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Departments of Anatomy and Cell Biology (G.M., T.B., O.K.V., S.O.D.) and Biochemistry and Molecular Biology (A.P.D., T.F.), University of Bergen, N-5009 Bergen, Norway
Address all correspondence and requests for reprints to: Dr. Stein Ove Døskeland, Department of Anatomy and Cell Biology, University of Bergen, Årstadveien 19, N-5009 Bergen, Norway. E-mail: stein.doskeland{at}med.uib.no
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The intracellular messenger cAMP has been reported to be a positive (4, 5) or negative (6, 7, 8) regulator of growth depending on the cell type. In epidermal growth factor (EGF)-stimulated primary cultured hepatocytes, an increment of cAMP induces a swift inhibition of the G1/S transition (9, 10) followed by a prolonged stimulation of cell proliferation (11, 12). In the prereplicative phase of liver regeneration, there is a burst of cAMP-dependent protein kinase (PKA) activation that is partly down-regulated during the phase of active regeneration (13). However, the role of cAMP during liver regeneration is still enigmatic.
Intracellular Ca2+ can affect cell growth (14), but little is known about the molecular mechanisms by which Ca2+ and calmodulin regulate cell proliferation (15). In the regenerating liver, there is an early increase in calmodulin synthesis (16), which may be induced by cAMP (17). Vasopressin increases cytosolic Ca2+ and activates the Ca2+/calmodulin-dependent protein kinase II (CaMKII) in isolated rat hepatocytes (18, 19). Vasopressin has been reported to modulate hepatocyte DNA replication in culture (20, 21, 22) and during liver regeneration (23).
We have previously found that the glucocorticoid, dexamethasone, sensitizes hepatocytes toward growth inhibition by cAMP (9, 24), and we wanted to know whether this represented a general pattern of synergy between stress mediators. Therefore, the relation was studied between the acute phase cytokine interleukin-1ß (IL-1ß) and the acute metabolic stress-induced messengers cAMP and Ca2+ with respect to modulation of hepatocyte proliferation. IL-1ß is one of several cytokines that has been reported to have a regulatory role in hepatocyte proliferation (25). Several signal transduction pathways have been reported to be involved in IL-1ß signaling (see Ref. 26 for review). In some cell lines, IL-1ß has been shown to increase endogenous cAMP (27). The links between cAMP and IL-1ß signaling transduction have been debated, and there are obvious differences among various cell types (28, 29). Recently, we reported that IL-1ß restricted hepatocyte DNA replication (30). The inhibition of DNA replication was counteracted by cAMP antagonists [Rp-8-bromo(Br)-cAMPS/Rp-8-chloro-cAMPS] or by microinjection of a cAMP-subresponsive regulatory subunit, suggesting that PKA has a permissive effect on the action of IL-1ß in hepatocytes.
The present study has obtained novel clues for growth modulatory mechanisms, particularly regarding the role of CaMKII. Activation of CaMKII by vasopressin (or microcystin) potentiated the inhibition of hepatocyte proliferation induced by cAMP or IL-1ß. The effect of IL-1ß on hepatocyte growth was not caused by an increase in the cAMP level or in PKA activity. CaMKII was activated by the microbial serine/threonine phosphatase inhibitor microcystin, based on phosphorylation of phenylalanine hydroxylase (PAH; EC 1.14.16.1), which is an endogenous marker of CaMKII activation (19, 31). The potencies of the CaMKII inhibitors KN93 (32) and KT5926 (33) were compared with respect to modulation of intact hepatocyte proliferation and the activity of isolated CaMKII, assayed under near-physiological conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P] ATP (3000
Ci/mmol), [3H]thymidine (40–60 Ci/mmol), and
[32P]orthophosphate (10 mCi/ml) were obtained from
Amersham (Little Chalfont, UK). IL-1ß (human; 100,000 U) was
purchased from Boehringer Mannheim (Mannheim, Germany).
Arginine-vasopressin (grade VI), cyclic
N6-benzoyladenosine 3',5'-monophosphate
(N6-benzoyl-cAMP), dexamethasone, the phosphate
acceptor heptapeptide (kemptide; Leu-Arg-Arg-Ala-Ser-Leu-Gly),
collagenase (type C-0130), and EGF were all obtained from Sigma
Chemical Co. (St. Louis, MO). (Rp)-8-bromoadenosine cyclic
3':5'-phosphorothioate was purchased from Biolog Life Science Institute
(Bremen, Germany). Microcystin-LR,
N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7),
and N-[2-(methylamino)-ethyl]-5-iso-quinolone-sulfonamide
(H-8) were obtained from Calbiochem (La Jolla, CA). Okadaic acid was
purchased from LC Services Corp. (Woburn, MA). Phenylalanine
hydroxylase was purified from rat liver as described previously (19).
