- Email: [email protected]
Arachidonic acid for loading induced prostacyclin and prostaglandin E2 release from osteoblasts and osteocytes is derived from the activities of different forms of phospholipase A2
Bone Vol. 27, No. 2 August 2000:241–247
Arachidonic Acid for Loading Induced Prostacyclin and Prostaglandin E2 Release From Osteoblasts and Osteocytes Is Derived From the Activities of Different Forms of Phospholipase A2 S. C. F. RAWLINSON, C. P. D. WHEELER-JONES, and L. E. LANYON Department of Veterinary Basic Sciences, The Royal Veterinary College, London, England
Introduction Mechanical loading of bone stimulates resident bone cells to produce prostacyclin (PGI2) and prostaglandin (PG)E2 by a mechanism that can be differentially regulated by ion channel blockers. We have investigated differences in the loadingrelated PG production mechanisms in rat ulnae explants loaded ex vivo. Loading and aluminium fluoride (AlF3, a nonselective activator of G-proteins) both increased PGI2 and PGE2 release into culture medium. Pertussis toxin (PTX) blocked loading-related release of PGE2, but not PGI2, while isotetrandrine, an inhibitor of G-protein-mediated activation of phospholipase (PL)A2, abolished the loading-related release of both PGs. This suggests both PTX-sensitive and -insensitive G-protein-dependent, PLA2-mediated mechanisms for loading-related PG production. Blockade of secretory (s)PLA2 activity prevented loading-related release of PGE2 and PGI2, whereas inhibition of cytosolic (c)PLA2 activity blocked loading-related release of PGE2 alone. cPLA2 was localized immuno-cytochemically to osteoblasts, but not to osteocytes. sPLA2 was localized to osteocytes and osteoblasts. Exogenous type-IA sPLA2 and type-IB sPLA2 stimulated significant increases in PGE2 and PGI2 release. PTX reduced the release of both PGs stimulated by type IA PLA2, but not type IB. Furthermore, inhibition of protein kinase C (PKC) activity blocked loading-related release of PGE2, but not that of PGI2. These data suggest that loadingrelated release of PGI2 and PGE2 utilizes arachidonic acid derived from the activity of different PLA2s. In osteocytes and osteoblasts, arachidonic acid for PGI2 synthesis is liberated by PTX-insensitive G-protein-dependent sPLA2 alone. In osteoblasts, arachidonic acid for PGE2 synthesis is released by PTX-sensitive, G-protein-dependent, cPLA2-mediated activity, which also requires upstream sPLA2 and PKC activities. (Bone 27:241–247; 2000) © 2000 by Elsevier Science Inc. All rights reserved.
The skeleton’s ability to adjust its architecture in relation to its local load-bearing environment implies that bone cells regulate bone modeling and remodeling activities in response to the consequences of loading in the bone tissue. Both prostacyclin (PGI2) and prostaglandin E2 (PGE2) have consistently been demonstrated to be produced by bone loaded in situ in organ culture.21,22 The loading-related release of these two prostanoids from resident osteocytes and osteoblasts can be independently modulated by selective ion channel blockers,23 suggesting that their generation is differentially regulated. Studies performed by Binderman et al. had suggested that mechanically stimulated PGE2 is produced from arachidonic acid derived from the action of phospholipase (PL)A2.4 Two distinct classes of PLA2 have since been described:16 high molecular weight cytosolic isoforms including the 85-kDa calcium-sensitive cPLA2,10,31 and low molecular weight “secretory” (14-kDa sPLA2)9,20,25 isoforms. G-proteins have also been demonstrated to be involved with PGE2 release from cells in monolayer culture, as shown by the effects of exogenous aluminium fluoride (AlF3), a nonselective activator of G-proteins.27 In the present study, we investigated the stimulatory effect of mechanical load and AlF3 on PG release from cultured rat ulnae. The involvement of G-proteins in the regulation of the mechanically related release of PGE2 and PGI2 was examined using pertussis toxin (PTX) and isotetrandrine, an inhibitor of Gprotein-mediated activation of PLA2. The results from these experiments suggested that the release of PGE2 and PGI2 from resident bone cells stimulated by mechanical load involve two different mechanisms, possibly at the level of PLA2. These mechanisms were analyzed further with selective inhibitors of cPLA2 and sPLA2 activity. cPLA2 and sPLA2 were also localized immuno-cytochemically. Materials and Methods
Key Words: Mechanical load; PLA2; Osteocytes; Osteoblasts; Prostaglandins.
Preparation of Tissue Ulnae from male rats (95 ⫾ 5 g) (Charles River, Margate, Kent, UK) were prepared in the manner described by Cheng et al.6 After a lethal injection of barbiturate, ulnae were removed and their shafts sectioned to an equal length by the removal of the proximal and distal extremities. Attendant soft tissue was removed, leaving the periosteum undisturbed, and the marrow was
Address for correspondence and reprints: Dr. S. C. F. Rawlinson, Department of Veterinary Basic Sciences, The Royal Veterinary College, London, NW1 0TU, England. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.
241
8756-3282/00/$20.00 PII S8756-3282(00)00323-9
242
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
Bone Vol. 27, No. 2 August 2000:241–247
flushed out. The explants were then cultured in a humidified incubator (5% CO2, 37°C) for 4 h in Dulbecco’s minimal essential medium plus 10% charcoal/dextran extracted fetal calf serum, 2 mmol/L L-glutamine, and 100 IU/mL penicillin and 100 g/mL streptomycin (Life Technologies Ltd., Paisley, Scotland). Loading After 4 h of preincubation, explants were transferred to the loading apparatus22 with fresh medium (4 mL), either with or without pharmacological agents, and allowed to equilibrate for 5 min in a humidified incubator. The explants were then subjected to intermittent axial load provided by a pneumatically operated actuator for 10 min (1 Hz). The load applied by the actuator was adjusted to engender maximum longitudinal tensile strains on the lateral midshaft surface of 3000 ε. This compares with peak strains at the same location in vivo of 1300 ε during walking and 2500 ε during more vigorous activities.18 Control bones were manipulated identically, that is, they were placed into the loading apparatus either with or without pharmacological agents, but not loaded. Measurement of prostaglandin concentrations in conditioned medium After removal of the bones from the loading apparatus, conditioned medium was collected and stored (⫺20°C) before determination of 6-keto-PGF1␣ (the stable hydrolytic product of PGI2) and PGE2 levels by enzyme immunoassay (Assay Designs Ltd., Ann Arbor, MI). None of the compounds that were added to the culture medium interfered with the immunoassays, except where indicated. Localization of phospholipase A2 Freshly dissected explants were immersed in 10% polyvinyl alcohol (Sigma, Poole, UK) and chilled by precipitate immersion in n-hexane (Sigma) at ⫺70°C. Undecalcified sections (10 m) were cut on a Bright’s microtome housed in a cryostat at ⫺30°C. Background blocking was accomplished by incubating sections for 1 h in 10% heat-inactivated normal goat serum in phosphatebuffered saline (PBS) (Sigma). Removal of serum by aspiration was followed by the application of primary antibody for either cPLA2 or sPLA2 (CG53Ab) for 12 h at 4°C. Control sections were incubated for this time in PBS alone. After washing 3 ⫻ 5 min in PBS, an FITC-conjugated second antibody (Sigma) was applied to the sections for 40 min. Three 5-min washes followed before mounting in glycerol (Merck, Poole, UK) and observation using an Olympus BHS microscope. The polyclonal cPLA2 and sPLA2 antisera employed in these studies were kindly provided by Dr. Ruth Kramer and Dr. Lisa Marshall (Eli Lily, Indianapolis, IN), respectively. Statistics Statistical levels of significance were determined on the raw data using the Student’s t-test (paired or unpaired, where appropriate), and a value of 0.05 was considered significant. PGE2 and PGI2 release are presented graphically. Each experiment was performed at least twice with the total number of bones subjected to each treatment being no less than eight.
