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Human Molecular Genetics, 2003, Vol. 12, No. 11 1211-1221
DOI: 10.1093/hmg/ddg146
© 2003 Oxford University Press

Upregulation of the Rac1/JNK signaling pathway in primary human schwannoma cells

Katherine Kaempchen, Kirsten Mielke{dagger}, Tamara Utermark, Sonja Langmesser and C. Oliver Hanemann*

Department of Neurology, Zentrum für klinische Forschung, University of Ulm, Helmholtzstr 8/1, 89081 Ulm, Germany

Received March 3, 2003; Revised March 27, 2003; Accepted April 4, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Schwann cells lacking the tumor-suppressor-protein merlin tend in man to build benign tumors (schwannoma). We observed that characteristic features of these cells which are relevant to tumorigenicity resemble those described in cells with high Rac activity. Moreover this small GTPase also phosphorylates merlin via PAK activation. We hypothesized that merlin deficiency might cause an activation of Rac and its dependent signaling pathways, in particular the pro-tumorigenic JNK pathway. We show an enhanced activation of Rac1 in primary human schwannoma cells, find both Rac and its effector PAK at the membrane where they colocalize, and describe increased levels of phosphorylated JNK in the nucleus of these cells. Further we describe regulation at post-transcriptional level with upregulated protein, but not mRNA levels for Rac1, and JNK1/2. We conclude that merlin regulates Rac activation, and suggest that this is important for human schwannoma cell dedifferentiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mutations of both alleles of the NF2 gene, coding for merlin, are the hallmark of an autosomal-dominantly inherited genetic disease (neurofibromatosis type II), which is characterized by the development of tumors of the nervous system, in particular schwannomas (1–4). Mutations are also found in all sporadic schwannomas and in 50% of all meningiomas (5–9). Merlin, a member of the ERM (ezrin–moesin–radixin) protein family of cytoskeleton-membrane-linkers (10), has been described as binding partner for cytoskeletal and transmembrane adhesion proteins, including CD44 and integrin chain ß1 (11–17). Moreover, the fact that lack of merlin expression in vivo results in the appearance of many glial tumors in man has established its role as tumor suppressor (18–21), although the mechanism by which it plays this role is as yet unclear. Recent work (16,22,23) suggests that merlin deficiency, similarly to merlin inactivation via phosphorylation by Rac1-activated PAK1 (24,25), results in a loss of cellular response to extracellular growth inhibitory signals, thus leading to uncontrolled cell growth. Additionally, schwannoma cells proliferate more in vitro than normal Schwann cells (26,27). However increased proliferation is unlikely to be sufficient to explain tumoral development. Besides proliferation, cell adhesion is also considered important in tumorigenesis. Rac, like other small GTPases of the Rho-GTPase family (Rho, Rac, Cdc42), is known to act directly on the cytoskeleton, inducing exactly those morphological changes (28–31) observed in merlin-deficient cells, namely cell spreading, and development of many membrane ruffles, lammellipodia and filopodia (32,27). Rac is moreover described to promote integrin dependent adhesion (33,34), another characteristic feature of schwannoma cells (T. Utermark, K. Kaempchen and C.O. Hanemann, manuscript in preparation). Further, inhibiting Rho and Rac1 GTPase activities reverses the abnormal cell morphology of human schwannoma cells (32).

As merlin deficiency and active Rac show so many similar effects on cell morphology and activity, we hypothesized that lack of merlin might promote Rac activation, thus enhancing classical Rac-dependent changes in the cell. If this were the case, Rac-dependent MAP kinase signaling pathways would be expected to be activated. The best described of these is the c-jun N-terminal kinase (JNK) pathway (22,35), already involved in oncogenesis (36).

The aim of the present work was to find out whether merlin absence in primary cultures of human schwannoma cells did indeed induce higher levels of Rac activity compared with those found in normal Schwann cells, and if so whether the JNK pathway would also be activated. For this to happen both Rac1 and its effector proteins, classically PAK (p21-activated kinase), must be found at the cell membrane, hence a series of localization studies establishing this fact. In a second step we studied the level(s) at which differential regulation took place.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Before carrying out further experiments on the cultured primary normal Schwann and schwannoma cells, lysates were tested by western blotting for merlin expression. As expected only Schwann cells were found to express merlin. Figure 1 shows merlin expression in three pairs of Schwann and schwannoma cell lysates which were used in subsequent experiments.



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  		Figure 1. Schwannoma cells do not express merlin. Western blot analysis for merlin (N-terminus) confirming that only normal Schwann cells (NF2+/+), and not schwannoma cells (NF2-/-) express merlin. NF2-/- A–C represent lysates from three different tumors (from different patients); in the same way NF2+/+ 1–3 are lysates from three different normal nerves.

