©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Novel (Rp)-cAMPS Analogs as Tools for Inhibition of cAMP-kinase in Cell Culture
BASAL cAMP-KINASE ACTIVITY MODULATES INTERLEUKIN-1beta ACTION (*)

(Received for publication, May 11, 1995)

BjT. Gjertsen (1)(§) Gunnar Mellgren (1)(§) Anne Otten (2) Erik Maronde (4) Hans-G. Genieser (3) Bernd Jastorff (4) Olav K. Vintermyr (1) G. Stanley McKnight (2) Stein O. D(¶)

From the  (1)Department of Anatomy and Cell Biology, University of Bergen, 19, N-5009 Bergen, Norway, the (2)Department of Pharmacology, School of Medicine, SJ-30, University of Washington, Seattle, Washington 98195, the (3)BIOLOG Life Science Institute, Bremen, Flughafendamm 9, P. O. Box 107125 D-28071 Bremen, Germany, and the (4)Universität Bremen, Institut für Organische Chemie, NW 2. Leobener Strasse, 28359 Bremen, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II.

(Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase.

The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI. It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1beta. This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1beta) signaling pathway.


INTRODUCTION

cAMP signaling via activation of cAMP-dependent protein kinase (cAK) (^1)is a major pathway of cell regulation. Both cAK type I and type II have catalytic (C) subunits whose activity is blocked when complexed with the regulatory (RI, RII) subunits. cAMP binding to R facilitates dissociative activation(1, 2, 3, 4, 5) . cAK can be specifically inhibited in intact cells by overexpression of R subunits with deficient cAMP-binding sites (6) or by introducing smaller peptides blocking the active site of C (for review see (7) ). A simpler and more versatile method is to expose cells to (Rp)-cAMPS(8, 9, 10) , which is a diastereoisomer of adenosine 3`,5`-phosphorothioate with sulfur in the equatorial position. (Rp)-cAMPS is believed to antagonize cAMP by binding to R without dissociating the kinase holoenzyme(11) . Cases where (Rp)-cAMPS has been a weak or incomplete cAMP antagonist have been ascribed to low affinity for the cAMP-binding sites of R, low lipophilicity, and inhibition of cyclic nucleotide phosphodiesterase, thereby increasing endogenous cyclic nucleotide(12, 13) .

In the present study improved cAMP antagonists were searched for by screening a number of (Rp)-cAMPS analogs with enhanced lipophilicity. The compounds were tested for ability to antagonize cAMP activation of isolated cAKI and cAKII under near physiological assay conditions and as cAMP antagonists in intact cells with different cAK isozyme expression. The cell systems were: 1) cAMP-dependent gene transcription and cell rounding in 3T3 fibroblasts overexpressing either RIalpha or RIIalpha(14, 15) , 2) cAKI-mediated apoptosis in IPC-81 promyelocytic leukemia cells(16, 17) , 3) inhibition of DNA replication mediated by cAK in primary rat hepatocytes(18) , and 4) inhibition of glucagon-induced modulation of protein phosphorylation in hepatocytes(19, 20) .

So far, little is known about the biological significance of the basal cAK activity in cells not stimulated with agents acting to increase the cAMP level. Study of fibroblasts microinjected with a peptide inhibitor of cAK suggests that decrease of the basal cAK activity is essential for cells to enter M-phase(21) . On the other hand, S-49 lymphoma cells which lack functional C subunit (22, 23, 24) appear to have a normal cell cycle transit. In the present study the basal cAK activity in hepatocytes was decreased by microinjection of RI or by treatment with (Rp)-8-Br-cAMPS. The basal phosphorylation was assessed by two-dimensional gel electrophoresis and autoradiography of proteins from P(i)-prelabeled hepatocytes. Evidence will be presented 1) that basal cAK activity exerts a tonic inhibition of DNA replication (G(1)/S transit) in primary rat hepatocytes, and 2) that basal cAK activity has a permissive effect on interleukin-1beta (IL-1beta) action on hepatocyte DNA replication. This appears to be a new type of link between the cAMP and IL-1beta signaling pathways, whose interdependence have been debated(25, 26, 27, 28, 29) .

The relative antagonistic effectiveness of (Rp)-analogs was not explained by their affinity for RI and RII only, and it was tested whether they differed in ability to stabilize the holoenzyme relative to the dissociated forms of cAK. This was done by assaying the ability of (Rp)-analogs to activate diluted cAKI and cAKII. It turned out that some (Rp)-analogs could act as partial agonists, and it was tested whether they under certain conditions could cooperate with cAMP to partially activate cAKII in intact cells.


EXPERIMENTAL PROCEDURES

Materials

(Rp/Sp)-N^6-Phenyl-cAMPS and (Rp)-8-Cl-cGMPS were synthesized according to the procedure described by Genieser et al.(30) . (Rp)-8-Br-cAMPS, (Rp)-8-Cl-cAMPS, (Rp/Sp)-8-chlorophenylthio-cAMPS, (Rp)-cAMPS, (Rp)-8-chlorophenylthio-cGMPS, (Rp)-8-Br-cGMPS, and N^6-phenyl-cAMP was from current stocks from BIOLOG Life Science Institute, Bremen. All (Rp)-phosphorothioates were free of their corresponding cyclic phosphates or (Sp)-phosphorothioates. Other cAMP analogs, prostaglandin E(1) (PGE(1)), and cholera toxin were obtained form Sigma. Human recombinant interleukin-1beta was from Boehringer, Mannheim, Germany. cAKI was purified from rabbit skeletal muscle or from rat IPC-81 cells(17) , cAKII from bovine myocardium or rat liver(31) , and recombinant bovine RIalpha from Escherichia coli(18) .

