©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Closely Related Isoforms of Protein Kinase C Produce Reciprocal Effects on the Growth of Rat Fibroblasts
POSSIBLE MOLECULAR MECHANISMS (*)

(Received for publication, April 27, 1994; and in revised form, September 28, 1994)

Christoph Borner (1) (2)(§) Marius Ueffing (3) Susan Jaken (4) Peter J. Parker (5) I. Bernard Weinstein (1)

From the  (1)Columbia-Presbyterian Center and Institute of Cancer Research, Columbia University, New York, New York 10032, the (2)Institute of Biochemistry, University of Fribourg, Rue du Musée 5, 1700 Fribourg, Switzerland, the (3)Institute of Clinical Molecular Biology and Tumorigenetics, Marchioninstr. 25, D-8000, Munich, Germany, the (4)W. Alton Jones Science Center, Lake Placid, New York, 12946, and the (5)Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously reported that two closely related protein kinase C (PKC) isoforms, PKCalpha and PKCbetaI, had divergent effects on the growth and transformation of the same parental R6 rat embryo fibroblast cell line (Housey, G. M., Johnson, M. D., Hsiao, W.-L. W., O'Brian, C. A., Murphey, J. P., Kirschmeier, P., and Weinstein, I. B.(1988) Cell 52, 343-354; Borner, C., Filipuzzi, I., Weinstein, I. B., and Imber, R. (1991) Nature 353, 78-80). Whereas cells that overexpress PKCbetaI lost anchorage dependence, grew to higher saturation densities, and generated small tumors when injected into nude mice, none of these properties were seen with cells that overexpress PKCalpha. In fact, the latter cells grew even slower and to lower saturation densities as compared to control cells. Here we investigate possible molecular mechanisms underlying the reciprocal effects of PKCalpha and PKCbetaI. Overexpression of both isoforms enhanced 12-O-tetradecanoyl phorbol-13 acetate-induced expression of the growth regulatory genes c-jun, c-myc, and collagenase and enhanced feedback inhibition of epidermal growth factor receptor binding and cellular levels of diacylglycerol. However, the cells overexpressing PKCbetaI differed from those overexpressing PKCalpha by displaying a decreased requirement for growth factors and by the production of a mitogenic factor. Thus, the basis for enhanced growth and transformation of cells overexpressing PKCbetaI may be the establishment of an autocrine growth factor loop. These findings may be relevant to the roles of specific isoforms of PKC in carcinogenesis and tumor growth.


INTRODUCTION

Protein kinase C (PKC) (^1)occupies a pivotal role in signal tranduction pathways that influence numerous cellular functions, including cell proliferation and tumorigenesis(1, 2, 3) . PKC-mediated signaling systems are initiated by the stimulation of cell-surface receptors by their respective ligands. This triggers the breakdown of phospholipids by phospholipase C (PLC) and D (PLD) enzymes to produce, among other products, diacylglycerol (DAG)(4) . DAG binds to and activates PKC. This activation is frequently accompanied by increased association of PKC with cellular membranes and the subsequent phosphorylation of various soluble and membrane-bound proteins (reviewed in Refs. 1, 3). Once phosphorylated, PKC substrates may provoke the activation of c-ras(6) , c-raf(7, 8) , MEK/MAP kinases(9) , and IkappaB/NFkappaB(10) . These latter proteins relay the agonist-evoked signal to the nucleus where gene expression and cell cycle processes are altered(11, 12) . Prominent nuclear changes that occur as a result of PKC activation consist of increased expression of immediate early response genes such as c-myc, c-fos/c-jun (AP-1) and others(13) . These genes encode transcription factors which alter the expression of secondary response genes whose protein products play major roles in proliferation and differentiation (reviewed in (14) ). In addition to its positive effects on signal transduction and gene expression, PKC can also act as a cellular guardian to prevent overstimulation of growth factor-elicited signaling pathways(1, 2, 3) . This is achieved through feedback inhibition of the activity of growth factor receptors, as well as inhibition of phospholipid hydrolysis and Ca transients.

In view of these complex and reciprocal functions of PKC, it is difficult to provide a simple description of the role of this enzyme in the multistage process of carcinogenesis. Nevertheless, it is now widely accepted that PKC constitutes the major cellular receptor for the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and related compounds(15) . Presumably, the activation of PKC by these compounds enhances the promotion phase of carcinogenesis by altering gene expression and stimulating the growth of initiated cells that carry mutations in oncogenes and/or tumor suppressor genes. The resulting clonal expansion also increases the likelihood of additional genetic changes(16, 17) . Since TPA can enter cells and directly stimulate PKC, it mimics the action of DAG while bypassing the normal agonist-mediated control of this enzyme(18) . In addition, since it is much more potent than DAG and only slowly metabolized, TPA causes marked and prolonged activation of PKC(5) . Activation of PKC by TPA is also associated with membrane translocation and marked phenotypic effects, including alterations in morphology and stimulation or inhibition of cell growth, depending on the cell system(19, 20, 21, 22, 23, 24) . At later time points, phorbol esters usually provoke proteolytic down-regulation of PKC, thereby terminating the effects of PKC activation(1, 3, 25) . This down-regulation abrogates the positive as well as the above-mentioned negative feedback effects of PKC on signal transduction. It is, therefore, not entirely clear whether phorbol esters contribute to tumorigenesis via PKC activation, PKC down-regulation, or a combination of the two processes.

