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Endocrinology Vol. 139, No. 1 89-94
Copyright © 1998 by The Endocrine Society

ARTICLES

Prostaglandin E2 Stimulates Sodium-Dependent Phosphate Transport in Osteoblastic Cells via a Protein Kinase C-Mediated Pathway1

Christian M. Veldman, Irene Schläpfer and Christoph Schmid

Division of Endocrinology and Metabolism, Department of Internal Medicine, University Hospital, CH-8091 Zurich, Switzerland

Address all correspondence and requests for reprints to: Dr. Christoph Schmid, Division of Endocrinology and Diabetes, Department of Internal Medicine, University Hospital, CH-8091 Zurich, Switzerland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eicosanoids are released by bone cells in response to physical, hormonal, and cytokine stimulation, and they can both inhibit and stimulate bone formation. We investigated the effect of PGE2 on sodium-dependent phosphate (NadPi) transport in a rat bone-derived cell line, PyMS. PGE2 and 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, increased NadPi uptake in a dose- and time-dependent manner. There was no change in Na-dependent alanine transport, and the effects of PGE2 and TPA were not associated with corresponding changes in cell number or protein content. Their effects on NadPi uptake were not additive. Calphostin C, an inhibitor of protein kinase C (10-8 M) completely blocked TPA- and PGE2-stimulated NadPi uptake. The results are consistent with a crucial role of protein kinase C activation in the short term stimulation of NadPi transport by PGE2 in PyMS cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EICOSANOIDS are multifunctional regulators of bone metabolism, with both inhibitory and stimulatory effects on bone formation (1). The effects of eicosanoids on bone are of particular interest because bone cells also produce eicosanoids. PGE2 is the major eicosanoid produced by rat osteosarcoma and mouse osteoblastic cell lines and is also the most active one. Moreover, agents that increase bone resorption (and subsequently bone formation), such as PTH and interleukin-1 (2), and mechanical stress (3, 4, 5) can stimulate endogenous eicosanoid production. In vivo studies showed that daily administration of PGE2 can increase bone mass in animals (6). PGE1 infusion is used to maintain the patency of the ductus arteriosis; after long term administration, these patients showed an increase in bone formation in the periosteal regions of the limbs and ribs (7).

In vitro, PGE2 stimulates both DNA and collagen synthesis in calvariae (1) and alkaline phosphatase activity in MC3T3-E1 cells (8). Furthermore, PGE2 increases alkaline phosphatase activity and mineralized bone nodule formation in calvarial cells (9). Stimulation of DNA synthesis by PGE2 appears to occur in bone tissue compartments rich in precursor cells, whereas no effect, inhibition, or delayed stimulation occurs in areas of highly differentiated osteoblasts (10, 11). Along this line, we found that PGE2 decreased DNA synthesis in calvaria cells (12), whereas it increased DNA synthesis in the periosteal cells of cultured fetal rat calvariae (11). Moreover, PGE2 increased DNA synthesis in less differentiated bone cells by a protein kinase C (PKC)-dependent event, whereas PGE2 and cAMP decreased DNA synthesis in more differentiated bone cells (10, 12).

