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Endocrinology Vol. 138, No. 11 4657-4664
Copyright © 1997 by The Endocrine Society

ARTICLES

Transforming Growth Factor-ß1 Inhibits Membrane Association of Protein Kinase C{alpha} in a Human Prostate Cancer Cell Line, PC31

Marilyn L. G. Lamm, Denise D. Long, Shannon M. Goodwin and Chung Lee

Department of Urology, Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Marilyn L. G. Lamm, Department of Urology, Northwestern University Medical School, Chicago, Illinois 60611. E-mail: mlamm{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The postreceptor signaling pathway(s) that mediates the effects of transforming growth factor-ß1 (TGF-ß1) is incompletely understood. The present study investigated the involvement of protein kinase C (PKC) in the growth-inhibitory action of TGF-ß1 in PC3, a human prostate cancer cell line. PKC{alpha}, the only conventional PKC isoform detected in PC3 cells, appeared to be constitutively active based on its presence in both Triton-soluble membrane fraction and cytosol. However, levels of membrane-associated PKC{alpha} were decreased by a growth-inhibitory dose of TGF-ß1. The response to TGF-ß1 was rapid (within 5 min), time dependent, isoform specific, and occurred without apparent changes in levels of total PKC{alpha} protein. TGF-ß1 also decreased the levels of membrane-associated PKC activity coincident with its inhibitory effect on PKC{alpha}’s membrane association. Inhibition of PKC activity appeared to be associated with growth inhibition in PC3 cells, because chelerythrine (a specific PKC inhibitor) likewise decreased cell proliferation. Taken together, our data suggest that inhibition of PKC activity, at least in part due to inactivation of PKC{alpha}, is an early event associated with TGF-ß1 postreceptor signaling that might mediate suppression of cell proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSFORMING growth factor-ß (TGF-ß) is a multifunctional regulator of cellular growth and differentiation (1, 2). It is capable of stimulating or inhibiting these processes depending on cell type and the extracellular milieu or culture conditions. In a variety of normal and transformed cells, particularly those of epithelial, endothelial, and hematopoetic origin, TGF-ß is a potent inhibitor of cell proliferation (1, 2). However, cells often escape the growth inhibitory influence of TGF-ß, and this condition may lead to uncontrolled growth or malignancies (3). A full understanding of TGF-ß signaling is indeed critical to normal and tumor cell biology.

The growth-inhibitory action of TGF-ß is initiated through activation of cell surface serine/threonine kinase receptors (type I and type II), which form a heteromeric complex on ligand binding (4, 5). In this functional conformation, the ligand-bound and constitutively phosphorylated type II receptor phosphorylates and activates the type I receptor to transduce the signal intracellularly (6, 7, 8). Following receptor activation, TGF-ß’s action has been linked to down-regulation of the expression and/or activity of cell cycle regulatory proteins such as c-myc, cyclins, cyclin-dependent kinases, and Rb, the product of the retinoblastoma gene (9, 10). These changes mediate the TGF-ß-induced arrest of the cell cycle in the late G1 phase (9, 10). However, the signaling events that occur downstream of TGF-ß receptor activation and upstream of nuclear events that are characteristic of TGF-ß’s growth inhibitory action are less clearly understood.

Studies have recently identified certain cytoplasmic proteins that may function as possible postreceptor downstream mediators of TGF-ß’s growth-inhibitory action. The proteins hMAD-3 and hMAD-4 (products of genes that are human homologs of the Drosophila Mothers against decapentaplegic, Mad, gene) can synergistically decrease cyclin A gene expression, an indicator of growth arrest (11). The activity of these proteins is regulated by TGF-ß receptors, and hMAD-3 (but not hMAD-4) has been shown to associate with the TGF-ß receptor complex consistent with the phosphorylation of hMAD-3 (11). Given that MAD homologs may be able to translocate from the cytoplasm to the nucleus (12, 13, 14), it has been postulated that an hMAD-3/hMAD-4 complex may function as a transcriptional regulator of TGF-ß-responsive genes (11). The {alpha} subunit of p21ras farnesyltransferase has also been shown to associate with the TGF-ß receptor (15). The cytoplasmic enzyme farnesyltransferase is involved in the membrane localization and, thereby, activation of ras, a 21-kDa guanine nucleotide-binding protein that functions upstream of mitogen-activated protein kinases (MAPKs), which mediate the mitogenic signals from growth factors (16, 17). A protein kinase member of the MAP kinase kinase kinase (MAPKKK) family whose activity can be stimulated in response to TGF-ß (TAK1: TGF-ß-activated kinase) has also been identified (18). The possible involvement of the ras-MAPK pathway in TGF-ß signaling is supported by the surprising evidence that shows that TGF-ß treatment results in a rapid activation of ras and MAPK in TGF-ß-sensitive but not in TGF-ß-insensitive intestinal epithelial cells (19, 20, 21).

Another signaling cytoplasmic protein that has been linked to the growth-inhibitory action of TGF-ß1 is protein kinase C (PKC). TGF-ß1, which inhibits mitogenesis induced by basic fibroblast growth factor in vascular smooth muscle cells, causes the translocation of PKC activity from the cytosolic to the membrane fractions, indicative of PKC activation (22). The phorbol ester phorbol 12-myristate 13-acetate (PMA), which generally activates PKC, has been shown to potentiate the growth-inhibitory effect of TGF-ß1 in prostate cancer cell lines PC3 and PC-3U (23). PKC activation has also been linked to TGF-ß’s effects on gene expression and cellular differentiation (24, 25, 26, 27). Other studies, however, have detected either no activation of PKC by TGF-ß or a suppressive effect of TGF-ß on PKC activity (28, 29, 30, 31). The seemingly conflicting role of PKC in TGF-ß signaling may be due, in part, to the heterogeneity of PKC isoforms in a given cell type.

