Carol C. Pilbeam

Pulsating Fluid Flow Stimulates Prostaglandin Release and Inducible Prostaglandin G/H Synthase mRNA Expression in Primary Mouse Bone Cells

Bone tissue responds to mechanical stress with adaptive changes in mass and structure. Mechanical stress produces flow of fluid in the osteocyte lacunar‐canalicular network, which is likely the physiological signal for bone cell adaptive responses. We examined the effects of 1 h pulsating fluid flow (PFF; 0.7 ± 0.02 Pa, 5 Hz) on prostaglandin (PG) E2, PGI2, and PGF2α production and on the expression of the constitutive and inducible prostaglandin G/H synthases, PGHS‐1, and PGHS‐2, the major enzymes in the conversion of arachidonic acid to prostaglandins, using mouse calvarial bone cell cultures. PFF treatment stimulated the release of all three prostaglandins under 2% serum conditions, but with a different time course and to a different extent. PGF2α was rapidly increased 5–10 minutes after the onset of PFF. PGE2 release increased somewhat more slowly (significant after 10 minutes), but continued throughout 60 minutes of treatment. The response of PGI2 was the slowest, and only significant after 30 and 60 minutes of treatment. In addition, PFF induced the expression of PGHS‐2 but not PGHS‐1. One hour of PFF treatment increased PGHS‐2 mRNA expression about 2‐fold relative to the induction by 2% fresh serum given at the start of PFF. When the addition of fresh serum was reduced to 0.1%, the induction of PGHS‐2 was 8‐ to 9‐fold in PFF‐treated cells relative to controls. This up‐regulation continued for at least 1 h after PFF removal. PFF also markedly increased PGHS activity, measured as the conversion of arachidonic acid into PGE2. One hour after PFF removal, the production of all three prostaglandins was still enhanced. These results suggest that prostaglandins are important early mediators of the response of bone cells to mechanical stress. Prostaglandin up‐regulation is associated with an induction of PGHS‐2 enzyme mRNA, which may subsequently provide a means for amplifying the cellular response to mechanical stress.

Prostaglandins in bone: bad cop, good cop?

Prostaglandins (PGs) are multifunctional regulators of bone metabolism that stimulate both bone resorption and formation. PGs have been implicated in bone resorption associated with inflammation and metastatic bone disease, and also in bone formation associated with fracture healing and heterotopic ossification. Recent studies have identified roles for inducible cyclooxygenase (COX)-2 and PGE(2) receptors in these processes. Although the effects of PGs have been most often associated with cAMP production and protein kinase A activation, PGs can engage an extensive G-protein signaling network. Further analysis of COX-2 and PG receptors and their downstream G-protein signaling in bone could provide important clues to the regulation of skeletal cell growth in both health and disease.

Prostaglandins: mechanisms of action and regulation of production in bone.

Prostaglandins (PGs), particularly PGE2, are produced by bone and have powerful effects on bone metabolism. PGs have an initial, transient, direct inhibitory effect on osteoclast function. However, the major long-term effect in bone organ culture is to stimulate bone resorption by increasing the replication and differentiation of new osteoclasts. PGs also stimulate osteoclast formation in cell culture systems. Stimulation of osteoclastic bone resorption may be important in mediating bone loss in response to mechanical forces and inflammation. PGs have a biphasic effect on bone formation. At relatively low concentrations or in the presence of glucocorticoids, the replication and differentiation of osteoblasts is stimulated and bone formation is increased. This increase is associated with an increase in production of insulin-like growth factor-I (IGF-I). However, at high concentrations or in the presence of IGF-I, PGE2 inhibits collagen synthesis. In osteoblastic cell lines this inhibition can be shown to occur at the level of transcription of the collagen gene. The stimulatory effect on bone formation has been demonstrated when PGs are administered exogenously, but it is not clear how endogenous PG production affects bone formation in physiological or pathologic circumstances. The production of PGs in bone is highly regulated. The major source appears to be cells of the osteoblast lineage. A major site of regulation is at the level of the enzyme PG endoperoxide synthase (cyclooxygenase or PGH synthase). PGE2 production and PGH synthase mRNA are increased by PTH and interleukin-1 and decreased by estrogen. Glucocorticoids probably act by a different mechanism, decreasing either arachidonic acid or PGH synthase activity. Many other factors including mechanical forces and growth factors influence PG production in bone. Thus endogenous PGs are probably important local regulators of bone turnover, and abnormalities in their production could play a role in the pathogenesis of osteoporosis.

Transcriptional induction of prostaglandin G/H synthase-2 by basic fibroblast growth factor.