 
Rac activity is upregulated in schwannoma
Correlating the morphology of schwannoma cells with that of cells presenting high levels of active Rac, we were curious to find out whether schwannoma cells really did present higher levels of the active GTPase. Thus we performed a pull-down assay on agarose beads bound to a GST fusion protein corresponding to the p21-binding domain (PBD residues 67–150) of human PAK1, testing for Rac-GTP in normal human Schwann and schwannoma cells in primary culture. This was considered appropriate as PAK has been described as the phosphorylating agent of merlin, and is activated by high affinity binding with Rac-GTP. Moreover the PBD or CRIB (Cdc42/Rac-interacting, binding) domain is a motive which is found in many Rac effector proteins, permitting the GTP form of the small GTPase to bind, and thus stimulate activation (37). No pharmacological activator of Rac was added to the cells prior to the assay, as we wished to investigate Rac activity under the same culture conditions as those in which the phenotypic differences were described. As can be observed in Figure 2A, we found massive Rac1 activity (Rac1 migrates to 21 kDa) in schwannoma cells, compared with the signal detected in normal Schwann cells. As depicted in Figure 2B, there was on average 21.6 times more Rac1-GTP in schwannoma cells compared with normal nerve cells (P=0.05; n=3). In this experiment, optical density levels of each western blot after loading control are normed to normal nerve values (NF+/+=1).



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  		Figure 2. Increased levels of active Rac1 in schwannoma cells. Western blot analysis for Rac1 after pull-down assay on PAK1 PBD (Pak binding domain)–GST coated agarose beads. (A) Band at 21 kDa shows only Rac1-GTP. M, Biotinylated protein marker; ctrl+, positive control, cells saturated with GTP{gamma}S; ctrl-, negative control, cells saturated with GDP; NF2-/-, merlin-deficient schwannoma cells; NF2+/+, merlin-positive Schwann cells; NF2, neurofibromatosis type 2. (B) Quantification analysis of band optical density: NF+/+=1, NF-/-=21.6 times more Rac1–GTP±SEM (P=0.05; n=3).

 
The positive control depicted (Fig. 2A) is somewhat fainter than the NF2-/- band as less protein lysate was used, thus keeping maximal amounts of lysate for the assay itself. The 30 kDa band seen on the blot probably corresponds to Rac-GTP still bound to part of the GST fusion peptide, and is thus recognized by the anti-Rac1 antibody.

Rac and PAK migrate to the membrane
Rac-GTP can activate its effectors when translocated to the membrane; this process is at least in part dependent on the cell's adhesion to the extracellular matrix (38). Only then can active Rac play a role in cell signaling (38). Moreover, when PAK is activated by Rac, it is phosphorylated at serine residues (39). In order for this to take place, it must be first transported to the cell membrane (40–42), where it is relevant to further stimulation of cell signaling cascades (43–45). We therefore studied the comparative localization of first Rac1 and then phosphorylated PAK in normal nerve and schwannoma cells by immunocytochemistry, using the same Rac1 antibody as for western blotting, and an antibody binding specifically to phosphorylated PAK (PAK1 serines, 198/203, PAK2 serines 192/197). We chose the latter antibody as serine residue phosphorylation is specific to GTPase activation, and because phosphorylation at these residues is expected to stabilize the molecule in its active state (46). A second phospho-PAK-antibody tested (phospho-PAK serine 144/serine 141) was found to give non specific staining in immunocytochemistry as tested with blocking peptides (data not shown). Confocal laser micrographs showed that a proportion of Rac1 staining did translocate to the membrane (Fig. 3C and D); moreover we found that schwannoma cells present activated PAK at high concentrations at the cell membrane, as well as in active membrane extensions such as membrane ruffles, lamelli and filopodia (Figs. 4Ac, d and Ba), whereas in both cases Schwann cells presented a basal cytoplasmic stain, without any predominance at the afore-mentioned areas (Figs. 3A, B and 4Aa, b). Further, as the phospho-PAK antibody had not yet been used for immunocytochemistry, we tested the specificity of the staining by pre-incubating the antibody with the specific blocking peptide. Our results showed a disappearance of the membrane stain in schwannoma cells, whereas the cytoplasmic stain remained in part (Fig. 4Bb). Thus, the membrane localization of active PAK we describe in Schwann, but not schwannoma cells is specific, and the cytoplasmic stain is to be ignored. We therefore conclude that both Rac and active PAK are to be found preferentially at the membrane of schwannoma cells, and suggest that it is highly likely that PAK is activated by the high levels of active Rac in these cells.



