Understanding the policy dynamics of COVID-19 in the UK: Early findings from interviews with policy makers and health care professionals.

The UK government response to COVID-19 has been heavily criticised. We report witnesses' perceptions of what has shaped UK policies and how these policies have been received by healthcare workers. Such studies are usually affected by hindsight. Here we deploy a novel prospective approach to capture real-time information. We are historians, social scientists and biomedical researchers who study how societies cope with infectious disease. In February 2020 we began regular semi-structured calls with prominent members of policy communities, and health care professionals, to elicit their roles in, and reactions to, the pandemic response. We report witnesses' perceptions that personal protective equipment (PPE) stocks were too small, early warnings have not led to sufficiently rapid policy decisions, and a lack of transparency is sapping public trust. Significant successes include research mobilisation. The early experiences and reactions of our witnesses suggest important issues for investigation, notably a perception of delay in decision making.

Effects of prostaglandin E2 and lipopolysaccharide on osteoclastogenesis in RAW 264.7 cells.

INTRODUCTION: Prostaglandins (PGs) can act on both hematopoietic and osteoblastic lineages to enhance osteoclast formation. METHODS: We examined PGE2 stimulated osteoclastogenesis in RAW 264.7 cells and the role of endogenous PGE2 in lipopolysaccharide (LPS) stimulated osteoclastogenesis. RESULTS: RANKL (1-100 ng/ml) increased formation of osteoclasts, defined as tartrate resistant acid phosphatase multinucleated cells, with peak effects at 30 ng/ml. Addition of PGE2 (0.01-1.0 microM) to RANKL (30 ng/ml) dose dependently increased osteoclast number 30-150%. Use of NS-398 (0.1 microM) or indomethacin (Indo, 1.0 micro M) to block endogenous PG synthesis had little effect on the response to RANKL alone but significantly decreased the response to PGE2. Addition of LPS (100 ng/ml) to RANKL increased osteoclast number 50%, and this response was significantly decreased by NS-398 and Indo. RANKL and PGE2 produced small, additive increases in COX-2 mRNA levels, while LPS produced a larger increase. PG release into the medium was not increased by RANKL and PGE2 but markedly increased by LPS. CONCLUSION: We conclude that RANKL stimulated osteoclastogenesis can be enhanced by PGE2 and LPS though direct effects on the hematopoietic cell lineage and that these effects may be mediated in part by induction of COX-2 and enhanced intracellular PG production.

Effects of prostaglandin E2 on gene expression in primary osteoblastic cells from prostaglandin receptor knockout mice.

Recent studies have shown that stimulation of osteoclastogenesis in cocultures of osteoblasts and spleen cells in response to prostaglandin E2 (PGE2) is markedly decreased when the osteoblasts are derived from cells lacking either the EP2 or the EP4 receptor. Induction of osteoclast formation requires upregulation of receptor activator of nuclear factor-kappaB ligand (RANKL) on cells of the osteoblastic lineage, which then binds to the RANK receptor on cells of the osteoclast lineage. Osteoprotegerin (OPG) is a decoy receptor for RANKL that can block its interaction with RANK. In addition, macrophage-colony stimulating factor (M-CSF) is essential for osteoclast formation. Finally, PGE2 can increase interleukin-6 (IL-6), which may further enhance osteoclastogenesis. To study the relative influence of the EP2 and EP4 receptors on response of these factors to PGE2, we examined mRNA levels for RANKL, OPG, M-CSF, and IL-6 in primary osteoblastic cell cultures derived from two lines of EP2 knockout mice (EP2-/-) and one line of EP4 knockout mice (EP4-/-) and the relevant wild-type controls (EP2+/+ and EP4+/+). The responses of cells from wild-type animals of all three lines were similar. After PGE2 treatment, RANKL mRNA levels were increased at 2 h, and this was sustained over 72 h. Basal RANKL expression was moderately reduced in EP2-/- cells and markedly reduced in EP4-/- cells. PGE2 increased RANKL mRNA in EP2-/- cells and EP4-/- cells, but the levels were significantly reduced compared with wild-type cells. There were no consistent changes in expression of M-CSF or OPG in the different genotypes or with PGE2 treatment. IL-6 mRNA was variably increased by PGE2 in both wild-type and knockout cells, although the absolute levels were somewhat lower in both EP2-/- and EP4 -/- cultures. Parathyroid hormone (PTH) increased RANKL and IL-6 and decreased OPG mRNA levels similarly in both wild-type and EP2-/- or EP4-/- cells. The major defect in the response to PGE2 in animals lacking either EP2 or EP4 receptors is a reduction in basal and stimulated RANKL levels. Loss of EP4 receptor appears to have a greater effect on basal RANKL expression than EP2.

