Cynthia B. Alander

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.

Extracellular Calcium Is a Potent Inducer of Cyclo‐oxygenase‐2 in Murine Osteoblasts Through an ERK Signaling Pathway

[Ca2+]e may be important in bone turnover. We found [Ca2+]e induces COX‐2 transcription and PGE2 production in primary calvarial osteoblasts through an ERK signaling pathway. Inhibition of PGE2 production inhibited the [Ca2+]e stimulation of osteoblastic differentiation but not the increase in cell number. Hence, some effects of [Ca2+]e on bone may be mediated by COX‐2.

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 mechanism 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 IC50s of approximately 3 nM for PGHS‐2 in Py1a cells. Indomethacin, flurbiprofen, naproxen, and piroxicam were relatively nonselective with IC50s ranging from 30 nM to 1 μM, while 6‐methoxy‐2 naphthyl acetic acid, the active metabolite of nabumetone, was inhibitory only at concentrations greater than 1 μM. 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.

Comparison of the effects of various lengths of synthetic human parathyroid hormone-related peptide (hPTHrP) of malignancy on bone resorption and formation in organ culture.

Parathyroid hormone-related peptide (PTHrP) has been shown to be the pathogenic agent in humoral hypercalcemia of malignancy (HHM), but the molecular forms that are secreted have not been fully characterized. PTHrP 1-34 has effects similar to parathyroid hormone (PTH), but C-terminal regions of the peptide, such as the 107-139 fragment found to inhibit resorption in a study by Fenton et al (1991), may have other biological activities not shared with PTH. We have compared the effects of the longer forms of recombinant human PTHrP (hPTHrP 1-84, 1-108, and 1-141) with hPTHrP 1-34 and synthetic bovine PTH (bPTH) 1-34 on bone resorption and formation in cultured neonatal mouse calvariae and fetal rat long bones. hPTHrP 1-84, 1-108, and 1-141 were qualitatively similar to hPTHrP 1-34 and PTH 1-34 in stimulating 45Ca release from both neonatal mouse calvariae and fetal rat long bones and in inhibiting the incorporation of [3H]-proline into collagenase digestible protein (CDP) and stimulating the incorporation of [3H]-thymidine (3H-TdR) in neonatal mouse calvariae. However, hPTHrP 1-108 and 1-141 were less potent at stimulating 45Ca release and inhibiting CDP labeling than hPTHrP 1-34, while hPTHrP 1-84 showed an intermediate potency. Since hPTHrP 1-108 and 1-141 were quite similar in potency, the difference cannot be attributed to an inhibitory effect of the 107-139 fragment. All the peptide lengths tested showed similar potency in stimulating [3H]-TdR incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prostaglandin E3 and eicosapentaenoic acid on rat bone in organ culture

To assess the possibility that diets rich in eicosapentaenoic acid (EPA) could have adverse effects on the skeleton, we examined the resorptive response to its major project, PGE3, and the effects and metabolism of EPA itself in cultured fetal rat long bones and neonatal rat calvaria. PGE3 stimulated bone resorption with a potency similar to that of PGE2. However, EPA was a much less effective precursor for PGE3 than was arachidonic acid (AA) for PGE2. In bones cultured with complement sufficient rabbit serum, which stimulates endogenous PGE release, addition of EPA had little effect on bone resorption while AA produced a substantial increase. Bones labeled with [3H]-AA and incubated with transforming growth factor-alpha (TGF-alpha), which stimulates endogenous PGE production, produced substantial amounts of PGE2, while bones labeled with [3H]-EPA and treated similarly produced less than 1/10th as much labeled PGE3. Thus, EPA appears to be a less effective precursor for the production of bone resorbing prostanoids than AA in cultured rat bone. However, since PGE3 is a potent stimulator of bone resorption, the possibility that dietary EPA can effect the production of bone resorbing prostanoids in man requires further study.

