Human parathyroid hormone fragment stimulates the de novo synthesis of prostaglandin endoperoxide synthase in chick calvaria

The human parathyroid hormone N-terminal fragment [hPTH-(1–34)] increases the conversion of exogenous unsaturated fatty acids to prostaglandins (PGs) in calvarial homogenates. Enzyme activities were completely blocked by indomethacin (5 × 10−7 M), a PG synthase inhibitor, and actinomycin D (5 μM), an inhibitor of transcription, by binding to DNA. In addition, a potent inhibitor of protein synthesis, cycloheximide (10 μM), totally inhibited the stimulating effect of hPTH-(1–34) on prostaglandin endoperoxide synthase (PG synthase, EC 1.14.99.1). The stimulatory effect of hPTH-(1–34) on PG synthase was also reduced by the addition of stannous chloride. However, epidermal growth factor (EGF), platelet-derived activating factor (PDGF), and ionophore A23187 did not show the same stimulating effect as hPTH-(1–34) on PG synthase in calvaria. The results further demonstrated that PG synthase is a membrane-bound enzyme in chick calvaria. In this communication, evidence is presented that hPTH-(1–34) stimulates the de novo synthesis of PG synthase as demonstrated by the increased activity in calvarial homogenates and microsomes.


Introduction
Parathyroid hormone (PTH) is a single chain 84-amino acid peptide (molecular weight of 9 500) secreted by the parathyroid glands. However, the structural requirements necessary for full biological activity are virtually satisfied by the NHe-terminal 34-amino acid fragment. Bone and kidney are the two principal target organs affected by PTH. PTH stimulates the breakdown of phospholipids from the rat tibia which occurs at the same time as calcium mobilization 2 and stimulates phosphoinositide turnover in mouse osteoblasts. In contrast, several reports have demonstrated that PTH stimulates the growth of bone 4'5 and cartilage. 6 '7 Endogenous skeletal prostaglandin (PG) production may participate in bone resorption and bone formation. PGs may not mediate the action of PTH on bone resorption. However, PGs do show a very close relationship with the action of PTH on skeletal tissues. [1][2][3][4][5][6][7][8][9][10][11][12][13] It has been demonstrated that the human parathyroid hormone N-terminal fragment [hPTH- ] stimulates PGE2 synthesis by chick calvaria in an organ culture. It appears that hPTH-  stimulates the bone cells to convert arachidonic acid to prostaglandin E 2 (PGE2), but does not activate the release of stored arachidonic acid. 13 Prostaglandin biosynthesis involves the initial conversion of arachidonic acid to cyclic endoperoxide intermediates designated PGG2 and PGH2 by prostaglandin endoperoxide synthase. 14 These rela-(C) 1993 Rapid Communications of Oxford ktd tively unstable compounds are then metabolized by other enzymes to form stable PGs, or are transformed into non-prostaglandin derivatives, is The mechanism by which hPTH-(1-34) increases the synthesis of PGE2 in chick calvaria appears to be related to the activation of enzyme activity in the biosynthesis of prostaglandins. 3 It is important to investigate whether hPTH-(1-34) can stimulate the de novo synthesis of PG synthase in chick calvaria. In this report, it was found that hPTH-(1-34) increases the conversion of exogenous unsaturated fatty acids to PGs in calvarial homogenates and microsomes. PG synthase is a membrane-bound enzyme which has been purified from sheep, 6'7 rat adipose tissue, 18  Methods: Preparation of calvarial homogenates Calvariae were dissected aseptically from. 17-day-old chick embryos and cultivated as previously described by Yang et al. 22 Routinely, the paired bones were preincubated in an organ culture system for 24 h, then the medium was replaced with fresh medium containing different tested additions for appropriate time periods. The paired bones from 20 chicks were randomly assigned to the experimental groups.
After this preincubation, paired bones from each group were harvested, weighed, and resuspended in 4ml of sodium phosphate buffer (10mM NaH2PO4, 10 mM Na2HPO4, 100/M dithiothreitol, pH 7.4) and then homogenized with a Polytron operated at full speed for 2 min. The homogenates were centrifuged at 400 g for 5 min at 4C to remove large unminced fragments. The supernatant was removed and incubated with 10/M cold arachidonic acid (AA) and 10 Ci 3H-arachidonic acid in an equal volume of 0.2 M Tris-chloride buffer (pH 8.5) containing 2mM glutathione, 1 mM hydroquinone, and 2 #M haemin at 37C for 10min. The reaction was terminated by the addition of 2 vol. of ethanol and was ready for extraction and purification of eicosanoids.
Isolation of microsomes from calvaria The procedures for isolating microsomes from calvariae were a modification of those of Stern and Vance. 2 All procedures were performed at 4C unless otherwise indicated. After a preincubation period of 36 h with or without hPTH-(1-34), the calvariae were harvested and resuspended in an appropriate volume of sodium phosphate buffer (10mM NaH2PO4, 10 mM Na2HPO4, 100 #M dithiothreitol, pH 7.4) and then homogenized with a Polytron operated at full speed for 2min.
