Porphyromonas gingivalis lipids inhibit osteoblastic differentiation and function

Abstract

Porphyromonas gingivalis produces unusual sphingolipids that are known to promote inflammatory reactions in gingival fibroblasts and Toll-like receptor 2 (TLR2)-dependent secretion of interleukin-6 from dendritic cells. The aim of the present study was to examine whether P. gingivalis lipids inhibit osteoblastic function. Total lipids from P. gingivalis and two fractions, phosphoglycerol dihydroceramides and phosphoethanolamine dihydroceramides, were prepared free of lipid A. Primary calvarial osteoblast cultures derived from 5- to 7-day-old CD-1 mice were used to examine the effects of P. gingivalis lipids on mineralized nodule formation, cell viability, apoptosis, cell proliferation, and gene expression. P. gingivalis lipids inhibited osteoblast differentiation and fluorescence expression of pOBCol2.3GFP in a concentration-dependent manner. However, P. gingivalis lipids did not significantly alter osteoblast proliferation, viability, or apoptosis. When administered during specific intervals of osteoblast growth, P. gingivalis total lipids demonstrated inhibitory effects on osteoblast differentiation only after the proliferation stage of culture. Reverse transcription-PCR confirmed the downregulation of osteoblast marker genes, including Runx2, ALP, OC, BSP, OPG, and DMP-1, with concurrent upregulation of RANKL, tumor necrosis factor alpha, and MMP-3 genes. P. gingivalis total lipids and lipid fractions inhibited calvarial osteoblast gene expression and function in vivo, as determined by the loss of expression of another osteoblast differentiation reporter, pOBCol3.6GFPcyan, and reduced uptake of Alizarin complexone stain. Finally, lipid inhibition of mineral nodule formation in vitro was dependent on TLR2 expression. Our results indicate that inhibition of osteoblast function and gene expression by P. gingivalis lipids represents a novel mechanism for altering alveolar bone homeostasis at periodontal disease sites.

FIG. 1.

Electrospray-MS characterization of total lipids of P. gingivalis and the highly enriched phosphoethanolamine dihydroceramide (PE DHC) or phosphoglycerol dihydroceramide (PG DHC) lipid fractions. Lipids were prepared and characterized by electrospray-MS as described in Materials and Methods. The upper frame shows the negative ions recovered in the total lipid extract of P. gingivalis lipids, the middle frame shows the ions recovered in the PE DHC lipid fraction, and the bottom frame shows the ions recovered in the PG DHC lipid fraction. A description of the MS interpretation of these ions and the structural deduction can be found in Nichols et al. (34). The PG DHC lipids include three component lipids that produce negative ions of 960, 946, and 932 m/z and, by analogy, the PE DHC lipids produce negative ions of 705, 691, and 677 m/z (34). Note that the dominant-negative ions previously reported for lipid A species of P. gingivalis (1,450, 1,690, and 1,770 m/z) (10) are not recovered in these lipid isolates.

FIG. 2.

Effects of P. gingivalis total lipids on the mineralized nodule formation and osteoblastic differentiation at day 21 calvarial osteoblast cultures. Cells were cultured with increasing concentrations of P. gingivalis total lipids for the 21-day culture period. Control cultures were not exposed to bacterial lipids. (A) Mineralized nodule formation revealed by von Kossa staining showed that the inhibitory effect of P. gingivalis total lipids was concentration dependent. (B) The inhibition of osteoblastic differentiation revealed by fluorescence of pOBCol2.3GFP showed that the inhibitory effect of P. gingivalis total lipids was also concentration dependent. Scale bar, 10 mm. (C) Quantitation of mineralized nodule formation or fluorescence expression of pOBCol2.3GFP in control osteoblast cultures or cultures exposed to increasing levels of P. gingivalis total lipids. Both von Kossa staining and GFP expression were quantified by summing pixels indicating either von Kossa staining or GFP fluorescence. The results in Fig. 2C represent the mean ± the standard deviation for n = 2 trials. One-factor analysis of variance showed significant differences between the treatment categories: von Kossa staining (P, 0.00181) and GFP fluorescence (P, 0.00007).

FIG. 3.