Effects of prostaglandin E2 on bone in mice in vivo

We have examined the effects of varying doses, schedules and routes of administration of prostaglandin E(2) (PGE(2)) on bone in mice. Male C57BL/6 mice treated with a high dose of PGE(2) (6 mg/kg/d) showed decreased trabecular bone volume (BV/TV) by 14 d, indicating increased bone resorption. However, there was also stimulation of bone formation at 14 d after 3 d treatment with PGE(2,) since mineral apposition rate (MAR) and bone formation rate (BFR/BS) were increased. In CD-1 male and female mice, PGE(2) (3mg/kg, 2/wk for 4 wk) increased MAR by 50% and BFR/BS by 100%, but there was no significant change in BV/TV. Tibial mRNA showed an increase in BMP-2 and RUNX-2 expression with PGE(2). Additional experiments using a higher dose or longer exposure did not increase bone mass. We conclude that exposure to high doses of PGE(2) in mice may be anabolic but is balanced by catabolic effects. Studies of PGE(2) in combination with an inhibitor of resorption could lead to development of a true anabolic model and permit assessment of the roles of specific PGE(2) receptors and signal transduction pathways.

Effects of two bacterial products, muramyl dipeptide and endotoxin, on bone resorption in organ culture

SummaryWe have compared two components of bacterial cell walls, muramyl dipeptide (MDP) and lipopolysaccharide (LPS), for their effects on bone resorption as measured by the release of previously incorporated45Ca. MDP is the smallest active component of peptidoglycan, whereas LPS is the active component of endotoxin. Fetal rat long bones were cultured for 5 days in a chemically defined medium supplemented with bovine serum albumin (BSA) or serum. LPS increased45Ca release at concentrations of 0.03–1.0 µg/ml. LPS further purified by electrolytic dialysis (ED-LPS) was active at 0.01 µg/ml. ED-LPS was ineffective at such low concentrations in the presence of serum. The response to MDP was more variable than that to LPS, but bone resorption was stimulated at concentrations of 10−7–10−5 M. MDP was less effective or inactive in medium supplemented with serum. Stereoisomers of MDP that do not have adjuvant activity caused minimal stimulation of bone resorption, whereas 6-0-steroyl MDP stimulated resorption at 10−8 M. The stimulation of bone resorption by LPS and MDP was not inhibited by indomethacin. Both LPS and MDP increased lysosomal enzyme release in proportion to their effects on45Ca release. LPS also markedly increased collagenase activity in the medium, but MDP did not. These results indicate that chemically different products of bacterial cell walls can stimulate bone resorption in vitro. These products may be distinguished by differences in dose response curve, serum inhibition, and collagenase release.

Effect of deletion of the prostaglandin EP2 receptor on the anabolic response to prostaglandin E2 and a selective EP2 receptor agonist

Studies using prostaglandin E receptor (EP) agonists indicate that prostaglandin (PG) E(2) can have anabolic effects through both EP4 and EP2 receptors. We previously found that the anabolic response to a selective EP4 receptor agonist (EP4A, Ono Pharmaceutical) was substantially greater than to a selective EP2 receptor agonist (EP2A) in cultured murine calvarial osteoblastic cells. To further define the role of the EP2 receptor in PG-mediated effects on bone cells, we examined the effects of EP2A and PGE(2) on both calvarial primary osteoblasts (POB) and marrow stromal cells (MSC) cultured from mice with deletion of one (Het) or both (KO) alleles of the EP2 receptor compared to their wild-type (WT) littermates. Deletion of EP2 receptor was confirmed by quantitative real-time PCR, Western blot and immunohistochemistry. The 1 month-old mice used to provide cells in these studies did not show any significant differences in their femurs by static histomorphometry. EP2A was found to enhance osteoblastic differentiation as measured by alkaline phosphatase mRNA expression and activity as well as osteocalcin mRNA expression and mineralization in the WT cell cultures from both marrow and calvariae. The effects were somewhat diminished in cultures from Het mice and abrogated in cultures from KO mice. PGE(2) effects were greater than those of EP2A, particularly in POB cultures and were only moderately diminished in Het and KO cell cultures. We conclude that activation of the EP2 receptor is able to enhance differentiation of osteoblasts, that EP2A is a true selective agonist for this receptor and that PGE(2) has an additional anabolic effect likely mediated by the EP4 receptor.