KT5926 was obtained from Kamiya Biomedical Co. (Thousand Oaks, CA), and
KN93 was purchased from Seikagaku America (Rockville, MD).
Procedure of cultivation
Rat liver hepatocytes were isolated from male Wistar rats
(150–250 g) by in vitro collagenase perfusion and processed
for primary in vitro culture as described previously (9).
The cells were grown on collagen gel in culture dishes at densities of
30,000 cells/cm2. The cells were cultured in a synthetic
serum-free medium (hepatocyte-selective medium) (12).
Determination of labeling index
The labeling index was determined after the addition of
[3H]thymidine (1.5 µCi/dish) and fixation in
glutaraldehyde 1–2 h later, as indicated in the figure legends. The
dishes were processed for autoradiography, and the fraction of labeled
cells was determined in separate microscopic fields (each of 0.1
mm2, with an average cell density of 31–60 nuclei/field)
from representative areas of the culture wells.
Determination of cAMP, the catalytic subunit of cAMP-dependent
kinase, and protein
For determination of cellular cAMP, the incubation medium was
quickly decanted and replaced with 1.5 ml ice-cold 5% (wt/vol) aqueous
trichloroacetic acid-0.1 M HCl. cAMP was measured by a
competitive binding assay (13).
The amount of catalytic subunit was determined by assaying its phosphotransferase activity (13). The kinase activity was linear with respect to the concentration of extract and the time of incubation. The kinase activity is expressed as nanomoles of phosphate incorporated into kemptide per min. Protein was determined as described previously (12).
CaMKII assay
The phosphotransferase activity of isolated CaMKII was assayed
at 30 C for 3–6 min in 45- to 60-µl reactions containing 15
mM HEPES (pH 7.2) with 20 µM phosphoacceptor
peptide (syntide-2), 10 µg/ml calmodulin, 100 mM KCl, 10
mM MgCl2, 1 mM CaCl2,
2.0 mM [-32P]ATP, and 0.5 mg/ml BSA. The
reaction was started by the addition of CaMKII, and the kinase
inhibitors were added immediately before CaMKII. The concentrations of
kinase inhibitors (KT5926 or KN93) are described in the text. The
amount of phosphate incorporated into substrate was determined by
spotting aliquots of the incubate on a phosphocellulose strip.
Two-dimensional gel electrophoresis and autoradiographic analysis
of phosphoproteins
After 44 h of culture, hepatocytes were shifted to a
phosphate-free medium and labeled with 32P (0.1 mCi/dish)
for a period of 1 h (final activity, 375 µCi/3 x
105 cells) before treatment with vasopressin or glucagon
for 0.5 h. The incubation was terminated by removing the medium
and quickly washing the monolayers in conditioned medium before the
addition of lysis buffer [9.8 M urea, 100 mM
dithioerythritol, 1.5% Pharmalyte (pH 3.5–10), 0.5% Pharmalyte (pH
5–6), 4% 3-((3-cholamidopropyl)dimethylammonio)-1-propane-sulfonate
(CHAPS), and 0.2% SDS]. Isolated hepatocytes were prelabeled with
32P for 1 h and exposed to vasopressin or microcystin
as described in the figure legends. Inhibitors of CaMKII were added 10
min before vasopressin/microcystin. Sample separation was achieved by
isoelectric focusing (80,000 volt-hours) in linear immobilized pH
gradients (IPG strips, pH 4.0–7.0, Pharmacia Biotechnology, Uppsala,
Sweden). After completion of the run, the strips were equilibrated for
12 min in solution [0.05 M Tris-HCl (pH 6.8), 2% (wt/vol)
SDS, 6.5 M urea, and 26% (wt/vol) glycerol] with 100
mM dithioerythritol and thereafter for 5 min in the same
solution with 0.24 M iodoacetamide. The strips were then
subjected to SDS-PAGE [13.5% (wt/vol) polyacrylamide separation
gel], and the gels were dried and exposed to DuPont NEF-496
autoradiography films. The density and the area of selected spots on
the autoradiographic film were determined after they were digitized by
scanning, using the NIH Image 1.57 program. Seven spots whose
phosphorylation appeared not to change after treatment served as
benchmarks. The radioactivity of spots well isolated from others could
be estimated directly by analysis in an Instant Imager 2024 (Packard
Instrument Co., Meriden, CT), and their relative intensities were the
same as those determined by autoradiography and scanning.
