Östen Ljunggren

Bradykinin stimulates production of prostaglandin E2 and prostacyclin in murine osteoblasts

The effect of bradykinin on prostaglandin production in mouse calvarial bones and in isolated osteoblasts has been examined. Bradykinin (1 mumol/l) stimulated prostaglandin formation in neonatal mouse calvarial bones incubated for 30 min. In isolated osteoblast-like cells from neonatal mice calvarial bones and in a cloned mouse calvarial osteoblastic cell lineage (MC3T3-E1) bradykinin stimulated the production of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of prostacyclin). The stimulation of PGE2 production occurred rapidly (30 s) and reached its maximum after 5-10 min. The stimulatory effect of bradykinin on PGE2 production in isolated osteoblast-like cells and in MC3T3-E1 cells was dose dependent with apparent half maximal stimulation seen at 10 and 3 nmol/l, respectively. Bradykinin-induced prostaglandin production was totally reversible after withdrawal of the agonist. Pretreatment with bradykinin (1 mumol/l) resulted in desensitization to a subsequent challenge with bradykinin (1 mumol/l), while pretreatment with bradykinin had no effect upon arachidonic acid (30 mumol/l) induced prostaglandin formation. Bradykinin-induced production of PGE2 was abolished by several structurally unrelated, competitive and non-competitive inhibitors of arachidonic acid metabolism as well as by corticosteroids. The mouse calvarial osteoblast-like cells also showed a PGE2 and 6-keto-PGF1 alpha response to thrombin, but not to parathyroid hormone (PTH), calcitonin and 1 alpha(OH)D3. The formation of cyclic AMP in mouse calvarial osteoblasts was enhanced by PTH, bradykinin, thrombin and arachidonic acid but not by calcitonin and 1 alpha(OH)D3. The cyclic AMP response to bradykinin, thrombin and arachidonic acid, but not that to PTH, was abolished by indomethacin. The degree of confluency of the cell cultures greatly influenced the amount of prostaglandins being produced. At higher cell density the amount of prostanoids synthesized per cell was substantially decreased in untreated control cultures as well as in bradykinin- and arachidonic acid-treated cells. These data suggest that osteoblasts are equipped with receptors for bradykinin coupled to prostaglandin production.

Prostaglandin E2 causes a transient inhibition of mineral mobilization, matrix degradation, and lysosomal enzyme release from mouse calvarial bonesIn Vitro

SummaryThe effect of prostaglandin E2 (PGE2) on the kinetic of bone resorptionin vitro was assessed by following the release of minerals and degradation of matrix in cultured mouse calvarial bones. PGE2 (1 and 3 μmol/liter) caused an initial inhibition of the release of45Ca, stable calcium, and inorganic phosphate from unstimulated calvarial bones. The effect was transient and after 24 and 48 hours the release of45Ca, stable calcium, and inorganic phosphate from PGE2-treated bones was enhanced. 0.3 μmol/liter of PGE2 stimulated the release of45Ca after 24 hours, but at this concentration no initial inhibition was observed. The initial inhibitory effect of PGE2 (1 μmol/liter) could be further increased by three structurally different inhibitors of cyclic AMP breakdown. PGE2 (1 μmol/liter) caused not only an initial inhibition of mineral release but also an initial inhibition of matrix degradation, as assessed by the release of3H from [3H]-proline labeled bones. In addition, PGE2 (3 μmol/liter), in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, caused a rapid (6 hours) inhibition of the release of the lysosomal enzymes β-glucuronidase and β-N-acetyl-glucosaminidase, without affecting the release of the cytosolic enzyme lactate dehydrogenase. Similar specific initial inhibition of lysosomal enzyme release was also seen in the presence of calcitonin and dibutyryl cyclic AMP, but not in the presence of parathyroid hormone (PTH). Neither PGE2 nor the phosphodiesterase inhibitors rolipram and Ro 20.1724, could inhibit the initial stages of PTH-induced45Ca release. Nor did PGE2 inhibit the stimulation of radioactive calcium mobilization induced by 1α(OH)-vitamin D3. Enhancement of cyclic AMP formation by forskolin, choleratoxin, and dibutyryl cyclic AMP in unstimulated bones resulted in an initial reduction of45Ca release (6 hours) followed by a delayed stimulation (96 hours). These data indicate that PGE2 has the capacity to reduce the activity of unstimulated, preexisting osteoclasts by a cyclic AMP-dependent process.

Evidence for BK1 bradykinin-receptor-mediated prostaglandin formation in osteoblasts and subsequent enhancement of bone resorption.

