Bradykinin (BK) stimulates bone resorption in vitro and synergistically potentiates interleukin-1 (IL-1)–induced bone resorption and prostaglandin (PG) formation, suggesting that kinins are important in inflammation-induced bone loss. The present study was undertaken to study 1) the role of the kinin B1 and B2 receptors in the synergistic interaction with IL-1 and tumor necrosis factor α (TNFα), 2) the molecular mechanisms involved in synergistic enhancement of PG formation, and 3) the effects of kinins on cytokine-induced expression of RANKL, RANK, and osteoprotegerin (OPG) (the latter being crucial molecules in osteoclast differentiation).
Formation of PGs, expression of enzymes involved in arachidonic acid metabolism, and expression of RANKL, RANK, and OPG were assessed in the human osteoblastic cell line MG-63 and in mouse calvarial bones. The role of NF-κB and MAP kinases was studied using pharmacologic inhibitors.
PGE2 formation and cyclooxygenase 2 (COX-2) protein expression were induced by IL-1β and potentiated by kinins with affinity for the B1 or B2 receptors, resulting in PGE2-dependent enhancement of RANKL. The enhancements of PGE2 formation and COX-2 were markedly decreased by inhibition of p38 and JNK MAP kinases, whereas inhibition of NF-κB resulted in abolishment of the PGE2 response with only slight inhibition of COX-2.
Kinin B1 and B2 receptors synergistically potentiate IL-1– and TNFα-induced PG biosynthesis in osteoblasts by a mechanism involving increased levels of COX-2, resulting in increased RANKL. The synergistic stimulation is dependent on NF-κB and MAP kinases. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including that in rheumatoid arthritis.
Inflammatory conditions such as rheumatoid arthritis and periodontal disease are associated with local loss of bone tissue, mainly due to activation of osteoclastic bone resorption. Based on the bone resorptive effects of cytokines such as interleukin-1 (IL-1), IL-6, IL-11, IL-17, tumor necrosis factor α (TNFα), TNFβ, leukemia inhibitory factor (LIF), and oncostatin M (OSM), and the inhibitory effect on bone resorption by cytokines such as IL-4, IL-10, IL-12, IL-13, IL-18, and interferon-γ (1), it has been suggested that inflammation-induced bone resorption is mediated by the concerted actions of stimulatory and inhibitory cytokines (2–4). In synovial fluid from patients with rheumatoid arthritis, kallikrein activity, as well as the activity of kininogens, has been demonstrated (5). In addition, increased kinin levels have been found in synovial fluid from patients with rheumatoid arthritis and in inflammatory exudates collected from dogs with periodontal disease (6–8).
These observations together with findings showing that bradykinin (BK) and kallidin (Lys-BK) can stimulate bone resorption in neonatal mouse calvariae prompted the suggestion that kinins should also be regarded as candidates responsible for osteoclastic activation in inflammatory conditions (9, 10). Interestingly, BK and Lys-BK not only stimulate bone resorption but also synergistically potentiate the bone resorptive effect of IL-1 (11). The latter effect of kinins is also associated with their synergistic potentiation of IL-1–induced prostaglandin E2 (PGE2) and PGI2 production (10, 11). In addition to playing an important role in inflammation-induced bone remodeling, kinins are involved in senescence-associated bone loss, since mice double mutant for Ins2Akita/+ and Bdkrb2−/− genes exhibit a complex phenotype, including kyphosis and osteoporosis (12).
The effects of kinins on target cells are linked to 2 subtypes of kinin receptors, the B1 receptor and the B2 receptor (10, 13, 14). B2 receptors are expressed constitutively in many different cell types, whereas B1 receptors are induced during inflammatory conditions (14, 15). The relative importance of signaling through the B1 and B2 receptors in vivo is not known, but it has been suggested that B1 receptors may be particularly important in inflammatory conditions, not only because of their induction but also due to the fact that des-Arg metabolites of BK and kallidin, acting preferentially on B1 receptors, are produced in inflammatory processes by carboxypeptidases.
We have found that activation of both the B1 and B2 receptors results in stimulation of bone resorption. We have also demonstrated that the kinin B1 and B2 receptors that stimulate PG biosynthesis can be found on osteoblasts isolated from neonatal mouse calvariae (16, 17) and on the human osteoblastic cell line MG-63 (18), observations which are consistent with the common view that osteoblasts are the target cells for several hormones and cytokines that stimulate bone resorption (19). It is not known if signaling through B1 receptors may interact with signaling through IL-1 receptors, similar to the synergistic interactions between B2 receptors and IL-1 (11). Moreover, the molecular mechanisms by which kinin receptors interact with IL-1 receptors, thus causing potentiation of PG formation and bone resorption, are not known. The potential mechanisms may include effects on cyclooxygenase 2 (COX-2) (PGH synthase 2), by which the conversion of arachidonic acid to the endoperoxide PGG2 is catalyzed and followed by a reduction of PGG2 to PGH2, and/or effects on the recently discovered PGE synthase (PGES), in which PGH2 is converted to PGE2 (20, 21). Several forms of PGES seem to exist, including microsomal inducible PGES-1 (mPGES-1) as well as mPGES-2 and cytosolic PGES (cPGES) (22, 23).