Homogenates were centrifuged at 12 000 g for 15 min. The resulting supernatants were harvested and centrifuged at 100000 g for 1 h to obtain cytosol and microsomal pellets. The supernatant was removed and the pellet was homogenized in fresh sodium phosphate buffer equivalent to one-half the volume of the total reaction mixture. The microsomes were further incubated with 10 #M cold arachidonic acid and 2/,Ci (9.4 nM) H-arachidonic acid in an equal volume of 0.2 M Tris-chloride buffer (pH 8.5) containing 2 mM glutathione, 1 mM hydroquinone, and 2/M haemin at 37C for 10 min. The reaction was terminated by the addition of 2 vol. of ethanol and was ready for extraction and separation of eicosanoids.
Extraction and purification of eicosanoids: The incubation precipitates from calvarial homogenates and microsomes were centrifuged at 800 g" for 10 min at 4C, and the supernatant layer was evaporated to aqueous phase. Ethanol was added to the residues to achieve a final concentration of 15% ethanol. The biological sample was further acidified to pH 3.5 with 1 M citric acid and applied to an ODS-silica column (Sep-Pak C18 cartridge). The Sep-Pak cartridge was attached to a 20 ml polypropylene Luerlok syringe and washed with 20 ml of ethanol and water successively. The column was washed with 20 ml of water, 20 ml 15% aqueous ethanol, and 20 ml of petroleum ether sequentially. The eicosanoids were eluted with 10ml of ethyl acetate. 24 The collected samples were dried by evaporation under a stream of nitrogen. The residues were then reconstituted in 1 ml of ethanol and filtered through a 0.45 #m filter (Millipore).
The samples were again dried by evaporation under a stream of nitrogen, reconstituted in 50 #1 of chloroform/methanol (2"1, v/v) and prepared for thin-layer chromatography.
Thin-layer chromatography: Thin-layer chromatography was performed by a method described previously. 13 After TLC, the radioactivity of the spots was determined by directly counting the scraped spots using liquid scintillation spectroscopy. The activity of prostaglandin endoperoxide synthase was determined by measuring the conversion of exogenous arachidonic acid to PGE2.

Results
Previous experiments have shown that a 36-h preincubation with hPTH-(1..-34) can activate endogenous PGE2 synthesis and mineral mobilization in intact chick calvaria. 13'2s In this report, it was further found that human PTH-(1-34) at a concentration of 0.6/g/ml stimulates PG synthase activity (about a three-fold increase) in chick calvaria. EGF at a concentration of 20 ng/ml and PDGF at a concentration of 20 mU/ml had no stimulatory effect on PG synthase activity (Fig. I(A) and Table 1). Ionophore A23187 at a concentration of 10 #M also had no eect on this enzyme activity (Fig. I(B) and Table 1).
The stimulatory effect of hPTH-(1-34) on PG synthase in chick calvaria, as well as the basal activity of PG synthase, was blocked by a cyclooxygenase inhibitor, indomethacin (5 10 -7 M), and a transcription blocker, actinomycin D (5/M). Moreover, the stimulatory effect of hPTH-(1-34) was completely abolished by a translation inhibitor, cycloheximide (10/M) (Fig.  I(B) and Table 1), as it brought the activity of PG synthase back to the basal level. It appears that hPTH-(1-34) stimulates the de novo synthesis of PG synthase in calvaria. Stannous chloride was also included in the experiments to reduce untransformed endoperoxide to prostaglandin F2 (PGF2). 4 The stimulatory effect of hPTH-(1-34) on PG synthase was blocked (statistically significant) by the addition of stannous chloride (1.9 mg/ml, 10/,zM) in ethanol; however, the total enzyme activity level was still higher than the basal levels ( Table 1).
We then tried to identify the location of PG synthase in chick calvaria. Human PTH-  stimulates the de novo synthesis of PG synthase in the microsomal fraction of the calvariae but not in    in the hPTH + INDO group. After this preincubation, the bones were harvested and homogenized with a Polytron and centrifuged at 400 x g for 10 min at 4C. The supernatant was harvested and incubated with 10/M cold arachidonic acid and 10/Ci aH-arachidonic acid for 10 min at 37C. The prostanoids were then extracted with a Sep-Pak C18 cartridge and separated by TLC, and the enzyme activity was determined as described (for details of these procedures refer to Materials and Methods). Data are represented as the mean +_ S.E. (disintegrations per min) for triplicate incubations of bone homogenates prepared from 20 chick calvariae. *Significantly different from control, as determined by Student's non-paired /-test (p < 0.05)" Significantly different from hPTH treatment alone, as determined by Student's non-paired /-test (p < 0.05). the cytosol fraction ( Fig. 1(C)). These findings further suggest that PG synthase in chick calvaria in a membrane-bound protein.