Determination of cAMP-binding Site Affinity, Kinase Activity, and Subunits of cAK

(Rp)-cAMPS and cAMP analogs were examined for their ability to compete with [^3H]cAMP for binding to site AI and BI of cAKI and sites AII and BII of the type II enzyme. The relative affinity for a binding site was expressed as K`(analog) = K(cAMP)/K(analog)(32) . Some analogs, like (Rp)-N^6-phenyl-cAMPS, (Sp)-N^6-phenyl-cAMPS, (Rp)-8-Cl-cAMPS, and (Rp)-8-Br-cAMPS had not been studied before, whereas others were available in higher quantities and higher purity than before(33) , allowing their affinity to be assessed more accurately and at physiological temperature (37 °C).

The phosphotransferase activity of isolated cAK was assayed (17) at 37 °C and pH 7.2 in 15 mM HEPES with 5 mM magnesium acetate, 70 µM phosphoacceptor peptide (Kemptide), 0.1 mM [-P]ATP, 0.1 mM EGTA, 1 mM dithioerythritol, 0.5 mg/ml bovine serum albumin, and 130 mM KCl. The concentration of C subunit was either 0.15 nM (incubation time 30 min) or 10 nM (incubation time 1 min). The cellular content of RI, RII, and C was determined as described earlier (17, 31) and related to protein content as determined by the Bio-Rad version of the Coomassie Blue dye binding assay, using -globulin as standard.

Cell Culturing: Assay for Cellular cAMP Responsiveness: Microinjection

Kirsten Ras-transformed NIH 3T3 rat fibroblasts were grown in Dulbecco's minimal essential medium with 10% newborn calf serum in 6-well dishes. Cells constitutively overexpressing mouse RIalpha or RIIalpha (15) were supplemented with 500 µg/ml geneticin (G418) until 3 days before further experimentation. Transient transfection with the cAMP-responsive reporter plasmid alpha168-luciferase (34) was by calcium phosphate precipitation (15) or lipofection(35) . The transfected cells were treated with cAMP analogs and assayed for luciferase activity as described by Otten et al.(15) . Cell rounding was scored after 1 h of incubation with cAMP analogs or forskolin, using a microscope with incubation chamber (125 magnification, 5% CO(2), 90% humidity). Three randomized sectors of each well were studied.

The IPC-81 promyelocytic leukemia cells were cultured in Dulbecco's minimal essential medium with 7% heat-inactivated horse serum. Cells in logarithmic growth phase (0.3 10^6/ml) were treated with apoptosis inducer (PGE(1), cholera toxin), which sometimes was added after 30 min of preloading with (Rp)-cAMPS analog. After 9 h of incubation, the cells were fixed in 3.5% formaldehyde with 10 µg/ml bisbenzimide H 33258 and scored for apoptosis based on fragmentation and hypercondensation of chromatin(17) .

Hepatocytes were isolated from male Wistar rats (150-250 g) by collagenase perfusion and grown on collagen gel with synthetic hepatocyte-selective medium(18) . The medium was supplemented with insulin (0.2 nM) and dexamethasone (100 nM) 2 h after seeding, and with epidermal growth factor (9 nM) 20 h after seeding. In some incubations (Rp)-cAMPS analog was added after 43.5 h and glucagon (2 nM) or IL-1beta (0.4 nM) after 44 h of culture. Hepatocyte replication was determined by pulse labeling of DNA with [^3H]thymidine (0.7 µCi/ml) from 55 to 56 h after seeding. Hepatocytes to be injected were cultured on dishes with grids. They received cytoplasmic injections 52 h after seeding (18) and were pulsed with [^3H]thymidine (0.7 µCi/ml) from 60-61 h in culture. Processing for autoradiography and determination of pulse labeling index were as detailed previously(18) .

Two-dimensional Gel Electrophoresis and Autoradiographic Analysis of Phosphoproteins

Hepatocytes were shifted to a phosphate-free medium and labeled with P(i) (0.1 mCi/dish) from 43 to 44.5 h after seeding. In some cultures (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS was added 43.5 h after seeding, and glucagon or interleukin-1beta added 44 h after seeding. The incubation was terminated (44.5 h) by removing the medium and quickly washing the monolayers in conditioned medium before 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% CHAPS, 0.2% SDS). Sample separation was by isoelectric focusing (80,000 Vh) in linear immobilized pH gradients (Immobiline Dry Strips, pH range 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% (w/v) SDS, 6.5 M urea, 26% 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-electrophoresis (13.5% polyacrylamide separation gel), the gels dried, and exposed to DuPont NEF-496 autoradiography films at -80 °C with intensifying screens. The density and the area of selected spots on the autoradiographic film were determined after having been 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 Instant Imager 2024 (Packard Instr. Co., Meriden, CT). The relative intensity of such directly evaluated spots was the same as that determined by autoradiography and scanning.


