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Endocrinology Vol. 140, No. 1 131-137
Copyright © 1999 by The Endocrine Society

ARTICLES

Parathyroid Hormone (PTH)-(1–34), [Nle8,18,Tyr34]PTH-(3–34) Amide, PTH-(1–31) Amide, and PTH-Related Peptide-(1–34) Stimulate Phosphatidylcholine Hydrolysis in UMR-106 Osteoblastic Cells: Comparison with Effects of Phorbol 12,13-Dibutyrate1

Amareshwar T. K. Singh, Joseph G. Kunnel, Paul J. Strieleman2 and Paula H. Stern

Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School (A.T.K.S., J.G.K., P.J.S., P.H.S.), and the Department of Basic and Behavioral Sciences, Northwestern University Dental School (J.G.K.), Chicago, Illinois 60611-3008

Address all correspondence and requests for reprints to: Amareshwar T. K. Singh, Ph.D., Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611-3008. E-mail: a-singh{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Studies were performed to determine the effects of PTH and related compounds on phosphatidylcholine (PC) hydrolysis in UMR-106 cells and the pathway by which the PTH effects occurred. The responses were compared with those of phorbol 12,13-dibutyrate (PDBu). Both bovine PTH-(1–34) [bPTH-(1–34)] and PDBu stimulated PC hydrolysis within 10 min. Significant effects were elicited by concentrations of 0.3–1 nM bPTH-(1–34) and 5 nM PDBu. Dose-dependent increases were seen at higher concentrations of both compounds, however, the response to bPTH-(1–34) was reduced at 30 nM. Bovine or human PTH-(1–34) and human PTH-related peptide-(1–34) [hPTHrP-(1–34)] were equipotent in their effects, whereas bovine [Nle8,18Tyr34]PTH-(3–34) amide [bPTH-(3–34)] and hPTH-(1–31) amide [hPTH-(1–31)] were less potent than bPTH-(1–34). bPTH-(3–34) did not antagonize the effects of bPTH-(1–34). Down-regulation of protein kinase C isozymes by 24-h treatment with PDBu completely prevented the stimulatory effect of PDBu on PC hydrolysis, but did not significantly affect the stimulatory effect of bPTH-(1–34). Both bPTH-(1–34) and PDBu stimulated transphosphatidylation of PC, indicating a phospholipase D-stimulated mechanism. The results suggest that in the UMR-106 cell line PTH can stimulate activation of PLD by a mechanism other than through protein kinase C.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH, A MAJOR systemic hormone, promotes the formation and resorption of bone. PTH stimulates osteoblasts through G protein-linked receptors. Although the major focus with regard to the effects of PTH on downstream signaling pathways has been on the activation of adenylyl cyclase (1), it is clear that PTH also activates membrane phospholipases (2). Activation of the phospholipid phosphatidylinositol bisphosphate (PIP2)-selective phospholipase C (PLC) by PTH to produce the signaling molecules diacylglycerol (DAG) and inositol trisphosphate (IP3) has been demonstrated in osteoblastic cells (3). DAG together with phosphatidylserine activate a number of protein kinase C (PKC) isozymes (4). Other isozymes additionally require calcium, which is released from endoplasmic reticulum stores by IP3 (4). Studies have shown that PTH causes a rapid translocation of PKC activity from the cytoplasm (inactive form) to the membrane (active form) in osteoblastic cells (5, 6, 7, 8). DAG, the lipid messenger molecule, can be generated by mechanisms in addition to phospholipase C-catalyzed hydrolysis of PIP2. Recently, attention has focused on the potential importance of phosphatidylcholine (PC) as a source of DAG (9). This can occur either through a PLC mechanism, with the direct production of DAG along with phosphocholine, or through a phospholipase D (PLD) mechanism, with the production of phosphatidic acid (PA), which can be further metabolized to DAG, along with choline. As the PC content in mammalian tissues is severalfold greater than that of PIP2, and the kinetics of its hydrolysis are slower, DAG production due to PC hydrolysis is likely to be slower and more prolonged than that resulting from PIP2 breakdown, producing a more continuous activation of PKC (10). It was therefore of interest to determine whether PTH might stimulate this pathway in osteoblasts in addition to stimulation of the hydrolysis of PIP2.

