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Infect Immun. Jun 1999; 67(6): 2841–2846.
PMCID: PMC96590

Inhibition of Osteoblastic Cell Differentiation by Lipopolysaccharide Extract from Porphyromonas gingivalis

Editor: J. R. McGhee

Abstract

Lipopolysaccharide from Porphyromonas gingivalis (P-LPS), an important pathogenic bacterium, is closely associated with inflammatory destruction of periodontal tissues. P-LPS induces the release of cytokines and local factors from inflammatory cells, stimulates osteoclastic-cell differentiation, and causes alveolar bone resorption. However, the effect of P-LPS on osteoblastic-cell differentiation remains unclear. In this study, we investigated the effect of P-LPS extract prepared by the hot-phenol–water method, on the differentiation of primary fetal rat calvaria (RC) cells, which contain a subpopulation of osteoprogenitor cells, into osteoblastic cells. P-LPS extract significantly inhibited bone nodule (BN) formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker, in a dose-dependent manner (0 to 100 ng of P-LPS extract per ml). P-LPS extract (100 ng/ml) significantly decreased BN formation to 27% of the control value and inhibited ALPase activity to approximately 60% of the control level on days 10 to 21 but did not affect RC cell proliferation and viability. P-LPS extract time-dependently suppressed the expression of ALPase mRNA, with an inhibitory pattern similar to that of enzyme activity. The expression of mRNAs for osteocalcin and osteopontin, matrix proteins related to bone metabolism, was markedly suppressed by P-LPS extract. Furthermore, P-LPS extract increased the expression of mRNAs for CD14, LPS receptor, and interleukin-1β in RC cells. These results indicate that P-LPS inhibits osteoblastic-cell differentiation and suggest that LPS-induced bone resorption in periodontal disease may be mediated by effects on osteoblastic as well as osteoclastic cells.

Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is an important periodontopathic bacterium. Its lipopolysaccharide (LPS), a major pathogenic component of the bacterial outer membrane, has multiple inflammatory actions and is involved in the destruction of periodontal tissues, including alveolar bone, the gingiva, and periodontal ligaments, in periodontal disease (19, 20, 28, 30, 32, 47, 49). LPS of P. gingivalis (P-LPS) stimulates the differentiation and activity of osteoclastic cells by mediating inflammatory cytokines and factors such as interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-α), and prostaglandin E2 (PGE2) and finally induces bone resorption (8, 20, 45, 58). LPS is also known to increase the release of inflammatory osteolytic factors from osteoblastic cells and to stimulate alveolar bone resorption by an indirect effect through the action on osteoblastic cells as well as a direct effect on osteoclastic cells (25, 36, 46). Physiological bone remodelling is controlled by a balance between bone formation and resorption. This balance is regulated by interaction between osteoblasts and osteoclasts and is mediated by calcitropic hormones, growth factors, and cytokines (10, 27, 33, 36, 45). Although the effect of LPS on osteoclasts and bone resorption and its mechanism of action (which involves binding with CD14, the LPS receptor) have been studied extensively by in vitro assays (2, 18, 19, 20, 31, 43, 53, 59), the mechanisms underlying its effects on osteoblasts and bone formation are not well known. Several reports have indicated that LPS and an extract of periodontopathic bacteria inhibit alkaline phosphatase (ALPase) activity, calcium and inorganic phosphate accumulation, and collagen synthesis in cultures of chicken and mouse calvaria and in MC3T3-E1 cells (a mouse osteoblastic cell line), suggesting that periodontal pathogens not only stimulate bone resorption but also inhibit bone formation (25, 29, 30). However, the effects of LPS on osteoblastic-cell differentiation and bone matrix production in periodontal disease have not been clearly elucidated.

