Biochimica et Biophysica Acta, 1176 (1993) 333-338 333 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00

BBAMCR 13371

Differential effects of activation of protein kinase C and cyclic-AMP-dependent protein kinase

on sodium-dependent phosphate uptake in NIH 3T3 cells

Zoltfin Olfih, Csaba Lehel and Wayne B. Anderson Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD (USA)

(Received 13 July 1992) (Revised manuscript received 16 November 1992)

Key words: Phosphate transport; Protein kinase C; cyclic-AMP-dependent protein kinase; Phorbol ester; Forskolin; (NIH 3T3 cell)

Activation of protein kinase C (PKC) by phorbol ester (PMA), or by diacylglycerol analogue (OAG) treatment of NIH 3T3 cells resulted in the rapid (within 2-5 min) stimulation (approx. 2-fold) of sodium-dependent phosphate (P~) transport. Conversely, preincubation of these cells with forskolin and cholera toxin, or incubation with 8-bromo-cAMP, to activate cAMP-dependent protein kinase (PKA), resulted in a decrease in Na +/P~ transport. Activation of either PKC or PKA did not change the Vma x of Pi uptake. However, activation of PKC did result in an increase, while activation of PKA caused a decrease, in the affinity for P~. These results indicate that there is differential regulation of Na+/Pi uptake in NIH 3T3 cells by activators of PKC (stimulated) and PKA (inhibited) as a consequence of changes in the affinity of the transporter for Pi.

Introduction

Phosphate ion (Pi) is an important regulator of intracellular metabolism. Its role in regulating glycoly- sis and oxidative phosphorylation is well established [1-3]. In addition, it has been suggested that increased Pi uptake may play an important role in the regulation of cell growth [4,5]. Thus, it is of interest to better understand the mechanism(s) involved in modulating the intracellular levels of Pi itself.

The intracellular concentration of Pi is known to be modulated by the synthesis and breakdown of phosphocreatine and ATP. The rates of Piuptake and efflux across the plasma membrane also influence the intracellular level of Pi, and several receptors and second messengers have been shown to regulate sodium-dependent P~ transport [6]. Studies by several groups have indicated that protein kinase C (PKC) and

Correspondence to: Z. Olfih, Laboratory of Cellular Oncoiogy, Na- tional Cancer Institute, Bldg. 36, Rm. 1D22, Bethesda, MD, 20892, USA. Abbreviations: PMA, phorbol-12-myristate 13-acetate; MPMA, phor- bol-4-O-methyl-12-myristate 13-acetate; Hepes, N-2-hydroxyethyl- piperazine-N '-2-ethanesulfonic acid.

cyclic-AMP-dependent protein kinase (PKA) both may be involved in the regulation of sodium-coupled P~ transport. However, these two protein kinases have been observed to have different effects on Pi uptake in different cell types [6]. Activation of PKA has been noted to inhibit sodium-dependent Pi transport in rab- bit proximal tubular [7], opossum kidney [8-10] and HeLa [11] cells. Yet, PKA may be without effect, or perhaps even be stimulatory, toward Pi transport in UMR-106 [12] and LLC-PK1 [13] cells. Conversely, activation of PKC has been reported to stimulate sodium-dependent Pi transport in LLC-PK1 [14,15], JTC-12P3 [15] and HeLa [16] cells, but to inhibit Pi uptake in opossum kidney cells [6,17].

Since the modulation of Pi uptake into the cell appears to play a prominent role in regulating such biological responses as cell proliferation, it is important to study the effects of PKC and PKA in mediating the sodium-dependent phosphate transporter(s) found in different cell types. NIH 3T3 cells have been utilized extensively as a model system to study cell growth regulation and oncogenic transformation. In this com- munication we have used NIH 3T3 cells to better characterize the rapid, short-term differential regula- tion of phosphate transport by the PKC and PKA signalling pathways.