PKC is a family of phospholipid-dependent serine/threonine kinases that regulate cell growth and differentiation (32, 33, 34). To date, 12 mammalian PKC isoforms have been identified and classified into three groups based on their structure and cofactor regulation: a) conventional PKC isoforms ({alpha}, ßI, ßII, and {gamma}) require phosphatidylserine, diacylglycerol, and Ca++ for activation; b) novel PKC isoforms ({delta}, {epsilon}, {eta}, {theta}, and µ) require phosphatidylserine and diacylglycerol for activation but are Ca++ independent; and c) atypical PKC isoforms ({zeta}, {lambda}, and {iota}) are activated by phospholipids but are Ca++/diacylglycerol independent. Because PKC isoforms exhibit specific patterns of cellular expression, localization, cofactor dependence, and substrate requirements, it has been implied that each member of the PKC family may play a unique role in signal transduction (32, 33). Therefore, to fully understand the role of PKC in TGF-ß signaling, it is necessary to define the involvement of specific PKC isoforms in this event.

The present study shows that TGF-ß1 is growth inhibitory in a human prostate cancer cell line, PC3. In these cells, TGF-ß1 also inhibits the membrane association of PKC{alpha} in a rapid, time-dependent, and isoform-specific manner. The inhibitory effect of TGF-ß1 on PKC{alpha} occurs coincident with a TGF-ß1-induced decrease in membrane-associated PKC activity. These data demonstrate that TGF-ß1-induced inhibition of PKC activity, at least in part due to inactivation of PKC{alpha}, is an early event associated with TGF-ß1 postreceptor signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
PC3 cells were obtained from American Type Culture Collection (Rockville, MD). Human TGF-ß1 was purchased from Collaborative Biomedical Products (Bedford, MA). Materials were purchased from the following sources: culture media reagents, Gibco BRL (Gaithersburg, MD); FBS, Summit Biotechnologies (Ft. Collins, CO); antispecific PKC isoform antibodies, Transduction Laboratories (Lexington, KY); chelerythrine, LC Laboratories (Woburn, MA); electrophoresis purity reagents and goat antimouse antibody, Bio-Rad Laboratories (Richmond, CA); prestained molecular weight markers, Diversified Biotech (Newton Center, MA); immunoblot ECL reagents, Amersham Corp. (Arlington Heights, IL); phorbol 12-myristate 13-acetate, protease inhibitors, and most other reagents, Sigma (St. Louis, MO). Radiochemicals were obtained as follows: [3H]thymidine (6.7 Ci/mM), Amersham Corp.; [{gamma}-32P]ATP (3000 Ci/mmol), New England Nuclear Research Products (Boston, MA).

Cell culture
PC3 cells were routinely maintained in culture medium [RPMI 1640 containing penicillin (100 U/ml) and streptomycin (100 µg/ml)] supplemented with 10% FBS and kept in a 37 C-5% CO2 incubator.

[3H]Thymidine incorporation
[3H]Thymidine incorporation was determined as previously described (35), with some modifications. Cells were seeded at approximately 2 x 104 per well (24-well plate) in culture medium supplemented with 10% FBS and allowed to adhere for 24 h. Then, medium was replaced with new culture medium supplemented with 1% FBS and containing TGF-ß1 or chelerythrine at preselected concentrations. Control vehicles for TGF-ß1 and chelerythrine were culture medium and 0.003% dimethylsulfoxide (DMSO), respectively.

Following 22 h in culture, 5 µCi/well [3H]thymidine was added, and incubation was continued for 4 h. Cells were then washed with cold culture medium and harvested through repeated pipetting. Cells were collected by centrifugation (16,000 x g at 4 C for 10 min) and incubated with ice-cold 10% trichloroacetic acid at 4 C for 60 min. The pellet obtained following subsequent centrifugation was incubated with 0.4 N NaOH at room temperature for 20 min and subjected to scintillation counting.

Cellular fractionation
Cells were seeded at approximately 106/75 cm2 flask in culture medium supplemented with 10% FBS and allowed to grow for 2–3 days. Then, medium was replaced with that supplemented with 1% FBS, and incubation was continued for 24 h. Cells then were washed twice with PBS and incubated in culture medium supplemented with 1% FBS containing TGF-ß1 or PMA at preselected concentrations. Control vehicle for PMA was 0.05% DMSO. Incubation was continued for varying periods of time.

At the end of the experiment, cells were washed twice with cold PBS and scraped into 1 ml cold homogenization buffer containing protease/phosphatase inhibitors [50 mM Tris-HCl, pH 7.5, 5 mM EDTA/EGTA, 10 mM MgCl2, 50 mM ß-glycerophosphate, 2 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium vanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin]. The cells were homogenized in a Dounce homogenizer with 30 strokes of the pestle, and the homogenate was centrifuged at 100,000 x g for 60 min at 4 C. The supernatant was used as cytosol fraction. The pellet was gently stirred for 30 min at 4 C in 100 µl homogenization buffer added with 1% Triton X-100. After centrifugation at 100,000 x g for 30 min at 4 C, the obtained supernatant was diluted to 1 ml with homogenization buffer and was used as the Triton-soluble membrane fraction.