Figure 1. Histograms showing the effects of mechanical load, ALF3 (10 mmol/L) or PTX (100 ng/mL) alone on PGE2 and PGI2 production from rat ulna explants, and load in the presence of PTX (100 ng/mL). Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005: a Compared with basal control; bcompared with control of the treatment.
Results Effect of mechanical load on PGE2 and PGI2 release Ulnar explants, subjected to a cyclical 1-Hz load producing peak levels of mechanical strain within the physiological range (3000 ε), responded with significantly increased release of PGE2 and PGI2 into the culture medium. (58%, p ⫽ 0.02; and 49%, p ⫽ 0.002, respectively, paired t-test, Figure 1). Effect of AlF3 on PGE2 and PGI2 release AlF3 (Sigma) (10 mmol/L), a nonselective stimulator of Gproteins,27 stimulated PGE2 release by 41% and PGI2 release by 50% (p ⫽ 0.04 and p ⫽ 0.02, respectively, unpaired t-test; Figure 1). Effect of PTX on basal and load-related PGE2 and PGI2 release Basal release of neither PGE2 nor PGI2 was altered by the presence of Gi/Go inhibitor PTX27 (Sigma) (100 ng/mL, unpaired t-test). The loading-related increase in the release of PGI2, compared with nonloaded PTX-treated control was also unaffected by PTX (72%, p ⬍ 0.005, paired t-test). However, com-
Bone Vol. 27, No. 2 August 2000:241–247
Figure 2. Histograms showing the effects of the inhibitor of G-proteinmediated activation of PLA2, isotetrandrine (5 g/mL); the cPLA2 inhibitor, AACOCF3 (50 mol/L); and sPLA2 inhibitor, manoalide (0.04 mmol/L), on loading-related PGE2 and PGI2 release from rat ulnar explants. Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005: acompared with basal control; bcompared with control of the treatment. Symbols beneath the graph indicate the presence (⫹) or absence (⫺) of treatment.
pared with PTX-treated, nonloaded controls, PTX blocked the loading-related release of PGE2 (Figure 1). Effect of isotetrandrine on basal and load-related PGE2 and PGI2 release Isotetrandrine (Calbiochem, Nottingham, UK) (5 g/mL), an inhibitor of G-protein-mediated activation of PLA2,30 had no effect on basal levels of either PGE2 or PGI2 release (unpaired t-test), but blocked the loading-related release of both PGE2 and PGI2 (Figure 2). Effect of PLA2 inhibitors on basal and load-related PGE2 and PGI2 release Arachidonyl-trifluoromethyl ketone (AACOCF3; Calbiochem) (50 mol/L), an inhibitor of cPLA2 activity,3 elevated background readings in the PGE2 immunoassay. However, after correction for this raised background level in the conditioned medium, it was determined that AACOCF3 reduced basal PGE2 release to below the level of detection. Mechanical loading did
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
243
Figure 3. Histograms showing the effects of type IB pancreatic PLA2 (50 units/mL) and type IA snake venom PLA2 (50 units/mL) on basal PGE2 and PGI2 release from rat ulnar explants. Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005: acompared with basal control; bcompared with control of the treatment.
not stimulate any increase in PGE2 release. In contrast, basal PGI2 release was significantly elevated by AACOCF3 (255%, p ⫽ 0.0001, unpaired t-test). Compared with AACOCF3treated nonloaded controls, AACOCF3 did not block the loading-related increases in PGI2 release (100%, p ⫽ 0.05, paired t-test) (Figure 2). Manoalide (Calbiochem) (0.04 mmol/L), an inhibitor of sPLA2 activity,15 significantly reduced basal levels of PGI2 released into the medium (⫺60%, p ⬍ 0.0001, unpaired t-test) and abolished loading-related increases in PGI2 production (paired t-test). In contrast, manoalide had no effect on basal PGE2 release but did block the loading-related increases in its release (Figure 2). Effect of exogenous sPLA2s on PGE2 and PGI2 release Type IB, pancreatic sPLA2 (Sigma) (50 units/mL) isolated from porcine pancreas significantly increased PGE2 release by 93%, and PGI2 release by 245% (p ⬍ 0.02 and p ⬍ 0.003, respectively, unpaired t-test; Figure 3a) after a 15-min exposure. Type IA, snake venom sPLA2 (Sigma) (50 units/mL) (from naja mossambica mossambica) significantly increased PGE2 release nearly fivefold, and PGI2 release 11-fold, (p ⬍ 0.005 and p ⬍ 0.003, respectively, unpaired t-test; Figure 3b) after a 15-min exposure.
244
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
Figure 4. Histograms showing the effects of PTX (100 ng/mL) on PGE2 and PGI2 release stimulated by 50 units/mL of type IB pancreatic PLA2 and 50 units/mL of type IA snake venom PLA2. Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005; acompared with basal control; bcompared with control of the treatment.
Bone Vol. 27, No. 2 August 2000:241–247
Figure 5. Histograms showing the effects of the PKC inhibitor, bisindolylmaleimide (400 nmol/L), on loading-related PGE2 and PGI2 release from rat ulnar explants. Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005: acompared with basal control; bcompared with control of the treatment.
Discussion Effect of PTX on exogenous PLA2-mediated PGE2 and PGI2 release PTX (100 ng/mL) had no significant effect on the stimulation of PGE2 or PGI2 release elicited by pancreatic PLA2. This contrasts with the effect of PTX on snake venom PLA2-mediated PG release. The levels of both of PGE2 and PGI2 release were significantly decreased (⫺28%, p ⫽ 0.04; and ⫺44%, p ⫽ 0.05, respectively, unpaired t-test) (Figure 4). Effect of bisindolylmaleimide on basal and load-related PGE2 and PGI2 release Bisindolylmaleimide I (Calbiochem) (400 nmol/L), an inhibitor of PKC activity,28 did not block loading-related PGI2 release compared bisindolylmaleimide-treated nonloaded control (88%, p ⬍ 0.02, paired t-test), but did block loading-related release of PGE2. (Figure 5). Immunolocalization of cPLA2 and sPLA2 Freshly dissected ulnae were cryosectioned and immunologically reacted to localize cPLA2 and sPLA2. sPLA2 reactivity was detected in both osteocytes and osteoblasts, whereas cPLA2 immunoreactivity could only be detected in osteoblasts (Figure 6).