RESULTS

Screening of (Rp)-cAMPS Analogs for Potential Usefulness as cAMP Antagonists

The (Rp)-cAMPS analogs are generally highly resistant to hydrolysis by mammalian cyclic nucleotide phosphodiesterases(12, 36) . The ones selected for the present study would be expected to penetrate well into cells since they were more lipophilic than (Rp)-cAMPS itself(36) , and many were congeners of non-(Rp)-analogs with proven ability to affect cAK in intact cells(16, 37) . The (Rp)-cAMPS analogs (at concentrations up to 0.5-1 mM) were screened for ability to antagonize glucagon-induced lowering of DNA replication in primary hepatocytes, which are particularly permeable to cAMP analogs (38) . It appeared that (Rp)-cAMPS, (Rp)-8Br-cAMPS, (Rp)-8-Cl-cAMPS, and (Rp)-8-chlorophenylthio-cAMPS could counteract glucagon. The following analogs were inactive or much less active in this respect: (Rp)-2-Cl-cAMPS, (Rp)-N^6-butyryl-cAMPS, (Rp)-N^6-phenyl-cAMPS, (Rp)-cGMPS, (Rp)-8-Br-cGMPS, (Rp)-8-Cl-cGMPS, (Rp)-8-chlorophenylthio-cGMPS, and (Rp)-8-aminobutylamino-cAMPS. (Rp)-8-Chlorophenylthio-cAMPS at concentrations above 0.3 mM had a nonspecific negative effect on DNA replication accompanied by cell swelling (data not shown). Based on these introductory experiments (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were selected for further scrutiny as possible new useful cAMP antagonists. (Rp)-8-Chlorophenylthio-cAMPS was also included in some experiments although it could induce cAK-independent cell toxicity.

Rp-8-Cl- and Rp-8-Br-cAMPS Are Particularly Efficient cAMP Antagonists toward Activation of cAKI

3T3 fibroblasts stably transfected with either RIalpha or RIIalpha (14, 15) served to probe if (Rp)-analog action was affected by the type of cAK expressed (Table 1). (^2)The analogs were tested for ability to antagonize forskolin-induced cell rounding, which is a recognized cAK-mediated process in fibroblasts(38, 39, 40) . It appeared that (Rp)-8-Cl-cAMPS, (Rp)-8-Br-cAMPS, and (Rp)-cAMPS antagonized forskolin with different relative potency depending on whether RI or RII was overexpressed. (Rp)-N^6-Phenyl-cAMPS failed to antagonize forskolin in any of the cell clones (Fig. 1). (Rp)-8-Chlorophenylthio-cAMPS failed to antagonize forskolin in either ``wild type'' or RI overexpressing cells, but was a potent, albeit only partial, antagonist in the type II cells (data not shown).




Figure 1: (Rp)-cAMPS analogs as antagonists of forskolin-induced cAMP actions in fibroblasts with or without overexpression of RI and RII. 3T3 fibroblasts were treated with forskolin (6 µM for wild type cells and 10 µM for the RIalpha and RIIalpha expressors) and various concentrations of the following phosphorothioate analogs of cAMP: (Rp)-cAMPS ((Rp)-cA; ), (Rp)-8-Cl-cAMPS (Rp8Cl-cA; ), (Rp)-8-Br-cAMPS (Rp8Br-cA; ) or (Rp)-N^6-phenyl-cAMPS (RpN^6P-cA; ; only data with 0.3 mM analog shown). The dotted lines in panel A show the concentrations of (Rp)-8-Br/Cl-cAMPS and of (Rp)-cAMPS required for half-maximal antagonism of forskolin. The percentage of rounded fibroblasts was scored as described under ``Experimental Procedures.'' A representative region from a dish with cells treated with forskolin alone is shown in panel D, and after treatment with forskolin and 300 µM (Rp)-8-Cl-cAMPS in panel E. The data represent the average of three separate experiments, each run in triplicate, the error bars indicating the S.E. Further details are given under ``Experimental Procedures.''



The fibroblast results suggested that (Rp)-8-Br/Cl-cAMPS should be better cAMP antagonists than (Rp)-cAMPS in cells expressing mainly cAKI. Apoptosis in IPC-81 leukemia cells is mediated by activation of cAKI(16) , which is the dominant isozyme in such cells (Table 1). IPC cell apoptosis was induced by cholera toxin or PGE(1), which acted via cAK since they were inefficient in a cell subclone (Fig. 2) with mutant cAMP-subresponsive cAK(17) . The apoptogenic actions of cholera toxin and PGE(1) were completely blocked by (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS. (Rp)-cAMPS was about 10-fold less potent and provided only partial protection (Fig. 2).


Figure 2: (Rp)-cAMPS analog antagonism of IPC leukemia cell apoptosis induced by cholera toxin or PGE(1). More than 80% of IPC-81 leukemia cells (open symbols) became apoptotic (panel C) after treatment for 9 h with 0.03 nM cholera toxin (CT; panel A) or 0.1 µM PGE(1) (panels B and C). Nearly all the cells were protected against CT and PGE(1) by (Rp)-8-Cl-cAMPS () and (Rp)-8-Br-cAMPS (; panel D), (Rp)-cAMPS () being less potent. The dotted lines show the concentrations of (Rp)-8-Br/Cl-cAMPS and of (Rp)-cAMPS required for half-maximal antagonism. The solid symbols (▾) represent data on the RI-mutated (Ala Asp), cAMP-subresponsive IPC cell line, in which cholera toxin gave a slight induction of apoptosis which was abolished by (Rp)-8-Br-cAMPS. The data presentation and statistics were as explained in the legend to Fig. 1.