Cell lysates
Cells (grown in 75 cm2 flasks as mentioned above) were washed with PBS and scraped into 0.5 or 1 ml lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, and protease/phosphatase inhibitors as mentioned above). Cells were then incubated in lysis buffer for 60 min at 4 C. The supernatant (total cell lysate) was obtained following centrifugation at 16,000 x g at 4 C for 20 min.

Immunoprecipitation of PKC{alpha}
Lysates were pre-cleared with protein A Sepharose (33% in lysis buffer) for 1 h at 4 C. Following centrifugation, the supernatant (containing ~100 µg protein) was incubated with or without antibody against PKC{alpha} (1:10 final dilution) for 3 h at 4 C. Then, 50 µl protein A Sepharose was added, and incubation was continued for 30 min. The pellet obtained following centrifugation was washed four times with buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS) and boiled in gel electrophoresis sample buffer. The supernatant (immunoprecipitate) was used for subsequent gel electrophoresis.

SDS-PAGE
Gel electrophoresis was performed using 5% stacking and 7.5% or 10% resolving polyacrylamide slab gels (36). Equal volumes of cytosol and Triton-soluble membrane fractions were loaded onto gel lanes. In some experiments, equal amounts of protein (as determined by protein assay; Ref.37) were used.

Immunoblot analysis
Proteins were transferred onto a nitrocellulose membrane overnight at 4 C in buffer consisting of 25 mM Tris, 192 mM glycine, pH 8.3, and 10% methanol (38). After transfer, the membrane was blocked for 3 h at room temperature in buffer (PBS and 0.1% Tween-20: PBS-T) containing 10% milk. The membrane was incubated with antispecific PKC isoform antibodies for 1 h at room temperature in PBS containing 5% BSA. Following washes in PBS-T containing 5% milk, the membrane was incubated with horseradish peroxidase-conjugated goat antimouse antibody for 30–60 min at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence using the ECL kit according to manufacturer’s directions (Amersham Corp.). Bands were quantitated by densitometric scanning.

PKC activity assay
PKC activity in Triton-soluble membrane fractions was measured as the ability to phosphorylate a specific PKC substrate, neurogranin peptide, using an assay kit from Promega (Madison, WI). The reaction was carried out for 5 min at 30 C. The biotinylated-[32P]-labeled neurogranin peptide was recovered from the reaction mix with the streptavidin matrix membrane, which was then subjected to scintillation counting. Basal PKC activity (measured in the absence of phospholipids) was subtracted from PKC activity measured in the presence of phospholipids to obtain enzymatic PKC activity. For a positive control, a partially purified preparation of PKC from rat brain (a gift from Dr. Evelyn T. Maizels, Northwestern University, Chicago, IL; Refs. 39, 40) was included in the assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TGF-ß1 on DNA synthesis
As shown in Fig. 1Go, TGF-ß1 inhibits the incorporation of [3H]thymidine in PC3 cells grown in culture medium supplemented with 1% FBS. [3H]Thymidine incorporation was decreased by approximately 50% with 10 ng/ml TGF-ß1, the highest concentration used in the present study. A decrease in cell number was likewise observed following incubation of PC3 cells with TGF-ß1 (data not shown).



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  		Figure 1. Inhibitory effect of TGF-ß1 on [3H]thymidine incorporation. Cells were incubated in culture medium supplemented with 1% FBS containing varying concentrations of TGF-ß1. At 22 h of culture, [3H]thymidine was added, and incubation was continued for 4 h as described in Materials and Methods. Radioactivity in acid-insoluble cell fraction was determined by scintillation counting. Each bar represents mean ± SEM of three experiments.

 
PKC isoform expression
Given the diversity in PKC isoforms (32, 33, 34), the expression of PKC isoforms in PC3 cells was examined using immunoblot analysis with antispecific PKC isoform antibodies. PKC isoforms in PC3 cell lysates were identified based on their molecular weights and comigration positions on gel electrophoresis relative to control macrophage or Jurkat cells. At least six PKC isoforms were detected in total lysates of PC3 cells: 1) conventional PKC{alpha}; 2) novel PKC{delta}, {epsilon}, µ; and 3) atypical PKC{iota} and {zeta} (data not shown).

Conventional PKCß and novel PKC{theta} were not detected even in overexposed immunoblots. These results are in agreement with an earlier report that PKCß and PKC{theta} RNAs were not detected in PC3 and androgen-dependent LNCaP prostate cancer cells; however, they were present in normal human prostate and DU145, another androgen-independent prostate carcinoma cell line (41).

Effect of TGF-ß1 on translocation of PKC{alpha}
It is generally considered that each member of the PKC family of enzymes may play a role in signal transduction (32, 33). Although several PKC isoforms were detected in PC3 cells, the present study initially determined the effect of TGF-ß1 on PKC{alpha}, the only conventional PKC isoform in PC3 cells. PKC{alpha} is a ubiquitous isoform that is critical in the signaling pathways of cell proliferation (34). One measure of PKC activation is the translocation or redistribution of PKC isoforms from the cytosolic fraction (where PKC is generally in its inactive state) to a membrane fraction (where PKC exhibits catalytic activity: Ref.42). Therefore, changes in the distribution patterns of PKC{alpha} in response to a growth-inhibitory dose of TGF-ß1 was investigated. This was accomplished through fractionation of PC3 cells into cytosolic and Triton-soluble membrane components followed by immunoblot analysis for the PKC isoform and densitometric scanning of autoradiograph bands.