Recently, it has been shown that expression of a TNF-related cytokine, RANKL, is a crucial factor in bone resorption (24, 25). RANKL is mainly expressed as a transmembrane protein on osteoblasts in the periosteum and on stromal cells in bone marrow, but also as a soluble cytokine. It activates the cognate receptor RANK on osteoclast progenitor cells, leading to stimulation of TNF receptor–associated factors (TRAFs) and downstream activation of several signaling molecules, including NF-κB, MAP kinases (MAPKs), activating protein 1 (AP-1), nuclear factor of activated T cells 2 (NFAT-2), and phosphatidylinositol 3-kinase, resulting in differentiation of the osteoclast progenitor cells to cells that finally fuse to multinucleated, bone-resorbing osteoclasts (24–26). The activation of RANK can be blocked by a decoy receptor, osteoprotegerin (OPG), which is released by osteoblasts/stromal cells.
The crucial role of the RANKL/RANK/OPG system is supported by observations that RANK- and RANKL-deficient mice lack osteoclasts and are osteopetrotic, similar to mice lacking TRAF-6, NF-κB (p50−/−/p52−/−), c-Fos, or NFAT-2 (24–26). Mice deficient in OPG exhibit abundant osteoclasts and an osteoporotic phenotype. RANKL is expressed in synovial tissue from patients with rheumatoid arthritis (27–29) and has been found to be crucial for osteoclast formation and bone loss in experimentally induced arthritis in mice (30). RANKL is also expressed in gingival tissue from patients with periodontal disease (31, 32), and increased RANKL/OPG is associated with bone loss in periodontitis (4, 31).
The aims of the present study were to study the interactions between kinins and several osteotropic cytokines known to stimulate bone resorption in order to observe their effects on PG production, expression of enzymes involved in the formation of PGs, and expression of RANKL, RANK, and OPG, and to evaluate the role of the transcription factor NF-κB and MAPKs, using the human osteoblastic cell line MG-63 and neonatal mouse calvarial bones.
MATERIALS AND METHODS
BK, Lys-BK, des-Arg9-BK (DABK), recombinant human COX-2, essentially fatty acid–free fetal bovine serum albumin (BSA), trypsin, gel loading solution, agarose, ethidium bromide, phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin A, Igepal CA-630, pyronin Y, dimethyl sulfoxide, actinomycin D, and phosphate buffered saline (PBS) were purchased from Sigma (St. Louis, MO), and the α-modification of minimal essential medium (α-MEM), fetal calf serum (FCS), TRIzol LS reagent kit, L-glutamine, and oligonucleotide primers were from Invitrogen (Stockholm, Sweden). Fluorescence-labeled probes (reporter fluorescent dye VIC or FAM at the 5′ end, and quencher fluorescent dye TAMRA at the the 3′ end), 384-well clear rxn plates, and TaqMan Universal polymerase chain reaction (PCR) master mix were from Applied Biosystems (Foster City, CA). B1 and B2 receptor agonists des-Arg10-Lys-BK (DALBK), Hoe 140 (D-Arg0[Hyp3,β-(2-Thienyl)-Ala5,D-Tic7,Oic8]-BK), des-Arg10-[Leu9]-Lys-BK, and des-Arg10-Hoe 140 were from Peninsula Laboratories (Belmont, CA) and Neosystem Laboratories (Strasbourg, France).
Recombinant human IL-1β, IL-1α, IL-6, IL-11, IL-17, TNFα, LIF, OSM, and transforming growth factor β (TGFβ) and immunoassay kits for mouse RANKL were from R&D Systems (Abdingdon, UK), and the radioimmunoassay kit for PGE2 was from DuPont/New England Nuclear Chemicals (Dreieich, Germany). The multiwell plastic culture and petri dishes used in all experiments were from Costar (Cambridge, MA). The first-strand complementary DNA (cDNA) synthesis kit was from Roche (Mannheim, Germany), and the RNAqueous-4PCR kit, RNAlater, and DNA-free kit were from Ambion (Europe) (Huntingdon, Cambridgeshire, UK). Synthetic bovine parathyroid hormone (PTH) was from Bachem (Bubendorf, Switzerland), and the bovine albumin standard, bicinchoinic acid (BCA) protein assay reagents, and SuperSignal chemiluminence kit were from Pierce Biotechnology (Rockford, IL). Antibodies used were rabbit polyclonal primary antibody against human COX-2 (Cayman Chemical, Ann Arbor, MI), goat polyclonal primary antibody against human actin (I-19; Santa Cruz Biotechnology, Santa Cruz, CA), and goat anti-rabbit and rabbit anti-goat IgG horseradish peroxidase (HRP)–conjugated secondary antibodies (Dakopatts, Glostrup, Denmark).
Pyrrolidine dithiocarbamate (PDTC) (an NF-κB inhibitor), SB203580 (a p38 MAPK inhibitor), SP600125 (a JNK MAPK inhibitor), PD98059 (an ERK MAPK inhibitor), methanol, and gelatin were from Merck (Darmstadt, Germany). The Microcon centrifugal filter devices were from Millipore (Molsheim, France), and glycine, Tris, blotting-grade blocker nonfat dry milk, 7.5–12% Ready Gel, sodium dodecyl sulfate (SDS) solution (10%), polyvinylidene difluoride (PVDF) membrane, and 10% Tween 20 solution were from Bio-Rad (Hercules, CA). Indomethacin was kindly supplied by Merck, Sharp and Dohme (Haarlem, The Netherlands), while 1,25-dihydroxyvitamin D3 was from Hoffman-LaRoche (Basel, Switzerland). Dr. Guy Drapeau (Centre de Recherche, Quebec, Canada) kindly provided the sar-[D-Phe8]-DABK and [Tyr-Gly-Lys-Aca-Lys]-DABK, while rabbit polyclonal primary antibody against human mPGES-1 (20) and human recombinant mPGES-1 were a kind gift from Dr. Per-Johan Jakobsson (Karolinska Institutet, Stockholm, Sweden).
