Stimulate Bone Resorption Research Articles

CAMP activation by PACAP/VIP stimulates IL‐6 release and inhibits osteoblastic differentiation through VPAC2 receptor in osteoblastic MC3T3 cells

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/vasoactive intestinal peptide (VIP) superfamily, stimulates cyclic AMP accumulation initiating a variety of biological processes such as: neurotropic actions, immune and pituitary function, learning and memory, catecholamine biosynthesis and regulation of cardiopulmonary function. Both osteoclasts and osteoblasts have been shown to express receptors for PACAP/VIP implicated in their role in bone metabolism. To further understand the role of PACAP/VIP family in controlling bone metabolism, we investigated differentiation model of MC3T3-E1 cells, an osteoblastic cell line derived from mouse calvaria. Quantitative RT-PCR analysis demonstrated that MC3T3-E1 cells expressed only VPAC2 receptor and its expression was upregulated during osteoblastic differentiation, whereas VPAC1 and PAC1 receptors were not expressed. Consistent with expression of receptor subtype, both PACAP and VIP stimulate cAMP accumulation in a time- and dose-dependent manner with the similar potency in undifferentiated and differentiated cells, while Maxadilan, a specific agonist for PAC1-R, did not. Furthermore, downregulation of VPAC2-R by siRNA completely blocked cAMP response mediated by PACAP and VIP. Importantly, PACAP/VIP as well as forskolin markedly suppressed the induction of alkaline phosphatase mRNA upon differentiation and the pretreatment with 2',5'-dideoxyadenosine, a cAMP inhibitor, restored its inhibitory effect of PACAP. We also found that PACAP and VIP stimulated IL-6 release, a stimulator of bone resorption, and VPAC2-R silencing inhibited IL-6 production. Thus, PACAP/VIP can activate adenylate cyclase response and regulate IL-6 release through VPAC2 receptor with profound functional consequences for the inhibition of osteoblastic differentiation in MC3T3-E1 cells.

Acute Effects Of Intense Exercise And Calcium Supplementation On Calcium Homeostasis And Bone Markers

Although exercise is accepted as having favorable effects on bone, under certain conditions exercise has been associated with a decrease in bone mineral density (BMD). One potential mechanism is an exercise-induced increase in parathyroid hormone (PTH), a potent stimulator of bone resorption, possibly triggered by dermal calcium loss. PURPOSE: To determine whether calcium (Ca) supplementation before or during exercise attenuates exercise-induced increases in PTH and C-terminal telopeptide of type I collagen (CTX, a marker of bone resorption), when compared with placebo. METHODS: Twenty competitive male athletes (cyclists and triathletes) completed three 35-km time trials on a cycle ergometer under differing Ca supplementation conditions: 1) 1000 mg of Ca 20 min before exercise and placebo during, 2) placebo before and 250 mg of Ca every 15 min during exercise (1000 mg total), or 3) placebo before and during the exercise bout. Supplementation was double-blinded and rides were performed in random order. PTH, CTX, ionized calcium (iCa), and bone-specific alkaline phosphatase (BAP) were measured before and immediately after exercise. RESULTS: PTH and CTX increased and iCa decreased significantly in response to exercise under all test conditions. Ca before exercise significantly attenuated the increase in PTH relative to placebo; there was a similar trend for Ca during exercise. There were no significant effects of Ca on changes in CTX, BAP, and iCa.TableCONCLUSION: Ca supplementation before, and perhaps during, exercise attenuated disruptions of Ca homeostasis. Further research is needed to determine the effects of increases in PTH and CTX on bone and whether Ca supplementation can diminish any exercise-induced demineralization.

Hypervitaminosis A and bone.

Animal, human, and in vitro data all indicate that excess vitamin A stimulates bone resorption and inhibits bone formation. This combination would be expected to produce bone loss and to contribute to osteoporosis development and may occur with relatively low vitamin A intake. It is possible that unappreciated hypervitaminosis A contributes to osteoporosis pathogenesis.