Fetal rat calvaria (RC) cells contain a subpopulation of osteoprogenitor cells which proliferate and differentiate into osteoblastic cells and finally form mineralized bone nodules (BNs) over the course of the culture period (5). Owen et al. (40) and Ohishi et al. (39) reported that RC cells show high activity of ALPase, a marker enzyme of osteoblastic-cell differentiation, and produce osteocalcin (OCN) and osteopontin (OPN). OCN is a major noncollagenous calcium binding protein in bone matrix and is synthesized by mature osteoblasts (17, 24, 56), and OPN is a prominent glycosylated phosphoprotein which exists in several organs including bone, kidneys, and mammary glands and is produced by osteoblasts at mineralized sites and also by osteoclasts in bone (9, 42, 50, 51, 56). Because both of these bone matrix proteins are regulated by calcitropic hormones and growth factors and are closely connected to BN formation and ALPase activity, their expression is considered to characterize mature osteoblasts and to be associated with mineralization of the bone matrix (9, 24, 38, 41, 56). These previous reports indicate that RC cell cultures provide a useful experimental model with which to investigate the effect of LPS on the differentiation of osteoblastic cells.

In the present study, we investigated the effects of P-LPS extract prepared by the hot-phenol–water method on osteoblastic markers including BN formation, ALPase activity, the expression of OCN and OPN mRNAs, and the expression of CD14 and IL-1β mRNAs by using a RC cell culture system. We discuss the actions of P-LPS extract on osteoblastic-cell differentiation and the mechanism by which it influences bone remodelling in periodontal disease.

MATERIALS AND METHODS

Cell culture.

RC cells were prepared as described by Bellows et al. (5). Briefly, rat calvariae were dissected from 21-day-old Wistar rat fetuses and their cells were isolated by sequential digestion (five times) with an enzyme mixture containing collagenase derived from Clostridium histolyticum (Sigma, Chemical Co., St. Louis, Mo.). The cells obtained from the last four digestion steps were inoculated into alpha minimal essential medium (α-MEM) containing 10% fetal calf serum and antibiotics. After 24 h of culture, the cells were trypsinized, seeded at a density of 6,000 cells/cm2 in the same medium containing 50 μg of ascorbic acid per ml and 2 mM β-glycerophosphate, and cultured for 4 to 21 days.

Preparation of P-LPS extract.

P. gingivalis 381 was cultured anaerobically in GAM broth (Nissui Seiyaku Co., Tokyo, Japan) at 37°C for 72 h. The P-LPS fraction was then extracted from the cells by the hot-phenol–water method (57). Briefly, the cultured bacterial cells were suspended in equal volumes of 90% phenol and distilled water, shaken for 20 min at 68°C, and immediately cooled in iced-water. After centrifugation at 8,000 × g for 20 min, the upper, aqueous phase was collected. The lower, phenol phase was mixed with distilled water and reextracted at 68°C. The aqueous phases were mixed, dialyzed against distilled water overnight, and centrifuged at 100,000 × g for 3 h at 4°C. The precipitate was washed twice with distilled water, lyophilized, treated with 2% Cetavlon (Nacalai Tesque, Inc, Kyoto, Japan) for 15 min at room temperature, and centrifuged at 3,000 × g for 20 min. The supernatant was lyophilized, dissolved in 0.5 M NaCl solution, and incubated with a 10-fold-greater volume of ethanol for 2 h at 4°C. After centrifugation at 8,000 × g for 20 min, the precipitate (LPS extract) was lyophilized and used for the following experiments.

Determinations of BN formation and ALPase activity.

For the assay of BN formation, RC cells were cultured with P-LPS extract (0 to 100 ng/ml) for 21 days, washed in phosphate-buffered saline (PBS), fixed with 10% neutral-buffered formalin, and stained in situ by the von Kossa technique (5). The number of mineralized BNs was counted under a dissecting microscope.

For the assay of ALPase activity, RC cells were cultured with or without P-LPS extract (100 ng/ml) for 4 to 21 days or with 0 to 100 ng of P-LPS extract per ml for 14 days. On the indicated day, the cells were washed in PBS, scraped into 50 mM Tris-HCl buffer (pH 7.4), sonicated, and centrifuged at 2,000 × g for 10 min. The ALPase activity in the supernatant was determined by the method of Lowry et al. (26) with p-nitrophenyl phosphate as a substrate.