Indomethacin and all kinins were dissolved in ethanol. The final concentration of ethanol did not exceed 0.1%, and this amount had no effect on basal production of PGE2.
Bone cell culture.
MG-63 cells are a human osteoblastic osteosarcoma cell line that expresses several osteoblastic phenotypes, including biosynthesis of type I collagen and osteocalcin (33). For experiments, cells were seeded at an initial density of 4–5 × 104 cells/cm2 in either 2-cm2 multiwell culture dishes for analysis of PG formation or 9.5-cm2 culture dishes for analysis of gene expression. In each dish, α-MEM/10% FCS was added and cells were cultured for 1–2 days until 80–90% confluent monolayers were obtained. The cells were then washed twice in PBS and once in serum-free α-MEM, and then incubated in α-MEM/1% FCS, with or without test substances, for 1–24 hours.
Culture of calvarial bones.
Calvarial bones from 5–7-day-old CsA mice were dissected and cut into 2 halves. The bones were preincubated for 18–24 hours in α-MEM containing 0.1% BSA and 1 μM indomethacin. Following preincubation, the bones were extensively washed and subsequently incubated for 24 hours, submerged in 24-well culture dishes containing 1.0 ml indomethacin-free medium with or without test substances. CsA mice were from our own inbred colony. Animal care and experiments were approved and conducted in accordance with accepted standards of humane animal care and use, as considered appropriate by the Animal Care and Use Committee of Umeå University.
Analysis of PG production.
PG biosynthesis in MG-63 cells and in calvarial bones was assessed by measuring the amount of PGE2 in the media at the end of the cultures (18).
After incubation with or without test substances, the MG-63 cells were washed twice in PBS, and total RNA was isolated using TRIzol LS reagent in accordance with the manufacturer's protocol (Invitrogen). At the end of the experiment, calvarial bones were homogenized (Ultra-Turrax; Jenke & Kunkel, Staufen, Denmark) and RNA was extracted using either TRIzol LS reagent or the RNAqueous-4PCR kit (Ambion).
One microgram of RNA from MG-63 cells was reverse transcribed into single-stranded cDNA with a first-strand cDNA synthesis kit using random p(dN)6 primers, and the cDNA was amplified utilizing a PCR core kit as previously described (34). For all genes, no amplification was detected in samples in which the reverse transcriptase reaction had been omitted (data not shown). The identity of the PCR products was confirmed using a QIAquick purification kit (Qiagen, Chatsworth, CA) and a Thermo Sequenase II DYEnamic ET Terminator Cycle Sequencing Premix Kit (Amersham Biosciences, Uppsala, Sweden), with sequences analyzed on a ABI 377XL DNA sequencer (Applied Biosystems). (The sequences of the specific primers, the GenBank accession numbers, the positions for the 5′ and 3′ ends of the nucleotides for the predicted PCR products, and the estimated sizes of the PCR products are listed in Appendix A on the Arthritis & Rheumatism Web site at http://www.mrw.interscience.wiley.com/suppmat/0004-3591/suppmat/.)
Quantitative real-time PCR.
Analyses of messenger RNA (mRNA) expression with quantitative real-time PCR were performed using the TaqMan Universal PCR Master Mix kit (Applied Biosystems) as described in detail previously (34). Following DNase treatment, total RNA (0.5–1 μg) was reverse transcribed into single-stranded cDNA using the first-strand cDNA synthesis kit with random p(dN)6 primers. Quantitative real-time PCRs were then carried out using TaqMan kinetics with fluorescence-labeled probes. The amplifications were performed using the ABI PRISM 7900 HT sequence-detection system and software (Applied Biosystems). For all genes, no amplification was detected in samples in which the reverse transcriptase reaction had been omitted (results not shown). (The sequences and concentrations of primers and probes, the GenBank accession numbers, and the numbers for the 5′ and 3′ ends of the nucleotides for the predicted PCR products are listed in Appendix A on the Arthritis & Rheumatism Web site at http://www.mrw.interscience.wiley.com/suppmat/0004-3591/suppmat/.) To control for variability in amplification due to differences in starting mRNA concentrations, RPL13A (human) and β-actin (mouse) were used as internal standards. Oligonucleotide primers and TaqMan probes were designed using Primer Express 2.0 (Applied Biosystems), based on the sequences from the GenBank database (http://www.ncbi.nlm.nih.gov).
Preparation of total cell lysates for protein analyses.
MG-63 cells were cultured to 80–90% confluent monolayers in 60-cm2 petri dishes, washed twice in PBS and once in serum-free α-MEM, and then incubated in α-MEM (without serum) with or without test substances for 4 or 24 hours. Following incubation, the cells were washed twice in PBS before adding the lysis buffer (1% Igepal CA-630, 0.1% SDS, 2 mM EDTA, 50 mM NaF, 0.1 mg/ml PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, in PBS). Protein concentrations of the cell lysates were measured using the BCA assay technique, with bovine albumin as standard.
