©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Phorbol Ester Inhibits the Phosphorylation of the Retinoblastoma Protein without Suppressing Cyclin D-associated Kinase in Vascular Smooth Muscle Cells (*)

(Received for publication, July 28, 1995; and in revised form, January 22, 1996)

Toshiyuki Sasaguri (§) Akio Ishida Chiya Kosaka Hiroshi Nojima (1) Jun Ogata

From the National Cardiovascular Center Research Institute and the Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To elucidate the role of protein kinase C in vascular smooth muscle cell proliferation, we examined the effects of phorbol 12-myristate 13-acetate (PMA) on G(1) events in human arterial cells. About 15 h after G(0) cells were stimulated with fetal bovine serum and basic fibroblast growth factor, [^3H]thymidine incorporation started. PMA (10 nM) inhibited the incorporation over 90% when added earlier than 3 h after stimulation, but had no effect when added 12 h or later. PMA inhibited the phosphorylation of the retinoblastoma protein (pRb), which normally began at about 9 h. PMA did not inhibit the gene expression of Cdk2, Cdk3, Cdk4, Cdk5, and cyclins G, C, and D, all of which began at 0-3 h. However, PMA reduced the expression of cyclins E and A, which usually began at 3-9 h and about 15 h, respectively. PMA inhibited the histone H1 kinase activity of Cdk2, which increased from about 9 h, whereas PMA did not inhibit the pRb kinase activities of cyclin D-associated kinase(s) and Cdk4, detectable from 0-3 h. These results suggested that the PMA-induced inhibition of pRb phosphorylation is not mediated by suppressing cyclin D-associated kinase(s) including Cdk4, but involves the suppression of Cdk2 activity that results from the reduced expression of cyclins E and A.


INTRODUCTION

Hyperplasia of vascular smooth muscle cells (VSMCs) (^1)plays a central role in the formation of atherosclerotic lesions and intimal thickening after angioplasty. It is also one of the pathological changes that occurs during the development of hypertension. Therefore, it is essential for understanding the etiology of these disorders to elucidate the mechanism regulating VSMC proliferation(1, 2, 3) .

VSMC proliferation is promoted by several biological substances, such as growth factors, cytokines, vasoactive peptides, catecholamines, and arachidonate metabolites(1, 2) . Most, if not all, of these substances activate protein kinase C (PKC), since upon stimulation of their receptors, phospholipase C is activated, which stimulates phosphoinositide turnover producing 1,2-sn-diacylglycerol (DAG), which then activates PKC(4, 5) . PKC activity is sustained by DAG produced from phosphatidylcholine by subsequently activated phospholipases C and D(6) . Lysophosphatidylcholine and fatty acids produced by phospholipase A(2) may also contribute to PKC activation(7) . Moreover, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, the products of phosphatidylinositol 3-kinase, reportedly activate some isoforms of the PKC family(8, 9) .

The role of the PKC pathway in mitogenesis remains undetermined. PKC activators such as phorbol esters and membrane permeable DAGs mimic competence factors (10, 11) and induce the expression of immediate early genes such as c-fos and c-myc(12) . PKC directly phosphorylates and activates Raf-1(13, 14) , which would lead to the activation of MAP kinase. Therefore, PKC may be involved in the early phases of mitogenesis as a positive mediator.

Nevertheless, phorbol ester inhibits the transition from G(1) to S phase in a variety of cell species including VSMCs(15, 16, 17, 18) . Since this effect is mimicked by repeated doses of membrane-permeable DAG and is prevented by the down-regulation of alpha- and -isoforms of PKC, we suggested that PKC mediates the G(1)/S inhibition induced by phorbol ester(18) . Moreover, PKC-mediated inhibition seems to operate as a physiological mechanism, since DNA synthesis is accelerated when PKC is down-regulated(18) . However, the mechanism by which PKC inhibits the G(1)/S progression remains to be investigated.

In the present study, we examined the effects of phorbol ester on cellular events during the G(1) and S phases, including the phosphorylation of the retinoblastoma protein (pRb) and the activation of cyclin-dependent kinases (Cdks), using VSMCs from human umbilical arteries.


EXPERIMENTAL PROCEDURES

Chemicals

Phorbol 12-myristate 13-acetate (PMA, Sigma) was dissolved in 100% Me(2)SO and stocked at -20 °C until use. The concentration of Me(2)SO added simultaneously with PMA (vehicle) was 0.1%. Other chemicals were of reagent grade.