Determinations of DNA content and cell viability.

The DNA content was determined fluorometrically by the method of Labarca and Paigen (23). Briefly, RC cells were cultured with 0 to 100 ng of P-LPS extract per ml for 14 or 21 days, washed in PBS, lysed in 0.15 N NaOH, neutralized with 0.15 N HCl, and reacted with a solution containing 1 μg of bis-benzimide (Hoechst 33258; Sigma Chemical Co.) per ml. The cellular DNA content was measured by determining the fluorescence spectrum at an excitation wavelength of 356 nm and an emission wavelength of 458 nm.

The viability of the RC cells cultured with 0 to 100 ng of P-LPS extract per ml for 21 days was assayed by using Alamar Blue solution (Wako Pure Chemical Industries, Osaka, Japan).

RNA isolation and Northern blot analysis.

RC cells were cultured with or without P-LPS extract (100 ng/ml) for 7, 14, or 21 days. In some experiments, the cells were cultured for 14 days and then treated with P-LPS extract for 6, 12, or 24 h. The total cellular RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform method (14). Aliquots (10 μg) of RNA were separated by electrophoresis in 1% agarose gels containing 2 M formaldehyde and transferred to nylon membranes (Hybond-N+; Amersham Life Science, Little Chalfont, United Kingdom). cDNA probes for rat ALPase (37), rat OCN (11), mouse OPN/2ar (48), and rat CD14 (55) were provided by G. A. Rodan (Merck Research Laboratories, West Point, Pa.), J. M. Wozney (Genetics Institute, Cambridge, Mass.), D. T. Denhardt (Rutgers University, Piscataway, N.J.), and S. Yamamoto (Oita Medical University, Oita, Japan), respectively. The cDNA of IL-1β was synthesized by the PCR amplification method with rat IL-1β primer (Clontech Laboratories, Inc., Palo Alto, Calif.). The cDNA probes were labeled with [α-32P]dCPT (Amersham Life Science) by using a random-primer DNA-labeling kit (Takara, Kyoto, Japan). Prehybridization was performed for more than 4 h at 42°C in 50% formamide–5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7])–5× Denhardt’s solution–0.5% sodium dodecyl sulfate–200 μg of salmon sperm DNA per ml. Hybridization was performed at 42°C for 18 to 24 h in the same solution with 106 dpm of 32P-labeled cDNA probes per ml. The membranes were washed twice in 2× SSPE–0.1% sodium dodecyl sulfate for 20 min at 42°C and exposed to X-ray films at −70°C for several days. The hybridization signals were determined with a chromato-scanner (CS-930; Shimadzu, Kyoto, Japan). The levels of ALPase, OCN, and OPN mRNAs were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.

RESULTS

Effect of P-LPS extract on BN formation and ALPase activity in RC cells.

In RC cells cultured with P-LPS extract (0 to 100 ng/ml) for 21 days, mineralized BNs appeared as dark dots after staining by the von Kossa technique (Fig. (Fig.1A).1A). These dark dots were more evident in nontreated control cultures than in cultures treated with 10 and 100 ng of P-LPS extract per ml. Figure Figure1B1B shows that P-LPS extract (0 to 100 ng/ml) decreased the number of mineralized BNs in a dose-dependent manner. At 0.1 ng/ml, P-LPS extract significantly diminished BN formation to 85% of the nontreated control value, while at 100 ng/ml it markedly decreased the number of BN to 27% of the control value.

FIG. 1
Effect of P-LPS extract on BN formation in RC cells. RC cells (6.0 × 104) were inoculated into 35-mm dishes and cultured in α-MEM with P-LPS extract (0 to 100 ng/ml) for 21 days. (A) The BNs formed during culture were stained by the von ...

