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Mar 20, 1992 - serum is platelet-derived growth factor (PDGF). MATERIALS AND METHODS. Chemicals and Biochemicals. Phorbol 12-myristate 13-.

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 6138-6141, July 1992 Cell Biology

Adhesion is required for protein kinase C-dependent activation of the Na+/H+ antiporter by platelet-derived growth factor MARTIN ALEXANDER SCHWARTZ*t AND CLAUDE LECHENE*t *Department of Cellular and Molecular Physiology, Harvard Medical School; and tDepartment of Medicine, Brigham and Women's Hospital, Boston,

MA 02115

Communicated by Judah Folkman, March 20, 1992

ABSTRACT Adhesion of normal, anchorage-dependent cells to a solid substratum leads to activation of the Na+/H+ antiporter and elevation of Intracellular pH. These effects are mediated by extracellular matrix proteins, such as fibronectin, and their receptors, the Integins. Experiments using ph cological inhibition and down-regulation of protein kise C (PKC) in C3H 10TY2cells show that platelet-derived growth factor induces activation of the Na+/H+ antiporter by means of a PKC-dependent pathway In adherent cells but cannot do so in poorly adherent cells. Poorly adherent cells are, however, able to elevate intracellular pH in response to a phorbol ester, inditing that PKC and subsequent steps in the pathway are functional. These results ndIate that couln of pla tderived growth factor to PKC activation requires cell adhesion.

MATERIALS AND METHODS Chemicals and Biochemicals. Phorbol 12-myristate 13acetate (PMA) and staurosporine were obtained from Sima.

Calphostin C (8) was a gift from Tatsuya Tamaoki, Kyowa Hakko Kogyo Co., Tokyo. Recombinant human BB PDGF was purchased from Upstate Biotechnology, Lake Placid, NY. 2',7'-Bis(2-carboxyethyl)-5-(and-6)carboxyfluorescein, acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes. Cell Culture. C3BH 10TY2 cells (9) were cultured in Dulbecco's modified Eagle's medium supplemented with 10%o calf serum (GIBCO) or 10%6 Serum Plus (JRH Biosciences, Lenexa, KS), which yielded similar results. For pH, measurements, cells were plated in 35-mm tissue-culture plastic dishes coated with polyHEMA, as described (2, 10). PolyHEMA was used at a concentration such that cells attached weakly but did not spread. Each dish also contained areas that had been scraped free of polyHEMA with a plastic pipet tip, where cells could attach and spread. Well-attached, spread cells could thereby be compared with weakly attached, round cells in the same dish at the same time. pHA Measurements. pHi was determined by microfluorimetry in individual cells loaded with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, as described (1, 2). Medium contained sodium bicarbonate and was gassed with C02, as before (2). For measurements of steady-state pHi, 15-20 cells were analyzed for each condition, and the means were calculated. ApH, signifes the difference in pHi between spread cells and round cells in the same dish.

Nonneoplastic, anchorage-dependent cells in culture require adhesion to a solid substratum to proliferate. Previous work in our laboratory has shown that cell spreading activates the Na+/H+ antiporter to induce a rapid and prolonged elevation of intracellular pH (pHi) (1, 2). The increase in pHi closely parallels increased DNA synthesis in nontransformed cells (2, 3), and the ability of transformed cells to grow without adhesion correlates with their ability to maintain an alkaline pHi without adhesion (4). Inhibition of Na+/H+ antiporter activity substantially and specifically inhibits DNA synthesis (3, 5). And, constitutive elevation of pHi by tranfection of nontransformed cells with a proton pump induces anchorageindependent proliferation (6). Thus, pHi or Na+/H+ antiporter activity may mediate some of the effects of adhesion on cellular proliferation. Although some controversy exists about the physiological relevance of results obtained without bicarbonate in the medium (5), it is generally accepted that prolonged maintenance of pH, at permissive levels is necessary, although not sufficient, for proliferation. Furthermore, these issues have no bearing on the present work because the described experiments used medium containing bicarbonate. Activation of the Na+/H+ antiporter by adhesion depends upon extracellular matrix proteins, such as fibronectin (1, 3) and is triggered by local clustering of the fibronectin receptor, integrin a5131, independent of cell shape changes (7). The aim in the experiments reported here was to identify the intracellular-signaling pathways that mediate the effect of extracellular matrix proteins and receptors on pHi. Measurements of the effects on pHi of serum and inhibitors and activators of protein kinase C (PKC) revealed that elevation of pHi by serum via PKC requires cellular adhesion to a solid substratum. Additional results suggest that the active component in serum is platelet-derived growth factor (PDGF).