Cell Culture and Cell Cycle Synchronization

VSMCs, obtained from the media of human umbilical arteries by explant culture, were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 20% (v/v) fetal bovine serum (HyClone) and 10 ng/ml human recombinant basic fibroblast growth factor (Amersham Corp.) (growth medium), and used at the third passage. Cultured cells were identified as VSMCs as described (19) . Cell cycle synchronization in the quiescent state (G(0)) was achieved by incubating the cells in serum-free DMEM containing 0.1% bovine serum albumin (BSA, Sigma) for 48 h. Thereafter, the synchronized cells were stimulated with growth medium to re-enter the cell cycle. The cells were counted using a Coulter counter (Z1), and DNA synthesis was assessed by means of [^3H]thymidine ([^3H]TdR) incorporation as described(20) .

Flow Cytometry

Cells were trypsinized, suspended in phosphate-buffered saline (PBS), and fixed with 85% (v/v) ethanol (-20 °C) for 30 min. After removing the ethanol, the cells were incubated in PBS containing RNase (172,000 units/ml, Sigma) for 30 min at 37 °C. The cells were stained for 30 min with 0.005% propidium iodide dissolved in PBS. Fluorescence was measured with a flow cytometer (Cyto ACE150, Japan Spectroscopic).

Immunoprecipitation

To prepare pRb immunoprecipitate, about 3 times 10^6 cells were lysed in 1 ml of 10 mM Tris/HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.2 mM Na(3)VO(4), 20 µg/ml leupeptin, 20 µg/ml phenylmethylsulfonyl fluoride, and 0.5% (v/v) Nonidet P-40 (buffer A), followed by centrifugation at 16,000 times g to remove the insoluble pellet. The supernatant was incubated with 50 µl of a 50% (v/v) suspension of protein G-conjugated Sepharose (Pharmacia Biotech Inc.) for 30 min at 4 °C, followed by centrifugation to remove the beads. The supernatant was incubated with 1 µg/ml anti-human pRb monoclonal antibody (XZ104, Pharmingen) for 1 h at 4 °C. Thereafter 50 µl of a 20% (v/v) suspension of protein G-Sepharose was added, and the incubation was continued for 1 h. Cdk2, Cdk4, and cyclin D immunoprecipitates were obtained using polyclonal antibodies to human Cdk2 (Upstate Biotechnology), Cdk4 (Pharmingen), and cyclin D (Upstate Biotechnology), respectively, after cells were sonicated for 10 s in 50 mM Hepes/NaOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 0.1 mM Na(3)VO(4), 20 µg/ml phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1% Tween 20, and 10% glycerol (buffer B). The precipitate, washed five times with buffer A or B, was immunoblotted or assayed for protein kinase.

Western Blotting

Immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride membrane as described(18) , and blotted with anti-human pRb monoclonal (PMG3-245, Pharmingen), Cdk4 polyclonal, and cyclin D polyclonal antibodies. Blots were visualized using the ECL Western blotting detection system (Amersham).

Cyclin-dependent Kinase Assay

To measure Cdk2 activity, the immunoprecipitate was suspended in the reaction buffer (50 µl) containing 20 mM Tris/HCl (pH 7.4), 10 mM MgCl(2), 1 mM dithiothreitol, 100 µg/ml histone H1 (Boehringer Mannheim), and 50 µM [-P]ATP (14.8 GBq/mmol, Amersham) and incubated for 20 min at 30 °C with occasional mixing. The reaction was stopped by adding an equal volume of 2% SDS, 10% 2-mercaptoethanol, 125 mM Tris/HCl (pH 6.8), 20% glycerol, and 0.04% bromphenol blue. The sample was boiled for 3 min and centrifuged to precipitate the beads, after which the supernatant was separated by SDS-PAGE and electroblotted to a membrane. Phosphorylated proteins were visualized using an image analyzer (BAS-2000, Fuji Film).

To measure the activities of cyclin D-associated kinase and Cdk4, glutathione S-transferase-fused murine pRb C-terminal (amino acid residues 754-921) (GST-Rb) prepared as described (21) from pGEX-3X containing the cDNA fragment provided by Hitoshi Matsushime, was used as the substrate. The immunoprecipitates obtained using the antibodies to cyclin D and Cdk4 were suspended in the buffer (40 µl) containing 50 mM Hepes/NaOH (pH 7.5), 10 mM MgCl(2), 1 mM dithiothreitol, 20 µg/ml GST-Rb, and 50 µM [-P]ATP (14.8 GBq/mmol), then incubated for 40 min at 30 °C. The following procedures were the same as those described for the Cdk2 assay.

