0022-3565/02/3003-1000 –1007$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 300:1000–1007, 2002
Vol. 300, No. 3
4353/964821
Printed in U.S.A.
The Protein Kinase A Inhibitor H89 Acts on Cell Morphology by
Inhibiting Rho Kinase
JOST LEEMHUIS, STEPHANIE BOUTILLIER, GUDULA SCHMIDT, and DIETER K. MEYER
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität, Freiburg, Germany
Received July 24, 2001; accepted November 8, 2001
This article is available online at http://jpet.aspetjournals.org
The small GTPases of the Rho family RhoA, Rac1, and
Cdc42 are molecular switches that organize the actin cytoskeleton (for review, see Van Aelst and D’Souza-Schorey,
1997; Hall, 1998; Luo, 2000; Ridley, 2000). In neural cells,
Rac1 and Cdc42 induce neurite formation, whereas RhoA
causes their retraction via stress fibers (Nobes and Hall,
1995, 1999; Tigyi et al., 1996; Amano et al., 1997; Santos et
al., 1997; Bito et al., 2000). Only in its GTP binding state can
RhoA interact with its effectors, which include ROCK-I
(p160ROCK), ROCK-II (ROK-Rho-kinase), protein kinase N
(PRK1/2-PKN), citron, citron kinase, mDia1, mDia2, rhophilin, and rhotekin (Leung et al., 1995; Ishizaki et al., 1996;
Matsui et al., 1996; Nakagawa et al., 1996; Van Aelst and
D’Souza-Schorey, 1997; Hall, 1998; Kaibuchi et al., 1999).
ROCKs increase the phosphorylation state of the myosin
light chain (Kimura et al., 1996; Amano et al., 1997), which
enhances the tension of stress fibers (Fukata et al., 2001).
The pharmacological agent Y-27632 inhibits both ROCKs
with an IC50 value in the submicromolar range (Uehata et
al., 1997; Ishizaki et al., 2000). Several serine/threonine kinase inhibitors recently have been reported to inhibit
ROCK-II when tested in a cell-free assay (Davies et al.,
2000). Also, H89, previously considered to be a selective
inhibitor of protein kinase A (PKA), has been shown to inhibit ROCK-II with an IC50 value of 270 nM (Chijiwa et al.,
1990; Davies et al., 2000).
The financial support of the Deutsche Forschungsgemeinschaft (Grant SFB
505/B6) is appreciated.
on neurite formation in the neuroblastoma-glioma line NG 10815, which expresses ROCK-I and ROCK-II. We found that H89
can indeed inhibit ROCKs and PKA. Because ROCKs act
downstream of RhoA, the inhibitory effect of H89 on ROCKs is
most prominent. The data indicate that H89 may not be used as
an antagonist of PKA in systems in which ROCKs play a role.
This finding is of special importance because PKA and the
two ROCKs have been reported to have opposite effects on
neurite formation. Whereas activated ROCKs can prevent or
retract such extensions (Leprince et al., 1996; Tigyi et al.,
1996; Bito et al., 2000), activated PKA can facilitate neurite
formation by inhibiting RhoA (Dong et al., 1998). Thus, the
inhibitory effect of H89 on ROCK-II or even both ROCKs may
mask the suppression of PKA activity. Consequently, H89
may facilitate neurite formation, although retraction is expected after inhibition of PKA. In the present study, we have
characterized the effects of H89 on the cytoskeleton in the
neuroblastoma ⫻ glioma cell line NG 108-15, which can form
neurite-like extensions (Gerber et al., 1978; Schecter, 1983).
Experimental Procedures
Materials. The cytotoxic necrotizing factor 1 (CNF1)-glutathione
S-transferase fusion protein was produced in Escherichia coli and
was purified as described previously (Schmidt et al., 1997). Y-27632
was a generous gift form Yoshitomi Pharmaceutical Industries
(Saitama, Japan). H89 and forskolin were bought from Calbiochem
(Bad Soden, Germany) and Tocris (Köln, Germany), respectively.
Cell Culture. Neuroblastoma ⫻ glioma hybrid cells NG 108-15
were cultured at 37°C with 8.6% CO2 in Dulbecco’s modified Eagle
medium with 4.5 g/l glucose (DMEM, PAN, Aidenbach, Germany),
supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin
Germany) and 100 IU/ml penicillin/100 ␮g/ml streptomycin. To induce neurite extensions, cells were cultured in Neurobasal medium
without serum (see also figure legends for respective time courses).
The medium was supplemented with B27, i.e., a mixture of substitu-
ABBREVIATIONS: CNF1, cytotoxic necrotizing factor 1; DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhanced green fluorescent
protein; FCS, fetal calf serum; MAP-2a,b, microtuble associated protein 2a,b; PKA, protein kinase A; PKI, heat-stable inhibitor peptide, which
selectively blocks the catalytic site of PKA; PKN, protein kinase N; RhoAN19, dominant negative RhoA; RhoAV14, constitutively active RhoA;
RhoAWT, wild-type RhoA; ROCK, Rho kinase.
1000
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
ABSTRACT
The small GTPase RhoA can retract cell extensions by acting
on two Rho kinases (ROCKs). Activated protein kinase A (PKA)
inhibits RhoA and induces extensions. The isoquinoline H89
inhibits PKA and thus should prevent the inactivation of RhoA.
