Phosphoinositides in mitogenesis: Neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation

Cell, Vol. 42, 479-466,

September

1965, Copyright

0092-6674/65/090479-10

0 1965 by MIT

$02.0010

Phosphoinositides in Mitogenesis: Neomycin Inhibits Thrombin-Stimulated Phosphoinositicle Turnover and Initiation of Cell Proliferation Darrell H. Carney, David L. Scott, Eric A Gordon, and Edward F. LaBelle Department of Human Biological Chemistry and Genetics The University of Texas Medical Branch Galveston, Texas 77550

Summary Thrombin stimulates 32Pi incorporation into phosphatidyllnositol 4-phosphate (PIP), phosphatidylinositol4,5-bis-phosphate (PIP,), and phosphatidylinositol (PI), and initiates DNA synthesis in hamster (NIL) fibroblasts at a half-maximal concentration of 125 rig/ml. Neomycin, which bind8 PIP, and PIP, inhibits both thrombin-stimulated initiation of cell proliferation and 32P incorporation into Pi at concentrations above 2 mM without affecting thrombin binding, thymidine uptake, or cellular protein synthesis. At lower concentrations, neomycin inhibits thrombin-stimulated release of inositol 1,4,5-trisphosphate (IP,), by selectively binding PIP,, but does not inhibit 32P incorporation into PI or initiation of DNA synthesis. Phosphoinositide recycling and diacylglyceroi release therefore appear necessary for initiation of cell proliferation by thrombin. IP,-stimulated Ca++ mobilization may not be required for thrombin mitogenesis, however, since neomycin can block IP3 release without inhibiting initiation. Introduction Highly purified human thrombin initiates cell proliferation by itself (Chen and Buchanan, 1975; Carney et al., 1978; Pohjanpelto, 1977; 1978; Zetter et al., 1977; PerezRodriguez et al., 1981) or in combination with epidermal growth factor (Cherington and Pardee, 1980; Gospodarowitz et al., 1978) or platelet-derived growth factor (Zetter and Antoniades, 1979). Thus, thrombin produced at the site of a wound may be an important growth regulator in wound healing and tissue repair. Several studies indicate that initiation of cell proliferation by thrombin involves interaction with high-affinity cell surface thrombin receptors (Carney and Cunningham, 1978a; 1978b; 1978c; Perdue et al., 1981; Van Obberghen-Schilling et al., 1982; Carney et al., 1984). These receptors are clustered on the surface of cells prior to thrombin binding, but do not associate with coated pits or participate in rapid receptor-mediated internalization of thrombin (Carney and Bergmann, 1982; Bergmann and Carney, 1982; Carney, 1983). It thus appears that thrombin initiates cell proliferation by a true transmembrane signal. Thrombin, like many other growth factors, stimulates amiloride-sensitive Na+/H+ exchange and activates the Na+, K+ ATPase (Pouyssegur et al., 1982; Stiernberg et al., 1984). Recent studies indicate that phorbol esters also activate Na+/H+ exchange (Besterman and Cuatrecasas, 1984). Moreover, stimulated phosphoinositide metabolism

has been implicated in initiation of cell proliferation by serum and a number of other growth factors. (For review see Michell, 1982; Marx, 1984; Nishizuka, 1984). Thus, it seemed possible that thrombin activates Na+IH+ exchange by stimulating phospholipase C cleavage of phosphoinositides to yield diacylglycerol. We have now shown that highly purified human a-thrombin stimulates 32P incorporation into PI and turnover of phosphoinositides in hamster (NIL) fibroblasts. The concentration dependence of thrombin stimulation correlates with initiation of cell proliferation. Moreover, inhibition of thrombin-stimulated phosphoinositide turnover by neomycin blocks thrombin initiation of DNA synthesis and cell proliferation. These studies, therefore, indicate that phosphoinositide turnover is a necessary event in the initiation of cell proliferation. Results Thrombin Stimulates Phosphorylation and Cycling of Phosphatidylinositides To determine whether thrombin stimulates phosphorylation of the phosphoinositides, serum-free quiescent cultures of hamster NIL cells were incubated for 30 min with 32Pi and then with either PBS or highly purified human a-thrombin (2 j@ml). As shown in Figure 1, thrombin stimulated 32Pi incorporation from two to eightfold into lipids that migrated on oxalate impregnated (Figure 1A) or acidic silicia gel (Figure 1B) thin layer chromatography plates with standard phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5bisphosphate (PIPI). In addition, if cells were prelabeled with 3H-inositol for 36 hr, 3H-labeled lipid bands were obsewed migrating with PI, PIP, and PIP, (data now shown). Thus it appears that these bands represent authentic phosphoinositides. On oxalate impregnated plates PI and PA migrate together, but in the acidic system that separates PI and PA, most of the radioactivity and virtually all of the thrombin-stimulated 32P incorporation appeared in PI rather than PA (Figure 1B). Thrombin also stimulated 32Pi incorporation into bands migrating with PC and to a lesser extent PE (Figures 1A and 1B). This stimulation into PC and PE was observed after 2 hr of thrombin treatment (Figure l), but not at early times. After 15 or 30 min it appeared that thrombin only increased cellular 32Pi incorporation into PI, PIP and PIP, (data not shown). In contrast to the two to eightfold stimulation of 32P incorporated into these specific lipids, 32P in aqueous extracts increased less than 20%. Thus, this stimulation does not simply reflect increased 32Pi uptake. In addition, a similar pattern of thrombin-stimulated j2Pi incorporation was observed in primary cultures of mouse embryo fibroblasts (data not presented). Thus, this stimulation is not restricted to either hamster cells or established strains of fibroblastic cells. To interpret the lipid labeling pattern, it is important to visualize the pathways for cycling and synthesis of these lipids (for review see Berridge, 1984). As shown in Figure

