Changes of Nuclear Protein Kinase C Activity and Isotype Composition in PC12 Cell Proliferation and Differentiation

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

224, 72–78 (1996)

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Changes of Nuclear Protein Kinase C Activity and Isotype Composition in PC12 Cell Proliferation and Differentiation PAOLA BORGATTI,* MERI MAZZONI,* CINZIA CARINI,* LUCA MARIA NERI,*,† MARCO MARCHISIO,* LUCIA BERTOLASO,* MAURIZIO PREVIATI,* GIORGIO ZAULI,* AND SILVANO CAPITANI*,1 *Institute of Human Anatomy, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy; and †Institute of Normal and Pathological Cytomorphology of C.N.R. c/o I.O.R., Institute of Research Codivilla Putti, Via di Barbiano 1/10, 40136 Bologna, Italy

eration and differentiation of the same cell type and that nuclear protein kinase C is crucial to the induction and persistence of the differentiated neuronal phenotype of PC12 cells. q 1996 Academic Press, Inc.

To establish whether protein kinase C was involved in the nuclear events underlying cell differentiation and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mitogenic stimulus. Western blot analysis with isoformspecific antibodies, performed on whole cell homogenates, cytoplasms, and purified nuclei, showed that PKC isotypes a, bI, bII, d, e, h, and z were expressed in PC12 cells and that all of them, except for bI, were found at the nuclear level, variably modulated depending on the cell treatment. Compared to serumstimulated cells, in which an early (1 day) and marked rise of protein kinase C activity was followed by a plateau, nerve growth factor-treated cells showed a progressive increase of protein kinase C activity coincident with the onset and maintenance of the differentiated phenotype. Western blot analysis of nuclei isolated from fully differentiated cells demonstrated an increase of protein kinase C a, paralleled by enhanced phosphotransferase activity along with the nerve growth factor treatment, and complete loss of the d isotype. In contrast, in nuclei of proliferating PC12 cells, after an early but modest increase at 1 day of mitogenic stimulation, protein kinase C activity reached a plateau. Isotype-specific analysis indicated a concomitant increase of protein kinase C bII, d, and z and the appearance of protein kinase C e and h at the nuclear level. Considering the relative intensity of the cytoplasmic and nuclear immunoreactive bands under the three conditions examined, clear-cut translocation to the nucleus occurred for PKC e and h in serum-stimulated cells. Additional nuclear accumulation of PKC by translocation from the cytoplasm was prominently induced for the z isoform after mitogenic stimulation and for PKC a during prolonged NGF treatment. Our data suggest that nuclear translocation and selective activation of distinct protein kinase C isoforms play a relevant role in the control of prolif-

INTRODUCTION

Protein kinase C (PKC) was originally described as a serine/threonine protein phosphotransferase modulated by calcium and lipid cofactors, including phosphatidylserine (PS), other anionic phospholipids, and diacylglycerol (DG) [1, 2]. It has been recently found that PKC represents a large gene family of isoenzymes, differing remarkably in their structure and expression in various tissues, in their mode of activation, cofactor requirement, and substrate specificity [3]. Therefore, PKC regulation appears rather complex and is still incompletely understood. The diversity of PKC-mediated cell responses is further increased by the evidence that only PKC a, bI, bII, and g out of the several isoforms identified so far are Ca2/ dependent [4] and that the endogenous activator DG can derive from turnover of either inositol lipids or phosphatidylcholine [5]. In addition, PKC autophosphorylation seems an essential step for enzyme activation [6–8], while the proteolytic cleavage of PKC by calpains, generating the constitutively active fragment PKM [9, 10], can also participate in long-term PKC activation. PKC has been shown to be involved in the proliferation and differentiation of a variety of cell types [3]. Usually, multiple isoforms are present in a single cell type, and accumulating evidence suggests that induction of proliferation or differentiation may correlate with the differential activation and intracellular localization of PKC isotypes [3, 11]. Although activation of PKC is often associated with insertion in the cell membrane [2], it has also been demonstrated that PKC is able to translocate to different cell compartments, and in particular to the nucleus of various cell types [12 – 18]. In order to investigate the involvement of PKC in