In kinase assays, H89 has been recently found to inactivate a
ROCK-II also. Because H89 may be able to exert opposite
effects on cell extensions, we have studied the effects of H89
H89 Acts on Cell Morphology by Inhibiting Rho Kinase
phate precipitate, 4 ␮g of vector DNA was dissolved in 60 ␮l of 250
mM CaCl2, then 60 ␮l of 2⫻ BBS (280 mM NaCl, 1.5 mM Na2HPO4,
and 50 mM BES, pH 7.1) was slowly added. The DNA/Ca2⫹-phosphate precipitate was added to 1 ml of incubation medium (35-mm
well). Cells were then incubated for 2.5 h at 2.5% CO2, which provided the low pH necessary for precipitate formation. The reaction
was stopped by washing the cells twice with prewarmed HEPESbuffered saline solution (135 mM NaCl, 4 mM KCL, 1 mM Na2HPO4,
2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 20 mM HEPES, pH
7.35). Afterward, the cells were grown in Neurobasal medium supplemented with B27 without serum for 16 h before they were used for
experiments.
Evaluation of Cell Morphology and Statistics. Cells bearing
extensions were examined using the 20-fold magnification of a Zeiss
Axiophot microscope. At least 15 cells per view were counted in 10
areas. The length of the extensions was determined using the confocal laser microscope and Laser sharp 2.1T software. For statistical
analysis Kruskal-Wallis and Mann-Whitney-U tests were used. If
samples showed normal distribution, analysis of variance was combined with Scheffe’s test.
Results
When incubated in DMEM medium with 10% FCS, NG
108-15 cells were polymorphic and had no extensions (data
not shown). Incubation in serum-free Neurobasal medium
plus B27 induced extensions in a time-dependent manner.
After 8 h, approximately 20% of the cells had extensions that
were ⱖ50 ␮m (Fig. 1, A and M). After 26 h, approximately
60% of the cells showed extensions that were ⱖ50 ␮m (e.g.,
Fig. 3A). Both incubation parameters were used for the subsequent experiments.
Y-27632 and H89 Induce Extensions in NG 108-15
Cells. Because inactivation of RhoA and of ROCKs has been
shown to facilitate neurite formation, we first compared the
effects of Y-27632 and H89 on the extensions of differentiating cells. The ROCKs inhibitor Y-27632 (5 ␮M) caused pronounced changes in NG 108-15 cell morphology, when it was
added during the last 4 h of the 8-h differentiation period.
Approximately 80% of the cells developed one to three extensions that were ⱖ 50 ␮m (Fig. 1, B and M). Whereas staining
of filamentous actin with phalloidine showed numerous filopodia in controls (Fig. 1D), cells treated with Y-27632 displayed actin structures similar to growth cones (Fig. 1E).
Also the neuron-specific proteins ␤-tubulin III and MAP-2
were expressed by the NG 108-15 cells. Immunoreactive material was uniformly dispersed in the controls (Fig. 1, G and
J). Treatment with Y-27632 produced densely filled extensions (Fig. 1, H and K). The effects of 10 ␮M H89 on the
morphology of the NG 108-15 cells corresponded to those of
Y-27632 (Fig. 1, C, F, I, and L). H89 and Y-27632 produced
extensions in approximately 80% of the cells at a concentration of 1 ␮M (Fig. 1M). At this concentration, Y-27632 has
been reported to inhibit the RhoA effectors ROCK-I, ROCKII, and PKN but not PKA (Uehata et al., 1997; Davies et al.,
2000), whereas H89 has no effect on PKN but inhibits PKA
and ROCK-II (Davies et al., 2000). Taken together, these
results suggested that Y-27632 and H89 induced extensions
by inhibiting one or both ROCKs.
Both ROCKs have been found in rat brain (Leung et al.,
1995, 1996; Matsui et al., 1996). To find out which ROCK was
involved in our experiments, the expression of both ROCKs
was studied in the NG 108-15 cells. Western blot analysis
showed immunoreactive bands indicating the presence of
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
ents that support neuronal differentiation (Brewer and Cotman,
1989). Neurobasal medium and B27 were bought from Invitrogen
(Carlsbad, CA).
Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 0.1%
(v/v) Triton X-100. Normal goat serum and normal donkey serum
only for ROCK-II were used to block unspecific reactions. Thereafter,
cells were incubated with one of the following primary antibodies:
monoclonal mouse anti-␤-tubulin-III antibody (Sigma, Deisenhofen,
Germany); monoclonal mouse anti MAP-2a,b antibody (Roche Molecular Biochemicals, Mannheim, Germany); polyclonal rabbit antiROCK-I antibody (Santa Cruz Biotechnology, Heidelberg, Germany);
and polyclonal goat anti-ROCK-II antibody (Santa Cruz Biotechnology). The resulting ␤-tubulin-III and MAP-2 immune complexes
were visualized with a CyTm 3-conjugated F(ab⬘)2 fragment of goat
anti-mouse IgG (Dianova, Hamburg, Germany). A CyTm 3-conjugated F(ab⬘)2 fragment of goat anti-rabbit was used to detect ROCK-I
and a CyTm 3-conjugated F(ab⬘)2 fragment of donkey anti-goat was
used to detect ROCK-II (Dianova). For actin staining, cells were fixed
with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 0.1% (v/v) Triton X-100. Afterward, the cells were
incubated with tetramethylrhodamine B isothiocyanate-conjugated
phalloidine (Biozol, München, Germany) and washed again with
PBS.