Inhibition of osteoclast formation and function by bicarbonate: Role of soluble adenylyl cyclase

High [HCO(3)(-)] inhibits and low [HCO(3)(-)] stimulates bone resorption, which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, and 24 mM HCO(3)(-) at pH 7.4 for 7-8 days and assayed for tartrate-resistant acid phosphatase (TRAP) and vacuolar-ATPase expression, and H+ accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by HCO(3)(-) in a dose-dependent manner. V-ATPase expression and H+ accumulation normalized to cell cross-sectional area or protein were not significantly changed. The HCO(3)(-) -induced inhibition of osteoclast growth and differentiation was blocked by either 2-hydroxyestradiol, an inhibitor of sAC or sAC knockdown by sAC specific siRNA. The model of HCO(3)(-) inhibiting osteoclast via sAC was further supported by the fact that the HCO(3)(-) dose-response on osteoclasts is flat when cells were saturated with 8-bromo-cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a 1-week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro-computed tomography (microCT), was higher in 24 mM HCO(3)(-) compared to 12 mM HCO(3)(-) treated calvaria. This HCO(3)(-) effect on BV/TV was blocked by 2-hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by HCO(3)(-), by acting as a HCO(3)(-) sensor.

Could insertion of the particles that induce osteolysis be a new treatment option in heterotopic ossification?

Heterotopic ossification (HO) is the formation of the bone outside the skeletal system, it is a common complication of surgery or trauma. The abnormal deposition or precipitation of calcium phosphate in tissues may be massive and clinically significant sometimes. Severe ossification often reduce joint function and induce pain. Unfortunately, to date, there is no satisfactory treatment available yet. Nowadays, periprosthetic osteolysis induced by wear debris is the most widely accepted cause of aseptic loosening after total joint replacement. A large number of researches have identified metal particles can stimulate bone resorption by targeting osteoclasts. So it is hypothesized that the insertion of metal particles in the region surrounding heterotopic bone could be a new treatment option in HO.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement

Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate differences in MMP and TIMP levels in the gingival crevicular fluid (GCF) at the resorption and apposition sides of orthodontically moved teeth, and to compare these with control teeth. GCF samples were collected from eight orthodontic patients wearing fixed appliances with superelastic nickel-titanium coil springs. The samples were analysed by gelatin zymography, which allows detection of both active and latent MMPs, and reverse zymography for analysis of TIMPs. Western blotting was performed to confirm the identity of MMPs. The data were analysed using either the one-way analysis of variance or the Kruskal-Wallis test. In general, higher levels of MMPs and TIMPs were found at both the resorption and apposition sides compared with the control teeth. Remarkably, partially active MMP-1 was found in GCF from both the resorption and the apposition side but was barely present at the control teeth. TIMP-1 was strongly increased at the apposition side. Gelatinases were mainly present at the resorption side, while gelatinolytic fragments were exclusively detected at the apposition side. MMP-9, which is known to be involved in bone degradation, and a 48 kDa gelatinase were increased at the resorption side. The small increase in TIMP-1 at the resorption side might stimulate bone resorption, whereas the large increase at the apposition side reduces bone resorption. The analysis of MMPs and TIMPs may contribute to the improvement of orthodontic treatment regimens.

Expression of RANK‐ligand in prostate cancer cell lines

The molecular mediators of bone remodelling, receptor activator of nuclear factor‐kappaB ligand (RANKL), receptor activator of nuclear factor‐kappaB (RANK) and osteoprotegerine (OPG), are believed to be involved in the cellular mechanisms by which tumours metastasize to bone. RANKL is a potent stimulator of osteoclastic bone resorption and is expressed in a variety of tumour cells. We have investigated if the membrane bound form of RANKL is expressed in prostate cancer cell lines, and whether this expression might be regulated by the presence of human osteoblasts. Three prostate cancer cell lines were co‐cultured with human osteoblast‐like cells (hOB) and RANKL expression on cell surface was measured by FACS. We found basal expression of RANKL on the cell surface, and in co‐culture with hOBs the number of cells expressing RANKL was increased between 2.5 and 4 times. These data suggest a signalling mechanism between bone cells and prostate cancer cells that might increase bone resorption and thereby promote bone metastases.

β2-Adrenoreceptor ligands regulate osteoclast differentiation in vitro by direct and indirect mechanisms

Pharmacological modulators of β-adrenoceptors can influence bone mineral density and fracture risk in humans. Studies reported that β-adrenoceptor ligands stimulate bone resorption by enhancing the expression of RANK-L, whereas the mechanisms by which β-adrenoreceptors regulate bone formation are poorly understood. Here we show that β2-adrenoceptor is predominantly expressed by bone cells, although low levels of β1- and β3-adrenoceptors were detectable. Noradrenaline and the selective β2-adrenoceptor agonists isoprenaline and salmeterol stimulated osteoclast formation and bone resorption in BM osteoblast co-cultures and increased expression of RANK-L by osteoblasts. All three ligands enhanced RANK-L induced osteoclast formation and increased osteoclast multinuclearity. There was no significant effect of noradrenaline or isoprenaline on osteoblast growth, differentiation or function. These findings confirm the importance of the sympathetic nervous system in the regulation of bone mass, and demonstrate that pharmacological agonists of β2-adrenoceptors directly and indirectly stimulate osteoclast formation, but have no direct effect on osteoblast growth, differentiation or function.