Northern Blotting

Northern blotting proceeded as described (20) . The coding regions of cDNAs for human Cdc2 (Cdk1), Cdk2, Cdk3, Cdk4, Cdk5, and cyclins C, D1, E, and G were produced independently by the polymerase chain reaction, using pairs of oligonucleotides (22-mer around the initiation or termination codons) synthesized according to the EMBL data base and subcloned into pCRII (Invitrogen) or pCR-ScriptSK(+) (Stratagene) vectors according to the manufacturer's protocol. The DNA sequence of each clone was determined to confirm the identity of the polymerase chain reaction products. The cDNAs for human cyclins A and B were gifts from Jonathon Pines, and those for murine cyclins D2 and D3 were from Hitoshi Matsushime.

Statistical Analysis

The results are expressed as the means ± standard deviation (S.D.) of the number of observations. The statistical significance was assessed by Student's t test for unpaired values.


RESULTS

G(1) Inhibition by PMA

We used human umbilical artery smooth muscle cells, because the cell cycle was the easiest to synchronize in this cell type among several VSMC species screened in a pilot study. Flow cytometry showed that the content of DNA was 2C in almost all the cells after serum starvation for 48 h, indicating that the cells were synchronized in the G(0) phase (Fig. 1). In control experiments, cells with an S phase DNA content began to increase about 15 h after supplementation with fetal bovine serum and basic fibroblast growth factor, with the maximal accumulation being obtained at 24 h. Cells with a G(2)/M phase DNA content (4C) appeared from about 30 h, peaking at 36 h. When PMA (10 nM) was added at time 0, cells with an S or G(2)/M phase DNA content were nearly completely absent, indicating that the cell cycle was arrested before the entry into S phase.


Figure 1: The effect of PMA on the cell cycle progression. VSMCs seeded at 8 times 10^5/dish (177 cm^2) were cultured in growth medium for 48 h, after which they were incubated in DMEM containing 0.1% BSA for 48 h to be synchronized to the G(0) phase. Cells were then stimulated with growth medium in the presence of 0.1% Me(2)SO (control) or 10 nM PMA. At the indicated times after stimulation, cells were trypsinized and stained with propidium iodide. The DNA content in about 1 times 10^4 cells was measured by flow cytometry. The x and y axes represent the intensity of fluorescence and cell number, respectively.



However, as shown in Fig. 1, some of the control cells did not enter S phase even after mitogenic stimulation. Since the cell viability was over 95% according to trypan blue exclusion, it was unlikely that these non-responding cells were injured or dead. Therefore, we considered that these cells were those remaining in G(0) phase due to contact inhibition, which cannot be avoided when using normal diploid cells. Nevertheless, we used these cells, because we felt that contamination with G(0) cells would not interfere with the interpretation of our experimental results.

The incorporation of [^3H]TdR started to increase about 15 h after mitogenic stimulation and reached a plateau at about 33 h (Fig. 2a), which corresponded well with the results of the flow cytometry (Fig. 1). There was little incorporation when cells were not stimulated, suggesting that entry into S phase was strictly dependent on mitogenic stimulation (Fig. 2a). To determine when PMA inhibits the cell cycle, PMA (10 nM) was applied at several time points during the G(1) and S phases (Fig. 2b). PMA added earlier than 3 h after stimulation inhibited [^3H]TdR incorporation over 90%. However, the effect was attenuated when PMA was added 6 h or later, and there was no effect when added 12 h or later, suggesting that PMA inhibited the G(1), but not S phase progression. The inhibition was maximal at a PMA concentration of 10 nM and partially reversed above this concentration (Fig. 2c).


Figure 2: The effect of PMA on DNA synthesis. a, cells seeded at 1 times 10^4/well (1.8 cm^2) were synchronized in the G(0) phase by serum starvation, after which they were incubated in growth medium (circle) or DMEM containing 0.1% BSA (bullet), in the presence of [^3H]TdR (37 kBq/well). The amount of incorporated radioactivity was determined at the indicated times. Data represent the means ± S.D. (n = 3). b, G(0) cells were labeled with [^3H]TdR in growth medium for 33 h, during which PMA (10 nM) or vehicle (0.1% Me(2)SO) was added at the indicated times. Data are the means ± S.D. (n = 3) of the PMA-induced inhibition (%) against the values obtained with vehicle. *, p < 0.01 versus vehicle-treated cells. c, G(0) cells were labeled with [^3H]TdR in DMEM containing 0.1% BSA (lane 1) or in growth medium (lanes 2-8) in the presence of various concentrations of PMA added at time 0. The amount of incorporated radioactivity was determined at 33 h. Lane 1, no stimulation (0.1% BSA); lane 2, growth medium; lane 3, growth medium + 0.1% Me(2)SO (vehicle); lanes 4-8, growth medium + PMA (0.1, 1, 10, 100, and 1000 nM, respectively). Data represent the means ± S.D. (n = 3). *, p < 0.01 versus lane 3.