Confocal Image Analysis. Cells were imaged using a Bio-Rad
(Hercules, CA) MRC 1024 (version 3.2) confocal system with a krypton-argon laser and an Axiovert 135TV microscope (Zeiss,
Oberkochen, Germany). CyTm 3 fluorescence was measured using an
excitation wavelength of 554 nm and an emission filter set at 576. A
40⫻ water objective lens was used. Images were obtained using laser
sharp 2.1T software and processed using Photopaint (Corel Corporation, Ottawa, Ontario, Canada).
Western Blot Analysis of ROCK-I and ROCK-II. NG108-15
cells were grown for 4 h in Neurobasal medium supplemented with
B27. They were lysed on ice for 15 min in 1 ml of lysis buffer (20 mM
Tris-HCl [pH 7.4], 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2
mM EDTA, 50 mM sodiumglycerophosphate, 20 mM sodium pyrophosphate, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 1 mM sodium orthovandate, 5 ␮g of aprotinin, and 5 ␮g of leupeptin per milliliter). The lysates were collected
with a rubber policeman, sonicated, and centrifuged (10 min, 2000g,
4°C). Rat brain was homogenized in lysis buffer, sonicated, and
centrifuged. After determination of the protein concentrations of the
supernatants (Bradford, 1976), aliquots containing 100 ␮g of protein
were separated on SDS-7.5% polyacrylamide gels and transferred to
a nitrocellulose filter. Western blot immunoanalysis was performed
using a polyclonal rabbit anti-ROCK-I (H-85) antibody or a polyclonal goat anti-ROCK-II (C-20) antibody (Santa Cruz Biotechnology). Specific immunoreactivity was detected by using an ECL kit
(Amersham International, Uppsala, Sweden).
Preparation of Transfection Vectors and Cell Transfection.
The coding regions of the constitutively active RhoA (RhoAV14), the
dominant negative RhoA (RhoAN19), and the wild-type RhoA
(RhoAWT) genes were excised from the plasmid GEX with BamHI/
EcoRI and inserted in frame into BglII/EcoRI sites of pEGFP-Cl
(CLONTECH, Heidelberg, Germany).
The Rous sarcoma virus-heat-stable inhibitor peptide (PKI) plasmid was a gift from T.J. Murphy (Atlanta, GA). The coding region of
PKI␣ was cloned from the Rous sarcoma virus-PKI plasmid by PCR
using the primers 5⬘-CGCGCGAATTCTATGGGAACTGATGTCGAAAC-3⬘ and 5⬘-CGCGCGGATCCCTATGACTCGGACTTAGCAG3⬘. The resulting DNA product was excised with BamHI/EcoRI and
inserted into BglII/EcoRI sites of pEGFP-C1. The construct was
verified by restriction digest analysis and sequencing.
Cells were transfected using a modified calcium-phosphate procedure (Köhrmann et al., 1999). Four hours before transfection, the
DMEM medium was replaced with Neurobasal medium supplemented with B27 without serum. To prepare the DNA/Ca2⫹-phos-
1001
1002
Leemhuis et al.
ROCK-I and of ROCK-II (Fig. 2). Immunocytochemistry was
applied to localize the respective kinases. Immunoreactivity
for ROCK-I was densely distributed in the cell body and
extensions, whereas the respective signal for ROCK-II was
rather faint. Apparently, NG 108-15 cells expressed both
ROCKs (Fig. 2).
Y-27632 and H89 Do Not Prevent the Retraction of
Extensions Caused by Constitutively Active RhoAV14
and Wild-Type RhoA. Activated RhoA can retract cell extensions (Nobes and Hall, 1995; Tigyi et al., 1996; Amano et
al., 1997; Santos et al., 1997; Schmidt et al., 1997; Nobes and
Hall, 1999). To test whether H89 inhibited morphological
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 1. Y-27632 and H89 change the morphology and the distribution of cytoskeletal proteins in NG 108-15 cells. Cells
were incubated in DMEM medium containing 10% FCS. One day after the last
passage, they were incubated in serumfree Neurobasal medium plus the supplement B27 for 4 h before they were treated
with 5 ␮M Y-27632 (B, E, H, and K) or 10
␮M H89 (C, F, I, and L) for another 4 h;
controls are shown in A, D, G, and J.
After fixation, cells were analyzed with
phase contrast (A–C) or stained for the
cytoskeletal proteins actin (D–F), ␤-tubulin (G–I), and MAP-2 (J–L). Bar ⬃ 50 ␮m.
M, concentration response curve of the
effects of 0.1, 0.5, 1, 5, 10, and 30 ␮M
Y-27632 (squares) or H89 (rhombes) on
the production of extensions ⱖ 50 ␮m.
The number of extension-bearing cells is
expressed as the percentage of total number of cells analyzed. Broken line, percentage of control cells. Mean ⫾ S.E.M.;
n ⱖ 150.
H89 Acts on Cell Morphology by Inhibiting Rho Kinase
1003
Fig. 2. NG 108-15 cells express both ROCKs. Upper panel shows Western
blot analysis: 100 ␮g of soluble proteins obtained from lysates of NG
108-15 cells or rat brain were separated on a SDS-7.5% polyacrylamide
gel and probed with specific polyclonal antibodies against ROCK-I and
ROCK-II. Lower panel, immunocytochemical evidence for the presence of
the ROCKs in NG 108-15 cells. Bar ⬃50 ␮m
Fig. 3. Y-27632 (5 ␮M) and H89 (10 ␮M) do not prevent the retraction of
extensions caused by transfection of NG 108-15 cells with constitutively
active RhoAV14 or RhoAWT. One day after the last passage, NG 108-15
cells were incubated for 4 h in serum-free Neurobasal medium plus B27.