In vitro and in vivo evidence for stimulation of bone resorption by an EP4 receptor agonist and basic fibroblast growth factor: Implications for their efficacy as bone anabolic agents

Prostaglandin E2 receptor subtype 4 agonists (EP4A) and basic fibroblast growth factor (FGF2) stimulate bone formation, but their effects on bone resorption are controversial. To provide additional insight into the skeletal effects of EP4A and FGF2, their regulation of expression of genes associated with bone formation and resorption in aged ovariectomized (OVX) rats and in cultured mouse bone marrow cells was determined. RNA was isolated from lumbar vertebrae of OVX rats (16 months of age) treated daily for 3 weeks with FGF2 or EP4A and processed for quantitative real time-PCR analyses. mRNA expression for the receptor activator of NF-κB ligand (RANKL) and cathepsin K (CTSK), but not osteoprotegerin (OPG), were upregulated by both FGF2 and EP4A. Addition of FGF2 and EP4A to the medium of cultured mouse bone marrow cells increased the formation of tartrate resistant acid phosphatase (TRAP) positive cells, upregulated the expression of RANKL and CTSK, and downregulated expression for OPG. EP4A also increased the formation of actin rings, an indicator of osteoclast activation, in a dose dependent manner in osteoclasts cultured on bone slices and triggered the formation of pits as revealed by a pitting assay. Gene expression for osterix (OSX) and IGF-2, genes associated with bone formation, was significantly greater in FGF2-treated OVX rats compared with EP4A-treated OVX rats. These findings at the molecular level are consistent with previous tissue-level histomorphometric findings, and at the doses tested, support the contention that FGF2 has a stronger bone anabolic effect than EP4A. The results of these in vivo and in vitro analyses clarify the effects of FGF2 and EP4A on bone formation and resorption, and provide insight into differences in the efficacy of two potential bone anabolic agents for restoration of lost bone mass in the osteopenic, estrogen-deplete skeleton.

Osteoprotegerin: regulator, protector and marker

Experimental and clinical trials in the field of bone biology helped to clarify the role of receptors, which belong to the tumor necrosis factor family, such as osteoprotegerin and receptor activator of nuclear factor kappaB (RANK), in the regulation of bone remodeling. The ligand of the receptor activator of nuclear factor kappaB (RANKL) is a stimulator of bone resorption, while osteoprotegerin is the soluble "decoy" receptor to RANKL, protecting thereby bone from resorption. Pathological states of bone remodeling (like osteoporosis) are associated with imbalance in the activity of osteoprotegerin and the receptor activator of nuclear factor kappaB. Recent studies, however, also indicate that the osteoprotegerin/RANKL/RANK system has important roles in the regulation of the immune and vascular system as well. In this review we summarize the function and regulation of osteoprotegerin, its role in pathological states--primarily in cardiovascular diseases--and its relevance as a marker of cardiovascular risk. Finally, we present our prospective trial performed among the chronic dialyzed patients, where we examined the association between the cardiovascular mortality, osteoprotegerin levels and the arterial stiffness.

Staphylococcus aureusInduces Expression of Receptor Activator of NF-κB Ligand and Prostaglandin E2in Infected Murine Osteoblasts

Osteomyelitis is an inflammatory disease of the bone that is characterized by the presence of necrotic bone tissue and increased osteoclast activity. Staphylococcus aureus is responsible for approximately 80% of all cases of human osteomyelitis. While the disease is especially difficult to treat, the pathogenesis of S. aureus-induced osteomyelitis is poorly understood. Elucidating the molecular mechanisms by which S. aureus induces osteomyelitis could lead to a better understanding of the disease and its progression and development of new treatments. Osteoblasts can produce several soluble factors that serve to modulate the activity or formation of osteoclasts. Receptor activator of NF-kappaB ligand (RANK-L) and prostaglandin E(2) (PGE(2)) are two such molecules which can promote osteoclastogenesis and stimulate bone resorption. In addition, previous studies in our laboratory have shown that osteoblasts produce inflammatory cytokines, such as interleukin 6, following infection with S. aureus, which could induce COX-2 and in turn PGE(2), further modulating osteoclast recruitment and differentiation. Therefore, we hypothesized that following infection with S. aureus, osteoblasts will express increased levels of RANK-L and PGE(2). The results presented in this study provide evidence for the first time that RANK-L mRNA and protein and PGE(2) expression are upregulated in S. aureus-infected primary osteoblasts. In addition, through the use of the specific COX-2 inhibitor NS 398, we show that when PGE(2) production is inhibited, RANK-L production is decreased. These data suggest a mechanism whereby osteoblasts regulate the production of RANK-L during infection.