1 The effects of the BK1 bradykinin (BK)‐receptor agonist des‐Arg9‐BK on bone resorption and prostaglandin formation in osteoblasts have been studied. 2 Des‐Arg9‐BK (1 μm) stimulated the release of 45Ca from prelabelled neonatal mouse calvarial bones and the formation of prostaglandin E2 (PGE2) in calvarial bones. The stimulatory effect on bone resorption and PGE2 formation could be totally inhibited by indomethacin, flurbiprofen and hydrocortisone. 3 The BK1 receptor antagonist des‐Arg9‐Leu8‐BK (10 μm) inhibited des‐Arg9‐BK (0.01–0.1 μm)‐induced release of 45Ca from prelabelled neonatal mouse calvarial bones, while leaving BK (0.1–1 μm)‐induced 45Ca release unaffected. 4 In isolated osteoblast‐like cells from neonatal mouse calvarial bones, des‐Arg9‐BK (1 μm) induced a slowly developing increase in PGE2 formation that was significantly different from untreated controls after 24 h. Treatment with BK caused a rapid burst (within minutes) of PGE2 formation. 5 Des‐Arg9‐Leu8‐BK (10 μm) selectively inhibited des‐Arg9‐BK (1 μm)‐induced PGE2 and prostacyclin formation in isolated osteoblast‐like cells incubated for 72 h. Des‐Arg9‐Leu8‐BK did not affect BK and Lys‐BK (1 μm)‐induced PGE2 and prostacyclin formation in isolated osteoblast‐like cells incubated for 72 h. 6 These data indicate that osteoblasts are equipped with BK1‐receptors mediating enhanced prostaglandin formation and subsequent bone resorption.

Comparison of human interleukin-1β and its 163–171 peptide in bone resorption and the immune response

Human interleukin-1 beta (IL-1 beta) caused a dose- and time-dependent enhancement of the release of 45Ca from prelabeled mouse calvaria in organ culture. In addition, IL-1 beta dose-dependently stimulated the formation of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha in the calvarial bones. However, IL-1 beta-induced 45Ca release was only partially inhibited by blocking the PGE2 response with indomethacin, suggesting that enhanced PGE2 formation in response to IL-1 beta is not necessary to obtain a bone resorptive effect, but that prostaglandins potentiate the action of IL-1 beta. The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, administered simultaneously with antigen (SRBC) to C3H/HeN male mice, induced a dose-dependent enhancement of specific antibody-producing cells in the spleen (PFC). The degree of PFC stimulation was comparable to that caused by native human IL-1 beta. In mouse bone cultures, neither 45Ca release nor prostanoid formation was stimulated by fragment 163-171. These data indicate that (1) IL-1 beta-induced stimulation of bone resorption is dissociable from IL-1 beta-induced increase of prostanoid biosynthesis and (2) the epitope of the IL-1 beta molecule involved in the immunostimulatory effects may be different from that involved in the stimulatory effects on bone resorption.

Thrombin increases cytoplasmic Ca2+ and stimulates formation of prostaglandin E2 in the osteoblastic cell line MC3T3-El

Using microfluorometric analysis in individual, fura-2 loaded cells, we found that thrombin (0.1-10 U/ml) caused a dose-dependent (EC50 approximately 0.5 U/ml), rapid (within seconds), transient increase in cytoplasmic Ca2+ in the osteoblastic cell line MC3T3-El. The thrombin induced rise in cytoplasmic Ca2+ was not dependent on extracellular Ca2+ and was unaffected by indomethacin. In MC3T3-El cells, thrombin (0.3-10 U/ml) caused a rapid and dose-dependent (EC50 approximately 0.5 U/ml) stimulation of PGE2 formation. The calcium ionophore A23187 (2 mumol/l) also rapidly stimulated an increase in cytoplasmic Ca2+ and the formation of PGE2 in MC3T3-El cells. These data indicate that thrombin mobilizes Ca2+ from intracellular stores and that Ca2+ may serve as a second messenger in thrombin induced stimulation of PGE2 biosynthesis in osteoblasts.

Haptoglobin synergistically potentiates bradykinin and thrombin induced prostaglandin biosynthesis in isolated osteroblasts

Haptoglobin of two different phenotypes (Hp 1-1 and Hp 2-1) dose-dependently (1-4 mg/ml) stimulated the formation of prostaglandin E2 (PGE2) in osteoblast-like cells isolated from neonatal mouse calvarial bones. The degree of stimulation obtained by haptoglobins (4 mg/ml) on PGE2 biosynthesis was in the same range as that caused by bradykinin (1 mumol/l). Pretreatment of osteoblasts with Hp 1-1 or Hp 2-1 (1-4 mg/ml) resulted in a dose-dependent, synergistic potentiation of the stimulatory effect of bradykinin (1 mumol/l) on PGE2 formation. Thrombin (7 U/ml) stimulated PGE2 formation in the osteoblast-like cells by a mechanism that was also synergistically potentiated by haptoglobin (2 mg/ml). These data show that haptoglobin per se stimulates PGE2 biosynthesis in isolated osteoblasts and, in addition, synergistically potentiates the effect of bradykinin and thrombin. Consequently, the enhanced production of haptoglobin seen in different inflammatory processes may contribute to the destruction of bone by inducing the formation of prostanoids capable of stimulating bone resorption.