Under basal conditions of culture, PKC{alpha} was constitutively localized in the membrane in addition to the cytosol (Fig. 2AGo). The occurrence of PKC{alpha}, or any other isoform, in its activated membrane-associated state is likely in response to the presence of mitogens or other factors in the serum-supplemented (1% FBS) culture medium. When PC3 cells were treated with a growth-inhibitory dose (10 ng/ml) of TGF-ß1 a rapid (within 5 min) and time-dependent decrease in the levels of immunoreactive membrane-associated PKC{alpha} and a concomitant increase in cytosolic PKC{alpha} were detected (Fig. 2Go, A and A'). The disparity in the levels of membrane-associated PKC{alpha} between control and TGF-ß1-treated cells was most evident following 60 min of incubation, i.e. the mean level of membrane-associated PKC{alpha} in TGF-ß1-treated cells was decreased by approximately 75% compared with that in control cells (n = 5 experiments).



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  		Figure 2. Short-term effect of TGF-ß1 on translocation of PKC{alpha} and PKC{iota}. Cells were incubated in culture medium supplemented with 1% FBS in absence or presence of a growth-inhibitory dose of TGF-ß1 (10 ng/ml) for times indicated. Subsequently, cells were homogenized and separated into cytosol and Triton-soluble membrane fractions as described in Materials and Methods. Proteins from equal volumes (50 µl) of cytosol and membrane fractions were separated by SDS-PAGE and subjected to immunoblot analysis using specific antibodies against PKC{alpha} (A) and PKC{iota} (B). A' and B', Immunoreactive bands from immunoblots were quantitated by scanning densitometry. Levels of PKC{alpha} and PKC{iota} in cytosol (•) or membrane ({blacksquare}) fractions of cells that were treated with TGF-ß1 for varying periods of time were compared relative to nontreated cells at incubation time 0. Similar profiles in distribution of PKC{alpha} and PKC{iota} were observed in four other experiments.

 
To determine whether the TGF-ß1 effect on PKC{alpha} was isoform specific, changes in the cellular distribution of the novel PKC{delta}, {epsilon}, and µ and of the atypical PKC{iota} and {zeta} were then examined. As with PKC{alpha}, these isoforms were also detected in both the cytosol and membrane fractions of PC3 cells under basal culture conditions; however, PKC{iota} was the only isoform observed to be constitutively localized in very high levels in both fractions (data not shown). TGF-ß1 treatment for up to 60 min did not cause major changes in the membrane-associated levels of the other PKC isoforms detected in PC3 cells, and this response is shown in detail for PKC{iota} in Fig. 2Go, B and B'. In contrast to its effect on PKC{alpha}, TGF-ß1 did not induce dramatic changes in the localization of PKC{iota}, which remained about equally distributed between the cytosolic and membrane fractions in both control and TGF-ß1-treated cells throughout the duration of the experiment (Fig. 2Go, B and B').

To determine whether the PKC{alpha} response was specific to TGF-ß1, PC3 cells were incubated with 100 nM PMA. In contrast to the response caused by TGF-ß1, PMA (an activator of PKC) caused an immediate (within 5 min) and almost complete loss of PKC{alpha} from the cytosol, which was sustained for at least 60 min (Fig. 3Go).



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  		Figure 3. Short-term effect of PMA on translocation of PKC{alpha}. Cells were incubated in culture medium supplemented with 1% FBS in absence or presence of 100 nM PMA for times indicated. Control vehicle was 0.05% DMSO. Cytosol and Triton-soluble membrane fractions were separated by SDS-PAGE and probed for PKC{alpha} by immunoblot analysis.

 
The inhibitory effect of TGF-ß1 on membrane association of PKC{alpha} persisted for at least 60 min. However, on prolonged incubation with TGF-ß1, PKC{alpha} was able to redistribute again to the membrane (Fig. 4Go). Densitometric scanning analysis of immunoreactive bands shown in Fig. 4Go revealed a 2-fold decrease in the levels of membrane-associated PKC{alpha} in TGF-ß1-treated cells following a 1 h incubation (228 and 114 densitometric units/mg protein for control and TGF-ß1-treated cells, respectively). By both 24 h and 48 h, the levels of membrane-associated PKC{alpha} in control and TGF-ß1-treated cells were almost similar (200 and 171 densitometric units/mg protein for control and TGF-ß1-treated cells, respectively).



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  		Figure 4. Long-term effect of TGF-ß1 on translocation of PKC{alpha}. Cells were incubated in culture medium supplemented with 1% FBS in absence or presence of TGF-ß1 (10 ng/ml) for times indicated. Equal amounts of protein (35 µg) from cytosol (C) and Triton-soluble membrane (M) fractions were separated by SDS-PAGE and probed for PKC{alpha} by immunoblot analysis.

 
There were no apparent differences in the total amounts of PKC{alpha} protein detected in total lysates as well as in PKC{alpha} immunoprecipitates (using PKC{alpha}-specific antibody) obtained from PC3 cells incubated with or without TGF-ß1 for 60 min (Fig. 5Go). These results indicate that the decrease in membrane-associated PKC{alpha} levels in TGF-ß1-treated cells was not due to any apparent changes in PKC{alpha} total protein that may have been caused by proteolysis. Additionally, Northern blot analysis of total RNA from control and TGF-ß1-treated cells did not reveal any apparent changes in levels of PKC{alpha} mRNA expression (data not shown).



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  		Figure 5. Effect of TGF-ß1 on PKC{alpha} total protein. Cells were incubated in culture medium supplemented with 1% FBS in absence or presence of TGF-ß1 (10 ng/ml) for 60 min. Equal amounts of protein from either total cell lysates (35 µg) or immunoprecipitates of control or TGF-ß1-treated cells were separated by SDS-PAGE and probed for PKC{alpha} by immunoblot analysis. Immunoprecipitates (IMMUNOPPT) were obtained using a specific anti-PKC{alpha} antibody as described in Materials and Methods. Jurkat cell lysates were used as a positive control.