334

Materials and Methods

Materials Fetal bovine serum was from ICN Flow (Costa Mesa,

CA), and all other cell culture materials were pur- chased from Gibco-BRL (Bethesda, MD). KHz32po 4 was from ICN Biomedicals (Irvine, CA). Forskolin (6/3-(/3'-(piperidino)propionyl)hydrochloride) was from RBI (Natick, MA). Phorbol, 4/3-phorbol, phorbol-12- myristate 13-acetate (PMA), and phorbol-4-O-methyl- 12-myristate 13-acetate (MPMA) were from Cal- biochem (La Jolla, CA). Bryostatin was generously provided by Dr. G. Robert Pettit, Arizona State Uni- verity. 8-Bromo-cAMP was from Sigma (St. Louis, MO) and 1-oleoyl-2-acetyl-glycerol (OAG) was purchased from Serdary Res. Lab. (London, Ontario, Canada) and the diacylglycerol kinase inhibitor R59022 was from Janssen Life Sciences (Beerse, Belgium).

Phosphate uptake All [32p]phosphate uptake experiments were per-

formed in triplicate and repeated at least three times, essentially as described by Escoubet et al. [18]. NIH 3T3 cells were grown to confluency in 24-well culture trays (Costar, Cambridge, MA) under conditions de- scribed previously [19]. Confluent populations of NIH 3T3 cells were deprived of serum by incubation in serum free medium for 18 h. Fresh serum-free culture medium (1 ml/well) was added back to the cells, and the cells then were pretreated for the indicated time periods with the various activators of PKC and PKA as described. The attached cells were washed once with uptake medium (10 mM Hepes-KOH buffer (pH 7.4), containing 0.1 mM KH2PO4, 137 mM NaC1, 5.4 mM KC1, 2.8 mM CaC1 z and 1.2 mM MgSO4). Uptake medium (200 /~I/well) supplemented w i t h KH232po 4 (5 #Ci/well; spec. act. 285 Ci/mg Pi) then was added to the cells to initiate [32p]phosphate transport. Fol- lowing incubation at 37°C for 2 min, [32p]phosphate transport was terminated by rapidly removing the up- take medium and washing the cells 4 times with ice-cold uptake medium without [32p]phosphate. Following ter- mination of Pi uptake the washed cells were solubilized in a lysis buffer (50 mM Tris-HC1 (pH 7.2) containing 150 mM NaC1, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA), and aliquots of the solubi- lized cell extracts were counted in a liquid scintillation counter. The cation specificity of Pi uptake was tested in the same uptake medium except that Na + was replaced with 137 mM Li +, K +, NH~-, or choline, as indicated. Sodium-dependent phosphate uptake was determined by subtracting the amount of Pi uptake noted in the absence of Na + (but with 137 mM choline chloride present in place of NaC1) from the amount of Pi uptake noted in the presence of 137 mM NaC1. The protein concentrations of the lysis-buffer-solubilized

cell extracts were determined with the use of protein assay reagent (BCA ®, Pierce) according to the proce- dure provided by the manufacturer, using BSA as protein standard.

Results

Although initial experiments were performed in serum-free growth medium (used to partially growth- arrest the cells in G o of the cell cycle by serum depriva- tion) there was an obvious need to switch to a less complex, more readily defined uptake medium to facil- itate the characterization of the regulatory properties of Pi transport. Therefore, subsequent experiments were performed in a simple isoosmotic solution con- taining equimolar (137 mM) cation concentrations of either Li +, Na +, K +, NH~- or choline chloride along with 0.1 mM K H z P O 4 t o define the cation require- ment(s) of Pi transport. The results presented in Fig. 1 show that Na +, and to some extent Li +, were able to support Pi uptake, while K +, NH~-, and choline were not effective. Interestingly, only Na+-dependent Pi

transport was stimulated by pretreatment of the cells with PMA to activate PKC. Phorbol ester activation of PKC was noted to be inhibitory toward P~ transport supported by Li +.

Incubation of confuent populations of serum-de- prived NIH 3T3 cells for the indicated time periods with 1 txM PMA to activate PKC induced a progres- sive, but transient, increase in short-term (2 min) Pi uptake through 20 min of preincubation (Fig. 2). The inactive phorbol analogue MPMA had no effect on

1200

1000 e-

~o ~00 O.