In the present study, we determined the effects of human (h) and bovine (b) PTH-(1–34) and a number of important PTH analogs, including [Nle8,18,Tyr34]bPTH-(3–34) amide [bPTH-(3–34)], hPTH-(1–31) amide [hPTH-(1–31)], and hPTH-related peptide-(1–34) [hPTHrP-(1–34)], on PC hydrolysis in UMR-106 osteoblasts. As PC hydrolysis can itself be activated by PKC-dependent mechanisms (4), we compared the effects of PTH and the phorbol ester, phorbol 12,13-dibutyrate (PDBu), which mimics DAG in activating DAG-sensitive PKC isozymes (4). The role of the PKC pathway in PTH-mediated PC hydrolysis in UMR-106 cells was further tested by prior down-regulation of PKC with PDBu, which down-regulates most classes of PKC isozymes (4). To determine whether PTH-stimulated PC hydrolysis in UMR-106 cells could be occurring through a PLD-mediated process, we determined the effect of bPTH-(1–34) on PC transphosphatidylation, a PLD-mediated process (11).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
hPTH-(1–34), bPTH-(3–34), and hPTHrP-(1–34) were purchased from Bachem (Torrance, CA). hPTH-(1–31) was obtained from Peninsula Laboratories, Inc. (Belmont, CA). bPTH-(1–34) was obtained from Bachem or Peninsula Laboratories, Inc.. PDBu was purchased from Sigma Chemical Co. (St. Louis, MO). [Methyl-3H]choline chloride ([3H]choline) and [9,10-(n)-3H]myristic acid ([3H]myristic acid) were purchased from Amersham (Arlington Heights, IL). UMR-106 osteoblastic osteosarcoma cells were purchased from American Type Culture Collection (Manassas, VA). 1,2-Dimyristoyl-sn-glycero-3-phosphoethanol, 1,2-dioleoyl-sn-glycero-3-phosphoethanol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol, and PA standards were obtained from Avanti Polar-Lipids, Inc. (Alabaster, AL).

Cell culture
UMR-106 cells were grown to confluence in 75-cm2 flasks in DMEM (Life Technologies, Gaithersburg, MD) containing glucose, L-glutamine, 100,000 U/ml penicillin G potassium, and 15% heat-inactivated horse serum (Life Technologies) at 37 C in a humidified 5% CO2 environment. Cells were then seeded on Primaria 35 x 10-mm sterile culture dishes and used the next day. Cells from passages 10–24 were used for the experiments.

Cell labeling and agonist treatments
In experiments to characterize PC hydrolysis, UMR-106 cells were incubated for 48 h at 37 C in a humidified 5% CO2 incubator in Primaria 35 x 10-mm sterile culture dishes with 2 ml DMEM containing [3H]choline (0.25 µCi/ml), glucose, L-glutamine, 100,000 U/ml penicillin, and 15% heat-inactivated horse serum. In preliminary experiments, it was demonstrated that nearly 95% of the incorporated label was in PC. Cells were washed with 2 ml DMEM, then incubated in 2 ml DMEM containing 20 mM HEPES buffer and 0.1% BSA (Sigma Chemical Co.) in the absence or presence of the indicated concentrations of agonists and/or inhibitors [bPTH-(1–34), hPTH-(1–34), bPTH-(3–34), hPTH-(1–31), hPTHrP-(1–34), or PDBu]. After incubation at the indicated times and concentrations, media were quickly removed, and radioactivity was determined. In preliminary experiments, it was determined that greater than 90% of the medium radioactivity was [3H]choline.