The relative antagonistic potency of (Rp)-8-Br/Cl-cAMPS and (Rp)-cAMPS in RI-dominated cells (Fig. 1A and 2) was mirrored in studies of isolated cAKI. cAMP activation of type I kinase under near physiological pH, ionic strength, and temperature was antagonized more potently by (Rp)-8-Cl-cAMPS and (Rp)-8-Br-cAMPS than by (Rp)-cAMPS, and the inverse was true for type II kinase. It was also noted that (Rp)-N^6-phenyl-cAMPS was a less potent cAKI antagonist than (Rp)-cAMPS (Fig. 3).


Figure 3: Differential antagonism of cAMP activation of isolated cAKI and II by (Rp)-cAMPS analogs. cAKI from rabbit muscle (panel A) or cAKII from bovine heart (panel B), each at 10 nM, was incubated for 1 min under phosphotransferase conditions with 0.3 µM cAMP and various concentrations (abscissa) of (Rp)-cAMPS (), (Rp)-8-Br-cAMPS (), (Rp)-8-Cl-cAMPS (), or, in the case of cAKI, (Rp)-N^6-phenyl-cAMPS (). The dotted lines show the concentrations of (Rp)-8-Br/Cl-cAMPS and of (Rp)-cAMPS required for half-maximal antagonism of cAMP. The data represent from 7 to 10 determinations from three separate experiments. The bars represent the S.E. Further details are given in ``Experimental Procedures.''



Rp-cAMPS Analogs Differ in Ability to Stabilize the Holoenzyme Form of cAK I and cAKII: Implications for Intact Cell Studies

(Rp)-8-Br-cAMPS was much more potent than (Rp)-N^6-phenyl-cAMPS as cAKI antagonist (Fig. 3A), implying that cAKI holoenzyme had higher affinity for (Rp)-8-Br-cAMPS than for (Rp)-N^6-phenyl-cAMPS. Free RI, on the other hand, had higher affinity for (Rp)-N^6-phenyl-cAMPS than for (Rp)-8-Br-cAMPS (Table 2). If a cyclic nucleotide binds with higher affinity to free R subunit than to R complexed with C subunit it will preferentially stabilize free R and thereby promote dissociation of cAK holoenzyme. This predicts that (Rp)-N^6-phenyl-cAMPS, relative to (Rp)-8-Br-cAMPS, should favor dissociation of cAKI holoenzyme.



This prediction was tested experimentally at cAK concentrations low enough (0.15 nM) to obtain significant dissociation of subunits even in the absence of cyclic nucleotide. It appeared that (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS hardly destabilized (dissociated) cAKI holoenzyme at all, and much less than (Rp)-N^6-phenyl-cAMPS. (Rp)-cAMPS and (Rp)-8-chlorophenylthio-cAMPS had intermediate destabilizing effects. For cAKII (Rp)-8-Br/Cl-cAMPS were about as dissociative as (Rp)-cAMPS (Fig. 4). These data provided a rational explanation for the high potency and selectivity of (Rp)-8-Br/Cl-cAMPS as antagonists of cAMP activation of cAKI, but also raised the possibility that (Rp)-analogs might act as weak partial agonists rather than as pure antagonists of cAMP.


Figure 4: (Rp)-cAMPS analog activation of dilute cAKI and II. cAKI from rabbit muscle (hatched bars) or cAKII from bovine heart (open bars), each at 0.15 nM, was incubated for 30 min under phosphotransferase assay conditions in the absence of cyclic nucleotide (CONTR) or with 50 µM of (Rp)-8-Br-cAMPS (Rp8Br-cA), (Rp)-8-Cl-cAMPS (Rp8Cl-cA), (Rp)-8-chlorophenylthio-cAMPS (Rp8CPT-cA), (Rp)-cAMPS ((Rp-cA), (Rp)-N^6-phenyl-cAMPS (RpN^6P-cA), or cAMP (cA). The data (representing determinations from three to four separate experiments) are given with standard error of the mean.