[ 800

=o

~. 200

Na ÷

ih Li +

• control

[ ] PMA

K ÷ NH4 + C h o l i n e

Fig. 1. Effect of monovalent cations on short-term phosphate uptake in NIH 3T3 cells. Each of the indicated cations (at a concentration of 137 mM) were used separately in chloride form in the uptake medium (minus NaCl as required) containing 0.1 mM KH232po 4 to determine Pi transport during a 2-min incubation at 37°C. Cells were either untreated (control, solid bars), or were pretreated with 1 /xM PMA for 10 min (hatched bars) prior to initiation of [32p]phosphate transport as described. Similar results were obtained in three inde- pendent experiments performed in triplicate. Each point represents

the mean +_ S.E. of triplicate determinations.

1 3 0 0

e -

.~'.~ 1,oo. < o

<1: " ~ 9 0 0 ' " I - . -

,', E

0 "

500 0 1 '0

0 PMA • F o r s k o l l n

i

2'0 3 0 Time, m i n

Fig. 2. Time-course of the effect of preincubation of NIH 3T3 cells with PMA, or forskolin, on short-term sodium-dependent phosphate transport. Confluent NIH 3T3 cells were preincubated with either 1 /~M PMA (o), 1 /zM MPMA ( . ) , or 50 /zM forskolin (e) for the indicated time periods prior to initiation of 32p i uptake (2 min) as described in Materials and Methods. Similar results were obtained in three independent experiments performed in triplicate. Values are

expressed as the mean _+ S.E. of triplicate determinations.

basal Pi uptake under the same conditions (Fig. 2). Further, similar kinetics for the increase in Pi uptake were observed when the cells were treated with the diacylglycerol analogue OAG (100 /zg/ml) and with bryostatin (10/zM) to activate PKC (Table I).

In contrast to the activation of PKC by PMA, prein- cubation of NIH 3T3 cells with forskolin for the time

TABLE I

Effect of activation of protein kinase C and cAMP-dependent protein kinase on short-term sodium-dependent phosphate transport in NIH 3T3 cells

Serum-deprived NIH 3T3 cells were preincubated for 10 min with the activators of PKC and with 8-Br-cAMP (1 mM) and for 4 h with the adenylate cyclase activator cholera toxin (0.5/zg/ml). The OAG (I00 p.g/ml) was added a second time following the initial 5-min incubation in an attempt to maintain the concentration of OAG within the ceils. To further protect the OAG added, cells also were treated with the diacylglycerol kinase inhibitor R59022 (5/.tM) along with OAG, as indicated. Following pretreatment of the cells for the time periods indicated, 2 rain [32p]phosphate uptake experiments were performed as described. Similar results were obtained in three independent experiments performed in triplicate. Data are expressed as mean + S.E.

Agent Change in basal Na +/Pi-uptake (% control)

activation inhibition

activators of PKC OAG OAG + R59022 bryostatin

activators of PKA 8-bromo-cAMP cholera toxin

121+6.1 140:1:5.2 164 + 6.7

22 + 2.9 36+4.8

335

O 1,. O .

E i .-

. m

E , D a .

O

E O .

tff < l - n. :D

III I-. ,< - r a. of) 0 -1- a.

1300

1 1 0 0 '

900

700 l

500 / 0

A

i a i

260 400 600 800 1000 [PMA], nM

400

300

200 l i

0 1 0 0 260 300 460 560 [ F o r s k o l i n ] , t.tM

Fig. 3. Effect of pretreatment of NIH 3T3 cells with increasing concentrations of PMA and forskolin on short-term sodium-depen- dent Pi uptake. Serum-deprived NIH 3T3 cells were pretreated with the indicated concentrations of PMA (panel A) or forskolin (panel B) for 10 min prior to determination of [32p]phosphate uptake during a 2-min time period. Values are expressed as the mean+ S.E.

of triplicate determinations.

