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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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 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 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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Prostaglandins are potent regulators of skeletal metabolism which can be produced by cells of the osteoblastic lineage.1 Regulation of prostaglandin production by mechanical forces, hormones, cytokines, and growth factors appears to be due in large part to effects on the transcriptional regulation of the inducible prostaglandin G/H synthase (PGHS-2) or cyclo-oxygenase-2.2,3 However, bone also contains the constitutive enzyme, PGHS-1 or cyclo-oxygenase-1. Selective inhibitors of PGHS-1 or PGHS-2 would facilitate the analysis of the relative roles of these two enzymes in physiologic and pathologic responses of the skeleton. Marked differences in the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on isolated preparations of the two enzymes have been reported,4–6 but in vivo studies indicate that the majority of NSAIDs inhibit both PGHS-1 and PGHS-2.7 Recently, compounds that may be selective inhibitors of PGHS-2 have been reported.8–11

To examine the regulation of the inducible and constitutive enzymes in osteoblasts, we have compared an immortalized rat osteoblastic cell line, Py1a,12 which expresses only PGHS-2 on Northern analysis, with a rat osteosarcoma cell line, ROS 17/2.8, which expresses only PGHS-1. ROS 17/2.8 has osteoblastic features and is constitutively active in prostaglandin synthesis.13 The availability of these cells has given us the opportunity to estimate the relative inhibitory potency (IC50) of different NSAIDs on prostaglandins produced only by the inducible or constitutive enzyme. The majority of NSAIDs tested in the present study showed similar activities against both enzymes. However, the compound NS-398, which was previously shown to be PGHS-2 selective,8–11 showed greater potency in Py1a than in ROS 17/2.8 cells. A smaller difference was also found with diclofenac, which is ordinarily classified as a nonselective NSAID. Changes in endogenous prostaglandin production had little functional effect on ROS 17/2.8 cells but did affect collagen and noncollagen protein synthesis in Py1a cells in which exogenous prostaglandins have been shown to inhibit collagen synthesis.12,14

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Materials

Murine PGHS-1 cDNA, which cross-reacts with rat PGHS-1 mRNA, was a gift of Drs. David DeWitt and William Smith (Michigan State University, East Lansing, MI, U.S.A.). Murine PGHS-2 cDNA, which also cross-reacts with rat mRNA, was obtained from Oxford Biomedical Research Inc. (Oxford, MI, U.S.A.). Murine glyceraldehyde-3-phosphate dihydrogenase (GAPDH) cDNA was amplified by PCR using an amplimer set from Clontech (Palo Alto, CA, U.S.A.). The prostaglandin E2 (PGE2) antibody was purchased from Dr. Lawrence Levine, Waltham, MA, U.S.A.). Enzyme-linked immunoassay (ELISA) kits for PGE2, PGF, and 6-keto-PGF were obtained from Elisa Technologies (Lexington, KY, U.S.A.). NS-398 was obtained from Cayman Chemical (Ann Arbor, MI, U.S.A.). Transforming growth factor β (TGF-β) was the gift of Genentech, Inc. (South San Francisco, CA, U.S.A.). Other prostanoids and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). The culture media were obtained from GIBCO BRL (Gaithersburg, MD, U.S.A.).

Cell cultures

ROS 17/2.8 and Py1a cells were grown to confluence in F-12 containing 5% fetal calf serum (FCS) and MC3T3-E1 cells in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS. Cells were then serum deprived for 24 h. The cells were treated for varying periods with TGF-β (10 ng/ml), arachidonic acid (AA, 10−5 M), or FCS. Combined treatment with AA and FCS was used to produce maximal induction of PGHS-2 and maximal prostaglandin production in Py1a cells. Although ROS 17/2.8 cells can produce large amounts of PGE2 simply with the addition of AA, these cells were treated in the same way as Py1a cells to minimize any differences in culture conditions.