Osteoblasts are responsible for the formation of the bone matrix and its mineralization, i.e. for the production of type I collagen, and for deposition of inorganic phosphate (and Ca) in bone. Phosphate (Pi) transport has been studied in monolayer cultures of osteoblastic osteosarcoma cells. Sodium-dependent Pi (NadPi) transport is a carrier-mediated process that is dependent on the presence of Na+ in the extracellular space (13). It has recently been shown that PTH (14), PTH-related peptide (PTHrP) (15), insulin-like growth factor I (IGF-I) (16), and transforming growth factor-{alpha} (17) stimulate NadPi transport in UMR-106–01 cells. PTH and PTHrP activate a common (PTH/PTHrP) receptor (18) that activates similar second messenger systems as the PGE2 receptors (19, 20, 21). Even though the NadPi transport studies had been conducted in the same cell line, UMR-106, they yielded apparently conflicting results. The PTHrP effects in UMR-106 cells found by Arao et al. (15) indicated that the PKC messenger system, rather than adenylate cyclase or cytosolic calcium, played a crucial role in the stimulation of NadPi transport by PTHrP in UMR-106 cells. In contrast, the stimulatory effect of PTH on NadPi transport in UMR-106 cells was suggested to involve PKA activation, as it was mimicked by forskolin, a stimulator of cAMP formation (14). PTH regulated NadPi transport in a biphasic way: it first increased the NadPi transport with a maximum (1.3-fold) after 4–6 h and inhibited NadPi transport thereafter up to ~18–20 h. The early pattern of the change in NadPi transport in response to forskolin was similar to that observed with PTH; however, no late inhibition occurred. PTH appeared to regulate NadPi transport in UMR-106 cells differently than did PTHrP in the same cell line. However, the effects of PGE2 on NadPi transport in bone cells have not been reported.

We used a rat bone-derived cell line, PyMS, to study the effect of PGE2 on NadPi transport in osteoblastic cells and to investigate whether pharmacological agents mimic or block the effect of PGE2 on NadPi transport. This cell line shares features of periosteal cells of calvariae in vivo in that DNA synthesis is stimulated by PGE2 (10, 11). Similar to rat Py1a (22, 23) and mouse MC3T3-E1 (8, 21) cells, PyMS cells represent less differentiated, preosteoblastic bone cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
PyMS cells were provided by Dr. A. Lichtler, University of Connecticut (Farmington, CT). These cells had been derived from fetal rat calvaria and immortalized by infection with polyoma virus mutant strains containing a functional gene encoding the large T antigen. PGE2, (Bu)2cAMP, 12-O-tetradecanoyl phorbol-13-acetate (TPA), A23187, indomethacin, and cycloheximide were obtained from Sigma Chemical Co. (St. Louis, MO); recombinant human IGF-I was purchased from Ciba Geigy (Basel, Switzerland); calphostin C and genistein were obtained from Calbiochem (La Jolla, CA); wortmannin was a gift from Dr. T. Payne (Sandoz, Basel, Switzerland); and recombinant human IGF-binding protein-3 (IGFBP-3) was obtained from Dr. A. Sommer (Celtrix, Santa Clara, CA). Gentamicin, glutamine, FCS, and trypsin were purchased from Life Technologies (Grand Island, NY); BSA was obtained from Serva (Heidelberg, Germany). All solutions of these agents were freshly prepared. PGE2, A23187, TPA, calphostin C, and genistein were dissolved in absolute ethanol to concentrations of 2 x 10-3, 10-2, 10-3, 10-4, and 10-4 M, respectively, and used at the concentrations indicated in Results. All media were adjusted to a final alcohol concentration of 0.1%.

Cell culture
The cells were passaged in Falcon tissue culture flasks (Becton Dickinson, Franklin Lakes, NJ) in DMEM supplemented with gentamicin (50 µg/ml), glutamine (2 mmol/liter), and FCS (5%) at 37 C in an atmosphere of 5% CO2 in air. Cell cultures between passages 14 and 39 were used in these studies. Cells grown to confluence were detached from the dishes with 0.25% trypsin and replated in Falcon multiwell tissue culture plates (35-mm diameter) at a density of 2 x 105 cells/well. Confluent monolayer cultures were used 4 days after seeding. Twenty-four hours before the transport studies, cells were rinsed with serum-free medium, and the growth medium was replaced by serum-free F-12 medium containing gentamicin (50 µg/ml), glutamine (2 mmol/liter), and charcoal-treated BSA at 1 g/liter. Cell monolayers were incubated for 10 min with indomethacin, calphostin C, genistein, and wortmannin, respectively, and TPA or PGE2 was added 10 min later. Aliquots of test agents were added directly to the medium. Cells were incubated as specified in Results.

