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1.
Figure 5

Figure 5. RAW 264.7 differentiated into TRAP+, multinucleated osteoclastic-like cells in response to exogenous RANKL or co-cultured with LPS-stimulated PDL cells. From: A dominant function of p38 Mitogen Activated Protein Kinase signaling in Receptor Activator of Nuclear Factor-κB Ligand expression and osteoclastogenesis induction by A. actinomycetemcomitans and E. coli LPS.

Top panel depicts RAW 264.7 cells only (5 × 104) cultured for 6 days without or with RANKL (50ng/mL) and RANKL/OPG (100ng/mL). Middle panel indicate co-culture experiments where vehicle control or LPS (1 μg/mL) treated mPDL cells were stimulated for 72h, then 5 × 104 cells were added to each well containing 5 × 104 RAW 264.7 cells. Co-cultures were maintained for 6 days, both in the presence and absence of OPG (100ng/mL) (right panels). All images are representative of three independent experiments.

Carlos Rossa, et al. J Periodontal Res. ;43(2):201-211.
2.
Figure 3

Figure 3. RANKL protein expression by LPS-stimulated PDL cells is dependent on p38 MAPK activity. From: A dominant function of p38 Mitogen Activated Protein Kinase signaling in Receptor Activator of Nuclear Factor-κB Ligand expression and osteoclastogenesis induction by A. actinomycetemcomitans and E. coli LPS.

PDL cells grown on 6-well plates were de-induced for 12 hours in culture medium containing 0.3% FBS and then stimulated with LPS from either E.coli or A. actinomycetemcomitans for 72 hours. The specific inhibitor for p38 MAPK (SB203580; 10 μM) was added to the culture medium 30 minutes before stimulation with LPS (1 μg/mL). (A) Western blot analysis of RANKL expression from PDL whole cell lysates. Positive control for RANKL expression is cell lysates from the human prostate cancer cell line (PC-3). (B) Density analysis of data from three independent experiments shows significant inhibition of LPS-induced RANKL protein by SB203580 (* p<0.05).

Carlos Rossa, et al. J Periodontal Res. ;43(2):201-211.
3.
Figure 4

Figure 4. LPS-stimulated PDL cells stimulate osteoclastogenesis, which is regulated by p38 MAPK pathway. From: A dominant function of p38 Mitogen Activated Protein Kinase signaling in Receptor Activator of Nuclear Factor-κB Ligand expression and osteoclastogenesis induction by A. actinomycetemcomitans and E. coli LPS.

Stimulation with RANKL induces RAW 264.7 cells to differentiate into multinucleated TRAP+ cells, whereas pre-treatment with OPG inhibits this effect *Indicates significant (p<0.05) difference from unstimulated cells and #indicates a significant decrease on the number of osteoclasts with OPG treatment (A). mPDL cells were stimulated with LPS from E. coli and A. actinomycetemcomitans Y4 and cultured 3 days. These cells were then co-cultured with RAW 264.7 cells for an additional 6 days and the number of multinucleated, TRAP+ cells was counted. Stimulation with LPS increased significantly the number of TRAP+ cells and this effect was inhibited by OPG *Indicates significant (p<0.05) difference from unstimulated cells and #indicates a significant decrease on the number of osteoclasts with OPG treatment. !Indicates p=0.056 for the significance of the decrease on the number of osteoclasts with OPG treatment in A. actinomycetemcomitans Y4 LPS-stimulated cells. (B). Co-culture of RAW cells with stable PDL cell lines over-expressing dominant negative mutants of MKK3 (MKK3dn-PDL) and MKK6 (MKK6dn-PDL), upstream activators of p38 MAPK, significantly decreased the number of TRAP+ cells *Indicates significant (p<0.05) difference from non-transfected PDL cells (C). Bar graphs indicate mean ± standard deviation of number of TRAP+, multinucleated cells counted in each well.

Carlos Rossa, et al. J Periodontal Res. ;43(2):201-211.
4.
Figure 1

Figure 1. Time course of RANKL mRNA expression induced by E.coli and A. actinomycetemcomitans LPS in PDL cells. From: A dominant function of p38 Mitogen Activated Protein Kinase signaling in Receptor Activator of Nuclear Factor-κB Ligand expression and osteoclastogenesis induction by A. actinomycetemcomitans and E. coli LPS.

RT-PCR shows that RANKL mRNA expression induced by LPS is biphasic, reaching an early peak after 4 h of stimulation and reached the maximum after 18 to 24 hours (A and B). E. coli LPS was a more potent inducer of OPG expression (A), which was already noticeable after 2 h of stimulation and remained essentially constant throughout the 24 h period. A. actinomycetemcomitans Y4 LPS increased OPG mRNA modestly, and this effect was delayed, since it took 18 hours of stimulation to be more evident. Stimulation for periods longer than 24 h did not induce further increases on either RANKL or OPG mRNA expression (data not shown). Also, we did not observe noticeable levels of RANKL mRNA expression by PDL cells in the absence of any stimulation (data not shown). Representative images of three independent experiments.

Carlos Rossa, et al. J Periodontal Res. ;43(2):201-211.
5.
Figure 2

Figure 2. p38 MAPK regulates preferentially RANKL in LPS-stimulated PDL cells. From: A dominant function of p38 Mitogen Activated Protein Kinase signaling in Receptor Activator of Nuclear Factor-κB Ligand expression and osteoclastogenesis induction by A. actinomycetemcomitans and E. coli LPS.

mPDL cells were grown to near confluency and de-induced in media containing 0.3% FBS for 8 h. The specific inhibitor for p38 MAPK (SB203580) was added to the culture medium at 10 μM 30 minutes before the 18 h stimulation with 1 μg/mL of LPS from E.coli and A. actinomycetemcomitans LPS. Total RNA was harvested and RT-PCR was performed and quantitated using GelDoc System. Results indicate that inhibition of p38 MAPK reduced LPS-induced RANKL mRNA expression, especially after E.coli LPS stimulation (A). No significant regulation of OPG mRNA expression was observed following p38 inhibition (B). The decrease on RANKL expression was sufficient to reduce the RANKL:OPG ratio (C). Figures are representative of three independent experiments and bar graphs indicate mean ± standard deviation of normalized fold changes of normalized gene expression. *Indicates significant difference (p<0.05) on mRNA expression in comparison to unstimulated cells. # indicates significant difference (p<0.05) on mRNA expression in comparison to LPS treated cells.

Carlos Rossa, et al. J Periodontal Res. ;43(2):201-211.

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