The Effect of PMA on the Phosphorylation of pRb

To elucidate the mechanism for the G(1) arrest by PMA, we examined the effect of PMA on the phosphorylation of pRb, since this phenomenon is a crucial milestone for the cell to advance into the S phase. Hyperphosphorylated pRb moves more slowly in SDS-polyacrylamide gels than the hypophosphorylated form. In quiescent cells, a 110-kDa protein, which seemed to be the hypophosphorylated form, was predominantly detected by immunoblotting (Fig. 3a). After mitogenic stimulation, a shift of the apparent molecular mass from 110 to 115 kDa on SDS-PAGE, which seemed to represent the increase of hyperphosphorylated pRb, began at about 9 h and lasted until 15-18 h. PMA (10 nM) inhibited the mobility shift (Fig. 3a). This effect peaked at 10 nM and was partially reversed at higher concentrations (Fig. 3b), similarly to the effect on [^3H]TdR incorporation. Once pRb was phosphorylated, however, PMA could not reverse the phosphorylation, because when added 18 h after mitogenic stimulation, when the mobility of pRb had shifted, PMA failed to reverse the shift from 115 to 110 kDa (Fig. 3c).


Figure 3: The effect of PMA on pRb phosphorylation. a, G(0) cells were incubated in growth medium in the presence of PMA (10 nM) or vehicle (0.1% Me(2)SO) for various times as indicated below, after which they were collected and lysed. The proteins precipitated by the anti-pRb antibody (XZ104) were separated by 7.5% SDS-PAGE and immunoblotted against another anti-pRb antibody (PMG3-245). Lanes 1 and 2, not stimulated with growth medium; lanes 3-18, incubated in growth medium for 3 h (lanes 3 and 4), 6 h (lanes 5 and 6), 9 h (lanes 7 and 8), 12 h (lanes 9 and 10), 15 h (lanes 11 and 12), 18 h (lanes 13 and 14), 21 h (lanes 15 and 16), and 24 h (lanes 17 and 18), respectively. Odd-numbered lanes, vehicle; even-numbered lanes, PMA. b, G(0) cells were incubated in DMEM containing 0.1% BSA (no stimulation) or in growth medium containing various concentrations of PMA as indicated. *, 0.1% Me(2)SO (vehicle) was added. Cells were lysed at 24 h and immunoblotted. c, after G(0) cells were stimulated with growth medium, PMA (10 nM) or vehicle (0.1% Me(2)SO) was added at 18 h. Cells were lysed at the indicated times and immunoblotted. To indicate the position of hypophosphorylated pRb, the immunoprecipitate from the G(0) cell lysate was also blotted (shown as time 0).



The Effects of PMA on the Expression of Cdks and Cyclins

To understand how PMA inhibits pRb phosphorylation, we examined the effects of PMA on the gene expression of Cdks by Northern blotting, since pRb may be the physiological substrate for Cdks, such as Cdc2, Cdk2, Cdk4, and Cdk6(22, 23, 24, 25) . As shown in Fig. 4, Cdc2 expression was not clear until 24 h, suggesting that it begins to be expressed after the entry into S phase. However, the expression of all of the other Cdks we tested, namely Cdk2, Cdk3, Cdk4, and Cdk5, started from the G(0) or early G(1) phase. The level of Cdk2 expression, which was low in quiescent cells, was elevated from early G(1) and further increased after the advance into S phase. The expression of Cdk3 appeared to be low in our cells, but it was detected at early G(1). Cdk4 was markedly expressed even in quiescent cells, but the level was not dramatically altered throughout the G(1) and S phases. The expression of Cdk5 was also marked from G(0) until the late G(1) phase, but this was attenuated after 12 h. PMA (10 nM) suppressed the expression of Cdc2, which seemed to result from the inhibition of the advance into S phase. On the other hand, the expression of Cdk2, Cdk3, Cdk4, and Cdk5 was not inhibited for the first 12 h after growth stimulation, indicating that PMA did not inhibit their expression during the G(1) phase. Further elevation of Cdk2 after the entry into S phase was lost in the presence of PMA, probably due to the inability of cells to go into this phase. In contrast, the amount of Cdk5 mRNA after 12 h did not decrease in the presence of PMA.


Figure 4: The effects of PMA on Cdk gene expression. G(0) cells were incubated in growth medium in the presence of PMA (10 nM) or vehicle (0.1% Me(2)SO). Total cellular RNA was extracted at indicated times. Equal amounts of RNA (20 µg/lane) were resolved by electrophoresis and hybridized with P-labeled cDNA fragments of Cdks. The sizes of the transcripts (in kilobase pairs) were: Cdc2, 1.6; Cdk2, 2.5; Cdk3, 2.7; Cdk4, 1.7; Cdk5, 1.4.