Cell 480

B

A

PE PA PIP

-

PIP*

-

PI -

PC *

PIP/PIP,

Origin PBS Figure Lipids

Thr

1. Effect of Thrombin

PBS

Thr

on the Incorporation

of szP into Fibroblast

Nonproliferating serum-free cultures of NIL fibroblasts were prepared in 80 mm dishes as described in Experimental Procedures. 3rP (15 &i/ml) was added to the cells for 30 min followed by addition of either PBS (20 PI) or thrombin (2 rg/ml) and the cells were incubated for 2 hr at 37%. The $,Pi was washed off the cells and the cells were scraped out of the dishes in 1 ml of PBS buffer. (A) The lipids were extracted from the cells and separated on oxalate impregnated thin-layer plates using chloroform-methanol-ammonia and autoradiography was performed as described in Experimental Procedures. An autoradiogram of the thin-layer plate is shown above together with the positions of standard lipids. (B) A parallel experiment similar to A, except that the lipid extracts were separated on thin-layer plates using a chloroformmethanol-acetic acid-water solvent mixture as described in Experimental Procedures.

2, there are phosphorylation-dephosphorylation cycles among PI, PIP and PIP,. These represent specific addition or removal of phosphates from the 4 and 5 position of inositol. In contrast, PI is only phosphorylated at the 1 position of inositol between inositol and diacylglycerol. Thus, the only way to stimulate 3zP incorporation into PI is to increase the phosphorylation of diacylglycerol to PA and then resynthesize PI from CDP-diacylglycerol and inositol. Our long-term 3H-inositol labeling experiments indicate that in NIL cells, as in other membranes, there is a very small amount of PIP and PIP, relative to the large PI pool (data not shown). Thus, we would expect newly synthesized 32P-labeled PI to collect in this pool and not contribute significantly to the increased 32P incorporation into PIP and PIP2. Since we initially observe an almost equivalent stimulation of incorporation into PIP,, PIP, and PI, thrombin appears to be stimulating phospholipase C cleavage of the phosphoinositides to diacylglycerol and inositol phosphates, and the cycling of diacylglycerol back to PI. We cannot, however, rule out the possibility that thrombin also stimulates the phosphoinositide kinases. To assure that the thrombin-stimulated phosphorylations represented phosphoinositide turnover, we prelabeled phosphoinositides in NIL cells with 3H-inositol for 36 hr prior to thrombin addition, and measured release of soluble inositol phosphates. As shown in Table 1, low mitogenie concentrations of thrombin (250 nglml) stimulated release of IP:, approximately fivefold when measured 30

set after thrombin addition. At this early time, there appeared to be less stimulation of IP and IP, release, indicating that thrombin initially activates phospholipase C cleavage of PIP, to produce diacylglycerol and IP3. Since these measurements are independent of 32P or free 3H-inositol pools they confirm that the increased 32P incorporation described above represents phosphoinositide turnover and not simply changes in the cellular 32Pi pools. Correlation Between Phosphoinositide Turnover and Mltogenesis To determine whether there was a correlation between thrombin-stimulated phosphorylation of phosphoinositides and initiation of DNA synthesis, we incubated quiescent serum-free cultures of NIL cells with various concentrations of thrombin. After 2 hr we measured 32Pi incorporation into phosphoinositides, and after 24 hr, 3Hthymidine incorporation into acid precipitable material. As shown in Figure 3, there is a dose-dependent increase in the amount of 32Pi incorporated into PI, PIP, and PIP,. The amount of 32P found in PI was increased approximately fourfold by thrombin and the amount of 32P found in PIP or PIP, was doubled (Figure 3A). It should be noted that half-maximal stimulation of incorporation into the phosphoinositides and half-maximal stimulation of thymidine incorporation both occurred at a thrombin concentration of approximately 125 nglml (compare Figures 3A and 38). Previous studies from our laboratory have shown similar dose-response curves for thrombin stimulation of cell proliferation and percent labeled nuclei (Carney et al., 1978; Crossin and Carney, 1981). Thus, there appears to be a close correlation between the ability of thrombin to stimulate phosphoinositide turnover and its ability to initiate DNA synthesis and cell proliferation. Is Phosphoinositide Cycling Necessary for Thrombin Mitogenesls? A stronger case could be made for a causal role of phosphoinositide turnover in mitogenesis if one could inhibit phosphoinositide phosphorylation or turnover and show an effect on initiation of proliferative events. In both cellfree and intact cell systems neomycin appears to selectively bind to PIP and PIP,, thus inhibiting their metabolism (Orsulakova et al., 1976; Schacht, 1976; Schibeci and Schacht, 1977; Stockhorst and Schacht, 1977; Lang et al., 1977; Lodhi et al., 1979; Downesand Michell, 1981). Therefore, we examined the effects of this drug on initiation of DNA synthesis and cell proliferation by thrombin. As shown in Figure 4, neomycin blocks thrombin stimulation of 3H-thymidine incorporation without inhibiting incorporation in control cultures. Moreover, when neomycin was added to cells 18 hr after thrombin’addition there was no inhibition of thymidine incorporation even at the highest concentrations of neomycin. This indicated that neomycin was not merely inhibiting thymidine transport or directly blocking DNA synthesis. Additional experiments showed that neomycin (5 mM) addition 2 to 4 hr after thrombin addition is almost as effective in inhibiting initiation of DNA synthesis as addition prior to thrombin, but neomycin is relatively ineffective if added 8 to 12 hr later