The above scenario is further complicated by the fact that PKC comprises a multigene family that encodes at least eleven distinct isoforms(1, 2) . These isoforms can be divided into three subgroups based on sequence homologies and cofactor requirements: conventional PKC (alpha, betaI, betaII, ) that are dependent on Ca for activity and membrane localization; non-conventional PKCs (, , /L, ) that are not dependent on Ca; and atypical PKCs (, , ) that are not dependent on Ca and are not stimulated by DAG or TPA (reviewed in Refs. 1, 2). The expression of these isoforms varies between tissues and cell types(1, 2, 26) . It seems likely, therefore, that individual isoforms differ with respect to their roles in growth control and other specific biologic effect, but possible redundancies in their functions may also exist. It has been difficult to examine this question since individual cells often express more than one isoform of PKC and at the present time isoform specific inhibitors are not available.

We have pursued a genetic approach to analyze the question of isoform specificity by utilizing gene transfer methods to develop derivatives of cell lines that stably overexpress high levels of a particular PKC isoform and then examining various biochemical and phenotypic properties of these derivatives. We previously reported that overexpression of PKCalpha and PKCbeta, two closely related conventional PKC isoforms, had opposite effects on the growth of the rat embryo fibroblast cell line R6(19, 22) .

In the present study we investigate the mechanisms underlying these divergent growth effects of PKCbetaI and PKCalpha. Our results provide some of the first direct evidence that, even when expressed in the same cell type, two isoforms of PKC can be redundant with respect to certain biologic effects but differ markedly with respect to other biologic effects.


EXPERIMENTAL PROCEDURES

Materials

Monoclonal anti-bovine PKCalpha (MC5), I-labeled sheep anti-mouse and donkey anti-rabbit secondary antibodies, [^3H]thymidine (80 Ci/mmol), and I-EGF (130 µCi/µg) were obtained from Amersham Corp. Polyclonal PKC- and PKC-specific antisera were from D. Fabbro, Ciba, Basel. A full characterization of these antibodies has previously been described(27) . Geneticin (G418) was purchased from Life Technologies, and TPA was from LC Services Corporation (Woburn, MA). DAG and cardiolipin were from Avanti Polar lipids. Escherichia coli DAG kinase was from Lipidex (Middleton, WI), [-P]ATP from New England Nuclear, Hydrofluor from National Diagnostics, Somervile, NJ, and bovine epidermal growth factor (EGF) from Collaborative Research (Bedford, MA). E64-d was from Sigma, and calpain inhibitors I and II were from Boehringer Mannheim. Human platelet-derived growth factor (PDGF) was kindly provided by Dr. R. Witte, Columbia University, New York. All other chemicals were reagent grade.

Plasmid Construction

The full-length cDNA encoding for bovine PKCalpha was excised from the clone bPKC206 (28) at BglII sites. This DNA fragment was subcloned into the BamHI site of the retroviral expression vector pZIP-Neo SV(X). A detailed description of the vector has been published elsewhere(29) .

Construction of R6 Cell Lines Overexpressing PKCbetaI and PKCalpha

The construction of the PKCbetaI-overexpressing fibroblast cell lines R6-PKC3betaI, R6-PKC5betaI, and 10T1/2-PKC4betaI as well as the vector control cell line R6-C1 have been described elsewhere(19, 30) . To obtain R6 cell lines overexpressing bovine PKCalpha, we transfected the bPKC206/pZIP-Neo SV(X) construct into early passage Rat 6 embryo fibroblasts by the calcium phosphate precipitation technique(31) . Following selection for resistance to G418, a panel of PKCalpha-transfected clones was isolated, expanded, and examined by Northern blot analysis for expression of the respective PKCalpha transcripts (data not shown). Clones expressing high (R6-bPKC4alpha), intermediate (R6-bPKC3alpha), or low (R6-bPKC7alpha) amounts of the expected 8.0-kilobase PKCalpha mRNA (LTR to LTR transcript), and control cells transfected with the retroviral vector alone (R6-SVXc1) were chosen for further studies. Pools of G418-resistant colonies were also collected, expanded, and used for our studies. Western blot analysis of total cellular extracts showed that when compared to R6-SVXc1 vector control cells, R6-bPKC4alpha, R6-bPKC3alpha, and R6-bPKC7alpha cells contained 41-, 25-, and 15-fold higher levels of the 81-kDa PKCalpha protein, respectively (data not shown). These results correlated with increased in vitro protein kinase and phorbol 12,13-dibutyrate (PDBu) binding activities (data not shown).

Cell Culture and Growth Curves

All cells were grown in serum-containing Dulbecco's modified Eagle's medium (DMEM, Life Technologies) as described(19, 30) . Starving of the cultures or growth to post-confluence were strictly avoided, unless they were required for special purposes such as DNA synthesis or foci formation assays. All experiments reported here were performed on early passage cells kept in culture for less than 6 weeks. The cells were generally harvested for RNA or protein when subconfluent.

Doubling times and saturation densities of the various cell derivatives were determined exactly as described (19, 22) with the modification that the cells were grown in 1% instead of 10% CS.

Immunoblot and Immunocytochemical Analysis

Total SDS extracts and native cytosolic and membrane fractions of cellular proteins were prepared exactly as described(27, 32) . Immunoblot analysis of these extracts were performed as previously published(27, 32) . Primary PKC isotype-specific antibodies were used on immunoblots as 1:500 (PKCalpha) or 1:2000 (PKC, ) dilutions(27) . I-Labeled sheep anti-mouse or donkey anti-rabbit reagents were used as secondary antibodies in a final dilution of 0.3 µCi/ml.

For immunocytochemical analysis, subconfluent R6 cell derivatives were fixed with paraformaldehyde, permeabilized with methanol, stained with M4 (PKCalpha-specific) or M7 (PKCalpha- and PKCbeta-specific) monoclonal antibodies and finally with fluorescein-conjugated second antibody, as described previously(33) .