We then examined the effects of PMA on the gene expression of cyclins, since association with a cyclin is required to activate Cdks. Cyclins G, C, D1, D2, and D3 were all induced from early G(1) (Fig. 5a). Cyclin G was expressed even in quiescent cells without significant changes in its mRNA levels throughout the G(1) and S phases. Cyclin C was also expressed in quiescent cells, but this increased after mitogenic stimulation. The expression of cyclins D1, D2, and D3 was low in quiescent cells, but all were induced from the early G(1) phase. PMA (10 nM) did not reduce the expression of any of these cyclins at least for the first 12 h, but rather tended to enhance that of cyclins G, C, D1, and D2 in the early G(1) phase. PMA attenuated the expression of cyclin D2 after 12 h.


Figure 5: The effects of PMA on the gene expression of cyclins. Northern blotting was performed as described in the legend to Fig. 4. a, cyclins expressed from the early G(1) phase. b, cyclins expressed later than early G(1) phase. The sizes of the transcripts (in kilobase pairs) were: cyclin G, 2.7; cyclin C, 2.4; cyclin D1, 4.5; cyclin D2, 6.5; cyclin D3, 2.2; cyclin E, 1.9; cyclin A, 2.9; cyclin B, 1.9; beta-actin, 2.1.



Fig. 5b demonstrates the mRNA expression of cyclins induced later than those shown in Fig. 5a. It was difficult to clearly determine when the expression of cyclin E began, since small amounts of the mRNA were detected 3-9 h after stimulation and before the expression was markedly enhanced. Cyclin A was expressed from about 15 h, which corresponded to around the G(1)/S border, and cyclin B was expressed a few hours later than cyclin A, probably after entry into S phase. In the presence of PMA (10 nM), however, the expression of all these mRNAs was suppressed.

The Effects of PMA on the Activities of Cdks

We considered that the suppressed expression of cyclins E and A would reduce Cdk2 activity, since this kinase is activated by associating with these cyclins(26, 27, 28, 29) . The activity of Cdk2, determined by the in vitro phosphorylation of histone H1, started to increase from about 9 h, and continued to increase until 24 h (Fig. 6a). PMA (10 nM) markedly inhibited this activation. The inhibitory effect was maximal when PMA was added at 0-6 h (Fig. 6b). PMA added at 15-21 h, which corresponded to the early S phase, also significantly inhibited Cdk2 activity. The inhibition was maximal at PMA concentration of 10 nM and partially reversed above this concentration, similarly to the effect on the [^3H]TdR incorporation (Fig. 6c).


Figure 6: The effect of PMA on the activity of Cdk2. a, G(0) cells were incubated in growth medium in the presence of 0.1% Me(2)SO (control) or 10 nM PMA. Cell lysates prepared at the indicated times were precipitated with anti-Cdk2 antibody for histone H1 kinase assay. After the enzyme reaction, assay mixtures were resolved by 12.5% SDS-PAGE, electroblotted, and autoradiographed. b, G(0) cells were incubated in growth medium for 24 h and analyzed as described in a. PMA (10 nM) was added at the indicated times after the release from G(0). NS and S, not stimulated and stimulated with growth medium in the absence of PMA, respectively. c, G(0) cells were incubated in growth medium for 24 h in the presence of various concentrations of PMA or vehicle, and analyzed as described in a. NS, not stimulated with growth medium.



Since Cdk2 phosphorylates pRb in vitro(23) , the PMA-induced inhibition of Cdk2 activity seemed to explain the inhibition of pRb phosphorylation. However, it remained to be determined whether PMA inhibits the cellular events that arise before Cdk2 activation. Cyclins of the D class and their major partner Cdk4 may play crucial roles in the G(1)/S transition by phosphorylating pRb(30, 31) . As shown in Fig. 4and Fig. 5, D-type cyclins and Cdk4 were expressed from the early G(1) phase, probably preceding the expression of cyclins E and A. We therefore tested whether PMA influences the activity of cyclin D-associated kinase(s), including Cdk4.

To confirm that PMA does not inhibit the expression of Cdk4 and D-type cyclins during the G(1) phase, the protein levels of Cdk4 and cyclin D were analyzed by Western blotting (Fig. 7). Consistent with the result of Northern blotting, PMA did not reduce their protein levels. Moreover, PMA did not reduce the amount of Cdk4 that co-precipitated with cyclin D, indicating that PMA did not inhibit the association of cyclin D with Cdk4.