Phosphatidylinositides 481

in Thrombin

Mitogenesis

Figure 2. Model of Phosphoinositide lism and Recycling

As shown, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP,) are interconverted by phosphokinases and phosphomonoesterases. In addition, these lipids can be cleaved by phospholipase C to produce diacylglycerol (DG) and inositol phosphates (IP IP,, and lP3. Resynthesis of and phosphorylation of PI occurs through phosphorylation of DG to phosphatidic acid (PA) and the combination of cytidine diphosphate-diacylglycerol (CDP-DG) with inositol (for review see Berridge, 1984).

DG I

+

lnositol

Table 1. Effect of Thrombin

l

q

on Release

of Soluble lnositol

fH-lnositol

966 f 262 f 120 f

(CPM/IO7

IPS

Phosphates

cells)

16.0

Thrombin (250 nglml)

Control Inositol-l-phosphate Inositol-I ,Sbisphosphate Inositol-1.4,5-trisphosphate

IP2 /

,p w

Metabo-

100 140 69

1436 422 592

f 264 k 37 * 144

Quiescent nonproliferating cultures of NIL ceils were prepared in 60 mm dishes and incubated for 36 hr in serum-free DV/F-12 medium containing 2 &i/ml SH-inositol. Cells were then incubated 30 set with or without thrombin (250 @ml), extracted with O°C TCA, and the inositol phosphates separated on Dowex columns as described in Experimental Procedures. Data represent the mean f one standard deviation from three separate experiments.

(E. A Gordon and D. H. Carney, unpublished). Thus, it appears that neomycin blocks thrombin-stimulated DNA synthesis by inhibiting an early initiation event. To ascertain that the inhibition of thymidine incorporation by neomycin represented a true inhibition of mitogenesis in these cells, we also looked at the effect of neomycin on thrombin-stimulated increases in percent labeled nuclei and cell number. As shown in Figure 5A, neomycin treatment caused a decrease in percent labeled nuclei from thrombin-stimulated levels of 49% to unstimulated control levels of approximately 10% with half-maximal inhibition between 2 mM and 5 mM neomycin. Neomycin also inhibited thrombin-stimulated increase in cell number with half-maximal inhibition again occuring between 2 mM and 5 mM neomycin (Figure 5B). Additional controls showed that neomycin did not affect binding of ‘*%thrombin to high-affinity cell surface receptors. It also did not inhibit uptake or incorporation of 3H amino acids into cellular protein at neomycin concentrations up to 15 mM, where thrombin stimulation of DNA synthesis was almost totally inhibited. In contrast other antibiotics, including streptomycin, which reportedly do not bind to PIP or PIP, (Schacht, 1976), inhibited DNA synthesis only at concentrations that inhibited cellular protein synthesis (data not shown). The effects of neomycin on thrombin-stimulated initiation of DNA synthesis may therefore be directly related to its inhibition of PIP and PIP, metabolism. If neomycin inhibition of thrombin-initiated cell proliferation is mediated through its effects on polyphosphoinosi-

8.0

0 8.0 6.0

2.0 I ‘98

I

I 31

Thrombin Figure 3. Effect of a-thrombin pholipids and the Incorporation

I

1 I I I I 125 500 2000 (rig/ml)

on the Incorporation of s2P into Phosof ‘H-thymidine into DNA

(A) Nonproliferating serum-free cultures of NIL cells were treated with ‘*P for 2 hr with the indicated a-thrombin concentrations as described in the legend of Figure 1A. The srPi was rinsed off the cells, the cells were scraped out of the dishes, and the lipids were extracted and separated by alkaline thin layer chromatography on oxalate impregnated silica gel as described in the legend of Figure 1A. The labeled lipids were scraped off the plates and the amount of 52P incorporated into PI (solid circles), PIP (open triangles), and PIP, (open squares) was determined as described. (B) Parallel cultures of serum-free NIL cells were treated with the indicated concentrations of a-thrombin for 24 hr and the initiation of DNA synthesis determined by measuring the incorporation of SH-thymidine into acid-precipitable material as described in Experimental Procedures.