RNA Isolation and Northern Blot Analysis

Total cellular RNA was isolated from untreated and TPA-treated control and PKC-overexpressing cells in parallel and transferred by capillary blotting onto Hybond-N nylon membranes (Amersham) as described previously(27) . The murine c-myc(34) was kindly supplied by L.-L. Hsieh, the c-fos and c-jun probes (35) were from T. Curran, and the human collagenase probe (36) was purchased from Oncogene Science, Uniondale, NY.

The relative abundance of RNA/lane was judged to be similar by comparing the ethidium bromide staining of the ribosomal bands. For further confirmation, the blots were hybridized with a probe for an endogenous housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH). In all cases, the ethidium bromide staining reflected the results obtained by the GAPDH probe.

Lipid Extraction and Analysis of DAG Formation

Control and PKC-overexpressing R6 cells were seeded in 100-mm plastic dishes, grown in DMEM plus 5% serum (in the absence of G418), and refed 48 h before harvesting at 50-80% confluence. 24 h or 30 min before harvesting (lipid extraction), cells on duplicate dishes were treated with 100 ng/ml TPA in 0.1% Me(2)SO. Control dishes received 0.1% Me(2)SO alone. Lipid extraction was performed exactly as described(37) . DAG was assayed by its conversion into [P]PA acid in vitro as described. A DAG standard (0.4 nmol) was run in parallel. DAG data were normalized to phospholipid phosphate determined by the method of Ames (38) and the DAG value expressed as a mol %.

Assays for Induction of DNA Synthesis

Serum-starved subconfluent vector control or PKC-overexpressing cell cultures were either stimulated with serum, single growth factors, or conditioned medium from PKC-overexpressing cells, and 15 h later [^3H]thymidine incorporation into the DNA of these cells was determined exactly as described(39) . All assays were done in triplicate wells, and the variability was less than 10%.

Conditioned medium (CM) was collected from PKC-overexpressing cells as follows. Complete medium was removed from subconfluent cultures, and the cultures were washed with DMEM and incubated for 30 h in DMEM in the absence of serum. This CM was collected, cleared of any cell debris by centrifugation at 5000 times g for 90 min, and a 100-200-µl aliquot was added to serum-starved control R6 cells to test for its capacity to induce DNA synthesis.

EGF-binding Assay

Cells were seeded in DMEM, 10% CS (without G418) in 24-well dishes at a density of 5 times 10^4 cells/well. 100 ng/ml TPA or 0.1% Me(2)SO (control) was added to the medium 24 h, 6 h, or 30 min prior to the binding assay. 48 h after seeding, monolayers were washed three times in DMEM buffered with 20 mM HEPES, pH 7.5, containing 0.1% bovine serum albumin, and then incubated with 1 nMI-EGF (1 µCi/well, 130 µCi/µg, 1.5 times 10^5 counts/min/ng) in the same medium for 2 h on ice. At the end of the incubation, cells were washed twice with medium and solubilized with 1 ml of 0.2 N NaOH, 1% SDS. After 60 min at 37 °C, radioactivity of the solubilized cells was determined. Nonspecific binding was determined by parallel incubation of each sample with a 100-fold excess of cold EGF. Specific binding was calculated as total minus nonspecific cell-associated radioactivity. Both specific and nonspecific binding were determined in triplicates for each time point.

Quantitation of Protein and RNA

Immunoblots, Northern blots, or thin layer plates were autoradiographed with Kodak XAR film at -70 °C and the relative abundance of protein, RNA, or lipid was quantitated by scanning preflashed autoradiographs on a laser densitometer (Molecular Dynamics) or by direct assays of the blots using Betascope counting (Betagen). Values were taken from the linear range of a I or P standard curve. Both methods gave similar results.


RESULTS

Activation and Down-regulation of PKC Isoforms

We have previously shown that rat PKCbetaI and mouse PKCalpha had distinct effects on the growth of a rat 6 (R6) embryo fibroblast cell line(19, 22) . Whereas overexpression of the former provoked enhanced growth and partial transformation(19) , overexpression of the latter led to a marked inhibition of cellular growth(22) . In this study we generated R6 cell lines which overexpress bovine PKCalpha to various extents. Consistent with our previous findings, bovine PKCalpha impeded growth in a dose-dependent manner (data not shown, see also Fig. 5).


Figure 5: Growth properties of PKCbetaI- and PKCalpha-overproducing R6 cells in low serum. Cells were seeded and treated as described under ``Experimental Procedures.'' Growth curves were determined in the absence (Ctrl) or presence of 16 or 160 nM TPA (10 or 100 ng/ml) in 1% calf serum. The doubling times (A) relate to the initial exponential phase of cell growth, the saturation density (B) to the number of cells/35-mm plate on day 28. The results are the average ± S.E. of three independent experiments.



To understand the basis for the disparate growth effects of PKCbetaI and PKCalpha, we performed a series of biochemical and molecular analyses on two PKCbetaI- and three PKCalpha-overexpressing cell lines. We show here the results of R6-PKC3betaI (overexpressing PKCbetaI 50-fold) and R6-bPKC4alpha cells (overexpressing bovine PKCalpha 40-fold) as representatives. Similar results were obtained with R6 cell derivatives overexpressing PKCbetaI or PKCalpha 10-20-fold (data not shown).

We first examined whether the growth differences can be explained by differential activation/translocation or down-regulation of the overexpressed or endogenously expressed PKC isoforms in response to the phorbol esters TPA or PDBu. This is because these agents were previously shown to activate PKC in vivo(18) and to exaggerate the disparate effects of PKCbetaI and PKCalpha on the growth of R6 cells(19, 22) .

