Cyclic AMP Potentiates the Retinoic Acid-induced Expression of ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261,No. 2, Issue of January 1 S . p 614+21,1986 nnted m U.S.A.

Cyclic AMP Potentiates the Retinoic Acid-induced Expressionof Tissue Transglutaminase in Peritoneal Macrophages* (Received for publication, January 29, 1985)

Michael P. Murtaugh, WilliamT. Moore, Jr.& and Peter J. A. Daviesf From the Department of Phrmacology, University of Texas Medical School at Houston, Houston, Texas 77025

Fresh serum and retinoids induce the expression of of tissue transglutaminase in macrophages appears to be due tissuetransglutaminaseincultured mouseresident to factors present in serum since it could be reproduced in peritonealmacrophages,Analoguesof cyclic AMP, uitro by culture of resident cells in serum-containing medium such as dibutyryl cyclic AMP, andagents that increase (12). Recently we have shown that removal of endogenous intracellular cyclic AMP levels enhance the induction. lipids from serum abolishes its ability to induce tissue transDibutyryl cyclic AMP alone has little effect on trans- glutaminase gene expression, and that readdition of retinoic glutaminase expression, but it increases sensitivity the acid to delipidized serum fully restores its transglutaminaseof macrophages to low concentrations of either seruminducing activity (13). or retinoic acid. Dibutyrylcyclic AMP potentiates the The possibility that retinoic acid in serum rapidly induced transglutaminase-inducingactivity of both free reti- macrophage transglutaminase expression is intriguing. Retinoic acid and retinoic acid bound to the serum retinolbinding protein. Pretreating macrophages with dibu- noic acid induces differentiation in several cell types, includtyryl cyclic AMPor retinoic acid does not prime the ing teratocarcinoma (14-16) and epithelial cells (17), and in myeloid leukemia cell lines (18, 19). In these instances the cells to respond to the other agent; instead, both agents response to retinoic acid usually requires several days for must be present simultaneously to obtain the synergistic induction of transglutaminase.Our studies suggest expression, and in all cases it is enhanced bycyclic AMP that the modulation of intracellular cyclic AMP levels analogues that increase intracellular cyclic AMP levels. Bemay have pronouncedeffects on retinoic acid-induced cause these studies indicated that cyclic AMP interacts with retinoic acid in regulating gene expression during differentiagene expression in myeloid cells. tion, we investigated whether cyclic AMP also potentiated the retinoic acid-induced expression of a specific gene product Transglutaminases are a group of calcium-requiring en- such as macrophage transglutaminase. We have found that zymes that catalyze the covalent cross-linking of proteins by dibutyryl cyclic AMP and agents that elevate intracellular the formation of t-(y-glutaminy1)-lysyl isopeptide bonds (1, cyclic AMP levels havedramatic effects on the serum-induced 2). These enzymes are widely distributed both inside cells and expression of macrophage transglutaminase. This effect apin extracellular fluids (3). The predominant form of transglu- pears to be due to the ability of cyclic AMP to sensitize taminase in mesenchymal cells and tissues is tissue transglu- macrophages to respond to low concentrations of retinoic acid taminase, an -80,000-dalton cytoplasmic protein that is in serum. The enhancement is due to a synergistic interaction thought to play an important role in the cross-linking of between cyclic AMP and retinoic acid on the expression of tissue transglutaminase. structural and membrane proteins (4-7). Dramatic alterations in cellular transglutaminase activity EXPERIMENTAL PROCEDURES are associated with the differentiation of normal and leukemic Materials-Ten-week-old ICR strain male mice were obtained from myeloid cells. Maturation of human peripheral blood monocytes into macrophages is accompanied by a marked increase Timco (now Harlan-Sprague-Dawley, Houston, TX). Mouse serum in the concentration of tissue transglutaminase (8). Differ- was obtained by clotting whole blood collected by cardiac puncture. 1640 media and methionine-free Dulbecco’s modified Eagle’s entiation of murine myeloblastic leukemia (Ml) cells into RPMI medium were obtained from Gibco (Grand Island, NY). Cyclic numacrophages is also accompanied by an increased levelof cleotides, trans-retinoic acid, and insulin were from Sigma. Cholera transglutaminase activity (9). The most striking increase in toxin and Pansorbin were from Calbiochem-Behring. Isobutyltransglutaminase activity is produced by the activation of methylxanthine was from Aldrich. 6-0-Steroyl muramyl dipeptide peritoneal macrophages. Macrophages elicited by a variety of was a generous gift of Dr. Gordon Jones (Syntex, PaloAlto, CA). 12inflammatory irritants contain 10-100 times more enzyme 0-Tetradecanoylphorbol 13-acetate was from Consolidated Midland (Brewster, NY). Sodium heparin was from Elkins-Sinn (Cherry Hill, activity than is found in unstimulated resident cells (10-12). NJ). Na’’’I and [%]methionine were obtained from Amersham Cop. This marked elevation in enzyme activity is due entirely to [3H]Putre~cinewas from New England Nuclear. Serum was delipithe induction of tissue transglutaminase (12). The induction dized using the method of Rothblat et at. (20). Serum retinol-binding *This research was supported by National Institutes of Health Grant AM27078 and American Cancer Society Grant BC-334. The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore behereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Supported by National Institutes of HealthNational Service Award 5T32 AM07282from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. Established Investigator of the American Heart Association.

protein was purified from human serum as described (21). Cell Culture-Resident peritoneal macrophages were obtained by lavage using 5 ml of ice-cold RPMI medium containing 10 units of heparin, 50 units of penicillin, and 50 pg of streptomycin/ml. Pooled cells werewashed by centrifugation a t 400 X g for 10 min and resuspended in RPMI medium. Mononuclear leukocytes were counted in a Coulter Counter (Coral Gables, FL). Cells were plated onto 16or 35-mm Nunc or Costar tissue culture plates (1-1.5 X lo6 cells/ cm2).Nonadherent cells were washed off after 1 h. The resultant cell monolayers were more than 95% macrophages by phagocytic uptake and staining for nonspecific esterase (22). Cultures were then incu-