Immunoisolation of 32P-labeled
phenylalanine hydroxylase and determination of
vasopressin/microcystin-induced phosphorylation
Hepatocyte suspension (5 x 106 cells/ml) was
incubated with carrier-free 32P at 37 C in a gyratory
shaking bath for 1 h. The cells were exposed to agents modulating
intracellular phosphorylation (vasopressin, microcystin, okadaic acid,
N6-benzoyl-cAMP, and glucagon with or without
phenylalanine) for 12 min. Some cells were treated with KT5926 or H-8
for 10 min before the addition of these agents. The endogenous protein
phosphorylation was quenched by mixing the hepatocytes with chilled
ammonium sulfate solution (saturated ammonium sulfate, 1 mM
ATP, 50 mM NaF, and 50 mM sodium phosphate, pH
7.0). The tubes containing the cells were then centrifuged at
15,000 x g for 5 min, and the cell pellet was kept in
liquid nitrogen until homogenization. For further description of the
method, see Ref. 19.
For immunoisolation, protein A-Sepharose suspended in 0.66 ml 50 mM sodium phosphate (pH 8.0) was mixed with 1 µg each of the monoclonal antibodies PH5 and PH7 and gently shaken for 30–60 min at 4 C. The adsorbent was then centrifuged at 12,000 x g for 15 sec, and the pellet was washed twice with phosphate buffer and resuspended in 0.6 ml homogenization medium. The resuspended beads were mixed with 160 µl cytosol containing 32P-labeled PAH and rocked in Eppendorf tubes for 1 h at 4 C. The protein A-Sepharose complexed to IgG-PAH was pelleted by centrifugation and washed four times with 1.25 ml homogenization buffer, four times with the same buffer without protease inhibitors containing 0.2% (wt/vol) Triton X-100, and finally once with the same buffer without Triton X-100. The pellet was finally heated (65 C, 10 min) in 115 µl sample buffer (19) and centrifuged (10 min, 12,000 x g). Aliquots of the final supernatant were applied to 1.5-mm polyacrylamide slab gels (10%) for SDS-PAGE. The proteins were silver stained, and the gels were dried and exposed to autoradiographic films. The stained gels and the respective autoradiograms were analyzed using an LKB Ultroscan XL (LKB, Rockville, MD), and the specific content of phosphate in the PAH was determined from the ratio between the absorbance of the autoradiogram and that of the silver-stained gel corresponding to the PAH band. Known amounts of rat PAH (19) were used for calibration of the silver staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were designed to probe
whether vasopressin sensitized the cells to low concentrations of
glucagon. Vasopressin enhanced the inhibitory effect of low
concentrations of glucagon; the EC50 of glucagon decreased
from 1.2 to 0.50 nM, but failed to inhibit further the
replication of cells treated with the strongest stimulators of
endogenous cAMP accumulation [such as >10 nM glucagon or
10 µM forskolin plus isobutylmethylxanthine (IBMX); Fig. 1A]. The glucagon-induced inhibition of DNA replication was
significantly enhanced in the presence of vasopressin
(P < 0.001; n = 15), as judged by the Wilcoxon
paired comparison test in which 15 pairs of hepatocyte cultures were
incubated with glucagon (0.5, 1, or 2 nM) in the absence
and presence of vasopressin. Similar results (P <
0.001; n = 15) were obtained when microcystin replaced
vasopressin.
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).
To exclude that vasopressin and microcystin acted through modulation of
glucagon signaling not related to cAMP or by inhibiting cAMP
degradation, they were tested for the ability to sensitize hepatocytes
toward the action of the cell-permeable, degradation-resistant cAMP
analog N6-benzoyl-cAMP. Vasopressin and
microcystin shifted the EC50 for
N6-benzoyl-cAMP from 7.1 to 0.80
µM without affecting the maximal inhibitory ability of
N6-benzoyl-cAMP (Fig. 1B). The inhibition of DNA
replication by 0.1–10 µM
N6-benzoyl-cAMP was significantly enhanced by
vasopressin (P < 0.001; n = 18) or microcystin
(P < 0.001; n = 14), as judged by the Wilcoxon
paired comparison test. Vasopressin or microcystin did not increase the
endogenous level of cAMP (data not shown). This indicated that the
observed sensitization was due to events downstream of cAMP.