Then cells were transfected for 2.5 h with a plasmid coding for EGFP
alone (A–C) or with a plasmid coding for EGFP/RhoAV14 (D–I) or EGFP/
RhoAWT (J–L). After a 16-h equilibration period, Y-27632 and H89 were
added for another 4 h. After fixation, cells were stained for filamentous
actin. EGFP fluorescence is shown in A through F and J through L; actin
staining is shown in G through I. Cells treated with Y-27632 or H89 are
shown in B, E, H, and K and C, F, I, and L, respectively. G through I show
cells of D through F at higher magnification. Bar ⬃50 ␮m.
Fig. 4. Quantification of the experiment shown in Fig. 3. Cells with
extensions ⱖ50 ␮m are shown as percent of total number of cells analyzed. Mean ⫾ S.E.M.; n ⱖ 150, a shows significant difference (P ⬍ 0.05)
to EGFP controls; b shows significant difference (P ⬍ 0.05) to EGFP cells
treated with Y-27632 and H89, respectively.
Gln-63 and thereby prevents the inactivation of the GTPase
(Flatau et al., 1997; Schmidt et al., 1997; Barth et al., 1999).
Therefore, we treated NG 108-15 cells with CNF1 to study
whether H89 and Y-27632 affected the resultant morphological effects. Approximately 60% of the NG 108-15 cells trans-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
effects of RhoA, we used NG 108-15 cells that transiently
expressed a fusion protein consisting of enhanced green fluorescent protein (EGFP) and RhoAV14 or overexpressed
RhoAWT. As controls, NG 108-15 cells were used that had
been transfected with the empty EGFP vector. The experiments were performed with fully differentiated cells which
best showed retraction of extensions caused by RhoAV14 or
RhoAWT. After 24 h in serum-free Neurobasal medium plus
B27, approximately 57% of the EGFP expressing cells
showed extensions. Addition of 5 ␮M Y-27632 or 10 ␮M H89
enhanced the number of cells with extensions to approximately 80% (Figs. 3, A, B, and C, and 4). In addition, both
agents elongated the extensions from 87 ␮m in controls to
approximately 140 ␮m (Table 1). Cells that expressed
RhoAV14 had small polymorphic cell bodies without extensions (Figs. 3D and 4). Tightly bundled stress fibers were also
observed (Fig. 3G). Treatment with 5 ␮M Y-27632 or with 10
␮M H89 did not produce any extensions in these cells (Figs.
3, E, F, H, and I, and 4). Y-27632 was also ineffective, when
used at concentrations of 10, 30, and 50 ␮M (data not shown).
However, both inhibitors enlarged the cell bodies (Fig. 3,
D–F) and strongly reduced the density of stress fibers (Fig. 3,
G–I) indicating that they indeed inhibited the relevant
ROCKs in these cells. However, inactivation of ROCKs was
not sufficient to overcome the retraction of extensions caused
by RhoAV14. Two explanations seemed possible for this lack
of activity. The mutated amino acid may have altered the
effector loop of RhoAV14 so that effectors were activated,
which caused neurite retraction independent of ROCKs. Alternatively, the overexpression of an active GTPase per se
may have activated such effectors. To test the latter hypothesis, we overexpressed wild-type RhoA in the NG 108-15
cells. Compared with EGFP controls, these cells displayed
fewer extensions, although they had more extensions than
cells transfected with RhoAV14 (Figs. 3J and 4). Also cells
expressing the RhoAWT did not respond to Y-27632 or H89
with increased neurite formation (Figs. 3, K and L, and 4).
This finding indicated that high intracellular concentrations
of active RhoA induced the retraction of extensions independent of ROCKs by recruiting other effectors.
Y-27632 and H89 Block the CNF1-Induced Retraction
of Extensions. Another way to activate RhoA is to treat the
cells with CNF1 of E. coli. This toxin deamidates RhoA at
1004
Leemhuis et al.
TABLE 1
Effect of 10 ␮M H89 and 5 ␮M Y-27632 on formation of cell extensions induced by 10 ␮M forskolin
Cells with extensions ⱖ50 ␮m and cells with branched extensions are shown as percentage of total number of cells analyzed. Mean ⫾ S.E.M.; n ⱖ 150. Length of longest
extension is given in micrometers; n ⫽ 48.
Percentage of Cells
with Extensions
Percentage of Cells
with Branched
Extensions
Length of Longest
Extension
57.1 ⫾ 2.8
80.8 ⫾ 3a
79.9 ⫾ 4.2a
83.9 ⫾ 1.3a
84.7 ⫾ 2.1a
82.2 ⫾ 2.4a
23.4 ⫾ 2.1
47.3 ⫾ 3.4a
44.9 ⫾ 2.2a
82.4 ⫾ 2.4a
60.4 ⫾ 2.5a,b
94.9 ⫾ 1.1a,b
87.0 ⫾ 6.1
144.2 ⫾ 10.0a
140.3 ⫾ 9.6a
99.3 ⫾ 7.1
155.0 ⫾ 10.3a,b
127.5 ⫾ 10.0a,b
␮m
Co
Y-27632
H89
Forskolin
Forskolin ⫹ H89
Forskolin ⫹ Y-27632
a
b
Significant difference (P ⬍ 0.05) to respective control.