periods indicated to stimulate adenylate cyclase and thus activate PKA, resulted in a pronounced, time-de- pendent decrease in short-term sodium-dependent phosphate uptake (Fig. 2). Incubation with 8-bromo cyclic AMP, an active analogue of cyclic AMP, and with cholera toxin to increase cyclic AMP levels, also resulted in inhibition of Pi uptake (Table I). Moreover, the effects of PMA (to activate PKC) and forskolin (to enhance PKA) on sodium-dependent Pi transport also were shown to be dose-dependent (Fig. 3). When NIH 3T3 cells were pretreated with each agent for 10 min prior to determination of Na÷/Pi uptake, half-maximal activation of Pi transport was observed with approx. 75 nM PMA, while half-maximal inhibition of Pi transport was noted with approx. 50/~M forskolin.

Studies were carried out to investigate which kinetic parameters of the sodium-dependent Pi transport sys-

336

tem were affected by the short-term activation of PKC and PKA (Fig. 4). Three separate experiments were carried out in duplicate to determine changes in the kinetic parameters of Na+/Pi uptake in the presence and absence of PMA and forskolin. The K m and Vma x

values are given as the mean + S.E. of the values of the kinetic parameters calculated from the results of the three separate duplicate experiments (n = 3). Preincu- bation of confluent populations of NIH 3T3 fibroblasts with 1 / zM PMA resulted in an increase in the affinity for Pi (Km = 0.15 ___ 0.02 mM, n = 3) of the Na+/Pi transporter relative to that noted in untreated cells (K m = 0.22 + 0.02 mM, n = 3). Conversely, preincuba- tion of these cells with 50 /zM forskolin for 10 min decreased the affinity for Pi (Kin = 0.29 + 0.01 mM, n = 3). Exposure of the cells for 10 min to either PMA or forskolin did not alter the Vma x (250 + 2.4 pmol Pi/min per mg protein, n = 3) of short-term (2 min) sodium-dependent Pi uptake (Fig. 4). To our knowl- edge, this is the first study to indicate a change in K m of Na-dependent Pi transport with the early (minutes) stimulatory effect of PMA activation of PKC.

The effect of long-term (hours) pretreatment of NIH 3T3 ceils with PMA on sodium-dependent Pi uptake is presented in Fig. 5. Prolonged treatment with PMA resulted in a progressive increase in Na+/Pi transport to give a 5-fold stimulation after 24 h of exposure. Of interest is the finding that the early

0.025

o control )

& PMA A ~ " oo=o . - - n

o.o15 ~ ~ ~ i > ,9 °,, ~,

E E E I-- v ~ v

0.010

0.005

(250 pmol PI/mln/mg protein)

0 .000 . , . , • , , , • ........ 0 5 1 0 1 5 2

1 / [ P i ]

Fig. 4. Effect of PMA and forskolin pretreatment of NIH 3T3 cells on the kinetic parameters (Km and Vma x) of sodium-dependent phosphate transport. Serum-starved NIH 3T3 cells were either un- treated (control) (o), or treated with 1 #M PMA (zx) or 50 p.M forskolin (e) for 10 min prior to initiation of [32plphosphate trans- port with phosphate concentrations between 0.05 and 2 mM, as indicated. Lineweaver-Burk linearization of the experimental data are shown, Lines were fitted to the data points by least-squares analysis with a Cricket Graph program run on a Macintosh Ilcx personal computer. Similar results were obtained in three indepen- dent experiments performed in duplicate. Symbols represent the mean + S.E. of the six determinations within the three experiments

performed in duplicate (n = 6).