Although vasopressin has been shown to act as a mitogen when added to
primary hepatocytes shortly after seeding (22, 34), the hormone did not
affect DNA replication when added to hepatocytes after 44 h of
culture (Fig. 1). Such a lack of mitogenic response has also been
reported for angiotensin II-induced elevation of intracellular
Ca2+ in rat liver T51B cells (35). That vasopressin had no
effect on hepatocyte proliferation by itself (Fig. 1) may be related to
the decreased number of vasopressin receptors with time in culture
(36). However, cultured hepatocytes maintain the ability to respond to
vasopressin by increasing cytosolic Ca2+ (36). The latter
observation is compatible with our finding that vasopressin could
induce specific changes in the protein phosphorylation pattern of
primary hepatocytes after 44 h of culture (data not shown).
Evidence that vasopressin modulates hepatocyte proliferation
through activation of CaMK
Vasopressin is known to increase the level of Ca2+
(37) and to induce phosphorylation of hepatocyte proteins known to be
substrates of CaMKII (18, 19). It was therefore tested whether
vasopressin depended on Ca2+/calmodulin to enhance cAMP
inhibition of hepatocyte DNA replication. Firstly, the hepatocytes were
treated with a general calmodulin antagonist, W7 (38), before the
addition of vasopressin and cAMP analog. W7 abolished the effect of
vasopressin (Fig. 2), suggesting that
calmodulin was required for the vasopressin effect. Next, KT5926, a
potent inhibitor of calmodulin-dependent protein kinases, including
CaMKII (33), counteracted the effect of vasopressin (Figs. 2 and 3A). Finally, KN93, a more selective
inhibitor of CaMKII (32), blocked the effect of vasopressin (Fig. 3B),
suggesting that a calmodulin-dependent protein kinase similar to CaMKII
is required for the action of vasopressin. A problem with ascribing the
inhibitor effects to CaMKII was the variation in IC50
values reported for the inhibition of CaMKII by KT5926 and KN93 (32, 33). Therefore, we decided to determine the IC50 values for
CaMKII inhibition in an assay with near-physiological pH (7.2),
temperature (37 C), salt composition (130 mM KCl), and ATP
level (3 mM). The IC50 values for inhibition of
CaMKII activity by KN93 and KT5926 in vitro (Fig. 4) correlated closely with those observed
for intact hepatocytes (Figs. 2 and 3).
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. These experiments were performed on
isolated hepatocytes, as the expression of PAH was down-regulated in
cultured hepatocytes (Døskeland, A. P., and S.O. Døskeland,
unpublished observations). The site phosphorylated (Ser16)
was the same in response to microcystin as that phosphorylated by
CaMKII in vitro (19). This was demonstrated by generation of
identical phosphopeptides of trypsinized PAH whether obtained after
incubation with phosphorylated CaMKII in vitro or from
hepatocytes treated with phosphatase inhibitor (data not shown). The
phosphorylation of PAH by microcystin (Table 1) and vasopressin (Fig. 5) was inhibited by KT5926. In agreement
with previous findings (19), okadaic acid, a potent inhibitor of
protein phosphatases 2A and 1 (44), stimulated the phosphorylation of
PAH by activation of CaMKII (Fig. 5). The phosphorylation of PAH by
okadaic acid was inhibited by KT5926 (Fig. 5). It was tested whether
KT5926 could inhibit the PKA-stimulated phosphorylation of PAH. As
shown in Fig. 5, KT5926 did not affect the phosphorylation of PAH in
response to 5 nM glucagon and 3 mM
extracellular phenylalanine. Phenylalanine was included because it
increases the intracellular rate of hydroxylase phosphorylation by PKA
and decreases the rate of phosphorylation by CaMKII (19). Exposure of
hepatocytes to 100 µM
N6-benzoyl-cAMP increased the phosphorylation of
PAH nearly 10-fold (9.9 ± 1.62) even in the presence of 30
µM KT5926 (9.4 ± 0.23), indicating that KT5926 did
not affect PKA-mediated phosphorylation of PAH. Moreover, the PKA
inhibitor H-8 did not affect vasopressin/microcystin-induced
phosphorylation of PAH, although it nearly blocked PKA-mediated
phosphorylation of PAH by N6-benzoyl-cAMP (data
not shown). As inhibitors of CaMKII (Fig. 4) could block the effects of
vasopressin (Figs. 2 and 3) and microcystin (data not shown) on
hepatocyte proliferation, it was tested whether they also blocked the
endogenous phosphorylation induced by these substances. Freshly
isolated hepatocytes preincubated with 32Pi
were treated with microcystin (0.5 µM) and KT5926 (30
µM) and subjected to two-dimensional gel electrophoresis
and autoradiography. A number of proteins showed changes in
phosphorylation state after exposure to microcystin. Several of them
were also changed in response to vasopressin. All protein
phosphorylations seen in response to vasopressin also appeared in
response to microcystin, indicating that microcystin mimicked
vasopressin and induced additional protein phosphorylation events as
well. Most of the protein phosphorylation events were sensitive to
treatment with KT5926 (Fig. 6 and Table 1), suggesting that a number of proteins are phosphorylated by the
Ca2+/calmodulin-dependent pathway(s) and are potential
mediators of the effects of vasopressin and microcystin.
