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Volume 17, Number 3, Issue of February 1, 1997 pp. 924-931
Copyright ©1997 Society for Neuroscience

Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II in PC12 Cells Alters Cell Growth, Morphology, and Nerve Growth Factor-Induced Differentiation

Thierry Massé1 and Paul T. Kelly2

1 Immuno-Virologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique-UMR 5537, Faculte de Medecine Lyon-Laënnec, 69372 Lyon Cedex 08, France, and 2 Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77225

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

To examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in cell differentiation and neuronal functions, stable transformants of PC12 cells were established that expressed levels of the alpha -subunit of CaMKII (alpha CaMKII) equivalent to mammalian neurons. The expression of the transfected alpha CaMKII gene or the endogenous beta CaMKII gene was monitored by RNase protection assays, and alpha CaMKII protein expression was determined by Western blots. Several PC12-derived clones expressed amounts of alpha CaMKII mRNA and alpha CaMKII protein similar to that of hippocampal tissues and several orders of magnitude greater than untransfected PC12 cells. CaMKII catalytic activity was four times higher in extracts from alpha CaMKII-overexpressing compared with untransfected PC12 cells. All clones overexpressing alpha CaMKII displayed altered cellular growth and adhesion properties including increased cell-to-substrate adhesion, decreased cell-to-cell adhesion, enhanced contact inhibition, and prolonged survival at confluency. Furthermore, the alpha CaMKII activity in overexpressing PC12 cells inhibited neurite elongation during NGF-induced differentiation. Inhibition of CaMKII activity in vivo with KN-62 caused the morphological phenotypes of alpha CaMKII-overexpressing cells to partially revert to that of untransfected PC12 cells. These results show that alpha CaMKII catalytic activity affects growth, morphology, and NGF-induced differentiation of PC12 cells.

Key words: cell Ca2+; cytoskeleton; growth cone; neurite; nerve growth factor; PC12 cells; Ca2+/calmodulin-dependent protein kinase

INTRODUCTION

Calcium/calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant neuronal protein kinases (Kelly, 1991). The activity of CaMKII was first identified in neuronal membrane fractions (Schulman and Greengard, 1978), and the alpha -subunit of CaMKII was later identified as the major postsynaptic density protein (mPSDp) (Kennedy et al., 1983; Rostas et al., 1983; Kelly et al., 1984; Sahyoun et al., 1985). The mPSDp displays characteristics of a structural protein, such as insolubility in chaotropic agents (Kelly and Cotman, 1976a; Feit et al., 1977), and a propensity to form disulfide bonds with itself and other synaptic proteins (Kelly and Cotman, 1976b). CaMKII is distinct from all known cytoskeletal/structural proteins (Kelly and Cotman, 1976a) and represents 30-40% of total proteins in PSDs, suggesting that alpha CaMKII could function as a structural/cytoskeletal protein at synapses (Kelly, 1991; Schulman and Hanson, 1993). Among the proteins present in PSDs, tubulin, actin, and fodrin are found in stoichiometric amounts, suggesting that alpha CaMKII could form complexes with these cytoskeletal proteins (Carlin et al., 1983; Sahyoun et al., 1985, 1986). Purified cytoplasmic CaMKII also appears to interact under in vitro conditions with polymers of purified actin (Ohta et al., 1986). However, the mechanisms regulating the accumulation of alpha CaMKII at synapses, as well as its function in the developing brain, are largely unknown (Kelly, 1991).

CaMKII phosphorylates a broad range of substrates and is expected to have pleiotropic regulatory actions in cellular differentiation and mature neuronal functions. For example, CaMKII in the neuronal nuclear matrix and PSDs are structurally related and phosphorylate a broad range of substrates in these cellular compartments (Sahyoun et al., 1984a,b). The activity of CaMKII also appears to be involved in nuclear membrane breakdown (Baitinger et al., 1990). Expression of a truncated, constitutively active form of CaMKII in mammalian cells leads to arrest of the cell cycle in G2 (Planas-Silva and Means, 1992). In addition, the activity of CaMKII in postsynaptic hippocampal neurons is necessary for the induction of long-term potentiation (Silva et al., 1992; Wang and Kelly, 1995), a cellular model of learning in mammals.