tides, these effects should occur at the same concentrations. As shown in Figure 6A, approximately two-thirds of the thrombin-stimulated 32P incorporation into PI is inhibited by neomycin at concentrations above 2.5 mM. As described below, at these concentrations neomycin binds

Cell 402

c

10.0

.-0 t-i 857 a1

8.0

uxEZ

6.0

gz ‘g u_ z

4.0

“Lof’/O.l

I 0.3 Neomycin

# 1.0

3.0

1 I 10.0

(mM)

Figure 4. Effect of Neomycin on Thrombin-Stimulated Initiation of DNA Synthesis Nonproliferating serum-free cultures of NIL cells were incubated for 24 hr in the presence of the indicated amounts of neomycin either with (solid circles) or without (open triangles) o-thrombin (250 nglml), and the incorporation of 3H-thymidine into acid-precipitable material determined as described in Experimental Procedures. Addition of neomycin 18 hr after thrombin addition (6 hr before measuring thymidine incorporation) appeared to have no effect (open squares). Error bars represent f one standard deviation from the mean of triplicate determinations. Neomycin treatment at the highest concentration (10 mM) does not decrease cell number or cell attachment to dishes during this period.

PIP and PIP2, inhibiting their cleavage by phospholipase C. Interestingly, the concentrations of neomycin required to half-maximally inhibit thrombin-stimulated phosphorylation of PI (Figure 6A) and initiation of DNA synthesis (Figure 4 and Figure 6C) were virtually identical (approximately 2 mM). Thus, there appears to be a direct correlation between the effects of neomycin on polyphosphoinositide turnover and initiation of DNA synthesis. Together these results indicate that increased phosphoinositide turnover generates a necessary signal for initiation of cell proliferation. Concentration Dependent Effects of Neomycin on Phospholnositide Metabolism and Potential Mltogenlc Signals Low concentrations of neomycin have been reported to bind PIP, preferentially to PIP (Lang et al., 1977; Lodhi et al., 1979). Such selective binding of PIP, should inhibit phospholipase C cleavage of PIP, and release of IPs, but not affect diacylglycerol released from cleavage of PI or PIP (see Figure 8). Thus it should be possible to use low concentrations of neomycin to evaluate the requirement for various parts of the phosphoinositide cycle. As shown in Figure 6B, neomycin at 0.6 mM caused an apparent accumulation of phosphorylated PIP, in thrombin-stimulated cells. At 1.25 mM neomycin there is also an apparent accumulation of PIP, indicating that at this concentration neomycin binds both PIP and PIP2. Only at these higher concentrations (above 1.25 mM) does neomycin inhibit the stimulation of diacylglycerol recycling to PI (see Figure 6A). Therefore, at low concentrations of neomycin (0.6

= 1.0 3 0.6

I 1.25 Neomycin

I 2.5

5

10

(mM)

Figure 5. Effect of Neomycin on Thrombin-Stimulated Initiation of DNA Synthesis and Cell Proliferation Quiescent populations of NIL cells were prepared as described and then incubated with the indicated concentrations of neomycin in the presence (solid circles) or absence (open circles) of 250 rig/ml thrombin. To measure labeled nuclei (A), cultures were incubated 24 hr with ‘H-thymidine (1 &i/ml) and then prepared for autoradiography as described in Experimental Procedures. Cell number was determined in parallel plates 40 hr after thrombin addition (B).

mM) there appears to be enough diacylglycerol released from PIP or PI to allow maximal recycling to PI. At this low concentration there is little, if any, inhibition of thrombinstimulated initiation of DNA synthesis or cell proliferation (Figures 4, 5, and 6C). Diacylglycerol released from PI or PIP may therefore be able to complete the initiation signal and inhibition is only observed when this lipid cycling is inhibited. If indeed, low neomycin concentrations bind PIP*, this effect should also be directly measurable by determining its effects on IPs release. As shown in Figure 7, neomycin concentrations of 0.6 mM and above almost totally inhibit thrombin stimulation of IP, release. Additional experiments have shown that these concentrations of neomycin also prevent thrombin-stimulated mobilization of Ca+* from intracellular stores as determined by Quin 2 fluorescence measurements (D. Carney, L. Muldoon, Ft. Dinnerstein, and M. Villareal, unpublished). Thus, 0.6 mM neomycin binds PIP* and blocks both IPs release and Ca++ mobilization without affecting diacylglycerol release and its cycling back to PI (see Figure 8). As discussed above, this low neomycin concentration had little, if any, effect on thrombin-stimulated DNA synthesis. Together these studies suggest that polyphosphoinositide cycling is necessary for initiation of cell proliferation and that production of diacylglycerol or lipid cycling may be more important than either IP1 release or Ca++ mobilization in generating proliferative signals.