Effects of P. gingivalis total lipids on cell viability, apoptosis, and proliferation. The concentration of P. gingivalis total lipids was 1,250 ng/ml in the indicated cultures. (A) Cell viability indicated by EthD-1 staining at day 7 showed no significant difference in cell death between lipid-treated and control osteoblast cultures (126.5 ± 15.6 versus 107.6 ± 12.0; P = 0.35). (B) Apoptosis observed at day 7 with annexin V staining, followed by flow cytometry, showed no significant difference between lipid-treated and control osteoblast cultures (5.9% ± 0.5% versus 6.3% ± 0.5%; P = 0.59). (C) Total cell counts per well at day 7 showed no significant difference in total cell recovery between lipid-treated and control osteoblast cultures ([306.6 ± 15.8] × 10³ versus [263.0 ± 25.3] × 10³; P = 0.18). (D) DNA quantitation showed no significant differences in total DNA recovered from control and lipid treated osteoblasts for day 7 cultures (4.3 ± 0.2 μg/ml versus 4.5 ± 0.5 μg/ml; P = 0.79), as well as day 21 cultures (8.5 ± 0.3 μg/ml versus 8.3 ± 0.4 μg/ml; P = 0.71).

FIG. 4.

Inhibition of the mineralized nodule formation by P. gingivalis total lipids depending on the week of lipid treatment in culture. (A) Cells were exposed to P. gingivalis total lipids (1,250 ng/ml) only during the specified culture intervals, including the first week only, the second week only, the third week only, the first and second week, the second and third week, or all 3 weeks. Control cultures were not exposed to P. gingivalis lipids. (B) Quantitation of mineralized nodule formation stained by von Kossa of day 21 cultures. The magnitude of inhibitory action of P. gingivalis total lipids was dependent on the stage of growth in cultures. Control cultures and cultures treated for the first week with lipids (&) were not significantly different from each other but both were significantly different from all other treatment groups, as determined by Scheffe contrasts among pairs of means. Cultures treated with lipids for the second week, the third week, or the combined first and second weeks (*) were not significantly different from each other but were significantly different from the remaining cell culture categories. Cultures treated for the second and third weeks or for all weeks (#) were not significantly different from each other but were significantly different from the remaining cell culture categories.

FIG. 5.

Effects of P. gingivalis total lipids on gene expression determined by real-time RT-PCR. Cells were cultured with P. gingivalis total lipids (1,250 ng/ml) or control medium for the entire 21-day culture period. Real-time RT-PCR was performed on total RNA isolated from day 21 cultures. Changes in gene expression, either as increased or decreased gene expression, in lipid-treated cultures are expressed as the fold change versus control cultures. Significant up or down expression of each gene was evaluated against parallel control cultures by using the Student t test, and P values are depicted opposite each histogram bar. Only the expression of the Col1a1 and RANKL genes was not significantly affected by lipid treatment of osteoblast cultures.

FIG. 6.

Effects of P. gingivalis lipid preparations on osteoblast function and gene expression in vivo. Mice that express the osteoblastic GFP reporter, pOBCol3.6GFPcyan, were lightly sedated, and the indicated lipid preparations (5 μg in 50 μl of PBS) were administered as a subcutaneous injection to the surfaces of the calvaria (two mice were treated with each lipid preparation, including the total lipid extract, PG DHC lipids and PE DHC lipids of P. gingivalis). Control mouse calvaria received only PBS. Mice were administered Alizarin complexone on day 6 and were sacrificed on day 7. Calvaria were cryosectioned and evaluated by fluorescence microscopy for osteoblast reporter (blue) and alizarin complexone (red) fluorescence (upper row). The right side of each calvarium section represents the cerebral cavity, and the left side represents the surface treated with the indicated lipid preparation. The inserts within each frame of the upper row depict magnified images of lipid-treated (left) and untreated (right) surfaces of each calvarium section. The exact same sections depicted in the upper row were then stained with hematoxylin and eosin (H&E), and photomicrographs were obtained (lower row). Multiple sections of each calvarium specimen were evaluated for fluorescence changes, and the images depicted above are representative of those observed in the replicate sections.

FIG. 7.

Engagement of TLR2 by P. gingivalis total lipids and major fractions of P. gingivalis lipids. Calvarial osteoblast cultures were established from the WT CD-1 or TLR2-null (TLR2^−/−) mice and treated with P. gingivalis total lipids, LPS, or two major P. gingivalis lipid fractions (PG DHC or PE DHC lipids) for the entire 21-day culture period. The von Kossa staining was performed at day 21 to reveal mineralized nodule formation in cultures. Control cultures did not receive P. gingivalis lipids, LPS, or lipid fractions. The inhibitory action of P. gingivalis total lipids was TLR2 dependent, and the PG DHC fraction, but not the PE DHC fraction, demonstrated TLR2-dependent inhibition of osteoblast mineralized nodule formation.