To know if (Rp)-analogs would be likely to act as partial agonists in intact cells, they were first tested for ability to activate isolated cAK at a concentration (50 nM) approaching that in intact cells (Table 1). Neither cAKI nor cAKII was activated more than 7% by any of the (Rp)-analogs (data not shown). This suggests that they will be incapable of significantly activating cAK intracellularly when acting alone. In the intact cell, however, the analogs coexist with endogenous cAMP, which might cooperate with (Rp)-analogs to activate cAK. The principle was demonstrated for isolated cAKII that (Rp)-analog bound to one site of R might cooperate with cAMP (not shown) or an agonistic cAMP analog (Fig. 5) bound to the other site. It was tested if the degradation-resistant site AI/AII-selective (Table 2) N^6-butyryl-cAMP could cooperate similarly with the site BII-selective (Rp)-8-Cl-cAMPS in intact fibroblasts. This turned out to be the case in RIIalpha-transfected fibroblasts (Fig. 6A), whereas (Rp)-8-Cl-cAMPS desensitized the RIalpha-transfected cells against N^6-butyryl-cAMP (Fig. 6B). The RII-transfected cells were activated synergistically also when the site BI/BII selective 8-methylamino-cAMP was combined with the site AI/AII preferring (Rp)-N^6-phenyl-cAMPS (Fig. 7A). Very little synergism was noted when the same analog pair was tested on the RI overexpressing fibroblasts (Fig. 7B). Since (Rp)-N^6-phenyl-cAMPS selects site A of both RI and RII (Table 2) its more modest synergism with 8-methylamino-cAMP in RI cells could not be explained by lack of cAMP-binding site complementarity, indicating that cells chiefly expressing the type I kinase may be less subject to the activating effect of (Rp)-cAMPS analogs.


Figure 5: (Rp)-8-Cl-cAMPS complementation of cAKII activation by N^6-benzoyl-cAMP. cAK type I (bullet) or type II () was incubated with, respectively, 0.2 and 0.1 µMN^6-benzoyl-cAMP (N^6B-cA) and various concentrations (abscissa) of (Rp)-8-Cl-cAMPS. The experimental details were otherwise similar to those of the experiments shown in Fig. 3.




Figure 6: (Rp)-8-Cl-cAMPS complements N^6-butyryl-cAMP action in fibroblasts predominantly expressing type II kinase. Activation of 3T3 fibroblast cAK was assessed by increased cAMP-responsive gene element-directed luciferase activity (, diamond, filled, ) and cell rounding (, bullet, ). The luciferase activity (left ordinate) is given as percent of the activity (routinely about 2 10^6 counts/min/mg extract protein) in extracts from cells treated with 10 µM forskolin and 300 µM isobutylmethylxanthine. The data are given after subtraction of rounded control cells (<7%) and luciferase activity in extracts from control cells (<8%). The upper panel (A) represents H1.9 fibroblasts overexpressing the RIIalpha subunit, and the lower panel (B) represents RN1.25 fibroblasts overexpressing the RIalpha subunit (for details of expression see Table 1). The dashed lines show the dose-response curve for N^6-butyryl-cAMP when combined with 250 µM (Rp)-8-Cl-cAMPS (Rp8Cl-cA; , ), the thin solid lines show the curve for N^6-butyryl-cAMP combined with 40 µM 8-chlorophenylthio-cAMP (8CPT-cA; diamond, filled, bullet), and the thick solid lines show N^6-butyryl-cAMP alone (, ). Due to the close correlation between cAMP-responsive gene element-directed gene expression and cell rounding both parameters are represented by common lines. The data shown are from representative experiments. Similar results were obtained when the concentration of 8-chlorophenylthio-cAMP was 30 or 60 µM rather than 40 µM (not shown). Further details of the assays for luciferase expression and cell rounding are given under ``Experimental Procedures.'' Analog site affinities are listed in Table 2.




Figure 7: (Rp)-N^6-phenyl-cAMPS complements 8-methylamino-cAMP action in fibroblasts predominantly expressing type II kinase. The upper panel (A) represents H1.9 fibroblasts overexpressing the RIIalpha subunit, and the lower panel (B) represents RN1.25 fibroblasts overexpressing the RIalpha subunit. The experimental set up was as described in the legend to Fig. 6, except that the cells were exposed to various concentrations (abscissa) of the site BI/BII-selective 8-methylamino-cAMP rather than the site AI/AII selective N^6-monobutyryl-cAMP. The dashed lines show the dose-response curve for 8-methylamino-cAMP when combined with 250 µM (Rp)-N^6-phenyl-cAMPS (Rp-N^6P-cA; ), the thin solid lines show the curve for 8-methylamino-cAMP combined with 80 µM 8-chlorophenylthio-cAMP (8-CPT-cA; diamond, filled, bullet), and the thick solid lines show 8-methylamino-cAMP alone (, ).



The validity of the observed (partially) agonistic actions of (Rp)-analogs (Fig. 4-7) depends crucially on their chemical purity. Early reports that (Rp)-cAMPS was a partial agonist for kinase activation (41, 42) were later dismissed as being due to contamination by (Sp)-cAMPS and cAMP(8, 43, 44) . High performance liquid chromatography analysis of the presently used (Rp)-cAMPS and (Rp)-N^6-phenyl-cAMPS after incubation with kinase for 1 h at 37 °C revealed single peaks comprising more than 99.1% of the UV-absorbing material applied. Comparison of the site AI mapping data for N^6-phenyl-cAMP and its (Rp)- and (Sp)-analogs reveals (Table 2) that at most 0.9% of the (Rp)-N^6-phenyl-cAMPS preparation could be (Sp-N^6-phenyl-cAMPS, even if all the binding activity were due to contaminating (Sp)-N^6-phenyl-cAMPS. Similarly, if all the binding activity were due to N^6-phenyl-cAMP this potential contaminant could at most be present at 0.1% of the concentration of (Rp)-N^6-phenyl-cAMPS. To know if a contamination of this extent could explain the activating properties of (Rp)-N^6-phenyl-cAMPS, it was made 0.1% in N^6-phenyl-cAMP and 1% in (Sp)-N^6-phenyl-cAMPS and assayed for kinase activation. Neither the concentration dependence nor the maximal level of activation were affected by such admixture of N^6-phenyl-cAMP and (Sp)-N^6-phenyl-cAMPS. Furthermore, (Rp)-cAMPS treated with phosphodiesterase had the unaltered ability to activate the kinases (data not shown). The results obtained were therefore due to the (Rp)-analogs and not contaminants. (Rp)-cAMPS has been reported to induce dissociation of type I isozyme only in the absence of MgATP(45) . Under the conditions of the present study (Fig. 4), (Rp)-cAMPS was activating whether ATP was 50 µM, 100 µM, or 1 mM (data not shown).