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Retinoic Acid

a n d CAMPInduce Macrophage Transglutaminase

bated at 37 "C in a C02/air incubator under the conditions described for individual experiments. Determination of Transglutaminase Activity-Macrophage monolayers were washed three times with RPMI medium, scraped from the well in a minimal volume of 20 mM Tris-HC1, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 15 mM 2-mercaptoethanol and disrupted by sonication for 30 s. Aliquots of the cell lysate (5-50 pg of protein) were incubated at 30 "C in atotal volume of 100 rl containing 20 mM Tris-HCl, pH 7.5, 15 mM 2-mercaptoethanol, 5 mMCaC12, 2 mg/ml N,N'-dimethyl casein, and 0.5 mM [3H]putrescine (90 dpm/pmol). At 5- and 10-min intervals, aliquots were spotted on Whatman3" filter paper. Filters were fixed and washed in trichloroacetic acid and ethanol, and protein-bound 3H was determined by liquid scintillation spectroscopy. Background values were obtained by using 5 mM EGTA' in place of 5 mM CaC12in thereaction. Alternatively, adherent cells were washed as described and lysed by the addition of 100 pl of the reaction mixture described above containing 0.05% Triton X-100. The lysate was then incubated in the well at 35 "C for 10 min at which time aliquots were spotted on filters and processed as described above. The protein concentration of cell lysates was determined by the Coomassie Blue binding assay of Bradford (23) using bovine yglobulin as the standard. ImmunochemicalDetection of Tissue Transglutaminase-To detect tissue transglutaminaseby immunoblot assay, cells were washed with RPMI medium, solubilized in 10 mM Tris-HC1, pH 7.5,1% SDS, 0.75 M 2-mercaptoethanol, and 2.5% sucrose, and boiled for 3 min. Samples containing 5-50 pg of cell protein were fractionated by electrophoresis in SDS-acrylamide gels as described previously (8, 12). Tissue transglutaminase was detected by autoradiography using a antibody (12) or unlabeled monospecific '251-anti-transglutaminase anti-transglutaminase antibody followed by affinity-purified '%I-labeled rabbit anti-goat IgC antibody. Autoradiographic bands were quantified by laser densitometric scanning (LKB, Uppsala, Sweden) using guinea pig liver tissue transglutaminase standards run in parallel lanes on the same gel. Metabolic Labeling and Immunoprecipitation-Approximately 0.3 X lo6 macrophages/l6-mm well were cultured overnight as described in the figure legends, washed with methionine-free Dulbecco's modified Eagle's medium, and incubated for 40 min in 100 ~1 of methionine-free Dulbecco's modified Eagle's medium containing 10 pCi of [35S]methionine.Labeling was stopped by aspirating the medium and washing one time with RPMI medium. Incubation in the chase phase was then continued under the prelabeling conditions until the cells were harvested for immunoprecipitation. To immunoprecipitate tissue transglutaminase, cells were washed in RPMI medium and lysed in 200 pl of 20 mM Tris-HC1, pH 7.5,150 mM NaC1, 2 mM EDTA, 1%aprotinin, 1%Triton X-100, and 1% sodium deoxycholate. Debris was removed by centrifugation in an Eppendorf microfuge. Equal amounts of cell protein were incubated with 10 fig of affinity-purified anti-transglutaminase antibody or 20 pg of preimmune IgG in a total volume of 400 containing 125 mM sodium phosphate, pH 7.6, 0.5 M NaCI, 1 mM EDTA, 0.5% bovine serum albumin, 1%aprotinin, and 0.1% soybean trypsin inhibitor. After 16 h a t 4 "C, immune complexes were adsorbed to killed and washed Staphylococcus aureus (Pansorbin) (24). Complexes were washed twice in 20 mM Tris-HC1, pH 7.5,150 mM NaC1,l mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, once in Tris-buffered saline, solubilized in SDS gel mixture, and electrophoresed on a 10%discontinuous acrylamide gel. Radioactive bands were located by fluorography (25) on Kodak X-Omat AR film using Cronex Lightning Plus intensifier screens(DuPont, Wilmington, DE).

615

wanted to identify physiological factors that might regulate the expression of the enzyme. Milhaud et al. (26) reported that analogues of cyclic AMP could influence the expression of'transglutaminase in Chinese hamster ovary cells,and therefore, we examined the effect of similar analogues on the induction of transglutaminase activity in macrophages. Freshly isolated mouse resident peritoneal macrophages were cultured in low concentrations of fresh mouse serum in the presence or absence of 1 mM dibutyryl cyclic AMP, and the levels of transglutaminase activity were determined (Fig. 1). Serum alone induced a small increase in transglutaminase activity, but the addition of dibutyryl cyclic AMP to the serum-containing media increased the induction of the enzyme markedly. To examine the specificity of the effect of dibutyryl cyclic AMP on transglutaminase expression, we compared the effectiveness of various concentrations of dibutyryl cyclic AMP, dibutyryl cyclic GMP, and Na butyrate on the serum-induced expression of transglutaminase activity (Fig. W ) .The effect of dibutyryl cyclic AMP was concentration-dependent; it was maximally effective at 0.5 mM (Fig. W ) . Over the same concentration range, dibutyryl cyclic GMP had no effect on the induction of transglutaminase. Sodium butyrate has been reported to increase transglutaminase activity in some cells (27), but addition of butyrate had no effect on the transglutaminase activity of cultured macrophages (Fig. 2 A ) . We also testedotheragents that elevate intracellular cyclic A.MP levels by independent molecular mechanisms. Cholera toxin, isobutylmethylxanthine, and 8-bromo-cyclic AMP were as effective as dibutyryl cyclic AMP in augmenting the induction of transglutaminase in macrophages (Fig. 2B). Dibutyryl Cyclic AMP Increases the Sensitivity of Macrophages toRetinoicAcid-induced Expression of Transglutam i m e Activity-Previous studies from our laboratory have shown that the ability of fresh serum to induce transglutaminase in macrophages is due to the presence of retinoids in the serum (13). To determine whether the effect of cyclic AMP on transglutaminase expression reflected an interaction