The inhibitory effects of NSAIDs were generally tested after 24 h of treatment with a stimulator and varying concentrations of the drug. The concentration that produced 50% inhibition of prostaglandin production (IC50) was estimated graphically from plots of PGE2 levels in treated cultures expressed as percent of control untreated cultures. Each NSAID was tested in at least three experiments.

Prostaglandin measurements

For estimation of IC50, the PGE2 concentration of the medium was measured by radioimmunoassay, as described previously.2 For analysis of the relative proportions of PGE2, PGF, and 6-keto-PGF, the metabolite of prostacyclin, produced by Py1a cells, ELISA kits were used. Values are the means of duplicate or triplicate analyses.

RNA extraction and Northern blot analysis

RNA extraction

Total RNA was extracted by the method of Chomczynski and Sacchi15 in 4 M guanidine isothiocyanate followed by phenol/chloroform-isoamyl alcohol (24:1). RNA was precipitated with isopropanol and washed with 80% ethanol.

After quantitation at 260 nm, 20–25 μg of total RNA was fractionated on a 1% agarose/2.2 M formaldehyde gel and transferred to nylon membrane (Genescreen; New England Nuclear, Boston, MA, U.S.A.) by positive pressure blotting (Posiblotter; Stratagene Inc., La Jolla, CA, U.S.A.). After prehybridization in a 50% formamide solution at 42°C, filters were hybridized overnight in a similar solution in rotating cylinders (Hybridizer; Techne, Inc., Princeton, NJ, U.S.A.) at 42°C.

Collagen and noncollagen protein synthesis

Cells treated with or without FCS and AA were cultured for 24 h and pulsed with 5 mCi/ml of [3H]proline (15–40 Ci/mm, Amersham, Arlington Heights, IL, U.S.A.) for the final 4 h of culture. The medium was removed and the cells scraped into extraction buffer (1 M NaCl, 2.25 mM EDTA, 1 mM N-ethyl-maleimide, and 0.2 mM phenylmethylsulfonylfluoride). The medium and cells were pooled and sonicated and the protein precipitated with 15% trichloroacetic acid (TCA). After repeated washing, the pellets were dissolved in 0.5 M NaOH and an aliquot was digested with purified bacterial collagenase to determine collagen-digestible protein (CDP) and noncollagen protein (NCP) labeling. The percentage of total protein synthesis represented by collagen (percentage collagen synthesis, PCS) was calculated after correcting for the relative abundance of proline in collagen compared with noncollagen protein. DNA content was determined by fluorimetry using diaminophenylindole.

DNA synthesis

Five μCi/ml of [3H]thymidine (New England Nuclear Corp.) was added to cultures for the last 2 h of the culture period. Cells were washed with cold phosphate-buffered saline and extracted with 1.0 ml of 10% trichloroacetic acid. Acid-extractable [3H]TdR was determined by measuring the radioactivity in the trichloroacetic acid washes. After the addition of 1 ml of 0.5 M NaOH to each well, cells were scraped from the dishes, put into glass tubes, and incubated overnight at 4°C. An aliquot of each cell digest was counted to quantitate thymidine incorporation (TdR) into DNA.

Statistics

The data were subjected to analysis of variance, and the significance of differences was determined by post hoc testing using Bonferroni's method.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Expression of PGHS-1 and PGHS-2

The expression of mRNA for the inducible and constitutive enzymes in ROS 17/2.8 and Py1a cells is compared with the murine osteoblastic cell line, MC3T3-E1, in Fig. 1. Both enzymes were easily detectable in MC3T3-E1 cells, but only PGHS-2 showed a substantial stimulatory response to AA, TGF-β, or the combination. No PGHS-1 mRNA was detectable in Py1a cells, but these cells expressed PGHS-2 mRNA when stimulated by AA. In this experiment, TGF-β alone was not effective, but in other studies it was able to induce the enzyme.

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Figure FIG. 1. Northern analysis of mRNA in osteoblastic cells. MC3T3-E1, Py1a, and ROS 17/2.8 cells were cultured to confluence, serum deprived for 24 h, and then treated with arachidonic acid (AA) or transforming growth factor β (TGF-β) singly or in combination for 4 h. mRNA was extracted and analyzed for inducible (PGHS-2) and constitutive (PGHS-1) prostaglandin G/H synthase as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Loading was assessed by ethidium bromide staining for 28S ribosomal RNA.