 
Effect of TGF-ß1 on PKC activity
The observed decrease in PKC{alpha} levels in the membrane fraction of PC3 cells (Fig. 2Go, A and A') was directly correlated with changes in PKC activity using an enzyme assay based on the phosphorylation of the neurogranin peptide, a specific PKC substrate. The levels of phospholipid-dependent activity of PKC in membrane fractions of PC3 cells were decreased rapidly (within 5 min) following treatment with a growth-inhibitory dose (10 ng/ml) of TGF-ß1 (Fig. 6AGo). TGF-ß1-induced inhibition of PKC activity lasted for at least 60 min (Fig. 6AGo). In contrast, the levels of membrane-associated PKC activity in cells treated with 100 nM PMA remained as high as that in control cells for up to 60 min (Fig. 6BGo).



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  		Figure 6. Effects on PKC Enzyme Activity. Cells were incubated in culture medium supplemented with 1% FBS in absence or presence of either 10 ng/ml TGF-ß1 (A) or 100 nM PMA (B) for times indicated. PKC activity in Triton-soluble membrane fractions of control and treated cells was determined by measurement of 32P phosphorylation of neurogranin, a specific PKC substrate, as described in Materials and Methods. Results are representative of two experiments.

 
Effect of chelerythrine on DNA synthesis
To determine whether inhibition of PKC activity might lead to growth suppression, PC3 cells were treated with an inhibitor of PKC activity. The benzophenanthridine alkaloid chelerythrine is a potent and highly selective inhibitor of PKC (43). As shown in Fig. 7Go, chelerythrine caused a decrease in [3H]thymidine incorporation in PC3 cells. An approximately 50% decrease in [3H]thymidine incorporation occurred following treatment of cells with 0.5 µM chelerythrine (Fig. 7Go). This is in agreement with the reported dissociation constant (Ki) (0.7 µM) for this inhibitor (43).



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  		Figure 7. Inhibitory effect of chelerythrine on [3H]thymidine incorporation. Cells were incubated in culture medium supplemented with 1% FBS containing varying concentrations of chelerythrine, a specific PKC inhibitor. Control vehicle was 0.003% DMSO. At 22 h in culture, [3H]thymidine was added as described in legend to Fig. 1Go. Each bar represents mean ± SE of triplicate determinations from one experiment. Results are representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates, for the first time, the ability of TGF-ß1 to inhibit PKC activity in PC3, a human prostate cancer cell line. The presence of type I and II TGF-ß receptors in PC3 cells has been previously established by our laboratory (44).

A growth-inhibitory dose of TGF-ß1 on PC3 cells caused a rapid (within 5 min) and time-dependent decrease in the levels of constitutively membrane-associated PKC{alpha} with a concomitant increase in cytosolic PKC{alpha}. This effect of TGF-ß1 appears to be specific and not due to an indiscriminate effect on PKC isoforms, because TGF-ß1 did not dramatically alter the membrane association of the other PKC isoforms detected in PC3 cells, as shown in detail for PKC{iota}. Moreover, the decrease in the levels of membrane-associated PKC{alpha} occurred in response to TGF-ß1 but not to PMA. Instead, PMA (a known activator of PKC) caused a rapid and almost complete loss of PKC{alpha} from the cytosol.

The mechanism(s) through which TGF-ß1 inhibits membrane association of PKC{alpha} is not known. TGF-ß1 may inhibit membrane association of PKC{alpha} by decreasing levels of intracellular Ca++. TGF-ß1 has been shown to inhibit intracellular Ca++ mobilization induced by platelet-derived growth factor in a mesangial cell line where TGF-ß1 is growth inhibitory (45). Depletion of intracellular Ca++ has been shown to inhibit the translocation of conventional PKC (46, 47, 48). The present observation that TGF-ß1 inhibited membrane association of the Ca++-dependent PKC{alpha} but not of the Ca++-independent PKC isoforms is in agreement with the possibility that TGF-ß1, in a manner yet unknown, decreases intracellular Ca++ levels in PC3 cells. TGF-ß1 may also inhibit membrane association of PKC{alpha} by causing enzyme dephosphorylation. It is established that for the conventional PKC{alpha} and PKCß, phosphorylation controls intrinsic catalytic potential (42). PKC{alpha} can be dephosphorylated and inactivated by a membrane-associated protein phosphatase 2A (49). Growth arrest by TGF-ß in human keratinocytes has been shown to involve acute activation of a protein phosphatase (50). Clearly, further work needs to be done to determine the mechanism(s) through which TGF-ß1 might inhibit the membrane association of PKC{alpha} in PC3 cells.

The translocation or redistribution of PKC isoforms from cytosol to membrane fractions has been used as a measure of PKC isoform activation (34, 42). Thus, the TGF-ß1-induced reverse translocation of PKC{alpha} from the membrane to the cytosol may be considered as leading to the inactivation of PKC{alpha}. In support of this view, present data demonstrate that TGF-ß1, but not PMA, inhibited the membrane-associated PKC activity in PC3 cells. The rapid onset of the effect of TGF-ß1 on inhibition of membrane-associated PKC activity coincided with the onset of TGF-ß1-induced decrease in membrane association of PKC{alpha}. These data, therefore, strongly indicate that TGF-ß1 suppresses PKC{alpha} activity in PC3 cells which contributes, at least in part, to the TGF-ß1-induced inhibition of PKC activity.

