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Dental Pulp Fibroblasts Contain Target Cells for Lysophosphatidic Acid

¹ Department of Oral Surgery, Vienna Medical University, Waehringerstraße 25a, A-1090 Vienna, Austria; and
² Ludwig Boltzmann Institute of Oral Implantology, Vienna, Austria;

View larger version (32K): [in a new window]   Figure 1. Expression profile of lysophosphatidic acid receptors LPA1, LPA2, and LPA3. LPA receptor expression profile in dental pulp fibroblasts (DPF). RNA was extracted and subjected to RT-PCR analysis. Amplification products were separated on 1.5% agarose gels, stained with ethidium bromide, and photographed. Lanes #1-6 represent amplification products of individual preparations of DPF.

View larger version (45K): [in a new window]   Figure 2. Effects of LPA on 3[H]-thymidine incorporation of DPF. (A) Dose-response curve of the indicated concentrations of LPA on 3[H]-thymidine incorporation of DPF. The Fig. shows the mean and standard deviation of results from 6 independent preparations, each performed in quadruplicate. *P < 0.05 and **P < 0.01 vs. unstimulated cells. (B) Effects of inhibitors of MAPK signaling U0126, SB203580, and SP600125 on 3[H]-thymidine incorporation by DPF of 6 individual preparations. Results are shown as mean and standard deviation. *P < 0.05 and **P < 0.01 vs. LPA alone. (C) Staining pattern of the nuclear antigen Ki67 in DPF incubated for 24 hrs with and without LPA at 10 µM. (D) Relative number of Ki67-positive cells in the indicated cultures. Data are means and standard deviation of results from triplicates of one donor. *P < 0.05.

View larger version (54K): [in a new window]   Figure 3. Effects of LPA on MAPK phosphorylation in DPF. Serum-starved DPF were exposed to LPA at a concentration of 10 µM for 5, 15, 45, and 135 min. Cell lysates were separated on a 10% SDS-PAGE and blotted onto a nitrocellulose membrane. Phosphorylated (pERK, pp38 MAPK, and pJNK) and unphosphorylated MAPKs were detected by Western blot analysis.

View larger version (71K): [in a new window]   Figure 4. Effects of LPA on alkaline phosphatase activity and expression of differentiation markers. (A) DPF were stimulated for 72 hrs with bone morphogenetic protein-7 (BMP-7) as indicated under serum-free conditions and stained for alkaline phosphatase activity where positive cells appear blue (AP+). (B) Amount of alkaline phosphatase activity per microscopic field, normalized to unstimulated controls. Data are given as means and standard deviation from results of two random selected preparations, each performed in triplicate. *P < 0.05 vs. unstimulated control; **P < 0.01 between marked groups. (C) Representative picture of a microscopic field that shows alkaline-phosphatase-positive cells given in blue. (D) RT-PCR analysis of mRNA isolated from DPF incubated with LPA for 72 hrs.