Immunocytochemical analysis on intact R6-PKC3betaI and R6-bPKC4alpha cells revealed that the overexpressed PKCalpha and PKCbetaI resided mainly in the cytoplasm of cells when the cells were grown under standard conditions (Fig. 1). Upon treatment of the cells with phorbol esters, both PKCalpha and PKCbetaI rapidly redistributed from the cytoplasm to the plasma membrane. This event, called translocation, has been shown to be associated with PKC activation(5) . There was also intensive membrane ruffling (Fig. 1) presumably associated with rearrangements of the underlying cytoskeleton. Since membrane ruffling was exaggerated in both the PKCalpha- and PKCbetaI-overexpressing cells, it is apparent that both of these isoforms can induce this morphologic change. With prolonged treatment of cells with TPA or PDBu, the translocation of PKC isoforms is usually associated with ``down-regulation'' of these enzymes due to proteolysis(25) . Isoforms of PKC differ in their susceptibility to down-regulation, and this is also a function of cell type (reviewed in (1) ). Here we found that whereas overexpressed PKCbetaI was almost completely depleted from R6-PKC3betaI cells, overexpressed PKCalpha is partially resistant to down-regulation in R6-bPKC4alpha cells at 24 h after treatment with phorbol esters (Fig. 1). This could be shown both by immunocytochemical analysis on intact cells (Fig. 1) as well as by Western blots on cytosolic and membrane fractions (Fig. 2A and (40) ). Appreciable amounts of PKCalpha but not PKCbetaI were still immunodetectable in cells treated with repetitive doses of TPA or PDBu for 28 days (data not shown).


Figure 1: Immunocytochemical analysis of R6 cells overproducing PKCbetaI or PKCalpha. Immunofluorescence of PKCbetaI or PKCalpha in intact R6-PKC3betaI, R6-bPKC4alpha, or R6-C1 vector control cells treated with or without 200 nM PDBu for the indicated time periods. Monolayer cultures were treated with PDBu where indicated, fixed with formaldehyde, permeabilized with methanol, stained with the monoclonal antibody M4 (specific for PKCalpha) or M7 (specific for PKCalpha/beta), and finally stained with a fluorescein-conjugated secondary antibody, as described previously (33) . All photomicrographs were exposed and printed for the same times to allow for quantitative comparisons.




Figure 2: Subcellular distribution and down-regulation of ectopically overexpressed PKCalpha and endogenously expressed PKC and PKC in R6 cells treated with TPA. Protein immunoblots were performed on cytosol (Cyt) and membrane (M) fractions of R6-PKC3betaI or R6-bPKC4alpha cells treated without (Ctrl) or with 100 ng/ml TPA for 30 min (30`) or 24 h. Cyt and M samples from both cell lines were analyzed on the same gel; autoradiographs are depicted separately for the sake of clarity. Immunodetection was performed with monoclonal PKCalpha- or polyclonal PKC- or PKC-specific antibodies as described under ``Experimental Procedures.'' A, overexpressed PKCalpha in R6-bPKC4alpha cells. B, endogenous PKC and PKC in R6-PKC3betaI and R6-bPKC4alpha cells as indicated. The apparent molecular masses in kilodaltons (kDa) of the PKC isoforms are indicated: p81 for PKCalpha, p76 for PKC, and p89 for PKC.



We next examined the possibility that overexpression of exogenous PKCalpha or PKCbetaI isoforms might alter the sensitivity of endogenous PKC isoforms to phorbol ester-induced translocation and down-regulation. We previously showed that R6 cells normally express four endogenous PKC isoforms, PKCalpha, PKC, PKC, and PKC(27) . Whereas PKCalpha and PKC are present at low abundance and are mainly confined to the cytosol, PKC and PKC are more abundant and are mainly membrane-bound(27) . This distribution of the endogenous isoforms of PKC was unaltered in either untreated R6-bPKC4alpha or R6-PKC3betaI cells (Fig. 2B). By contrast, PKC and PKC exhibited differential extents of down-regulation in R6-bPKC4alpha or R6-PKC3betaI cells following treatment with TPA. While they were almost completely down-regulated in cells overexpressing PKCalpha, they were highly resistant to TPA-induced down-regulation in cells overexpressing PKCbetaI (Fig. 2B).

Taken together these results indicate that the overexpressed PKCalpha and PKCbetaI isoforms undergo similar translocation when the respective cells are treated with phorbol esters. In addition, their overexpression does not perturb the subcellular distribution of endogenous PKC isoforms. By contrast, in response to long term phorbol ester treatment, the overexpressed isoforms are differentially down-regulated and they appear to influence the down-regulation of endogenous PKC and PKC isoforms by an unknown mechanism. In any case, neither PKCalpha- nor PKCbetaI-overexpressing R6 cells are fully depleted of PKC following prolonged phorbol ester treatment, thus enabling these cells to mainain PKC-mediated responses for prolonged periods of time (see below).

Inducibility of Early and Secondary Response Genes in PKC-Overexpressing Cells

Several ``immediate early'' gene products that are thought to be implicated in cell proliferation are induced by certain growth factors, and there is evidence that PKC plays a role in mediating the induction of these genes(13, 14) . A set of ``secondary response'' genes that are the targets of immediate early gene products are also crucial for cell proliferation; thus these genes are indirectly controlled by TPA. It was of interest, therefore, to examine the basal levels of expression and TPA-inducibility of the mRNAs for the immediate early genes c-fos, c-myc, and c-jun and for the secondary response gene collagenase, in subconfluent monolayer cultures of R6-PKC3betaI, R6-bPKC4alpha, and vector control R6-C1 cells.