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In ROS 17/2.8 cells, PGHS-1 mRNA was present at high levels and no PGHS-2 mRNA was detected by Northern analysis. Freshly added FCS also induced PGHS-2 in MC3T3-E1 and Py1a cells, as previously demonstrated,3,16 but not in ROS 17/2.8 (data not shown).

Prostaglandin production

To induce PGHS-2 expression and to supply exogenous substrate for both PGHS-1 and −2, cells were treated with FCS and AA. All three cell lines produced substantial amounts of PGE2, as measured by immunoassay. In previous studies, we showed that this was the major prostaglandin produced by ROS 17/2.8 and MC3T3-E1.13,17 However, the predominant product of Py1a cells after stimulation with FCS and AA was PGF. PGI2, as measured by its metabolite, 6-keto-PGF, and PGE2 were produced in somewhat smaller amounts (Table 1).

Table Table 1. Incorporation of [3H]Proline into Collagenase Digestible (CDP) and Noncollagen Protein (NCP), Percent Collagen Synthesis (PCS), and Prostaglandin Production in Py1a Cells Cultured in 5% Fetal Calf Serum (FCS) with Arachidonic Acid (AA, 10−5 M) with and Without Indomethacin (Indo)
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Indomethacin produced a parallel inhibition of all three products and this was associated with changes in cellular function (see below). Similar results were obtained with Py1a cells treated with TGF-β and AA (data not shown).

Effects of NSAIDs

The estimated IC50's for the NSAIDs tested are listed in Table 2 and the effects shown graphically for NS-398 and indomethacin in Fig. 2. In Py1a cells, NS-398 was a potent inhibitor of PGHS-2 with an IC50 of approximately 3 nM. In contrast, the IC50 in ROS 17/2.8 cells expressing only PGHS-1 was about 80 nM. Of the other NSAIDs tested, only diclofenac showed any appreciable difference in potency in the two cell types, with an IC50 for PGHS-2 in Py1a of 3 nM and 30 nM for PGHS-1 in ROS 17/2.8. 6-methoxy-2-naphthyl acetic acid (6-MNA), the active metabolite of nabumetone, which was previously reported to have some selectivity for PGHS-2, was a weak inhibitor with an estimated IC50 of more than 1000 nM. Indomethacin showed similar values of IC50 of 30 nM for both PGHS-2 and PGHS-1.

Table Table 2. Approximate IC50s for NSAIDs in 24 h Cultures of Py1a or ROS 17/2.8 Cells Stimulated with 5% FCS and AA (10−5 M)
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Figure FIG. 2. Effects of NS-398 and indomethacin on PGE2 production in Py1a (•) and ROS 17/2.8 (▪) cells. Cultures were treated with FCS and AA (10−5 M) for 24 h in the presence and absence of the indicated concentrations of NSAIDs. Points are means and vertical lines SE for the PGE2 concentration, calculated as percent of the value in the absence of NSAIDs for four to eight cultures. Graphic estimates of IC50 are indicated in Table 1.

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Effects of endogenous prostaglandins on cell function

Stimulation of prostaglandin synthesis in Py1a cells with AA and FCS resulted in a reduction in collagen synthesis as measured by the incorporation of [3H]proline. This is similar to the effects of exogenous prostaglandins on this cell line.12,14 Addition of indomethacin resulted in a relative increase in proline incorporation into collagen and an increase in percent collagen synthesis (Tables 1 and 3). In cultures treated with AA plus TGF-β, an increase in percent collagen synthesis was also observed with indomethacin (Table 3). The values for percent collagen synthesis were similar to those obtained in Py1a cells, cultured serum-free with bovine serum albumin (BSA), which do not produce measurable PGs.12,14 ROS 17/2.8 cells showed a lower level of percent collagen synthesis (0.9–1.3) with no significant effect of indomethacin. With respect to [3H]thymidine incorporation, indomethacin appeared to reduce the response in cultures treated with AA plus TGF-β, suggesting that some of the mitogenic effect of TGF-β might be mediated by endogenous prostaglandins. In contrast, there was no inhibition of TdR incorporation by indomethacin in cultures treated with FCS.