Effects of global or targeted deletion of the EP4 receptor on the response of osteoblasts to prostaglandin in vitro and on bone histomorphometry in aged mice

Because global deletion of the prostaglandin EP4 receptor results in neonatal lethality, we generated a mouse with targeted EP4 receptor deletion using Cre-LoxP methodology and a 2.3 kb collagen I a1 promoter driving Cre recombinase that is selective for osteoblastic cells. We compared wild type (WT), global heterozygote (G-HET), targeted heterozygote (T-HET) and knockout (KO) mice. KO mice had one targeted and one global deletion of the EP4 receptor. All mice were in a mixed background of C57BL/6 and CD-1. Although there were one third fewer G-HET or KO mice at weaning compared to WT and T-HET mice, G-HET and KO mice appeared healthy. In cultures of calvarial osteoblasts, prostaglandin E(2) (PGE(2)) increased alkaline phosphatase (ALP) activity in cells from WT mice, and this effect was significantly decreased in cells from either G-HET or T-HET mice and further decreased in cells from KO mice. A selective agonist for EP4 receptor increased ALP activity and osteocalcin mRNA levels in cells from WT but not KO mice. A selective COX-2 inhibitor, NS-398, decreased osteoblast differentiation in WT but not KO cells. At 15 to 18 months of age there were no differences in serum creatinine, calcium, PTH, body weight or bone mineral density among the different genotypes. Static and dynamic histomorphometry showed no consistent changes in bone volume or bone formation. We conclude that expression of the EP4 receptor in osteoblasts is critical for anabolic responses to PGE(2) in cell culture but may not be essential for maintenance of bone remodeling in vivo.

Effects of thionapthene 2-carboxylic acid and related compounds on bone resorption in organ culture

SummaryWe have compared the effects of thiophene 2-carboxylic acid (TCA) and a number of sulfur-and nitrogen-containing analogs for their ability to inhibit bone resorption in organ cultures of fetal rat long bones. Four compounds,—thionapthene-2-carboxylic acid (TNCA), dibenzo-thiophene-4-carboxylic acid, indole-2-carboxylic acid and carbazole-1-carboxylic acid—caused a doserelated inhibition of PTH-stimulated bone resorption, although TCA was ineffective in this system. TNCA at 3×10−4M or 10−4M was the most potent inhibitor of PTH-stimulated bone resorption and was selected for further study. TNCA also inhibited stimulation of resorption by prostaglandin E2 and 1,25-dihydroxyvitamin D. Unlike calcitonin, the effect of TNCA was persistent and did not show escape. Moreover, TNCA could inhibit resorption in bones that had previously escaped from calcitonin. TNCA did not appear to be a nonspecific toxin, since it did not decrease incorporation of [3H] thymidine or [3H]proline into fetal rat long bones. The fact that resorption in unstimulated cultures was only decreased when the control rates were high also argues against nonspecific toxicity. Moreover, this suggests that TNCA will be most effective under conditions of accelerated bone resorption when an inhibiting effect is most desirable. heterocyclic sulfur-containing compound, thiophene-2 carboxylic acid (TCA) in the rat. Subsequent studies showed that this compound could inhibit bone resorption in organ cultures of neonatal mouse calvaria [3]. We have compared TCA with a number of analogs for their ability to inhibit PTH stimulated bone resorption in organ culture. TCA itself was found to be relatively ineffective, whereas several of the analogs were inhibitory at concentrations of 3×10−4 or 10−5M. One of the most potent compounds, thionapthene 2-carboxylic acid (TNCA) was selected for further study. TNCA was a potent inhibitor not only of PTH stimulated bone resorption, but of resorption stimulated by prostaglandin E2 (PGE2) and 1,25-dihydroxyvitamin D (1,25-(OH)2D3). It was less effective in inhibiting resorption in control unstimulated cultures. TNCA did not appear to act as a nonspecific toxin in that it did not decrease [3H]-thymidine or [3H]-proline incorporation into bones. The inhibitory effect of TCA was persistent, unlike that of calcitonin, which shows escape after initial inhibition. Moreover, TNCA had a powerful inhibitory effect when it was added to bones that had previously escaped from CT.