Transport studies
Before the start of experiments, monolayers were washed at room temperature with 1 ml transport buffer A or B containing 140 mM NaCl or choline chloride, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl, and 15 mM HEPES, adjusted to pH 7.4, with Tris-HCl. Transport buffer B, containing choline chloride instead of NaCl, was used for measuring Na+-independent solute uptake. The 32P uptake or [14C[alanine uptake studies were initiated by adding 1 ml transport buffer containing 0.1 mM KH2PO4 (1 µCi 32PO4/ml; 200 mCi/mmol) or 0.1 mM L-alanine (1 µCi L-[14C]alanine/ml; 158 mCi/mmol), respectively (both from Amersham, Aylesbury, UK). Ten minutes later, the buffer was removed, and the dishes were quickly rinsed three times with 1 ml ice-cold transport buffer. The cells were then solubilized with 1 ml 2% SDS, and the radioactivity of a 0.5-ml aliquot was counted in a liquid scintillation counter.

Determination of cell number, protein content, and [3H]thymidine incorporation into DNA
After 4 days of culture (24 h in test medium as described above), cells were detached from some of the dishes with trypsin, counted in a hemocytometer, and lysed from parallel dishes into 2 ml 0.1% Triton X-100 for the determination of protein content by a modification of the Lowry procedure (24). For [3H]thymidine incorporation into DNA, cells were plated and grown as described above, but exposed to test medium for 18 h as indicated, then pulsed with [methyl-3H]thymidine (Amersham; 80 Ci/mmol; 1 µCi/dish) for 3 h at 37 C and rinsed three times with cold PBS, DNA was precipitated and washed three times in situ with 10% trichloroacetic acid, and incorporated radioactivity was measured in a liquid scintillation counter (12).

Statistical analysis
Results were obtained by pooling data from several independent experiments in which exactly the same points were tested and are expressed as the mean ± SE. The significance of the differences was assessed by two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of Pi transport, sodium dependency
Pi uptake by confluent PyMS cells increased linearly with incubation time for up to at least 30 min in the presence of extracellular Na (not shown). There was little uptake of Pi in the absence of Na. After a 10-min incubation, Na-independent Pi uptake amounted to less than 12% of total Pi uptake, and it did not increase with time (not shown). NadPi transport was calculated by subtraction of the Na-independent component from total Pi uptake in the presence of Na. The kinetic parameters of the NadPi transport system were obtained from Lineweaver-Burk plots. The apparent affinity constant (Km) was 106 ± 7 µmol/liter. Based on this observation, the subsequent studies (Figs. 1Go and 2Go and Tables 1–4GoGoGoGo) were conducted at a Pi concentration of 0.1 mM, i.e. close to the Km of the NadPi transport system.



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  		Figure 1. NadPi transport in cells exposed to PGE2 for 24 h. NadPi transport was measured for 10 min at a Pi concentration of 0.1 mM. Data presented are the mean ± SE of three experiments performed in triplicate. Significance (*, P < 0.05; **, P < 0.005) is indicated in comparison with vehicle values.

 


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  		Figure 2. Effects of PGE2 and TPA on NadPi transport: time course. Cells were incubated in serum-free medium and exposed to 10-6 M PGE2 and 10-7 M TPA for 0.5, 2, 6, and 24 h before measuring NadPi transport per 10 min. The Pi concentration was 0.1 mM. Data are the mean ± SE of three experiments performed in triplicate. Significance (*, P < 0.05; **, P < 0.005) is indicated in comparison with vehicle values.

 

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  		Table 1. Effects of PGE2, IGF-I, and their combination on NadPi transport, Na-dependent alanine transport, and DNA synthesis after 2 and 24 h

 

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  		Table 2. NadPi transport and DNA synthesis in PyMS cells exposed to 10-6 M PGE2 and IGF-I in the presence or absence of 10-6 M indomethacin or 10-8 M IGFBP-3, respectively, for 24 h (A) and exposed to 10-7 M TPA, 10-6 M A23187, and 0.5 mM (Bu)2cAMP for 24 h (B)

 

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  		Table 3. Kinetic parameters, cell number, protein content, and NadPi transport exposed to PGE2 and/or TPA for 24 h

 