Regulation of fibroblast growth factor 2 and fibroblast growth factor receptors by transforming growth factor beta in human osteoblastic MG-63 cells.

Fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) are important regulators of bone cell function. Although FGF-2 is a major modulator of bone cell function, its expression and regulation in human osteoblasts have not been investigated. We examined FGF-2 messenger RNA (mRNA) expression and regulation in the human osteosarcoma MG-63 cells. Northern analysis revealed that MG-63 cells expressed FGF-2 mRNA transcripts of 7, 4, 2.2, and 1.3 kilobases (kb). In the absence of serum, treatment with transforming growth factor beta (TGF-beta; 0.1-10 ng/ml) increased all FGF-2 mRNA transcripts. Maximal increase was seen with 1 ng/ml of TGF-beta. TGF-beta increased FGF-2 mRNA expression within 2 h and this was sustained for 24 h. Phorbal myristate acetate (PMA; 1 microM) also increased FGF-2 mRNA at 6 h. Time course studies showed that TGF-beta did not significantly alter FGFR1 or FGFR2 mRNA expression in MG-63 cells. Western blotting with anti-human FGF-2 revealed that MG-63 cells synthesize three isoforms of FGF-2 protein of approximately 18, 22/23, and 24 kDa, which were increased after either 6 h or 24 h of treatment with TGF-beta. Increased FGF-2 mRNA and protein expression in response to TGF-beta was markedly reduced by the protein kinase A (PKA) inhibitor H-89. Immunogold labeling of MG-63 cells treated with TGF-beta showed increased labeling for FGF-2 and FGFR2 in the nuclei. In contrast, TGF-beta treatment significantly decreased FGFR1 labeling in the nuclei. These data show that TGF-beta regulates FGF-2 gene expression in human osteosarcoma cells. Furthermore, TGF-beta modulates the cellular localization of FGF-2 and its receptors.

Effects of selective prostaglandin EP4 receptor antagonist on osteoclast formation and bone resorption in vitro.

Prostaglandin estradiol (PGE(2)) stimulates bone resorption by a cyclic AMP (cAMP)-dependent mechanism that involves prostaglandin E receptors of the EP2 and EP4 subtypes. We tested a potent selective EP4 antagonist (EP4RA), which blocks PGE(2) binding to EP4 receptors. We examined the effects of EP4RA on osteoclastogenesis in murine marrow cultures, on cAMP production in primary osteoblastic (POb) cell cultures, and on bone resorption in organ cultures. EP4RA (1 micromol/L) decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNC) by 46%-48% in cultures treated with 0.1-1.0 micromol/L PGE(2) and by 96% in cultures treated with 0.01 micromol/L PGE(2). EP4RA also decreased TRAP(+) MNC formation by 60% in 1,25-dihydroxyvitamin D (1,25D)-treated cultures and by 62% in parathyroid hormone (PTH)-treated cultures. A chemically related analog of EP4RA that lacks antagonist activity did not inhibit TRAP(+) MNC formation. EP4RA decreased cAMP production in PGE(2)-treated POb by 44% but did not block cAMP response to PTH. EP4RA inhibited the increase in receptor activator of NF-kappaB ligand (RANKL) mRNA levels produced by PGE(2). In fetal rat long bone cultures, EP4RA decreased 45Ca release from control, unstimulated cultures by 12%-25% and from PGE(2)-stimulated cultures by 22%-37%. Because EP4RA partially inhibited osteoclastogenesis not only in response to PGE(2) but also in response to 1,25D and PTH, these results suggest that activation of the EP4 receptor may play a general role in osteoclastic bone resorption. EP4RA showed partial inhibition of PGE(2)-stimulated osteoclastogenesis at 1 micromol/L, but almost complete inhibition at 0.01 micromol/L PGE(2). This could be due to the limited efficacy of the antagonist at high concentrations of PGE(2), or an alternative pathway, such as activation of the EP2 receptor.