The ability of TGF-ß1 to inhibit PKC activity appears to be linked to its growth-inhibitory action. In support of this view, present data demonstrate that [3H]thymidine incorporation in PC3 cells was also inhibited by chelerythrine, presumably through its potent and highly selective inhibition of PKC activity (43). At a high dose (5 µM), chelerythrine has been shown to cause cell death in androgen-independent DU145 prostate cancer cells but not in androgen-dependent LNCaP prostate cancer cells (51). Earlier studies in prostate cancer cells have shown that phorbol esters such as PMA inhibit cell proliferation (52, 53). PMA treatment in these studies, however, was done for at least 24 h. It is known that phorbol esters initially activate PKC, but that on prolonged treatment, they down-regulate and inactivate PKC (32, 42). Therefore, the decrease in cell proliferation following PMA treatment may actually be an outcome of PMA-induced PKC inactivation.

Data from the present study are in direct contrast to an earlier report that suggested that TGF-ß may be an endogenous activator of the growth-inhibitory pathway of PKC (22). Both TGF-ß and PMA inhibited the basic fibroblast growth factor-induced mitogenesis in vascular smooth muscle cells, and they both caused the translocation within minutes of total PKC activity from the cytosol to the membrane (22). The ability of TGF-ß to either stimulate or inhibit PKC activity may be dependent on the cell type, i.e. normal smooth muscle cells vs. prostate cancer cells. Alternatively, TGF-ß’s effect may also be dictated by the type of PKC isoform involved in the process. Whereas PC3 cells possess only conventional PKC{alpha}, vascular smooth muscle cells have both PKC{alpha} and PKCß (22). Translocation of specific PKC isoforms in response to TGF-ß1 was not determined in the previous study (22).

The mechanism through which TGF-ß1-induced inhibition of PKC{alpha} activity may lead to cell growth arrest remains to be elucidated. TGF-ß1 may cause the inhibition of a PKC{alpha}-dependent phosphorylation of a protein(s) critical for the transduction of the mitogenic signal. Indeed, a study has shown that TGF-ß1 inhibited the conventional PKC isoform-dependent phosphorylation of a protein in response to PMA stimulation in mouse epidermal cells (30). PKC{alpha} is a ubiquitous isoform that is critical in the signaling pathways of several mitogens (34). Inhibition of the activity of PKC{alpha} by TGF-ß1 could directly disrupt the propagation of the mitogenic signals and, thereby, lead to TGF-ß1-induced suppression of cell proliferation. PKC{alpha} has also been implicated in the regulation of cell differentiation (34). Inhibition of PKC{alpha} activity by TGF-ß1 could affect differentiated cellular functions such as extracellular matrix formation (34), and this may help maintain cells in their growth-arrested state.

Understanding the mechanism(s) of TGF-ß1-induced inhibition of cell proliferation is critical for tumor cell biology. Transformed cells often escape the growth-inhibitory influence of TGF-ß, leading to malignancies, even as they continue to secrete TGF-ß (3). Although some of these malignant cells have defects in TGF-ß receptors (44, 54), those that have the normal functional compliment of receptors may have mutations in their postreceptor TGF-ß signaling pathway. Knowledge of the downstream intermediates or events that participate in TGF-ß1 signaling should help in the design of anticancer therapies.

In summary, TGF-ß1 inhibited cell proliferation in PC3, an androgen-independent human prostate cancer cell line. In these cells, a growth-inhibitory dose of TGF-ß1 also inhibited the membrane association of PKC{alpha} in a rapid, time-dependent and isoform-specific manner. The inhibitory effect of TGF-ß1 on PKC{alpha} occurred coincident with a TGF-ß1-induced decrease in membrane-associated PKC activity. Inhibition of PKC activity appeared to be associated with growth inhibition in PC3 cells, because chelerythrine, a specific PKC inhibitor, also decreased cell proliferation in these cells. Taken together, our data suggest that TGF-ß1-induced inhibition of PKC activity, at least in part due to inactivation of PKC{alpha}, is an early event associated with TGF-ß1 postreceptor signaling that might mediate suppression of cell proliferation.


   Acknowledgments
 
We thank Dr. Evelyn T. Maizels (Northwestern University Medical School, Chicago, IL) for the generous gift of a partially purified PKC from rat brain and for helpful critical suggestions. We also thank David Zelner and Sharon Sintich (Northwestern University Medical School, Chicago, IL) for their help with the densitometric scanning analysis and the preparation of some of the figures used in this article.