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) or microcystin (data not shown). It
was also noticed that IL-1ß (0.3 nM) and glucagon (2
nM) acted with remarkably similar kinetics to restrict the
hepatocyte G1/S transition. The onset of inhibition and the
rate of recovery of DNA replication were the same (data not shown).
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). This
supports the idea that increased PKA activity is necessary for
vasopressin action. In Fig. 7B, it is also shown that treatment of
hepatocytes with vasopressin and a low concentration of
N6-benzoyl-cAMP (10 nM) sensitized
the cells even more than vasopressin by itself; the EC50
for IL-1ß was less than 0.05 nM.
That IL-1ß apparently acted like cAMP on hepatocyte proliferation in
the absence and presence of vasopressin raised the question of whether
IL-1ß could increase hepatocyte cAMP. The cell content of cAMP was,
therefore, determined at various time points after the addition of 0.4
nM IL-1ß. No detectable increase in cAMP was observed
after treatment with IL-1ß, against a 4-fold increase in cells
treated with 1 nM glucagon (Fig. 8). Moreover, cAMP phosphodiesterase
inhibitor (IBMX) acted in a synergistic manner with glucagon to
increase endogenous cAMP, whereas no such synergism was detected with
IL-1ß. It is possible that IL-1ß acted by up-regulating the amount
of active catalytic (C) subunit or down-regulating the inhibitory
regulatory subunits (RI and RII) of PKA. Determination of PKA subunits
in hepatocytes 7 h after exposure to IL-1ß failed to reveal any
change in their levels (data not shown). It is, therefore, concluded
that IL-1ß did not inhibit hepatocyte proliferation through
activation of PKA.
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The effects of IL-1ß and vasopressin on the hepatocyte
G1/S transition in the presence of sub-basal, basal, or
moderately increased PKA activity are summarized in Fig. 9.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vasopressin and norepinephrine act by increasing hepatocyte Ca2+ and activation of CaMKII (18). It was, therefore, of interest to probe how the Ca2+-activated stress pathway interacts with the IL-1ß and cAMP pathways. Interactions between cAMP and Ca2+ signaling pathways are well known, such as in the regulation of carbohydrate metabolism in liver (47). It is, however, a novel observation that activation of CaMKII by vasopressin and microcystin potentiates cAMP- and IL-1ß-mediated inhibition of hepatocyte DNA replication, and it should be noted that CaMKII (activated by vasopressin) inhibited hepatocyte proliferation only in the presence of increased PKA activity and/or after exposure to IL-1ß. The biological significance of the IL-1ß/cAMP synergism for the intact liver could be to halt cell proliferation under conditions of combined general stress (IL-1ß) (46) and metabolic stress (cAMP).
The fact that the action of IL-1ß on proliferation was abolished by
the specific PKA antagonist (Rp)-8-Br-cAMPS could be
explained by an IL-1ß-mediated activation of PKA. Such a link between
IL-1ß and cAMP signal transduction pathways has previously been shown
in other cell types (27). However, IL-1ß did not increase the cAMP
level (Fig. 7) or the PKA activity (data not shown), indicating that
PKA is not the primary target for IL-1ß in hepatocytes. Based on
these results, our working hypothesis is that the cAMP and IL-1ß
signaling pathways converge at a point downstream of PKA. Previously,
we reported that basal PKA activity has a permissive, rather than a
direct, mediator role on IL-1ß action (30). In the present study it
is shown that inhibition of basal PKA activity by
(Rp)-8-Br-cAMPS counteracted the effects of both IL-1ß and
vasopressin.