Significant difference (P ⬍ 0.05) to respective group treated with forskolin.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
fected with the empty EGFP vector showed extensions when
they were preincubated for 24 h in serum-free Neurobasal
medium plus B27 (Fig. 5J; see also previous experiment).
Treatment with CNF1 (100 ng/ml) for 4.5 h induced large
polymorphic cell bodies. Now, only 11.8% of the cells had
extensions (Fig. 5, A and J). However, 5 ␮M Y-27632 or 10
␮M H89 added to the incubation medium 30 min before
CNF1 blocked the CNF1 induced retraction of extensions,
more than 60% of the CNF1-treated cells still showed extensions (Fig. 5, B, C, and J).
To confirm that the effects of CNF1 were related to RhoA,
NG 108-15 cells were transfected with an expression plasmid
coding for a fusion protein consisting of EGFP and RhoAN19.
Such cells showed numerous extensions that were rod- or
cone-like (Fig. 5D). They did not respond to CNF1 toxin with
retraction of their extensions (Fig. 5, G and J). Additional
treatment with 5 ␮M Y-27632 or 10 ␮M H89 only slightly
enhanced the number of extension-bearing cells (Fig. 5, H–J).
CNF1 is endocytozed in a clathrin-independent manner
and translocates into the cytosol via an acidic dependent
procedure in which RhoA may be involved (Contamin et al.,
2000). Thus, inactivation of RhoA may reduce the cellular
uptake of CNF1. Therefore, we studied a RhoA-independent
CNF1 effect, i.e., the toxin-induced enlargement of cell bodies, which is mediated by Rac1 activation (S. Boutillier, unpublished observation). Indeed, CNF1 increased the largest
diameter of the cell bodies from 39.1 ⫾ 1.9 ␮m in pEGFP
controls to 52.5 ⫾ 0.9 ␮m (P ⬍ 0.05; n ⫽ 24). Transfection of
negative RhoA slightly enhanced the cell diameter to 38.5 ⫾
2.2 ␮m. In these cells, CNF1 further increased the diameter
to 46.9 ⫾ 1.7 ␮m (P ⬍ 0.05; n ⫽ 24). Apparently, CNF1 was
present in effective concentrations in the cells transfected
with negative RhoA. Taken together, these results indicated
that CNF1 induced retraction of extensions by activating
RhoA and subsequently ROCKs that could be inhibited by
Y-27632 and H89.
PKA and the Formation of Extensions in NG 108-15
Cells. Because these data did not exclude the possibility that
H89 exerted an additional effect via PKA, we studied the
actions of a selective PKA inhibitor on the morphological
changes induced by CNF1. The PKI that selectively blocks
the catalytic site of PKA (Day et al., 1989) was used for these
experiments. NG 108-15 cells were transfected with expression plasmids coding for EGFP alone or an EGFP/PKI fusion
protein. The resulting cells had similar morphologies (Fig. 6,
A, D, and G). In both cell types, CNF1 (100 ng/ml) retracted
the extensions (Fig. 6, B, E, and G).
Fig. 5. Retraction of cell extensions caused by CNF1 (100 ng/ml) is
prevented by 5 ␮M Y-27632 or 10 ␮M H89 and by transfection of cells
with RhoAN19. NG 108-15 cells were transfected with plasmids coding
for EGFP alone (A–C) or for EGFP/RhoAN19 (D–I). EGFP immunofluorescence is shown in A through I. Sixteen hours after transfection,
cells were treated with Y-27632 (B, E, and H) or H89 (C, F, and I) for
4.5 h. CNF1 was given 0.5 h after the drugs for 4 h (A–C and G–I). Bar
⬃50 ␮m. Quantification of the effects of CNF1 on cell extensions is
shown in J. Cells with extensions ⱖ 50 ␮m are shown as percent of
total number of cells analyzed. Cells transfected with EGFP alone
(open columns); cells transfected with EGFP/RhoAN19 (striped columns). Mean ⫾ S.E.M.; n ⱖ 150; a shows significant difference (P ⬍
0.05) to EGFP controls; b shows significant difference (P ⬍ 0.05) to
EGFP cells treated with CNF1.
H89 Acts on Cell Morphology by Inhibiting Rho Kinase
1005
inhibitors affected this action (Table 1). In controls, 22% of
the cells exhibited branched neurites. This number was enhanced by forskolin to 82%, whereas H89 and Y-27632 had
smaller effects, i.e., 48 and 52%, respectively. Combination of
forskolin and Y-27632 caused 95% of the cells to form
branched extensions. In contrast, H89 reduced this number
to 60%, indicating that H89 was able to diminish this effect of
forskolin.
Discussion
To prove that PKI indeed inhibited cellular PKA, we tested
its effect on morphological changes induced by 10 ␮M forskolin. When applied for 4 h, forskolin induced long and slim
extensions with multiple branches (Fig. 6, C and G). PKI
completely prevented these effects of forskolin (Fig. 6, F and
G). Taken together, these results showed that PKA was not
involved in the retraction of cell extensions caused by CNF1.
In addition, they indicated that the observed effects of H89
were only due to its inhibitory effect on ROCKs.