In serum-free mouse osteoblastic MC3T3-E1 cells, basic fibroblastic growth factor (bFGF) induced mRNA and protein for prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in arachidonic acid (AA) conversion to prostaglandins. mRNA accumulation peaked at 1 h with bFGF 1 nM. In cells stably transfected with a 371-bp PGHS-2 promoter-luciferase reporter, bFGF stimulated luciferase activity, which peaked at 2-3 h with bFGF 1-10 nM. In the presence of exogenous AA, bFGF stimulated PGE2 production, which paralleled luciferase activity. In serum-free neonatal mouse calvarial cultures, bFGF stimulated PGE2 production in the absence of exogenous AA. bFGF stimulated PGHS-2 mRNA accumulation, which peaked at 2-4 h and then decreased; there were later mRNA elevations at 48 and 96 h that were inhibited by indomethacin. In both MC3T3-E1 cells and neonatal calvariae, bFGF produced smaller and slower increases in PGHS-1 mRNA levels than for PGHS-2. bFGF stimulated bone resorption in mouse calvariae with a maximal increase of 80% at 1 nM. Stimulation was partially inhibited by nonsteroidal anti-inflammatory drugs. We conclude that bFGF rapidly stimulates PGE2 production in osteoblasts, largely through transcriptional regulation of PGHS-2, and that prostaglandins mediate some of bFGFs effects on bone resorption.

Ovariectomy enhances and estrogen replacement inhibits the activity of bone marrow factors that stimulate prostaglandin production in cultured mouse calvariae.

To examine PG production in estrogen deficiency, we studied effects on cultured neonatal mouse calvariae of bone marrow supernatants (MSup) from sham-operated (SHAM), ovariectomized (OVX), or 17 beta-estradiol (OVX+E)-treated mice. MSups were obtained 3 wk after OVX when bone density had decreased significantly. 10-60% MSup increased medium PGE2 and levels of mRNA for inducible and constitutive prostaglandin G/H synthase (PGHS-2 and PGHS-1) and cytosolic phospholipase A2 in calvarial cultures. OVX MSups had twofold greater effects on PGHS-2 and medium PGE2 than other MSups. IL-1 receptor antagonist and anti-IL-1 alpha neutralizing antibody decreased MSup-stimulated PGHS-2 mRNA and PGE2 levels and diminished differences among OVX, sham-operated, and OVX+E groups. In contrast, antibodies to IL-1 beta, IL-6, IL-11, and TNF alpha had little effect. There were no significant differences in IL-1 alpha concentrations or IL-1 alpha mRNA levels in MSups or marrow cells. PGHS-2 mRNA in freshly isolated tibiae from OVX mice was slightly greater than from sham-operated. We conclude that bone marrow factors can increase PG production through stimulation of PGHS-2; that OVX increases and estrogen decreases activity of these factors; and that IL-1 alpha activity, together with additional unknown factors, mediates the differential MSup effects.

Fluid Flow Induction of Cyclo-Oxygenase 2 Gene Expression in Osteoblasts Is Dependent on an Extracellular Signal-Regulated Kinase Signaling Pathway†‡

Mechanical loading of bone may be transmitted to osteocytes and osteoblasts via shear stresses at cell surfaces generated by the flow of interstitial fluid. The stimulated production of prostaglandins, which mediates some effects of mechanical loading on bone, is dependent on inducible cyclo‐oxygenase 2 (COX‐2) in bone cells. We examined the fluid shear stress (FSS) induction of COX‐2 gene expression in immortalized MC3T3‐E1 osteoblastic cells stably transfected with −371/+70 base pairs (bp) of the COX‐2 5′‐flanking DNA (Pluc371) and in primary osteoblasts (POBs) from calvaria of mice transgenic for Pluc371. Cells were plated on collagen‐coated glass slides and subjected to steady laminar FSS in a parallel plate flow chamber. FSS, from 0.14 to10 dynes/cm2, induced COX‐2 messenger RNA (mRNA) and protein. FSS (10 dynes/cm2) induced COX‐2 mRNA within 30 minutes, with peak effects at 4 h in MC3T3‐E1 cells and at ≥8 h in POBs. An inhibitor of new protein synthesis puromycin blocked the peak induction of COX‐2 mRNA by FSS. COX‐2 promoter activity, measured as luciferase activity, correlated with COX‐2 mRNA expression in both MC3T3‐E1 and POB cells. FSS induced phosphorylation of extracellular signal‐regulated kinase (ERK) in MC3T3‐E1 cells, with peak effects at 5 minutes. Inhibiting ERK phosphorylation with the specific inhibitor PD98059 inhibited FSS induction of COX‐2 mRNA by 55‐70% and FSS stimulation of luciferase activity by ≥80% in both MC3T3‐E1 and POB cells. We conclude that FSS transcriptionally induces COX‐2 gene expression in osteoblasts, that the maximum induction requires new protein synthesis, and that induction occurs largely via an ERK signaling pathway.