(Rp)-8-Br/Cl-cAMPS Can Decrease Basal cAMP-dependent Phosphorylation in Hepatocytes and Thereby Relieve a Tonic Inhibition of DNA Synthesis

Hepatocyte DNA replication in primary culture in synthetic medium with epidermal growth factor can be negatively modulated by glucagon or other agents activating cAKI or (to a lesser extent) cAKII(18) . It was noted that (Rp)-8-Br/Cl-cAMPS not only prevented the glucagon-induced depression of DNA synthesis (Fig. 8B), but also increased the DNA synthesis in control hepatocytes (Fig. 8A). The DNA replication in (Rp)-8-Br/Cl-cAMPS-treated cells was significantly higher than in controls (p < 0.005), as judged by the Wilcoxon paired-comparison test, where 15 pairs of hepatocyte cultures were incubated with or without 0.3-0.4 mM (Rp)-8-Br/Cl-cAMPS. These data suggested two novel features: 1) (Rp)-8-Br/Cl-cAMPS can decrease even the basal level of cAK activity, and 2) hepatocyte DNA replication is under moderate tonic inhibition by the basal cAK activity. Point 1 above was corroborated by the finding that the basal phosphorylation state of proteins whose phosphorylation was increased by a moderate concentration (2 nM) of glucagon could be decreased below basal by (Rp)-8-Br/Cl-cAMPS (see spots 1-3 in the autoradiograms of Fig. 9, A-D and Table 3). Conversely, proteins whose phosphorylation was decreased by glucagon showed increased phosphorylation after treatment with (Rp)-8-Br/Cl-cAMPS (see spots 4 and 5 in the autoradiograms of Fig. 9, A-D, and Table 3). Point 2) was corroborated by the demonstration that basal DNA replication was stimulated also by microinjection of RIalpha subunit (Fig. 10), whose only known intracellular action is to inhibit C subunit activity.


Figure 8: (Rp)-cAMPS analog effects on basal, interleukin-1beta-inhibited, and glucagon-inhibited hepatocyte DNA replication. Primary rat hepatocytes treated with 0.4 nM interleukin-1beta (IL-1beta) or 2 nM glucagon from 44 to 56 h after seeding had about half as many [^3H]thymidine-labeled nuclei (ordinate) as control cells. The effects of IL-1beta (panel A) or glucagon (panel B) were completely overcome by (Rp)-8-Cl-cAMPS (panels A and B; ) and (Rp)-8-Br-cAMPS (panels A and B; ), whereas the effect of glucagon was only partially overcome by (Rp)-cAMPS (panel B; ). Panel C illustrates the low percentage of [^3H]thymidine-labeled nuclei in hepatocytes treated with 2 nM glucagon, and panel D shows the much higher labeling when the cells had been cotreated with 150 µM (Rp)-8-Cl-cAMPS and 2 nM glucagon. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS not only antagonized IL-1beta and glucagon, but also increased the labeling index above the level in control cells (panel A, solid symbols). The data are given with standard error of the mean for analog concentrations tested in three or four separate experiments.




Figure 9: Autoradiograms of two-dimensional PAGE of extracts from P(i)-prelabeled cultured rat hepatocytes: effects of (Rp)-8-Br-cAMPS, glucagon, and interleukin-1beta. Hepatocytes were labeled with P(i) from 43 h after seeding and treated with 300 µM (Rp)-8-Br-cAMPS from 43.5 to 44.5 h (panels B and D), 2 nM glucagon from 44 to 44.5 h (panels C and D), 0.4 nM interleukin-1beta (IL-1beta) from 44 to 44.5 h (panel E), or left untreated as control (panel A). The proteins were separated by two-dimensional gel electrophoresis and autoradiographic spots subjected to computer analysis as described under ``Experimental Procedures.'' The arrows in panel A (spots 1-14) point to proteins whose phosphorylation state changed after treatment with either glucagon, IL-1beta, or (Rp)-8-Br-cAMPS. Treatment with (Rp)-8-Br-cAMPS (panel B) decreased the phosphorylation of three proteins (spots 1-3) and increased the phosphorylation of two proteins (spots 4 and 5). Glucagon (panel C) stimulated the phosphorylation of eight proteins (spots 1-3 and 10-14), whereas the phosphorylation of two proteins (spots 4 and 5) was inhibited. (Rp)-8-Br-cAMPS appeared to abolish the phosphorylation response to 2 nM glucagon (panel D). Four phosphoproteins (spots 6-9) were uniquely affected by IL-1beta (panel E). The autoradiograms shown are representative examples from four to nine separate experiments.