1 To whom correspondence and reprint requests should be addressed. Fax: 39-532-207351.

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the nuclear events accompanying cell proliferation and differentiation, we chose, as a model system, the rat pheochromocytoma PC12 cell line [19]. In the presence of high serum concentrations, PC12 cells survive and multiply, showing a round morphology and a transformed phenotype. When treated with nerve growth factor (NGF), most PC12 cells are able to differentiate into sympathetic-like neurons with the extension of long, axon-like neurites. Of note, NGF also has a slight mitogenic effect during the first 24 h of treatment [20] and rescues PC12 cells from apoptotic death induced by serum withdrawal [21, 22]. Like other neurotrophins, NGF exerts its effects utilizing a family class of receptor tyrosine kinases, the trks, prominently expressed in neuronal cells [23]. However, even though key steps of the signaling cascade initiated by trk, including tyrosine phosphorylation of phospholipase C g1 [24] and activation of MAP kinase [22, 25], have been identified, the nuclear events which trigger the differentiation program await full elucidation. In the present study we have investigated the presence, catalytic activity, subcellular distribution, and isoform composition of PKC in PC12 cells before and after continuous treatment with mitogenic concentrations of serum or differentiating doses of NGF. We report that distinct PKC isoforms are involved in PC12 proliferation and differentiation and that nuclear PKCs play a pivotal role in the induction and persistence of the differentiated neuronal phenotype. MATERIALS AND METHODS Cell culture and fractionation. PC12 pheochromocytoma cells [19], kindly provided by Dr. Lloyd Greene (Columbia University, NY), were cultured in D-MEM (Dulbecco’s MEM, Gibco, Grand Island, NY), supplemented with 10% heat-inactivated horse serum (HS, Sigma, St. Louis, MO), 5% heat-inactivated fetal calf serum (FCS, Gibco), 50 mg/ml streptomycin, and 50 U/ml penicillin, at 377C in a humidified atmosphere of 7.5% CO2 in air. In differentiation experiments, PC12 cells were seeded at low density (5 1 103/cm2) and serum-starved (D-MEM plus 1% HS) for 24 h before adding 100 ng/ ml NGF (UBI, Lake Placid, NY). The medium (D-MEM plus 1% HS plus 100 ng/ml NGF) was replaced every other day up to 10 days of culture. The cells were either employed for fractionation studies or in situ immunocytochemistry. Nuclei and cytoplasm were obtained essentially as described earlier [26]. The cell fractionation buffer contained 10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 2 mM MgCl2 , 0.5% nonidet P-40 (NP-40), 2 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mM benzamidine, 10 mg/ml soybean trypsin inhibitor (STI), 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM Na3VO4 , 20 nM okadaic acid (all from Sigma), 15 mg/ml calpain inhibitor I, and 30 mg/ml calpain inhibitor II (Calbiochem, La Jolla, CA). The purity of isolated nuclei was carefully assessed by morphological and biochemical criteria as previously described [12, 27]. In addition, cytoplasmic contamination was also ruled out by immunochemical analysis with anti-cytoskeleton antibodies [28] employing monoclonal anti-actin (Amersham, Buckingamshire, UK) and anti-tubulin (Sigma). Further analysis of nuclear purity was performed by using a monoclonal antibody against PLC-d1, which, by both cytochemical and immunochemical