Expression of high levels of alpha CaMKII in a cell model is important to understand its potential role as an enzyme and/or a structural protein (Sahyoun et al., 1985, 1986). The studies herein have examined the hypothesis that alpha CaMKII overexpression alters the general cell growth, differentiation, and morphology of neurons. Our goal was to develop a cellular model that displays high levels of CaMKII expression characteristic of many CNS neurons. The PC12 cell model described herein allowed us to examine a number of cellular changes associated with alpha CaMKII expression and NGF-induced cell differentiation and determine which cellular properties require alpha CaMKII catalytic activity versus its potential function as a cytoskeletal/structural protein (T. Massé and P. Kelly, unpublished observations). A role for CaMKII in cellular morphology has been reported in neuroblastoma NB2a and NG-108 cells (Goshima et al., 1993) or PC12 cells (Tashima et al., 1996) using experimental strategies somewhat like ours. We found that overexpression of alpha CaMKII in PC12 cells greatly enhances cell survival in confluent cultures, increases cell-to-substrate adhesion, and greatly inhibits neurite outgrowth during NGF-induced cellular differentiation. A preliminary account of these results appeared elsewhere (Massé et al., 1993).

MATERIALS AND METHODS

Cell culture, transfection, and clonal selection. PC12 cells were grown in DMEM (5-10% CO2) supplemented with 10% horse serum and 5% fetal calf serum (FCS). Differentiation was induced by incubating cells with 50 nM NGF (generously provided by William Mobley, UCSF) in DMEM + 1% FCS.

The cDNA encoding alpha CaMKII (rat brain) was subcloned into the eukaryote expression vector pRc/cytomegalovirus (CMV, Invitrogen) containing the early CMV promoter and genes for neomycin and ampicillin resistance (pCMV-alpha CaMKII). PC12 cells were plated on poly-L-ornithine-coated tissue culture plates and transfected 24 hr later with plasmids linearized with ScaI (cutting in the ampicillin gene, increasing the likelihood of integrating the full-length plasmid with intact alpha CaMKII and neomycin resistance genes). Transfections were done by the Ca2+ phosphate method (Graham and van der Eb, 1973); precipitates were left on cells for 16 hr, washed with DMEM, and incubated in complete medium for 24 hr before adding the neomycin analog G418. Stable transfectants were selected over 2 weeks in medium containing G418 (0.5-3.0 mg/ml). Clones were isolated and maintained under G418 selection for 2 additional weeks; clones overexpressing alpha CaMKII (alpha PC12) were identified by immunofluorescence.

RNase protection. [32P]cRNAs were prepared from pCMV-alpha CaMKII or pCMV-beta -CaMKII (beta -CaMKII cDNA was a gift from Dr. Rachael Neve) linearized with ClaI or HinfI, respectively. SP6 RNA polymerase (Melton et al., 1984) was used to generate 32P-labeled probes, which were hybridized overnight at 55°C to total cellular RNA prepared from rat hippocampus, untransfected PC12 cells, or alpha CaMKII-overexpressing PC12 cells. Single-strand cRNAs were digested at room temperature for 1 hr with 40 µg/ml RNase A + 3000 U/ml RNase T1; [32P]cRNA fragments were sized on polyacrylamide/urea sequencing gels.