Cell lysates were mixed with sample buffer (200 mM Tris HCl, pH 6.7, 20% glycerol, 10% β-mercaptoethanol, 5% SDS, 0.01% pyronin Y) and boiled for 3 minutes. Protein samples were then loaded on Tris HCl polyacrylamide gels (7.5–12%) and electrophoresis was performed. Electrophoresed proteins were then blotted onto a PVDF membrane, which was blocked (1% milk, 1% BSA in Tris buffered saline [TBS] for actin; 5% milk, 0.1% Tween 20 in TBS [TTBS] for mPGES-1; and 3% gelatin in TBS for COX-2) either overnight (with actin or COX-2) or for 2 hours (with mPGES-1) at room temperature.
For detection by Western blotting, the membrane was incubated with primary antibody (1:200 in 1% milk, 1% BSA, 0.05% TTBS for actin; 1:1,000 in 2% milk, 0.05% TTBS for mPGES-1; and 1:200 in 1% gelatin, 0.05% TTBS for COX-2) either for 60 minutes at room temperature (with actin or COX-2) or overnight at 4°C (with mPGES-1). After incubation, the membranes were washed 3 times for 10 minutes in 0.05% TTBS, followed by incubation with either goat anti-rabbit or rabbit anti-goat HRP-conjugated secondary antibodies (1:1,000 in 1% milk, 1% BSA in TTBS for actin; 1:1,000 in 2% milk, 0.05% TTBS for mPGES-1; and 1:2,000 in 1% gelatin in TTBS for COX-2) for 60 minutes at room temperature. Finally, the membranes were washed extensively with either TTBS or TBS, followed by development using a chemiluminence detection kit in accordance with the manufacturer's protocol (Pierce Biotechnology). The COX-2 and mPGES-1 bands migrated to the same positions as those of recombinant COX-2 and mPGES-1, respectively.
RANKL protein analysis.
Calvarial bones were dissected and cultured for protein analysis in the same manner as described above. RANKL protein in calvarial bones and culture media was analyzed by a commercially available enzyme-linked immunosorbent assay as previously described (35).
Data in multiple treatment groups were compared using one-way analysis of variance (ANOVA), with Levene's homogenecity test and, subsequently, Bonferroni's or Dunnett's T3 post hoc test. Results are the mean ± SEM. Most of the cumulative data are from 2–3 independent experiments, with values based on 4 wells/group. Results in time-course studies are representative of 2 independent experiments. In concentration-dependent studies, the results from several experiments were pooled by recalculating the data in each experiment relative to untreated controls, which were arbitrarily set to 100%. The real-time PCR results are representative of 2–3 independent experiments, and the values are based on the findings in MG-63 cells in 4 petri dishes (20 cm2) per group or 4 wells (9.5 cm2) per group or the findings in 5 calvarial halves per group, with each sample analyzed in triplicate.
Synergistic interactions between BK, IL-1β, and TNFα and effects on PG production.
Cotreatment with BK (1 μM) and IL-1β (1.5–1,500 pg/ml) resulted in a large synergistic, concentration-dependent stimulation of PGE2 biosynthesis (Figure 1a). TNFα (0.1–30 ng/ml) also caused a concentration-dependent stimulation of PGE2 formation (Figure 1b; inset), which was synergistically potentiated by BK (Figure 1b). BK caused a stimulation of PGE2 biosynthesis as early as 30 minutes after treatment (Figure 1c; inset), but this response was not further augmented during the 24-hour experimental period (Figure 1c). The synergistic stimulation obtained by cotreatment with IL-1β and BK was clearly observed at 24 hours, but not at 4 hours (Figure 1c). The effects of IL-1β and the interactions between IL-1β and BK were abolished by indomethacin (1 μM) (results not shown).
Involvement of kinin receptor subtypes in the synergism between kinins and cytokines.
The B2 receptor agonists BK and Lys-BK and the B1 receptor agonists DABK, DALBK, and Sar[D-Phe8]-DABK all stimulated PGE2 formation and synergistically potentiated the effect of IL-1β (Table 1). Similarly, the B1 receptor agonist [Tyr, Gly, Lys, Aca, Lys]-DABK synergistically potentiated the stimulatory effect of IL-1β (results not shown). The effects of DABK, DALBK, and Sar[D-Phe8]-DABK were clearly inhibited by des-Arg10-Hoe 140 and des-Arg10[Leu9]-Lys-BK (Table 1). However, the latter 2 compounds did not inhibit the effects of BK and Lys-BK. Hoe 140 clearly inhibited the effects of BK and Lys-BK, and also showed a small but significant inhibitory effect on the stimulation of PGE2 formation as induced by DABK, DALBK, and Sar[D-Phe8]-DABK; these findings are consistent with previous observations in some other cell types (for review, see ref.18).
Table 1. Effects of different BK receptor antagonists on PGE2 biosynthesis in the human osteoblastic cell line MG-63 stimulated with different kinins and kinin analogs in the absence and presence of IL-1β*
Values are the mean ± SEM pg/105 cells for 4 wells incubated with or without test substances for 24 hours. BK = bradykinin; PGE2 = prostaglandin E2; IL-1β = interleukin-1β; Lys-BK = kallidin; DABK = des-Arg9-BK; DALBK = des-Arg10-Lys-BK.
Antagonists were added to a final concentration of 3 μmoles/liter.
P < 0.01 versus untreated (in the absence of stimulator and antagonist) controls.
P < 0.01 versus stimulator-treated cultures in the absence of antagonist.