This series of experiments suggest that in resident cells of long bone explants, arachidonic acid for loading-related PGE2 and PGI2 synthesis is derived by different mechanisms. The evidence for this conclusion is drawn from the differences in the loadingrelated release of PGE2 and PGI2 in the presence of selective pharmacological inhibitors. Both mechanical loading and AlF3 (a nonspecific activator of G-proteins) stimulated PGI2 and PGE2 release above basal levels. PTX, which had no significant effect on basal release of either prostanoid, blocked the loading-related increase of PGE2, but not PGI2. Loading in the presence of isotetrandrine (an inhibitor of G-protein-mediated activation of PLA2) blocked loading-related release of both PGE2 and PGI2. These results suggest that the loading-related release of PGE2 and PGI2 is dependent on (both PTX-sensitive and PTX-insensitive) G-protein-mediated PLA2 activities, and therefore the liberation of arachidonic acid for loading-related PGE2 and PGI2 release could be derived from the activity of different forms of PLA2. Consistent with this is the finding that blockade of osteoblast cPLA2 activity by AACOCF3 abolished the loading-related release of PGE2, but not that of PGI2. Loading-related PGI2 release was unaffected by bisindolylmaleimide, an inhibitor of PKC activity. However, functional PKC activity is required for loading-related cPLA2-derived PGE2 release, because inhibition of PKC activity blocked the loading-related release of PGE2. This is consistent with the
Bone Vol. 27, No. 2 August 2000:241–247
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
245
Figure 6. Digitally scanned photomicrographs of (a, b) cPLA2 immuno-localization and (c, d) sPLA2 immuno-localization in undecalcified cryostat sections of rat ulnae. cPLA2 can be seen in the osteoblast cell layer on the bone surface, but is absent in the osteocytes of the bone matrix. sPLA2 is present in the osteoblastic cell layer and also in osteocytes of the bone matrix.
observation that cPLA2 activity can be enhanced by PKCmediated phosphorylation.14 It further supports the notion that the source of arachidonate for loading-related PGE2 synthesis is different from that for synthesis of PGI2. Interestingly, inhibition of osteoblastic and osteocytic sPLA2 activity with manoalide blocked the loading-related increases of both PGE2 and PGI2. This implies that active sPLA2 is also a requirement for the loading-related cPLA2-mediated PGE2 release from osteoblasts. Both exogenous type IA and type IB PLA2 applied to bone explants elevated PGI2 and PGE2 release into culture medium. This is consistent with the demonstration of an sPLA2 receptor in osteoblastic MC3T3-E1 cells.29 Treatment with PTX significantly reduced the release of PGE2 and PGI2 by type IA snake venom PLA2, but PTX did not block type IB pancreatic PLA2mediated PG release. Because PTX did not inhibit loadingrelated PGI2 release, while isotetrandrine did, it is possible that arachidonate for loading-related PGI2 synthesis is derived from type IB sPLA2 isoform (the PTX-insensitive G-protein-dependent pathway). Differences in PLA2-derived free arachidonic acid for loading-related synthesis of PGE2 and PGI2 may correlate with either (or both) the various consequences of mechanical loading to which resident bone cells are subjected and with the sites of PG production. PGE2 has been demonstrated in resident osteoblasts and its loading-related release is dependent on cPLA2 and active sPLA2, which are also localized to osteoblasts. cPLA2 was not detected in osteocytes. PGI2 (as 6-keto-PGF1␣), and PGI2 synthetase, however, are present in both osteocytes and osteoblasts,17,19 and the loading-related release of PGI2 is dependent on sPLA2 activity, which is localized to these cells. Mechanically induced PG release from isolated bone cell monolayer culture systems has been reported previously. Physiological levels of uniaxial mechanical strain (3400 ε) applied to isolated primary rat osteoblasts stimulated increases in PGI2, but not PGE2, release.32 However, fluid shear strains have been demonstrated to increase PGE2 release from isolated osteoblasts, a process that is dependent on PTX-sensitive G-proteins and sensitive to inhibitors of PKC.26,27 Our data relating to PGE2 release from osteoblast cells in situ are in accordance with those reports. Isolated osteocytes from embryonic chick calvariae release PGE2 and PGI2 in response to pulsed fluid flow.1,2,11,12 These
findings of increased PGE2 release from isolated osteocytes are, however, not consistent with the lack of positive immunostaining for PGE2 in osteocytes of adult mammalian bone samples in situ.17,21 Such discrepancies could stem from the species, age, and site from which the osteocytes are derived. Alternatively, the preferential survival of immature osteocytes in the isolation procedure from the lacunae-canalicular network, and their subsequent maintenance in monolayer culture, might account for these osteoblast-like characteristics.
Figure 7. Schematic to represent a possible signaling pathway between osteocytes and osteoblasts via the agency of sPLA2. sPLA2 derived from osteocytes activates a pertussis toxin-sensitive G-protein receptor residing in the osteoblast membrane, or directly activates a pertussis toxinsensitive G-protein in the cytoplasm. This signal is relayed to cPLA2, via a PKC-dependent mechanism, causing the release of arachidonic acid for conversion to PGE2 via cyclooxygenase.
246
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
It has been postulated that the osteocyte network, in situ, assesses the local mechanical environment in bone tissue, and by integrating the alterations in the local levels and distribution of bone surface strains and fluid shear forces, coordinates osteoblast behavior accordingly.13,5 If osteocytes are to be effective in influencing bone remodeling, there must be osteocyte-to-osteoblast communication. Gap junctions between osteoblasts and osteocytes have been reported,7,8 and are obvious candidates through which intracellular messages could pass. However, extracellular molecules might also fulfill a communicative role. That the early increases in loading-related PGE2 release from osteoblastic cPLA2 are dependent on sPLA2 activity suggests that sPLA2 acts in an autocrine or paracrine manner. A transcellular, ligand-binding mechanism29 would provide a communicative mechanism between osteocytes and osteoblasts via the agency of osteocyte-derived sPLA2. Such a mechanism of communication has been previously proposed in mast cell/fibroblast coculture experiments where sPLA2 released from activated mast cells stimulates the release of PGE2 from fibroblasts.24 A scheme based on the coculture model24 to illustrate the possible mechanism of communication in resident bone cells is presented in Figure 7. In conclusion, these results confirm a role for regulatory G-proteins in loading-related PGE2 and PGI2 release from osteoblasts and osteocytes in situ. Experiments with PTX and isotetrandrine suggest the involvement of both PTX-sensitive and PTX-insensitive G-protein-mediated PLA2s in the liberation of arachidonic acid for synthesis of these prostanoids. sPLA2 is responsible for the liberation of arachidonic acid for PGI2 production in osteoblasts and osteocytes. cPLA2 activity appears to be regulated by a PKC-dependent pathway and sPLA2 is also necessary to facilitate the liberation of arachidonic acid for PGE2 release from osteoblastic cPLA2. If osteocyte-derived sPLA2 were to act as a paracrine mediator of osteoblast activity, this would represent a mechanism for osteocyte-to-osteoblast cell communication advocated previously.5,13
Bone Vol. 27, No. 2 August 2000:241–247
8. 9.
10.
11.
12.
13. 14.
15.
16.
17.
18.
19.
20.
Acknowledgment: These studies were supported by a grant awarded by The Wellcome Trust.
21.
References
22.