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analysis, was shown to be sharply cytoplasmic in PC12 and other cell types [26, 29]. Immunochemical analysis. Isoform specific anti-PKC a, bI, bII, g, d, e, h, z, and u antibodies and related control peptides were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). For Western blot analysis, PC12 whole cell lysates, cytoplasmic fractions, and isolated nuclei (50 mg protein) were separated by 0.1% SDS, 7.5% polyacrylamide gel electrophoresis (PAGE), according to Laemmli [30]. Proteins were then electroblotted onto nitrocellulose membranes [31]. The membranes were incubated in phosphatebuffered saline (PBS, Gibco), supplemented with 3% (w/v) nonfat dry milk for 30 min at room temperature, and then incubated overnight at 47C with anti-PKC a, bI, bII, g, d, e, h, u, and z isoform-specific antibodies (diluted 1:500). After four washings with PBS/0.1% Tween 20/0.1% bovine serum albumin (BSA), peroxidase-conjugated antirabbit IgG (diluted 1:1500) was applied to the membrane for 60 min at room temperature, washed, and revealed with the enhanced chemiluminiscence Western blotting detection reagent (Amersham). The specificity of immunoreactive bands was assessed by preincubating the single polyclonals with peptide antigens. Protein molecular weight markers (Sigma) were run with each gel. Assay of protein kinase C activity. One hundred micrograms of PC12 cell and nuclear lysates were incubated at 307C for 10 min in a reaction mixture containing 50 mM Tris–HCl, pH 7.4, 5 mM MgCl2 , 0.5 mM DTT, 100 mM Na3VO4 , 250 mM CaCl2 , 4 mg/ml DG, 100 mg/ml PS, 13 mM ATP, 1 mCi of [g-32P]ATP in a final volume of 50 ml. PKC activity was assayed by measuring the incorporation of 32 Pi into a serine-substituted peptide (RFARKGSLRQKNVHEVKN), corresponding to amino acids 19–36 of PKC (UBI). The reactions were stopped with an appropriate volume of 41 Laemmli treatment buffer (TB) [30], boiled for 5 min, and electrophoresed on 18% polyacrylamide/0.1% SDS. The gels were stained with Coomassie R-250, destained, and autoradiographed on Kodak X-OMAT S films. Under these conditions, the peptide was clearly separated and migrated to the bottom of the gel according to the calculated molecular weight (2.342 kDa, dephosphorylated form). The peptide spots were excised and radioactivity was counted in a liquid scintillation counter. Evaluation of bromodeoxyuridine (BrdU) incorporation and differentiation into sympathetic-like neurons. PC12 cells were seeded at low density (5 1 103/cm2) on poly-L-lysine- and collagencoated glass coverslips, and BrdU uptake was evaluated as previously described [32]. The proportion of differentiated cells was determined following the method described by Greene [21, see also Ref. 33]. A cell was considered differentiated when neurite length reached at least twice that of the cell body. Neurite length was determined on printed micrographs considering at least 100 randomly selected cells. Protein determination. The protein content of cell homogenates and isolated nuclei was determined with the Bio-Rad protein assay. Statistical analysis. The results were expressed as means { standard deviation (SD) of the means, obtained from three or more experiments performed in duplicate.

RESULTS

Nuclei were isolated from serum-starved, serumtreated, or NGF-treated PC12 cells following a previously established procedure [12, 16], which produced, in all cases, nuclei free of cytoplasmic contamination and lacking the nuclear membrane as shown by electron microscopy (Fig. 1). The purity of nuclear preparations was further evaluated by Western blot analysis with antibodies against actin, tubulin, and PLC-d1. These proteins, largely represented in whole cell homogenates, were absent from isolated nuclei (Fig. 2).

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FIG. 1. Ultrastructural analysis of nuclei isolated from serum-treated (a) or 10 days NGF-treated (b) PC12 cells in the presence of 0.5% NP-40. Note that the use of detergent in the fractionation procedure completely removed the nuclear membrane. Bar, 1 mm.

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FIG. 2. Western blot analysis of actin, tubulin and PLC-d1 associated with whole PC12 cell lysates and isolated nuclei probed with specific monoclonal antibodies. RB: rat brain (positive control); C: cells; N: isolated nuclei. Molecular weight markers are indicated on the left, and the apparent size of the three proteins is indicated on the right (kDa).

Taking into account the fact that PC12 cells show a significant percentage (1 in 106) of spontaneous mutations which can markedly alter their growth culture properties [19, 21], we first characterized the ability of our PC12 cell clone to respond to either mitogenic or differentiative stimuli. Therefore, serum-starved PC12 cells were supplemented with either high serum concentrations (10% HS plus 5% FCS) or 100 ng/ml NGF. The serum-starvation procedure (24 h in D-MEM plus 1% HS) greatly reduced the percentage of cells incorporating BrdU without significantly affecting cell survival. When serum-starved PC12 cells were placed in complete medium, a sharp increase in BrdU uptake was noticed after 24 h of culture, followed by a plateau. On the other hand, the addition of 100 ng/ml of NGF to serum-starved PC12 cells transiently increased the percentage of cells incorporating BrdU after the first 24 h of culture, followed by a progressive decline, which paralleled the homogeneous differentiation of most PC12 cells into sympathetic-like neurons with the emissions of long neurites (data not shown). Since all PKC isoforms analyzed thus far possess the same substrate specificity, reflecting the conservation of the substrate binding site [3], our assay performed on a serine-substituted peptide of the amino-terminal region of PKC allowed us to investigate the whole catalytic activity. The mitogenic response induced in serum-starved PC12 cells by the addition of 10% HS / 5% FCS was accompanied by a concomitant rapid increase in PKC activity in total cell homogenates and nuclei, which reached a maximal level after 24 h (Fig. 3). In the presence of NGF, total cell lysates showed a transient and marked peak of PKC activity after the first day of NGF treatment followed by a sharp decline and a secondary progressive increase (Fig. 3a). On the other hand, in isolated nuclei a constant increase from Day 1 onward occurred (Fig. 3b), which closely paralleled the secondary increase observed in total cell lysates (Fig. 3a). Basically similar results were obtained