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RESULTS

Dibutyryl Cyclic AMP Potentiates the Induction of Transglutaminase Activity in Macrophages-Our initial interest in developing these studies was in identifying factors that could regulate the expression of transglutaminases in cultured peritoneal macrophages. We had found that fresh serum was a potent inducer of macrophage transglutaminase activity and The abbreviations used are: EGTA, ethylene glycol bis@-aminoethyl ether)-N,N,N',N' -tetraacetic acid; SDS, sodium dodecyl sulfate; RA-RBP, retinoic acid bound to serum retinol-binding protein; Bt2cAMP,dibutyryl cyclic AMP; DLMS, delipidized mouse serum.

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FIG. 1. Effect of dibutyryl cyclic AMP on the induction of macrophage transglutaminase by serum. Resident macrophages were cultured for 18 h in medium alone, medium containing 2.5% mouse serum, or medium containing 2.5% mouse serum and 1 mM dibutyryl cyclic AMP. Cells were assayed at 35 "C in the culture wells as described under "Experimental Procedures." Data represent the means of duplicate samples.

Retinoic Acid and CAMPInduce Macrophage Transglutaminase

616 A

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FIG. 2. Effect of dibutyryl cyclic AMP, dibutyryl cyclic GMP, sodium butyrate, and agents that elevate intracellular cyclic AMP on the induction of macrophage transglutaminase activity. A, mouse resident peritoneal macrophages were cultured for 18 h in RPMI 1640 medium and 2.5% mouse serum containing dibutyryl cyclic AMP (closed circles), dibutyryl cyclic GMP (open circles), or sodium butyrate (closed squares) a t the indicated concentrations. Enzyme activity was determined as described under “Experimental Procedures.” Data represent the means of two to four observations. B, cells were incubated for 24 hin 2.5% mouse serum containing cholera toxin (C. T.,1 pg/ml), isobutylmethylxanthine ( I B M X ,0.2 mM), or 8-bromo-cyclicAMP @-Br-cAMP, 1mM). Transglutaminase activity was determined as described under “Experimental Procedures.” The datashown are means of four to six observations. Standard errors were less than or equal to 10% of the mean in all cases.

nM Retinoic

minase activity (closed circles, Fig. 3). Even 0.1 nM retinoic acid is sufficient to produce a significant increase in transglutaminase activity if dibutyryl cyclic AMP is present in the media, and 100 nM retinoic acid and 1 mM dibutyryl cyclic AMP induced an 8-fold increase in enzyme activity. These studies showed that retinoic acid and dibutyryl cyclic AMP alone were sufficient to promote transglutaminase expression in the macrophages. However, even in the presence of dibutyryl cyclic AMP, retinoicacid is nota very potent inducer of transglutaminase.Concentrations of 100 nM or above are required to induce largeincreases inenzyme activity.We have found that macrophages are much more sensitive to retinoic acid if it is bound to the serum RBP than if it is free in solution (13). We therefore investigated whether dibutyryl cyclic AMP was as effective in promoting the transglutaminase inducing activityof RA-RBP as it had been in promoting the activity of free retinoic acid. Macrophages were cultured in serum-free media with either 1 or 10 nM RA-RBP in the presence or absence of 1 mM dibutyryl cyclic AMP (Table I, Experiment I). In this experiment, the cells cultured in media alone had a basal enzyme activity of 498 pmol/min/mg. Addition of 1 nM RA-RBP induced a small increase in transglutaminase activity, to 865 pmol/min/mg. The combination of 1 nM RA-RBP plus dibutyryl cyclic AMP, however, induced a dramatic increase in enzyme activity, to 4270 pmol/min/ mg. This same potentiation could be seen in cells exposed to 10 nM RA-RBP. On its own 10 nM RA-RBP was only slightly more effective than 1 nM RA-RBP in inducing transglutaminase expression, but in combinationwith dibutyryl cyclic AMP it induced a very large increase in transglutaminase activity. These studies demonstrated that dibutyryl cyclic AMP not only increased the response of macrophages to free retinoic acid but italso could increasetheir response to very low levels of RA-RBP. The most effective form of retinoic acid for the induction of tissue transglutaminase is retinoic acid reconstituted with delipidized serum (13).As can be seen in Table I, Experiment 11, macrophages cultured in 1 nM retinoicacid plus 0.1% delipidized mouse serum showalargeincreaseinenzyme activity.Cells culturedin media alonehad basalenzyme

acid

FIG. 3. Effect of dibutyryl cyclic AMP on the level of transglutaminase activity in macrophages treated with retinoic acid. Resident peritoneal macrophages were cultured for 20 h in RPMI medium containing retinoic acid alone ( o ” 0 ) or retinoic Enzyme activity was acid and 1 mM dibutyryl cyclic AMP (M). determined as described under “Experimental Procedures.” Data represent the means of multiple determinations.