Table Table 3. Incorporation of [3H]Thymidine into DNA or [3H]Proline into CDP or NCP in Py1a Cells Cultured with AA (10−5 M) and TGF-β (10 ng/ml) or 5% FCS with or Without Indomethacin (Indo, 10−6M)
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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In this study, we have taken advantage of the observation that two rat osteoblastic cell lines, Py1a and ROS 17/2.8, differ in their expression of the critical enzymes for prostaglandin production. In Py1a, an osteoblastic cell line derived from rat calvaria and immortalized by transfection with polyoma virus T antigen,12 only the inducible PGHS-2 mRNA is detectable. In ROS 17/2.8, a rat osteosarcoma-derived cell line with osteoblastic phenotype,13 we are able to detect only the mRNA for the constitutive enzyme PGHS-1. A murine osteoblastic cell line, which we have studied previously, MC3T3-E1, expresses mRNA and proteins for both enzymes.3 Both enzymes are present in organ cultures of bone,2 but they have not yet been localized by immunocytochemistry or in situ hybridization.

In the present studies, we tested seven NSAIDs for their inhibitory effects on prostaglandin production stimulated by FCS and AA in the two cell types bearing different PGHS isoenzymes. The only differential effects we observed were with NS-398, a compound that has been shown selectively to inhibit PGHS-2 in other cell systems, and with diclofenac, which was not considered previously to be a selective inhibitor of either enzyme. It is of interest that diclofenac has been shown to decrease bone resorption in postmenopausal women.18 These results differ from those obtained by looking at instantaneous inhibition of cyclo-oxygenase activity with isolated enzymes.4 In those studies, indomethacin and piroxicam preferentially inhibited PGHS-1, while flurbiprofen showed no differential effect and 6-MNA preferentially inhibited PGHS-2. In our studies, 6-MNA, which is the active metabolite of nabumetone, was relatively ineffective in inhibiting prostaglandin synthesis in either cell line. There are a number of possible explanations for the differences that we observed in these prolonged cell culture experiments and those observed with isolated enzymes. Selective inhibition of PGHS-2 appears to be related to a time-dependent increase in the inactivation of these enzymes.19 Lack of uptake by the cell could explain the lack of efficacy of 6-MNA and differences in uptake might also affect the differences observed with other NSAIDs. There are also possible interactions involving autoamplification of the enzymes.16 While these are not relevant in the present models in which only one form is expressed, they could be explored in bone cells that express both enzymes.

Currently, selective inhibitors of PGHS-2 are being explored for their possible clinical use. If compounds that can safely produce long-term inhibition in vivo can be obtained, this will make it possible to explore the relative importance of the constitutive and inducible enzyme in the regulation of bone turnover. Based on our studies of the response of the two enzymes to cytokines, hormones, growth factors, and mechanical forces, the major regulator does appear to be PGHS-2. However, the constitutive enzyme, PGHS-1, may be important in initial responses to perturbations that result in the release of AA. In any event, the availability of two osteoblastic cell lines from the same species that have differential expression of the two enzymes will be useful in assessing new inhibitors for their potential effects on bone.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