Knockout of the murine prostaglandin EP2 receptor impairs osteoclastogenesis in vitro.

Prostaglandin E2 (PGE2) stimulates the formation of osteoclast-like tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in vitro. This effect likely results from stimulation of adenylyl cyclase, which is mediated by two PGE2 receptors, designated EP2 and EP4. We used cells from mice in which the EP2 receptor had been disrupted to test its role in the formation of TRAP + MNC. EP2 heterozygous (+/-) mice in a C57BL/6 x 129/SvEv background were bred to produce homozygous null (EP2 -/-) and wild-type (EP2 +/+) mice. PGE2, PTH, or 1,25 dihydroxyvitamin D increased TRAP+ MNC in 7-day cultures of bone marrow cells from EP2 +/+ mice. In cultures from EP2 -/- animals, responses to PGE2, PTH, and 1,25 dihydroxyvitamin D were reduced by 86%, 58%, and 50%, respectively. A selective EP4 receptor antagonist (EP4RA) further inhibited TRAP+ MNC formation in both EP2 +/+ and EP2 -/- cultures. In cocultures of spleen and calvarial osteoblastic cells, the response to PGE2 or PTH was reduced by 92% or 85% when both osteoblastic cells and spleen cells were from EP2 -/- mice, by 88% or 68% when only osteoblastic cells were from EP2 -/- mice and by 58% or 35% when only spleen cells were from EP2 -/- mice. PGE2 increased receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) messenger RNA expression in osteoblastic and bone marrow cell cultures from EP2 +/+ mice 2-fold but had little effect on cells from EP2 -/- mice. Spleen cells cultured with RANKL and macrophage colony stimulating factor produced TRAP+ MNC. PGE2 increased the number of TRAP+ MNC in spleen cell cultures from EP2 +/+ mice but not in cultures from EP2 -/- mice. EP4RA had no effect on the PGE2 response in spleen cell cultures. PGE2 decreased the expression of messenger RNA for granulocyte-macrophage colony stimulating factor in spleen cell cultures from EP2 +/+ mice but had little effect on cells from EP2 -/- mice. These data demonstrate that the prostaglandin EP2 receptor plays a role in the formation of osteoclast-like cells in vitro. A major defect in EP2 -/- mice appears to be in the capacity of osteoblastic cells to stimulate osteoclast formation. In addition, there appears to be a defect in the response of cells of the osteoclastic lineage to PGE2 in EP2 -/- mice.

Identification of multiple cis-acting elements mediating the induction of prostaglandin G/H synthase-2 by phorbol ester in murine osteoblastic cells.