TABLEI Effect of dibutyryl cyclic AMP onretinoic acid-induced expression of trans.elutaminase activity in mouse peritoneal macrophages Culture conditions

Transglutaminase activity“

Experiment I Media aloneb 498 -+ 136 1 nM RA-RBP 865 f 128 with retinoic acid, we examined the effect of dibutyryl cyclic 1 nM RA-RBP + 1 mM Bt2cAMP 4270 f 241 AMP on retinoic acid-induced expressionof tissue transglu10 nM RA-RBP 935 2 102 4810 f 483 10 nM RA-RBP + 1 mM BtzcAMP taminase. Experiment I1 The abilityof retinoic acid t o induce macrophage transgluMedia alone‘ 437 f 60 taminase depends on the culture conditions. retinoic Free acid RA 1 nM + 0.1% DLMS 1335 2 134 and retinoic acidbound to albumin are weak inducers of 1 nM RA + 0.1% DLMS + 1mM Bt2cAMP 4030 f 219 macrophage transglutaminase, whereas retinoic acid bound to 100 RAnM + 0.1% DLMS 3685 f 210 the serum retinol-binding protein (RA-RBP) or reconstituted 100 nMRA + 0.1% DLMS + 1 mM Bt2cAMP 7850 f 561 with delipidized serum is a much more effective inducer of Transglutaminase activity was determined as described under the enzyme (13). Because of the simplicity of studying trans- “Experimental Procedures” and is expressed as pmol/min/mg of cell glutaminase induction in a chemically defined medium, we protein. Data represent the mean and range of duplicate determinafirst examined the effect of dibutyryl cyclic AMP and free tions. Freshly isolated resident peritoneal macrophages were cultured retinoic acid on macrophages cultured in media alone (Fig. 3). Free retinoic acid is a very poorinducer of transglutamin- in RPMI 1640 media for 20 h. Retinoic acid and purified RBP were reconstituted in equimolar amounts (RA-RBP) and added to RPMI ase in macrophages; even at levels as high as 100 nM, there is at theindicated concentrations. only a small increase inenzyme activity (open circles, Fig. 3). e Resident peritoneal macrophages were cultured for 16 h in RPMI In the presenceof dibutyryl cyclic AMP, however, these same 1640 media alone in the absence or presence of retinoic acid (RA), delipidized mouse serum, and dibutyryl cyclic AMP as indicated. levels of retinoic acid induce a large increase in transgluta-

Retinoic Acid and CAMPInduce Macrophage Transglutaminase

617

activities of 437 pmol/min/mg, and those exposed to 1 nM ously to retinoic acid and dibutyryl cyclic AMP (Fig. 5 ) . In retinoic acid and DLMS had an activity of 1337 pmol/min/ this particular experimentwe compared the inductionof the mg. Addition of dibutyryl cyclic AMP greatly increased the enzyme in macrophages incubated in media alone (X), 1 mM induction by this low level of retinoic acid. The transgluta- dibutyryl cyclic AMP alone (open squares), 1 nM RA-RBP minase activity in these cells (4030 pmol/min/mg) was more alone (open circles), or 1 nM RA-RBP plus 1 mM dibutyryl than 9-fold higher than in control cells. A similar potentiation cyclic AMP (closed circles). The level of transglutaminase was observed in cells treated with 100 nM retinoic acid and activity infreshly isolated macrophageswas very low and rose DLMS. Under these conditions the retinoid alone increased slowly over 24 h when the cells were cultured in serum-free media. The addition of 1 mM dibutyryl cyclic AMP or 1 nM the enzyme activity to 3685 pmol/min/mg and the addition of dibutyryl cyclic AMP resulted in a further doubling of the RA-RBP to the media resulted in a small increase in this induction. The transglutaminase activity of cells exposed to accumulation of the enzyme so that at 24 h the total level of 100 nM retinoic acid and 1 mM dibutyryl cyclic AMP in enzyme activity was 2-3-fold higher than in the controlcells. DLMS was as high or higher than the maximal inductions If the dibutyryl cyclic AMP and the RA-RBP were added simultaneously to the cells, there was a muchmore rapid achieved with cells cultured in fresh serum. As a final step we also examined the effect of dibutyryl induction of the enzyme. Transglutaminase activity was incyclic AMP on the induction of transglutaminase activityby creased within 3 h, and the enzyme continued to accumulate varying concentrationsof fresh mouse serum (Fig. 4).In spite rapidlyfor 18-24h. This rapid induction of macrophage of considerable variability in the activityof different batches transglutaminase in the presence of dibutyryl cyclic AMP is of fresh mouse serum, concentrationsabove 1% are generally identical to the time course for the induction of the enzyme required to induce significant increases in the transglutamin- in response to retinoic acid in delipidized serum (13) or fresh ase activity of cultured macrophages (open circles, Fig. 4) and mouse serum (12). The preceding study examined theeffect of the simultane10-20% serumis generally required to inducea maximal ous addition of retinoic acid and dibutyryl cyclic AMP on induction of the enzyme (open circles, Fig. 4, inset). In the presence of dibutyryl cyclic AMP (closed circles, Fig. 4), mac- macrophage transglutaminase expression. In studies on the rophages are much more sensitive to serum; concentrations induction of differentiation in humanmyeloid leukemia (HLof serum below 1% now induce a large increase inmacrophage 60) cells, Olsson et al. (28) suggested that retinoic acid may transglutaminase activity, and 5% serum induces a maximal prime cells to respond tocyclic AMP. To test thispossibility effect. It is apparent from the inset inFig. 4 that the primary in macrophages, we examined the effect of first exposing the effect of the dibutyryl cyclic AMP is to shift the serum dose- cells either to retinoic acid or dibutyryl cyclic AMP on their response curve to the left, indicating that the addition of subsequent response to the other agent. Resident peritoneal dibutyryl cyclic AMP to the media increases the sensitivity macrophages were incubated in 1 mM dibutyryl cyclic AMP of the cells tothe transglutaminase-inducing activity of or 1 nM RA-RBP for 6 h. The cells were then washed twice for a further 6 h in either agent serum. These findings are inaccord with our previous obser- and the incubation continued vations that dibutyrylcyclic AMP increases the sensitivity of alone or both agents together, as shown in Table 11. In the macrophages t o retinoic acid. case of cells preincubated in dibutyrylcyclic AMP if the cells Effect of Dibutytyl Cyclic AMP on theKinetics of the I n d u c - were simply incubatedin dibutyryl cyclic AMP for 12 h,there tion of Macrophage Transglutaminase Activity-The preced- was a small increase(130 pmol/min/mg) in transglutaminase ing studies demonstrated that dibutyryl cyclic AMP poten- activity. If the cells were first incubated in dibutyryl cyclic cyclic AMP was washed offand tiated retinoid-induced expression of macrophage transglu- AMP and then the dibutyryl taminase activity, but they gave little insight into the mech- the incubation continued for a n additional 6 h in 1 nM RAanisms that might be involved in this interaction. To inves- RBP alone, therewas a small increase (405 pmol/min/mg) in tigate this issue, we first examined the kinetics for the induc- transglutaminase activity. This induction,however, was much (1419pmol/ tion of tissue transglutaminase in cells exposed simultane- less than the increase in enzyme activity obtained 2500