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  		Figure 3. Translocation of Rac1 to the membrane of schwannoma cells. Confocal laser micrographs showing Rac1 (panels to the right, cy-3 labeled second antibody) staining in Schwann cells (NF2+/+) (A, B) with a cytoplasmic localization, and schwannoma cells (NF2-/-), (C, D) with a preferential translocation to the membrane (arrows), and active membrane extensions (arrow heads). (D) Detail of (C). The panels to the left show actin cytoskeleton for reference (marked with phalloidin-alexa-fluor 488). (A) and (C) are taken with a x40 oil lens, (B) and (D) with a x100 oil lens (each picture is a summation of eight Z-sections taken through the cell). The scale bar represents 15 µm.

 


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  		Figure 4. Active PAK translocates to the membrane of schwannoma cells. (A) Confocal laser micrographs showing the localization of phosphorylated PAK1/2 in Schwann cells (NF2+/+), with faint cytoplasmatic staining (a, b); and schwannoma cells (NF2-/-) showing increased staining at the membrane (arrows) and at areas of membrane activity, such as lammellipodia/filopodia (arrow heads, c, d). In green is stained actin cytoskeleton (alexa-fluor 488), in red phospho-PAK (cy-3). The top panels are micrographs taken with a x40 oil lens, the bottom panels are details of top migrographs taken with a x100 oil lens (each picture is a summation of eight Z-sections taken through the cell). The scale Bar represents 15 µm. (B) Specific staining of phospho-PAK at the membrane (arrows) and in membrane extensions (arrow heads) of schwannoma cells (a) disappears when the primary antibody is pre-incubated with the specific blocking peptide (b), whereas the faint cytoplasmic stain does not. The latter is to be considered unspecific. Micrographs taken with a x40 oil lens. The scale bar represents 15 µm.

 
Rac1 and active PAK colocalize at the membrane of schwannoma cells
As both Rac1 and phosphorylated PAK translocated to the membrane of schwannoma cells, we studied in further experiments the colocalization of these two proteins in schwannoma cells. As for the Rac pull-down assay presented above, one can suggest that as only Rac-GTP binds PAK, areas of colocalization are identical to those of active Rac localization. Figure 5 shows a high degree of colocalization (yellow) between Rac1 and phosphorylated PAK at the membrane of a schwannoma cell. This supports the hypothesis that the very high levels of active Rac1 found in schwannoma cells also find a functional outlet by activating the preferred effector PAK.



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  		Figure 5. Rac1 and activated PAK colocalize at the membrane of schwannoma cells. Confocal laser micrograph taken through a x100 oil lens, showing localization of Rac1 (marked with alexa-fluor 488, green) and phosphorylated PAK (marked with Cy-3, red) at the membrane of a schwannoma cell. The right hand panel shows a colocalization (yellow) of these proteins, suggesting that the translocated Rac is active and can bind and activate its effector. The scale bar represents 15 µm.

 
More phosphorylated form of JNK1 and 2 speaks for enhanced activity of the Rac/JNK pathway
The different splicing forms of JNK1 and JNK2 migrate to 46 kDa (JNK1ß and JNK2ß) and 54 kDa (JNK1{alpha} and JNK2{alpha}), respectively, thus creating two distinct bands on a gel. JNKs are activated by phosphorylation of Thr183 and Tyr185 in the catalytic domain of the enzyme. Therefore we determined the activity of JNKs by an anti-active-JNK antibody recognizing only the dually phosphorylated form of the kinase. In normal human Schwann cells the active forms of JNKs were virtually absent, whereas these were strongly detected in schwannoma cells. Both bands corresponding to the 46 and 54 kDa splicing forms of JNK1/2ß and JNK1/2{alpha}, respectively, exhibit a similar band intensity (Fig. 6A, top blot). Optical density measurements comparing phospho-JNK western bands of schwannoma and Schwann cell lysates after loading control show a significant increase in tumor cells (54 kDa band on average 27.9 times more intense in tumor cells, P=0.05; 45 kDa band, mean 2.8 times more intense, P=0.01; n=3 in both cases; Fig. 6B). The data obtained by western blotting was supported by immunocytochemical staining of normal Schwann and schwannoma cells with the anti-phospho-JNK antibody. Nuclear immunoreactivity of dually phosphorylated JNKs was found in 16.7% of normal Schwann and 36.7% of schwannoma cells, (Fig. 7B; n=3), and this difference was found to be statistically significant (P=0.002). Moreover, immunocytochemical staining of phospho-JNK was restricted to the nucleus, suggesting a predominant activity as transcription factor activator (Fig. 7A). We then investigated the activation of c-Jun, the most classical protooncogenic transcription factor linked to the JNK pathway. As the active, phosphorylated form of c-Jun is present at only low levels in the cell, and primary cultures provide very limited material, an immunocytochemical approach was chosen. Nuclear immunoreactivity was found in 25.9% of normal Schwann and 31.9% of schwannoma cells, however these results were not significantly different.