RESULTS in pH, was measured cells in 35-mm dishes containing areas coated with the nonadhesive polymer polyHEMA, where cells adhered weakly and stayed spherical, and areas of bare tissue-culture plastic, where cells adhered well and spread. Adhesion of fibroblasts under these conditions is mediated primarily by fibronectin and its receptor, integrin a5p1Previous work had shown that for 10T½ cells in high serum, the ApHi was =0.15 pH unit (7), similar to other cell types (1-3). In 10TY2 cells, ApHi partially depended on serum. Cells in 0.25% serum had a ApHi approximately half that of cells in 10% serum: in low serum, ApHi was 0.07 + 0.02 SD (n = 9) vs. 0.15 0.01 SD (n = 9) in high serum. Eliminating serum completely had no further effect on ApHi but reduced cell

viability and was, therefore, avoided. The increase in ApHi in

Abbreviations: pHi, intracellular pH; PMA, phorbol 12-myristate 13-acetate; PDGF, platelet-derived growth factor; PKC, protein kinase C; ApHi, difference in pHi between spread cells and round cells in the same dish. tPresent address: Committee on Vascular Biology, Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.


Cell Biology: Schwartz and Lechene high serum was because serum increased pHi in spread cells but not in round cells (Fig. 1A). The effect of serum on ApHi appeared to depend on PKC because exposure to 200 nM PMA for 24 hr to down-regulated PKC lowered pHi in spread cells in serum but did not affect pHi in round cells or in cells in low serum (Fig. 1B). In vitro assays demonstrated complete loss of calcium- and phospholipid-dependent kinase activity in cell extracts after similar treatment of 10TY2 cells with PMA (data not shown), confirming that PKC was down-regulated. Involvement of PKC was supported by additional experiments. When pHi was measured in cells in 10% serum after stimulation for 30 min with PMA, pH, in round cells increased in a dose-dependent manner, whereas pH, in spread cells was unchanged (Fig. 2). The half-maximum for the effect was at 21 ± 3 nM (n = 4). The reported Kd for binding to PKC varies somewhat because PMA is very hydrophobic and tends to be adsorbed nonspecifically, but 20 nM is within the range of reported values (11). At saturation, ApH, was reduced to 0.065 ± 0.017 SD (n = 6). The effect of calphostin C (8), an inhibitor reported to be highly specific for PKC, was also examined. Calphostin C caused a dose-dependent decrease in the pHi of spread cells, whereas pH, of round cells was unchanged (Fig. 3). The half-maximal effect occurred at 54 ± 10 nM (n = 3), in agreement with the reported Ki of 50 nM (8) for inhibition of PKC. At saturation, ApH, was reduced to 0.075 ± 0.006 SD (n = 8). Time-course measurements revealed that the effect of calphostin C was complete in 15 min (tl2 = 9 ± 2 min, n = 3). This time course is consistent with previous data

Proc. NatL. Acad. Sci. USA 89 (1992)













Bw *


10% serum

spread round

0.25% serum

FIG. 1. Effect of serum in cells with and without PKC. (A) pHi in untreated spread cells or round cells in the same dish in 10%/ serum or 0.25% serum. Similar results were obtained in eight experiments. (B) pHi in cells treated for 24 hr with 200 nM PMA to down-regulate PKC. pHi was again measured in spread cells and round cells in 10%b serum or 0.25% serum. Similar results were obtained in three