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FIG. 4. Effect of dibutyryl cyclic AMP on the serum-induced expression of macrophage transglutaminase activity. Resident macrophages were cultured for 20 h in RPMI 1640 medium containing various concentrations of mouse serum alone ( O “ 0 ) or C#ls )were . harserum plus 1 mM dibutyryl cyclic AMP (U vested and transglutaminase activity was assayed as described under “Experimental Procedures.” Inset, a comparable experiment using higher concentrations of mouse serum, Data represent the means of duplicate determinations.

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FIG. 5. Time course of transglutaminase induction by dibutyryl cyclic AMP and retinoic acid-RBP. Resident macrophages were cultured for various lengths of time inRPMI 1640 medium alone ( X ) or containing 1 nM retinoic acid-RBP and 1 mM dibutyryl cyclic AMP (O), 1 nM retinoic acid-RBP (O), or 1 mM Transglutaminase activities were measured dibutyryl cyclic AMP (0). as described under “Experimental Procedures.” Data represent the means of duplicate determinations from duplicate culture wells. RASRBP, retinoic acid bound to serum retinol-binding protein.

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Retinoic Acid and CAMPInduce Macrophage Transglutaminase

TABLEI1 Effectof dibutyryl cyclic AMP and retinoic acid-RBPpretreatment followed by each agent alone or both togethr on macrophage transglutaminase

butyvl cyclic AMP (closed squares) were no more active than control cells (open squares) when challenged with the combination of dibutyryl cyclic AMP plus serum. Thus, bothaspects of the experiment suggested that pre-exposure to dibutyryl Induced cyclic AMP does not alter the kinetics for the. subsequent Preincubation” Incubation transglutaminase condition condition induction of the enzyme. activitvb The reciprocal set of experiments is shown in Fig. 6B. In pmol/rnin/mg this experiment macrophages were preincubated for 10 h in BhcAMP BtSAMP 130 7 1.5% fresh serum before being challenged by addition of 1419 f 10 BtZcAMP RA-RBP + B ~ c A M P dibutyryl cyclic AMP alone or dibutyryl cyclic AMP plus 405 f 17 BtSAMP RA-RBP RA-RBP serum. In this particular experiment preincubation in serum RA-RBP 306 f 7 RA-RBP B ~ c A M P+ RA-RBP 2058 f 27 alone (closed circles and squares) did induce a significant rise RA-RBP BtcAMP 960 f 18 in cellular transglutaminase activity when compared to cells Resident peritoneal macrophages were cultured for 6 h in RPMI preincubated in media alone (open circles and squares). If the media containing 1 mM dibutyryl cyclic AMP or 1 nM retinoic acid- cells were washed well, however,and thepreincubation media RBP holoprotein. The cells were washed with media and incubated replaced with media containing dibutyryl cyclic AMP alone, an additional 6 h in 1 mM dibutyryl cyclic AMP or 1 nM retinoic there was no further induction of the enzyme regardless of acid-RBP as indicated in the second column. whether the cells had been pre-exposed to serum (closed Transglutaminase activity wasdetermined as describedunder “Experimental Procedures.”Data are the means k S.D. of duplicate circles) or not (open circles). Also, if both sets of cells were determinations from duplicate dishes and represent the amount of challenged with the combination of dibutyryl cyclic AMP plus activity induced duringthe 12-h pretreatment and incubation period. serum, then both responded promptly with an equivalent These values were obtained by subtracting the level of enzyme activity induction of the enzyme. Thus, preincubation with serum also present in cells cultured in media alone (964 pmol/min/mg). did not alter the kinetics of the induction of transglutaminase activity. These studies confirmed the previous findings with min/mg) if the cells were exposed to both dibutyryl cyclic purified RA-RBP that thesimultaneous presence of dibutyryl AMP and RA-RBP during the second incubation period. cyclic AMP and the retinoid (or serum) was required for the These results suggest that pre-exposure of macrophages to optimal induction of transglutaminase activity. dibutyryl cyclic AMP does not prime them to respond to a Effect of Dibutyryl Cyclic AMP on the Accumulation of subsequent challenge with retinoic acid. Tissue Transglutaminase in Macrophages-Up to this point We also tested the reciprocal possibility that pre-exposing we had focused on the ability of dibutyryl cyclic AMP to cells to retinoic acid might subsequently alter their response promote the induction of transglutaminase activity in macto dibutyryl cyclic AMP. In this experiment, incubation for rophages. Our studies did not distinguish, however, between 12 h with 1 nM RA-RBP resulted in a small induction (306 several different molecular mechanisms that might account pmol/min/mg) of macrophage transglutaminase activity. If for this process. To study the induction in more detail, we the cells were first incubated with the RA-RBP and then used an immunochemical approach to measure the levels of excess RA-RBP washed off and dibutyryl cyclic AMP added, tissue transglutaminase in cells treated with low levels of there was a significantly greater induction of transglutamin- serum in the presence or absence of dibutyryl cyclic AMP. ase activity. Although the activity in these cells (960 pmol/ Fig. 7 compares the overall profile of Coomassie Blue-stained min/mg) was substantially greater than in cells incubated in proteins (panel A ) or tissue transglutaminase immunoblots dibutyryl cyclic AMP alone (130 pmol/min/mg), it was still (panel B ) from macrophages treated with 2.5% serum in the much less than the level obtained in the presence of both presence or absence of 1 mM dibutyryl cyclic AMP. The dibutyryl cyclic AMP and RA-RBP (2058 pmol/min/mg). induction of tissue transglutaminase canbe easily seen by the Thus, it also seems to be true that pre-exposing cells to RA- increased prominence of a band at 78,000 kDa (shown by the RBP does not prime them for the ability to respond to arrow) in the cells treated with serum plus dibutyryl cyclic dibutyryl cyclic AMP. Optimal induction of the enzyme ap- AMP. To confirm thatthis new band was indeed tissue pears to require the simultaneous presence of both retinoic transglutaminase, parallel samples were transferred to nitroacid and cyclic AMP. cellulose filters and probed with an iodinated antibody to It was possible that the preincubation experiments de- tissue transglutaminase. A single band of tissue transglutascribed aboveoverlooked subtle effects of either dibutyryl minase can be seen in the cells treated with a low concentracyclic AMP or retinoic acid on the kinetics of transglutamin- tion of serum alone, and the amount of this protein was ase induction. To evaluate this we carried out adetailed study dramatically increased in the cells treated with serum plus on the time course for the induction of transglutaminase dibutyryl cyclic AMP. Detailed inspection of the overall patactivity in cells preincubated in either dibutyryl cyclic AMP tern of polypeptide bands from the control and dibutyryl (Fig. 6A) or a low concentration of fresh mouse serum (Fig. cyclic AMP-treated cells revealed that tissue transglutamin6B). Fresh mouse serum was used in these experiments as a ase was the only identifiable protein whose expression was substitute for RA-RBP because of the limited amounts of the altered by dibutyryl cyclic AMP. The ability of dibutyryl cyclic AMP to potentiate the inpurified RA-RBP available to us. In the case of cells preincubated in dibutyryl cyclic AMP (panel A ) , the cells preincu- duction of tissue transglutaminase could reflect either its bated with 1 mM dibutyryl cyclic AMP for 10 h (closed circles ability to promote the synthesis of the enzyme or its ability and squares) had slightly higher levels of total cellular trans- to retard its degradation. To distinguish between these two glutaminase activity than cells incubated in media alone (open possibilities, we compared the rate of tissue transglutaminase circles and squares). The cells first treated with dibutyryl turnover in macrophages pulse-labeled with [35S]methionine cyclic AMP and then switched to serum (closed circles) were and chased with unlabeled methionine in the presence or no more active than cells that had been preincubated in media absence of a low level of serum plus dibutyryl cyclic AMP. alone (open circles) in terms of transglutaminase expression. Tissue transglutaminase was then identified by immunopreFurthermore, the cells that had been preincubated with di- cipitatingthe enzymefrom the metabolically labeledcell ~~~~