Figure 10: Microinjected RIalpha increased basal hepatocyte DNA replication and counteracts interleukin-1beta inhibition of DNA synthesis. Primary rat hepatocytes cultured for 52 h were microinjected with mutant RIalpha or exposed to (Rp)-8-Br-cAMPS. 0.4 nM Interleukin-1beta (IL-1beta) was added to some of the cultures 30 min later. After 60 h in culture the cells were pulsed (1 h) with [^3H]thymidine and processed for autoradiography. Both injection of RIalpha and addition of (Rp)-8-Br-cAMPS counteracted the inhibitory effect of IL-1beta on DNA replication. The labeling index increased also in control cells after injection of RIalpha or addition of (Rp)-8-Br-cAMPS. The standard error of the mean of three to six experiments is indicated by error bars, each involving the microinjection of 360-460 cells for every experimental condition. Further details are given under ``Experimental Procedures.''



An eventual involvement of cGMP-dependent protein kinase was addressed by testing whether (Rp)-8-Cl-cGMPS or (Rp)-8-Br-cGMPS could counteract the glucagon action or increase basal DNA replication. None of the (Rp)-cGMPS analogs were efficient in either respect (data not shown). This observation argues against the cGMP kinase being the target for (Rp)-8-Br/Cl-cAMPS. Furthermore, microinjected R (Fig. 10) is completely specific for cAK.

Evidence That the Basal Hepatocyte cAK Activity Has a Permissive Role for the Antiproliferative Interleukin-1beta Signaling

Interleukin-1beta mimicked glucagon by decreasing hepatocyte DNA replication in a (Rp)-8-Br/Cl-cAMPS-sensitive manner (Fig. 8A). The IL-1beta action was also neutralized by microinjected cAMP-subresponsive RI subunit (Fig. 10). The cytokine was therefore presumed to act via activation of cAK. Surprisingly, no increase of cAMP was found in cells treated for 1, 2, 5, 10, 30, 60, or 120 min with IL-1beta. All determinations of cAMP in such cells were within the range of cAMP levels observed in control cells (3.5-4.3 pmol/mg protein) in parallel incubates. For comparison, the cAMP level in hepatocytes treated with 2 nM glucagon ranged from 12 to -22 pmol/mg protein. Furthermore, no mimicry of glucagon action was observed for phosphorylation of hepatocyte proteins. On the contrary, the cytokine enhanced the phosphorylation of a group of protein whose phosphorylation was unaffected by glucagon (spots 6-9 in Fig. 9; Table 3and Table 4). The most plausible explanation was that cAK had a permissive rather than a direct mediator role for IL-1beta action on DNA replication. The IL-1beta-induced phosphorylations (Fig. 9E, Table 4) were not affected by (Rp)-8-Br/Cl-cAMPS (Table 4), indicating that decreased cAK activity did not interfere with the part of interleukin-1beta signaling inducing increased protein phosphorylation. A permissive action of basal cAK activity for optimal IL-1beta signaling appears to be a new regulatory principle for interaction between these pathways.




DISCUSSION

The novel compounds (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were superior to the parent compound (Rp)-cAMPS as cAMP antagonist for isolated cAKI (Fig. 3) and in cells predominantly expressing cAKI, like IPC leukemia cells (Fig. 2), RI-transfected fibroblasts^2 (Fig. 1), and primary hepatocytes (Fig. 8). The available evidence indicates that (Rp)-8-Br/Cl-cAMPS in these cells acted solely by decreasing the cAK activity: 1) in RI-transfected fibroblasts the relative antagonistic potencies of (Rp)-8-Br-cAMPS, (Rp)-8-Cl-cAMPS, and (Rp)-cAMPS (Fig. 1A) were about as for isolated cAKI (Fig. 3A). 2) IPC cells treated with (Rp)-8-Br/Cl-cAMPS were as resistant to cAMP elevating agents as mutant IPC cells with cAMP-subresponsive cAK (Fig. 2). 3) The effect of (Rp)-8-Br/Cl-cAMPS on hepatocyte DNA replication was reproduced by microinjected R-subunit (Fig. 10), which is a completely specific inhibitor of the C subunit of cAK. 4) Halogenated (Rp)-cGMPS analogs, which interact preferentially with the cGMP-kinase(46) , could not mimic the effects of (Rp)-8-Br/Cl-cAMPS on hepatocyte DNA replication. 5) (Rp)-8-Br/Cl-cAMPS selectively counteracted the action of the classical cAMP elevating agent glucagon with regard to protein phosphorylation in hepatocytes (Fig. 9; Table 3and Table 4). It is therefore proposed that (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as first line cAMP antagonist, particularly for studies in cells expressing predominantly cAKI.