Figure 7: The effect of PMA on the association between cyclin D and Cdk4. G(0) cells were incubated in growth medium in the presence of PMA (10 nM) or vehicle and lysed 7.5 h after the release from G(0). Lysates were immunoprecipitated (IP) with antibodies to Cdk4 or cyclin D as indicated. Precipitates were separated by 10% SDS-PAGE, and immunoblotted with antibodies to Cdk4 or cyclin D as indicated. The position of the IgG heavy chain (about 50 kDa) is also shown.



The activities of cyclin D-associated kinase and Cdk4 were measured by the in vitro phosphorylation of GST-Rb, since pRb may be the physiological substrate for these enzymes and the histone H1 kinase activity of Cdk4 was very low (not shown). The activity that co-precipitated with cyclin D, of which the level was low in quiescent cells, increased during the G(1) phase to peak at 7.5 h (Fig. 8a). After it dropped once at 9 h, the activity again increased from 12 until 24 h, when the activity was maximal during the observation period. In the presence of PMA (10 nM), the elevation in the G(1) phase was enhanced and lasted for a much longer period, although there was no second increase. The activity of Cdk4 was relatively high even in quiescent cells and not markedly changed during the G(1) phase, although it significantly increased at 12-24 h (Fig. 8b). PMA also enhanced Cdk4 activity for the first 9 h.


Figure 8: The effects of PMA on the activities of cyclin D-associated kinase and Cdk4. a, G(0) cells were incubated in growth medium in the presence of PMA (10 nM) or vehicle for the indicated periods. Cell lysates were then prepared and precipitated with anti-cyclin D antibody. After the kinase reaction, assay mixtures were resolved by 10% SDS-PAGE. Proteins were transferred to membranes and autoradiographed. The arrows indicate the positions of GST-Rb (46 kDa). b, the procedure was the same as described in a, except that anti-Cdk4 antibody was used for immunoprecipitation.




DISCUSSION

The cellular event mediating the cell cycle arrest induced by PMA should take place during G(1), but not S phase, since PMA added after the advance into S phase failed to inhibit DNA replication. This was consistent with flow cytometry studies on melanoma and U937 cells, which showed the inability of phorbol ester to block S phase progression(32, 33) . Therefore, we studied the events that arise prior to the G(1)/S transition.

Several afferent signals converge onto pRb, including positive as well as negative signals that inhibit the G(1)/S transition. In turn pRb transmits the integrated signal to a number of downstream effectors, such as E2F transcription factors(31) . The pRb function is regulated by phosphorylation, which occurs in the middle of the G(1) phase, close to and perhaps simultaneously with the R (restriction) point(34) . Hypophosphorylated pRb actively represses the advance into S phase, whereas the hyperphosphorylated form loses this ability(31) . Transforming growth factor-beta (TGF-beta), cAMP, and cell-cell contact inhibit pRb phosphorylation(31, 35) . PMA also inhibits pRb phosphorylation in leukemia and lymphoma cell lines, and vascular endothelial cells(35, 36, 37) . Although mechanisms for the inhibition by the former three signals have been proposed as described below, it remains to be determined how PMA inhibits pRb phosphorylation.

In our VSMCs, PMA added simultaneously with mitogens inhibited the in vivo phosphorylation of pRb in a dose-dependent fashion similar to that of its effect on DNA synthesis. However, once pRb was phosphorylated, PMA failed to reverse it. Consistently, the inhibition of DNA synthesis was abolished, when PMA was added later than 12 h after the release from G(0), when pRb had already undergone phosphorylation. Therefore, PMA seemed to arrest cells at the G(1) phase by inhibiting an event that occurs before pRb phosphorylation.

Cdk2 phosphorylates pRb in vitro and it may be a mediator of G(1)/S transition(23, 38) . Cdk2 activity increased from about 9 h after the release from G(0). Therefore, the onset of the activation appeared to correspond to that of pRb phosphorylation. The mobility of pRb appeared to have shifted by 15-18 h, whereas Cdk2 activity continued to increase until 24 h. The progressive elevation of Cdk2 activity after completion of the mobility shift could contribute to the maintenance of phosphorylation, or induce the phosphorylation of other sites on pRb that is undetectable by the mobility shift. PMA inhibited Cdk2 activity in a dose-dependent manner again similar to that for the inhibition of DNA replication. Therefore, suppressed Cdk2 activity may explain the PMA-induced inhibition of pRb phosphorylation. Once phosphorylated, pRb may not be dephosphorylated even when Cdk2 activity is suppressed, because PMA added in the S phase inhibited Cdk2 activity but did not reverse the mobility shift of pRb. Coupling with either cyclins E or A is prerequisite for Cdk2 activation(26, 27, 28, 29) . The time course of cyclin E expression appeared to correspond well with that of Cdk2 activation, whereas cyclin A expression apparently occurred later than cyclin E, and therefore, than pRb phosphorylation. PMA inhibited the expression of these two cyclins, but not Cdk2. Therefore, it was likely that the inhibition of Cdk2 activity resulted from the reduced gene expression of cyclin E and, possibly, cyclin A.