BK, Lys-BK, DABK, and DALBK all synergistically potentiated the stimulatory effect of IL-1β (50 pg/ml) in a concentration-dependent manner (Figure 1d). The synergistic interaction between IL-1β (50 pg/ml) and [Tyr, Gly, Lys, Aca, Lys]-DABK (10 nM) was inhibited in a concentration-dependent manner by both des-Arg10-Hoe 140 and Hoe 140, with the latter being 10-fold less potent (Figure 1e). Hoe 140 and des-Arg10-Hoe 140 did not inhibit the stimulatory effect of IL-1β alone (Figure 1e).
Effects of BK, in the absence and presence of osteotropic cytokines, on PG production.
We screened for interactions between BK and a wide range of different cytokines known to stimulate bone resorption. BK (1 μM), IL-1α (100 pg/ml), IL-1β (100 pg/ml), TNFα (10 ng/ml), and TGFβ (30 ng/ml) significantly stimulated the biosynthesis of PGE2 in MG-63 cells after treatment with these compounds for 24 hours (results not shown). In addition, cotreatment of MG-63 cells for 24 hours with BK and IL-1α, IL-1β, or TNFα resulted in a synergistic stimulation of PGE2 formation (results not shown). No such interaction was observed between BK and TGFβ. Treatment with IL-6 (100 ng/ml), IL-11 (10 ng/ml), IL-17 (30 ng/ml), OSM (10 ng/ml), or LIF (10 ng/ml) did not stimulate PGE2 formation, and none of these cytokines interacted with BK (results not shown).
Effects of kinin B1 and B2 receptor agonists, IL-1β, and TNFα on the expression of enzymes involved in arachidonic acid release and metabolism.
Stimulation of MG-63 cells for 24 hours with BK (3 μM) resulted in a small (1.5–1.9 fold), reproducible, and statistically significant (P < 0.05) enhancement of COX-2 mRNA (Figures 2a and b). In contrast, DALBK (3 μM) did not significantly increase COX-2 mRNA at 24 hours (Figures 2a and b). Treatment of the MG-63 cells with IL-1β (100 pg/ml) or TNFα (10 ng/ml) for 24 hours caused a more robust (13–25 fold) enhancement of COX-2 mRNA (Figures 2a, b, d, and e). Cotreatment of the cells for 24 hours with BK and either IL-1β or TNFα resulted in a potentiation of COX-2 mRNA (Figures 2a and b). Similarly, cotreatment with DALBK (3 μM) and either IL-1β or TNFα for 24 hours caused increased stimulation of COX-2 mRNA (Figures 2a and b). In contrast to the effects on COX-2 message, the expression of COX-1 mRNA was not significantly affected by BK, DALBK, or IL-1β, neither in the absence nor in the presence of kinins (Figure 2c).
The stimulatory effects of BK and IL-1β on COX-2 mRNA were time-dependent and transient, with maximal effects seen at 4 hours (Figure 2d). The time-course study revealed that, although DALBK did not cause any increase of COX-2 mRNA at 24 hours, this BK B1 receptor agonist caused significant enhancement of COX-2 mRNA at 4 hours and at 8 hours (Figure 2e). At 4 hours, BK (3 μM) caused a 6-fold enhancement, and DALBK (3 μM) caused a 4-fold enhancement of COX-2 mRNA, while IL-1β (100 pg/ml) caused a 50-fold enhancement of COX-2 mRNA. The potentiation caused by cotreatment with kinins and IL-1β was time-dependent and maximal at 4 hours, resulting in 66- and 89-fold stimulation of COX-2 mRNA by IL-1β plus BK and IL-1β plus DALBK, respectively (Figures 2d and e).
The stimulatory effects of BK and IL-1β, as well as the stimulatory effect of cotreatment with BK and IL-1β, on COX-2 mRNA expression in MG-63 cells at 4 hours were unaffected by indomethacin (1 μM) (Figure 2f), whereas indomethacin abolished the effects of BK, as well as the effects of IL-1β with or without BK, on PGE2 formation (results not shown). Similarly, the synergistic potentiation of COX-2 mRNA caused by IL-1β and DALBK was unaffected by indomethacin (results not shown). In accordance with these observations, enhancement of COX-2 mRNA expression by TNFα, TNFα plus BK, or TNFα plus DALBK was unaffected by indomethacin (results not shown).
To determine whether the enhancement of the steady-state level of COX-2 mRNA was dependent on an altered stability of the mRNA, we preincubated MG-63 cells with or without IL-1β, in the absence and presence of BK, for 24 hours and then treated the cells with the transcription inhibitor actinomycin D (5 μg/ml) for 24 hours. In these experiments, expression of the housekeeping gene RPL13A remained stable for 8 hours, whereas COX-2 mRNA levels declined in a time-dependent manner. IL-1β did not affect the half-life of COX-2 mRNA. However, BK significantly enhanced the decline of COX-2 mRNA in cells cotreated with IL-1β and BK (Figure 2g).
COX-2 protein expression, as assessed by Western blot analysis, was undetectable in unstimulated cells and in cells stimulated by BK (3 μM) or DALBK (3 μM) (results not shown). In contrast, the expression of COX-2 protein was clearly enhanced by IL-1β (100 pg/ml) at 4 hours and at 24 hours (Figure 2h). At both time points, cotreatment with IL-1β and BK or with IL-1β and DALBK resulted in COX-2 protein levels that were substantially higher than the levels observed in cells stimulated with IL-1β alone.
