THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 9, Issue of March 25, pp. 5929-5933, 1991 Printed in U. S . A.

Granulocyte-Macrophage Colony-stimulating Factor Induces Transcriptional Activation of Egr-1 in Murine Peritoneal Macrophages*

(Received for publication, July 26,1990)

Jingwen Lius, Jill Lacy$, Vikas P. Sukhatmell, and David L. ColemanS(1 From the $Department of Internal Medicine, Yale University School of Medicine, Department of Veterans Affairs Medical Center, West Haven, Connecticut 06516, the §Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520; ana! the (Departments of Medicine and Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, Uniuersity of Chicago, Chicago, Illinois 60637

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematopoietic growth factor that induces both growth and differentiation of tissue macrophages. The subcellular mechanism of action of GM-CSF is unknown. We have examined the effect of GM-CSF on the immediate early response gene, Egr- I , in murine peritoneal macrophages. Our data dem- onstrate that recombinant GM-CSF (25 unitslml) pro- duces a 12-fold increase in Egr-I mRNA within 30 min. Pretreatment with cycloheximide (10 wg/ml) had no effect on the ability of GM-CSF to increase Egr-1 mRNA. In nuclear runoff studies, GM-CSF increased the transcription rate of Egr-I by 10-fold at 10 min. The maximal effect on Egr-1 transcription occurred at 26 min (13-fold) and decreased by 45 min. The half- life of Egr-1 mRNA in GM-CSF-treated macrophages is 13-21 min. We were unable to calculate the half-life in control cells, however, because of the short half-life and low level of constitutive expression of Egr-1 mRNA. Endogenous protein kinase C activity in mac- rophages was depleted by treatment with 12-0-tetra- decanoylphorbol-13-acetate for 24 h. GM-CSF in- creased Egr-1 mRNA in protein kinase C-depleted macrophages, whereas the stimulatory effect of 12-0- tetradecanoylphorbol-13-acetate on Egr-1 was blocked. These data show that GM-CSF rapidly in- creases transcription of Egr-I mRNA. The effect of GM-CSF on Egr-1 mRNA does not require de novo protein synthesis or protein kinase C. These findings provide a basis for investigating the molecular mech- anism of action of GM-CSF in tissue macrophages.

Granulocyte-macrophage CSF’ is a 23,000-Da glycoprotein that regulates the growth and differentiation of several cell lineages (1,2). In most responsive cell types, GM-CSF induces both activation and proliferation (reviewed in Ref 1). The subcellular mechanism of action of GM-CSF in macrophages

* This work was supported by grants from the Medical Research Service of the Department of Veterans Affairs. The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom reprint requests should be s e n t Medical Service 111/ I, Dept. of Veterans Affairs Medical Center, 950 Campbell Ave., West Haven, C T 06516.

’ The abbreviations used are: CSF, colony-stimulating factor; DMEM, Dulbecco’s modified Eagle’s medium; GM-CSF, granulocyte- macrophage colony-stimulating factor; TPA, 12-0-tetradecanoyl- phorbol-13-acetate; kb(s), kilohases.

is unknown. Recently, the human GM-CSF receptor has been cloned from human placenta (3). The predicted protein se- quence does not contain a tyrosine kinase domain, but does contain sequence homology with the receptors for prolactin, interleukin 6, interleukin 2 f l chain, and erythropoietin (3).

Recent studies have identified a subgroup of immediate early response genes that encode proteins with structural and/ or functional features common to transcriptional regulatory proteins (45). Thus, early response genes may link the rapid changes in second messengers to more sustained alterations in gene expression. Four major classes of immediate early transcription factor genes have been identified the jun family (c-jun, jun-B, and jun-D) (6-8), the fos family (c-fos, fra-1, and fos-B) (9-ll), a member of the steroid hormone receptor superfamily (12), and the early growth response family (Egr- I , Egr-2, Egr-3, and Egr-4) (5, 13).’ These genes are induced within minutes of cell stimulation and their transcription does not require protein synthesis. The importance of the immediate early response genes is derived from their probable role as nuclear mediators of signal transduction processes. Although GM-CSF has been shown to increase c-fos mRNA in a macrophage cell line, its effect on immediate early gene expression in tissue macrophages is unknown (14).