Prostaglandins and Bone Metabolism

Publisher Summary This chapter focuses on the effects of prostaglandins (PG) and other eicosanoids on bone resorption and formation. Eicosanoids are oxygenated 20-carbon fatty acids derived from polyunsaturated eicosatrienoic, eicosatetranoic (arachidonic), and eicosapentanoic fatty acids. The production of PGs involves three major steps which include hormone-or stress-activated mobilization of AA, conversion of AA to prostaglandin endoperoxide H 2 (PGH 2 ), and conversion of diffusible PGH 2 by tissue-specific isomerases and reductases to PGE 2 , PGD 2 , PGF 2α , prostacyclin (PGI 2 ), and thromboxane. The committed step in the conversion of AA to PGs is catalyzed by a bifunctional enzyme, which converts free AA to PGG 2 in a cyclooxgenase reaction followed by reduction of PGG 2 to PGH 2 in a peroxidase reaction. This enzyme, formally named prostaglandin endoperoxide H synthase or prostaglandin G/H synthase (PGHS), is popularly called cyclooxgygenase (COX) in reference to its first function. The individual PG synthases are differentially distributed in tissues and are believed to influence PG production largely by determining the predominant type of prostanoid synthesized in a particular tissue. PGE 1 and PGE 2 stimulate osteoclast formation in marrow cultures. The PG enhancement of stimulated osteoclast formation in marrow culture may reflect the increased formation of new osteoclastic precursors, while stimulated resorption in organ culture may be more dependent on activation of a pool of available osteoclastic precursors. When added to isolated osteoclasts in vitro, PGE 2 transiently inhibits bone resorption. PGs can have both stimulatory and inhibitory effects on bone formation. PGs can increase both periosteal and endosteal bone formation in the rat and produce substantial increases in bone mass, similar to the effects of PTH. At high concentrations, PGs can inhibit collagen synthesis in cell and organ culture. This inhibitory effect occurs largely via transcriptional inhibition of collagen and are mediated by the FP receptor rather than an EP receptor.

Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In Vivo

We tested the hypothesis that induction of cyclo‐oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP‐2 induced COX‐2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP‐2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX‐2 promoter‐luciferase reporter construct (Pluc) and in MC3T3‐E1 cells transfected with Pluc. Deletion analysis identified the −300/−213‐bp region of the COX‐2 promoter as necessary for BMP‐2 stimulation of luciferase activity. Mutation of core‐binding factor activity 1 (muCbfa1) consensus sequence (5′‐AACCACA‐3′) at −267/−261 bp decreased BMP‐2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX‐2 knockout (−/−) and wild‐type (+/+) mice, the absence of COX‐2 expression reduced the BMP‐2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP‐2 induced osteoclast formation in cells from COX‐2+/+ mice but not in cells from COX‐2−/− mice. In vivo, BMP‐2 (10 μg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (μCT), was decreased by 78% in COX‐2−/− mice compared with COX‐2+/+ mice. We conclude that BMP‐2 transcriptionally induces COX‐2 in osteoblasts via a Cbfa1 binding site and that the BMP‐2 induction of COX‐2 can contribute to effects of BMP‐2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP‐2 stimulation of ectopic bone formation in vivo.

Strontium Ranelate Promotes Osteoblastic Differentiation and Mineralization of Murine Bone Marrow Stromal Cells: Involvement of Prostaglandins

Strontium ranelate is a new anti‐osteoporosis treatment. This study showed that strontium ranelate stimulated PGE2 production and osteoblastic differentiation in murine marrow stromal cells, which was markedly reduced by inhibition of COX‐2 activity or disruption of COX‐2 gene expression. Hence, some anabolic effects of strontium ranelate may be mediated by the induction of COX‐2 and PGE2 production.

Fracture incidence in postmenopausal women with primary hyperparathyroidism

BACKGROUND The association of bone loss and increased fractures in postmenopausal women with minimally symptomatic hyperparathyroidism has not been clearly defined. This study was done to determine the frequency of fractures in postmenopausal women with hyperparathyroidism. METHODS Forty-six postmenopausal women who had undergone parathyroidectomy for hyperparathyroidism during a 5-year period (1986 to 1991) were interviewed, and their medical records were examined to determine their fracture history. Forty-four postmenopausal women without hyperparathyroidism were contacted by random digit dialing and interviewed as controls. RESULTS The groups were comparable with regard to age, weight, height, race, and age at menopause. Medical conditions and medication use were also similar, except for more reports of hypothyroidism in the hyperparathyroidism group (p = 0.05). Only 13% of women presented for treatment because of bone concerns, either fractures (9%) or low bone density (4%). However, on interview, 48% of the patients with hyperparathyroidism reported fractures compared with 25% of the controls (p = 0.02), a difference that remained even when those presenting with bone disease were excluded (p = 0.05). Of those with fractures, multiple fractures occurred in 36% of patients with hyperparathyroidism compared with 9% of controls and generally occurred after minor rather than major trauma (92% versus 45%, p = 0.002). Appendicular skeletal sites were reported for 86% of hyperparathyroidism groups and 92% of control groups fractures. Moreover, 50% of patients with hyperparathyroidism reported height loss compared with 27% of the control group (p = 0.05). CONCLUSIONS This study shows that postmenopausal women with hyperparathyroidism reported more fractures and height loss than the control group, even when patients with hyperparathyroidism who presented because of bone disease were excluded.