With the exception of c-fos, the basal levels of all of these mRNAs were increased in both R6-bPKC4alpha and R6-PKC3betaI cells when compared to the control cells (Fig. 3). Thus, overexpression of PKCalpha and PKCbetaI increases the levels of expression of both immediate early and secondary response genes. This could be mediated by the 10-20% of the overexpressed isoforms that were tightly associated with the membrane fraction ( Fig. 2and (40) ) because these membrane-associated PKCs might reflect activated molecules(41) . Treatment of the control cells with 100 ng/ml TPA caused transient increases in the levels of c-fos, c-myc, c-jun, and collagenase mRNAs, with maximal induction at 30 min, and 2, 3, and 6 h, respectively (Fig. 3). In TPA-treated R6-PKC3betaI cells, the kinetics of induction of these mRNAs were similar but the induced levels of c-myc, c-jun, and collagenase mRNAs were higher and more sustained than in control cells, presumably due to the intense and prolonged activation of PKCbetaI in these cells (Fig. 3). A different result was obtained with c-fos mRNA in R6-PKC3betaI cells; the level of induction obtained with TPA was not increased, but it persisted for a longer period of time than in the control cells (Fig. 3). Surprisingly, R6-bPKC4alpha cells displayed changes in the levels of all of these mRNAs, in response to TPA, that were very similar to those seen in R6-PKC3betaI cells (Fig. 3). These results indicate that the reciprocal effects of PKCalpha and PKCbetaI overexpression on growth and cell transformation are not due to differences in the regulation of this set of genes.


Figure 3: Basal and TPA-induced mRNA levels of immediate early and secondary genes in PKCbetaI- and PKCalpha-overexpressing R6 cells. RNA Northern blots showing the levels of c-myc, c-fos, c-jun, and collagenase transcripts in control R6-C1, R6-PKC3betaI, and R6-bPKC4alpha cells treated without (0) or with 100 ng/ml TPA for the indicated times. Total RNA was isolated from subconfluent cell cultures and hybridized to corresponding P-labeled cDNA probes as described under ``Experimental Procedures.'' To assure equal RNA loading, these samples were also hybridized to a GAPDH cDNA probe.



Down-regulation of EGF-Receptor Binding and DAG Production in PKC-Overexpressing Cells

Activated PKC can phosphorylate the EGF receptor on a critical threonine residue (Thr) in its cytoplasmic domain(42) . This results in down-regulation of EGF receptor binding and decreased EGF stimulation of mitogenesis(42) . Activation of PKC also diminishes the hydrolysis of phosphatidylinositol 4,5-bisphosphate and therefore the production of DAG, presumably by phosphorylation and inactivation of a PI-specific PLC(43) . It was of interest, therefore, to examine both of these negative feedback responses in our cell systems.

Analysis of the control cell line R6-SVXc1 revealed a high level of saturable binding of I-EGF to EGF receptors. Following exposure to 100 ng/ml TPA for 30 min, this binding was markedly reduced (Fig. 4A). This reduction was due to a decreased affinity of the EGF receptor for its ligand and not a decrease in the number of receptors (data not shown). By 24 h of TPA treatment, EGF binding returned to the control level (Fig. 4A), presumably reflecting extensive down-regulation of the endogenous isoforms of PKC(27) .


Figure 4: Enhanced negative feedback regulation of EGF binding and DAG production in PKCbetaI- and PKCalpha-overexpressing R6 cells. I-EGF binding (A) and cellular levels of DAG (B) were determined for subconfluent control R6-SVX, R6-PKC3betaI, and R6-bPKC4alpha cells following treatment without (Ctrl) or with 100 ng/ml (160 nM) TPA for 30 min (30`), 6 h and 48 h, as described under ``Experimental Procedures.'' All values shown are means of three independent assays ± SE.



In untreated R6-bPKC4alpha cells, EGF binding was similar to that of the control cells. Incubation of these cells with TPA for 30 min decreased EGF binding by an extent that was similar to that seen with the control cells (Fig. 4A). However, EGF binding did not return to the control level in long term TPA-treated R6-bPKC4alpha cells (Fig. 4A). Since in both the control and the PKCalpha-overexpressing cell lines the levels of EGF receptor protein and mRNA were not affected by prolonged treatment with the TPA (data not shown), the latter result is probably due to persistent phosphorylation of the EGF receptor by PKCalpha in the overexpresser cells. This interpretation is in agreement with the relative resistance of the overexpressed PKCalpha to TPA-induced down-regulation ( Fig. 1and Fig. 2A) and may indeed contribute to the growth inhibition mediated by TPA in PKCalpha-overexpressing cells. Prolonged inhibition of EGF receptor binding was also seen in TPA-treated R6-PKC3betaI cells (Fig. 4A). In these cells, even the basal level of EGF binding was slightly decreased (Fig. 4A). Presumably, these effects also reflect prolonged phosphorylation of the EGF receptor by the overexpressed PKCbetaI, although the persistent levels of PKC and PKC in TPA-treated R6-PKC3betaI cells (Fig. 2B) may also contribute to these effects.

When we analyzed cellular levels of DAG, we found that the untreated control cells had a relatively high level of DAG. This level was markedly decreased in control cells treated with TPA for 30 min (Fig. 4B). By 48 h, however, it had returned to the original level, presumably reflecting down-regulation of the endogenous isoforms of PKC(27) . The cellular levels of DAG in untreated R6-bPKC4alpha and R6-PKC3betaI cells were much lower than that in the control cells and remained low following treatment with TPA for either 30 min or 48 h (Fig. 4B). Thus, high levels of either PKCalpha or PKCbetaI exert a negative effect on cellular levels of DAG, presumably by inhibiting the activity of PI-specific PLC, although other explanations have not been excluded.