The tumor promoter phorbol 13-myristate 12-acetate (PMA), the best characterized protein kinase C agonist, frequently regulates gene expression via activation of Fos/Jun (AP-1) complexes. PMA rapidly and transiently induces prostaglandin G/H synthase-2 (PGHS-2) expression in murine osteoblastic MC3T3-E1 cells, but no functional AP-1 binding motifs in the 5'-flanking region have been identified. In MC3T3-E1 cells transfected with -371/+70 bp of the PGHS-2 gene fused to a luciferase reporter gene (Pluc), PMA stimulates luciferase activity up to eightfold. Computer analysis of the sequence of the PGHS-2 promoter region identified three potential AP-1 elements in the -371/+70 bp region, and deletion analysis suggested that the sequence 5'-aGAGTCA-3' at -69/-63 bp was most likely to mediate stimulation by PMA. Mutation of the putative AP-1 sequence reduces the ability of PMA to stimulate Pluc activity by 65%. On electrophoretic mobility shift analysis (EMSA), PMA induces binding to a PGHS-2 probe spanning this sequence, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Mutation of this AP-1 site also causes a small (22%) but significant reduction in the serum stimulation of Pluc activity in transiently transfected MC3T3-E1 cells. On EMSA, serum induces binding to a PGHS-2 probe spanning the AP-1 site, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Joint mutation of this AP-1 site and the nearby CRE site at -56/-52 bp, previously shown to mediate serum, v-src and PDGF induction of PGHS-2 in NIH-3T3 cells, blocks both PMA and serum induction of Pluc activity in MC3T3-E1 cells. Hence, the AP-1 and CRE binding sites are jointly but differentially involved in both the PMA and serum stimulation of PGHS-2 promoter activity.

Involvement of CCAAT enhancer binding protein transcription factors in the regulation of prostaglandin G/H synthase 2 expression by interleukin-1 in osteoblastic MC3T3-E1 cells.

Interleukin-1 (IL-1) stimulates prostaglandin production in bone by a rapid and transient activation of prostaglandin G/H synthase 2 (PGHS-2) gene expression. In osteoblastic MC3T3-E1 cells, IL-1 caused a transient increase in PGHS-2 messenger RNA (mRNA), which peaked at 2 h. IL-1 caused a 2- to 4-fold activation of a 371-base pair (bp) murine PGHS-2 promoter/luciferase construct in stable transfectants. This response mapped to a proximal promoter element(s) located between -150 and -40 bp. This region contains a putative CCAAT enhancer binding protein (C/EBP) site (centered at -135 bp), which shows enhanced binding of C/EBPbeta and C/EBPdelta by mobility shift analysis after IL-1 treatment. A transient cotransfection approach was used to examine the effects of C/EBPbeta and C/EBPdelta overexpression. IL-1 caused a maximal 3- to 7-fold stimulation of PGHS-2 promoter activity after 2.5 h. Overexpression of murine C/EBPbeta and C/EBPdelta caused a dose-dependent increase in basal and IL-1-stimulated luciferase activity. C/EBPdelta caused a greater enhancement of basal and IL-1-stimulated promoter activity than C/EBPbeta, suggesting that C/EBPdelta is a stronger transactivator. Overexpression of p20C/EBPbeta, a dominant negative inhibitor of C/EBP function, blocked the stimulation of PGHS-2 promoter activity by IL-1 and blocked the ability of overexpressed C/EBPbeta and C/EBPdelta to increase basal and IL-1-stimulated promoter activity. Mutagenesis of the C/EBP site reduced, but did not abolish, the stimulation of PGHS-2 promoter activity by IL-1 and blunted the effect of overexpressed C/EBPdelta on basal and IL-1-stimulated promoter activity. These results suggest an essential role for C/EBPbeta and C/EBPdelta in the induction of PGHS-2 gene expression by IL-1 in osteoblastic cells.

Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture.

We examined the effect on osteoclast formation of disrupting the prostaglandin G/H synthase genes PGHS-1 and-2. Prostaglandin E(2) (PGE(2)) production was significantly reduced in marrow cultures from mice lacking PGHS-2 (PGHS-2(-/-)) compared with wild-type (PGHS-2(+/+)) cultures. Osteoclast formation, whether stimulated by 1,25-dihydroxyvitamin D(3) (1,25-D) or by parathyroid hormone (PTH), was reduced by 60-70% in PGHS-2(-/-) cultures relative to wild-type cultures, an effect that could be reversed by providing exogenous PGE(2). Cultures from heterozygous mice showed an intermediate response. PGHS inhibitors caused a similar drop in osteoclast formation in wild-type cultures. Co-culture experiments showed that supporting osteoblasts, rather than osteoclast precursors, accounted for the blunted response to 1,25-D and PTH. This lack of response appeared to result from reduced expression of RANK ligand (RANKL) in osteoblasts. We cultured spleen cells with exogenous RANKL and found that osteoclast formation was 50% lower in PGHS-2(-/-) than in wild-type cultures, apparently because the former cells expressed high levels of GM-CSF. Injection of PTH above the calvaria caused hypercalcemia in wild-type but not PGHS-2(-/-) mice. Histological examination of bone from 5-week-old PGHS-2(-/-) mice revealed no abnormalities. Mice lacking PGHS-1 were similar to wild-type mice in all of these parameters. These data suggest that PGHS-2 is not necessary for wild-type bone development but plays a critical role in bone resorption stimulated by 1,25-D and PTH.