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  		Figure 6. Enhanced activation of JNK in schwannoma cells, as well as increased expression of JNK1 and 2 suggests two levels of regulation. (A) Expression levels of phospho-JNK (top blot), JNK1 (middle blot) and JNK2 (bottom blot) by western blot analysis. Each isoform can present two bands at 46 kDa (JNKß) and 54 kDa (JNK{alpha}). NF2-/-, merlin-deficient schwannoma cells; NF2+/+, merlin-positive Schwann cells; ctrl+, positive control, PC12 cells incubated with sorbitol. (B) Table showing relative quantities of JNK1, JNK2, and phospho-JNK in schwannoma cells (NF2-/-), where that in NF2+/+ is 1.

 


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  		Figure 7. Active JNK translocates preferentially to the nuclei of schwannoma cells. (A) Nuclear localization of phospho-JNK in schwannoma cells (NF2-/-). Immunocytochemical staining of cells fixed in 4% paraformaldehyde, revelation with ABC–DAB. (B) Mean percentage of phospho-JNK positive nuclei (±SD; P< 0.01) in immunocytochemical staining. NF2-/-, merlin-deficient schwannoma cells; NF2+/+, merlin-positive Schwann cells; ctrl+, positive.

 
The regulation does not only lie in enhanced phosphorylation, but also in post-transcriptional regulation of both Rac and diverse members of the Rac/JNK pathway
In order to examine whether merlin deficiency also leads to enhanced expression, we studied the regulation of signaling molecules first at mRNA and then protein level. RT–PCR showed the JNK3 isoform to be virtually absent in both Schwann and schwannoma cells (data not shown). Comparative mRNA levels were determined by means of quantitative RT–PCR on a LightCycler (Roche) and we show a lack of differential regulation at transcriptional level for Rac1 (P=0.11, n=3), JNK1 (P=0.52, n=4), JNK2 (P=0.75, n=4), and c-Jun (P=0.37, n=4). Table 1 details the crossing points in LightCycler analysis for NF2-/- and NF2+/+ material, after correction with the house-keeping gene, showing again no significant difference. Thus we suggest that transcriptional regulation of the Rac–JNK pathway does not occur in schwannoma cells.


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  		Table 1. No transcriptional regulation of Rac1, JNK1 and 2 or c-jun
 
We further investigated the translational level of regulation. The quantity of Rac1 present was studied by SDS–PAGE of total protein lysate and western blotting. As is obvious from Figure 8A, the quantity of Rac1 was found to be increased in schwannoma cells, thus indicating an overexpression of Rac1 at protein level. Figure 8B shows a graph representing optical density measurements; all results are normed to normal nerve values after loading control. There is on average 2.8 times more total Rac1 in tumor cells (P=0.004; n=6). However, when comparing the difference between normal Schwann and schwannoma cells, it is clear that the higher expression of Rac protein (times 2.8) cannot alone explain the enormous difference in active Rac presence (times 21.6); statistical investigation performed on this data shows the difference to be significant (P=0.03).



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  		Figure 8. Increased expression of total Rac1 in schwannoma cells. (A) Western blot of Rac1 expression. NF2-/-, merlin-deficient schwannoma cells; NF2+/+, merlin-positive Schwann cells. (B) Optical density measurements, all results are normed to normal nerve values (NF2+/+)=1, after loading control. Total Rac1 in NF2-/- cells: 2.8±SEM (P=0.004; n=6).