In vitro phosphorylation. Cells were harvested by scraping in PBS, pelleted (1000 × g/10 min) at 4°C, resuspended in 150 µl of ice-cold homogenization buffer containing (in mM): 10 Tris-HCl pH 7.5, 1 EGTA, 1 EDTA, 0.5 dithiothreitol (DTT), 0.1 phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin and 20 µg/ml soybean trypsin-inhibitor, and sonicated for 5 sec. Protein concentrations were determined by the micro-BCA method (Pierce). Phosphorylation reactions were initiated by adding 5 or 10 µg of cellular extract protein (in 10 µl) to 40 µl of a solution, such that the following final concentrations were obtained (in mM): 25 HEPES, pH 7.4, 10 MgCl2, 0.5 DTT, 2 CaCl2, 1.5 µM CaM, and 50 µM ATP and 3.5 µCi 32P-gamma -ATP (3000 Ci/mmole). Certain reactions contained 20 µM autocamtide-3, a peptide substrate for CaMKII (Hanson and Schulman, 1992). To determine Ca2+-independent CaMKII activity in cell extracts, Ca2+/CaM was replaced with 0.5 mM EGTA or 0.5 mM EGTA + 50 µM of a pseudosubstrate peptide inhibitor of CaMKII [Ala286(281-302)] (Hanson and Schulman, 1992). Phosphorylation reactions (31°C for 45 or 60 sec) were linear with respect to time and protein concentration (5-10 µg) and were terminated by the addition of 4× SDS sample buffer and heated at 85°C for 5 min or spotted on phosphocellulose filters, as described elsewhere (Waxham et al., 1990).

RESULTS

alpha CaMKII overexpression in PC12 cells

In initial experiments, NB2a and NG-108 cells proved unsuitable as cellular models for the overexpression of alpha CaMKII. First, we were unable to select clones expressing high levels of alpha CaMKII (e.g., similar to rat hippocampal neurons). Second, we found that the expression of alpha CaMKII under the regulation of several promoters (e.g., CMV, RSV, and SV-40) was inhibited when cellular differentiation of these cell lines was induced by 8-bromo-cAMP or retinoic acid (data not shown). In contrast, stable PC12 cell transformants were routinely obtained that expressed high levels of alpha CaMKII that approximated its expression in hippocampal neurons. In addition, short-term growth (~4 hr) of alpha CaMKII-overexpressing PC12 cells in NGF produced a transient increase in alpha mRNA levels (data not shown), which was expected because the CMV promoter contains a serum response element that can be stimulated by NGF.

Independent clones (~100) overexpressing alpha CaMKII were selected and studied. The level of alpha CaMKII expression in untransfected versus transformed PC12 (alpha PC12) cells was determined using RNase protection assays. Total RNA from rat hippocampus and alpha CaMKII-overexpressing cells showed protected fragments of the expected size for alpha CaMKII mRNA (Fig. 1A,a). The levels of alpha CaMKII mRNA in several alpha CaMKII-overexpressing cell lines were comparable with levels in rat hippocampus. In contrast, very low alpha -mRNA signals were detected after RNase protections using RNA prepared from untransfected PC12 cells. Densitometry of autoradiograms showed that untransfected PC12 cells contained ~100- to 250-fold less alpha -mRNA than alpha PC12 cells. A probe specific for beta -CaMKII mRNA demonstrated that its concentration was similar in untransfected and alpha PC12 cells, but ~2 times lower than rat hippocampal RNA. Western blot analysis showed high levels of alpha CaMKII in most G418 resistant clones after transfection, whereas alpha CaMKII was undetectable in untransfected PC12 cells (Fig. 1B). Many PC12 clones expressed levels of alpha CaMKII comparable with rat hippocampus (i.e., ~2% of total protein) (Erondu and Kennedy, 1985). Although most of the results presented herein were with the two alpha PC12 clones shown in Figure 1, we observed the same overall phenotype for numerous clones that expressed as little as 25% as much alpha CaMKII.

Fig. 1. Analysis of alpha CaMKII mRNA and protein expression in PC12 cells and hippocampus. A, RNase protection analysis of alpha CaMKII (a) and beta CaMKII (b) mRNAs in normal and alpha CaMKII-overexpressing PC12 cells or hippocampal tissue from adult rat brain. Total cellular RNA from two different PC12 cultures, two independent clones overexpressing alpha CaMKII, or hippocampi were analyzed. Equivalent amounts of RNA were hybridized with subunit-specific cRNA probes and digested with RNase A + T1 (see Materials and Methods). Autoradiograms show cRNA fragments protected by mRNAs from alpha CaMKII- overexpressing cells (lanes 1, 2), untransfected cells (lanes 3, 4), and hippocampus (lanes 5, 6). B, Western blot analysis of alpha CaMKII (top) and actin (bottom) in untransfected PC12 cells (lane 2), alpha CaMKII-overexpressing PC12 cells (lanes 1, 4-8), and rat brain hippocampus (lane 3). Equivalent amounts of total cellular protein (50 µg) were analyzed with monoclonal antibodies specific for alpha CaMKII or actin.
[View Larger Version of this Image (30K GIF file)]