Low dose estrogen and calcium have an additive effect on bone resorption in older women.

Previous studies have shown that treatment with estrogen or calcium decreases bone turnover in older women. The mechanisms by which estrogen and calcium exert their effects are probably different. We therefore examined the possibility of an additive or synergistic effect of combined treatment with calcium and low dose estrogen on bone turnover in older women, using biochemical markers. Thirty-one healthy women over 70 yr of age were randomized to 12 weeks of treatment with either micronized 17beta-estradiol [0.5 mg/day Estrace (E2)] or 1500 mg/day elemental calcium, given as carbonate plus vitamin D (800 IU/day; Ca+D). At the end of the initial 12-week treatment period, both groups received both Ca+D and E2 for an additional 12 weeks. Eleven older women were followed for 36 weeks without any treatment and served as a control group. Serum and urine were collected at baseline, at 12 and 24 weeks on treatment, and at 12 weeks after treatment was terminated for measurement of biochemical markers of bone turnover. Markers of bone formation were bone alkaline phosphatase, osteocalcin, and type I procollagen peptide; markers of bone resorption were urinary cross-linked C-telopeptides and N-telopeptides of type I collagen, serum cross-linked N-telopeptides of type I collagen, urinary free deoxypyridinoline cross-links, and serum bone sialoprotein. Repeated measures ANOVA was used to determine changes in bone turnover measures over time by group. All markers of bone resorption decreased with initial treatment and decreased further with combination therapy (P < 0.001). Markers of bone formation decreased with Ca+D treatment, but not with E2 alone; there was no additional effect of combination therapy on formation markers compared to Ca+D alone. Neither markers of formation nor resorption changed in the control group. These results suggest that there is an additive effect of low dose estrogen and calcium on bone resorption, but not on bone formation, in older women. Thus, the combination of low dose estrogen plus calcium is likely to be more effective in older women than either treatment alone.

Regulation of prostaglandin G/H synthase-2 expression by interleukin-1 in human osteoblast-like cells.

Interleukin-1 (IL-1) is an important factor in bone metabolism, and its actions may be mediated in part via prostaglandins. Prostaglandin G/H synthase (PGHS), a critical enzyme in the synthesis of prostaglandins, has two isoforms, PGHS-1, which is generally constitutively expressed, and PGHS-2, which is inducible. This study examines the effects of IL-1 on PGHS-2 mRNA expression in human osteosarcoma MG-63 cells, the human osteoblast-like initial transfectant (HOBIT) cell line, and primary human osteoblastic (HOB) cells. IL-1 induced PGHS-2 mRNA expression in MG-63 cells within 1 h, and expression was maintained for 24 h. There was a dose-related increase in PGHS-2 mRNA levels with 1-100 ng/ml of IL-1. Induction of PGHS-2 protein and media prostaglandin E2 (PGE2) paralleled induction of PGHS-2 mRNA levels. IL-1 similarly induced PGHS-2 mRNA expression and PGE2 production in HOBIT and HOB cells. Among other potential agonists, phorbol myristate acetate (PMA) was a potent inducer of PGHS-2 expression, while forskolin (FSK), serum, and prostaglandins had little effect. Cycloheximide enhanced effects of both IL-1 and PMA, suggesting that de novo protein synthesis is not required for induction of PGHS-2. Twenty-four hours of PMA pretreatment blocked the induction of PGHS-2 by PMA but not by IL-1, suggesting that IL-1 induction of PGHS-2 mRNA is not dependent on the protein kinase C pathway. Although FSK alone had little effect, it enhanced induction of PGHS-2 mRNA by IL-1. PGHS-1 was constitutively expressed and showed little change with treatment. In summary, we show that IL-1 is a potent inducer of PGHS-2 expression and PGE2 production in human osteosarcoma cells as well as in osteoblastic cells derived from normal human bone.