 
For the JNK1 isoform, the 46 kDa band (JNK1 ß) is predominantly present with an increased intensity in tumoral Schwann cells, on average 1.3 times more in tumor cells. This result was, however, not significant. The 54 kDa band shows an average 2.8 times higher intensity in schwannoma cells (P=0.03; n=4; Fig. 6A, middle blot, and B). For JNK2 only the 54 kDa band was recognizable in both cases with an obviously higher band intensity in schwannoma cells; on average there was 1.5 times more JNK2 in tumor cells (P=0.01, n=4; Fig. 6A, bottom blot, and B). Again the difference at protein level is not as marked as that of the active form, signaling that regulation takes place at two different levels. Here no statistical test was possible, as the anti-active JNK recognizes both JNK1 and 2. Figure 6B shows a table representing how many times more of each protein there is in tumor cells compared with normal Schwann cells. It is evident that the JNK{alpha} isoforms in particular are not only slightly more highly expressed, but also preferentially more highly activated in tumor cells. c-Jun was also tentatively investigated by western blotting (data not shown). It was, however, not possible to detect sufficient levels of the protein with the available material to suggest regulation at this level.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mutations in both alleles of the merlin/schwannomin gene lead to the development of multiple benign glial tumors, most frequently schwannomas. The already described particularities of schwannoma cell morphology, adhesion and increased proliferation in vitro much resemble those of cells overexpressing the small GTPase Rac, and these are all considered to be relevant to tumorigenesis (47–49). Moreover some of these characteristics are reversible by introduction of dominant negative Rac (32). It has also been suspected that merlin and other ERM proteins have a role in the regulation of the Rho–GTPase family, because of their binding to Rho–GDI (guanine nucleotide dissociation inhibitor) (50,51). Therefore we hypothesized that merlin deficiency might result in enhanced Rac activity, and contribute to schwannoma genesis. This was further stimulated by recent findings of Shaw and co-workers (22) in merlin deficient (NF2-/-) mouse fibroblasts. This group was able to show that merlin is phosphorylated in the presence of constitutively active Rac, and further that merlin overexpression in these cells inhibits JNK activity. However no appreciably higher levels of activated Rac were detected in NF2-/- mouse fibroblasts. Nevertheless the results gathered using Shaw's model might not be directly comparable to human NF2 tumors. Indeed mice heterozygous for merlin develop a variety of metastatic tumors but no schwannomas (20), the latter of which are almost exclusively found in NF2 patients (5,6). Thus in this study we investigated the activity of Rac in human schwannoma compared with normal Schwann cells in primary culture, as well as that of the JNK isoforms downstream of Rac. We demonstrate massively enhanced Rac1 activity, and a higher expression of Rac1 at protein level in human schwannoma cells, and show that expression is not sufficiently upregulated to explain the height of activation level. It is important to note that we analyzed Rac activity by measuring the amount of active Rac–GTP bound to its effector PAK, which is described as the physiological effector of merlin phosphorylation (24,25). We further showed in schwannoma cells a translocation of both Rac1 and active PAK to the membrane in areas considered important for Rac activity, therefore suggesting that the increased levels of active Rac are also able to activate their preferred effector. Finally we are able to support this theory by presenting a distinct colocalization of Rac1 with the activated form of its preferred effector PAK at the membrane of schwannoma cells. Thus we believe that we provide first evidence that merlin is not only downstream of Rac but is also involved in regulating Rac activity.

One way in which this might be regulated has recently been described (16,52–54). In normal Schwann cells, both ezrin and merlin are phosphorylated when the cells are sub-confluent (16). Ezrin binds preferentially to CD44 in these conditions (open conformation), and permits down-stream signaling from the growth-factor receptors bound to the CD44 platform. Signaling is possibly in part promoted by the fact that phosphorylated ezrin can bind Rho-GDI (51), and prevent it from blocking the GDP form of GTPases (here Rac) in the cytosol, thus enhancing GTPase activation. However phosphorylated merlin is also able to bind Rho-GDI, although this binding has not been shown to influence GDI activity (50), thus by regulatory competition it might inhibit the positive feed-back loop. Merlin further regulates the system by switching places with ezrin in high density cultures (both ezrin and merlin are then in their hypophosphorylated form), and inhibiting signaling (16). This leads to contact inhibition of growth. Further it can in this case also internalize growth factor receptors (52). In Schwannoma cells lacking merlin these regulatory mechanisms would fail, thus leading to uncontrolled growth in response to external factors, and loss of contact inhibition of growth. This would also explain the slightly higher proliferation of schwannoma cells, in particular as active Rac is required for cell proliferation (55,56).

Our data on Rac activity is further supported by the fact that Rac-GTP promotes integrin-mediated adhesion (33,34,57) via the alteration of the avidity state of integrin receptors by lateral clustering. Conforming to this we have shown in earlier work (T. Utermark, K. Kaempchen and C.O. Hanemann, manuscript in preparation), that in primary culture human schwannoma cells adhere better to laminin, and that this increased adhesion is dependent on the upregulation of the integrin receptors {alpha}6ß1 and {alpha}6ß4, and increased clustering, signaling high avidity. This data combined with recently published work by del Pozo et al. (58), showing that integrins induce the activation of Pak by Rac by stimulating the transfer of Rac to the membrane and dissociating Rac–GDP from Rho–GDI, could additionally explain how lack of merlin may induce increased Rac activity.