The expression of mPGES-1 mRNA was unaffected by BK (3 μM) or DALBK (3 μM) at 2–24 hours, whereas IL-1β (100 pg/ml) caused a time-dependent, statistically significant enhancement of mPGES-1 mRNA at 4–24 hours (Figures 3a and b). Costimulation of the MG-63 cells with IL-1β and either BK (Figure 3a) or DALBK (Figure 3b) resulted in a time-dependent potentiation by the 2 kinins at 4–24 hours. The enhancements of mPGES-1 mRNA expression were unaffected by indomethacin (Figures 3a and b; insets). Similarly, TNFα enhanced mPGES-1 mRNA, an effect that was potentiated by BK and unaffected by indomethacin (results not shown). With the addition of the transcription inhibitor actinomycin D, we were able to demonstrate that IL-1β had no effect on the half-life of mPGES-1 mRNA, but that BK clearly enhanced the decline of mPGES-1 mRNA in cells cotreated with IL-1β and BK (Figure 3c).
Western blot analysis demonstrated that treatment with IL-1β for 24 hours enhanced the expression of mPGES-1 protein. Moreover, this enhancement was not potentiated by BK or DALBK (Figure 3d). The expression of mRNA for mPGES-2 or cPGES in MG-63 cells was not regulated by IL-1β, neither in the absence nor in the presence of BK or DALBK (Figure 3e).
Furthermore, at 4 hours, BK (3 μM), DALBK (3 μM), and IL-1β (100 pg/ml), with or without kinins, did not affect the expression of cytosolic phospholipase A2 (cPLA2) mRNA (results not shown), and there was still no effect of BK, DALBK, and IL-1β on cPLA2 mRNA even after 24 hours (Figure 3f). However, cotreatment with IL-1β and either BK or DALBK caused a small (∼1.8 fold) but statistically significant increase of cPLA2 mRNA expression.
Importance of NF-κB and p38 MAPK in the synergistic interactions between kinins and IL-1β.
The NF-κB inhibitor PDTC (30 μM) substantially inhibited the release of PGE2 that was induced by cotreatment with IL-1β and either BK or DALBK (Figure 4a). The enhancement of COX-2 mRNA induced by cotreatment with IL-1β and kinins was only slightly decreased by PDTC (Figure 4b). The increase in mPGES-1 mRNA in cells treated with kinins and IL-1β was decreased by 40–50% in PDTC-exposed cells (Figure 4c).
The p38 MAPK inhibitor SB203580 (10 μM) abolished the release of PGE2 that was induced by cotreatment with IL-1β and either BK or DALBK (Figure 4d). The enhancement of COX-2 mRNA caused by cotreatment with kinins and IL-1β was inhibited by 80% by SB203580 (Figure 4e). The expression of mPGES-1 mRNA in cells cotreated with kinins and IL-1β was decreased by 50% by the SB203580 inhibitor (Figure 4f).
The MAPK JNK inhibitor SP600125 (30 μM) inhibited the release of PGE2 that was induced by cotreatment with IL-1β and either BK or DALBK (Figure 4g). The enhancement of COX-2 mRNA caused by cotreatment with kinins and IL-1β was inhibited by 70% by SP600125 (Figure 4h). The expression of mPGES-1 mRNA in cells cotreated with kinins and IL-1β was also decreased by 70% by SP600125 (Figure 4i).
Inhibition of ERK MAPK by PD98059 (30 μM) markedly inhibited the release of PGE2 that was induced by cotreatment with IL-1β and either BK or DALBK, without significantly affecting the enhancements of COX-2 and mPGES-1 mRNA (results not shown).
Effects of kinin B1 and B2 receptor agonists and IL-1β on PGE2 formation and COX-2 mRNA expression in mouse calvariae.
To assess whether the effects of kinins and IL-1β observed in the human osteoblastic MG-63 cell line can be obtained in intact mouse calvarial bones, we studied the effects on COX-2 mRNA expression and, in parallel, the production of PGE2 in treated calvarial bone samples. Costimulation with IL-1β (100 pg/ml) and either BK (3 μM) or DALBK (3 μM) caused a synergistic potentiation of IL-1β–induced PGE2 formation, resulting in a 250-fold stimulation of PGE2 formation compared with the untreated control (Figure 5a). BK and IL-1β significantly stimulated the expression of COX-2 mRNA in calvarial bones exposed to the agonists for 24 hours (Figure 5b). In contrast, DALBK alone did not cause any significant enhancement of COX-2 mRNA at 24 hours (Figure 5b), which is consistent with the findings obtained in the MG-63 cells in the 24-hour experiments (Figures 2a and e). In calvarial bones exposed for 24 hours to IL-1β in the presence of BK or DALBK, both kinins potentiated the stimulatory effect of IL-1β on COX-2 mRNA (Figure 5b).
Indomethacin abolished the enhanced PGE2 formation in calvariae costimulated with IL-1β and BK or with IL-1β and DALBK (results not shown). Moreover, IL-1β induced COX-2 mRNA expression in the calvarial bones, and the synergistic up-regulation of COX-2 mRNA caused by BK or DALBK was significantly decreased by indomethacin (Figure 5c). IL-1β in the presence of indomethacin still caused a significant 2.4-fold enhancement of COX-2 mRNA (Figure 5c).
Effects of B1 and B2 receptor agonists and IL-1β on RANKL, RANK, and OPG mRNA expression in mouse calvariae.