(Rp)-Analogs could decrease even the basal cAK activity, as judged by depressed basal phosphorylation of proteins whose phosphorylation increased in response to elevated cAMP (Fig. 9; Table 3). That basal cAK activity can be responsible for protein phosphorylation is supported by studies of a subline (kin) of S-49 lymphoma cells with genetically deficient expression of functioning C subunit(47) , and a possible decrease of phosphorylation of pyruvate kinase observed in hepatocytes in suspension treated with (Rp)-cAMPS(20) . (Rp)-8-Br-cAMPS enhanced the phosphorylation of proteins whose phosphorylation was negatively regulated by cAMP (Fig. 9; Table 3). This indicates that the basal cAK activity is responsible also for negative regulation of protein phosphorylation, which therefore must be a phenomenon sensitive to very slight activation of cAK. The presumably indirect mechanisms whereby cAK depresses protein phosphorylation can involve inhibition of other kinases (48, 49) or stimulation of phosphatases(49, 50, 51, 52) .

Since kin S-49 cells appear to have normal proliferation rate and cell cycle traverse (22, 24, 47) the basal cAK activity cannot be universally essential for cell cycle traverse. Evidence has been presented, however, that down-regulation of cAK activity is required for induction of fibroblasts mitosis(21) . The present study pointed to two other proliferation-related actions of the basal cAK activity in primary hepatocytes. First, their DNA replication was enhanced when the basal cAK activity was decreased by either (Rp)-8-Br-cAMPS or by microinjection of R subunit ( Fig. 8and Fig. 10). This indicates that the basal cAK activity exerted a tonic inhibition of G(1)/S transition rate in epidermal growth factor-stimulated hepatocytes. Second, the inhibitory action of IL-1beta on hepatocyte DNA replication was nearly abolished when the basal cAK activity was inhibited ( Fig. 8and 10). This indicates that the basal cAK activity permits the IL-1beta-induced inhibition of DNA replication. The links between cAMP and IL-1beta-signaling pathways have been debated, and obviously differ between cell types(25, 26, 27) . There is evidence for increased cAMP in response to IL-1beta (28) and for IL-1beta-induced activation of cAK without any increase of cAMP(29) . The present study added the more subtle concept of permissive action of cAK for IL-1beta action to the list of links between the two signaling pathways. Such permissive actions of the basal cAK activity may be more common than hitherto realized, since few studies have attempted to decrease the basal cAK activity. Hopefully, the advent of the improved (Rp)-cAMPS analogs may help elucidate more such cases. It may be noted that without the clear evidence that cAK-dependent phosphorylation events were unaffected by IL-1beta (Fig. 9; Tables III and IV) the data of Fig. 8A would easily have been misinterpreted as proving a mediator role of cAK activation for IL-1beta action. Obviously, that an effect is blocked by inhibitors of cAK is not conclusive evidence that the observed effect was mediated by increased activation of cAK.

A novel and unexpected observation was that adenine modifications affected the action of (Rp)-cAMPS analogs on the equilibrium between the dissociated and holoenzyme forms of cAK, the usefulness of (Rp)-8-Br/Cl-cAMPS as cAKI antagonists being related to the very low dissociation of cAKI isozyme in the presence of these (Rp)-analogs (Fig. 4). Unfortunately, all available (Rp)-analogs induced significant dissociation of cAKII (Fig. 4), and, under certain conditions, could contribute to partial activation of cAKII in intact cells (Fig. 6A and 7A). (Rp)-Analogs should therefore be used with caution in studies of cells predominantly expressing cAKII. Ongoing experiments are aimed at synthesizing novel (Rp)-analogs with improved agonist activity toward cAKII. These efforts will be guided by the observations of the present study (Fig. 4).


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of Norway, the Novo Nordisk Foundation, National Institutes of Health Grants ML 44948 and GM 32875, Grant Ja 246/6 from the Deutsche Forschungsgemeinschaft, Der Fonds der Chemischen Industrie, and the EU Human Capital and Mobility program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors are equal.

To whom correspondence should be addressed. Tel.: +47-55206376; Fax: +47-55206360.

(^1)
The abbreviations used are: cAKI and cAKII, isozyme type I and II of cAMP-dependent protein kinase; R and C, the regulatory and catalytic subunits, respectively, of cAMP-dependent protein kinase; RI and RII, the regulatory subunits of cAMP-dependent protein kinase isozymes I and II, respectively; AI, BI, AII and BII, the cAMP-binding sites in RI and RII, respectively; Sp-cAMPS and Rp-cAMPS, the diastereoisomers (Sp)-cAMPS and (Rp)-cAMPS of adenosine 3`,5`(cyclic)phosphoro[thioate]; Rp-8-Br/Cl-cAMPS, Rp-8-Br-cAMPS as well as Rp-8-Cl-cAMPS; IL-1beta, interleukin-1beta; PGE(1), prostaglandin E(1); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

(^2)
The RI/RII ratio was higher in the RI-transfected (RN1.25) fibroblasts than in IPC cells (Table I). Nevertheless, isozyme II-directed cAMP-analog pairs synergized significantly in activating cAMP-responsive gene activity and cell rounding in RN1.25 cells (Fig. 6B), but not in inducing IPC cell apoptosis (16). This demonstrates that endogenous holoenzyme-associated RII was still present in RN1.25 cells overexpressing RIalpha, as supported by the finding of a substantial amount of type II holoenzyme by DEAE chromatography of RN1.25 cell extracts (data not shown). The RN1.25 cells thus cannot be considered a pure type I system, but they have much more type I kinase than the RIIalpha expressing H1.9 cell line, which showed very little type I directed analog synergism (Fig. 7A).


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