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  		Table 4. Effects of PGE2, TPA, and IGF-I in combination with calphostin C, genistein, and wortmannin on NadPi transport after 2 h

 
PGE2
NadPi transport was increased in cells that had been exposed to PGE2 for 24 h (Figs. 1Go and 2Go and Tables 1–3GoGoGo). The lowest concentration that produced a significant stimulation was 10-8 M, and maximal stimulation occurred at 10-6 M (Fig. 1Go). The effect of PGE2 on NadPi transport was studied over a range of Pi concentrations from 0.02–2 mM. Analysis of the data using the Lineweaver-Burk plot indicated that PGE2 increased the maximal velocity (Vmax) of the NadPi transport system at an extracellular Na concentration of 140 mmol/liter, without affecting its Km (Table 3Go).

To determine whether the effect of PGE2 on NadPi transport is specific, we looked at Na-dependent alanine transport. Alanine uptake increased linearly with incubation times for up to at least 30 min in the presence of extracellular Na (not shown). After a 10-min incubation, Na-independent alanine transport amounted to less than 6% of the total uptake, and it did not increase with time (not shown). Na-dependent alanine transport was calculated by subtraction of the Na-independent component from the total alanine uptake in the presence of Na. PGE2 at 10-6 M increased Na-dependent alanine transport after 24 h only when expressed per dish (1.1 ± 0.02-fold; not shown), not when expressed per protein (Table 1Go) or cell number (not shown), whereas in dishes treated in parallel, PGE2 increased NadPi transport 2.0 ± 0.04-fold (Tables 2AGo and 3Go). The effect of PGE2 was specific for NadPi transport.

PGE2 stimulated NadPi uptake from 204 ± 4 pmol Pi/dish·10 min (control) to 246 ± 7 pmol Pi/dish·10 min (1.2-fold) within 2 h (Fig. 2Go and Tables 1Go and 4Go), to 324 ± 8 pmol Pi/dish·10 min (1.6-fold) after 6 h (Fig. 2Go), and to 392 ± 5 pmol Pi/dish·10 min after 24 h at 10-6 M, i.e. 1.9-fold (Fig. 2Go and Tables 1–3GoGoGo). Kinetic values for NadPi transport determined after 2-h treatment with PGE2 and TPA in selected experiments (not shown) indicated an increase in the Vmax corresponding to the increase in NadPi transport and no change in Km, as shown for the 24 h point in Table 3Go. PGE2 at 10-6 M stimulated DNA synthesis ~3 ± 0.2-fold (Tables 1Go and 2AGo), and slightly increased cell number and protein content per culture dish (Table 3Go).

Indomethacin, IGF-I and IGFBP-3
Indomethacin was added to inhibit the possible amplification of locally induced eicosanoids by exogenous PGE2 (21). Indomethacin (10-6 M) had no effect on basal and PGE2- (Table 2AGo) and TPA-stimulated (not shown) NadPi transport or on Na-dependent alanine transport during 24 h of treatment. Indomethacin (10-6 M) had no effect on basal and PGE2-stimulated DNA synthesis (Table 2AGo).

Because locally produced IGF-I could be a mediator of selected PGE2 effects (26), we examined IGF-I and PGE2 together after 2- and 24-h incubations. The effects of IGF-I and PGE2 were detected after 2 and 24 h, and these effects were additive (Table 1Go). The stimulatory effect of 10-9 M IGF-I, but not that of 10-6 M PGE2, was blocked by 10-8 M IGFBP-3 (Table 2AGo). IGF-I at 10-9 M stimulated DNA synthesis 7-fold (Table 1Go). The effects of IGF-I and PGE2 were synergistic (Table 1Go). The stimulatory effect of 10-9 M IGF-I, but not that of 10-6 M PGE2 was blocked by 10-8 M IGFBP-3 (Table 2AGo).