The Ca2+/CaM-stimulated CaMKII activity in alpha PC12 cell extracts was ~50% greater than the activity measured in hippocampal extracts, as determined by the in vitro phosphorylation of autocamtide-3 (see Materials and Methods). In addition, alpha PC12 cells contained 4.1 ± 0.6 (n = 6) times higher Ca2+/CaM-stimulated CaMKII activity compared with untransfected cells. The Ca2+-<UNL>in</UNL>dependent CaMKII activity in cell extracts was determined in assays containing EGTA, with or without a pseudosubstrate inhibitor of CaMKII [Ala286(281-302)]. Although alpha PC12 cells contained significantly more Ca2+/CaM-dependent CaMKII activity compared with untransfected PC12 cells, the percentage of total CaMKII activity that was Ca2+-<UNL>in</UNL>dependent in alpha PC12 cells (9.8 ± 0.4%, n = 10) was very similar to the Ca2+-<UNL>in</UNL>dependent activity in extracts from untransfected cells (9.3 ± 1.1%). We also examined the distribution of CaMKII activity in crude soluble (S1) and particulate (P1) fractions prepared from alpha PC12 and untransfected PC12 cell homogenates (Table 1). These results indicated that alpha PC12 cells contained ~3.8 ± 1.1 (n = 4) times more Ca2+/CaM-stimulated CaMKII activity compared with untransfected cells. The percentage of total CaMKII activity that was Ca2+-independent in S1 and P1 fractions from alpha PC12 cells (5.1 ± 1.7%, n = 4) was 50% lower compared with untransfected cells (9.3 ± 0.6%, n = 4). These results indicate that although the percentage of Ca2+-independent CaMKII activity in alpha PC12 cells is slightly lower than untransfected PC12, the absolute amount of Ca2+-independent activity is higher (three- to fourfold) in alpha PC12 cells.

Table 1. Substrate phosphorylation (CPMs)

Experimental group EGTA Ca2+/CaM Ca2+-independent activity (%)
Hippocampus Ha 2000 21,200 9.4
PC12 Cells S1 540 5880 9.2
P1 2040 21,830 9.3
 alpha CaMKII S1 2640 38,900 6.8
PC12 Cells P1 5120 56,810 9.0
CaMKII 1240 129,800 1.0
a PC12 cell and tissue homogenates (H) were prepared in homogenization buffer (see Materials and Methods) using a Dounce homogenizer; homogenates were centrifuged 10,000 × g (10 min/4°C) to prepare crude particulate (P1) and soluble (S1) fractions. Values (average of duplicates) are from a representative experiment; phosphorylation reactions used 5 µg of extract protein (0.1 µg for CaMKII); CaMKII was purified as described elsewhere (Waxham et al., 1990). A background value for each phosphorylation condition was obtained with reactions to which extract protein was not added (background was subtracted from each experimental value).

The in vitro phosphorylation of endogenous proteins in cell extracts was analyzed by SDS-PAGE and autoradiography. Scanning densitometry of autoradiograms showed that the Ca2+/CaM-dependent phosphorylation of several proteins was four- to sixfold greater in alpha PC12 cell extracts compared with untransfected PC12 cells (data not shown). These results confirm that alpha CaMKII-overexpressing PC12 clones contain similar levels of functional CaMKII activity compared with hippocampal tissue, which makes them a suitable model to study the role of CaMKII in neuronal functions.