1. Ajubi, N. E., KleinNulend, J., Alblas, M. J., Burger, E. H., and Nijweide, P. J. Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes. Am J Physiol 39:E171–E178; 1999. 2. Ajubi, N. E., Klein-Nulend, J., Nijweide, P. J., Vrijheid Lammers, T., Alblas, M. J., and Burger, E. H. Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes-a cytoskeleton-dependent process. Biochem Biophys Res Commun 225:62– 68; 1996. 3. Bartoli, F., Lin, H. K., Ghomashchi, F., Gelb, M. H., Jain, M. K., and Apitzcastro, R. Tight-binding inhibitors of 85-KDa phospholipase A2 but not 14-KDa phospholipase A2 inhibit release of free arachidonate in thrombin stimulated human platelets. J Biol Chem 269:15625–15630; 1994. 4. Binderman, I., Zor, U., Kaye, A. M., Shimshoni, Z., Harell, A., and Somjen, D. The transduction of mechanical force into biochemical events in bone cells may involve activation of phospholipase A2. Calcif Tissue Int 42:261– 266; 1988. 5. Burger, E. H., Klein-Nulend, J., Vanderplas, A., and Nijweide, P. J. Function of osteocytes in bone: their role in mechanotransduction. J Nutr 125:S2020 – S2023; 1995. 6. Cheng, M. Z., Zaman, G., and Lanyon, L. E. Estrogen enhances the stimulation of bone collagen synthesis by loading and exogenous prostacyclin, but not prostaglandin E2, in organ cultures of rat ulnae. J Bone Miner Res 9:805– 816; 1994. 7. Donahue, H. J., McLeod, K. J., Rubin, C. T., Andersen, J., Grine, E. A., Hertzberg, E. L., and Brink, P. R. Cell-to-cell communication in osteoblastic
23.
24.
25.
26.
27.
28.
29.
30.
networks: cell line-dependent hormonal regulation of gap junction function. J Bone Miner Res 10:881– 889; 1995. Doty, S. B. Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 33:509 –512; 1981. Emadi, S., Mirshahi, M., Elalamy, I., Nicolas, C., Vargaftig, B. B., and Hatmi, M. Cellular source of human platelet secretory phospholipase A2. Br J Haematology 100:365–373; 1998. Ghrib, F., Pyronnet, S., Bastie, M. J., FagotRevurt, P., Pradayrol, L., and Vaysse, N. Arachidonic-acid-selective cytosolic phospholipase A2 is involved in gastrin-induced AR4-2J-cell proliferation. Int J Cancer 75:239 – 245; 1998. Klein-Nulend, J., Burger, E. H., Semeins, C. M., Raisz, L. G., and Pilbeam, C. C. Pulsating fluid flow stimulates prostaglandin E2 release and inducible prostaglandin-G/H synthase messenger RNA expression in primary mouse bone cells. J Bone Miner Res 10:S405–S405; 1995. Klein-Nulend, J., Vanderplas, A., Semeins, C. M., Ajubi, N. E., Frangos, J. A., Nijweide, P. J., and Burger, E. H. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9:441– 445; 1995. Lanyon, L. E. Control of bone architecture by functional load bearing. J Bone Miner Res 7:S369 –S375; 1992. Lin, L. L., Lin, A. Y., and Knopf, J. L. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci USA 89:6147– 6151; 1992. Mayer, A. M. S., Glaser, K. B., and Jacobs, R. S. Regulation of eicosanoid biosynthesis in vitro and in vivo by the marine natural product manoalide: a potent inactivator of venom phospholipases. J Pharmacol Exp Ther 244:871– 878; 1988. Mayer, R. J. and Marshall, L. A. New insights on mammalian phospholipase A2s: comparison of arachidonoyl selective and arachidonoyl-nonselective enzymes. FASEB J 7:339 –348; 1993. Miyauchi, M., Takata, T., Ogawa, I., Ito, H., Kobayashi, J., Nikai, H., and Ijuhin, N. Immunohistochemical demonstration of prostaglandins in various tissues of the rat. Histochem Cell Biol 105:27–31; 1996. Mosley, J. R., March, B. M., Lynch, J., and Lanyon, L. E. Strain magnitude related changes in whole bone architecture in growing rats. Bone 20:191–198; 1997. Okiji, T., Morita, I., Kawashima, N., Kosaka, T., Suda, H., and Murota, S. Immunohistochemical detection of prostaglandin I2 synthase in various calcified tissue-forming cells in rat. Arch Oral Biol 38:31–36; 1993. Pruzanski, W., Stefanski, E., Vadas, P., and Ramamurthy, N. S. Inhibition of extracellular release of proinflammatory secretory phospholipase A2 (sPLA2) by sulfasalazine: a novel mechanism of anti-inflammatory activity. Biochem Pharmacol 53:1901–1907; 1997. Rawlinson, S. C. F., El Haj, A. J., Minter, S. L., Tavares, I. A., Bennett, A., and Lanyon, L. E. Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: a role for prostacyclin in adaptive bone remodeling? J Bone Miner Res 6:1345–1351; 1991. Rawlinson, S. C. F., Mosley, J. R., Suswillo, R. F. L., Pitsillides, A. A., and Lanyon, L. E. Calvarial and limb bone cells in organ and monolayer culture do not show the same early responses to dynamic mechanical strain. J Bone Miner Res 10:1225–1232; 1995. Rawlinson, S. C. F., Pitsillides, A. A., and Lanyon, L. E. Involvement of different ion channels in osteoblasts’ and osteocytes’ early responses to mechanical strain. Bone 19:609 – 614; 1996. Reddy, S. T. and Herschman, H. R. Transcellular prostaglandin production following mast cell activation is mediated by proximal secretory phospholipase A2 and distal prostaglandin synthase 1. J Biol Chem 271:186 –191; 1996. Reddy, S. T., Winstead, M. V., Tischfield, J. A., and Herschman, H. R. Analysis of the secretory phospholipase A2 that mediates prostaglandin production in mast cells. J Biol Chem 272:13591–13596; 1997. Reich, K. M. and Frangos, J. A. Protein kinase C mediates flow-induced prostaglandin E2 production in osteoblasts. Calcif Tissue Int 52:62– 66; 1993. Reich, K. M., McAllister, T. N., Gudi, S., and Frangos, J. A. Activation of G-proteins mediates flow-induced prostaglandin E2 production in osteoblasts. Endocrinology 138:1014 –1018; 1997. Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. Staurosporine, a potent inhibitor of phospholipid/Ca2⫹ dependent protein kinase. Biochem Biophys Res Commun 135:397– 402; 1986. Tohkin, M., Kishino, J., Ishizaki, J., and Arita, H. Pancreatic type phospholipase A2 stimulates prostaglandin synthesis in mouse osteoblastic cells (MC3T3-E1) via a specific binding site. J Biol Chem 268:2865–2871; 1993. Tsunoda, Y. and Owyang, C. The regulatory site of functional GTP-binding
Bone Vol. 27, No. 2 August 2000:241–247 protein-coupled to the high-affinity cholecystokinin receptor and phospholipase A2 pathway is on the G subunit of GQ protein in pancreatic acini. Biochem Biophys Res Commun 211:648 – 655; 1995. 31. Underwood, K. W., Song, C. Z., Kriz, R. W., Chang, X. J., Knopf, J. L., and Lin, L. L. A novel calcium-independent phospholipase A2, cPLA2␥, that is prenylated and contains homology to cPLA2. J Biol Chem 273:21926 –21932; 1998. 32. Zaman, G., Suswillo, R. F. L., Cheng, M. Z., Tavares, I. A., and Lanyon, L. E.
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
247
Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture. J Bone Miner Res 12:769 –777; 1997.