TPA, A23187, and (Bu)2cAMP
To determine which of the distinct signal transduction pathways influences NadPi transport in PyMS cells, PGE2 receptors were bypassed by pharmacological agents. NadPi transport was increased in PyMS cells that had been exposed to TPA for 24 h (Fig. 2Go and Tables 2BGo and 3Go). The lowest concentration that produced a significant stimulation was 10-9 M, and maximal stimulation occurred at 10-7 M (not shown). TPA stimulated NadPi uptake after 24 h at 10-7 M 1.5 ± 0.03-fold (Tables 2BGo and 3Go and Fig. 2Go). A stimulatory effect of TPA on NadPi transport was seen after 0.5 h (1.5 ± 0.1-fold), and maximal stimulation (2.0 ± 0.06-fold) was detected after 2 h (Fig. 2Go). The stimulatory effects of PGE2 and TPA on NadPi transport were not additive after 24 h (Table 3Go) or after 0.5, 2, and 6 h (not shown). In fact, PGE2 and TPA in combination increased NadPi transport less markedly than PGE2 alone after 24 h (Table 3Go). Treatment of the cells with 10-7 M TPA for 24 h abolished subsequent stimulation of NadPi transport by 10-6 M PGE2, but not by 10-9 M IGF-I (not shown). TPA at 10-7 M markedly decreased DNA synthesis to 27 ± 4% of the control value (Table 2BGo), but had no significant effect on cell number and protein content per culture dish (Table 3Go).

NadPi transport was slightly increased in cells that had been exposed to A23187, but not in cells exposed to 0.1 and 0.5 mM (Bu)2cAMP, for 24 h. The concentration of A23187 that produced significant stimulation (1.1-fold) was 10-6 M (Table 2BGo), the highest concentration tested. Na-dependent alanine transport was measured in parallel with NadPi transport. It was not affected by exposing cells to TPA, A23187, and (Bu)2cAMP for 24 h (not shown). A23187 at 10-6 M stimulated NadPi transport in PyMS cells to the same small extent after 0.5, 2, 6, and 24 h. (Bu)2cAMP at 0.5 mM did not affect NadPi transport in PyMS cells after 0.5, 2, 6 (not shown), and 24 h (Table 2BGo). At the concentration that had a slight effect on NadPi transport, 10-7 M A23187 markedly stimulated DNA synthesis ~13 ± 0.4-fold (Table 2BGo). (Bu)2cAMP at 0.1 (not shown) and 0.5 mM (Table 2BGo) decreased DNA synthesis to ~40 ± 2% of the control value.

Calphostin C, genistein, and wortmannin
We used blockers of signal pathways as an additional approach to determine which of the second messenger systems was relevant in mediating increased NadPi transport in response to PGE2. Calphostin C interacts with the site of diacylglycerol and phorbol esters and is an inhibitor of PKC, genistein is an inhibitor of tyrosine kinases, and wortmannin is a blocker of phosphatidylinositol (PI)-3 kinase. Calphostin C at 10-8 M completely blocked PGE2- and TPA-stimulated, but not IGF-I-stimulated, NadPi transport after 2 h (Table 4Go). Genistein at 10-8 M partly blocked IGF I-stimulated, but not PGE2- and TPA-stimulated, NadPi transport after 2 h (Table 4Go). Wortmannin at 10-7 M partly blocked IGF-I-stimulated, but not basal or PGE2- or TPA-stimulated, NadPi transport after 2 h (Table 4Go). Na-dependent alanine transport was not affected by exposing PyMS cells to calphostin C, genistein, or wortmannin for 2 h (not shown). Cell death was a major problem in cell cultures exposed to 10-8 M calphostin C or 10-8 M genistein for 18–24 h. At 10-8 M, calphostin C decreased basal NadPi transport to 87 ± 4% of the control value after 6 h and to 35 ± 12% after 24 h, and decreased PGE2-stimulated NadPi transport to 84 ± 8% of the control value after 6 h and to 38 ± 15% after 24 h in two independent tests (not shown). No concentration of calphostin C was found that selectively decreased PGE2- or TPA-stimulated, but not basal, NadPi transport after 6 or 24 h. Wortmannin at 3 x 10-8 and 10-7 M partly blocked IGF-I-stimulated, but not basal or PGE2- or TPA-stimulated, NadPi transport after 24 h and decreased basal, PGE2-stimulated, TPA-inhibited, and IGF-I-stimulated DNA synthesis (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PGE2 stimulated NadPi transport in bone-derived PyMS cells. The stimulation was dose related and time dependent. The specificity of the effect is demonstrated by the finding that the uptake of Pi in the absence of extracellular Na is unaffected, suggesting that the change in NadPi uptake cannot be accounted for by a nonspecific increase in diffusional movement of Pi. PGE2 tended to increase the Na-dependent alanine transport, another Na-dependent transport system, within 24 h only when expressed per culture dish, not when expressed per protein content or cell number, indicating that the effect of PGE2 is specific for NadPi transport in PyMS cells. The increase in NadPi transport after incubation with PGE2 was due to an increase in the apparent Vmax with no change in the apparent Km for Pi.