ALPase activity in the nontreated RC cells increased until day 10, and this level was maintained up to day 21 (Fig. (Fig.2).2). When RC cells were treated with 100 ng of P-LPS extract per ml, inhibition of enzyme activity became significant on day 7 (P < 0.01) and consistent inhibition, to approximately 60% of the control level, was observed on days 10 to 21. To determine the dose response of ALPase activity, RC cells were cultured with P-LPS extract (0 to 100 ng/ml) for 14 days (Fig. (Fig.3).3). ALPase activity was significantly inhibited at 0.1 ng of P-LPS extract per ml and decreased in a dose-dependent manner, reaching 61% of the control level at 100 ng of P-LPS extract per ml. This pattern of inhibition of ALPase activity was similar to that of P-LPS extract inhibition of BN formation in RC cells. We also investigated the effect of LPS extracts derived from other bacteria on ALPase activity; enzyme activity was inhibited to 56.6, 57.9, and 57.8% of the control level by LPS extracts (100 ng/ml) from Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum, respectively, whereas the LPS from Escherichia coli had no significant effect.

FIG. 2
Effect of P-LPS extract on ALPase activity in RC cells. RC cells were cultured with (solid circles) or without (open circles) 100 ng of P-LPS extract per ml for the period indicated, and ALPase activity was determined as described in Materials and Methods. ...
FIG. 3
Dose effect of P-LPS extract on ALPase activity in RC cells. RC cells were cultured in α-MEM with P-LPS extract (0 to 100 ng/ml) for 14 days, and enzyme activity was determined. Values are means and standard errors for quadruplicate samples. *, ...

Effect of P-LPS extract on proliferation and cell viability in RC cells.

The effect of P-LPS extract on cell proliferation was investigated by determining the cellular DNA content. On days 14 and 21, the DNA content of nontreated RC cells was 12.8 ± 1.5 and 21.6 ± 0.7 μg/well, respectively, and the DNA content of RC cells treated with 100 ng of P-LPS extract per ml was 13.8 ± 0.6 and 21.9 ± 0.5 μg/well, respectively. P-LPS extract at 1 to 100 ng/ml did not significantly affect the DNA content of RC cells. Moreover, by day 21, P-LPS extract (1, 10, and 100 ng/ml) had no influence on cell viability. The percent viabilities for each P-LPS extract dose were 91, 91, and 108% of the nontreated control values, respectively. These results indicate that P-LPS extract has no effect on the proliferation or viability of RC cells.

Effects of P-LPS extract on the expression of ALPase, OCN, OPN, CD14 and IL-1β mRNAs.

The effects of P-LPS extract on the expression of ALPase, OCN, and OPN mRNAs were investigated by using RC cells treated with 100 ng of P-LPS extract per ml for 7, 14, or 21 days (Fig. (Fig.4).4). The densities of the bands for each mRNA were measured by densitometric scanning and expressed relative to those for nontreated control cultures, which were assigned a density of 1.00. The expression of ALPase mRNA was time-dependently decreased by P-LPS extract, reaching densities of 0.81, 0.59, and 0.44 on days 7, 14, and 21, respectively. In the control culture, the expression of OCN mRNA rose markedly during the latter half of the culture period. P-LPS extract caused a slight decrease in OCN mRNA expression (to 0.71 of control) on day 7 and almost completely suppressed OCN mRNA expression (to 0.12 and 0.08 of the control value) on days 14 and 21, respectively. The OPN mRNA level in the control cells also rose from day 7 to day 14, and this high level was maintained until day 21. The expression was time-dependently inhibited by P-LPS extract, reaching 0.93, 0.58, and 0.25 on days 7, 14, and 21, respectively.

FIG. 4
Effect of P-LPS extract on ALPase, OCN, and OPN mRNA levels in RC cells. Total RNA was extracted from RC cells cultured with (L) or without (C) 100 ng of P-LPS extract per ml for 7, 14, or 21 days as described in Materials and Methods. The levels of ALPase, ...