   Footnotes
 
1 This work was supported by NIH Grants HD-28048, CA-69851, and CA-60553 (to C.L.). Back

Received March 13, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Roberts AB, Sporn MB 1990 The transforming growth factors-ß. In: Sporn MB, Roberts AB, eds. Handbook of experimental pharmacology. Peptide growth factors and their receptors. Springer-Verlag, Heidelgerg FRG; vol 95:419–472
  2. Massague J, Cheifetz S, Laiho M, Ralph DA, Weis FMB, Zentella A 1992 Transforming growth factor-ß. Cancer Surv 12:81–103[Medline]
  3. Filmus J, Kerbel RS 1993 Development of resistance mechanisms to the growth-inhibitory effects of transforming growth factor-beta during tumor progression. Curr Opin Oncol 5:123–129[Medline]
  4. Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wang X-F, Massague J 1992 TGFß signals through a heteromeric protein kinase receptor complex. Cell 71:1003–1014[CrossRef][Medline]
  5. Franzen P, ten Dijke P, Ichijo H, Yamashita H, Schulz P, Heldin C-H, Miyazono K 1993 Cloning of a TGFß type I receptor that forms a heteromeric complex with the TGFß type II receptor. Cell 75:681–692[CrossRef][Medline]
  6. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J 1994 Mechanism of activation of the TGF-ß receptor. Nature 370:341–347[CrossRef][Medline]
  7. Bassing CH, Yingling JM, Howe DJ, Wang T, He WW, Gustafson ML, Shah P, Donahoe PK, Wang X-F 1994 A transforming growth factor ß type I receptor that signals to activate gene expression. Science 263:87–89[Abstract/Free Full Text]
  8. Carcamo J, Weis FMB, Ventura F, Wieser R, Wrana JL, Attisano L, Massague J 1994 Type I receptors specify growth-inhibitory and transcriptional responses to transforming growth factor ß and activin. Mol Cell Biol 14:3810–3821[Abstract/Free Full Text]
  9. Derynck R 1994 TGF-ß-receptor-mediated signaling. Trends Biochem Sci 19:548–553[CrossRef][Medline]
  10. Massague J, Polyak K 1995 Mammalian antiproliferative signals and their targets. Curr Opin Gen Dev 5:91–96[CrossRef][Medline]
  11. Zhang Y, Feng X-H, Wu R-Y, Derynck R 1996 Receptor-associated Mad homologues synergize as effectors of the TGF-ß response. Nature 383:168–172[CrossRef][Medline]
  12. Graff JM, Bansal A, Melton DA 1996 Xenopus Mad proteins transduce distinct subsets of signals for the TGFß superfamily. Cell 85:479–487[CrossRef][Medline]
  13. Hoodless PA, Haerry T, Abdollah S, Stapleton M, O’Connor MB, Attisano L, Wrana JL 1996 MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell 85:489–500[CrossRef][Medline]
  14. Liu F, Hata A, Baker JC, Doody J, Carcamo J, Harland RM, Massague J 1996 A human Mad protein acting as a BMP-regulated transcriptional activator. Nature 381:620–623[CrossRef][Medline]
  15. Wang T, Danielson PD, Li B-Y, Shah PC, Kim SD, Donahoe PK 1996 The p21ras farnesyltransferase {alpha} subunit in TGF-ß and activin signaling. Science 271:1120–1122[Abstract]
  16. Satoh T, Nakafuku M, Kaziro Y 1992 Function of ras as a molecular switch in signal transduction. J Biol Chem 267:24149–24152[Free Full Text]
  17. Davis RJ 1993 The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553–14556[Free Full Text]
  18. Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N, Taniguchi T, Nishida E, Matsumoto K 1995 Identification of a member of the MAPKKK family as a potential mediator of TGF-ß signal transduction. Science 270:2008–2011[Abstract/Free Full Text]
  19. Mulder KM, Morris SL 1992 Activation of p21ras by transforming growth factor ß in epithelial cells. J Biol Chem 267:5029–5031[Abstract/Free Full Text]
  20. Yan Z, Winawer S, Friedman E 1994 Two different signal transduction pathways can be activated by transforming growth factor ß1 in epithelial cells. J Biol Chem 269:13231–13237[Abstract/Free Full Text]
  21. Hartsough MT, Mulder KM 1995 Transforming growth factor ß activation of p44mapk in proliferating cultures of epithelial cells. J Biol Chem 270:7117–7124[Abstract/Free Full Text]
  22. Weiss RH, Yabes AP, Sinaee R 1995 TGF-ß and phorbol esters inhibit mitogenesis utilizing parallel protein kinase C-dependent pathways. Kidney Int 48:738–744[Medline]
  23. Franzen P, Ichijo H, Miyazono K 1993 Different signals mediate transforming growth factor-ß1-induced growth inhibition and extracellular matrix production in prostatic carcinoma cells. Exp Cell Res 207:1–7[CrossRef][Medline]
  24. Markovac J, Goldstein GW 1988 Transforming growth factor beta activates protein kinase C in microvessels isolated from immature rat brain. Biochem Biophys Res Comm 150:575–582[CrossRef][Medline]
  25. Robertson PL, Markovac J, Datta SC, Golstein GW 1988 Transforming growth factor beta stimulates phosphoinositol metabolism and translocation of protein kinase C in cultured astrocytes. Neurosci Lett 93:107–113[CrossRef][Medline]
  26. Halstead J, Kemp K, Ignotz RA 1995 Evidence for involvement of phosphatidylcholine-phospholipase C and protein kinase C in transforming growth factor-ß signaling. J Biol Chem 270:13600–13603[Abstract/Free Full Text]
  27. Lafon C, Mazars P, Guerrin M, Barboule N, Charcosset J-Y, Valette A 1995 Early gene responses associated with transforming growth factor-ß1 growth inhibition and autoinduction in MCF-7 breast adenocarcinoma cells. Biochem Biophys Acta 1266:288–295[Medline]
  28. Guerrin M, Darbon JM, Guilbaud N, Monsarrat B, Valette A 1990 Transforming growth factor beta (TGF-ß) reverses phorbol diester resistance of a breast adenocarcinoma (MCF-7) subline. Biochem Biophys Res Comm 166:687–694[CrossRef][Medline]