Finally, we tested how the two inhibitory actions of H89 on
PKA and ROCKs affected the morphological changes induced
by 10 ␮M forskolin (Table 1). Compared with controls, forskolin significantly enhanced the number of cells that showed
extensions. Additional application of 5 ␮M Y-27632 or 10 ␮M
H89 had no further effect. Compared with controls, H89 and
Y-27632 did not only induce extensions but also elongated
them by approximately 67% (Table 1). In contrast, forskolin
had no significant effect on this parameter. When Y-27632 or
H89 were applied together with forskolin, they significantly
elongated the neurites to values observed after single application of the kinase inhibitors. This indicated that the elongation was due to ROCK inhibition. Forskolin has been
shown to cause branching of neurites in NG 108-15 cells
(Weeks et al., 1991). Therefore, we tested whether the kinase
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 6. The protein kinase A inhibitor PKI does not change the effect of
CNF1 on cell morphology but prevents that of forskolin. Cells were
transfected with plasmids coding for EGFP alone (A–C) or for an EGFP/
PKI fusion protein (D–F). Sixteen hours later, cells were treated for 4 h
with vehicle (A and D), CNF1 (100 ng/ml) (B and E) or forskolin (5 ␮M) (C
and F). Bar ⬃50 ␮m. Quantitative analysis of cells showing extensions
after treatment with CNF1 or with forskolin is given in G. Cells with
extensions ⱖ50 ␮m are shown as percent of total number of cells analyzed. Mean ⫾ S.E.M.; n ⱖ 150, a shows significant difference (P ⬍ 0.05)
to EGFP; b shows significant difference (P ⬍ 0.05) to PKI; c shows
significant difference (P ⬍ 0.05) to EGFP ⫹ forskolin.
In the present study, we tested whether the established
PKA inhibitor H89 also inhibited morphologically relevant
ROCKs. By comparing its effect on neurite formation in NG
108-15 cells with that of Y-27632, which inhibits ROCKs but
not PKA (Uehata et al., 1997; Ishizaki et al., 2000), we found
that both agents indeed exerted nearly identical effects. In
undifferentiated cells, they produced neurite-like extensions
and caused similar changes in the distribution of the cytoskeletal proteins F-actin, neurotubulin, and MAP-2. In differentiated cells, which already have long extensions, their
effect on neurite formation was less pronounced. However,
they blocked the retraction caused by the E. coli toxin CNF1,
which activates RhoA. Experiments with dominant negative
RhoA confirmed that CNF1 indeed caused the neurite retraction by acting via RhoA. They suggested that Y-27632 and
H89 indeed inhibited the effect of CNF1 by acting on the
relevant ROCKs. ROCK-I and ROCK-II have been found in
central nervous system or cell lines derived from it and both
isoforms induced neurite retraction (Nakagawa et al., 1996;
Hirose et al., 1998; Katoh et al., 1998). In NG 108-15 cells we
found both isoforms. This finding makes it possible that H89
does not only act on ROCK-II as has been reported before
(Davies et al., 2000), but also on ROCK-I. In these experiments, we obtained no evidence that H89 exerted its action
by inhibiting PKA. Use of PKI, a selective inhibitor peptide of
PKA, corroborated this conclusion. PKI did not affect neurite
formation, when transfected into NG 108-15 cells. PKI also
did not alter the retraction caused by CNF1. Thus, our results confirmed and extended biochemical data that H89 can
block ROCK(s) (Davies et al., 2000). In view of a previous
report that H89 can antagonize ␤-adrenoceptors (Penn et al.,
1999), the present data further limit its usefulness.
Although these results suggested that ROCKs mediated
the retraction of neurites in the NG 108-15 cells, we observed
that RhoA can exert such an effect independent of ROCKs.
Thus, neurite retraction caused by constitutively active RhoA
was neither blocked by Y-27632 nor by H89. Moreover, both
agents were not able to prevent the retraction caused by
overexpression of wild-type RhoA. The formation of stress
fibers induced by RhoA depends on the presence of active
ROCKs (Nakano et al., 1999). Because Y-27632 and H89
prevented the formation of stress fibers by constitutively
active RhoA, both agents effectively inhibited the ROCKs.
This finding indicated that the neurite retraction was independent of stress fibers and that additional effectors were
involved. mDia has been described as a RhoA effector that
acts on actin polymerization via profilin (Watanabe et al.,
1997). Both ROCK and mDia seem to be necessary for the
formation of regularly organized stress fibers (Nakano et al.,
1999). Whether mDia can act independently of RhoA and
thereby cause neurite retraction is unknown and has to be
1006
Leemhuis et al.
References
Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, and Kaibuchi K (1997) Formation of actin stress fibers and focal adhesions enhanced by
Rho-kinase. Science (Wash DC) 275:1308 –1311.
Barth H, Olenik C, Sehr P, Schmidt G, Aktories K, and Meyer DK (1999)
Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing
factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho. J Biol Chem
274:27407–27414.
Bito H, Furuyashiki T, Ishihara H, Shibasaki Y, Ohashi K, Mizuno K, Maekawa M,
Ishizaki T, and Narumiya S (2000) A critical role for a Rho-associated kinase,
p160ROCK, in determining axon outgrowth in mammalian CNS neurons. Neuron
26:431– 441.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
72:248 –254.
Brewer GJ and Cotman CW (1989) Survival and growth of hippocampal neurons in
defined medium at low density: advantages of a sandwich culture technique or low
oxygen. Brain Res 494:65–74.
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K,
Toshioka T, and Hidaka H (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor
of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]5- isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol
Chem 265:5267–5272.
Contamin S, Galmiche A, Doye A, Flatau G, Benmerah A, and Boquet P (2000) The
p21 Rho-activating toxin cytotoxic necrotizing factor 1 is endocytosed by a clathrinindependent mechanism and enters the cytosol by an acidic-dependent membrane
translocation step. Mol Biol Cell 11:1775–1787.