O PMA

5000 ' • PMA+CHX

A MPMA

UJ '~ -t CHX ~ 4000 ' II PMA . . . . . itine

I-- 1:3- [] a m ~ 0. '-~ O1 UJ E 3000"

<:.E ==E

2°°0 R

E lOOO •

o = = 0 5 1 '0 1 5 2 0 2 5

Time, hours

Fig. 5. Time-course of the effect of long-term (hours) pretreatment of NIH 3T3 cells on phosphate uptake. Serum-deprived NIH 3T3 cells were pretreated with 1 p.M PMA (o), 1 #M PMA+5 /zg/ml cycloheximide (e), 1 /zM MPMA (A), 1 /.~M PMA +5 /zg/ml a-amanitine (11), and 5 /zg/ml a-amanitine (D) or 5 /zg/ml cyclo- heximide (+) alone for the indicated time periods prior to initiation of 32p uptake (30 min incubation). Each point represents the mean _+

S.E. of triplicate determinations.

increase in sodium-dependent Pi transport noted at 30 and 60 min of exposure to PMA were not blocked by the presence of a-amanitine (an inhibitor of transcrip- tion) and cycloheximide (an inhibitor of translation). The presence of these inhibitors did exhibit a partial (30%) inhibition of the progressive increase in Na÷/Pi uptake observed with 4 h of PMA treatment. The presence of either a-amanitine or cycloheximide com- pletely inhibited the ability of PMA to further enhance Na+/Pi uptake at time intervals above 4 h. The long- term (through 24 h) exposure of NIH 3T3 cells to the less active phorbol ester analog MPMA, or to either a-amanitine or cyloheximide alone, had no effect on sodium-dependent Pi uptake (Fig. 5). These results suggest that short-term (through 60 min) exposure of cells to PMA appears to enhance sodium phosphate cotransport by direct activation of PKC to induce the phosphorylation of transporter protein(s). The progres- sive increase in sodium-dependent Pi uptake noted with prolonged (hours) exposure to PMA appears to require de-novo protein synthesis and insertion of new transporters in the membrane.

Discussion

The regulation of sodium-dependent Pi uptake has been studied extensively in renal cell systems (see Ref. 6). More recently, sodium-dependent P~ uptake has been demonstrated in other cell types as well (see Ref. 11). These studies have implicated membrane signal transduction systems and protein kinase activities in the regulation of Pi transport. Here we have carried

out studies to characterize the effects and counterac- tions on Pi uptake in NIH 3T3 cells of two protein kinase systems known to play a key role in cellular regulation: PKC and PKA. Treatment of NIH 3T3 cells with the phorbol ester tumor promoter PMA, with bryostatin, or with the diacyglycerol analogue OAG to activate PKC caused the rapid (within 2-5 min) stimu- lation of short-term sodium-dependent Pi uptake. Con- versely, exposure of NIH 3T3 cells to forskolin or cholera toxin to elevate cyclic AMP levels, or with the cyclic AMP analogue 8-bromo cyclic AMP, to activate PKA resulted in the rapid inhibition of sodium-depen- dent P~ transport. These are the first studies to demon- strate that sodium-dependent phosphate uptake in NIH 3T3 cells is rapidly (within minutes) stimulated by activation of PKC and inhibited by activation of PKA.

As mentioned previously, studies by other investiga- tors clearly indicate that sodium-coupled phosphate transport systems present in different cell types exhibit different patterns of response to activation of PKA and PKC. One cell model which shows similarities with that reported here for NIH 3T3 ceils (stimulation by PKC and inhibition by PKA) is the HeLa cell system [11,16]. However, even between the NIH 3T3 and HeLa cell models there is an apparent difference in the regula- tion of Pi transport by PKC and PKA. The early (within minutes) stimulation and inhibition of sodium- dependent Pi uptake in NIH 3T3 cells mediated by the activation of PKC and PKA, respectively, was found to be a consequence of changes in the K m for phosphate, with no apparent change in Vma ~. In contrast, activation of PKC in HeLa cells was observed to increase the Vma x for phosphate uptake, while the K m for phosphate remained constant. In studies with MDCK cells, Es- coubet et al. [18] observed that PMA treatment of these cells had a biphasic effect on sodium-dependent Pi uptake (i.e., inhibition at early (1 h) followed by stimulation at late (15 h) time periods). The early inhibitory effect of PMA activation of PKC on Pi transport in MDCK cells was a result of a decrease in Vma x, but no change in affinity (K m) for phosphate. The late (15 h) stimulatory effect of PMA in MDCK cells appeared to be through an increase in the affinity for P~, without a significant change in Vma x.