Bradykinin stimulates prostaglandin E2 formation in isolated human osteoblast-like cells

The effect of bradykinin on prostaglandin E2 formation in cells from human trabecular bone has been studied. The cells responded to parathyroid hormone with enhanced cyclic AMP formation and were growing as cuboidal-shaped, osteoblast-like cells. In these isolated human osteoblast-like cells, bradykinin (1 μmol/l) caused a rapid (5 min) stimulation of prostaglandin E2 formation. This finding indicates that human osteoblasts are equipped with receptors for bradykinin linked to an increase in prostaglandin formation.

Forskolin sensitizes parathyroid hormone-induced cyclic AMP response, but not the bone resorptive effect, in mouse calvarial bones

The effect of forskolin on parathyroid hormone (PTH) stimulated bone resorption, as assessed in vitro by the release of 45Ca from prelabelled neonatal mouse calvarial bones, and cyclic AMP formation in mouse calvarial bones and osteoblast-like cells was investigated. Much higher concentrations (100-300-times) of PTH were required to stimulate cyclic AMP accumulation than to stimulate mineral mobilization in murine calvarial bones. PTH, in the absence of phosphodiesterase inhibitor, stimulated cyclic AMP formation in mouse calvarial bones at and above concentrations of 3-10 nmol/l with EC50 at 10-15 nmol/l. In the presence of forskolin (1 or 10 mumol/l) the minimal concentration required to obtain a cyclic AMP response to PTH was decreased by a factor of 30-100 and the EC50 value was decreased to 1-2 nmol/l. Similar results were seen in osteoblast-enriched cells. In addition, the magnitude of the PTH-induced cyclic AMP response was substantially potentiated by forskolin, both in calvarial bones and in isolated osteoblasts. Forskolin, in the absence of PTH, stimulated cyclic AMP levels in mouse calvaria at and above 1 mumol/l. In the presence of PTH, the response to forskolin was potentiated over the whole dose-response curve with apparent EC50 value at 1-2 mumol/l of forskolin. Forskolin (1 mumol/l) did not affect the magnitude of the 45Ca release response to PTH in 24 or 48 h cultures. In 96 h cultures, forskolin, in an additive manner, potentiated the effect of PTH on calcium mobilization. These results show that forskolin, in mouse calvarial bones and in isolated osteoblasts, in addition to directly stimulating cyclic AMP, can enhance receptor-mediated activation of adenylate cyclase. The finding that forskolin did not synergistically potentiate PTH-induced bone resorption suggests that there is no simple relationship between PTH-induced cyclic AMP formation and stimulation of bone resorption.

INHIBITORY EFFECTS OF GAMMA -INTERFERON ON BRADYKININ-INDUCED BONE RESORPTION AND PROSTAGLANDIN FORMATION IN CULTURED MOUSE CALVARIAL BONES

The effects of mouse recombinantγ-interferon (γ-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and prostaglandin E2 (PGE2) have been studied using cultures of neonatal calvarial bones and analyzing the release of45Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects ofγ-IFN and indomethacin on formation of PGE2 in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 μmol/l) totally abolished bradykinin (1 μmol/l) induced45Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE2 (1 μmol/l).γ-IFN (1000 U/ml) almost totally inhibited45Ca release stimulated by bradykinin (1 μmol/l), but the inhibitory effect could only be partially overcome by PGE2.γ-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg9-bradykinin (1 μmol/l) on45Ca release. The stimulatory effects of PGE2 (1 μmol/l) on radioactive calcium mobilization was partially inhibited byγ-IFN (1000 U/ml), whereas indomethacin (1 μmol/l) was without effect. The inhibitory effect ofγ-IFN on45Ca release stimulated by bradykinin and PGE2 was dose-dependent with threshold for action at 3–30 U/ml. Comparative dose-response curves showed thatγ-IFN was most potent as inhibitor of bradykinin induced45Ca release. Bradykinin (1 μmol/l) significantly stimulated PGE2 formation by a mechanism that was completely inhibited by indomethacin (1 μmol/l).γ-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE2 formation. These data show that i)γ-IFN is a potent inhibitor of bone resorption induced by bradykinin and bradykinin analogues and ii) that the mechanism of action can be mainly explained by an inhibition of kinin induced prostaglandin biosynthesis. The results, however, also show thatγ-IFN can inhibit bone resorption by mechanisms unrelated to prostaglandin formation.