In some experiments, RC cells were cultured for 14 days and then treated with 100 ng of P-LPS extract per ml for 6 to 24 h. The cellular expression of CD14 mRNA was distinctly increased by treatment with P-LPS extract at 6 h (Fig. (Fig.5A).5A). Furthermore, P-LPS extract markedly stimulated IL-1β mRNA expression in RC cells at 6 h (Fig. (Fig.5B).5B). These results show that marked expression of CD14 and IL-1β mRNAs can be induced in RC cells by relatively short exposure to P-LPS extract.

FIG. 5
Effect of P-LPS extract on CD-14 and IL-1β mRNA levels in RC cells. RC cells were cultured for 14 days and then incubated with (L) or without (C) P-LPS extract (100 ng/ml) for 6, 12, or 24 h. After RNA preparation, CD-14 (A) and IL-1β ...

DISCUSSION

RC cells form mineralized BNs through three distinct stages: proliferation, bone matrix maturation, and mineralization (40). The number of mineralized BNs is reflected in the appearance of differentiated osteoprogenitor cells (6). The RC cell culture system has previously been used to investigate the effects of many osteotropic hormones and factors on osteoblastic-cell differentiation (3, 7, 35, 39, 40). Periodontopathic bacteria and their products are thought to influence osteoblastic-cell differentiation and bone formation as well as bone resorption during inflammatory bone destruction in periodontal disease (18, 19, 20, 29, 30, 36, 46, 53). However, few studies have investigated the effects of LPS and periodontopathic bacterial products on BN formation and osteoblastic-cell differentiation. Loomer et al. (25) reported that a metabolic product and sonicated extract derived from P. gingivalis significantly suppressed collagen synthesis and calcium and inorganic phosphate accumulation in the periosteal tissues of embryonic chicken calvaria and inhibited ALPase activity to approximately 40 to 55% of the control level. The inhibitory actions of these bacterial products on ALPase activity were similar to the effects of P-LPS extract in RC cells observed during the present study. Murata et al. (34) examined the effects of sonicated extracts from several bacteria, including P. gingivalis, A. actinomycetemcomitans, P. intermedia, and E. coli, on ALPase activity in MC3T3-E1 cells. The extracts of A. actinomycetemcomitans and P. intermedia had a clear suppressive effect on enzyme activity, whereas extracts of P. gingivalis and E. coli had no effect. This differed from our results, in which P-LPS extract inhibited ALPase activity in RC cells. The reason for this difference is not clear, but it may be due to a difference in the methods used to purify the bacterial products. The LPS extract used in our study was prepared from P. gingivalis by the hot-phenol–water method and Cetavlon treatment. This extract may contain bacterial components other than LPS, which may affect osteoblastic-cell differentiation. We investigated the purity of P-LPS extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. P-LPS extract showed a ladderlike pattern of bands (data not shown), and its pattern was similar to that of LPS purified from P. gingivalis as described by Chen et al. (12). To confirm the suppressive effect of P-LPS on osteoblastic-cell differentiation, we further purified P-LPS extract by treatment with RNase A, DNase I, and proteinase K and then with phenol-chloroform-petroleum ether (2:5:8, by volume) and acetone. The purified P-LPS inhibited ALPase activity of RC cells to the same degree as the original P-LPS extract did, whereas other fractions in the purification procedure slightly suppressed the enzyme activity (data not shown). These results demonstrated that the inhibition of osteoblastic-cell differentiation was attributed mainly to P-LPS. Several studies have indicated that LPS and extracts derived from P. gingivalis have both direct and indirect effects on osteoclastic-cell activity and bone resorption (20, 46, 53). We have confirmed a similarity in the inhibitory effects of LPS extracts from P. gingivalis, A. actinomycetemcomitans, P. intermedia, and F. nucleatum on ALPase activity in RC cells, suggesting that some periodontal pathogens not only stimulate osteoclastic-cell differentiation and bone resorption but also inhibit osteoblastic-cell differentiation during periodontal bone remodelling.