The rapid (within minutes) effects of activation of PKC and PKA on sodium-dependent P~ uptake in NIH 3T3 cells suggests that these protein kinases might act by directly phosphorylating the transporter. Alterna- tively, one or both of the kinases might act through modification of another membrane component(s) which then serves to influence phosphate uptake. However, in addition to these short-term effects, prolonged incu- bation of cells with PMA also can result in significant changes in Pi transport. As noted by Escoubet et al. [18] prolonged (15 h) treatment of MDCK cells with PMA resulted in significant stimulation of Pi uptake.

337

This long-term effect of PMA was dependent on pro- tein synthesis. Prolonged (overnight) treatment of HeLa cells with PMA to first activate and then down-regulate PKC, however, was observed to have little effect on the basal level of phosphate uptake [16]. In contrast, we have noted that treatment of NIH 3T3 ceils for 24 h with 1 p.M PMA induced a significant (more than 5-fold) increase in basal phosphate transport over un- treated control cells (Fig. 5). This long-term effect of PMA to increase sodium-dependent Pi transport in NIH 3T3 cells was partially (30%) decreased by both a-amanitine (to inhibit transcription) and cyclohex- imide (to inhibit translation), following a 4-h treatment with PMA, while the presence of these inhibitors com- pletely blocked the ability of PMA to further enhance Na÷/Pi transport at exposure times to PMA longer than 4 h. Thus, while both short-term and long-term exposure of NIH 3T3 cells to PMA can result in significant stimulation of [32p]orthophosphate uptake, the mechanisms involved may differ. The short-term (minutes) response to stimulate Na+/Pi uptake ap- pears to involve the direct activation of PKC by PMA. In contrast, since prolonged (hours) treatment of NIH 3T3 cells with 1 /~M PMA results in the progressive down-regulation of PKC, the increase in sodium-de- pendent P~ transport noted at time periods beyond 4 h of PMA treatment does not appear to involve a direct action of PKC. Rather, the long-term (hours) increase in the Na+/Pi uptake noted with prolonged exposure to PMA appears to involve an induced increase in the de-novo synthesis of some component(s) of the Na÷/Pi transport system, as indicated by the inhibition of this response in the presence of transcription and protein synthesis inhibitors.

It is apparent that the regulatory effects of PKC and PKA on phosphate uptake differ significantly in differ- ent cell types, although the reasons for these diverse effects remain unclear. One possibility is that different isotypes (or modified forms) of sodium-phosphate transporters exist in the different cell types. Another reason might be the presence of different PKC isotypes [20] in the various cells which could differentially medi- ate the PMA signal. Or, differences in membrane composition between the cell types might influence the regulatory properties of the sodium-dependent phos- phate symporter.

In conclusion, short-term sodium-dependent Pi up- take in NIH 3T3 cells is stimulated by activation of PKC and inhibited by activation of PKA. The rapid (within minutes) alterations in Pi uptake appear to be due to changes in the K m for Pi, and not to changes in Vma x. Thus, stimulation of PKC and PKA through hormone-induced production of their second messen- ger activators (diacylglycerol and cyclic AMP, respec- tively) probably serve as a short-term regulators of the intracellular level of Pi. Other studies have suggested

338

r e g u l a t o r y ' c ross - t a lk ' b e t w e e n the P K C a n d P K A sig- na l l i ng pa thways to d i f fe ren t i a l ly r e g u l a t e ce l lu la r sys- t e m s [21,22]. T h e p r e s e n t resu l t s i nd ica t e tha t the r ap id

c h a n g e s in s o d i u m - d e p e n d e n t Pi u p t a k e in N I H 3T3 cells in r e s p o n s e to ac t iva t ion of P K C a n d P K A pre- sen ts a n exce l l en t sys tem to f u r t h e r s tudy the an t - agonis t i c effects o f t hese two s ignal t r a n s d u c t i o n pa th - ways.

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