alpha CaMKII overexpression alters cell growth and adhesion properties

A majority of clones overexpressing alpha CaMKII displayed very similar growth properties. Within 30-60 min after plating, alpha PC12 cells attached and spread on substratum; all were flat and phase-contrasted within 24 hr. Under similar conditions, untransfected PC12 cells displayed small, round, and phase-bright morphologies, which by 24 hr, remained the predominant phenotype (data not shown). Over the course of 3-5 d in culture, untransfected PC12 cells flattened and grew as tight aggregates, whereas alpha PC12 cells remained flat and separated from one another, with distinct cell borders (Fig. 2). In contrast, clones selected for overexpression of beta -galactosidase (under control of the CMV promoter) displayed morphological properties (± NGF) that were similar to untransfected PC12 cells (data not shown).
Fig. 2. General morphology of normal (A) and alpha CaMKII-overexpressing (B) PC12 cells. Phase-contrast micrographs were obtained on living cells plated at low density on untreated tissue culture plastic and grown for 5 d in standard medium containing 15% serum. Scale bar, 100 µm.
[View Larger Version of this Image (90K GIF file)]

When untransfected PC12 cells were plated on plastic bacteriological Petri dishes, <10% of the cells attached within 24 hr, and most cells formed suspension aggregates (data not shown). In contrast, ~50% of alpha PC12 cells attached to bacteriological plastic within 60 min of plating, and all attached within 24 hr (data not shown). The morphology and growth cones of G418-resistant alpha PC12 clones expressing very low or undetectable levels of alpha CaMKII were similar to that of untransfected PC12 cells. These differences in cell morphology indicated that alpha CaMKII overexpression results in increased cell-to-substrate adhesion and decreased cell-to-cell contact.

Table 2. NGF-induceda neurite extension of alpha CaMKII-overexpressing PC12 cells

Culture condition No neurites (%) Short neurites (%) Long neurites (%)
Untransfected cells (n = 652) 6 22 72
Untransfected cells + KN-62 (n = 401) 7 26 67
 alpha CaMKII-overexpressing cells (n = 543) 68 26 6
 alpha CaMKII-overexpressing cells + KN-62 (n = 728) 24 43 33
a Untransfected or alpha CaMKII-overexpressing PC12 cells were cultured in NGF (50 nM) in the presence or absence of KN-62 (10 µM). Neurites were analyzed by phase-contrast microscopy after 2 d of differentiation in NGF. Neurite extension was assessed by counting cells with short neurites (length <=  1 body diameter) or long neurites (length >=  1 cell body diameter); data are expressed as the percentage of total cells with long, short, or no neurites.

During the first 3 d after trypsinization and replating, the initial replication rate of alpha PC12 cells was ~50% slower than untransfected PC12 cells (Fig. 3). However, during exponential growth (3-6 d after replating), both alpha PC12 and untransfected PC12 cells displayed similar growth rates. alpha PC12 cells stopped dividing at lower cell densities. The slower initial replication rate and the inhibition of replication at lower cell density both suggest that contact inhibition is enhanced in alpha PC12 cells. alpha PC12 cells displayed a high capacity to survive after reaching confluency compared with untransfected cells (Fig. 3). Untransfected PC12 cells underwent significant cell death 2-3 d after reaching confluency, even though half of the culture medium was replaced with fresh medium every 2 d. In contrast, alpha PC12 cells showed little cell death for >7-10 d after reaching confluency; in several experiments, confluent cultures of alpha PC12 cells displayed no apparent cell death for 3-4 weeks (data not shown).

Fig. 3. Cellular growth curves of untransfected (dashes) and two independent clones of alpha CaMKII-overexpressing PC12 cells (a, b). After trypsinization of confluent cultures, equivalent numbers of cells (5 × 105) were plated on tissue culture plastic and grown for the indicated times; half of the medium was replaced with fresh medium every 2 d. Cells were trypsinized, pelleted, and resuspended in PBS plus Trypan blue for counting. The number of total viable cells are given as the mean of triplicate cultures for each condition. Error bars indicate SEM.
[View Larger Version of this Image (16K GIF file)]