Growth of PKC-overexpressing Cells in Low Serum

The finding that R6-PKC3betaI cells grew faster and to higher saturation densities than the control or PKCalpha overexpresser cells suggested that the former cells may be less dependent on external growth factors.

Therefore, we carried out growth studies in the presence of a limiting concentration of CS. When grown in 1% CS, control R6-SVXc1 cells exhibited a doubling time of 23 h (Fig. 5A) and grew to a saturation density of about 3 times 10^6 (Fig. 5B). TPA did not significantly influence these growth properties (Fig. 5). R6-bPKC4alpha cells were more compromised with respect to growth rate and saturation density when compared to the control cells, and the presence of low or high doses of TPA led to a further growth inhibition (Fig. 5). On the other hand, the doubling time of R6-PKC3betaI cells was shorter than that of control or PKCalpha-overexpressing cells, and the former cells reached a higher saturation density, especially in the presence of TPA.

These results indicate that PKCalpha overexpression may increase the dependence of R6 cells on growth factors, hence the growth inhibitory effect. By contrast, R6-PKC3betaI cells appear to have a decreased growth factor requirement, especially in the presence of TPA. We have also found that R6-PKC3betaI cells survive for a longer period of time in serum-free medium than control or R6-bPKC4alpha cells (data not shown).

Growth Factor Production by PKC-overexpressing Cells

The studies shown in Fig. 5indicated that the R6-PKC3betaI cells grew better in 1% CS than either the R6-bPKC4alpha or the control cells. In previous studies we obtained evidence that R6 cells that overexpress PKCbetaI elaborate a growth factor(39, 44, 45) . It was of interest, therefore, to compare the dependence of these two types of cell lines on specific growth factors. Cell cultures were starved of serum for 30 h to obtain quiescent G(o) cells. The cells were then exposed to 10% CS or various growth factors, and the extent of induced DNA synthesis was determined by [^3H]thymidine incorporation. [^3H]Thymidine was added to the cultures at 15 h following the serum or growth factor additions, and the extent of incorporation of radioactivity into DNA was measured at 18 h. We found that maximal induction of DNA synthesis was observed in the R6-PKC3betaI, R6-bPKC4alpha, and R6-SVXc1 (or R6-C1) cells during this time period (data not shown). The extent of DNA synthesis in response to 10% CS was taken as the positive control value (100%) for each cell line. The addition of EGF or PDGF to the quiescent vector control cells did not result in significant induction of DNA synthesis (Fig. 6A). This result is consistent with the dependence of normal fibroblasts on multiple growth factors for mitogenesis(44) . A similar finding was obtained with the R6-bPKC4alpha cells (Fig. 6A) and also other PKCalpha-overexpressing cell lines (R6-bPKC3alpha, R6-bPKC7alpha) (data not shown). On the other hand, EGF or PDGF alone were able to stimulate DNA synthesis in quiescent cultures of R6-PKC3betaI cells, to 60-70% of the level obtained with 10% CS (Fig. 6A).


Figure 6: Effects of serum, single growth factors, and the conditioned medium of PKCbetaI-overexpressing cells on the DNA synthesis of R6 cells. A, induction of DNA synthesis (measured as [^3H]thymidine incorporation) 15-18 h following the addition of 10% serum (CS) or a single dose of EGF (5 ng/ml) or PDGF (10 ng/ml) to serum-starved control R6-C1, R6-PKC3betaI, or R6-bPKC4alpha cells. In the absence of any additions, [^3H]thymidine incorporation with R6-C1 cells was less than 1% of the control value (plus 10% serum); with PKC-overexpressing cells, the corresponding value was between 5 and 7.5%. B, induction of DNA synthesis following addition of CM from R6-PKC3betaI (CMbeta3), R6-PKC5betaI (CMbeta5), 10T1/2-PKC4betaI (CMbeta4), or R6-bPKC4alpha (CMalpha) cells to serum-starved R6-SVXc1 vector control cells. The additions were in the presence or absence of 5 ng/ml EGF. [^3H]Thymidine incorporation assays were done in triplicates in 96-microwell plates (10^4 cells/well), and the results are expressed as mean values/well. The error bars indicate the standard deviations.



R6-PKC3betaI cells might represent a clone which fortuitously contains an activated oncogene that abrogates the need for a second growth factor. This seems to be unlikely since we found that other clones that overexpress PKCbetaI also displayed a decreased requirement for growth factors. (^2)Therefore, we examined the possibility that the cells that overexpress PKCbetaI might excrete a growth factor. Serum-free CM was collected from cultures of R6-bPKC4alpha and R6-PKC3betaI cells and tested on quiescent serum-starved R6-SVXc1 cells for its capacity to stimulate DNA synthesis. The CM from the PKCbetaI-overexpressing cells induced DNA synthesis to a level that was 30% of that obtained with 10% CS, and in combination with EGF the extent of DNA synthesis was about 70% of that obtained with 10% CS (Fig. 6B). On the other hand, the CM from the PKCalpha-overexpressing cell line when tested either alone or in combination with EGF did not significantly stimulate DNA synthesis in the serum-starved vector control cells (Fig. 6B). Thus, R6 cells that express high levels of PKCbetaI generate and secrete a growth factor(s) (see also Refs. 39, 44, 45), but this is not the case for R6 cells that express high levels of PKCalpha.

We wondered whether the amount of secreted growth factor correlated with the extent of PKCbetaI activation. We therefore exposed R6-PKC3betaI cells to various doses (10-300 nM) of TPA for 0-24 h and then collected the respective conditioned media. Surprisingly, none of these media were capable of inducing DNA synthesis of quiescent R6-SVXc1 cells better than the medium from untreated R6-PKC3betaI cells (data not shown).