Davies SP, Reddy H, Caivano M, and Cohen P (2000) Specificity and mechanism of
action of some commonly used protein kinase inhibitors. Biochem J 351:95–105.
Day RN, Walder JA, and Maurer RA (1989) A protein kinase inhibitor gene reduces
both basal and multihormone-stimulated prolactin gene transcription. J Biol
Chem 264:431– 436.
Dong JM, Leung T, Manser E, and Lim L (1998) cAMP-induced morphological
changes are counteracted by the activated RhoA small GTPase and the Rho kinase
ROKalpha. J Biol Chem 273:22554 –22562.
Flatau G, Lemichez E, Gauthier M, Chardin P, Paris S, Fiorentini C, and Boquet P
(1997) Toxin-induced activation of the G protein p21 Rho by deamidation of
glutamine. Nature (Lond) 387:729 –733.
Fukata Y, Amano M, and Kaibuchi K (2001) Rho-Rho-kinase pathway in smooth
muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends
Pharmacol Sci 22:32–39.
Gerber LD, Stein S, Rubinstein M, Wideman J, and Udenfriend S (1978) Binding
assay for opioid peptides with neuroblastoma ⫻ glioma hybrid cells: specificity of
the receptor site. Brain Res 151:117–126.
Hall A (1998) Rho GTPases and the actin cytoskeleton. Science (Wash DC) 279:509 –
514.
Hansen TO, Rehfeld JF, and Nielsen FC (2000) Cyclic AMP-induced neuronal differentiation via activation of p38 mitogen-activated protein kinase. J Neurochem
75:1870 –1877.
Heidemann SR, Joshi HC, Schechter A, Fletcher JR, and Bothwell M (1985) Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and
microtubule stability of PC12 cells. J Cell Biol 100:916 –927.
Hirose M, Ishizaki T, Watanabe N, Uehata M, Kranenburg O, Moolenaar WH,
Matsumura F, Maekawa M, Bito H, and Narumiya S (1998) Molecular dissection
of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in
neuroblastoma N1E-115 cells. J Cell Biol 141:1625–1636.
Ishizaki T, Maekawa M, Fujisawa K, Okawa K, Iwamatsu A, Fujita A, Watanabe N,
Saito Y, Kakizuka A, Morii N, and Narumiya S (1996) The small GTP-binding
protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous
to myotonic dystrophy kinase. EMBO J 15:1885–1893.
Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, and Narumiya S (2000) Pharmacological properties of Y-27632, a specific inhibitor of rhoassociated kinases. Mol Pharmacol 57:976 –983.
Kaibuchi K, Kuroda S, and Amano M (1999) Regulation of the cytoskeleton and cell
adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem
68:459 – 486.
Katoh H, Aoki J, Ichikawa A, and Negishi M (1998) p160 RgoA-binding kinase ROKa
induces neurite retraction. J Biol Chem 273:2489 –2492.
Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng
J, Nakano T, Okawa K, et al. (1996) Regulation of myosin phosphatase by Rho and
Rho-associated kinase (Rho-kinase). Science (Wash DC) 273:245–248.
Köhrmann M, Haubensak W, Hemraj I, Kaether C, Lessmann VJ, and Kiebler MA
(1999) Fast, convenient, and effective method to transiently transfect primary
hippocampal neurons. J Neurosci Res 58:831– 835.
Lang P, Gesbert F, Delespine-Carmagnat M, Stancou R, Pouchelet M, and Bertoglio
J (1996) Protein kinase A phosphorylation of RhoA mediates the morphological
and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J 15:510 –
519.
Leprince P, Bonvoisin C, Rogister B, Mazy-Servais C, and Moonen G (1996) Protein
kinase- and staurosporine-dependent induction of neurite outgrowth and plasminogen activator activity in PC12 cells. Biochem Pharmacol 52:1399 –1405.
Leung T, Chen XQ, Manser E, and Lim L (1996) The p160 RhoA-binding kinase ROK
alpha is a member of a kinase family and is involved in the reorganization of the
cytoskeleton. Mol Cell Biol 16:5313–5327.
Leung T, Manser E, Tan L, and Lim L (1995) A novel serine/threonine kinase binding
the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem 270:29051–29054.
Luo L (2000) Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci 1:173–180.
Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito M, Nakano T,
Okawa K, Iwamatsu A, and Kaibuchi K (1996) Rho-associated kinase, a novel
serine/threonine kinase, as a putative target for small GTP binding protein Rho.
EMBO J 15:2208 –2216.
Ming GL, Song HJ, Berninger B, Holt CE, Tessier-Lavigne M, and Poo MM (1997)
cAMP-dependent growth cone guidance by netrin-1. Neuron 19:1225–1235.
Nakagawa O, Fujisawa K, Ishizaki T, Saito Y, Nakao K, and Narumiya S (1996)
ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein
serine/threonine kinase in mice. FEBS Lett 392:189 –193.
Nakano K, Takaishi K, Kodama A, Mammoto A, Shiozaki H, Monden M, and Takai
Y (1999) Distinct actions and cooperative roles of ROCK and mDia in Rho small G
protein-induced reorganization of the actin cytoskeleton in Madin-Darby canine
kidney cells. Mol Biol Cell 10:2481–2491.
Nobes CD and Hall A (1995) Rho, Rac, and Cdc42 GTPases regulate the assembly of
multimolecular focal complexes associated with actin stress fibers, lamellipodia,
and filopodia. Cell 81:53– 62.