alpha CaMKII overexpression alters cellular response to NGF

Because PC12 cells overexpressing alpha CaMKII displayed distinct growth properties under standard culture conditions, we examined their behavior during NGF-induced cell differentiation. All clones overexpressing alpha CaMKII displayed very similar morphologies during NGF-induced cell differentiation. Within 1-2 hr after adding NGF (50 nM) and decreasing serum from 15 to 1%, alpha PC12 cells flattened and formed numerous growth cone-like structures, which did not extend from the perimeter of the cell body (Fig. 4). Even after 2-3 d in NGF, alpha PC12 cells still displayed large growth cones with little or no neurite extension (Figs. 4A, 5). Untransfected PC12 cells, or G418 selected clones that did not overexpress alpha CaMKII, rapidly extended many long neurites in NGF (Fig. 4B). At least 4-5 d in NGF was required for a significant number of alpha PC12 cells to extend neurites longer than 1 cell body diameter (see below). Untransfected PC12 cells grown in NGF for 4 d displayed long and slender neurites that appeared to contact the substrate only once or twice throughout their length (Fig. 4B). This apparent weak substrate adhesion of untransfected PC12 neurites was supported by their fragility during media changes or histochemical processing. In contrast, the very short neurites of alpha PC12 cells, when present, appeared flat, wide at their base, and more strongly adherent to the substratum compared with neurites of untransfected cells (see Fig. 4A). Thus, the overexpression of alpha CaMKII appeared to inhibit neurite elongation, promote growth cone flattening, and increase the adhesion of cells and neurites to substrate.
Fig. 4. Phase-contrast morphology of alpha CaMKII-overexpressing (A, C, D) and untransfected (B) PC12 cells after NGF differentiation under the following conditions: NGF differentiation for 2 d (A), NGF differentiation for 5 d (B), NGF differentiation in KN-62 (10 µM) for 2 d (C), and NGF differentiation in KN-62 (10 µM) for 5 d (D). Scale bar, 100 µm.
[View Larger Version of this Image (218K GIF file)]

Fig. 5. Time-lapse microscopy of growth cone dynamics. Phase-contrast micrographs of alpha CaMKII-overexpressing PC12 cells after culture in NGF for 2 d. Sequential micrographs of the same cells every 3 hr showed that growth cones are not stable during a 6 hr period (white arrowheads); after growth cone retraction/collapse, they reappear at the same location on the cell surface (black arrowheads). Scale bar, 50 µm.
[View Larger Version of this Image (84K GIF file)]

To determine whether these alterations in cell morphology and inhibition of neurite extension required alpha CaMKII activity, alpha PC12 cells were cultured in KN-62, an inhibitor of CaMKII (IC50 of ~1 µM when assayed using purified CaMKII) (see Tokumitsu et al., 1990). Cell morphologies were analyzed by phase-contrast microscopy after differentiation in NGF + or - KN-62 (1-10 µM). Neurite extension was assessed by counting cells presenting short neurites (length <=  1 body diameter) and cells with long neurites (length >=  1 cell body diameter). After 2 d in NGF, 72% of untransfected PC12 cells displayed long neurites and 22% short neurites (Table 1). After 2 d in NGF + KN-62 (10 µM), KN-62 had no detectable effect on the percentage of untransfected cells displaying short (26%) versus long neurites (67%). In contrast, alpha PC12 cells differentiated very little after 2 d in NGF, with only 6% of the cells displaying long neurites and 26% showing short neurites. The remaining cells (68%) displayed large growth cones immediately adjacent to the cell body with no apparent neurites. In contrast, alpha PC12 cells differentiated in NGF + KN-62 (10 µM) for 2 d displayed highly differentiated morphologies, with 33% displaying long neurites and 43% short neurites (Table 1, Fig. 4C). Because this KN-62 concentration is 10 times higher than its in vitro IC50 (see above), we examined the effects of lower concentrations of inhibitor. alpha PC12 cells differentiated in NGF + 1 µM KN-62 displayed morphologies that were very similar to alpha PC12 cells differentiated in NGF alone (data not shown), whereas cells grown in NGF + 5 µM KN-62 displayed long neurites very similar to cells grown in NGF + 10 µM KN-62. KN-62 was added to cultures only once at the start of each experiment (1-2 hr after NGF addition), and its stability in cultures was not assessed; most experiments used 10 µM KN-62. Phase-contrast microscopy also showed that cell-to-substrate adhesion of alpha PC12 cells decreased during differentiation in NGF + KN-62. The number of growth cones per cell was not affected by KN-62 in either untransfected or alpha PC12 cells. Thus, KN-62 significantly altered the morphology and neurite extension of alpha CaMKII-overexpressing cells grown in NGF such that they appeared similar to untransfected PC12 cells during NGF-induced differentiation.