Phosphatidylinositides 483

in Thrombin

Mitogenesis

Discussion Increased phosphorylation and turnover of phosphoinositides and other lipids occurs in many systems in response to various hormones and growth factors. We have shown that addition of highly purified human thrombin to hamster (NIL) fibroblasts in serum-free medium stimulated phosphoinositide turnover, phosphorylation of the polyphosphoinositides PIP and PIP2, and release of soluble IP,. The concentration of thrombin required for halfmaximal stimulation of these phosphorylations correlates with the concentration required to initiate DNA synthesis and cell proliferation in these cells. More importantly, we demonstrate for the first time that neomycin, a drug that binds PIP, and PIP and thus inhibits polyphosphoinositide metabolism, blocks thrombin initiation of DNA synthesis and cell proliferation. These neomycin studies, therefore, provide evidence that phosphoinositide cleavage and cycling is necessary as an early event in initiation of cell proliferation.

0 8.0 6.0 4.0 2.0

04

’

0 ‘0.6

I

1

I

1.2

2.5

5.0

Neomycin

I 10.0

I 20.0

(mM)

Figure 6. Effect of Neomycin on Thrombin-Stimulated Phosphatidylinositide Phosphorylation and Initiation of DNA Synthesis Nonproliferating cultures of NIL cells were prepared and incubated for 2 hr as described in Figure 1A with or without thrombin in the presence of the indicated amounts of neomycin. Incorporation of 32Pi into PI (A, solid circles), PIP (6, open triangles), and PIP? (B, open squares) was determined and expressed as thrombin-stimulated CPM by subtracting PBS control values at each neomycin concentration. The incorporation of ‘H-thymidine into DNA of parallel thrombin-stimulated cells was also measured as a function of increasing neomycin concentrations as described in Experimental Procedures (C).

0

1 0

0.3

0.6

1.25

Neomycin Figure 7. Effect of Neomycin lnositol Trisphosphate

2.5

5.0

10.

(mM)

on Thrombin-Stimulated

Release

of ‘H-

Nonproliferating cultures of NIL cells were incubated for 36 hr with SHmyoinositol (2 $Xml). pretreated for 30 min with indicated concentrations of neomycin, then with thrombin (250 nglml) or PBS for 30 set, and the soluble inositol phosphates were separated on Dowex columns as described in Table 1 and Experimental Procedures. Each point represents the mean of duplicate or triplicate determinations from three separate experiments. Control levels of IP, were as reported in Table 1 and were unaffected by neomycin.

Stimulation of Phosphoirrositide Turnover by Thrombin and Other Growth Factors In both fibroblasts and platelets, it appears that thrombin action stimulates phospholipase C cleavage of PIP, to release diacylglycerol and IP3 (Rittenhouse-Simmons, 1979; Lapetina et al., 1981; Billah and Lapetina, 1982a; 1982b; Prescott and Majerus, 1983; Agranoff et al., 1983). In platelets, much of the diacylglycerol is cleaved by diacylglycerol lipase to release arachidonate (RittenhouseSimmons, 1980; Prescott and Majerus, 1983; Neufeld and Majerus, 1983). From our studies, it appears that fibroblasts may cycle diacylglycerol through PA and CDPdiacylglycerol to regenerate PI (see Figure 2). Thus the response of fibroblasts to thrombin may be slightly different from that of platelets. Phosphoinositide metabolism is affected by a number of agents involved in regulating cell.proliferation. For example, early studies showed that transformation correlated with increased phosphoinositide turnover (Koch and Diringer, 1973; Diringer and Friis, 1977). More recent studies have shown that Rous sarcoma virus protein pp60v-src and avian Sarcoma virus protein ~68”~~ stimulate phosphorylation of phosphoinositides (Sugimoto et al., 1984; Macara et al., 1984). In addition, tumor promoters act like diacylglycerol, which is released by phosphoinositide turnover, in activation of protein kinase C (Castagna et al., 1982; Niedel et al., 1983). In contact inhibited cells, addition of serum, partially purified serum fractions, or prostaglandins increases PI phosphorylation and the turnover of prelabeled PI (Pasternak, 1972; Ristow et al., 1875; Macphee et al., 1984). It is unclear, however, whether these agents stimulate all or only part of the phosphoinositide cycle. Epidermal growth factor stimulates Ca++ dependent J2Pi incorporation into PI and PA in A-431 cells (Sawyer and Cohen, 1981), but not in 3T3 cells where it stimulates cell proliferation (Macphee et al., 1984). Since polyphosphoinositide turnover is generally Ca++ independent, these results suggest that there may be more than one mechanism for activating parts of this cycle and that

Cell 404

Figure 8. Model of Thrombin-Stimulated Phosphoinositide Metabolism: Effects of Neomycin As indicated, thrombin stimulates phosphorylation of the polyphosphoinositides, the cleavage of the phosphoinositides to diacylglycerol and inositol phosphates, and the recycling of diacylglycerol back to PI (See Figure 2 for abbreviations). Neomycin (Neo) blocks phosphoinositide metabolism by selectively binding PIP? and PIP This blocks phospholipase C cleavage of PIP, and PIP as well as the indicated kinase and esterase activities. Low neomycin concentrations preferentially bind to PIP?, inhibiting release of IP,; however, diacylglycerol is still released from PIP or PI cleavage (see text for details). Our results indicate that neomycin only inhibited initiation of DNA synthesis at concentrations that inhibited diacylglycerol release and recycling to PI. Thus, lipid recycling or diacylglycerol activation of protein kinase C may be necessary to initiate cell proliferation, whereas release of IP, may not.