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Hour5 01 Incubation

FIG.6. Effect of pretreatment with dibutyryl cyclic AMP or serum on the subsequent induction of macrophage transglutaminase bymouse serum. A, resident peritoneal macrophages were cultured for 1 0 h in RPMI 1640 media with (0.m) or without (0, 0 ) 1 mM dibutyryl cyclic AMP. Both setrc of cultures were then rinsed and incubated an additlonal 0-14 h in media containing 1% mouse serum with (0, m) or without (0, 0 )1 mM dibutyryl cyclic AMI’.At theindicatedtimesthe cellswerewashed andtransglutaminaseactivity was determined as described under “Experimental Procedures.”I?, two sets of mouse resident peritoneal macrophages were cultured for 10 h in RPMI 1640 media with (0, or without ( 0 , O ) 1.5% mouse serum. The medium was removed, and the cells were washed and cultured for an additional 14 h in media containing 1 mM dibutyryl cyclic AMP (0,O) or 1.5% mouse serum (0..). At the indicated times cells were washed and transglutaminase activity was determined as described under “Exoerimental Procedures.” Values shown are the means k S.E. of replicate determinations from duplicate wells.

.)

A

controlserum + BtsAMP ” o 1224 o 12 24 Hours chase

B

t

20

1-

w

+

BtzcAMP

-

+

BtzcAMP AMP ontheserum-in-

FIG.7. Effectofdibutyrylcyclic duced expression of tissue transglutaminase. A, resident macrophages were incubated lor 48 h in HPMI 1640 medium containing 2.5% mouse serum with or without 1 mM dibutyryl cyclic AMP. Cell lysates, prepared as described under “Experimental Procedures” and containing 40 pg of protein, were fractionated by electrophoresis on a 10% SDS-acrylamide gel, andproteinbands werevisualized by staining withCoomassie Blue. The arrow denotes the position of tissue transglutaminasea t M,78,000. Molecular weight standards are bovine fibronectin (220,000), phosphorylase a (94,000), bovine serum albumin (67,000), ovalbumin (45,000). and soybean trypsin inhibitor (20,000). H , samples containing 5 p g of cell protein from the experiment in panel A were electrophoresed ona 10% SDS-acrylamide gel, electroblotted onto nitrocellulose paper, and probedwith ‘*‘I-antitransglutaminase antihody as described under “Experimental Procedures.”

