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Regulation of PLAP-1 Expression in Periodontal Ligament Cells

S. Yamada1, Y. Ozawa1, M. Tomoeda1, R. Matoba2, K. Matsubara2, and S. Murakami1,*

1 Department of Periodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan; and
2 Taisho Laboratory of Functional Genomics, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0101, Japan


Figure 1
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  		Figure 1. Real-time RT-PCR analysis of PLAP-1 mRNA expression in the process of cytodifferentiation of human PDL cells. (A) ALPase activity of PDL cells. Human PDL cells were cultured in the presence of 10 mM ß-glycerophosphate and 50 µg/mL ascorbic acid. Triangle shows ALPase activity of PDL cells cultured in the presence of FGF-2 (20 ng/mL) from day 15 in the cultured period. Circles show ALPase activities of PDL cells cultured without FGF-2. The values are given as means ± standard deviations (SD) of triplicate assays. *P < 0.05, n = 3 vs. respective PDL cells on day 20. (B) Down-regulation of PLAP-1 mRNA of PDL cells by FGF-2. RNA from the same samples in panel A was reverse-transcribed into cDNA. Expression of specific mRNAs was detected by real-time PCR with primers specific for PLAP-1, Biglycan, Decorin, and HPRT. Results are presented as the ratio of the amount of each SLRP mRNA divided by HPRT mRNA. The values are given as means ± standard deviations (SD) of triplicate assays.

 

Figure 2
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  		Figure 2. Effects of various cytokines on PLAP-1 mRNA expression in PDL cells. After FCS deprivation for 48 hrs, human PDL cells were stimulated with indicated cytokines for another 48 hrs. Cells were harvested, and RNA was isolated for real-time RT-PCR analysis. Results are presented as the ratio of the amount of PLAP-1 mRNA divided by HPRT mRNA. The values are given as means ± standard deviations (SD) of quadruplicate assays. *P < 0.005, n = 4, compared with None.

 

Figure 3
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  		Figure 3. mRNA induction of PLAP-1 and mineralization-related genes in PDL cells by BMP-2. After FCS deprivation for 48 hrs, human PDL cells were stimulated with 100 ng/mL of BMP-2 for another 48 hrs. Cells were harvested, and RNA was isolated for RT-PCR analysis. The numbers of PCR cycles were: 24 for PLAP-1, 21 for Osteonectin, 37 for Osteopontin, 37 for Osteocalcin, 37 for Bone sialoprotein, and 27 for HPRT. The sizes of PCR products are shown on the right of each panel. Similar results were obtained in 3 separate experiments, and representative data are shown.

 

Figure 4
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  		Figure 4. Induction of PLAP-1 protein on PDL cells by BMP-2. (A) Alignment of N-terminal of PLAP-1, Biglycan, and Decorin protein. The peptide sequence for antigen to immunize rabbits is shown in the dark-shaded box. (B) Experiment I: Immunocytochemical detection of PLAP-1 protein on PDL cells stimulated with BMP-2. Human PDL cells cultured in the glass-bottomed dish were stimulated with the different concentrations of BMP-2 for 48 hrs. The cells were washed with PBS and incubated with the anti-PLAP-1 polyclonal antibody or pre-immune rabbit serum, followed by the biotinylated goat anti-rabbit IgG mAb and then with streptavidin-Alexa Fluor 488. (a) No BMP-2 stimulation. (b) BMP-2 stimulation (200 ng/mL). (c) BMP-2 stimulation (400 ng/mL). (d) BMP-2 stimulation (400 ng/mL), staining with pre-immune rabbit serum. Experiment II: Dose-dependent inhibition of PLAP-1 staining with the PLAP-1 antigenic peptide. The anti-PLAP-1 polyclonal antibody was pre-incubated with different concentrations of the PLAP-1 peptide before BMP-2-stimulated (in 400 ng/mL) PDL cells were stained. (e) No PLAP-1 peptide. (f) Pre-incubation with the PLAP-1 peptide (30 mg/1 mL of anti-PLAP-1 antibody). (g) Pre-incubation with the PLAP-1 peptide (15 mg/1 mL of anti-PLAP-1 antibody). Experiment III: No cross-reaction of the anti-PLAP-1 antibody to Decorin or to Biglycan. The anti-PLAP-1 antibody was pre-incubated with either recombinant Decorin or Biglycan before BMP-2-stimulated (in 400 ng/mL) PDL cells were stained. (h) No pre-incubation. (i) Pre-incubation with recombinant Decorin (30 mg/1 mL of anti-PLAP-1 antibody). (j) Pre-incubation with recombinant Biglycan (30 mg/1 mL of anti-PLAP-1 antibody). (k) Pre-immune rabbit serum staining. Representative data of 3 independent experiments are shown. Scale bars = 50 µm.

 




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