Mice lacking the type I interleukin-1 receptor do not lose bone mass after ovariectomy.

We measured the effects of ovariectomy on the bone mass of mice that lacked type I interleukin-1 receptor (IL-I R1 -/- mice) in two genetic backgrounds (C57BL/6 x 129/Sv and C57BL/6) to investigate the role of interleukin-1 in the actions of estrogen on bone. At three weeks after surgery, ovariectomized wild-type mice decreased trabecular bone volume in the proximal humerus by 70% in a C57BL/6 x 129/Sv background and 48% in a C57BL/6 background compared to sham-operated controls. In contrast, there was no significant decrease in trabecular bone mass in IL-1 R1 -/- mice after ovariectomy. The estrogen status of all groups was confirmed by measurement of uterine wet weight. These results demonstrate that a functional IL-1 response pathway is required for mice to lose trabecular bone mass after ovariectomy in this model and they imply that IL-1 is an important mediator of the effects of ovariectomy on bone mass. Hence, therapeutic interventions that block the effects of IL-1 on bone may be beneficial for treating diseases of rapid bone loss such as post-menopausal osteoporosis.

Transforming growth factor-beta1 regulation of prostaglandin G/H synthase-2 expression in osteoblastic MC3T3-E1 cells.

Transforming growth factor-beta (TGFbeta) plays an important role in bone development and remodeling. TGFbeta stimulates PGE2 production, enhances interleukin-1-stimulated PGE2 production, and can stimulate PG-mediated bone resorption. We found that TGFbeta induced prostaglandin G/H synthase (PGHS-2) messenger RNA (mRNA) and PGE2 production in neonatal mouse calvarial cultures and in primary cells derived from these calvariae. We used MC3T3-E1 cells, an immortalized osteoblastic cell line derived from mouse calvariae, to examine the mechanism of PGHS-2 induction. PGHS-2 mRNA was rapidly induced by TGFbeta (10 ng/ml) in MC3T3-E1 cells; mRNA levels peaked at 4-8 h and were still elevated at 24 h. Induction of PGHS-2 protein and PGE2 production correlated with PGHS-2 mRNA levels. In contrast, TGFbeta had much less effect on PGHS-1 mRNA levels. Unlike the response to other agonists, PGHS-2 mRNA induction by TGFbeta was not enhanced by cycloheximide pretreatment, suggesting a requirement for new protein synthesis. To study transcriptional regulation, cells were stably transfected with a PGHS-2 promoter-luciferase reporter construct containing 371 bp of the 5'-flanking region and 70 bp of untranslated DNA from the PGHS-2 gene. TGFbeta-stimulated luciferase activity paralleled PGHS-2 mRNA induction. Stimulation of luciferase activity and PGHS-2 mRNA levels by other agonists, including interleukin-1, TGF alpha, forskolin, and phorbol 13-myristate 12-acetate, were enhanced by TGFbeta. A 90% drop in luciferase activity occurred with deletion of the region from -371 to -213 bp of the PGHS-2 promoter. The PG response to TGFbeta in MC3T3-E1 cells appears to be mediated primarily by transcriptional regulation of PGHS-2 expression through one or more cis-acting elements located between -371 and -213 bp in the 5'-flanking region of the PGHS-2 gene.

Parathyroid hormone increases prostaglandin G/H synthase-2 transcription by a cyclic adenosine 3',5'-monophosphate-mediated pathway in murine osteoblastic MC3T3-E1 cells.