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when histone H1 was used as an alternative substrate to explore PKC activity (data not shown). We next investigated whether qualitative changes of specific PKC isoforms occurred after the different cell treatments. Cellular, cytoplasmic, and nuclear fractions obtained from serum-starved PC12, PC12 treated for 2 days with 10% HS / 5% FCS, or PC12 cells induced to differentiate for 7 days with 100 ng/ml of NGF were compared by Western blot analysis with anti-PKC a, bI, bII, g, d, e, h, u, z antibodies (Fig. 4). As expected [3, 4], the apparent molecular weight of the different PKC isoforms ranged from 74 (d) to 92 kDa (e). We were not able to detect any PKC g and u in either cellular or nuclear homogenates. Compared to serum-starved cells, total cell lysates showed an increase of PKC a and e after both mitogenic and differentiative treatment. PKC a, in particular, was highly overexpressed

FIG. 3. PKC catalytic activity in (a) whole PC12 cell lysates and (b) isolated nuclei. The assay was performed at different culture times after the addition of mitogenic serum concentrations (10% HS / 5% FCS) or 100 ng/ml NGF to PC12 cells serum-starved for 24 h. Data are expressed as means { SD of four separate experiments performed in duplicate.

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PKC e and h, which were absent in nuclei from serumstarved and NGF-treated cells, and the increase of the levels of PKC bII, d, and z. Considering the relative intensity of the cytoplasmic and nuclear immunoreactive bands under the three conditions examined, clear-cut translocation to the nucleus occurred for PKC e and h in serum-stimulated cells. Nuclear accumulation of PKC following additional translocation from the cytoplasm was prominently induced for the z isoform after mitogenic stimulation and for PKC a during prolonged NGF treatment. DISCUSSION

FIG. 4. Representative Western blot analysis of PKC isoforms in PC12 whole cell homogenates (1), cytoplasms (2), and nuclei (3). The three fractions were obtained from serum-starved (S, 1% HS for 24 h), serum-stimulated (SS, 10% HS / 5% FCS for 48 h), and NGFtreated (NGF, 100 ng/ml for 7 days) PC12 cells. The apparent molecular weight of the PKC isoforms a, bI, bII, d, e, h, and z were 80, 79, 80, 74, 92, 84, 79 kDa, respectively. Molecular weight markers were b-galactosidase (116 kDa), phosphorylase b (97.4 kDa), and bovine albumin (66 kDa).

in differentiated cells. Clear-cut changes were observed for PKC bI, which lacked in quiescent and proliferating cells and appeared after NGF treatment. PKC h was overexpressed after serum stimulation and completely down-modulated in terminally differentiated cells. In addition, PKC d and z were increased in serum-stimulated cells. The differences in the expression of PKC isoforms among serum-starved, serum-treated, or NGFtreated PC12 cells were even more remarkable in isolated nuclei (Fig. 4). After long-term NGF treatment, only PKC a, bII, and z were present to a detectable extent in the cell nucleus. Compared to the basal levels found in serum-starved cells, nuclear PKC a largely increased, while nuclear PKC d decreased below the detection limit. In contrast, the addition of high serum concentrations to serum-starved PC12 cells induced the appearance at the nuclear level of