Measurement of transphosphatidylation
To determine the effects of agonists on the transphosphatidylation activity of the cells, UMR-106 cells were incubated with [3H]myristic acid (1.0 µCi/ml DMEM containing glucose, L-glutamine, 100,000 U/ml penicillin, and 15% heat-inactivated horse serum) at 37 C in a humidified 5% CO2 environment for 24 h. In preliminary experiments (Sanders, J. L., and P. J. Strieleman, unpublished), it was determined that under these conditions approximately 70% of the label incorporated into phospholipids was in PC, with the following approximate amounts in other phospholipids: 10% in phosphatidylethanol, 9% in sphingomyelins, 4% in phosphatidylserine, 4% in phosphatidylinositol, and less than 1% each in phosphatidylglycerol, PA, and lysophospholipids. After the labeling, cells were washed with 2 ml DMEM, then treated with 10 nM bPTH-(1–34) or 25 nM PDBu in 2 ml DMEM containing 20 mM HEPES buffer and 0.1% BSA in the absence or presence of 1% absolute ethanol for 30 min at 37 C. Media were quickly removed, and cells were scraped into 1.0 ml ice-cold methanol. Lipids were extracted by the method of Folch (12). Phosphatidylethanol was separated from other phospholipids by chromatography on thin layer silica gel plates employing chloroform-methanol-acetic acid (65:15:3, vol/vol/vol) as the mobile phase, using the modified method of Kates (13). Authentic phosphatidylethanol standards, 1,2-dimyristoyl-sn-glycero-3-phosphoethanol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol, 1,2-dioleoyl-sn-glycero-3-phosphoethanol, and PA (Avanti Polar Lipids) were run concurrently on the same plate. Lipids were visualized by exposure to iodine vapor. The thin layer chromatograms were then sprayed with En3Hance (DuPont, Wilmington, DE) to increase surface autoradiography and exposed to an imaging film (Eastman Kodak Co., Rochester, NY) for 72 h at -80 C. The regions of the TLC plate corresponding to the phosphatidylethanols were scraped into scintillation vials and counted by liquid scintillation spectrophotometry.

Down-regulation of PKC
To test the effects of agonists in UMR-106 cells in which DAG-activated PKC isozymes had been down-regulated, the cells were subcultured at 10 x 104 cells/culture dish in DMEM containing 15% heat-inactivated horse serum, penicillin, and [3H]choline chloride (0.25 µCi/ml) for 24 h at 37 C in a 5% CO2 environment. PDBu (1.0 µM) or dimethylsulfoxide (DMSO; vehicle; Sigma Chemical Co.) was added to the cells at 24 h, and incubations continued for an additional 24-h period. Cells were about 90% confluent on the day of the assay. Pretreatment of cells was terminated by aspirating the medium and washing cells twice with 2 ml DMEM containing 20 mM HEPES (pH 7.4) and 0.1% BSA while maintaining culture dishes at 37 C on a plate warmer. This procedure has been shown by us to down-regulate PKC{alpha}, -ß, -{delta}, -{epsilon}, and -{eta} isozymes (14). Immediately after washing, 2 ml fresh medium were added to each dish, followed by the addition of 10 nM bPTH-(1–34), 500 nM PDBu, or DMSO in 20-µl volumes to the appropriate culture dishes. Dishes were then incubated for 60 min at 37 C in a CO2 incubator, and medium radioactivity was determined.

Statistical analysis
Analyses were performed by ANOVA with Newman-Keuls posttest to determine the effects of agonists or inhibitors on PC hydrolysis in UMR-106 cells. Multiple linear regression was used when dose-response curves were compared. P < 0.05 was considered significant. The results are expressed as the mean ± SE of at least three determinations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course: bPTH-(1–34) and PDBu
Untreated UMR-106 cells prelabeled with [3H]choline released [3H]choline into the culture medium over a 1-h incubation (Fig. 1AGo). Addition of bPTH-(1–34) (10 nM) to the cells elicited a rapid initial increase in [3H]choline release from the UMR-106 cells (Fig. 1AGo). The effect was significant at 10 min, and the increase over the control value was maintained for up to 1 h (Fig. 1AGo). In some experiments in which earlier time points were examined, significant effects were detected as early as 2 min (data not shown). UMR-106 cells incubated in the presence of 500 nM PDBu showed similar early responses (Fig. 1BGo). However, in contrast to bPTH-(1–34), there was a progressive increase in PC hydrolysis over time, with the slope of the time-course curve being significantly different from that in the controls by linear regression analysis (P < 0.05).



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  		Figure 1. Time course of 10 nM bPTH-(1–34) (A)- or 500 nM PDBu (B)-stimulated release of [3H]choline from UMR-106 cells. Results are the mean and SE of three determinations. *, P < 0.05 vs. respective time-matched controls.