First of all, we checked whether extracellular mediators were involved in the PGE2 effect on NadPi transport. Indomethacin was added to determine whether the effects of exogenous PGE2 were amplified by locally induced eicosanoids based on observations in murine osteoblastic MC3T3-E1 cells and in rat osteoblastic Py1a cells (23). The effects of PGE2 on NadPi transport in PyMS cells were not sensitive to indomethacin pretreatment, indicating that increased endogenous PGE2 production under the influence of PGE2 does not play a role. Studies of cultured osteoblastic cells from rat calvariae had shown that PGE2 increased IGF-I production (25, 26). However, PyMS cells barely produce IGF-I (27), and IGF-I production is not increased in response to PGE2. Furthermore, the increase in NadPi transport in response to PGE2 is unlikely to be mediated via increased local IGF-I production, because the effects of IGF-I and PGE2 on NadPi transport are additive. Moreover, IGFBP-3, which blocks the stimulatory effects of IGF-I on PyMS cells, did not affect the stimulatory effect of PGE2 on NadPi transport and DNA synthesis. Locally released PGE2 and IGF-I do not appear to be involved in the observed PGE2-stimulated NadPi transport, and the rapid onset of action points to a direct effect of PGE2.

At least two types of PGE2 receptors are expressed by osteoblasts (21, 28). Bone cells appear to express subtypes EP1 and EP2, but not EP3 messenger RNA (28). EP1 activation is coupled to increased phosphatidylinositol turnover and calcium mobilization, and EP2 mediates increased cAMP formation (20). Bone cells, including the rat Py1a line (similar to PyMS cells), also express PGF receptors (29) to which the PKC signaling pathway may also be coupled. To determine which of the distinct signal transduction pathways influences NadPi transport in PyMS cells, PG receptors were bypassed by pharmacological agents. Direct activation of PKC by TPA caused a dose- and time-dependent increase in NadPi uptake by PyMS cells through an increase in the apparent Vmax. The response to TPA was qualitatively similar to that to PGE2; however, a shorter time of incubation of TPA was required to achieve significant and maximal stimulation of NadPi transport. PGE2 and TPA appear to regulate Pi membrane transport by the same or a similar mechanism. This hypothesis is consistent with the observation that the effects of PGE2 and TPA on NadPi transport in PyMS cells are neither additive nor synergistic at 24 h or any other time period (not shown). The combination of 10-6 M PGE2 and 10-7 M TPA was less effective in stimulating NadPi transport after 24 h than was 10-6 M PGE2 alone. The stimulatory effect of TPA on NadPi transport may vanish because prolonged treatment of the cells with phorbol esters depletes the cellular PKC pools. Consistent with down-regulation of the PKC pathway by TPA, treatment of the cells with 10-7 M TPA for 24 h abolished subsequent stimulation of NadPi transport by 10-6 M PGE2 (but not by 10-9 M IGF-I). However, prolonged (24-h) treatment with TPA leads to profound changes in cell function, including a marked decrease in DNA synthesis compared with the control value. The increase in NadPi transport in response to PGE2 is unlikely to be due to an increase in cAMP formation or an increase in cytosolic calcium levels. (Bu)2cAMP had no effect and the calcium ionophore A23187 only a marginal effect on NadPi transport in PyMS cells. This is in sharp contrast to their effects on DNA synthesis; A23187 increased and (Bu)2cAMP inhibited DNA synthesis in PyMS cells. Stimulation of DNA synthesis by PGE2 and inhibition by Bt2cAMP were also found by Suda et al. in MC3T3-E1 cells (21).