Discussion
The action of PTH on bone metabolism may involve several 'second messengers', including cAMP, 26 calcium, 2v the Na+-Ca 2+ exchange mechanism 28 and prostaglandins. 29 In previous publications, it was demonstrated that hPTH-(1-34) stimulates calcium mobilization 25 and PGE2 synthesis 13 in chick calvaria. In this communication, it has been further found that hPTH-(1-34) stimulates the de novo synthesis of PG synthase in chick calvaria. The authors speculate that PG synthase may be involved in the action of hPTH-(1-34) on bone resorption or bone formation which are coupling factors controlling bone metabolism.
Since PGE2 was the predominant eicosanoid produced by chick calvaria, the activity of PG synthase was determined by measuring the conversion of exogenous arachidonic acid to PGE2. PGF2 was the second major prostanoid to appear on TLC. Trace amounts of PGB2, PGD2, an unknown product that eluted before 6-keto-PGFl (more polar than 6-keto-PGFl), an unknown product that eluted after PGE2 (less polar than PGE2), and an unknown product eluted after PGD2 (less polar than PGD2) were also detected.
Epidermal growth factor is a 53-amino acid polypeptide isolated from male mouse submaxillary glands, which stimulates PGE2 synthesis in mouse calvaria. 3'31 Tashjian et al. 2 found that a low concentration of PDGF stimulates bone resorption via the enhanced local production of PGE2. In previous observations, it was found that the use of different concentrations of EGF, PDGF, bradykinin and ionophore do not stimulate PGE2 synthesis in chick calvaria. It was also noted that ionophore at a concentration of 10/.tM has no deleterious effects on chick calvaria (unpublished data). Lack of responsiveness to these agonists may be due to a difference in species. In this study, the results further demonstrated that EGF, PDGF and ionophore have no stimulatory effects on the de novo synthesis of PG synthase in chick calvaria. On the other hand, it was found that hPTH-(1-34) does not have a stimulatory effect on PG synthesis in rat calvaria, whereas hPTH-(1-34) does show a minor effect (not statistically significant) in mouse calvaria (unpublished data). These observations all support the highly specific action of hPTH-(1-34) on chick calvaria.
The temporal sequence of PG synthase synthesized by human dermal fibroblasts can be separated into an early transcriptional stage and a subsequent translational stage. 33 The results in Table 1 indicate that the DNA transcription effect of hPTH-(1-34) on PG synthase can be blocked by the addition of actinomycin D, and the translational effect can be blocked by the addition of cycloheximide. At this moment, the authors are not able to conclude whether the stimulatory effect of hPTH-(1-34) on PG synthase occurs at the early transcriptional stage and/or the translational stage. It is possible that both stages are affected by hPTH-(1-34). However, it has been reported that hPTH-(1-34) stimulates the synthesis of DNA in the central bone of calvaria, as The biosynthesis of this enzyme in chick calvaria may be regulated by a specific gene.
Isolation and determination of this specific gene might be a better way to clarify the real mechanism of hPTH-(1-34) acting on chick calvaria.
The incubation mixtures were treated with stannous chloride (1.9 mg/ml, 10/,M) in ethanol in order to reduce prostaglandin endoperoxide to PGF2. 4 The results of these experiments do not show a complete reduction of PGE2 production. The synthesis of both PGE2 and PGF2 may preclude the possibility of non-enzymic reduction of prostaglandin endoperoxide to these prostaglandins. The data also reveal that part of the stimulatory effect of hPTH-(1-34) on PG synthase is blocked by the addition of stannous chloride. The basal levels of this enzyme activity are not changed by SnCl2, and a certain degree of the stimulatory effect of hPTH-(1-34) still remains (Table 1). In view of the partial inhibition by SnC12, it is quite possible that part of the increased activity may be due to induction of the PG endoperoxide E isomerase by hPTH-(1-34) that converts PGH2 to PGE2. PG synthase contains both cyclooxygenase and peroxidase activities within a single protein. Cyclooxygenase component converts arachidonic acid to a hydroperoxy endoperoxide (PGG2). The PGG2 then reacts with PG synthase in a peroxidation reaction to give compound I (PGHS I), which subsequently converts to compound II (PGHS II). Native PG synthase is then regenerated. 34 Hsuanyu and Dunford 35 have demonstrated that the whole peroxidase cycle is rapidly completed (within seconds), and a mixture of compound I and compound 1I was achieved at a very early stage rather than pure compound I. Although the inducible form of PG synthase is now considered to be PGHS II, the authors are not able to conclude which form of PG synthase was activated by hPTH-  in the present study. PG synthase has been discovered to be a membrane-bound protein in other tissues. [16][17][18][19][20][21] However, this enzyme has never been proved to be a membrane-bound protein in skeletal tissues. This communication strongly supports the view that PG synthase is a membrane-bound enzyme in chick calvaria.