Amongst the downstream targets of Rac1 are JNKs (c-Jun N-terminal kinases; SAPKs, stress-activated protein kinases) (59–61). Studies from JNK knock-out mice provide evidence that JNK2 is critical for tumor promotion (36). We were able to demonstrate that primary human Schwann as well as schwannoma cells express only the JNK1 and JNK2 isoforms and more importantly that the activity of JNK is increased in schwannoma cells. We also find an enhanced expression of both isoforms. JNK1 shows two bands in western analysis, whereas only the JNK2{alpha} band is present in both Schwann and schwannoma cells. From this we can deduce that at least JNK1 activity is increased, as the phospho-JNK western presents both bands. We cannot, however, tell for sure whether JNK2 is also more highly activated. Further investigation is required at this level as it cannot be ruled out that both isoforms mediate separate signals by phosphorylating different substrates (62,63). However immunodepletion experiments cannot be performed in primary human cells. Two main hypotheses present themselves for the upregulation of the JNK pathway: firstly a direct upregulation by the enhanced activity of Rac, which is able to phosphorylate early members of the JNK cascade such as MLK-3; secondly, as merlin is phosphorylated by PAK, it offers under normal conditions a stochiometric competition to the JNK pathway in binding active PAK. Therefore, lack of merlin might lead to a misbalance in favor of JNK signaling. We further studied the activation of the classical proto-oncogenic transcription factor linked to the JNK pathway, c-jun, but were not able to detect any increased activity. Because the levels of phospho-JNK were increased, and we also find this form almost exclusively in the nucleus, we suggest that in schwannoma cells other as yet not defined transcription factors are activated by the JNK pathway. A second possibility is that the method we use here in primary culture, because of the limited amount of material available, is not sensitive enough to detect activation of a nuclear protein. This we suggest, as Shaw et al. (22), and Kim et al. (64) both describe, increased activation of the AP-1 complex (as studied in fibroblasts with a luciferase reporter) in cells lacking merlin, and conversely a decrease in activity after reintroduction of merlin. Low transfection rates in primary Schwann cells would, however, make such an approach unreliable in this case.

In summary we find in schwannoma cells lacking merlin expression superior activation both of Rac1, its effector PAK and JNKs 1 and 2, and suppose them linked via already described signaling pathways. This is further supported by the fact that we find Rac1 and its activated effector to translocate to the membrane of schwannoma cells where they colocalize. We moreover show that this higher activation is further enhanced by their upregulated expression at protein level. The mechanism by which loss of merlin leads to an elevated protein expression is still unclear and demands further work. Our data, however, suggest a post-transcriptional device; either activation of certain transcription factors might regulate the expression of proteins involved in post-transcriptional regulation, or the disorganization of the actin cytoskeleton caused by merlin deficiency might directly affect regulatory mechanisms.

The results of our study open the possibility that members of the signaling cascade may in future be used as therapeutic targets for highly specific anti-schwannoma or even anti-meningioma drugs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Preparation of human Schwann cell cultures
Normal human Schwann cells and schwannoma cells were isolated as previously described (27,65). Peripheral nerves were obtained from surgical patients not carrying any disease predisposing to a peripheral neuropathy. Schwannomas were kindly provided by NF2 patients after informed consent. Diagnosis of NF2 was based on clinical criteria defined by the NIH Consensus Conference on Neurofibromatoses (66). Cells were collected and resuspended in proliferation medium: DMEM, 10% FCS, 0.5 µM forskolin, 10 nM ß1-heregulin177–244 (Mark Sliwkowski, Genentech, San Francisco, CA, USA), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma), 2.5 µg/ml insulin (Sigma). Cells were seeded into six-well plates or eight-well labtek slides (Nunc, Rochester, NY, USA), coated with 1 mg/ml poly-L-lysine (Sigma) and 4 µg/ml natural mouse laminin (Gibco), at a density of 10 000 cells/cm2. Proliferation medium was changed every 3–4 days and cells were passaged when confluent or at the latest after 8 days in culture. All RNA and protein preparations were done using cells in the second to fourth passage, as then the number of contaminating fibroblasts was negligible (27). This was in each case tested by staining the cultures for S100 in the first passages.

Nerves and tumors
In total, material derived from 13 normal nerves and 14 tumors, all from different patients, was used in the quoted experiments. ‘n’ numbers in experiments always represent one normal nerve and one schwannoma preparation (i.e. n=3 means that three different normal nerves have been compared with three schwannoma).