Finally, to ensure that the production of the growth factor results from the overexpression of PKCbetaI and not just from a rare mutagenic event, we collected the conditioned media of another R6 cell clone overexpressing PKCbetaI 20-fold (R6-PKC5betaI) as well as from a mouse 10T1/2 fibroblast cell clone overexpressing PKCbetaI 11-fold (10T1/2-PKC4betaI). In both cases we detected a growth stimulatory activity in the conditioned medium although this activity was somewhat less than that of R6-PKC3betaI cells (Fig. 6B).


DISCUSSION

The purpose of the present study was to explore molecular mechanisms which might explain why, when stably overexpressed in a rat embryo fibroblast cell line, PKCbetaI and PKCalpha, two closely related PKC isoforms, exert different effects on growth control. We find that both isoforms, when overexpressed, provoke similar extents and kinetics of immediate early gene induction and negative feedback responses despite the fact that PKCbetaI stimulates and PKCalpha inhibits growth. However, R6 cells overexpressing PKCbetaI secrete a growth factor which is not produced by R6 cells that overexpress PKCalpha. This growth factor may act in an autocrine fashion to stimulate autonomous growth and transformation of cells overexpressing PKCbetaI.

The reciprocal effects of overexpression of PKCalpha and PKCbetaI on R6 cells is somewhat surprising because the two isoforms share considerable homology and cofactor requirements, and in subcellular systems they phosphorylate similar substrates(1, 2, 3, 28, 40) . The differences in their effects on growth do not appear to be due to different extents of activation of the two isoforms in the R6 derivatives, since both isoforms underwent complete membrane translocation in response to treatment with TPA. The two isoforms, however, exhibit different sensitivities to down-regulation in response to TPA and, in addition, appear to influence the down-regulation of the endogenous PKC isoforms PKC and PKC. It is therefore possible that the high transformation state of TPA-treated R6-PKC3betaI cells is not only due to the initial activation of overexpressed PKCbetaI but also to the persistent presence and/or activation of endogenous PKC. Consistent with this idea is our recent finding that overexpression of PKC in R6 cells is associated with enhanced cellular growth and transformation(23) . By contrast, PKCalpha appears to be an isoform which impedes with the growth of fibroblasts, and the partial resistance of this PKC isoform to TPA-induced down-regulation even enhances the growth inhibitory effect in TPA-treated cells. To obtain a more concise picture of the role of PKC isoforms in cell growth, we attempted to block TPA-induced down-regulation of the overexpressed and endogenous PKC isoforms in R6-PKC3betaI and R6-bPKC4alpha cells by cellular treatment with cell-permeable calpain inhibitors (E64-d and calpain inhibitor I and II). Although down-regulation of the PKC isoforms could be protracted by 3-6 h, no complete blockage was observed. In addition, prolonged exposure of fibroblasts to single or repetitive doses of calpain inhibitors turned out to be cytotoxic (data not shown).

Another reason for the differential growth effects of PKCalpha and PKCbetaI may be their association with distinct subcellular structures, thereby having access to different substrates and signaling pathways. There are reports that PKCalpha can be detected in the nucleus (46) and in focal adhesion plaques (33) in fibroblasts and that PKCbeta can be detected in the nucleus of liver (47) and leukemic cells(48) . However, neither the nuclei, nor focal adhesion plaques were enriched in PKCalpha or PKCbetaI in the R6 cell derivatives used in the present studies. (^3)Differential association with other subcellular structures, such as the Golgi, mitochondria, endo- or exocytotic vesicles, or gap junctions, have, however, not been ruled out. We are currently searching for isoform-specific cell substrates by comparing phosphoprotein maps (40) between phorbol ester-treated R6-bPKC4alpha and R6-PKCbetaI cells. It will also be of interest to examine whether putative down-stream effectors of PKC such as c-raf(7, 8) , MEK/MAP kinases(9, 49) , ras-GAP (6, 50) , S6 kinases(51) , or glycogen synthase kinase 3beta (GSK-3) (52) are differentially phosphorylated and/or activated in response to phorbol ester treatment of R6-bPKC4alpha and R6-PKC3betaI cells.

It is astonishing to find that overexpression of both PKCalpha and PKCbetaI has similar effects on the induction of immediate early genes, given the fact that higher expression of these gene products has been shown to be associated with enhanced proliferation(11, 13, 14) . An additional regulation of the transcriptional activity of these gene products, however, often occurs by post-translational phosphorylation. It will therefore be important to compare the phosphorylation state and gel shift activity of c-jun, c-fos, and c-myc between untreated and TPA-treated R6-bPKC4alpha or R6-PKC3betaI cells. In addition, differences in the expression of other immediate early gene products (11, 13, 14, 52) or proteins that regulate phases of the cell cycle, such as the retinoblastoma gene product(53) , p53(54) , cdc2-like kinases(55) , or specific cyclins (55) , remain to be explored.

PKC has been shown to negatively influence growth by feedback inhibiting EGF binding and DAG production through phosphorylation of the EGF receptor and PLC, respectively(1, 2, 3, 42, 43) . In the present study, we found that TPA-induced inhibition of EGF receptor binding and TPA-induced decreased cellular levels of DAG were enhanced in the cells that overexpressed either PKCalpha or PKCbeta. Therefore, these negative feedback inhibitory effects cannot explain why overexpression of these two isoforms exert reciprocal effects on the growth of these cells. It has recently been recognized that DAG may also be generated by a PLD-mediated hydrolysis of phosphatidylcholine (reviewed in (56) ). In addition, Pai et al.(57) observed enhanced activation of PLD, and therefore DAG production, by TPA and other agonists in R6 cells overexpressing PKCbetaI (the presently described R6-PKC3betaI). At first glance, these results may contradict our present finding that DAG levels are decreased in TPA-treated PKCbetaI-overexpressing cells. In the previous study, the induction of DAG production by the PLD-mediated pathway is, however, transient and takes place at very early time points following PKC activation (5-15 min). Longer treatments of R6-PKC3betaI cells with phorbol ester (>30 min) also led to a diminuation of DAG levels below control levels(57) , as reported in the present study.