These studies were supported by grants AR-18063, AR-38933, and DK-48361 from the National Institutes of Health. We thank Ms. Lisa Godin for her careful preparation of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Kawaguchi H, Pilbeam CC, Harrison JR, Raisz LG 1995 The role of prostaglandins in the regulation of bone metabolism Clin Orthop 313:3646.
  • 2
    Kawaguchi H, Raisz LG, Voznesensky OS, Alander CB, Hakeda Y, Pilbeam CC 1994 Regulation of the two prostaglandin G/H synthases by parathyroid hormone, interleukin-1, cortisol, and prostaglandin E2 in cultured neonatal mouse calvariae Endocrinology 135:11571164.
  • 3
    Pilbeam CC, Kawaguchi H, Hakeda Y, Voznesensky O, Alander CB, Raisz LG 1993 Differential regulation of inducible and constitutive prostaglandin endoperoxide synthase in osteoblastic MC3T3-E1 cells J Biol Chem 268:2564325649.
  • 4
    Meade EA, Smith WL, DeWitt DL 1993 Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs J Biol Chem 268:66106614.
  • 5
    Smith WL, Meade EA, DeWitt DL 1994 Interactions of PGH synthase isozymes-1 and -2 with NSAIDs Ann NY Acad Sci 744:5057.
  • 6
    DeWitt DL, Meade EA, Smith WL 1993 PGH synthase isoenzyme selectivity: The potential for safer nonsteroidal antiinflammatory drugs Am J Med 95:40S44S.
  • 7
    O'Neill GP, Kennedy BP, Mancini JA, Kargman S, Ouellet M, Yergey J, Falgueyret JP, Cromlish WA, Payette P, Chan CC et al. 1995 Selective inhibitors of COX-2 Agents Actions 46:159168.
  • 8
    Futaki N, Yoshikawa K, Hamasaka Y, Arai I, Higuchi S, Iizuka H, Otomo S 1993 NS-398, a novel non-steroidal anti-inflammatory drug with potent analgesic and antipyretic effects, which causes minimal stomach lesions Gen Pharmacol 24:105110.
  • 9
    Futaki N, Takahashi S, Yokoyama M, Arai I, Higuchi S, Otomo S 1994 NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro Prostaglandins 47:5559.
  • 10
    Gierse JK, Hauser SD, Creely DP, Koboldt C, Rangwala SH, Isakson PC, Seibert K 1995 Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase Biochem J 305:479484.
  • 11
    Masferrer JL, Zweifel BS, Manning PT, Hauser SD, Leahy KM, Smith WG, Isakson PC, Seibert K 1994 Selective inhibition of inducible cyclooxygenase 2 in vivo is antiinflammatory and nonulcerogenic Proc Natl Acad Sci USA 91:32283232.
  • 12
    Raisz LG, Fall PM, Petersen DN, Lichtler A, Kream BE 1993 Prostaglandin E2 inhibits alpha 1(I)procollagen gene transcription and promoter activity in the immortalized rat osteoblastic clonal cell line Py1a Mol Endocrinol 7:1722.
  • 13
    Rodan SB, Wesolowski G, Rodan GA 1986 Clonal differences in prostaglandin synthesis among osteosarcoma cell lines J Bone Miner Res 1:213220.
  • 14
    Fall PM, Breault DT, Raisz LG 1994 Inhibition of collagen synthesis by prostaglandins in the immortalized rat osteoblastic cell line Py1a: Structure-activity relations and signal transduction mechanisms J Bone Miner Res 9:19351943.
  • 15
    Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 162:156159.
  • 16
    Pilbeam CC, Raisz LG, Voznesensky O, Alander CB, Delman BN, Kawaguchi H 1995 Autoregulation of inducible prostaglandin G/H synthase in osteoblastic cells by prostaglandins J Bone Miner Res 10:406414.
  • 17
    Harrison JR, Lorenzo JA, Kawaguchi H, Raisz LG, Pilbeam C 1994 Stimulation of prostaglandin E2 production by interleukin-1α and transforming growth factor α in osteoblastic MC3T3-E1 cells J Bone Miner Res 9:817823.
  • 18
    Bell NH, Hollis BW, Shary JR, Eyre DR, Eastell R, Colwell A, Russell RG 1994 Diclofenac sodium inhibits bone resorption in postmenopausal women Am J Med 96:349353.
  • 19
    Copeland RA, Williams JM, Giannaras J, Nurnberg S, Covington M, Pinto D, Pick S, Trzaskos JM 1994 Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase Proc Natl Acad Sci USA 91:1120211206.