Western blotting
Protein was prepared from subconfluent cells using 100 µl denaturing lysis buffer [1% TritonX-100; 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 2 mM EDTA, 10% glycerol, 0.1% SDS, 1% sodium deoxycholate, 1 : 500 small peptide inhibitor mix (Sigma), 1 : 100 100 mM PMSF stock]/4x105 cells. Protein concentrations were determined with a detergent compatible protein assay (Biorad, Munich, Germany) according to the manufacturer's protocol. Proteins were separated by SDS–PAGE on a polyacrylamide gel in non-reducing sample buffer (4xsample buffer: 200 mM Tris–HCl pH 6.8, 8% SDS, 40% glycerol, bromophenol blue) and transferred onto a polyvinylidene fluoride (Immobilon P 1500, Millipore) or nitrocellulose (Amersham) membrane. Equivalent loading of protein was confirmed in a separate experiment by coomassie blue, and membranes were stained after use with Colloidal Gold Total Protein staining (Biorad). Blocking was performed in TBS, 0.1% Tween (TBS-T), 5% milk and 2% BSA. The membranes were incubated overnight at 4°C with antibodies against either rabbit polyclonal anti-merlin N-terminus (1 : 250, Santa Cruz), mouse monoclonal anti-Rac1 (Transduction Laboratories, 1 : 500), rabbit polyclonal anti-JNK1 (Pharmingen, 1 : 1500 dilution), rabbit polyclonal anti-JNK2=SAPK{alpha} (Stressgen, 1 : 750 dilution), rabbit polyclonal anti-phospho-JNK (Promega, 1 : 2500 dilution), or rabbit polyclonal anti-c-Jun (Calbiochem, 1 : 1000 dilution), and incubated with appropriate secondary antibodies. ECL (Amersham) was used for detection. Each experiment was repeated three times, using different pairs of normal Schwann and schwannoma cells.

For phospho-JNK experiments subconfluent cells were lysed under denaturating conditions. They were harvested by scraping in a small volume of 1x PBS. Cells were resuspended in DLB-buffer [10 mM Tris–HCl pH 7.4, 1% SDS, 1% phosphatase-inhibitor cocktail II (Sigma)], incubated for 5 min at 99°C, and sonicated. Western blotting was performed as described above, except that the membrane was blocked in TBS-T with 4% milk.

Rac 1 assay
Once 75–80% confluent, FCS was reduced to 2%. The next day cells were trypsinized, and resuspended in 250 µl denaturating lysis buffer (described above) per well (two wells per probe) and incubated for 30 min on ice. Protein concentration was determined as above; equivalent total protein concentration of probes was ascertained as described. Schwannoma and normal Schwann cells were mixed (one well each), lysed together and split for positive and negative controls. Thus, in total, half as much protein was loaded in controls. These were prepared by saturating cells with either 100x GTP{gamma}S (10 mM) or 100x GDP (100 mM) according to the manufacturer's protocol (Rac/CDC42 assay kit, Upstate Technologies). To each probe 5 µl of PAK-1 agarose beads were added. After 60 min incubation on ice, agarose beads were collected, resuspended in 15 µl 2x non-reducing sample buffer (described above), and boiled for 5 min. Western blotting was performed as stated above, also with anti-Rac1 antibody. Differences to afore stated protocols: anti-Rac1 (Transduction laboratories) 1 : 250 dilution used overnight, washes in TBS-T (NO blocking agents).

Optical density measurements and statistical analysis
Each western blot was scanned, and optical density measurements of whole bands were performed, after correction of background levels with the Gel-Pro Analyser 4.0® software. Results were normed to normal nerve (normal nerve band intensity=100%), so as to permit comparison between blots. Statistical analysis (paired two-tailed t-tests) was performed on this data, and results were considered significant when P<=0.05.

Immunocytochemistry
After 24–48 h incubation, cells seeded on eight-well labtek slides (Nunc) were fixed in 4% paraformaldehyde for 15 min at room temperature. For DAB staining endogenous peroxidases were inactivated. The slides were blocked in 5% BSA–PBS. Cells were incubated overnight with primary antibody diluted in 1% BSA/PBS: phospho-c-Jun Ser 63 (1 : 1000; Cell Signaling) and phospho-JNK (1 : 1000; Biosource). Controls were incubated without primary antibody addition. An appropriate secondary biotinylated antibody was added 1 : 200 in PBS, and incubated for 1 h at room temperature. Detection was performed with the ABC–DAB method [ABC Vector Elite kit and diaminobenzidine (DAB) tablets (Sigma)] according to the manufacturer's instructions. The slides were counterstained with haematoxilin, and coverslipped. 300–500 cells per preparation were counted at random, and the percentage of positive cells was established (non-stained nuclei could be detected in diffraction microscopy within the same sight-field).

