Stimulation of prostaglandin E2 and bone resorption by recombinant human interleukin 1 alpha in fetal mouse bones

Recombinant human interleukin 1 alpha (rhIL-1 alpha) stimulates prostaglandin E2 and bone resorption in cultured forearm bones of fetal mouse in a dose-dependent manner: the minimal rhIL-1 alpha to elicit a significant bone resorption was 1.6 ng/ml (89 pM). The half maximal concentrations to elicit bone resorption and thymocyte proliferation were 3.3 ng/ml (183 pM) and 0.31 ng/ml (17 pM), respectively. The bone resorbing activity induced by IL-1 was partially inhibited by indomethacin and hydrocortisone, and completely inhibited by anti-IL 1-antibody. There was a good correlation between PGE2 production and bone resorption induced by IL-1 alpha. These results suggest that rhIL-1 alpha stimulates bone resorption at approximately 10 times the concentrations necessary for thymocyte proliferation and that PGE2 produced in the bone is at least in part involved in osteoclastic bone resorption.     

Recombinant human interleukin 1 alpha and beta stimulate mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

The effects of interleukin 1 (IL-1) on MC3T3-E1 cells (clonal osteoblast-like cells established from mouse calvaria) were studied to elucidate the mechanism of IL-1-induced bone resorption. Recombinant human interleukin 1 alpha (rhIL-1 alpha) and beta (rhIL-1 beta) stimulated PGE2 production in MC3T3-E1 cells in a dose dependent manner. rhIL-1 alpha and 1 beta also stimulated MC3T3-E1 cells to produce macrophage-colony stimulating activity (M-CSA) in a dose-dependent manner. Indomethacin completely abolished PGE2 production but did not affect CSA. These results suggest that bone resorption induced by IL-1s is at least in part mediated by PGE2 produced by osteoblasts, and that M-CSA produced by osteoblasts may synergistically potentiate bone resorption by recruiting osteoclast precursors.     

Up-regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.     

The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.     

Role of prostaglandin E produced by osteoblasts in osteolysis due to bone metastasis

Prostaglandin E2 (PGE2) is produced in bone mainly by osteoblasts and stimulates bone resorption. Osteolytic bone metastasis of cancers is accompanied by bone resorption. In this study, we examined the roles of PGE2 in osteolysis due to bone metastasis of breast cancer. Injection of human breast cancer cells, MDA-MB-231 (MDA-231), into nude mice causes severe osteolysis in the femur and tibia. The expression of cyclo-oxygenase-2 (COX-2) and the receptor activator of NF-kappaB ligand (RANKL), a key molecule in osteoclast differentiation, mRNAs was markedly elevated in bone with metastasis. When MDA-231 cells were cocultured with mouse calvaria, COX-2-induced PGE2 production and bone resorption progressed. The contact with MDA-231 cells could induce the expression of COX-2 and RANKL in osteoblasts by mechanisms involving MAP kinase and NF-kappaB. The blockage of PGE2 signal by indomethacin and EP4 antagonist abrogated the osteoclast formation induced by the breast cancer cells. Here, we show a PGE-dependent mechanism of osteolysis due to bone metastasis.     

Effects of TGF-beta, TNF-alpha, IL-beta and IL-6 alone or in combination, and tyrosine kinase inhibitor on cyclooxygenase expression, prostaglandin E2 production and bone resorption in mouse calvarial bone cells

Cyclooxygenase-2 (COX-2) and tyrosine kinase, which are involved in the biosynthesis of prostaglandin E(2) (PGE(2)) in mouse calvarial osteoblasts, are stimulated by cytokine interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-6 (IL-6). IL-1beta and IL-6 and, to a lesser extent, TNF-alpha, enhances COX-2 mRNA levels in calvarial osteoblasts. Simultaneous treatment with IL-6 and IL-1beta and TNF-alpha resulted in enhanced COX-2 mRNA levels accompanied by the cooperative stimulation of PGE(2) biosynthesis compared to cells treated with IL-1beta or TNF-alpha or IL-6 alone. In contrast, the presence of TGF-beta reduced COX-2 mRNA level, PGE(2) biosynthesis and bone resorption induced by IL-1beta, TNF-alpha, IL-6 or a combination thereof. However, neither IL-1beta, TNF-alpha, IL-6 nor a combination of IL-1beta, TNF-alpha, IL-6 enhanced COX-1 mRNA levels in calvarial osteoblasts. A novel Src tyrosine kinase inhibitor, Herbimycin A (HERB), reduced COX-2 mRNA levels as well as PGE(2) production induced by IL-1beta, TNF-alpha and IL-6 or a combination of IL-1beta, TNF-alpha, IL-6, whereas COX-1 mRNA levels remained unaffected. Finally, HERB was found to inhibit in vitro bone resorption. These results indicate that the cooperative effects of IL-beta, TNF-alpha, IL-6 on PGE(2) production are due to the enhanced expression of the COX-2 gene and that tyrosine kinase(s) are involved in COX-2 signal transduction in mouse calvarial osteoblasts. Thus, the Src family of kinase inhibitors may be useful in treating diseases associated with elevated bone loss.     