Nobes CD and Hall A (1999) Rho GTPases control polarity, protrusion, and adhesion
during cell movement. J Cell Biol 144:1235–1244.
Penn RB, Parent JL, Pronin AN, Panettieri RA Jr, and Benovic JL (1999) Pharmacological inhibition of protein kinases in intact cells: antagonism of beta adrenergic
receptor ligand binding by H-89 reveals limitations of usefulness. J Pharmacol
Exp Ther 288:428 – 437.
Ridley AJ (2000) Rho, in GTPases (Hall A ed) pp 89 –136, Oxford University Press,
Oxford.
Santos MF, McCormack SA, Guo Z, Okolicany J, Zheng Y, Johnson LR, and Tigyi G
(1997) Rho proteins play a critical role in cell migration during the early phase of
mucosal restitution. J Clin Invest 100:216 –225.
Schecter A (1983) Light and ultrastructural characteristics of neuroblastoma glioma
hybrid NG 108-15 cells. Life Sci 33:719 –722.
Schmidt G, Sehr P, Wilm M, Selzer J, Mann M, and Aktories K (1997) Gln 63 of Rho
is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature (Lond)
387:725–729.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
tested in future experiments. Transient transfections with
expression vectors can result in high concentrations of the
respective protein. Therefore, it is possible that the RhoA
proteins produced by the NG 108-15 cells in large and unphysiological amounts recruited and activated other RhoA
effectors or even nonRhoA effectors which then caused neurite retraction. This speculation is in agreement with our
finding that Y-27632 and H89 prevented the neurite retraction induced by activation of endogenous RhoA with CNF1.
Neurites are induced or elongated if a respective driving
force prevails over the retraction caused by actin filaments.
This driving force is probably provided by Rac1 and Cdc42,
i.e., other members of the Rho family of GTPases (Nobes and
Hall, 1995; Tigyi et al., 1996; Amano et al., 1997; Santos et
al., 1997; Nobes and Hall, 1999; Bito et al., 2000). The
strength of the driving force may depend on the cell type and
on factors in the incubation medium that activate or inhibit
the GTPases. Such agents may explain why we observed
neurite elongation induced by Y-27632 or H89 in NG 108-15
cells maintained in Neurobasal medium, whereas such an
effect was less or not at all present in other studies (Chijiwa
et al., 1990; Hansen et al., 2000).
Activation of PKA initiates neurite formation and guides
axonal pathfinding (Heidemann et al., 1985; Chijiwa et al.,
1990; Ming et al., 1997; Song et al., 1997; Shimomura et al.,
1998; Wang and Murphy, 1998; Hansen et al., 2000). PKA
phosphorylates RhoA at serine 188 (Lang et al., 1996) and
thereby reduces the binding and activation of ROCKs (Dong
et al., 1998) leading to reduced tension of actin fibers and
thus to neurite outgrowth. H89 decreases the activity of PKA
and thus disinhibits RhoA, whereas it can directly inhibit
ROCKs. Therefore, we also tested which of both possible
effects of H89 prevails. Because ROCKs are downstream of
RhoA, it was to be expected that their inhibition will dominate the resultant morphology. This was indeed observed
when forskolin and H89 were tested together. In conclusion,
the present results suggest H89 may not be used as an
antagonist of PKA in systems in which RhoA and its effector
ROCK play a role.
H89 Acts on Cell Morphology by Inhibiting Rho Kinase
Shimomura A, Okamoto Y, Hirata Y, Kobayashi M, Kawakami K, Kiuchi K, Wakabayashi T, and Hagiwara M (1998) Dominant negative ATF1 blocks cyclic AMPinduced neurite outgrowth in PC12D cells. J Neurochem 70:1029 –1034.
Song HJ, Ming GL, and Poo MM (1997) cAMP-induced switching in turning direction
of nerve growth cones. Nature (Lond) 388:275–279.
Tigyi G, Fischer DJ, Sebök A, Marshall F, Dyer DL, and Miledi R (1996) Lysophosphatidic acid-induced neurite retraction in PC12 cells: neurite-protective effects of
cyclic AMP signaling. J Neurochem 66:549 –558.
Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H,
Yamagami K, Inui J, Maekawa M, and Narumiya S (1997) Calcium sensitization
of smooth muscle mediated by a Rho-associated protein kinase in hypertension.
Nature (Lond) 389:990 –994.
Van Aelst L and D’Souza-Schorey C (1997) Rho GTPases and signaling networks.
Genes Dev 11:2295–2322.
Wang X and Murphy TJ (1998) Inhibition of cyclic AMP-dependent kinase by ex-
1007
pression of a protein kinase inhibitor/enhanced green fluorescent fusion protein
attenuates angiotensin II-induced type 1 AT1 receptor mRNA down-regulation in
vascular smooth muscle cells. Mol Pharmacol 54:514 –524.
Watanabe N, Madaule P, Reid T, Ishizaki T, Watanabe G, Kakizuka A, Saito Y,
Nakao K, Jockusch BM, and Narumiya S (1997) p140mDia, a mammalian homolog
of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand
for profilin. EMBO J 16:3044 –3056.
Weeks BS, Papadopoulos V, Dym M, and Kleinman HK (1991) cAMP promotes
branching of laminin-induced neuronal processes. J Cell Physiol 147:62– 67.
Address correspondence to: Prof. Dieter K. Meyer, Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität, Albertstrasse 25, D-79104 Freiburg, Germany. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017