To further examine the apparent inhibition of neurite outgrowth in alpha CaMKII-overexpressing PC12 cells, cells were grown in NGF for 2 d and then time-lapse micrographs were taken at 3 hr intervals. Figure 5 shows that growth cones routinely form, grow in size, and then retract in <3 hr without extending neurites (growth cones marked with black arrowheads). Growth cones also appear to retract and reappear at the same location on a cell (growth cones marked with black and white arrowheads). In contrast, neurites of untransfected PC12 cells grown in NGF extended long distances from the cell body (Fig. 4B). These neurites do not retract during the course of 1 cell cycle (Waymire et al., 1978). These results indicate that the activity of alpha CaMKII in overexpressing cells inhibits neurite elongation but not growth cone formation.

DISCUSSION

One goal of these studies was to develop a cell model that displays certain phenotypes of CNS neurons, such as the expression of high levels of the alpha CaMKII. This goal was achieved using PC12 cells transfected with alpha CaMKII cDNA under control of the strong CMV promoter. Comparison of many G418-resistant PC12 clones expressing high versus low or undetectable levels of alpha CaMKII showed that the properties described above were positively correlated with alpha CaMKII overexpression. alpha CaMKII overexpression resulted in increased cell-to-substrate adhesion (i.e., larger and flatter cells), decreased cell-to-cell adhesion, and growth saturation at lower cell densities. Furthermore, alpha CaMKII overexpression inhibited neurite outgrowth during NGF-induced differentiation.

A correlation between alpha CaMKII overexpression and cell morphology has been reported in neuroblastoma (NB2a) and NG-108 cells (Goshima et al., 1993) or PC12 cells (Tashima et al., 1996) using experimental strategies similar to ours. The major differences between our study and the latter studies are the cell models and differentiation protocols. We observed that transfected NB2a and NG-108 cells did not express very high levels of alpha CaMKII compared with hippocampal or cortical tissues, which we believe is important to assess the enzymatic and structural roles of alpha CaMKII in neuronal functions. In addition, we used NGF as a morphogen, whereas the other studies used membrane-permeable cyclic nucleotide analogs (i.e., dibutyryl-cAMP or 8-bromo-cAMP), and cAMP analogs may cause a short-term induction of CRE-containing promoters (e.g., c-fos) followed by a downregulation or refractory period (Gius et al., 1990; Foulkes et al., 1991; Marksitzer et al., 1995) (Massé and Kelly, unpublished observations). It is also possible that long-term activation of cAMP pathways may enhance other phosphorylation pathways, including those mediated by CaMKII. For example, the phosphorylation of inhibitor-1 by PKA will increase the inhibition of type-1 protein phosphatase that dephosphorylates many proteins phosphorylated by CaMKII (Shields et al., 1985). In our studies, the differentiation of alpha PC12 cells with dibutyryl- or 8-bromo-cAMP decreased the levels of alpha CaMKII mRNA and protein. In contrast, growth of overexpressing PC12 cells in NGF for 1-5 d did not significantly change their cellular content of alpha CaMKII or its in vitro catalytic activity assayed with cell extracts.

In agreement with Goshima et al. (1993), we found that alpha CaMKII overexpression enhances cell-to-substrate adhesion. However, our studies with PC12 cells show that increased adhesion does not stimulate neurite outgrowth, suggesting that high levels of alpha CaMKII activity inhibit other mechanisms involved in growth cone motility. With regard to the properties of NGF- versus cAMP-induced differentiation, we observed that the retarded neurite outgrowth of alpha PC12 cells was reversed by the CaMKII inhibitor KN-62; this reversal was not observed by Tashima et al. (1996). Because the inhibition of neurite extension in alpha PC12 cells was reversed with KN-62, it seems unlikely that our results were attributable to selecting G418-resistant PC12 clones that were simply defective in extending neurites.