 
Dose-response curves: bPTH-(1–34) and PDBu
Figure 2Go illustrates the effects of 60-min treatment with a range of concentrations of bPTH-(1–34) (A) and PDBu (B) on the release of [3H]choline into the culture medium from prelabeled UMR-106 cells. bPTH-(1–34) elicited a dose-dependent increase in the release of [3H]choline (Fig. 2AGo). In different experiments, significant effects were obtained at 0.3–1 nM bPTH-(1–34). The effect of bPTH-(1–34) on PC hydrolysis was maximal at 10 nM and declined at 30 nM. PDBu also elicited a dose-dependent increase in the release of [3H]choline (Fig. 2BGo). A significant response was obtained with 5 nM PDBu, and 250 nM produced a maximal effect.



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  		Figure 2. Concentration dependence of the effects of bPTH-(1–34) (A) or PDBu (B) on the release of [3H]choline from UMR-106 cells. The incubation time was 1 h. Results are the mean and SE of 9 determinations for bPTH-(1–34) and 8–12 determinations for PDBu. *, P < 0.05 vs. control; #, P < 0.05 vs. 10 nM bPTH-(1–34).

 
Effects of PTH and analogs
bPTH-(1–34), hPTH-(1–34), and PTHrP-(1–34), tested at 0.1–10 nM, showed similar potencies on [3H]choline release into the culture medium (Fig. 3AGo). Multiple linear regression analysis showed no difference among the three curves. bPTH-(3–34) was less potent than bPTH-(1–34), having no effect at 10 nM but eliciting significant stimulation at 100 nM (Fig. 3BGo). To test whether bPTH-(3–34) could antagonize the effect of bPTH-(1–34) on PC hydrolysis, as it can inhibit PTH-stimulated cAMP production (15), we tested the effects of bPTH-(1–34) and bPTH-(3–34) added together. The combination of 1 nM bPTH-(1–34) and 100 nM bPTH-(3–34) gave a response that was greater than that to either of the individual treatments alone (Fig. 3BGo), indicating that bPTH-(3–34) is not an antagonist of bPTH-(1–34) on this pathway. hPTH-(1–31) was less potent than bPTH-(1–34), having little effect at 1 or 10 nM but eliciting a significant response at 100 nM (Fig. 3CGo).



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  		Figure 3. Effects of 0.1–1 nM bPTH-(1–34), hPTH-(1–34), and hPTHrP-(1–34) (A); 1 nM bPTH-(1–34), 10 and 100 nM bPTH-(3–34), and 1 nM bPTH-(1–34) plus 100 nM bPTH-(3–34) (B); and 1 and 10 nM bPTH-(1–34) and 1–100 nM hPTH-(1–31) (C) on the release of [3H]choline from UMR-106 cells. Results are the mean and SE of three determinations for A, four for B, and three for C. Incubations were performed for 30 min in A and for 1 h in B and C. *, P < 0.05 vs. control; #, P < 0.05 vs. 100 nM bPTH-(3–34).

 
Effects of bPTH-(1–34) and PDBu on transphosphatidylation in UMR-106 cells
To test whether PLD could be a mediator of the PC hydrolysis in UMR-106 cells, the cells were prelabeled with [3H]myristic acid and then treated with 10 nM bPTH-(1–34) or 25 nM PDBu in the absence or presence of 1% absolute ethanol to determine effects on the formation of [3H]phosphatidylethanol, the product of PLD-mediated transphosphatidylation of the phospholipids (Fig. 4Go). As indicated in Materials and Methods, [3H]myristic acid is predominantly incorporated into PC. In the presence of ethanol, bPTH-(1–34) and PDBu elicited similar significant increases in [3H]phosphatidylethanol. In the absence of ethanol, there was essentially no difference between the control and bPTH-(1–34)- or PDBu-treated groups.



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  		Figure 4. bPTH-(1–34) (10 nM) or 25 nM PDBu induced transphosphatidylation in UMR-106 cells. Results are the mean and SE of three determinations. Incubations were performed for 30 min. *, P < 0.05 vs. control (+Eth).

 
Phorbol down-regulation of PKC
Twenty-four-hour pretreatment of [3H]choline-prelabeled UMR-106 cells with 1 µM PDBu completely prevented the acute (1 h) effect of 500 nM PDBu on PC hydrolysis, but did not significantly prevent the acute (1 h) response to 10 nM bPTH-(1–34) (Fig. 5Go). The amounts of label in the total PC fraction were not significantly different in the control and down-regulated cells at the start of the acute incubation (data not shown).