IL-1β (300 pg/ml), PTH (1 nM), and 1,25-dihydroxyvitamin D3 (1 nM) significantly enhanced RANKL mRNA in calvarial bones, with a more pronounced effect caused by IL-1β (21-fold stimulation) compared with PTH (11-fold) and 1,25-dihydroxyvitamin D3 (8-fold) (Figure 5d). In contrast, BK and DALBK did not stimulate RANKL mRNA. However, the stimulation caused by IL-1β was significantly potentiated by both BK and DALBK (Figure 5d).
The enhancement of RANKL mRNA induced by IL-1β and the combinations of IL-1β and either BK or DALBK was significantly decreased by indomethacin (Figure 5e). Interestingly, considering the fact that indomethacin only partially inhibits IL-1β–induced 45Ca release (36), it was found that IL-1β, in the presence of indomethacin, still caused a significant 2.7-fold enhancement of RANKL mRNA (Figure 5e).
RANKL protein expression in calvarial bones and culture media was significantly enhanced by IL-1β and 1,25-dihydroxyvitamin D3, whereas BK had no effect (Figures 5f and g). Consistent with the mRNA data, IL-1β caused a more robust enhancement of RANKL protein. Moreover, BK synergistically potentiated the IL-1β–induced expression of RANKL protein.
The expression of OPG mRNA was unaffected by treatment for 24 hours with BK, DALBK, and IL-1β, in the absence and in the presence of kinins (Figure 5h). In contrast, as expected, PTH and 1,25-dihydroxyvitamin D3 decreased the expression of OPG mRNA.
Finally, the expression of RANK mRNA was unaffected by BK, DALBK, and IL-1β, in the absence and in the presence of kinins, and was unaffected by PTH (Figure 5i). In contrast, 1,25-dihydroxyvitamin D3 caused a significant enhancement of RANK mRNA, in agreement with previous findings (37).
The finding that BK and Lys-BK, as well as several B1 receptor agonists, synergistically stimulated PGE2 formation induced by IL-1β or TNFα suggests that signaling through both the B1 and B2 receptors leads to interactions with IL-1 and TNFα receptor signaling, resulting in synergistic stimulation. The synergistic interaction, as assessed by PGE2 formation, was delayed, since no synergism was observed at 4 hours but was clearly observed after 24 hours of costimulation. These observations indicate the possibility that the mechanism causing the synergism involves induction of gene expression. We therefore examined the expression of COX-2 and mPGES-1, the 2 most important enzymes involved in conversion of arachidonic acid to PGE2.
Costimulation with IL-1β or TNFα and either BK or DALBK resulted in potentiation of COX-2 expression by the kinins, in both human osteoblastic MG-63 cells and mouse calvarial bones, a response that could be observed at both the mRNA level and the protein level. The enhancement of steady-state levels of COX-2 mRNA was most likely due to enhanced transcription, since cotreatment with IL-1β and BK decreased the half-life of COX-2 mRNA. Since PGE2 has been shown to increase COX-2 mRNA (38, 39), we evaluated whether the large amounts of PGE2 produced by costimulation with kinins and cytokines were involved in the increased expression of COX-2. Indomethacin did not affect the induction of COX-2 mRNA in MG-63 cells. In contrast, synergistic stimulation of COX-2 mRNA in calvarial bones by IL-1β and kinins was substantially inhibited by indomethacin. These observations indicate that interactions between B2 and B1 receptors and IL-1 and TNFα receptors leads to either a PGE2-independent or a PGE2-dependent induction of COX-2 mRNA expression depending on the cell type. This observation is consistent with the findings that BK can induce COX-2 by NF-κB–dependent or –independent mechanisms in different cell types (40, 41).
Both BK and DALBK potentiated IL-1β– and TNFα-induced expression of mPGES-1 mRNA in MG-63 cells; these effects were not dependent on PGE2 formation, and there were no effects on mPGES-2 or cPGES mRNA. The effect was associated with a decreased half-life of mPGES-1 mRNA, indicating that the effect of kinins was due to potentiation of cytokine-induced transcription of mPGES-1 mRNA. The enhanced mPGES-1 mRNA caused by IL-1β resulted in increased mPGES-1 protein levels. However, the potentiation by B1 and B2 agonists on IL-1β–induced mPGES-1 mRNA did not result in any potentiation of protein levels.
Our results suggest that enhanced expression of COX-2 is part of the mechanism by which kinins synergistically potentiate IL-1β– and TNFα-induced PGE2 formation, a view further substantiated by the observation that the potentiation of COX-2 expression preceded the synergistic effect on PGE2 formation. However, kinin-induced potentiation of mPGES-1 seems not to be involved in the interactions between kinins and IL-1β. It is likely that the increased levels of PGG2/PGH2, formed by the enhanced COX-2 in cells costimulated by kinins and IL-1β, can be sufficiently converted to PGE2 by the IL-1β–induced increase of mPGES-1, since the catalytic efficiency of this enzyme is unusually high (42).
In attempts to study the intracellular signaling mechanisms involved in the interactions between kinins and IL-1β, we have observed that IL-1β and TNFα stimulate the NF-κB pathway in MG-63 cells, as assessed by electrophoretic mobility shift assay (EMSA) and by protein analysis of IκBα (Brechter AB, et al: unpublished observations). EMSA analyses have also demonstrated that both cytokines activate AP-1 in MG-63 cells. We therefore evaluated the importance of the transcription factor NF-κB and 3 MAPKs related to activation of AP-1.