In the present study, we have examined the effect of GM- CSF on the expression of the immediate early response gene, Egr-1 (5) (also known as Zif 268 (15), Krox 24 (16), TIS 8 (17), NGFl-A (18), and d-2 (19)). Egr-1 cDNA was identified through a differential screening strategy to detect genes ex- pressed rapidly following addition of serum to murine fibro- blasts (5). Egr-I mRNA is 3.4 kb and is induced by both mitogenic and differentiation signals in diverse cell types (5, 15-19). The Egr-1 protein is an 80-kDa nuclear protein that binds in the presence of zinc to the DNA consensus sequence CGCCCCCGC (20,21). The DNA-binding properties are con- ferred by three zinc finger domains of the Cys2-His2 subclass in the carboxyl terminus (5, 22). Recently, the Egr-1 protein has been shown to activate transcription of a transient re- porter construct containing four repeats of the Egr-1 DNA- binding sequence upstream of a thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene (23).:’

Consensus sequences that comprise potential binding sites for transcriptional regulatory factors have been identified in the 5“flanking region of the Egr-1 gene (24, 25). In addition to six serum response elements which contain the core CC(A or T),GG sequence found in the fos and actin serum response elements (26,27), Egr-1 contains core Sp-1-binding sites (24),

’ S. Patwardhan and V. Sukhatme, unpublished observations. :’ A. Gasher and V. Sukhatme, unpublished observations.

5929

5930 Granulocyte-Macrophuge CSF Increases Egr-1

two AP-1 binding sites similar to that found in the TPA response element (17, 28), and two potential cyclic AMP response elements that contain 6 and 7 bases, respectively, out of the 8 found in the prototype cyclic AMP response elements (24, 28). Although the Egr-1 serum response ele- ments have been shown to mediate transcriptional activation of Egr-I, the role of the putative TPA and cyclic AMP response elements in mediating transcriptional activation of Egr-1 is unknown.

A recent report, published during the preparation of this manuscript, showed that GM-CSF increased Egr-1 (TIS 8 ) mRNA by 30 min in human neutrophils and in a myeloid cell line (29). The mechanism of the effect of GM-CSF on Egr-1 expression in these cells has not been defined. In view of the transcriptional regulatory properties of Egr-1 protein and the paucity of clues to the subcellular mechanism of action of GM-CSF in tissue macrophages, we have examined the effect of GM-CSF on Egr-I mRNA in murine peritoneal macro- phages.

EXPERIMENTAL PROCEDURES

Animals-Six- to eight-week-old BALB/c mice (male/female) were purchased from the NIH breeding colony and maintained in the Animal Care Facility of the Department of Veterans Affairs Medical Center, West Haven, CT.

Media and Reagents-Except where otherwise noted, all chemicals were purchased from Sigma. DMEM (GIBCO), supplemented with 1 mM glutamine, 50 pg/ml streptomycin, 100 units/ml penicillin, and 0.05 mM 2-mercaptoethanol, was used to support the cultures of peritoneal macrophages. Purified murine recombinant GM-CSF was purchased from BACHEM Bioscience Inc. (Philadelphia). The GM- CSF preparation had an activity of 7.0 X lo7 units/mg. One unit of activity was defined by the manufacturer as half-maximal stimulation of NSF-60 cells. The stock recombinant GM-CSF preparation was free of detectable endotoxin (< 50 pg/ml) in the limulus amebocyte lysate assay (30).

Macrophage Isolation and Culture-Murine thioglycolate-elicited peritoneal exudate cells were collected and purified by adherence as described previously (31). Peritoneal exudate cells (40-50 X lo6) were incubated a t 37 "C in 5% CO, for 3 h in 150-mm plastic tissue culture plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) to permit adherence. Nonadherent cells were removed (31), and the peritoneal macrophages were incubated overnight in DMEM (serum- free) for the RNA blot and nuclear runoff assays. The macrophage proliferation assays were conducted after 72 h of culture in DMEM with 2% fetal calf serum exactly as described previously (32).