Osteoprotegerin levels increased by interleukin-1beta in human periodontal ligament cells are suppressed through prostaglandin E(2) synthesized de novo

Osteoprotegerin (OPG) is a novel tumor necrosis factor receptor superfamily that inhibits osteoclast differentiation, activity, and survival. Interleukin-1beta (IL-1beta) increases OPG expression. IL-1beta also increases prostaglandin E(2) (PGE(2)) production and stimulates bone resorption. In the present study, we examined the involvement of PGE(2) in IL-1beta-induced increases in OPG levels in human periodontal ligament cells (HPL cells) in an effort to clarify apparently conflicting IL-1beta actions on bone resorption and understand IL-1beta-induced increases in secretion of OPG and PGE(2) in HPL cells. 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, a mRNA synthesis inhibitor, partly inhibited the increase in OPG mRNA levels induced by IL-1beta. Cycloheximide, a protein synthesis inhibitor, enhanced the stimulatory effect of IL-1beta. Etodolac, a selective cyclooxygenase-2 inhibitor, suppressed the increase in PGE(2) levels. Furthermore, etodolac reinforced the promotion of OPG expression by IL-1beta at the mRNA and protein levels. PGE(2) added to cultures of HPL cells decreased OPG mRNA levels in a dose- and time- dependent manner. These findings suggest that the increase in OPG levels induced by IL-1beta in HPL cells is suppressed through PGE(2) synthesized de novo.     

Autocrine/paracrine regulation of apoptosis in epithelial cells by prostaglandin E2

This study was designed to investigate the effect of IL-1alpha-induced up-regulation of cyclooxygenase-2 (COX-2) on prostaglandin E(2) (PGE(2)) secretion and the subsequent phenotypic effects of PGE(2) on epithelial cells. The effect of IL-1alpha on COX-2 expression was investigated in the T24 bladder epithelial cell line following treatment with 0, 0.05, 0.5, 1 or 10 ng/ml IL-1alpha for 1, 2, 4 or 6 h. Quantitative PCR confirmed up-regulation of expression of COX-2 with maximal expression observed following treatment with 0.5 ng/ml IL-1alpha for 1 h. Co-treatment of the cells with 0.5 ng/ml IL-1alpha in the presence or absence of 100 ng/ml IL-1 receptor antagonist (RA) abolished the up-regulation in COX-2 expression confirming that the effect of IL-1alpha is mediated via its membrane-bound receptors. Treatment with 0.5 ng/ml IL-1alpha resulted in a time-dependent increase in PGE(2) secretion with maximal secretion detected at 24 and 48 h after stimulation with IL-1alpha. Co-treatment of the cells with IL-1alpha and IL-1RA or the COX-2 enzyme inhibitor NS398 abolished the IL-1alpha mediated secretion of PGE(2). Treatment of T24 cells with 100 nM PGE(2) resulted in a significant elevation in cAMP generation confirming the expression of functional PGE(2) receptors. Finally, the effect of exogenous treatment with PGE(2) on apoptosis of T24 cells was assessed using cell death detection ELISA. T24 cells were treated with camptothecin to induce apoptosis in the presence or absence of 50 or 100 nM PGE(2) or 10 microM forskolin. Treatment of T24 cells with increasing doses of camptothecin alone resulted in a significant increase in the induction of apoptosis (P<0.01). However, co-treatment of the cells with 50 or 100 nM PGE(2) or 10 microM forskolin resulted in the inhibition of induction of the apoptotic pathway by camptothecin. These data demonstrate that PGE(2) inhibits apoptosis of epithelial cells possibly via cAMP-dependent pathway.     

Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells

Communication between endothelial and bone cells is crucial for controlling vascular supply during bone growth, remodeling, and repair but the molecular mechanisms coordinating this intercellular crosstalk remain ill-defined. We have used primary human and rat long bone-derived osteoblast-like cells (HOB and LOB) and human umbilical vein endothelial cells (HUVEC) to interrogate the potential autocrine/paracrine role of vascular endothelial cell growth factor (VEGF) in osteoblast:endothelial cell (OB:EC) communication and examined whether prostaglandins (PG), known modulators of both OB and EC behavior, modify VEGF production. We found that the stable metabolite of PGI2, 6-keto-PGF(1alpha) and PGE2, induced a concentration-dependent increase in VEGF release by HOBs but not ECs. In ECs, VEGF promoted early ERK1/2 activation, late cyclooxygenase-2 (COX-2) protein induction, and release of 6-keto-PGF1alpha. In marked contrast, no significant modulation of these events was observed in HOBs exposed to VEGF, but LOBs clearly exhibited COX-dependent prostanoid release (10-fold less than EC) following VEGF treatment. A low level of osteoblast-like cell responsiveness to exogenous VEGF was supported by VEGFR2/Flk-1 immunolabelling and by blockade of VEGF-mediated prostanoid generation by a VEGFR tyrosine kinase inhibitor (TKI). HOB alkaline phosphatase (ALP) activity was increased following long-term non-contact co-culture with ECs and exposure of ECs to VEGF in this system further increased OB-like cell differentiation and markedly enhanced prostanoid release. Our studies confirm a paracrine EC-mediated effect of VEGF on OB-like cell behavior and are the first supporting a model in which prostanoids may facilitate this unidirectional VEGF-driven OB:EC communication. These findings may offer novel regimes for modulating pathological bone remodeling anomalies through the control of the closely coupled vascular supply.     

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.     

Hormonal regulation of PGE2 and COX-2 production in rabbit uterine cervical fibroblasts.

Prostaglandins (PGs) cause uterine contraction to initiate labor at term. We investigated the effect of progesterone and 17beta-estradiol on the production of PGE2 in rabbit uterine cervical fibroblasts. When the cervical fibroblasts were treated with interleukin-1alpha (IL-1alpha), the level of PGE2 was augmented in a time- and dose-dependent manner. The IL-1alpha-augmented PGE2 level was almost completely suppressed by progesterone and 17beta-estradiol at the physiological concentration (0.01 microM), whereas a slight decrease in the basal level of PGE2 was observed in the cervical fibroblasts treated with both hormones at a pharmacological concentration (1 microM). In addition, the level of PGE2 augmented by IL-1alpha was due to the increase of cyclooxygenase (COX) activity, which was inhibited by progesterone and 17beta-estradiol as well as by indomethacin and a specific COX-2 inhibitor, NS-398, but not by the well-known COX-1 inhibitor, aspirin. Furthermore, progesterone and 17beta-estradiol suppressed the IL-1alpha-augmented COX-2 production but not the constitutive production of COX-1 in rabbit uterine cervical fibroblasts. These results suggest that progesterone and 17beta-estradiol prevent the initiation of labor by inhibiting PGE2 production after the suppression of COX-2 production during pregnancy in the rabbit.     

IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.     

Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts

VEGF is a highly specific stimulator of endothelial cells and may play an important role in angiogenesis in the process of tissue regeneration. We previously showed that cyclooxygenase-2 (COX-2) expressed in mesenchymal cells of the ulcer bed is involved in the ulcer repair process. To clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we investigated the relation between COX-2 expression and VEGF production in human gastric fibroblasts in vivo and in vitro. Gastric fibroblasts were cultured in RPMI 1640 with and without IL-1alpha or IL-1beta in the presence or absence of NS-398, a selective COX-2 inhibitor. Supernatant VEGF and PGE(2) concentrations were measured by enzyme-linked immunosorbent assay. COX-2 expression in fibroblasts was determined by Western blot analysis. VEGF and COX-2 expression in surgical resections of human gastric ulcer tissue was examined immunohistochemically. IL-1 dose dependently enhanced VEGF release in cultured gastric fibroblasts after a 24-h stimulation. IL-1 also stimulated PGE(2) production in gastric fibroblasts via COX-2 induction. NS-398 significantly suppressed VEGF and PGE(2) release from IL-1-stimulated gastric fibroblasts; concurrent addition of PGE(2) restored NS-398-inhibited VEGF release. COX-2 and VEGF immunoreactivity were colocalized in fibroblast-like cells in the ulcer bed of gastric tissues. These results suggest that COX-2 plays a key role in VEGF production in gastric fibroblasts stimulated by IL-1 in vitro and that angiogenesis induced by the COX-2-VEGF pathway might be involved in gastric ulcer healing.