PTH increased PG synthase-2 transcription in osteoblastic MC3T3-E1 cells at 30 min, as assessed by nuclear run-on assays. To determine the signaling pathways used by PTH, the activity of a construct containing the PG synthase-2 gene between nucleotides -963 and +70 linked to a luciferase reporter was analyzed in stably transfected MC3T3-E1 cells. Agents that activate the cAMP-protein kinase A or protein kinase C pathways increased PG synthase-2 promoter activity. In contrast, the calcium ionophore ionomycin was ineffective. The protein kinase A inhibitor H89 blocked PTH stimulation of PG synthase-2 promoter activity, whereas an overnight pre-incubation with phorbol ester to down-regulate protein kinase C did not. PTH-(3-34), a peptide that has greatly reduced ability to activate the cAMP-protein kinase A pathway, did not increase PG synthase-2 transcription or promoter activity. PTH could induce PG synthase-2 messenger RNA accumulation and PG synthase-2 transcription in the presence of cycloheximide. In addition, PTH-stimulated PG synthase-2 transcription was maintained at a high level at 2 h in the presence of cycloheximide. We conclude that PTH rapidly increases PG synthase-2 transcription in MC3T3-E1 cells, mainly through a cAMP-protein kinase A-mediated pathway without the need for protein synthesis. In contrast, the attenuation of increased PG synthase-2 transcription by PTH requires de novo protein synthesis.

Differential effects of nonsteroidal anti-inflammatory drugs on constitutive and inducible prostaglandin G/H synthase in cultured bone cells.

The production of prostaglandins by osteoblasts is an important mechanisms for the regulation of bone turnover. Bone cells contain both inducible and constitutive prostaglandin G/H synthase (PGHS-2 and PGHS-1) and these are differentially regulated. Nonsteroidal anti-inflammatory drugs (NSAIDs), which selectively inhibit one of these enzymes, would be useful in assessing their relative roles in bone metabolism. By Northern analysis, only PGHS-2 is expressed by the immortalized rat osteoblastic cell line, Py1a, while only PGHS-1 is expressed by the rat osteosarcoma cell line, ROS 17/2.8. We tested the relative inhibitory potency (IC50) of seven different NSAIDs on these two cell lines. A recently described selective inhibitor of PGHS-2, NS-398, was approximately 30 times more potent in inhibiting PGHS-2 than PGHS-1, and diclofenac was approximately 10 times more potent. Both had IC50's of approximately 3 nM for PGHS-2 in Py1a cells. Indomethacin, flurbiprofen, naproxen, and piroxicam were relatively nonselective with IC50's ranging from 30 nM to 1 microM, while 6-methoxy-2 naphthyl acetic acid, the active metabolite of nabumetone, was inhibitory only at concentrations greater than 1 microM. These results indicate that the presently available NSAIDs are unlikely to distinguish completely between effects mediated by PGHS-2 or PGHS-1. However, the cell systems employed could provide a model for the analysis of new compounds with greater selective activity.

Transcriptional induction of cyclooxygenase-2 in osteoblasts is involved in interleukin-6-induced osteoclast formation.

Interleukin-6 (IL-6) induces osteoclast-like cell (osteoclast) formation in a dose-dependent fashion in cocultures of mouse bone marrow cells and osteoblastic cells when soluble IL-6 receptor (sIL-6R) is present. Simultaneous treatment with submaximal doses of IL-1alpha and IL-6 with sIL-6R caused marked induction of osteoclast formation and PGE2 synthesis. These effects were suppressed by adding neutralizing antibodies against IL-1alpha or IL-6R and were totally abolished by adding nonsteroidal antiinflammatory drugs, such as indomethacin and a selective cyclooxygenase-2 (COX-2) inhibitor (NS398). In mouse osteoblastic cells, both IL-1alpha and IL-6 with sIL-6R markedly induced messenger RNA expression of COX-2, but not COX-1, as determined by Northern blot analysis, and luciferase activity in cells stably transfected with a COX-2 promoter-luciferase fusion construct. IL-6 and sIL-6R, when added separately, did not stimulate COX-2 messenger RNA expression. Simultaneous addition of IL-1alpha and IL-6 with sIL-6R to osteoblast cultures cooperatively induced transcription of COX-2, which was associated with a marked increase in COX activity measured by the conversion of arachidonic acid into PGE2. The increased PGE2 synthesis by osteoblasts may play an important role in osteoclastogenesis induced by submaximal doses of IL-1 and IL-6.