For immunofluorescence, after fixing, cells were permeabilized in 4% paraformaldehyde, 0.03% Triton-X100 in PBS each for 10 min at room temperature and blocked in 10% goat serum for 1 h at room temperature, before they were incubated with the primary antibody [rabbit polyclonal anti-phospho-PAK1/2 (PAK1 serines 198/203, PAK2 serines 192/197) 1 : 500, Cell Signaling Technology, or mouse monoclonal anti-Rac1 1 : 250, Transduction Laboratories in 5% goat serum] overnight at 4°C. The secondary antibody (goat anti-mouse-Cy3 1 : 500, Dianova; in 2% goat serum) was added with Phalloidin-alexa-fluor-488 (1 : 100, Molecular Probes) for 40 min at room temperature, followed by counterstaining with DAPI (1 : 5000, Sigma). For colocalization studies both primary antibodies were incubated together at the described dilutions; secondary antibodies consisted of a goat anti-rabbit-Cy3 1 : 500 and a goat anti-mouse-alexa-fuor 488 (1 : 200, Molecular Probes). No phalloidin was added in this case. Cells were coverslipped using VectaShield mounting medium (Vector Laboratories) and stored at 4°C. As control the first antibody was omitted once in every experiment. Analysis of the cells was carried out on a Leica confocal microscope (argon–krypton laser) with Leica TCS NT software. Pictures were taken either with a x40 or a x100 oil lens, and each canal taken separately, keeping the other closed to avoid ‘shine-through’ effects. Eight Z-sections were taken throughout the cell width. Cells from three different normal nerves, and schwannoma cells from three different donors/patients were used in each case; experiments were repeated three times.

For blocking experiments the anti phospho-PAK (PAK1 serines 198/203, PAK2 serines 192/197) antibody was pre-incubated with a 50 times higher concentration of the relevant specific peptide which had been used to manufacture the antibody (kindly provided by Cell Signaling) in PBS for 2 h at 37°C. In the control experiment the antibody was pre-incubated with PBS only. Immunocytochemistry was then preformed as described.

RNA preparation
Total RNA isolation was performed as previously described by Chomczynski (67) and homogenizing by application on Qiashredder (Qiagen, Hilden, Germany), followed by treatment with DNase I. RNA integrity was checked on an agarose gel.

Quantitative RT–PCR
One microgram of total RNA was used for cDNA synthesis using the First Strand cDNA Synthesis Kit for RT–PCR (Roche, Mannheim, Germany). To examine cDNA synthesis efficiency, a PCR of a house-keeping gene (histone H2Az) was performed. For LightCycler reactions the LightCycler Fast Start DNA Master SYBR Green I Kit (Roche) was used according to the manufacturer's protocol with 4 mM MgCl2 and 0.30 µM primer. For normalization a house-keeping gene (huHPRT) was run with every reaction.

The primers (TIB molbiol, Berlin, Germany) used were the following: JNK1 (312 bp)—sense, 5'-CCAGTCAGGCAAGGGATTT-3'; antisense, 5'-CGATGATGATGATGGATGCTGAGAG-3'; JNK2 (200 bp)—sense, 5'-CGCCACTCCTTCTCAGTCTTC-3'; antisense, 5'-CCATCAACTCCCAAGCATTTC-3'; JNK3 (339 bp)—sense, 5'-AACCAGTTCCTACAGTGTGGAAGTG-3'; antisense, 5'-CTGAATCACTTGACATAAGTTGGC-3'; c-jun (107 bp)—sense, 5'-AGGGAACAGGTGGCACAG-3'; antisense, 5'-CCCGACGGTCTCTCTTCA-3'; Rac1 (307 bp)—sense, 5'-GCCGATTGCCGATGTGTT-3'; antisense, 5'-CTCGGATCGCTTCGTCAAA-3'.

The LightCycler program was the following: 95°C, 10 min; 95°C, 10 s; 55°C, 8 s and 72°C, 14 s for 45 cycles. Melting curves were examined for product control and crossing points were used for expression analysis after correction with the house-keeping gene. Experiments were repeated four times independently, using different pairs of RNA from normal Schwann and schwannoma cells.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +49 73150033640; Fax: +49 73150033609; Email: oliver.hanemann{at}medizin.uni-ulm.de Back

{dagger} Present address: Institute of Physiological Biochemistry, Medical School Hannover, 30625 Hannover, Germany. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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Y. Nakai, Y. Zheng, M. MacCollin, and N. Ratner
Temporal control of Rac in Schwann cell-axon interaction is disrupted in NF2-mutant schwannoma cells.
J. Neurosci., March 29, 2006; 26(13): 3390 - 3395.
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