  29. Ito M, Yasui W, Kyo E, Yokozaki H, Nakayama H, Ito H, Tahara E 1992 Growth inhibition of transforming growth factor ß on human gastric carcinoma cells: receptor and postreceptor signaling. Cancer Res 52:295–300[Abstract/Free Full Text]
  30. Ohtsuki M, Massague J 1992 Evidence for the involvement of protein kinase activity in transforming growth factor-ß signal transduction. Mol Cell Biol 12:261–265[Abstract/Free Full Text]
  31. Nishikawa K, Yamamoto S, Nagumo H, Otsuka C, Kato R 1993 Suppressive effect of transforming growth factor-ß on the phosphorylation of endogenous substrates by conventional and novel protein kinase C in primary cultured mouse epidermal cells. Biochem Biophys Res Comm 193:384–389[CrossRef][Medline]
  32. Nishizuka Y 1992 Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258:607–614[Abstract/Free Full Text]
  33. Dekker LV, Parker PJ 1994 Protein kinase C—a question of specificity. Trends Biochem Sci 19:73–77[CrossRef][Medline]
  34. Nishizuka Y 1995 Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9:484–496[Abstract]
  35. Lee C, Sutkowski DM, Sensibar JA, Zelner D, Kim I, Amsel I, Shaw N, Prins GS, Kozlowski JM 1995 Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone. Endocrinology 136:796–803[Abstract]
  36. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  37. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin-phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  38. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354[Abstract/Free Full Text]
  39. Maizels ET, Miller JB, Cutler, Jr, RE, Jackiw V, Carney EM, Kern L, Hunzicker-Dunn M 1990 Calcium-independent phospholipid/diolein- dependent phosphorylation of a soluble ovarian Mr 80 000 substrate protein: biochemical characteristics. Biochim Biophys Acta 1054:285–296[Medline]
  40. Lamm MLG, Ekstrom RC, Maizels ET, Rajagopalan RM, Hunzicker-Dunn M 1994 The effect of protein kinases on desensitization of the porcine follicular membrane luteinizing hormone/chorionic gonadotropin-sensitive adenylyl cyclase. Endocrinology 134:1745–1754[Abstract]
  41. Powell CT, Brittis NJ, Stec D, Hug H, Heston WDW, Fair WR 1996 Persistent membrane translocation of protein kinase C{alpha} during 12-O-tetradecanoylphorbol-13-acetate-induced apoptosis of LNCaP human prostate cancer cells. Cell Growth Diff 7:419–428[Abstract]
  42. Newton AC 1995 Protein kinase C: structure, function, and regulation. J Biol Chem 270:28495–28498[Free Full Text]
  43. Herbert JM, Augereau JM, Gleye J, Maffrand JP 1990 Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Comm 172:993–999[CrossRef][Medline]
  44. Kim IY, Ahn H-J, Zelner DJ, Shaw JW, Sensibar JA, Kim J-H, Kato M, Lee C 1996 Genetic change in transforming growth factor ß (TGF- ß) receptor type I gene correlates with insensitivity to TGF-ß1 in human prostate cancer cells. Cancer Res 56:44–48[Abstract/Free Full Text]
  45. Baffy G, Sharma K, Shi W, Ziyadeh FN, Williamson JR 1995 Growth arrest of a murine mesangial cell line by transforming growth factor ß1 is associated with inhibition of mitogen-induced Ca2+ mobilization. Biochem Biophys Res Comm 210:378–383[CrossRef][Medline]
  46. Martin TFJ, Hsieh K-P, Porter BW 1990 The sustained second phase of hormone-stimulated diacylglycerol accumulation does not activate protein kinase C in GH3 cells. J Biol Chem 265:7623–7631[Abstract/Free Full Text]
  47. Trilivas I, McDonough PM, Brown JH 1991 Dissociation of protein kinase C redistribution from the phosphorylation of its substrates. J Biol Chem 266:8431–8438[Abstract/Free Full Text]
  48. Ha K-S, Exton JH 1993 Differential translocation of protein kinase C isozymes by thrombin and platelet-derived growth factor. J Biol Chem 268:10534–10539[Abstract/Free Full Text]
  49. Hansra G, Bornancin F, Whelan R, Hemmings BA, Parker PJ 1996 12-O-Tetradecanoylphorbol-13-acetate-induced dephosphorylation of protein kinase C{alpha} correlates with the presence of a membrane-associated protein phosphatase 2A heterotrimer. J Biol Chem 271:32785–32788[Abstract/Free Full Text]
  50. Gruppuso PA, Mikumo R, Brautigan DL, Braun L 1991 Growth arrest induced by transforming growth factor ß1 is accompanied by protein phosphatase activation in human keratinocytes. J Biol Chem 266:3444–3448[Abstract/Free Full Text]
  51. Krongrad A, Bai G 1994 c-fos promoter insensitivity to phorbol ester and possible role of protein kinase C in androgen-independent cancer cells. Cancer Res 54:6073–6077[Abstract/Free Full Text]
  52. Wang X-H, Whyzmuzis CA, An S, Chen Y, Wu JM, Schneidau TA, Mallouh C, Tazaki H 1994 Regulation of cell growth and the c-myc proto-oncogene expression by phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) in the androgen-independent human prostatic JCA-1 cells. Biochem Mol Biol Int 34:47–53[Medline]
  53. Novichenko N, Konno S, Nakajima Y, Hsieh TC, Xu W, Turo K, Ahmed T, Chiao JW 1995 Growth attenuation in a human prostate cell line mediated by a phorbol ester (43889). Proc Soc Exp Biol Med 209:152–156[Abstract]
  54. Park K, Kim SJ, Bang YI, Park JG, Kim NK, Roberts AB, Sporn MB 1994 Genetic changes in the transforming growth factor (TGF-ß) TßR-II gene in human gastric cancer cell: correlation with sensitivity to growth inhibition by TGF-ß. Proc Natl Acad Sci USA 91:8772–8776[Abstract/Free Full Text]



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