“ -

1

2

3

4

”

5

6

FIG. 8. Pulse-chase analysis of transglutarninase ntability after induction by serum and dibutyryl cyclic AMP. Resident peritoneal macrophages were cultured in HPMl 1640 rnedin alone (lanes 1-3) or media containing 1% mouse serum and 1 mM dihutvryl cyclic AMP ([ones 4-6). After 16 h the cells were pulse-labeled with [‘%]methionine as described under “Experimental Procedures.” The cells were immediately lysed ( 0 h; lanps 1 and 4 ) or they were incubated for 12 h (lones 2 and 5 ) or 24 h (lanes 3 and 6 ) in the prelabeling media. Tissue transglutaminase (arrow) was immunoprecipitated from equal amounts of cell lysate and identified by electrophoresis and fluorography as described under “Experimental Procedures.“

extract. Fig. 8, lunes 1-3, shows the transglutaminase band (shown by the arrow) in the control cells either immediately after pulse-labeling (lane 1 ) or following 12 or 24 h of chase (lunes 2 and 3, respectively). It is apparent that the intensity culturedinserum plusdibutyryl cyclic AMP.The tissue of the transglutaminase band is relatively constant over the transglutaminase band is much more intense in these immu24-h chase period, indicating that the rateof tissue transglu- noprecipitates, indicating that the rate of enzyme synthesis taminase turnover in these cells was very slow. The half-life was significantly increased under the conditions of enzyme for total macrophage proteins either in the presence or ab- induction (lune 4 uersu.? lane 1 ) . Again, there was little desence of dibutyryl cyclic AMP (determinedby trichloroacetic crease in the intensity of the tissue transglutaminase band acid precipitation of radio-labeled proteins) was 8.4 h (data over the 24-h chase period (lanes 5 and 6), suggesting that the qot shown). Lanes 4-6 show the results obtained with cells enzyme also turned over very slowly in the presence of the

620

Retinoic Acid and CAMPInduce Macrophage Transg1utamina.w

ranges from2 to 10 nM. Our studiessuggest that thebiological activity of these levels, at least in thecase of the inductionof tissue transglutaminase, may depend critically on the intracellular levels of cyclic AMP in the target cells. Thus, we suspect that agents that modify intracellular cyclic AMP levels (in macrophages these would include prostaglandins and catecholamines(33-36)) may also modify the susceptibility of the cells to regulation by serum retinoids. As part of these studies, we have attempted to gain some flours d incubation insight into the mechanisms involved in the interaction of FIG. 9. Effect of actinomycin D on the induction of trans- retinoic acid and cyclic AMP. One issue we have evaluated is glutaminase by serum and dibutyryl cyclic AMP. Resident Peritoneal macrophages werecultured for 5 h in 1% mouse serum and the role of serum proteins in the effect of dibutyryl cyclic 1 mM dibutyryl cyclic AMP. Actinomycin D (0.1pg/ml) was added AMP on the inductionof macrophage transglutaminase activ(arrow)to Some of the cultures (O),and the incubation was continued ity. This concern was prompted by ourfindingthatthe for 17 h.Transglutaminaseactivity wasdetermined as described binding of retinoic acid to the serum RBP greatly increased of replicate its effectiveness as an inducer of macrophage transglutaminunder “Experimental Procedures.” Data shown are means determinations. Standard deviations were less than 10% of their ase activity (13). While the mechanism of this effect is not respective means. clear, othershave shown that the binding of retinoids to RBP can facilitate their delivery to susceptible cells and tissues dibutyryl cyclic AMP plus serum. The abilityof dibutyryl cyclic AMP to promote the induc- (37-40). Thus, it was possible that the ability of dibutyryl tion of tissue transglutaminase was blocked by actinomycin cyclic AMP to potentiate the transglutaminase-inducing activity of free retinoic acid reflectedits ability to reproduce the D. Two sets of macrophages were cultured for 5 h in the effects of RBP. To investigate this we prepared the purified presence of serum plus dibutyryl cyclic AMP, and then acticomplex of retinoic acid and RBP andshowed that dibutyryl nomycin D was added to one set and not to the other. The cyclic AMP also greatly potentiateditstransglutaminaseexperiment was continued for a further 17 h, and then the transglutaminase activity of the cells was assayed. As can be inducing activity. Dibutyryl cyclic AMP also potentiated the rose over the first action of retinoic acid reconstituted with delipidized serum seen inFig. 9, the transglutaminase activity 5 h of the incubation, but the addition of actinomycin D to and fresh serum. These findingssuggest that the primary the cells completely blocked further accumulation of the en- effect of dibutyryl cyclic AMP is toincrease the sensitivityof macrophages to retinoic acid itself independent of how the zyme (closed circle, Fig. 9). Thus, the enhanced expression of retinoid is presented to the cell. tissue transglutaminase that occurred in response t o dibutyryl How cyclic AMP and retinoids synergise one another in the cyclic AMP appears to depend on the capacity of the cells to control of gene expression isstillnot clear. Ithasbeen carry out denovo RNA synthesis. suggested that retinoidsmight prime cells to respond to cyclic AMP by inducing the cyclic AMP-dependent protein kinase DISCUSSION (28). Retinoic acid has been shown to increase the activityof We have used the induction of tissue transglutaminase in cyclic AMP-dependent protein kinase inseveral cultured cell myeloid cells as a probe to study the mechanisms involved in lines (41,42), although we have failed to detect similareffects retinoid action (13,29). One important aspect of these studies in macrophages.* To determine whether theprocess we were has been to identify the physiological factors thatregulate the studying involved a similar priming of the cells by retinoic response of cells to retinoids. Several studies have recently acid, macrophageswere preincubated inretinoic acidor serum reported that the abilityof retinoids toinduce differentiation and thensubsequently challenged with dibutyrylcyclic AMP. in a variety of normal and malignantcell types can begreatly The interpretationof these experiments issomewhat compliincreased by the addition of agents that elevate intracellular cated by the fact that itis hard to remove retinoids bound to cyclic AMP (15,28,30,31). We therefore investigated whether cells by simple washing. Therefore, there is a n unavoidable cyclic AMP also potentiated the action of retinoids at the carry-over of retinoids from the preincubation phase into the level of expression of a specific gene product, in this case the second exposure with dibutyryl cyclic AMP. Nonetheless, in expression of tissuetransglutaminaseincultured macro- spite of theselimitations,thepreincubationexperiments phages. Our results in this regard are very clear-cut. Using showed clearlythat serial exposure of the cells to retinoic acid several different means to elevate the levels of intracellular (or serum) and dibutytylcyclic AMP was much less effective cyclic AMP, itis apparent thatcyclic AMP greatly potentiates than simultaneous exposure of the cells to both agents. Simretinoid-induced expression of tissue transglutaminase. Diilarly, in the reciprocal experiments, the serial exposure of butyryl cyclic AMP on its own has a small but reproducible the cells first to dibutyryl cyclic AMP and then to retinoic effect on the basal levels of enzyme activity, a n effect that acid (or serum) was also not nearly as effective as the simulhas beenpreviously reported in Chinese hamsterovary fibro- taneous exposure to both agents. These results suggest that blasts (26) and human promyelocytic leukemia (HL-60) cells there must be a fairly direct interaction between the cyclic (29). Retinoids or fresh serum, on the other hand, can have a nucleotide and the retinoid in terms of the activationof gene very large effect on the inductionof tissue transglutaminase. expression. This could involve a synergistic potentiation of The enzyme levels can increase 100-fold in cells exposed to signal transduction to the nucleus or possiblecooperation optimal concentrations of either retinoic acid or fresh serum between the two agents at thelevel of gene expression itself. (12, 13). Dibutyryl cyclic AMP has very little effect on the Cyclic AMPhas beenshown topotentiatethehormonemaximal induction of the enzyme, but it greatly increases the regulatedexpression of severalgenes inmammalian cells sensitivity of the cells to low levels of retinoic acid or serum. including phosphoenolpyruvate carboxykinase and prolactin This may be biologically important because the levels of (43), and retinoid-regulated gene expression could well be retinoic acid in normal serum are quite low. DeRuyter et al. P. J. A. Davies, unpublished observation. (32) have reported that retinoicacid in normal human serum