In vascular endothelial cells, cyclin A appeared to be expressed from the late G(1); therefore, we suggested that PMA caused G(1)/S arrest by reducing cyclin A expression(39) . In VSMCs, however, there was little expression of cyclin A until the cells entered the S phase. This difference may have resulted from the difficulty in synchronizing endothelial cells in the G(0) phase. There was little incorporation of [^3H]TdR until 15 h after growth stimulation in VSMCs, whereas the incorporation gradually increased from 4 h in endothelial cells. We consider that the endothelial cells assumed to be synchronized in the G(0) phase contained a substantial number of G(1) cells, which expressed cyclin A earlier than truly G(0)-synchronized cells.

The onset of Cdk2 activation appeared to be simultaneous with that of pRb phosphorylation, implying that these two events were closely linked. However, Cdk2 activation did not clearly precede pRb phosphorylation. It was conceivable, therefore, that the initiation of pRb phosphorylation is mediated by other kinases, and that PMA inhibits their activities. Cdks associated with D-type cyclins, such as Cdk4 and Cdk6, also phosphorylate pRb in vitro(21, 24, 25) . They may be the kinases most prominently involved in the phosphorylation of pRb (31) . In contrast to Cdk2, the cyclin D-associated kinase activity increased in the early to middle G(1) phase forming a peak 7.5 h after the release from G(0), thus clearly preceding the initiation of pRb phosphorylation. PMA did not suppress this activity, but rather enhanced it in the early to middle G(1) phase. PMA did not inhibit the activity of Cdk4, either. Consistently, PMA did not inhibit the expression of cyclins D1, D2, and D3, and Cdk4 at least for 12 h after mitogenic stimulation. Moreover, PMA did not impair the association of cyclin D with Cdk4. Therefore, it was unlikely that the PMA-induced inhibition of pRb phosphorylation resulted from the inhibition of cyclin D-dependent kinases. It remains to be investigated why Cdk4 activity was relatively high even in quiescent cells and its dependence on growth stimulation appeared lower than the activity that co-precipitated with cyclin D.

However, if cyclin D-dependent kinases such as Cdk4 and Cdk6 are responsible for pRb phosphorylation, our results are contradictory, in that PMA inhibited the in vivo phosphorylation of pRb regardless of its inability to suppress the in vitro phosphorylation of GST-Rb induced by cyclin D-associated kinase. Although we could not resolve this discrepancy, there are some explanations. Collaboration between cyclin D and cyclin E might be required for pRb phosphorylation. This has been suggested by a report on pRb phosphorylation in Saccharomyces cerevisiae(40) . This phosphorylation required specific combinations of yeast G(1) cyclins, Cln3 and either Cln1 or Cln2. The functions of Cln3 and Cln2 in pRb phosphorylation were complemented by human cyclin D1 and cyclin E, respectively. Therefore, it is possible that in our cells, reduced cyclin E restrains pRb phosphorylation, even in the presence of active cyclin D-Cdk complexes.

Alternatively, PMA could activate pRb phosphatase. Although studies on the mechanism of pRb dephosphorylation are so far limited, evidence suggests that one of the pRb phosphatases is a type 1 protein phosphatase (PP1)(41) . The pRb directly binds to the catalytic subunit of PP1, and the complex is detected in G(0)/G(1), mid-G(1), and M phase cells, suggesting the importance of PP1 for the maintenance of hypophosphorylated pRb from late M to mid-G(1) phase(42) . Therefore, if PMA can stimulate pRb phosphatase, our apparently contradictory results would be coincident, since pRb may be able to interact with pRb phosphatase in vivo, but not in our in vitro pRb kinase assay system, which lacks pRb phosphatase.

Negative growth signals generated by TGF-beta, cAMP, and cell-cell contact may be mediated by inhibitor proteins of Cdks(43, 44, 45) . Several Cdk inhibitors have been found in mammalian cells, including p21, p27, p16, and p15(46) . Although there are only a few reports on their mechanisms of action, p27 may inhibit Cdk4 and Cdk2 by preventing the phosphorylation with Cdk-activating kinase(44, 47) . p16 interrupts the association of cyclin D1 with either Cdk4 or Cdk6(48, 49) . However, PMA inhibited neither the activities of cyclin D-associated kinase and Cdk4 nor the binding of cyclin D to Cdk4, suggesting that the effect of PMA is not mediated by these inhibitors.