The effect of β-blockers on bone metabolism as potential drugs under investigation for osteoporosis and fracture healing

Background: β-Adrenergic receptor antagonists (β-blockers) have a well-recognised antihypertensive action that is mediated through a reduction in cardiac output and in the release of renin from the kidneys and inhibition of the action of endogenous catecholamines on β-adrenergic receptors. This class of drugs has been shown to reduce the incidence of cardiovascular disease. Recent evidence suggests that β-blockers may also have an effect on bone structure, metabolism and fracture healing. Objective: This paper reviews in vitro and in vivo data that suggest β-blockers have primarily an anabolic effect on bone metabolism. Results: The sympathetic nervous system has a catabolic effect on bone, and in vitro studies have shown that adrenergic agonists stimulate bone resorption. The β-blocker propranolol has been shown to increase bone formation in ovariectomised female rats. Also, recent observational clinical studies provide evidence to show that β-blockers are associated with reduction in fracture risk in both men and women. Conclusion: Although there are some controversial studies, most research concludes that β-blockers show promise in the treatment of osteoporosis and fracture healing.

Effects of RANKL, osteoprotegerin, calcium and phosphorus on survival and activation of Muscovy duck osteoclasts in vitro

The aim of this study was to determine whether receptor activator of nuclear factor NF-κB ligand (RANKL), osteoprotegerin (OPG) and a calcium:phosphorus (Ca:P) ratio of 2:1 could affect survival and activation of Muscovy duck osteoclasts (OCs). Bone marrow cells were obtained from 5-day-old Muscovy ducks and cultured with (Group A) No added factors, (B) 30 ng/mL soluble RANKL (sRANKL), (C) 30 ng/mL sRANKL and 10 ng/mL OPG, (D) 10 ng/mL OPG, (E) 50 ng/mL OPG, (F) 100 ng/mL OPG and (G) 30 ng/mL sRANKL, 6 mmol/L Ca and 3 mmol/L P. sRANKL promoted the survival of OCs on day 2, whereas the number of OCs decreased with addition of OPG in a dose-dependent manner. OPG and Ca:P (2:1) both inhibited OC survival induced by RANKL. RANKL stimulated bone resorption by OCs, whereas OPG, but not Ca:P (2:1), inhibited the activity of OCs induced by RANKL. RANKL promotes the survival and activation of OCs from Muscovy ducks, whereas OPG and, to a lesser extent, Ca:P (2:1) reduce the life span and inhibited the activation of OCs induced by RANKL.

RANKL, osteopontin, and osteoclast homeostasis in a hyperocclusion mouse model

The biological mechanisms that maintain the position of teeth in their sockets establish a dynamic equilibrium between bone resorption and apposition. In order to reveal some of the dynamics involved in the tissue responses towards occlusal forces on periodontal ligament (PDL) and alveolar bone homeostasis, we developed the first mouse model of hyperocclusion. Swiss-Webster mice were kept in hyperocclusion for 0, 3, 6, and 9 d. Morphological and histological changes in the periodontium were assessed using micro-computed tomography (micro-CT) and ground sections with fluorescent detection of vital dye labels. Sections were stained for tartrate-resistant acid phosphatase, and the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteopontin (OPN) was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). Traumatic occlusion resulted in enamel surface abrasion, inhibition of alveolar bone apposition, significant formation of osteoclasts at 3, 6 and 9 d, and upregulation of OPN and RANKL. Data from this study suggest that both OPN and RANKL contribute to the stimulation of bone resorption in the hyperocclusive state. In addition, we propose that the inhibition of alveolar bone apposition by occlusal forces is an important mechanism for the control of occlusal height that might work in synergy with RANKL-induced bone resorption to maintain normal occlusion.