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  		Figure 5. Effect of down-regulation of PKC by 24-h pretreatment with PDBu (1 µM) on the release of [3H]choline from UMR-106 cells treated for 1 h with bPTH-(1–34) or PDBu. Results are the mean and SE of five or six determinations. *, P < 0.05 vs. PDBu response in DMSO-pretreated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PKC, a key enzyme in cell signaling, is activated by DAG, a second messenger produced by the hydrolysis of membrane phospholipids by various phospholipases in response to a number of agonists. Two phospholipids that have been shown to be important sources of DAG are the phosphorylated inositol phospholipid PIP2 and the major membrane phospholipid PC. The kinetics and magnitude of the DAG production from these two sources have different characteristics. The increase in DAG produced by the breakdown of inositol phospholipids is usually small and transient because the pool is small, with PIP2 representing less than 1% of the membrane phospholipid. In contrast, PC accounts for about 50% of the total phospholipid in mammalian tissues (9), and the phosphodiester cleavage of PC leads to a sustained increase in the amount of DAG resulting in the prolonged activation of PKC, which is crucial for responses that involve cell proliferation or differentiation (16, 17). Based on generation of the phospholipid products, primarily arachidonic acid and DAG, phospholipase A2 and PLC were originally thought to be the phospholipases whose activation in response to agonists caused PC hydrolysis (11). There is now increasing evidence that a major mechanism of PC hydrolysis may be through the activation of PLD, producing PA. This PA is then further metabolized by a phosphohydrolase, resulting in much of the DAG yield (9, 10, 18). The importance of PLD in PC hydrolysis was recognized with the demonstration that several agonists exhibited rapid lipid transphosphatidylation activity (9, 10, 18), a reaction specific to PLD.

Because PTH is a critical regulator of normal bone-remodeling processes, stimulating both formation and resorption through receptors on osteoblastic cells, determining the signaling pathways activated by PTH in osteoblastic cells has been of considerable interest. Identified phosphorylation sites within the second (19) and third (20) intracellular loops of the PTH receptor are specifically involved in G protein-mediated processes that lead to the activation of adenylyl cyclase to increase cAMP or in the activation of PLC to cause breakdown of PIP2. PTH stimulates both cAMP-activated protein kinase A (PKA) and PKC signaling pathways (1, 2, 3, 6, 7, 8, 21). PTH signaling through PKA has been associated with c-fos gene activation in SaOS2 osteoblastic cells (22) and UMR-106 cells (23) and with bone sialoprotein gene expression in embryonic osteoblasts (24). The PLC-mediated breakdown of PIP2 would result in the production of DAG, an activator of many of the PKC isozymes (4). PTH activation of PKC has been associated with c-fos gene activation (25) and sodium-dependent phosphate transport (26) in UMR-106 cells. Both pathways appear to be involved in PTH-stimulated osteoblast-mediated osteoclast differentiation (27) and calcium efflux from cultured calvaria (28).

In the current studies, the participation of PLD-mediated PC breakdown in PTH action was examined, as this process can provide another source of DAG for the activation of PKC. Hydrolysis of the PC pool can result in a more prolonged production of DAG. As the effect of PTH on DAG production in osteoblastic cells and bone is a maintained response (3, 29), in contrast to the transient increase in inositol trisphosphate (3), it was conceivable that PTH would stimulate PC hydrolysis. The current results indicate that bPTH-(1–34) or hPTH-(1–34) and the agonists tested, hPTHrP-(1–34), bPTH-(3–34), and hPTH-(1–31), stimulate PC hydrolysis in UMR-106 osteoblastic osteosarcoma cells. The response is relatively rapid, occurring within the first 10 min. Significant effects were elicited by concentrations of bPTH-(1–34) as low as 1 nM. There was a dose-dependent increase in the response as the concentration of bPTH-(1–34) was raised to 10 nM. At the highest concentration tested (30 nM), the effect decreased. This could have been the result of activation of another pathway that antagonized the response. As the bPTH-(1–34) concentration required to elicit the decline was extremely high, it was not investigated further. The similar dose dependence of the agonist effects of hPTHrP-(1–34) on PC hydrolysis was comparable to that in other systems in which the two peptides have similar potencies (30). bPTH-(3–34) was a weaker agonist than bPTH-(1–34) on PC hydrolysis in the UMR-106 cells, but it had no antagonist activity, similar to its action on calcium transients (31), phosphatidylinositol hydrolysis (31), DAG production (29), PKC (6, 8), and bone resorption (29), but in contrast to its effects on adenylyl cyclase, where it is inactive (6, 8, 31) or acts as an antagonist (14). hPTH-(1–31) also stimulated PC hydrolysis in our studies. In other studies, hPTH-(1–31) stimulated adenylyl cyclase, but not PLC (32).