Alternative mechanisms have been suggested for TGF-beta and cAMP. TGF-beta reduces the levels of Cdk2 and Cdk4 in human keratinocytes (50) and of Cdk4 in mink lung epithelial cells(51) , and cAMP inhibits the expression of cyclin D in murine macrophages(52) . However, in contrast, PMA did not inhibit, but sometimes enhanced the cellular events arising in the G(0) to early G(1) phase, that included the expression of cyclins G, C, D, Cdk2, Cdk3, Cdk4, and Cdk5. This corresponded to other reports that phorbol ester stimulates the G(0)/G(1) transition by mimicking competence factors(10) . Significantly, these contrasts between PMA and other growth inhibitory signals indicate that PMA is a unique inhibitory agent that suppresses pRb phosphorylation in a different manner from others. PMA inhibits the proliferation of a lymphoma cell line by inducing TGF-beta(53) . However, this may not be so in our cells due to the above reason, and moreover, because neutralizing antibodies to TGF-beta did not attenuate the PMA-induced inhibition of G(1)/S transition. (^2)

We showed that PKC mediates the phorbol ester-induced inhibition of G(1)/S transition, since the inhibition was mimicked by repeated doses of membrane-permeable DAG and abolished by down-regulation of PKC by a preincubation with phorbol ester(18, 39) . The PMA dose-effect relationship also suggested the involvement of PKC, because the maximal inhibitory effects on DNA synthesis, pRb phosphorylation, and Cdk2 activity were always obtained around 10 nM. This was in agreement with the reported concentrations for PKC activation. This inhibition was always partially reversed at concentrations over 100 nM, which we assumed to result from the down-regulation of PKC.

It seems that the PKC pathway inhibits the cell cycle of VSMCs at the mid-to-late G(1), but not the early G(1) phase. Cells remain competent for proliferation while PKC is being activated. We thus speculated that PKC is a physiological regulator of the G(1) phase, hindering DNA synthesis just before the R point by preventing pRb phosphorylation until the circumstances are favorable for advance into the remainder of the cell cycle. Alternatively, PKC could contribute to cell differentiation, since several lines of evidence have suggested that the prevention of pRb phosphorylation and the resulting cell cycle arrest are necessary for subsequent cell differentiation(31) . To test these possibilities, it is essential to understand how PKC activity is controlled during the cell cycle and cell differentiation.

In addition, we found that vascular endothelial cells are arrested in the G(2), as well as the G(1) phase by PMA and DAG (54) . If this is also a phenomenon universal among various cell species, it would suggest that PKC operates as a regulator of the cell cycle clock at two points, namely in the G(1) and G(2) phases. There are multiple phosphorylation sites on pRb (55) , and phosphorylation progresses by multiple steps, not only in the G(1), but also in the G(2) phase(56) . Hence, the G(2) inhibition could also involve the inhibition of pRb phosphorylation. It is essential to characterize each phosphorylation site in terms of the regulation of pRb function.


FOOTNOTES

*
This study was supported in part by grants from the Ministry of Education, Science, and Culture (a grant-in-aid for scientific research), the Ministry of Health and Welfare (Research Grant for Cardiovascular Diseases 6B-1), the Science and Technology Agency (Special Coordination Funds for Promoting Science and Technology (Encouragement System of COE)), the Uehara Memorial Foundation, and the Yamanouchi Foundation for Research on Metabolic Disorders. 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: Dept. of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565, Japan. Tel.: 81-6-833-5012; Fax: 81-6-872-7485; sasaguri{at}ri.ncvc.go.jp.

(^1)
The abbreviations used are: VSMC, vascular smooth muscle cell; PKC, protein kinase C; pRb, the retinoblastoma protein; Cdk, cyclin-dependent kinase; PMA, phorbol 12-myristate 13-acetate; DAG, 1,2-sn-diacylglycerol; TGF-beta, transforming growth factor-beta; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TdR, thymidine; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.

(^2)
T. Sasaguri, unpublished data.


ACKNOWLEDGEMENTS

We are indebted to Jonathon Pines for providing the cDNAs of cyclins A and B and to Hitoshi Matsushime for the plasmids encoding GST-Rb and cyclins D2 and D3. We also thank Steven I. Reed and Li-Huei Tsai for sending the plasmids encoding cyclin E and Cdk2, respectively, which were used in preliminary studies.


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