not all of the cycle is involved in generating a mitogenic signal. In 3T3 cells maintained in platelet-poor plasma, platelet-derived growth factor stimulates arachidonate release, prostaglandin production, PI turnover, and an increase in release of diacylglycerol and inositol phosphates (Shier, 1980; Habenicht et al., 1981; Shier and Durkin, 1982; Berridge et al., 1984). It is possible, however, that prostaglandin production or the effects of other plasma factors play a role in these changes. Inhibition of either phospholipase A2 activity, which would block arachidonate release and prostaglandin production (Shier and Durkin, 1982) or synthesis of PI (Ristow et al., 1980) appears to inhibit mitogenesis. Therefore, several changes in phospholipid metabolism generated by combinations of these factors may be necessary to complete a growth signal. It appears that none of these signals alone is sufficient to initiate cell proliferation. For example, exogenous diacylglycerol addition to 3T3 cells by itself does not initiate, but acts synergistically if added with insulin and other growth factors (Rozengurt et al., 1984). Since thrombin initiates proliferation in serum-free medium it is possible that more than one type of signal is generated by thrombin interaction with these cells. Therefore, it is important to determine which, if any, of these phosphoinositide related events are necessary and/or sufficient for thrombin to initiate cell proliferation. Two of these potential signals, IP3 and diacylglycerol, have been considered in some detail. A number of growth factors stimulate phospholipase C dependent release of IPJ (Berridge et al., 1984; Vincentini and Villereal, 1984), which stimulates mobilization of intracellular Ca++ (Streb et al., 1983; Joseph et al., 1984; Dawson and Irvine, 1984; Burgess et al., 1984; and Prentki et al., 1984; Mix et al., 1984; Moolenaar et al., 1984). Yet this is avery short lived signal. Thrombin stimulation of IPJ release is maximal at 30 set and is mostly attenuated by 2 to 3 min. In other studies using Quin 2 fluorescence, we recently found that thrombin also causes Ca++ mobilization in these cells be-

ginning 30 set to 1 min after thrombin addition and lasting approximately 3 min (D. Carney, L. Muldoon, Ft. Dinnerstein, and M. Villereal, unpublished). Therefore, it appears that thrombin stimulation of IP, release causes Ca++ mobilization as it does in other systems. The short duration of this signal may mean that IP, stimulated Ca++ mobilization itself activates Ca++ dependent forms of phospholipase C to favor cleavage of PIP or PI, thus regulating further IP, and Ca++ release (Majerus et al., 1984). In this way, lipid cycling and release of diacylglycerol continues in a “short-cycle” without IP, release or Ca++ mobilization. If phosphoinositide turnover is necessary for initiation, mitogenesis may require continued high levels of diacylglycerol released from cleavage of PIP,, PIP, or PI to activate protein kinase C (Takai et al., 1979; Kishimoto et al., 1980; Sando and Young, 1983). To evaluate the potential importance of these signals in thrombin mitogenesis we have used neomycin to selectively block IP, release. Selective Effects of Neomycin on PI Turnover and Thrombin Mitogenesis Neomycin inhibits phosphoinositide turnover in a number of tissues by directly binding to PIPI and PIP (Orsulakova et al., 1978; Schacht, 1978; Schibeci and Schacht, 1977; Stockhorst and Schacht, 1977; Lang et al., 1977; Lodhi et al., 1979; Downes and Michell, 1981). At low concentrations, neomycin selectively binds PIP* over PIP (Lodhi et al., 1979; Lang et al., 1977). Under these conditions phospholipase C may still cleave PI or PIP to release diacylglycerol, but will not cleave PIP2 to release IP, (see Figure 8). In our experiments low concentrations of neomycin (0.8 mM) caused an accumulation of 32P labeled PIP? and blocked release of 3H-IP,, but did not inhibit 52Pi incorporation into PI. Thus, release of diacylglycerol from PIP or PI is sufficient to allow maximal diacylglycerol recycling back to PI. In contrast, at higher concentrations where neomycin binds both PIP* and PIP approximately two-thirds of the 32Pi incorporation into PI is inhibited. Thus, with this