Osteoporosis and Inflammation

Osteoporosis represents a major healthcare burden, affecting approximately 10 million people aged over 50 years in the United States and with another 30 million or more at risk. One of the major contributing factors to osteoporosis is withdrawal of estrogen during menopause in women. Human and animal experiments have implicated pro-inflammatory cytokines as primary mediators of the accelerated bone loss at menopause including interleukin-1, tumor necrosis factor-alpha, and interleukin-6. Increased production of pro-inflammatory cytokines is associated with osteoclastic bone resorption in a number of disease states including rheumatoid arthritis, periodontitis, and multiple myeloma; estrogen withdrawal is associated with increased production of pro-inflammatory cytokines, and exposure of bone cultures to supernatants from activated leukocytes is associated with increased bone resorption. A major advance has been the discovery of RANKL, its receptor RANK, and the endogenous inhibitor osteoprotegerin. The binding of RANKL to RANK is essential for the differentiation and activation of osteoclasts and mediates the actions of essentially all known stimulators of osteoclastic bone resorption. RANKL expression is heightened in post- compared with pre-menopausal women, and this effect is attenuated by estrogen replacement therapy. RANKL is also a therapeutic target; a human antibody with high specificity and affinity to RANKL is currently under clinical evaluation for the treatment of osteoporosis in post-menopausal women and of metastatic bone disease in cancer patients with bone metastasis. Early data are promising.

IL-1β and TNF-α Regulate IL-6-type Cytokines in Gingival Fibroblasts

Interleukin-6 (IL-6)-type cytokines are pleiotropic molecules capable of stimulating bone resorption and expressed by numerous cell types. In the present study, we tested the hypothesis that gingival fibroblasts may exert local osteotropic effects through production of IL-6 and related cytokines. IL-6-type cytokine expression and regulation by IL-1beta and tumor necrosis factor-alpha (TNF-alpha) were studied in fibroblasts from the non-inflamed gingiva of healthy individuals. Constitutive mRNA expression of IL-6, IL-11, and leukemia inhibitory factor (LIF), but not of oncostatin M (OSM), was demonstrated, as was concentration-dependent stimulation of IL-6 and LIF mRNA and of protein by IL-1beta and TNF-alpha. IL-11 mRNA and protein were concentration-dependently stimulated by IL-1beta. The signaling pathway involved in IL-6 and LIF mRNA stimulation involved MAP kinases, but not NF-kappaB. The findings support the view that resident cells may influence the pathogenesis of periodontal disease through osteotropic IL-6-type cytokine production mediated by activation of MAP kinases. IL-1alpha (interleukin-1alpha); IL-1beta (interleukin-1beta); IL-6 (interleukin-6); IL-11 (interleukin-11); LIF (leukemia inhibitory factor); OSM (oncostatin M); alpha(1)-coll. I (alpha(1)-collagen I); ALP (alkaline phosphatase); BMP-2 (bone morphogenetic protein-2); OC (osteocalcin); BSP (bone sialoprotein); TNFR I (tumor necrosis factor receptor I); TNFR II (tumor necrosis factor receptor II); IL-1R1 (interleukin-1 receptor 1); GAPDH (glyceraldehyde-3-phosphate dehydrogenase); RPL13A (ribosomal protein L13A); mRNA (messenger ribonucleic acid); cDNA (complementary deoxyribonucleic acid); PCR (polymerase chain-reaction); BCA (bicinchoninic acid); ELISA (enzyme-linked immunosorbent assay); alpha-MEM (alpha modification of Minimum Essential Medium); and FCS (fetal calf serum).

Is hyperhomocysteinemia a risk factor for osteoporosis?

Hyperhomocysteinemia (HHCY) has been accepted as a risk factor not only for cardiovascular diseases (CVDs) but also for osteoporosis. Furthermore, clinical data suggest that HHCY also increases fracture risk, but have no or only minor effects on bone mineral density (BMD). Measurement of biochemical bone turnover markers indicates a shift of bone metabolism toward bone resorption. Animal studies confirm these observations showing a reduced bone quality and stimulation of bone resorption in hyperhomocysteinemic animals. In addition, homocysteine (HCY) has been found to accumulate in bone tissue by collagen binding. These results indicate a causal involvement of HHCY in osteoporosis. The first experimental studies revealed that several pathomechanisms might be involved. Cell culture studies demonstrate that high HCY and low vitamin B levels stimulate osteoclasts but not osteoblasts, indicating again a shift of bone metabolism toward bone resorption. In addition, HHCY seems to have adverse affects on extracellular bone matrix by disturbing collagen crosslinking. Well-performed large cell culture studies confirm the results obtained with exogenous HCY administration. In conclusion, existing data suggest that HHCY (and possibly vitamin B deficiencies) adversely affects bone quality by a stimulation of bone resorption and disturbance of collagen crosslinking.