The most striking difference we observed between R6-bPKC4alpha and R6-PKC3betaI cells was that the latter cells exhibited a decreased requirement for growth factors, most likely due to the secretion of an autocrine acting growth factor(s) into their medium. Studies in progress suggest that the autocrine growth factor is a novel protein with a molecular mass of about 68 kDa.^2 Its production appears to be constitutive in at least four different PKCbetaI-overexpressing fibroblast cell lines, two R6 fibroblast derivatives (R6-PKC3betaI and R6-PKC5betaI) (Fig. 6B), one 10T1/2 derivative (10T1/2-PKC4betaI) (Fig. 6B), and one NIH/3T3 derivative (NIH-PKC6betaI) (data not shown). All these cell clones exhibit transformed phenotypes, albeit to different degrees (Refs. 19, 30, and data not shown). Neither various growth conditions (high/low serum, growth at post-confluence) nor cellular treatments with defined growth factors or phorbol esters resulted in an enhanced secretion of the autocrine growth factor into the conditioned medium of these cells. A possible explanation for this finding may be that the growth factor gene promoter becomes demethylated (and thereby activated) as soon as the introduced PKCbetaI has started to function within a cell. Alternatively, the growth factor may be constitutively produced by normal fibroblasts but reside at an intracellular site in an inactive, non-secreted form. A small amount of active PKCbetaI would then trigger maturation and/or secretion of the growth factor (for example through a PKC-evoked signaling pathway). To resolve these issues definitely, we are awaiting the molecular cloning and further characterization of the novel growth factor.

The notion that the growth factor secreted from PKCbetaI-overexpressing cells contributes to the transformed phenotype of these cells is further bolstered by the recent observation that highly transformed R6 cells overexpressing PKC also produce an autocrine-acting growth factor. (^4)Although the two factors may not be the same molecular entity, this latter finding indicates that the establishment of an autocrine growth factor loop may be a general mechanism by which certain PKC isoforms transform cells. By contrast, R6 cells overexpressing PKCalpha are obviously incapable of producing a mitogenic factor. Apart from three different clones of R6 cells overexpressing PKCalpha to various extents (R6-bPKC4alpha, R6-bPKC3alpha, and R6-bPKC7alpha), we have also examined Balb/c and NIH3T3 fibroblasts overexpressing large amounts of either bovine or mouse PKCalpha (22) (data not shown). In addition, we have treated all the above cell lines with TPA for various time periods to try to stimulate growth factor production in a PKC-dependent manner. In no instances was a growth stimulatory activity detected in the conditioned medium of PKCalpha-overexpressing cells.

It should be stressed that the growth inhibition of R6-PKC4alpha cells is not solely due to the inability of these cells to produce an autocrine growth factor. We infer this because the conditioned medium of R6-PKC3betaI cells could neither stimulate the DNA synthesis nor overcome the TPA-induced growth inhibition of R6-PKC4alpha cells (data not shown). This finding suggests that PKCalpha is linked to a intracellular signaling pathway which negatively regulates the cell cycle. We are currently in progress to identify the molecular players of such a signaling pathway.

Taken together, the present study indicates that two rather similar isoforms of PKC can produce both redundant effects (similar changes in morphology, expression of early response genes, and regulation of negative feedback circuits) and also opposite effects (growth inhibition or growth stimulation, respectively) when stably expressed at high levels in the same cell type. They may, therefore, play different roles in the process of carcinogenesis and the growth of established tumors.


FOOTNOTES

*
This work was supported by Swiss National Science Foundation Grant 31-36152.92 and 31-34600.92 (to C. B.), the DAAD genetechnology section of the German Federal Government (to M. U.), Grant CA 02111 from the National Cancer Institute, and an award from the Markey Charitable Trust (to I. B. W). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 413-782-6347; Fax: 413-782-6341.

(^1)
The abbreviations used are: PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; DAG, diacylglycerol; EGF, epidermal growth factor; GSK-3, glycogen synthase kinase-3; Me(2)SO, dimethyl sulfoxide; PI, phosphatidylinositol; CS, calf serum; PDGF, platelet-derived growth factor; PKA, cAMP-dependent protein kinase; MAP kinase, mitogen activated protein kinase; MEK, MAP kinase kinase; TPA, 12-O-tetradecanoyl phorbol 13-acetate; PDBu, phorbol 12,13-dibutyrate; GAPDH, glyceraldehyde phosphate dehydrogenase; CM, conditioned medium; DMEM, Dulbecco's modified Eagle's medium.

(^2)
M. Ueffing, unpublished results.

(^3)
S. Jaken, unpublished results.

(^4)
I. B. Weinstein, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to D. Fabbro for the antisera to PKC and PKC, R. Imber, and I. Filipuzzi for cloning the bovine PKCalpha cDNA into pZIP-Neo SV(X); Koji Nomoto for helping with the DAG kinase assay;, and T. Curran, L.-L. Hsieh and R. Witte for providing us with the c- fos/c- jun and c- myc cDNA probes and PDGF, respectively. We thank Barbara Castro and John Rozankowski for valuable assistance in preparing the manuscript.


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