Phosphatidylinositides 485

in Thrombin

Mitogenesis

system we can begin to dissect the requirement for phosphoinositide recycling and the relative importance of diacylglycerol and IP, release in this process. Comparing the concentration of neomycin necessary to inhibit initiation of DNA synthesis by thrombin with its effect on thrombin-stimulated 32Pi incorporation into the phosphoinositides suggests a direct correlation between neomycin inhibition of polyphosphoinositide turnover and its inhibition of thrombin mitogenicity. The half-maximal inhibition for thrombin-stimulated DNA synthesis occurred at approximately 2 mM neomycin-the same concentration that half-maximally inhibited formation of 32P-labeled PI. At these concentrations, neomycin had no effect on thymidine transport, thrombin binding to its receptors, or cellular protein synthesis. In contrast, other antibiotics that do not bind polyphosphoinositides (Schacht, 1976) only inhibited thymidine incorporation at concentrations where they also inhibited protein synthesis and were toxic to control cultures. Thus, the inhibitory effects of neomycin on thrombin-stimulated initiation of DNA synthesis appear to specifically relate to neomycin inhibition of phosphoinositide cycling. Low concentrations of neomycin (0.6 mM), which inhibited cleavage of PIP, and release of IP,, did not inhibit initiation of DNA synthesis by thrombin. Additional studies have shown that this concentration of neomycin also blocks thrombin-stimulated Ca++ mobilization from intracellular stores (unpublished). Since diacylglycerol is still released from PI or PIP (as described above) these results suggest that diacylglycerol production or lipid recycling generates an initiation signal in the absence of IP, stimulated Ca++ mobilization. This suggests that at least this form of Ca++ mobilization is not necessary, perhaps because other mechanisms may also lead to increases in intracellular Ca”. In contrast, at higher neomycin concentrations where diacylglycerol production and lipid cycling were inhibited, initiation of DNA synthesis was also inhibited. Thus, stimulation of diacylglycerol production or lipid cycling to PI appears to be necessary for thrombin to initiate cell proliferation. If diacylglycerol release is necessary for thrombin mitogenesis one might predict that protein kinase C plays a central role in the action of thrombin. We have recently discovered that thrombin increases the frequency of transformed foci development in monolayers of 3T3 cells and promotes transformation of cells exposed to carcinogens (D. Morris, J. Ward, and D. Carney, unpublished). These effects of thrombin may also be generated through activation of protein kinase C. Further studies are underway to determine what other signals might be involved in these processes. Experlmental

Procedures

cell Culture NIL ceils are an established strain of Syrian hamster fibroblasts provided to us by Frank Solomon, which are maintained and grown in a 1:l mixture of Dulbecco-Vogt modified Eagles medium and Ham’s F-12 medium (DV/FiP. Grand Island Biological Co., Grand Island, New York) supplemented with 10% calf serum (Irvine Scientific, Irvine,

California), penicillin (100 U/ml), and streptomycin (100 &ml). Nonproliferating quiescent cultures were prepared as follows. NIL cells were subcultured using an EDTAtrypsin solution (phosphate-buffered saline containing 0.02% EDTA and 0.05% trypsin) into 60 mm culture dishes or 24 well plates (Falcon Plastics, Becton-Dickinson, Oxnard, California) at a density of 6.5 x l(r cells per cm*. After attachment of the cell monolayer, serum-containing medium was removed and replaced with serum-free DVlFlP medium. The cells were then incubated at 3pC for 10 to 15 min, and the medium was changed to fresh serum-free DV/FlZ medium. To further eliminate the influence of any remaining serum factors and bring the cells to a quiescent state, these cultures were incubated in serum-free medium for 48 hr prior to use in lipid labeling or growth initiation experiments. As described previously, these cultures are arrested in a G,IG, state and are mitogenically responsive to thrombin or epidermal growth factor without additional growth factors (Crossin and Carney, 1981). Phosphollpid Lsbellng, Extraction, and Separatlon ‘*Pi (2-30 &i/ml, Amersham, Arlington Heights, Illinois) or ‘H-myoinositol(15 rCi/ml, Amersham) was added to quiescent cultures of NIL cells and incubated for various periods at 3pC, either with or without purified human thrombin (gift of Dr. John W. Fenton, II; Fenton et al., 1977) and with or without neomycin (Upjohn, Kalamazoo, Michigan). Where indicated, medium was removed from the cells and the incubations stopped by one of two procedures. In the first procedure, the cells were rinsed four times with 4OC PBS and scraped from the plates with a rubber policeman in a total volume of 1 ml PBS. In the second procedure, 2 ml of 4OC 10% trichloroacetic acid was added to the cells and frozen by dipping the plates into a bath of dry ice methanol. In both cases, phospholipids were extracted from the cells by the procedure of Agranoff et al. (1983). First OOC methanol (2 ml) was added to the cell suspension and the tubes were mixed thoroughly; then 1 ml HCI (2.4 M) was added, plus chloroform (3 ml), and the tubes were mixed again. The resulting two phase system was centrifuged at 1000 x g for 5 min and the lower chloroform phase was removed to clean test tubes while the upper aqueous phase was treated with 2 ml more chloroform, mixed, and centrifuged. Next 0.5 ml HCI (2.4 M) and 4 ml methanol-HZ0 (1 :I) were added to the combined chloroform phases, the tubes mixed and centrifuged, and the final chloroform phases with the purified lipids separated from the aqueous phases. Chloroform was removed from the lipids by evaporation under NI. The phospholipids were separated from each other by thin layer chromatography on silica gel 60 plates (MCB Manufacturing Chemists, Inc., Cincinnati, Ohio) using one of two TLC systems. In the first system, the plates were developed with potassium oxalate (1% in H20), air dried, activated for 15 min at 115OC, and then the lipids were spotted onto the plates and the plates developed in chloroform-methanol4 M NH,OH (9:7:2; procedure of Gonzalez-Sastre and Folch-Pi, 1968). In the second system the plates were spotted with lipid and developed in a solvent of chloroform-methanol-acetic acid-H,0 (65:43:1:3). The positions of radiolabeled lipids on the plates were determined by autoradiography under X-ray film. Plates containing 3H-labeled lipids were sprayed with Autofluor (National Diagnostics, Somerville, New Jersey) before autoradiography. Silica gel containing radioactive lipids was scraped from the TLC plates into scintillation vials and the radioactivity measured in a Beckman liquid scintillation spectrometer. Measurement of W-lnosltol Phosphate Release Cytosolic levels of 3H-inositol phosphates were determined using a modification of the procedure described by Vincentini and Villereal (1984). Nonproliferating monolayer cultures of NIL fibroblasts were prepared as described above in 60 mm dishes. Six to 16 hr after switching the cells to serum-free DVlF-12 medium, the medium was removed and replaced with serum-free DVIF-12 medium containing 2 #Ci/ml ‘Hmyoinositol (Amersham). After 36 hr the cells were treated with thrombin or neomycin as indicated. Preliminary experiments indicated that thrombin-stimulated IP, release was maximal 30 set after thrombin addition. Therefore, in all experiments presented, reactions were stopped 30 set after thrombin addition by quickly aspirating the medium, dipping the plates in O°C PBS, and adding 2 ml of OOC trichloroacetic acid (TCA) (10%) to each plate. After 5 min the TCA was removed from each plate, and the plates were rinsed with an additional 2 ml of 0°C TCA.