Both the NF-κB inhibitor PDTC and specific inhibitors of the p38, JNK, and ERK MAPKs substantially inhibited PGE2 release induced by cotreatment with either BK or DALBK and IL-1β, indicating that both NF-κB and MAPKs are involved. The effect of PDTC was associated with a marginal decrease of COX-2 mRNA and a partial inhibition of mPGES-1 mRNA, which indicates that other mechanisms also are involved in the large inhibition of PGE2 release. In contrast, the inhibition by the p38 and JNK MAPK inhibitors was associated with substantial inhibitions of COX-2 and mPGES-1 mRNA, whereas the ERK inhibitor did not affect COX-2 or mPGES-1 mRNA, indicating that the p38 and JNK MAPKs are important signal-transducing pathways in the interactions between kinins and IL-1β leading to PGE2 release and COX-2 expression. Consistent with these findings, we have observed that cotreatment of MG-63 cells with IL-1β and BK results in enhanced phosphorylation of p38 and JNK, but not of ERK (Brechter AB, et al: unpublished observations).
Consistent with observations in other cell types (38, 43), stimulation of B2 receptors significantly enhanced COX-2 mRNA in MG-63 cells and in mouse calvarial bones. In addition, this study was the first to show that a BK B1 receptor agonist significantly enhanced COX-2 mRNA in both the human osteoblastic cell line MG-63 and in mouse calvariae. The effect was more delayed and more transient for the B1 agonist compared with the B2 agonist. The findings that BK-induced PGE2 formation is maximal at 30–60 minutes and does not further increase in 24-hour experiments indicate that the BK-induced expression of COX-2 is not crucial for the PGE2 response to BK.
We have also previously shown that the PGE2 response induced by B1 receptor agonists in primary mouse calvarial osteoblasts (16) and in MG-63 cells (18) is delayed by several hours. One explanation for this may be that B1 receptor agonists do not activate cPLA2, a view supported by our observation that DABK, in contrast to BK, did not cause any rapid rise of intracellular calcium in the mouse osteoblastic cell line MC3T3-E1 (44). Our novel observation in the present study, showing that DALBK causes a delayed enhancement of COX-2 mRNA expression, indicates that the delayed PGE2 response after stimulation with B1 receptor agonists may be due to the COX-2 induction. Another possibility explaining the delayed PGE2 response to DALBK might be that B1 receptor agonists cause an autoamplification of its own receptor (45). However, using quantitative real-time PCR and radioligand binding, we have observed that DABK and DALBK do not up-regulate B1 receptor expression in MG-63 cells (Brechter AB, et al: unpublished observations).
Having established the potentiation by kinins on enzymes involved in PG formation, we next sought to determine if the interactions also might involve effects on the expression of molecules involved in osteoclast differentiation and activity. Since the RANKL/RANK/OPG system has been shown to be important in inflammation-induced bone resorption, including that in rheumatoid arthritis (27–29, 46) and in periodontal disease (4, 31), we focused on these molecules. Since RANKL mRNA expression in the MG-63 cells was scarce, we used neonatal mouse calvarial bones for these experiments, the rationale being that activation of BK receptors synergistically potentiates IL-1–induced bone resorption in the calvarial bones (11).
IL-1β caused a significant enhancement of RANKL mRNA and protein in the calvarial bones. In contrast to the effects of PTH and 1,25-dihydroxyvitamin D3, which decreased OPG mRNA, IL-1β did not affect OPG mRNA expression. This observation in the calvarial bones is in contrast to observations in MG-63 cells, primary human osteoblastic cells, and human bone marrow stromal cells, in which IL-1β and IL-1α enhance OPG (47–50). It is likely that the lack of effect by IL-1β on OPG in mouse calvariae is the explanation as to why IL-1β, although causing a more prominent effect on RANKL, is not a more effective stimulator of bone resorption in mouse calvariae as compared with PTH or 1,25-dihydroxyvitamin D3 (results not shown). Whereas BK did not affect RANKL mRNA or protein expression, cotreatment of the calvarial bones with IL-1β and BK caused a synergistic potentiation of IL-1β–induced expression of RANKL mRNA and protein. Cotreatment did not affect OPG mRNA, nor did cotreatment affect RANK mRNA. These observations suggest that the increased expression ratio of RANKL to OPG in osteoblasts is the most likely explanation for the synergistic potentiation of bone resorption caused by kinins.
In the processes of inflammation, several proinflammatory molecules are present that may act either solely or in concert. Our results show that kinins, acting via both the B1 and the B2 receptors, interact with the signaling of IL-1 and TNFα receptors, causing synergistic potentiation of cytokine-induced PGE2 formation by a mechanism involving increased expression of COX-2. Most importantly, these interactions also increase the expression of RANKL, the most crucial activator of osteoclast differentiation and activity in inflammatory disorders such as rheumatoid arthritis and periodontal disease.
Dr. Lerner had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Brechter, Lerner.
Acquisition of data. Brechter.
Analysis and interpretation of data. Brechter, Lerner.
Manuscript preparation. Brechter, Lerner.
Statistical analysis. Brechter.
We thank Mrs. Inger Lundgren and Mrs. Ingrid Boström for skillful technical assistance, Dr. Per-Johan Jakobsson (Karolinska Institutet, Stockholm, Sweden) for providing the rabbit polyclonal primary antibody against human mPGES-1 and the human recombinant mPGES-1, and Dr. Guy Drapeau (Centre de Recherche, Quebec, Canada) for providing the kinin analogs.