We used not only stimulators, but also blockers, of signal pathways as an additional approach to see which of the second messenger systems were relevant in mediating increased NadPi transport in response to PGE2 (and TPA). We compared the effects of calphostin C, genistein, and wortmannin on IGF-I-, PGE2-, and TPA-stimulated NadPi transport. Calphostin C completely prevented the stimulation of NadPi transport by both PGE2 and TPA, but not by IGF-I, indicating that PGE2 and TPA regulate NadPi transport by a PKC-mediated pathway. Genistein and wortmannin did not affect TPA- and PGE2-stimulated, but partly blocked IGF-I-stimulated, NadPi transport, indicating that the stimulation of NadPi transport by PGE2 and TPA is not mediated via tyrosine kinase- or PI-3 kinase-dependent mechanisms. Genistein-sensitive tyrosine kinases and the PI-3 kinase, however, appear to play a role in the regulation of IGF-I-stimulated NadPi transport. We have previously observed that IGF-I rapidly (within 30 min) stimulated NadPi transport and suggested that it could stimulate exocytotic insertion of Pi transporter-containing vesicles in the plasma membrane because its effects were completely prevented by colchicine, a microtubule-depolymerizing agent (30). The results with the blockers used in this study were obtained mainly in 2-h incubation tests, where the data are consistent with highly specific effects of these blockers on PGE2-, TPA-, and IGF-I-stimulated NadPi transport. Basal NadPi transport was not affected by the blockers within 2 h, but markedly decreased after 6 and 24 h. Exposing the cell cultures for longer time (18–24 h) to 10-8 M genistein or 10-8 M calphostin C caused cell death. Incubation with wortmannin was tolerated for 24 h, but the specificity of its effect was possibly lost because the basal rate of DNA synthesis was decreased. Remarkably, neither basal nor PGE2-stimulated NadPi uptake was reduced.

It was considered important to know the influence of PGE2 (and TPA) on DNA synthesis of PyMS cells under our experimental conditions and to compare the results with the data for NadPi transport. PGE2 stimulated DNA synthesis 3-fold in PyMS cells, whereas TPA decreased DNA synthesis to 27% of the control value. PGE2 increased DNA synthesis in preosteoblasts (11) and MC3T3-E1 (21) cells. Because TPA mimicked the early effect of PGE2 on NadPi transport, PGE2 and TPA in the short term seem to affect similar pathways with regard to NadPi uptake; however, the long term effects of PGE2 and TPA are not equal and do not elicit signaling that results in comparable effects on DNA synthesis. Wortmannin decreased DNA synthesis, but not NadPi transport; therefore, PI-3 kinase-dependent pathways appear to be required for DNA synthesis but not for the stimulation of NadPi transport by PGE2. In agreement with our findings, wortmannin blocked insulin-induced, but not TPA-induced, activation of mitogen-activated protein kinase in rat adipocytes (31), indicating that TPA activates mitogen-activated protein kinase without involvement of the PI-3 kinase. Taken together, the effect of PGE2 on NadPi transport is not simply related to changes in cell growth, as it is clearly dissociated from its effect on DNA synthesis.

In conclusion, our data suggest that PGE2 stimulates NadPi transport in PyMS cells in the short term by using the PKC-mediated signal transduction system.


   Acknowledgments
 
The authors are grateful to Drs. E. R. Froesch and M. Gosteli-Peter for correcting this manuscript.


   Footnotes
 
1 This work was supported by Grant No. 32-46808.96 from the Swiss National Science Foundation. Back

Received June 6, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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