Cell 486

The combined TCA washes were then extracted five times with ethyl ether to remove the TCA, the aqueous phases applied to 1 ml columns of Dowex 1 x 8-50 resin (formate form, 20-50 mesh), and the columns eluted with H,O to remove free SH-inositol, then with 0.1 M formic acid containing 0.2 M ammonium formate to remove )H-inositol-l-phosphate (IQ, 0.4 M ammonium formate to remove aH-inositol-l$-bisphosphate (IP,), and 1 M ammonium formate to remove ‘H-inositol-1,4,5trisphosphate (IP,).

Measurement

of DNA Synthesfs

and Cell Proliferation

The effects of neomycin and thrombin on DNA synthesis were determined by measuring the incorporation of SH-thymidine (1 &i/ml; ICN Pharmaceuticals, Irvine, California) during a 30 min incubation generally from 23.5 to 24 hr after thromdin addition (Stiernberg et al.,-1983; Stiernberg et al., 1984). Following incubation, cells were rinsed with PBS followed by extraction and rinsing five times with 10% TCA. The acid-precipitable material was dissolved overnight in 0.5 ml KOH (0.5 N) at 23OC, 0.25 ml HCI (1 N) was added, and the solution was counted in 10 ml of Beckman RediSolve-HPb (Beckman Instruments, Houston, Texas) scintillation fluid. DNA synthesis was also assessed after thrombin and neomycin treatment using SH-thymidine autoradiography of cells labeled for 24 hr (beginning immediately after thrombin addition) to determine the percentage of labeled nuclei. Following incubation as described above, cell monolayers were rinsed and extracted with 10% TCA, fixed with 3% paraformaldehyde, and covered with a thin film of lllford K-5 autoradiographic emulsion. After 10 days, the emulsion was developed by immersing plates in MicrodolX developer. They were then fixed and incubated 25 min with Hoechst stain (1 pg/ml in PBS) to fluorescently label cell nuclei. Random fields from each plate were scored (approximately 2000 nuclei) using a Leitz fluorescence microscope to determine the percentage of nuclei that had incorporated 3Hthymidine. Cell number was determined using a Coulter Electronics particle counter after removing cells from dishes with trypsin EDNA solution as described previously (Carney et al., 1978).

Measurement

of Protein

Synthesis

The effect of neomycin and other antibiotics on protein synthesis in quiescent cultures of NIL cells was determined by incubating nonproliferating cultures of NIL cells with a mixture of ‘H amino acids (ICN Pharmaceuticals, Inc., Irvine, California) at 2 &i/ml for 2 hr at JPC. Amino acid incorporation was stopped by addition of warm 10% TCA followed by five rinses of warm TCA. The acid precipitable material was then dissolved in 0.5 ml of KOH and counted in scintillation fluid as described above.

Acknowledgments The authors wish to thank John W. Fenton, II, for gifts of highly purified human thrombin produced through funding by NIH grant HL-13160, and Helen Amato and Becky Hansen for their excellent secretarial skills. This work was supported by NIH grant AM-25807 to D. H. C. and AM-25244 to E. F. L. D. L. S. was a recipient of a Summer Undergraduate Research Award from the Graduate School of Biomedical Sciences, E. A. G. received graduate support from the University of Texas, and D. H. C. is the recipient of a Research Career Development Award (CA-00805) from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

November

7, 1984; revised

June

as sec-

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Formation thrombin

of lysophosor ionophore

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