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The multigene family of PKC enzymes is involved in the control of crucial biological events, such as cell proliferation and differentiation. PKC is present in virtually all tissues and constitutes one of the major transducers of receptor-mediated stimuli. The different cellular responses induced by PKC activation vary widely depending upon the different cell types considered, the kind of agonist employed, and the length of stimulation [2, 4]. In PC12 cells, the role played by PKC in proliferation and differentiation is still largely unsettled. For instance, in NGF-mediated differentiation both PKCdependent [34–39] and PKC-independent mechanisms have been reported [33, 40, 41]. Our data indicate that when PC12 cells were induced to differentiate into sympathetic-like neurons by the addition of NGF, deep changes in PKC activity characterized the onset and maintenance of the differentiated state. The most relevant feature was the progressive increase in the activity of nuclear PKC, which paralleled the prolonged rate of neurite appearance occurring after the initial 24- to 36-h lag period of NGFinduced neuritogenesis [21]. On the other hand, the transient peak of PKC activity observed only in whole cell lysates after the first day of NGF treatment correlated well with the slight mitogenic effect exerted initially by NGF. Consistently, when serum-starved cells were supplemented with high serum concentrations (10% HS plus 5% FCS), an early increase of the activity of nuclear and cellular PKC was followed by a plateau. The increase of nuclear PKC activity was accompanied by clear-cut modifications in isoform composition, which resulted, compared to both serum-starved and serum-treated PC12 cells, in a quite distinct PKC arrangement. In this respect, at the whole cell level, it was already suggested that some PKC isoenzymes, which are expressed in tissue and differentiation stagespecific patterns [3, 41], may be involved in the NGFmediated differentiation of PC12 cells [37, 42]. Recently, it has been reported that during NGF-induced differentiation total cellular PKC activity increases, PKC a is highly expressed, and PKC d translocates to the particulate fraction, playing an essential role in

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neuritogenesis [43]. We have documented here, for the first time, the existence of striking differences in the composition of PKC isoforms in differentiated versus proliferating PC12 cells at both cellular and nuclear levels. The large increase in catalytic activity observed after 7–10 days of NGF treatment was paralleled by the appearance of PKC bI and up-regulation of PKC a and e. PKC a was the only isoform which prominently increased in comparison to starved and mitogenically stimulated cells. Similarly, an increase in PKC a has been associated with megakaryocytic differentiation of the K562 hematopoietic cell line [44] and with NGFinduced differentiation of PC12 [43], although the role of nuclear PKC a was not investigated in those studies. On the other hand, PKC d, e, h, and z appeared to be up-regulated in proliferating cells at cytoplasmic or nuclear levels, and most of them were variably downregulated in differentiated PC12 cells. Of note, activation of PKC z has been coupled to mitogenic stimuli in a different cell system [45], while it has been recently demonstrated that PKC d, e, h [46], and z [47] are potently and selectively activated by the products of phosphatidylinositol 3-kinase [48], which plays a key role in many signal transduction pathways mediated by receptor-associated tyrosine kinases. As a whole, our data support the contention that signal transduction pathways lead to quite divergent responses, such as proliferation and differentiation in PC12 cells, by the usage of different PKC isoenzymes. The progressive increase of cellular and nuclear PKC activity in differentiated PC12 cells strengthens the notion that PKC may modulate programs of cell differentiation by inducing phosphorylation of specific transcription factors and phenotype-related cellular proteins. It has been proposed that PKC may cycle in and out of the nucleus, transmitting information by phosphorylating various proteins that have key functions in cellular activities, and may associate with the nucleus after an increase in diacylglycerol [18, 49]. Although the mechanisms controlling the migration of PKC molecules to the nucleus are largely unknown [50], it is noteworthy that PKC a is a Ca2/- and phospholipiddependent enzyme. The presence of specific PKC isoforms at the nuclear level could potentially be modulated by the products of the inositol lipid cycle, which our group and other groups have demonstrated to be present in the nucleus of different cell types [26, 29, 49, 51–53]. It has also been proposed that Ca2/- and phospholipid-dependent isoforms contain domains, not shared by other isoforms, that can facilitate nuclear localization [50]. However, since nuclear PKC bII, d, e, h, and z appeared to be involved in the response of PC12 cells to mitogenic stimulation, it is conceivable that other, probably more subtle, mechanisms may ac-

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count for the selective recruitment of specific PKC isoforms at the nuclear level. We thank Maurizio Stroscio for photographic assistance. This work was supported by CNR (Target Project ACRO and Grant 94.00413.CT12), MURST (40% and 60%), and AIRC. L.B. is the recipient of a fellowship from AIRC.

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Received November 28, 1995 Revised version received January 16, 1996

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