Little is known about the role of CaMKII in regulation of cell proliferation. The expression of a constitutively active CaMKII has been reported to arrest cells in G2 (48), whereas in HeLa cells (49) and NIH-3T3 cells (50), cellular proliferation was inhibited after prolonged exposure to the CaMKII inhibitor KN93. Calmodulin and CaMKII seem to be required for G1/S transition in several cell types (49). The latter observations do not exclude CaMKII as a negative modulator of hepatocyte proliferation after activation of PKA. In the present study it is shown that activated CaMKII acts together with cAMP and IL-1ß to inhibit hepatocyte G1/S transition.
Convergent regulation by the Ca2+ and cAMP signaling systems was reported previously (51), and various CaMKs have been shown to have different effects on the activation of cAMP response element (CRE)-binding protein (CREB) (52). CaMKII-mediated phosphorylation of CREB at Ser133 and Ser142 does not stimulate transcription in vitro (52). Contradictory data exist, however, regarding the involvement of CaMKII in CRE gene activation in intact cells. Recently, one report suggested that CaMKII activates CRE genes by phosphorylation of activating transcription factor-1 (53), whereas another report suggested that CaMKII was unable to activate activating transcription factor-1 (54). An interesting observation is, however, that low level stimulation of PKA appears to be required for CREB activation through non-PKA-mediated pathways (55). A similar mechanism could explain the necessity of PKA activation for inhibition of DNA replication by vasopressin.
cAMP has been reported to inhibit the proliferation of some cell types, such as Rat1 fibroblasts, by interfering with signaling through the mitogen-activated protein kinase pathway (7). However, protein kinase members of the mitogen-activated protein kinase cascade are activated within a few minutes after growth factor binding (56), i.e. long before the G1/S restriction point. It thus appears unlikely that these targets are involved in the cAMP-mediated inhibition of the hepatocyte G1/S transition. The progression from G1 to S phase in mammalian cells is thought to be regulated by different G1 cyclins and their associated catalytic subunits, the cyclin-dependent kinases (see Ref. 57 for review; 58). In human diploid fibroblasts, growth factor-induced expression of cyclin D is repressed upon treatment with cAMP analogs (59, 60), and in the astrocytic cell line, cyclin D1 and E expression is inhibited by cAMP (61). Moreover, it should be noted that cAMP has been shown to arrest macrophages in mid-G1 by increasing the level of p27kip1, an inhibitor of cyclin-dependent kinase-4 activation (62). Thus, it is possible that cAMP-mediated inhibition of hepatocyte G1/S transition could be due to effects on a G1 cyclin or on the activity of a cyclin-dependent kinase.
The microcystin-related phosphatase inhibitor okadaic acid mimics the activation of CaMKII in isolated hepatocytes with regard to phosphorylation of PAH (19). In this paper it has been shown that both microcystin and vasopressin increase the phosphorylation of PAH. The effects of vasopressin and microcystin on hepatocyte proliferation were selectively counteracted by inhibitors of CaMKII (KN93 and KT5926). Phosphorylation of several common endogenous proteins by vasopressin and microcystin were sensitive to treatment with KT5926. The results suggest that vasopressin and microcystin act through activation of CaMKII.
In conclusion, this paper reports novel synergistic antiproliferative effects of activated PKA, CaMKII, and the IL-1ß signaling pathways. Basal activity of PKA was required for IL-1ß action, whereas increased PKA activity, as observed during the prereplicative phase of liver regeneration (13), was required for the actions of vasopressin and microcystin.
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Acknowledgments |
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Footnotes |
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2 Present address: UNIGEN Center for Molecular Biology, Norwegian
University of Science and Technology, N-7005 Trondheim, Norway.
Received March 19, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or combined
with either 1 µM vasopressin (
) or 1 µM
microcystin (
). Some cultures were also supplemented with 10
µM forskolin and 50 µM IBMX in addition to
150 nM glucagon (indicated by +F/I on the
abscissa) in the absence (•) or presence of
vasopressin (
) or microcystin (
). The hepatocytes were pulsed
with [3H]thymidine (0.15 µCi/dish) from 54–56 h of
culture before fixation and preparation for autoradiography.
Half-maximal inhibition of hepatocyte DNA replication
(EC50) is indicated by the dotted lines. The
bars represent the SEM of three separate
experiments.
) and KN93 (
) and 1
µM vasopressin-10 nM
N6-benzoyl-cAMP (
) at 43.5 h of
culture, i.e. 30 in before treatment with IL-1ß. Other
cultures were treated with 300 µM
(Rp)-8-Br-cAMPS (
) or were left unsupplemented (
), 50 µM IBMX (
),
or 1 nM glucagon and 50 µM IBMX (