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 PGF2alpha 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. PGF2alpha 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.

Molecular cloning and expression of a rat prostaglandin E2 receptor of the EP2 subtype.

Prostaglandin E2 (PGE2) is a potent local mediator of cell growth and differentiation in various tissues. The receptors for PGE2 have been classified into four pharmacological subtypes, EP1, EP2, EP3, and EP4, based on the responses to selective agonists and antagonists. We have cloned a functional cDNA for the rat EP2 receptor subtype from a rat lung cDNA library. The rat EP2 receptor cDNA encodes 357 amino acid residues having high homology with the human and mouse EP2 receptors and containing seven putative transmembrane domains. In COS-7 cells transfected with rat EP2 cDNA, specific [3H]PGE2 binding was found with a dissociation constant of 14.9 nM, and this binding was inhibited by unlabeled PGE2 and PGE2 alpha. PGE2 and butaprost, an EP2 selective agonist, were effective in increasing the cAMP level in the COS-7 cell transfectants. Northern blot and RT-PCR analysis showed widespread distribution of the EP2 receptor in various tissues. Higher EP2 expression was found in fetal long bones and calvariae than in adult by RT-PCR and in situ hybridization, suggesting a role for this receptor in rapidly growing skeletal tissue.

Lack of evidence for an increase in interleukin-6 expression in adult murine bone, bone marrow, and marrow stromal cell cultures after ovariectomy.

Interleukin-6 (IL-6) has been implicated as a mediator of postmenopausal bone loss. In vitro studies of bone and bone marrow cells have suggested that estrogen regulates bone turnover by controlling the production of IL-6, a potent stimulator of osteoclastogenesis and bone resorption. To investigate this hypothesis in an in vivo model, we examined the effect of ovariectomy or estrogen replacement on IL-6 mRNA and protein expression in adult mouse bone and bone marrow in vivo and in marrow stromal cell cultures. Eight-week-old CD-1 mice were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized and subcutaneously implanted with 21-day slow-release pellets containing 10 micrograms of 17 beta-estradiol (O + E). Placebo pellets were implanted in the SHAM and OVX mice. Uterine weights at 1, 2, or 3 weeks after surgery were significantly decreased (68-76%) in OVX animals compared with SHAM or O + E. In mice sacrificed at 1 or 3 weeks after surgery, we found by nonquantitative reverse transcribed polymerase chain reaction (RT-PCR), that SHAM, OVX, and O + E calvariae (CALV) constitutively expressed IL-6 mRNA. In contrast, IL-6 mRNA was either barely detectable or absent in the tibia (TIB) and bone marrow (BM). In the mice sacrificed 3 weeks after surgery, we determined by quantitative RT-PCR that IL-6 mRNA in the CALV from the OVX and O + E groups were decreased by 81 and 92%, respectively, compared with SHAM. IL-6 protein levels in the flushed bone marrow (BMSups) were detectable and were not significantly different among the groups. In bone marrow cells that were cultured for 1 week, basal levels of IL-6 protein were low and did not differ significantly among the SHAM, OVX, or O + E groups sacrificed 1, 2, or 3 weeks after surgery. After the addition of hrIL-1 alpha, IL-6 protein levels increased 100- to 1300-fold over control. IL-6 levels in cells from animals sacrificed 2 weeks after surgery were significantly lower in the hrIL-1 alpha-stimulated OVX and O + E groups than in hrIL-1 alpha-stimulated SHAM cell cultures. In conclusion, in this model we could find no increase in IL-6 production with in vivo estrogen withdrawal in calvaria, long bones, bone marrow, or marrow stromal cell cultures. If increases in IL-6 expression are involved in the effects of estrogen withdrawal on bone, the magnitude of these changes are relatively small and below the limits of detection of the assays that we employed.