HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (7) Citing Articles via Google Scholar Google Scholar Articles by Fukushima, H. Articles by Okabe, K. Search for Related Content PubMed PubMed Citation Articles by Fukushima, H. Articles by Okabe, K.

Parathyroid-hormone-related Protein Induces Expression of Receptor Activator of NF-B Ligand in Human Periodontal Ligament Cells via a cAMP/Protein Kinase A-independent Pathway

¹ Department of Physiological Science and Molecular Biology and
² Department of Oral Growth and Development, Fukuoka Dental College, Tamura 2-15-11, Sawara-ku, Fukuoka 814-0193, Japan;

View larger version (26K): [in a new window]   Figure 1. Effects of PTHrP on TRAP+ cell formation in co-cultures of human PDL cells with mouse spleen cells. (A) Mouse spleen cells (1 x 105 cells/well) were co-cultured with PDL cells (1 x 105 cells/well) in the presence or absence of PTHrP (10–8 M), TGF-ß (10 ng/mL), or EGF (10 ng/mL) in the presence of Dex (10–7 M) for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in three independent experiments. *P < 0.01 vs. control cultures. (B) Mouse spleen cells (1 x 105 cells/well) were co-cultured with PDL cells (1 x 105 cells/well) in various concentrations of PTHrP together with Dex for 10 days. TRAP+ cells were counted and expressed as the number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in three independent experiments. *P < 0.01 vs. control cultures. (C) Microscopic view of TRAP+ cells. Resorption areas were stained with Mayer’s hematoxylin (lower panels). Bar = 100 µm. (D) Human PDL cells were treated with PTHrP (10–8 M) in the presence of Dex for the indicated time. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (E) Co-cultures were pre-treated with or without either OPG (100 ng/mL) or sRANK (100 ng/mL) for 30 min, followed by incubation with PTHrP (10–8 M) in the presence of Dex for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PTHrP-treated cultures.

View larger version (42K): [in a new window]   Figure 2. Inhibition of the cAMP/PKA signaling pathway did not inhibit PTHrP-induced TRAP+ cell formation in co-cultures. (A) Co-cultures were pre-treated with or without Rp-cAMP (100 µM), H89 (1 µM), or PKI (1 µM) for 30 min, followed by incubation with either PTHrP (10–8 M) or PGE2 (10–8 M), together with Dex (10–7 M), for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PGE2-treated cultures. (B) Microscopic view of TRAP+ cells. Bar = 100 µm. (C) Human PDL cells were pre-treated with PKI (1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (D) Human PDL cells were pre-treated with or without PKI (0.1, 1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) or PGE2 (10–8 M) for 20 min together with Dex. Total cell lysates were used for PKA assay, with the PepTag PKA assay kit. Similar results were obtained in 3 independent experiments. (E) PKA activity was measured by densitometry. Each column indicates the relative value of phosphorylation by PKA vs. the intensity of the phosphorylation signal without PTHrP. *P < 0.01 vs. PTHrP or PGE2-treated cultures. (F) Co-cultures of mouse bone marrow cells (1 x 105 cells/well) and mouse calvaria cells (1 x 105 cells/well) were pre-treated with or without H89 (1 µM) or PKI (1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) for 5 days. TRAP+ multinucleated cells (MNCs) were counted. Data shown are the number of TRAP+ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. (G) Microscopic view of TRAP+ cells and MNCs. Bar = 100 µM.

View larger version (24K): [in a new window]   Figure 3. PKC pathway is partially involved in PTHrP-induced TRAP+ cell formation in co-cultures. (A) Co-cultures of mouse spleen cells and human PDL cells were pre-treated with or without PD98059 (10 µM) (ERK inhibitor), NS-398 (10 µM) (COX2 inhibitor), SB203580 (10 µM) (p38 MAPK inhibitor), and Ro-32-0432 (1 µM) (PKC inhibitors) for 30 min, followed by incubation with PTHrP (10–8 M) together with Dex for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PTHrP-treated cultures. (B) Human PDL cells were pre-treated with or without SB203580 (10–7, 10–6 M) for 30 min, followed by incubation with PTHrP (10–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (C) We cultured mouse bone marrow cells (BMCs) with M-CSF for 3 days to prepare bone marrow macrophages (BMMs). BMMs were pre-treated with or without SB03580 (10–7, 10–6 M) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 3 days. Data shown are number of TRAP+ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. RANKL-treated cultures. (D) Co-cultures of mouse spleen cells and human PDL cells were pre-treated with or without Ro-32-0432 (0.1 and 1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) together with Dex for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PTHrP-treated cultures. (E) Human PDL cells were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (F) We cultured mouse BMCs with M-CSF for 3 days to induce BMMs. BMMs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 3 days. Mouse BMCs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 5 days. Data shown are number of TRAP+ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. RANKL-treated cultures. (G) Mouse BMCs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with M-CSF (100 ng/mL) for 3 days. After 3 days, cells were stained with FITC-conjugated anti-c-fms antibody, and c-fms+ cells were counted by flow cytometry (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. M-CSF-treated cultures.

View larger version (32K): [in a new window]   Figure 4. Schematic representation of PTHrP-induced osteoclast formation. PTHrP induces RANKL expression in PDL cells by a mechanism independent of the cAMP/PKA signaling pathway. PKC contributes to induction of RANKL mRNA induced by PTHrP in PDL cells as well as regulation of osteoclast precursor cell number. PGE2 induces RANKL expression in PDL cells via the cAMP/PKA signaling pathway.