The synthesis of bone matrix proteins such as collagen, OCN, and OPN is an important precursor stage for BN and bone formation. Periodontal pathogens are thought to inhibit bone matrix production during alveolar bone destruction in periodontal disease, and P-LPS has been reported to suppress collagen synthesis in rat calvaria organ cultures (30, 36, 45). We found that P-LPS extract decreased the gene expression of OCN and OPN, two noncollagenous proteins closely related to bone remodelling. OCN is thought to be a specific marker of osteoblastic activity and mineralization of the bone matrix, because it is synthesized specifically by osteoblasts before being released into the blood or accumulated in bone and because its synthesis is regulated by many calcitropic hormones and factors (17, 24, 38, 41, 56). OPN is synthesized mainly by osteoblasts and osteoclasts and is deposited in bone. Its production is controlled by many calcitropic factors, suggesting that it plays an important role in bone formation and resorption (9, 42, 50, 51, 56). The expression of both OCN and OPN mRNAs in RC cells was weak for the first 7 days but increased markedly on day 14, when mineralization of BNs was starting to occur in the cultures. P-LPS extract strongly blocked OCN mRNA expression during the latter phase of culture. It also moderately inhibited OPN gene expression on day 14 and produced a further marked reduction on day 21. In contrast, Cheng et al. (13) indicated that LPS increased OPN mRNA expression in both MC3T3-E1 cells and primary osteoblastic cells derived from fetal rat calvaria. They suggested that this elevation might be related to bone resorption by means of osteoclastic-cell activation through osteoblastic cells. The reasons for the difference between these results and ours is unclear; however, one possible explanation is a difference in experimental conditions. It is known that continuous exposure of rat bone to IL-1, a cytokine closely associated with the mechanism of action of LPS, inhibits bone formation whereas transient treatment stimulates formation (45). This suggests that the observed difference in the effect of P-LPS on OPN mRNA expression might be due to a difference in the treatment period. Because infected periodontal tissues are continuously exposed to periodontopathic bacteria, we think that the continuous action of LPS would cause marked inhibition of OCN and OPN production and resultant suppression of mineralized BN formation.

LPSs stimulate macrophages, fibroblasts, and osteoblastic cells in inflamed periodontal tissues to secrete osteolytic cytokines and factors including IL-1, IL-6, TNF-α and PGE2, and P-LPS causes alveolar bone destruction through these inflammatory components (1, 16, 21, 22, 52, 58). These LPS-stimulated actions are mediated by binding between LPS and CD14, an LPS receptor that exists predominantly on the surfaces of monocytes/macrophages and neutrophils (43, 58, 59). LPS induces bone resorption via the CD14 pathway, because an anti-CD14 antibody and an anti-sense CD14 oligonucleotide inhibited LPS-stimulated IL-1 and IL-6 gene expression, osteoclastic-cell differentiation, and bone resorption in mouse embryonic calvarial cells (2, 31). We also observed a clear elevation in the production of CD14 and IL-1β mRNAs when RC cells were treated with P-LPS extract (100 ng/ml) for 6 h. Stashenko et al. (52) and Evans et al. (15) indicated that IL-1β decreases ALPase activity in rat and human osteoblastic cells. IL-1β also inhibits OCN and type I collagen synthesis in several types of osteoblastic cells and plays an inhibitory role in the regulation of bone formation (44, 54). Because these effects of IL-1β are similar to those of P-LPS extract on ALPase activity and OCN production, P-LPS might inhibit osteoblastic cell differentiation via an autocrine pathway involving CD-14 and IL-1β in RC cell cultures. The actions of other osteolytic factors such as IL-6, TNF-α, and PGE2 in RC cells are unknown. We therefore intend to elucidate the details of the mechanism underlying LPS-induced inhibition of osteoblastic-cell differentiation in the future.

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