PMA (riM) FIG. 2. Effect of PMA on pH,. (A) pH, was measured in spread cells and round cells in 10%1 serum 20-30 min after adding the indicated concentration of PMA. (B) ApHi was calculated from data in A. Similar results were obtained in four experiments.

showing that the effect of adhesion on pHi is rapidly reversed when the stimulus is terminated (1, 7). The PKC inhibitor staurosporine (12) gave very similar results (data not shown). Taken together, experiments with PMA and calphostin C indicate that in the presence of 10%6 serum, spread cells maintain a higher steady-state pHi than round cells due to PKC activation. This effect is responsible for z50% of the difference in pHi between spread cells and round cells. In low serum, where ApH, was initially low, neither PMA nor calphostin C had any further effect on ApH, (data not shown). Additional experiments demonstrated that 10%o plateletpoor human plasma was unable to elevate pHi in spread cells and increase ApH, (data not shown). Together with other studies showing that PDGF elevates pH, via PKC (13), this result suggested that the component in serum most likely responsible for activating the Na+/H+ antiporter was PDGF. To test this hypothesis, we examined cells in high serum that had been depleted of PDGF by passage through heparinSepharose (14): pH; in these cells was similar to cells in low serum (Fig. 4), and ApH, was 0.08 + 0.01 SD (n = 5), consistent with the idea that PDGF might be required for serum to activate the Na+/H+ antiporter and increase ApH;. The effect of adding PDGF to cells in low serum was also examined. Time-course measurements showed that the pH, increase in individual cells was somewhat heterogeneous, as seen by others (15); however, mean pH, was elevated in spread cells but not in round cells when measured 0.5 hr after PDGF addition (Fig. 4); pH; remained constant for at least 24 hr (data not shown). In these experiments, ApH, increased from 0.07 ± 0.02 without PDGF to 0.14 ± 0.016 SD (n = 5) after PDGF. These results show that PDGF or another

Proc. NatL Acad. Sci. USA 89 (1992)

Cell Biology: Schwartz and Lechene








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. * -r......


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FIG. 4. Role of PDGF. pHi was measured in spread cells or round cells in medium with the indicated concentration of serum or with PDGF at 25 ng/ml. Similar results were obtained in five experiments.

0.12 0.10




Calphostin C (pM) FIG. 3. Effect of calphostin C on pH1. (A) pHi was measured 30 min after adding the indicated concentration of calphostin C to spread cells and round cells plated 24 hr earlier in medium/101% serum. 0, Spread cells; *, round cells. (B) ApHi was calculated from the data in A. Similar results were obtained in four experiments.

growth factor with similar biological activities, origin, and heparin-binding properties is the major component in serum responsible for activating the Na+/H+ antiporter in spread cells but not in round cells.

DISCUSSION The main conclusion from these results is that serum cannot increase pH, via PKC in round cells, as it does in spread cells. This interpretation is based on experiments using activation, down-regulation, and inhibition of PKC. PDGF is probably the active factor in serum because the effect is not seen in its absence and because the effect is restored by adding purified PDGF. Furthermore, PDGF is well known to initiate inositol phospholipid hydrolysis (16) and to activate the Na+/H+ antiporter via PKC (13). Adhesion can still elevate pH, to some extent, independent of growth factors and PKC. The serum- and PKC-independent effect is responsible for part of the increase in pH, in 10T'/2 cells and all of the increase in capillary endothelial cells (3). The pathways involved in this component have not been identified. Failure of the round cells to respond to serum or PDGF could be due, in principle, to a defect anywhere in the pathway-from the binding of growth factor with its receptor to the antiporter itself. A number of steps in this pathway can, however, be ruled out. (i) It is unlikely that loss of surface expression of growth factor receptors can account for the unresponsiveness of round cells. Although PDGF binding has not been directly measured, PDGF is still able to induce phosphorylation of proteins on tyrosine, indicating that re-