Date Received: November 8, 1999 Date Revised: March 16, 2000 Date Accepted: March 28, 2000
Arachidonic Acid for Loading Induced Prostacyclin and Prostaglandin E2 Release From Osteoblasts and Osteocytes Is Derived From the Activities of Different Forms of Phospholipase A2 S. C. F. RAWLINSON, C. P. D. WHEELER-JONES, and L. E. LANYON Department of Veterinary Basic Sciences, The Royal Veterinary College, London, England
Introduction Mechanical loading of bone stimulates resident bone cells to produce prostacyclin (PGI2) and prostaglandin (PG)E2 by a mechanism that can be differentially regulated by ion channel blockers. We have investigated differences in the loadingrelated PG production mechanisms in rat ulnae explants loaded ex vivo. Loading and aluminium fluoride (AlF3, a nonselective activator of G-proteins) both increased PGI2 and PGE2 release into culture medium. Pertussis toxin (PTX) blocked loading-related release of PGE2, but not PGI2, while isotetrandrine, an inhibitor of G-protein-mediated activation of phospholipase (PL)A2, abolished the loading-related release of both PGs. This suggests both PTX-sensitive and -insensitive G-protein-dependent, PLA2-mediated mechanisms for loading-related PG production. Blockade of secretory (s)PLA2 activity prevented loading-related release of PGE2 and PGI2, whereas inhibition of cytosolic (c)PLA2 activity blocked loading-related release of PGE2 alone. cPLA2 was localized immuno-cytochemically to osteoblasts, but not to osteocytes. sPLA2 was localized to osteocytes and osteoblasts. Exogenous type-IA sPLA2 and type-IB sPLA2 stimulated significant increases in PGE2 and PGI2 release. PTX reduced the release of both PGs stimulated by type IA PLA2, but not type IB. Furthermore, inhibition of protein kinase C (PKC) activity blocked loading-related release of PGE2, but not that of PGI2. These data suggest that loadingrelated release of PGI2 and PGE2 utilizes arachidonic acid derived from the activity of different PLA2s. In osteocytes and osteoblasts, arachidonic acid for PGI2 synthesis is liberated by PTX-insensitive G-protein-dependent sPLA2 alone. In osteoblasts, arachidonic acid for PGE2 synthesis is released by PTX-sensitive, G-protein-dependent, cPLA2-mediated activity, which also requires upstream sPLA2 and PKC activities. (Bone 27:241–247; 2000) © 2000 by Elsevier Science Inc. All rights reserved.
The skeleton’s ability to adjust its architecture in relation to its local load-bearing environment implies that bone cells regulate bone modeling and remodeling activities in response to the consequences of loading in the bone tissue. Both prostacyclin (PGI2) and prostaglandin E2 (PGE2) have consistently been demonstrated to be produced by bone loaded in situ in organ culture.21,22 The loading-related release of these two prostanoids from resident osteocytes and osteoblasts can be independently modulated by selective ion channel blockers,23 suggesting that their generation is differentially regulated. Studies performed by Binderman et al. had suggested that mechanically stimulated PGE2 is produced from arachidonic acid derived from the action of phospholipase (PL)A2.4 Two distinct classes of PLA2 have since been described:16 high molecular weight cytosolic isoforms including the 85-kDa calcium-sensitive cPLA2,10,31 and low molecular weight “secretory” (14-kDa sPLA2)9,20,25 isoforms. G-proteins have also been demonstrated to be involved with PGE2 release from cells in monolayer culture, as shown by the effects of exogenous aluminium fluoride (AlF3), a nonselective activator of G-proteins.27 In the present study, we investigated the stimulatory effect of mechanical load and AlF3 on PG release from cultured rat ulnae. The involvement of G-proteins in the regulation of the mechanically related release of PGE2 and PGI2 was examined using pertussis toxin (PTX) and isotetrandrine, an inhibitor of Gprotein-mediated activation of PLA2. The results from these experiments suggested that the release of PGE2 and PGI2 from resident bone cells stimulated by mechanical load involve two different mechanisms, possibly at the level of PLA2. These mechanisms were analyzed further with selective inhibitors of cPLA2 and sPLA2 activity. cPLA2 and sPLA2 were also localized immuno-cytochemically. Materials and Methods
Key Words: Mechanical load; PLA2; Osteocytes; Osteoblasts; Prostaglandins.
Preparation of Tissue Ulnae from male rats (95 ⫾ 5 g) (Charles River, Margate, Kent, UK) were prepared in the manner described by Cheng et al.6 After a lethal injection of barbiturate, ulnae were removed and their shafts sectioned to an equal length by the removal of the proximal and distal extremities. Attendant soft tissue was removed, leaving the periosteum undisturbed, and the marrow was
Address for correspondence and reprints: Dr. S. C. F. Rawlinson, Department of Veterinary Basic Sciences, The Royal Veterinary College, London, NW1 0TU, England. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.
241
8756-3282/00/$20.00 PII S8756-3282(00)00323-9
242
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
Bone Vol. 27, No. 2 August 2000:241–247
flushed out. The explants were then cultured in a humidified incubator (5% CO2, 37°C) for 4 h in Dulbecco’s minimal essential medium plus 10% charcoal/dextran extracted fetal calf serum, 2 mmol/L L-glutamine, and 100 IU/mL penicillin and 100 g/mL streptomycin (Life Technologies Ltd., Paisley, Scotland). Loading After 4 h of preincubation, explants were transferred to the loading apparatus22 with fresh medium (4 mL), either with or without pharmacological agents, and allowed to equilibrate for 5 min in a humidified incubator. The explants were then subjected to intermittent axial load provided by a pneumatically operated actuator for 10 min (1 Hz). The load applied by the actuator was adjusted to engender maximum longitudinal tensile strains on the lateral midshaft surface of 3000 ε. This compares with peak strains at the same location in vivo of 1300 ε during walking and 2500 ε during more vigorous activities.18 Control bones were manipulated identically, that is, they were placed into the loading apparatus either with or without pharmacological agents, but not loaded. Measurement of prostaglandin concentrations in conditioned medium After removal of the bones from the loading apparatus, conditioned medium was collected and stored (⫺20°C) before determination of 6-keto-PGF1␣ (the stable hydrolytic product of PGI2) and PGE2 levels by enzyme immunoassay (Assay Designs Ltd., Ann Arbor, MI). None of the compounds that were added to the culture medium interfered with the immunoassays, except where indicated. Localization of phospholipase A2 Freshly dissected explants were immersed in 10% polyvinyl alcohol (Sigma, Poole, UK) and chilled by precipitate immersion in n-hexane (Sigma) at ⫺70°C. Undecalcified sections (10 m) were cut on a Bright’s microtome housed in a cryostat at ⫺30°C. Background blocking was accomplished by incubating sections for 1 h in 10% heat-inactivated normal goat serum in phosphatebuffered saline (PBS) (Sigma). Removal of serum by aspiration was followed by the application of primary antibody for either cPLA2 or sPLA2 (CG53Ab) for 12 h at 4°C. Control sections were incubated for this time in PBS alone. After washing 3 ⫻ 5 min in PBS, an FITC-conjugated second antibody (Sigma) was applied to the sections for 40 min. Three 5-min washes followed before mounting in glycerol (Merck, Poole, UK) and observation using an Olympus BHS microscope. The polyclonal cPLA2 and sPLA2 antisera employed in these studies were kindly provided by Dr. Ruth Kramer and Dr. Lisa Marshall (Eli Lily, Indianapolis, IN), respectively. Statistics Statistical levels of significance were determined on the raw data using the Student’s t-test (paired or unpaired, where appropriate), and a value of 0.05 was considered significant. PGE2 and PGI2 release are presented graphically. Each experiment was performed at least twice with the total number of bones subjected to each treatment being no less than eight.
