA. After 1-day of serum deprivation, NP cells were treated with vehicle (control) or 200 nM PMA. The same blots were stripped and reprobed with antibodies for total and phosphorylated proteins of ERK or p38. β-actin was detected as a loading control. (C) NP cells were transfected with the c-fos reporter plasmid. The transfected cells were then treated with PMA with or without the MAPK inhibitors PD98059 (PD, 30 or 50 μM), SKF86002 (SKF, 5 or 20 μM), SB202190 (SB, 1 or 10 μM), or SP600125 (SP, 1 or 10 μM) for 24 h. *P < 0.05 indicates significant differences between groups; n.s. = not significant.
Figure 5.
Effect of PMA on PKC regulation of c-fos expression in NP cells.
(A) Real-time RT-PCR analysis of PKCγ and PKCδ mRNA levels in NP cells treated with PMA (200 nM). (B) NP cells were cotransfected with c-fos and increasing concentrations of the WT-PKCγ or WT-PKCδ expression plasmids (100–500 ng). *P < 0.05 indicates significant differences between groups. n.s. = not significant. (C) Detection of c-fos protein expression by immunofluorescence microscopy. NP cells were transfected with or without WT- PKCγ or WT-PKCδ expression plasmids, fixed, and stained with an antibody against c-fos (green). Scale bar= 50 μm (original magnification 20×).
Figure 6.
Effect of c-fos on aggrecan (Agg) and collagen type II (Col2) expression and cell proliferation in NP cells.
(A) NP cells or stable AP-1 reporter cell were cotransfected with the Agg (400 ng) or Col2 (400 ng) reporter constructs and increasing concentrations of the WT-c-fos plasmid (100–500 ng), the DN-c-fos (100–500 ng) plasmid, or empty backbone. *P < 0.05 indicates differences between groups. Error bars represent the SD. (B) Detection of Agg protein expression by immunofluorescence microscopy. NP cells were cultured with (a) or without (b) WT-c-fos for 24 h, fixed, and stained with an antibody against Agg (green). Scale bar= 100 μm(C) Real-time-RT-PCR analysis of c-fos, Agg or Col2 expression in cells treated with vehicle (control) or a WT-c-fos expression plasmid. Total RNA was extracted from transfected cells and real-time RT-PCR was used to determine c-fos, Agg, Col2 and GAPDH RNA levels as an internal control. (D) The NP cells were treated with c-fos (1 ng/mL) in 96-well plates. Cell proliferation was evaluated using the MTT viability assay 12 or 24 h after treatment. n.s. = not significant.
Figure 7.
Proposed model for the regulation of c-fos expression by PKC signaling and MAPK signaling in NP cells.