Pengaruh Induksi Lipopolisakarida (LPS) terhadap Osteopontin Tulang Alveolaris Tikus pada Masa Erupsi Gigi

Tooth eruption is a complex process that involves osteoclasts to form the eruption pathway. Lipopolysaccharide (LPS) is a bacterial component that plays multifunctional roles in the inflammatory reactions, and one of these roles is powerful stimulation of bone resorption. Osteopontin (OPN) i a phosphorylated glycoprotein the expression of which is associated with migration, attachment and signaling of osteoclast. The objective of this study was to investigate the effect of LPS on rat alveolaris bone osteopontin during the period of tooth eruption. Fifty five Wistar male albino rats, five days of age were divided into three groups. The first group was not subjected to any treatment. The second group was inducted with LPS at five days of age. The third group was inducted with LPS at nine days of age. Expression of OPN was analysed by immunohistochemical (IHC) approach. The results showed significant differences in OPN expression (p<0.05) within the treated subjects. It was concluded that LPS induction during tooth eruption increases the level of alveolar bone osteopontin.

Mechanisms for formation of myeloma bone disease

Myeloma cells stimulate bone resorption by enhancing osteoclast formation and suppress bone formation by inhibiting osteoblast differentiation. Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta as well as RANK ligand play a major role in the enhancement of bone resorption in myeloma. Myeloma cell-derived soluble Wnt inhibitors as well as TGF-beta released from the bone tissues through enhanced bone resorption are thought to suppress osteoblast differentiation. Such pathognomonically skewed cellular components in the bone marrow create a microenvironment suitable for myeloma cell growth and survival (a myeloma niche) , which should be targeted to suppress myeloma expansion along with amelioration of bone lesions.

Kinin B1 and B2 receptor expression in osteoblasts and fibroblasts is enhanced by interleukin-1 and tumour necrosis factor-α. Effects dependent on activation of NF-κB and MAP kinases

Pro-inflammatory mediators formed by the kallikrein–kinin system can stimulate bone resorption and synergistically potentiate bone resorption induced by IL-1 and TNF-α. We have shown that the effect is associated with synergistically enhanced RANKL expression and enhanced prostaglandin biosynthesis, due to increased cyclooxygenase-2 expression. In the present study, the effects of osteotropic cytokines and different kinins on the expression of receptor subtypes for bradykinin (BK), des-Arg10-Lys-BK (DALBK), IL-1β and TNF-α have been investigated. IL-1β and TNF-α enhanced kinin B1 and B2 receptor binding in the human osteoblastic cell line MG-63 and the mRNA expression of B1 and B2 receptors in MG-63 cells, human gingival fibroblasts and intact mouse calvarial bones. Kinins did not affect mRNA expression of IL-1 or TNF receptors. EMSA showed that IL-1β and TNF-α activated NF-κB and AP-1 in MG-63 cells. IL-1β stimulated NF-κB via a non-canonical pathway (p52/p65) and TNF-α via the canonical pathway (p50/p65). Activation of AP-1 involved c-Jun in both IL-1β and TNF-α stimulated cells, but c-Fos only in TNF-α stimulated cells. Phospho-ELISA and Western blots showed that IL-1β activated JNK and p38, but not ERK 1/2 MAP kinase. Pharmacological inhibitors showed that NF-κB, p38 and JNK were important for IL-1β induced stimulation of B1 receptors, and NF-κB and p38 for B2 receptors. p38 and JNK were important for TNF-α induced stimulation of B1 receptors, whereas NF-κB, p38 and JNK were involved in TNF-α induced expression of B2 receptors. These data show that IL-1β and TNF-α upregulate B1 and B2 receptor expression by mechanisms involving activation of both NF-κB and MAP kinase pathways, but that signal transduction pathways are different for IL-1β and TNF-α. The enhanced kinin receptor expression induced by the pro-inflammatory cytokines IL-1β and TNF-α might be one important mechanism involved in the synergistic enhancement of prostaglandin formation caused by co-treatment with kinins and one of the two cytokines. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including periodontitis and rheumatoid arthritis.