ceptors must be present and functional (H. McNamee, D. Ingber, and M.A.S., unpublished work). Also, we have measured binding of epidermal growth factor to round 3T3 cells and basic fibroblast growth factor to round capillary endothelial cells; in both cases, binding was unchanged relative to spread cells, even though the round cells were unable to respond mitogenically (D. Ingber and M.S., unpublished work). Thus, binding of PDGF to its receptor appears to be uncoupled from subsequent steps in the pathway. (ii) It is also unlikely that PKC or any step subsequent to PKC activation is inhibited in round cells because adding the exogenous activator PMA elevates pH, in round cells and decreases ApH,. Apparently, then, a step in the pathway between the PDGF receptor and PKC must be impaired in round cells. Because PDGF activates PKC by regulating hydrolysis of phosphatidylinositol diphosphate (13, 16), the best explanation is that PDGF-induced hydrolysis of lipids requires adhesion. Fig. 5 summarizes this view. In support of this hypothesis, it has been found that in suspended cells, PDGF fails to trigger release of calcium (17) or release of water-soluble inositol phosphates (H. McNamee, D. Ingber, and M.S., unpublished work). PDGF has also been shown to induce hydrolysis of phosphatidylcholine to produce a second species of diacylglycerol, particularly at longer times (18). This result suggests that phosphatidylcholine hydrolysis may also require adhesion. Some evidence, however, suggests that this diacylglycerol

FIG. 5. Regulation of inositol phospholipid breakdown by adhesion. The PDGF receptor (PDGFR) activates phospholipase C (PLC) to induce breakdown of phosphatidylinositol diphosphate (PIP2). Diacylglycerol (DAG) then activates PKC, which leads to activation of the Na+/H+ antiporter. Either activation of PLC by the PDGFR or hydrolysis of PIP2 appears regulated by adhesion. FN, fibronectin; IP3, inositol 1,4,5-triphosphate.

Cell Biology: Schwartz and Lechene differs from phosphatidylinositol diphosphate-derived diacylglycerol with respect to PKC regulation (19). Furthermore, phosphatidylcholine hydrolysis has not been demonstrated to lead to activation of the antiporter. Thus, whether adhesion is also needed for hydrolysis of phosphatidylcholine in response to PDGF remains an open question. The idea that activation of PKC by a soluble growth factor requires cell adhesion may have wider significance. In a number of systems adhesion has been found to be permissive for the action of growth factors or hormones on cell growth, differentiation, and gene expression (20, 21). Our results show that adhesion to extracellular matrix allows a growth factor to act at the level of a well-defined cellular-signaling pathway. These results, therefore, represent one step toward understanding the effect of extracellular matrix on cellular signaling at the molecular level. We thank Erin Rupp and Glen Seidner for skilled technical assistance. We also thank Dr. Tatsuya Tamaoki for generously providing the calphostin C. This work was supported by funds from the Lucille B. Markey Charitable Trust and the Elsa U. Pardee foundation (M.A.S.) and U.S. Public Health Service Grant 5P441 RR02604 (C.L.). 1. Schwartz, M. A., Both, G. & Lechene, C. (1989) Proc. NatI. Acad. Sci. USA 86, 4525-4529. 2. Schwartz, M. A., Jr., Cragoe, E. J. & Lechene, C. P. (1990) J. Biol. Chem. 265, 1327-1332. 3. Ingber, D. E., Prusty, D., Frangioni, J. J., Jr., Cragoe, E. J., Lechene, C. P. & Schwartz, M. A. (1990) J. Cell Biol. 110, 1803-1811. 4. Schwartz, M. A., Rupp, E. E., Frangioni, J. V. & Lechene,

C. P. (1990) Oncogene 5, 55-58.

Proc. Natl. Acad. Sci. USA 89 (1992)


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