Figure 1. Histograms showing the effects of mechanical load, ALF3 (10 mmol/L) or PTX (100 ng/mL) alone on PGE2 and PGI2 production from rat ulna explants, and load in the presence of PTX (100 ng/mL). Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005: a Compared with basal control; bcompared with control of the treatment.
Results Effect of mechanical load on PGE2 and PGI2 release Ulnar explants, subjected to a cyclical 1-Hz load producing peak levels of mechanical strain within the physiological range (3000 ε), responded with significantly increased release of PGE2 and PGI2 into the culture medium. (58%, p ⫽ 0.02; and 49%, p ⫽ 0.002, respectively, paired t-test, Figure 1). Effect of AlF3 on PGE2 and PGI2 release AlF3 (Sigma) (10 mmol/L), a nonselective stimulator of Gproteins,27 stimulated PGE2 release by 41% and PGI2 release by 50% (p ⫽ 0.04 and p ⫽ 0.02, respectively, unpaired t-test; Figure 1). Effect of PTX on basal and load-related PGE2 and PGI2 release Basal release of neither PGE2 nor PGI2 was altered by the presence of Gi/Go inhibitor PTX27 (Sigma) (100 ng/mL, unpaired t-test). The loading-related increase in the release of PGI2, compared with nonloaded PTX-treated control was also unaffected by PTX (72%, p ⬍ 0.005, paired t-test). However, com-
Bone Vol. 27, No. 2 August 2000:241–247
Figure 2. Histograms showing the effects of the inhibitor of G-proteinmediated activation of PLA2, isotetrandrine (5 g/mL); the cPLA2 inhibitor, AACOCF3 (50 mol/L); and sPLA2 inhibitor, manoalide (0.04 mmol/L), on loading-related PGE2 and PGI2 release from rat ulnar explants. Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005: acompared with basal control; bcompared with control of the treatment. Symbols beneath the graph indicate the presence (⫹) or absence (⫺) of treatment.
pared with PTX-treated, nonloaded controls, PTX blocked the loading-related release of PGE2 (Figure 1). Effect of isotetrandrine on basal and load-related PGE2 and PGI2 release Isotetrandrine (Calbiochem, Nottingham, UK) (5 g/mL), an inhibitor of G-protein-mediated activation of PLA2,30 had no effect on basal levels of either PGE2 or PGI2 release (unpaired t-test), but blocked the loading-related release of both PGE2 and PGI2 (Figure 2). Effect of PLA2 inhibitors on basal and load-related PGE2 and PGI2 release Arachidonyl-trifluoromethyl ketone (AACOCF3; Calbiochem) (50 mol/L), an inhibitor of cPLA2 activity,3 elevated background readings in the PGE2 immunoassay. However, after correction for this raised background level in the conditioned medium, it was determined that AACOCF3 reduced basal PGE2 release to below the level of detection. Mechanical loading did
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
243
Figure 3. Histograms showing the effects of type IB pancreatic PLA2 (50 units/mL) and type IA snake venom PLA2 (50 units/mL) on basal PGE2 and PGI2 release from rat ulnar explants. Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005: acompared with basal control; bcompared with control of the treatment.
not stimulate any increase in PGE2 release. In contrast, basal PGI2 release was significantly elevated by AACOCF3 (255%, p ⫽ 0.0001, unpaired t-test). Compared with AACOCF3treated nonloaded controls, AACOCF3 did not block the loading-related increases in PGI2 release (100%, p ⫽ 0.05, paired t-test) (Figure 2). Manoalide (Calbiochem) (0.04 mmol/L), an inhibitor of sPLA2 activity,15 significantly reduced basal levels of PGI2 released into the medium (⫺60%, p ⬍ 0.0001, unpaired t-test) and abolished loading-related increases in PGI2 production (paired t-test). In contrast, manoalide had no effect on basal PGE2 release but did block the loading-related increases in its release (Figure 2). Effect of exogenous sPLA2s on PGE2 and PGI2 release Type IB, pancreatic sPLA2 (Sigma) (50 units/mL) isolated from porcine pancreas significantly increased PGE2 release by 93%, and PGI2 release by 245% (p ⬍ 0.02 and p ⬍ 0.003, respectively, unpaired t-test; Figure 3a) after a 15-min exposure. Type IA, snake venom sPLA2 (Sigma) (50 units/mL) (from naja mossambica mossambica) significantly increased PGE2 release nearly fivefold, and PGI2 release 11-fold, (p ⬍ 0.005 and p ⬍ 0.003, respectively, unpaired t-test; Figure 3b) after a 15-min exposure.
244
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
Figure 4. Histograms showing the effects of PTX (100 ng/mL) on PGE2 and PGI2 release stimulated by 50 units/mL of type IB pancreatic PLA2 and 50 units/mL of type IA snake venom PLA2. Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005; acompared with basal control; bcompared with control of the treatment.
Bone Vol. 27, No. 2 August 2000:241–247
Figure 5. Histograms showing the effects of the PKC inhibitor, bisindolylmaleimide (400 nmol/L), on loading-related PGE2 and PGI2 release from rat ulnar explants. Results are mean ⫾ SEM. Levels of significance: *p ⬍ 0.05, **p ⬍ 0.005: acompared with basal control; bcompared with control of the treatment.
Discussion Effect of PTX on exogenous PLA2-mediated PGE2 and PGI2 release PTX (100 ng/mL) had no significant effect on the stimulation of PGE2 or PGI2 release elicited by pancreatic PLA2. This contrasts with the effect of PTX on snake venom PLA2-mediated PG release. The levels of both of PGE2 and PGI2 release were significantly decreased (⫺28%, p ⫽ 0.04; and ⫺44%, p ⫽ 0.05, respectively, unpaired t-test) (Figure 4). Effect of bisindolylmaleimide on basal and load-related PGE2 and PGI2 release Bisindolylmaleimide I (Calbiochem) (400 nmol/L), an inhibitor of PKC activity,28 did not block loading-related PGI2 release compared bisindolylmaleimide-treated nonloaded control (88%, p ⬍ 0.02, paired t-test), but did block loading-related release of PGE2. (Figure 5). Immunolocalization of cPLA2 and sPLA2 Freshly dissected ulnae were cryosectioned and immunologically reacted to localize cPLA2 and sPLA2. sPLA2 reactivity was detected in both osteocytes and osteoblasts, whereas cPLA2 immunoreactivity could only be detected in osteoblasts (Figure 6).