Several pathways exist by which agents can activate PLD-stimulated hydrolysis of PC. One mechanism is through PKC, the signaling process by which phorbol esters, which can function as DAG mimetics (4), activate this pathway (33). The specific phosphorylation step by which this kinase activates the hydrolysis has not been defined. PDBu-stimulated PC hydrolysis and transphosphatidylation were demonstrated in UMR-106 cells in the current study. PC hydrolysis was seen with PDBu concentrations at as low as 5 nM and at incubation times as brief as 10 min. There was a continuing progressive increase in medium [3H]choline over a 1-h incubation. The effect of PDBu to stimulate PC hydrolysis was not seen in UMR-106 cells pretreated for 48 h with a high concentration (1 µM) of the phorbol ester. In our previous studies (14), we have shown that this 48-h regimen produces maximal, although not complete, down-regulation of the conventional and novel PKC isozymes, with no effects on the atypical PKCs, which lack the motifs to bind DAG and are insensitive to PDBu (4).

Like PDBu, bPTH-(1–34) stimulated transphosphatidylation, indicating that it also activated PLD in the UMR-106 cells. However, in contrast to the effects of PDBu, the effects of bPTH-(1–34) on PC hydrolysis did not result in progressive accumulation over the 1-h incubation. Also, the effects of bPTH-(1–34) were unaffected by down-regulation of PKC isozymes by the 24-h treatment with PDBu. This suggests that the effect of bPTH-(1–34) on PC hydrolysis could be mediated differently from the effect of PDBu and probably through a PKC-independent pathway. Although PTH has been shown to activate PKC in bone cells (5, 6, 7, 8), the effect of PDBu to increase PLD could be mediated through a PKC isozyme that is unaffected by PTH. It is interesting that similar to our present findings with bPTH-(1–34), most agonists that have been examined for their effects on PC breakdown in osteoblastic cells also seem to effect this via a PKC-independent pathway. In other studies, PGE2 (34), PGF2{alpha} (35), PGD2 (36), platelet-derived growth factor (37), basic fibroblast growth factor (38), thrombin (39), endothelin (40), ATP (41), and sodium fluoride (42) were found to stimulate phosphatidylcholine hydrolysis through PKC-independent mechanisms. Only thromboxane A2 used a PKC pathway, as demonstrated by the sensitivity of the effects of this autacoid to PKC inhibitors (43). Alternative signaling pathways by which PTH could stimulate PC hydrolysis would be through PC-specific phospholipase C (11) or through the small GTP-binding proteins, ADP-ribosylation factor (44) and RhoA (45). In future studies, we will examine the roles of these pathways in PTH-mediated PC hydrolysis in osteoblastic cells.

In various tissues, roles for choline and PA, the direct products of PLD-catalyzed hydrolysis of phosphatidylcholine, and the consequences of other PLD-catalyzed reactions have begun to be defined. The functional importance in bone of PLD-mediated PC hydrolysis remains to be determined. There could also be cross-talk between PLD and the well established effects of PTH on signaling through PKA; however, the nature of this interaction cannot be readily predicted. In HL60 cells (46) and C6 glioma cells (47), increases in cAMP led to increased expression and activity of PLD. However, in Rat-1 fibroblasts transfected with {alpha}-adrenergic receptor subtypes, the cAMP increase elicited by phenylephrine acted as an inhibitory modulator of PLD activity (48). Further studies on the interactions among the PTH signaling pathways in osteoblasts and their functional consequences could provide new insights into our understanding of bone remodeling.


   Footnotes
 
1 This work was supported by NIH Grant AR-11262 (to P.H.S.). A portion of this study was presented at the 1997 Annual Meeting of American Society for Bone and Mineral Research, Cincinnati, OH. Back

2 Present address: Biological Sciences Collegiate Division, The University of Chicago, Chicago, Illinois 60637. Back

Received March 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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