Macrophage Retinoic Acid and CAMPInduceTransglutaminase

62 1

another example of dual regulatory mechanisms for the con- 11. Schroff, G., Neumann, C., and Sorg, C. (1981) Eur. J. Immunol. 11,637-642 trol of gene expression. 12. Murtaugh, M. P., Mehta, K., Johnson, J., Myers, M., Juliano, R. Our interest in the interaction of cyclic AMP and retinoids J., andDavies, P. J. A. (1983) J. Bid. Chem. 258,11074-11081 stemmed from reports from several laboratories that cyclic 13. Moore, W. T., Jr., Murtaugh, M. P., and Davies, P. J. A. (1984) J , Biol. Chem. 2 5 9 , 12794-12802 AMP potentiated the ability of retinoids to induce differentiation in several transformed cell lines (28,44). For instance, 14. Strickland, S., and Mahdavi, V. (1978) Cell 1 5 , 393-409 both the morphologic differentiation and the expression of 15. Rizzino, A., and Crowley, C. (1980) Proc. Natl. Acad. Sci. U. S. A. 7 7 , 457-461 specific proteins that follow the exposure of human promye- 16. Linder, S., Krondahl, U., Sennerstam, R., and Ringertz,N. R. locytic leukemia (HL-60) cells to retinoic acid are expressed (1981) Exp. Cell Res. 1 3 2 , 453-460 more rapidly or atlower concentrations of retinoic acid in the 17. Fuchs, E., and Green, H. (1981) Cell 25,616-625 presence of exogenous analogues of cyclic AMP (29). Simi- 18. Breitman, T. R., Selonick, S. E., and Collins, S. J. (1980) Proc. Natl. Acad. Sci. U. S. A . 77, 2936-2940 larly, in teratocarcinoma cells, the extentof retinoid-induced differentiation of the cells can be dramatically increased by 19. Olsson, I. L., and Breitman, T. R. (1982) Cancer Res. 4 2 , 39243927 agents that elevate intracellularlevels of cyclic AMP (14, 30, 20. Rothblat, G. H., Arborgast, L. Y., Ouellett, L., and Howard, B. 31,45). However, it is often hard to deduce at what level the V. (1967) I n Vitro (Rockuille) 12,554-557 interaction between cyclic AMP and retinoic acid occurs. It 21. Peterson, P. A. (1971) J. Biol. Chem. 2 4 6 , 34-43 is possible that both retinoic acid and cyclic AMP induce 22. Tucker, S. B., Pierre, R. V., and Jordon,R. E. (1978) J. Immunol. Methods 14, 267-269 specific sets of proteins and the interactions between these Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 sets of proteins result in a more differentiated cell. Alterna- 23. 24. Richert, N. D., Davies, P. J. A., Jay, G., and Pastan, I. H. (1979) tively, cyclic AMP and retinoic acid could cooperate at the J. Virol. 3 1, 695-706 level of the expression of specific genes, ix. the presence of 25. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 4 6 , 83-88 cyclic AMP and retinoic acid together favors the expression of particular genes critical for the development of differentia- 26. Milhaud, P. G., Davies, P. J . A,, Pastan, I., and Gottesman, M. (1980) Biochim. Biophys. Acta 630,476-484 tion. Our resultsfavor this latter model for we have been able 27. P. J., Orr, G. R., Patterson, M. K., Conway, E., to demonstrate that cyclic AMP can potentiate the expression Birckbichler, Carter, H. A., and Maxwell, M. D. (1983) Biochim. Biophys. of a specific retinoic acid-inducible geneproduct, tissue transActa 763, 27-34 glutaminase. Webelieve that tissue transglutaminasemay be 28. Olsson, I. L., Breitman, T . R., and Gallo, R. C. (1982) Cancer Res. 42,3928-3933 representative of a group of genes important for the function of differentiated cells that arecoordinately regulated by cyclic 29. Davies, P. J . A,, Murtaugh, M. P., Moore, W. T., Jr., Johnson, G. S., and Lucas, D. (1985) J. Biol. Chern. 260, 5166-5174 AMP and retinoic acid. Acknowledgments-We thank Mary Sobieski for her assistance in carrying out some of the experiments and Sandra Hobbs for typing the manuscript. REFERENCES

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