This series of experiments suggest that in resident cells of long bone explants, arachidonic acid for loading-related PGE2 and PGI2 synthesis is derived by different mechanisms. The evidence for this conclusion is drawn from the differences in the loadingrelated release of PGE2 and PGI2 in the presence of selective pharmacological inhibitors. Both mechanical loading and AlF3 (a nonspecific activator of G-proteins) stimulated PGI2 and PGE2 release above basal levels. PTX, which had no significant effect on basal release of either prostanoid, blocked the loading-related increase of PGE2, but not PGI2. Loading in the presence of isotetrandrine (an inhibitor of G-protein-mediated activation of PLA2) blocked loading-related release of both PGE2 and PGI2. These results suggest that the loading-related release of PGE2 and PGI2 is dependent on (both PTX-sensitive and PTX-insensitive) G-protein-mediated PLA2 activities, and therefore the liberation of arachidonic acid for loading-related PGE2 and PGI2 release could be derived from the activity of different forms of PLA2. Consistent with this is the finding that blockade of osteoblast cPLA2 activity by AACOCF3 abolished the loading-related release of PGE2, but not that of PGI2. Loading-related PGI2 release was unaffected by bisindolylmaleimide, an inhibitor of PKC activity. However, functional PKC activity is required for loading-related cPLA2-derived PGE2 release, because inhibition of PKC activity blocked the loading-related release of PGE2. This is consistent with the
Bone Vol. 27, No. 2 August 2000:241–247
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
245
Figure 6. Digitally scanned photomicrographs of (a, b) cPLA2 immuno-localization and (c, d) sPLA2 immuno-localization in undecalcified cryostat sections of rat ulnae. cPLA2 can be seen in the osteoblast cell layer on the bone surface, but is absent in the osteocytes of the bone matrix. sPLA2 is present in the osteoblastic cell layer and also in osteocytes of the bone matrix.
observation that cPLA2 activity can be enhanced by PKCmediated phosphorylation.14 It further supports the notion that the source of arachidonate for loading-related PGE2 synthesis is different from that for synthesis of PGI2. Interestingly, inhibition of osteoblastic and osteocytic sPLA2 activity with manoalide blocked the loading-related increases of both PGE2 and PGI2. This implies that active sPLA2 is also a requirement for the loading-related cPLA2-mediated PGE2 release from osteoblasts. Both exogenous type IA and type IB PLA2 applied to bone explants elevated PGI2 and PGE2 release into culture medium. This is consistent with the demonstration of an sPLA2 receptor in osteoblastic MC3T3-E1 cells.29 Treatment with PTX significantly reduced the release of PGE2 and PGI2 by type IA snake venom PLA2, but PTX did not block type IB pancreatic PLA2mediated PG release. Because PTX did not inhibit loadingrelated PGI2 release, while isotetrandrine did, it is possible that arachidonate for loading-related PGI2 synthesis is derived from type IB sPLA2 isoform (the PTX-insensitive G-protein-dependent pathway). Differences in PLA2-derived free arachidonic acid for loading-related synthesis of PGE2 and PGI2 may correlate with either (or both) the various consequences of mechanical loading to which resident bone cells are subjected and with the sites of PG production. PGE2 has been demonstrated in resident osteoblasts and its loading-related release is dependent on cPLA2 and active sPLA2, which are also localized to osteoblasts. cPLA2 was not detected in osteocytes. PGI2 (as 6-keto-PGF1␣), and PGI2 synthetase, however, are present in both osteocytes and osteoblasts,17,19 and the loading-related release of PGI2 is dependent on sPLA2 activity, which is localized to these cells. Mechanically induced PG release from isolated bone cell monolayer culture systems has been reported previously. Physiological levels of uniaxial mechanical strain (3400 ε) applied to isolated primary rat osteoblasts stimulated increases in PGI2, but not PGE2, release.32 However, fluid shear strains have been demonstrated to increase PGE2 release from isolated osteoblasts, a process that is dependent on PTX-sensitive G-proteins and sensitive to inhibitors of PKC.26,27 Our data relating to PGE2 release from osteoblast cells in situ are in accordance with those reports. Isolated osteocytes from embryonic chick calvariae release PGE2 and PGI2 in response to pulsed fluid flow.1,2,11,12 These
findings of increased PGE2 release from isolated osteocytes are, however, not consistent with the lack of positive immunostaining for PGE2 in osteocytes of adult mammalian bone samples in situ.17,21 Such discrepancies could stem from the species, age, and site from which the osteocytes are derived. Alternatively, the preferential survival of immature osteocytes in the isolation procedure from the lacunae-canalicular network, and their subsequent maintenance in monolayer culture, might account for these osteoblast-like characteristics.
Figure 7. Schematic to represent a possible signaling pathway between osteocytes and osteoblasts via the agency of sPLA2. sPLA2 derived from osteocytes activates a pertussis toxin-sensitive G-protein receptor residing in the osteoblast membrane, or directly activates a pertussis toxinsensitive G-protein in the cytoplasm. This signal is relayed to cPLA2, via a PKC-dependent mechanism, causing the release of arachidonic acid for conversion to PGE2 via cyclooxygenase.
246
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
It has been postulated that the osteocyte network, in situ, assesses the local mechanical environment in bone tissue, and by integrating the alterations in the local levels and distribution of bone surface strains and fluid shear forces, coordinates osteoblast behavior accordingly.13,5 If osteocytes are to be effective in influencing bone remodeling, there must be osteocyte-to-osteoblast communication. Gap junctions between osteoblasts and osteocytes have been reported,7,8 and are obvious candidates through which intracellular messages could pass. However, extracellular molecules might also fulfill a communicative role. That the early increases in loading-related PGE2 release from osteoblastic cPLA2 are dependent on sPLA2 activity suggests that sPLA2 acts in an autocrine or paracrine manner. A transcellular, ligand-binding mechanism29 would provide a communicative mechanism between osteocytes and osteoblasts via the agency of osteocyte-derived sPLA2. Such a mechanism of communication has been previously proposed in mast cell/fibroblast coculture experiments where sPLA2 released from activated mast cells stimulates the release of PGE2 from fibroblasts.24 A scheme based on the coculture model24 to illustrate the possible mechanism of communication in resident bone cells is presented in Figure 7. In conclusion, these results confirm a role for regulatory G-proteins in loading-related PGE2 and PGI2 release from osteoblasts and osteocytes in situ. Experiments with PTX and isotetrandrine suggest the involvement of both PTX-sensitive and PTX-insensitive G-protein-mediated PLA2s in the liberation of arachidonic acid for synthesis of these prostanoids. sPLA2 is responsible for the liberation of arachidonic acid for PGI2 production in osteoblasts and osteocytes. cPLA2 activity appears to be regulated by a PKC-dependent pathway and sPLA2 is also necessary to facilitate the liberation of arachidonic acid for PGE2 release from osteoblastic cPLA2. If osteocyte-derived sPLA2 were to act as a paracrine mediator of osteoblast activity, this would represent a mechanism for osteocyte-to-osteoblast cell communication advocated previously.5,13
Bone Vol. 27, No. 2 August 2000:241–247
8. 9.
10.
11.
12.
13. 14.
15.
16.
17.
18.
19.
20.
Acknowledgment: These studies were supported by a grant awarded by The Wellcome Trust.
21.
References
22.