Although alpha CaMKII overexpression altered the growth and neurite extension properties of PC12 cells, it did not compromise their ability to respond to NGF; growth cones formed rapidly after adding NGF, and the relative abundance of NGF receptors in PC12 cells as determined immunohistochemically changed very little in alpha CaMKII-overexpressing cells (data not shown). Neurite elongation appeared to be inhibited by CaMKII catalytic activity. This inhibition seemed to be attributable to growth cone instability of alpha PC12 cells grown in NGF, because growth cones emerged transiently and repetitively without forming neurites (Massé and Kelly, unpublished observations). alpha CaMKII overexpression did not dramatically change the percentage of Ca2+-independent CaMKII activity in cells relative to total Ca2+/CaM-stimulated CaM-kinase activity. Although alpha PC12 cells contained three- to fourfold higher absolute levels of Ca2+-independent CaMKII activity compared with untransfected cells, we believe this activity is unlikely to contribute to the distinct phenotype of alpha PC12 cells. This is based on the fact that the CaMKII inhibitor KN-62 is competitive with Ca2+/CaM and does not inhibit Ca2+-independent CaMKII activity (Tokumitsu et al., 1990). Our results show that the apparent inhibition of Ca2+/CaM-dependent CaMKII activity with KN-62 resulted in growth cone stabilization and neurite extension and suggest that alpha CaMKII activity in alpha PC12 cells inhibits neurite outgrowth by disrupting one or more steps in the mechanism of neurite elongation, possibly through increased phosphorylation of specific protein substrates (Tokui et al., 1990; Lapadula et al., 1991). Mechanisms that regulate neurite extension and growth cone motility most likely require a dynamic balance between Ca2+/CaM-dependent protein kinase and phosphatase activities. Recent observations show that the Ca2+/CaM-dependent protein phosphatase calcineurin functions in neurite outgrowth and directed filopodia motility in dorsal root ganglion neurons (Chang et al., 1995).

Results published recently suggest that KN-62 may have additional actions besides inhibiting CaM kinases. For example, KN-62 appears to inhibit Ca2+ influx in adrenal chromaffin cells (Maurer et al., 1996); however, KN-62 had no observable effect on cell morphology. Additional data suggest that KN-62 blocks voltage-gated Ca2+ channels in adrenal chromaffin cells (Marley and Thomson, 1996), whereas it does not affect Ca2+ currents in hippocampal CA1 neurons (Wyllie and Nicoll, 1994). It is also important to note that CaM-kinase activity can upregulate Ca2+ channel activity (Wang and Best, 1992; Kitamura et al., 1993), which suggests that cells treated with KN-62 may indirectly downregulate Ca2+ channel influx attributable to KN-62's inhibition of CaM-kinase.

alpha CaMKII-overexpressing cells displayed altered growth properties, including decreased replication rates, enhanced contact inhibition, and greatly enhanced survival after reaching confluency, compared with untransfected PC12 cells. This result suggests that alpha CaMKII may enhance cell-to-cell signaling that regulates cell replication as well as participates in maintaining metabolic homeostasis during quiescence. Because the expression of alpha CaMKII in postmitotic neurons in vivo remains high in the adult brain (Rostas et al., 1983; Burgin et al., 1990), our discovery that alpha CaMKII overexpression greatly enhances cell survival at confluency suggests that CaMKII contributes to cellular homeostasis and longevity of neurons in the brains of adult and aging humans. The mechanisms underlying such roles for CaMKII in neuronal function probably involve the regulation of gene expression (Sheng et al., 1990; Lerea and McNamara, 1993). However, it is tempting to propose that certain features of the alpha CaMKII-overexpression phenotype is attributable to a direct regulation of cytoskeletal functions by CaMKII (Iwig et al., 1995) (Massé and Kelly, unpublished observations).

FOOTNOTES

Received May 2, 1996; revised Oct. 31, 1996; accepted Nov. 11, 1996.

  

This work was supported by grants from the Human Science Frontiers Program (T.M.) and National Institutes of Health (P.T.K.). We thank Kim Huber and Tony Moore for helpful comments.

Correspondence should be addressed to Dr. Paul T. Kelly, Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, Houston TX 77225.


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