1. Ajubi, N. E., KleinNulend, J., Alblas, M. J., Burger, E. H., and Nijweide, P. J. Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes. Am J Physiol 39:E171–E178; 1999. 2. Ajubi, N. E., Klein-Nulend, J., Nijweide, P. J., Vrijheid Lammers, T., Alblas, M. J., and Burger, E. H. Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes-a cytoskeleton-dependent process. Biochem Biophys Res Commun 225:62– 68; 1996. 3. Bartoli, F., Lin, H. K., Ghomashchi, F., Gelb, M. H., Jain, M. K., and Apitzcastro, R. Tight-binding inhibitors of 85-KDa phospholipase A2 but not 14-KDa phospholipase A2 inhibit release of free arachidonate in thrombin stimulated human platelets. J Biol Chem 269:15625–15630; 1994. 4. Binderman, I., Zor, U., Kaye, A. M., Shimshoni, Z., Harell, A., and Somjen, D. The transduction of mechanical force into biochemical events in bone cells may involve activation of phospholipase A2. Calcif Tissue Int 42:261– 266; 1988. 5. Burger, E. H., Klein-Nulend, J., Vanderplas, A., and Nijweide, P. J. Function of osteocytes in bone: their role in mechanotransduction. J Nutr 125:S2020 – S2023; 1995. 6. Cheng, M. Z., Zaman, G., and Lanyon, L. E. Estrogen enhances the stimulation of bone collagen synthesis by loading and exogenous prostacyclin, but not prostaglandin E2, in organ cultures of rat ulnae. J Bone Miner Res 9:805– 816; 1994. 7. Donahue, H. J., McLeod, K. J., Rubin, C. T., Andersen, J., Grine, E. A., Hertzberg, E. L., and Brink, P. R. Cell-to-cell communication in osteoblastic
23.
24.
25.
26.
27.
28.
29.
30.
networks: cell line-dependent hormonal regulation of gap junction function. J Bone Miner Res 10:881– 889; 1995. Doty, S. B. Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 33:509 –512; 1981. Emadi, S., Mirshahi, M., Elalamy, I., Nicolas, C., Vargaftig, B. B., and Hatmi, M. Cellular source of human platelet secretory phospholipase A2. Br J Haematology 100:365–373; 1998. Ghrib, F., Pyronnet, S., Bastie, M. J., FagotRevurt, P., Pradayrol, L., and Vaysse, N. Arachidonic-acid-selective cytosolic phospholipase A2 is involved in gastrin-induced AR4-2J-cell proliferation. Int J Cancer 75:239 – 245; 1998. Klein-Nulend, J., Burger, E. H., Semeins, C. M., Raisz, L. G., and Pilbeam, C. C. Pulsating fluid flow stimulates prostaglandin E2 release and inducible prostaglandin-G/H synthase messenger RNA expression in primary mouse bone cells. J Bone Miner Res 10:S405–S405; 1995. Klein-Nulend, J., Vanderplas, A., Semeins, C. M., Ajubi, N. E., Frangos, J. A., Nijweide, P. J., and Burger, E. H. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9:441– 445; 1995. Lanyon, L. E. Control of bone architecture by functional load bearing. J Bone Miner Res 7:S369 –S375; 1992. Lin, L. L., Lin, A. Y., and Knopf, J. L. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci USA 89:6147– 6151; 1992. Mayer, A. M. S., Glaser, K. B., and Jacobs, R. S. Regulation of eicosanoid biosynthesis in vitro and in vivo by the marine natural product manoalide: a potent inactivator of venom phospholipases. J Pharmacol Exp Ther 244:871– 878; 1988. Mayer, R. J. and Marshall, L. A. New insights on mammalian phospholipase A2s: comparison of arachidonoyl selective and arachidonoyl-nonselective enzymes. FASEB J 7:339 –348; 1993. Miyauchi, M., Takata, T., Ogawa, I., Ito, H., Kobayashi, J., Nikai, H., and Ijuhin, N. Immunohistochemical demonstration of prostaglandins in various tissues of the rat. Histochem Cell Biol 105:27–31; 1996. Mosley, J. R., March, B. M., Lynch, J., and Lanyon, L. E. Strain magnitude related changes in whole bone architecture in growing rats. Bone 20:191–198; 1997. Okiji, T., Morita, I., Kawashima, N., Kosaka, T., Suda, H., and Murota, S. Immunohistochemical detection of prostaglandin I2 synthase in various calcified tissue-forming cells in rat. Arch Oral Biol 38:31–36; 1993. Pruzanski, W., Stefanski, E., Vadas, P., and Ramamurthy, N. S. Inhibition of extracellular release of proinflammatory secretory phospholipase A2 (sPLA2) by sulfasalazine: a novel mechanism of anti-inflammatory activity. Biochem Pharmacol 53:1901–1907; 1997. Rawlinson, S. C. F., El Haj, A. J., Minter, S. L., Tavares, I. A., Bennett, A., and Lanyon, L. E. Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: a role for prostacyclin in adaptive bone remodeling? J Bone Miner Res 6:1345–1351; 1991. Rawlinson, S. C. F., Mosley, J. R., Suswillo, R. F. L., Pitsillides, A. A., and Lanyon, L. E. Calvarial and limb bone cells in organ and monolayer culture do not show the same early responses to dynamic mechanical strain. J Bone Miner Res 10:1225–1232; 1995. Rawlinson, S. C. F., Pitsillides, A. A., and Lanyon, L. E. Involvement of different ion channels in osteoblasts’ and osteocytes’ early responses to mechanical strain. Bone 19:609 – 614; 1996. Reddy, S. T. and Herschman, H. R. Transcellular prostaglandin production following mast cell activation is mediated by proximal secretory phospholipase A2 and distal prostaglandin synthase 1. J Biol Chem 271:186 –191; 1996. Reddy, S. T., Winstead, M. V., Tischfield, J. A., and Herschman, H. R. Analysis of the secretory phospholipase A2 that mediates prostaglandin production in mast cells. J Biol Chem 272:13591–13596; 1997. Reich, K. M. and Frangos, J. A. Protein kinase C mediates flow-induced prostaglandin E2 production in osteoblasts. Calcif Tissue Int 52:62– 66; 1993. Reich, K. M., McAllister, T. N., Gudi, S., and Frangos, J. A. Activation of G-proteins mediates flow-induced prostaglandin E2 production in osteoblasts. Endocrinology 138:1014 –1018; 1997. Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. Staurosporine, a potent inhibitor of phospholipid/Ca2⫹ dependent protein kinase. Biochem Biophys Res Commun 135:397– 402; 1986. Tohkin, M., Kishino, J., Ishizaki, J., and Arita, H. Pancreatic type phospholipase A2 stimulates prostaglandin synthesis in mouse osteoblastic cells (MC3T3-E1) via a specific binding site. J Biol Chem 268:2865–2871; 1993. Tsunoda, Y. and Owyang, C. The regulatory site of functional GTP-binding
Bone Vol. 27, No. 2 August 2000:241–247 protein-coupled to the high-affinity cholecystokinin receptor and phospholipase A2 pathway is on the G subunit of GQ protein in pancreatic acini. Biochem Biophys Res Commun 211:648 – 655; 1995. 31. Underwood, K. W., Song, C. Z., Kriz, R. W., Chang, X. J., Knopf, J. L., and Lin, L. L. A novel calcium-independent phospholipase A2, cPLA2␥, that is prenylated and contains homology to cPLA2. J Biol Chem 273:21926 –21932; 1998. 32. Zaman, G., Suswillo, R. F. L., Cheng, M. Z., Tavares, I. A., and Lanyon, L. E.
S. C. F. Rawlinson et al. Regulation of PGE2 and PGI2 release by PLA2s
247
Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture. J Bone Miner Res 12:769 –777; 1997.
Date Received: November 8, 1999 Date Revised: March 16, 2000 Date Accepted: March 28, 2000