Preparation of Probes-The full-length murine Egr-1 probe (3.1 kh) was purified from plasmid pUC 13 after digestion with EcoRI (5). The human y-actin cDNA probe (1.0-kb insert) was purified from a plasmid kindly provided by B. Forget (Yale University) after digestion by PstI and Xbal. Murine c-fos cDNA (3.7-kb insert) was purified from a plasmid vector (provided by J. Li, Yale University) after digestion with BamHI. The plasmids containing the human HLA class I (B locus) gene (kindly provided by S. Weissman, Yale Univer- sity) and murine cyclophilin gene (kindly provided by R. Handschu- macher, Yale University) were used in the nuclear runoff. assay without purification of the probes. For RNA blot analysis, the purified inserts were radiolabeled to a specific activity of lo9 cpm/pg by random primer extension (33) and purified by spin column.

RNA Isolation and RNA Blot Analysis-Total RNA was isolated by ultracentrifugation through a cesium chloride gradient as described (34). Total RNA (25-40 pg/lane) was loaded and separated by elec- trophoresis through a 6.7% formaldehyde, 1% agarose gel. Gels were stained with ethidium bromide (0.1 pg/ml gel solution) to confirm that equivalent amounts of RNA had been applied in each lane. The

50% formamide, 5 X SSC, 50 mM NaH,PO, (pH 6.5), 1 X Denhardt's filters were prehybridized for 2-4 h a t 42 "C in a solution containing

solution, 100 pg/ml denatured salmon sperm DNA. Hybridization and washing of the filters were conducted as previously described (35). The filters were exposed to Kodak XAR film with an intensifying screen at -70 "C for 1-3 days and later reprobed with y-actin or HLA class 1 to corroborate that equal amounts of RNA were present in each lane.

Nuclear Runoff Transcription Assay-Runoff transcription assays

were performed by using a modification of a method described pre- viously (35). After overnight incubation in DMEM with 0.5% fetal calf serum, the macrophages were incubated with fresh, warm DMEM at 37 "C in 5% CO, for 2 h, and then 50 units/ml of GM-CSF or diluent were added for varying periods of time (5-45 min). After incubation, the plates containing macrophages were placed on ice, washed three times with phosphate-buffered saline and overlaid with lysis solution (35). The monolayers were gently scraped with a rubber policeman and the cells were lysed by trituration in lysis buffer. Nuclei were washed once in lysis buffer and stored at -70 "C in aliquots of 225 pl as described (35). More than 90% of the nuclei were

60 pl of reaction buffer (35) plus 250 pCi of [a-32P]UTP and incubated intact by microscopic examination. Nuclei (225 pl) were mixed with

for 30 min at 30 "C. After incubation, nuclei were pelleted for 20-30 s in a microfuge, resuspended in 100 pl of 10 mM Tris (pH 8), 10 mM NaCl, 6 mM MgClz with 200 units of RNAse-free DNase I (Boehringer Mannheim), and incubated for 30 min a t 37 "C. After addition of proteinase K (1 mg/ml, Boehringer Mannheim), nuclei were vortexed thoroughly and incubated for 60 min a t 37 "C. The reaction was extracted twice with phenol/chloroform, and the radiolabeled RNA was precipitated with isopropyl alcohol and 2.3 M ammonium acetate

diolabeled RNA (lo7 cpm) from each sample (35) was hybridized to to remove unincorporated nucleotides. An equivalent amount of ra-

denatured DNA probes (1 pg each) and immobilized on nitrocellulose filter paper in 2 ml of hybridization buffer (35) for 60 h at 60 "C. The filters were washed (35), dried, and exposed to Kodak XAR film for 1-3 days. Quantitation of the amount of RNA hybridized to each probe was determined by densitometry using a Microscan 1000 Den- sitometer (Technology Resources Inc., Nashville, TN).

Determination of mRNA Half-life-To determine the half-life of Egr-I mRNA, macrophages were treated with GM-CSF (25 units/ ml) or control buffer for 30 min, and actinomycin D (10 Fg/ml) was added. Total RNA was isolated and analyzed by RNA blot for Egr-I mRNA level. The concentration of Egr-I was determined from den- sitometric analysis of each band and expressed in relative units. The half-life of mRNA (tLh) as determined as previously described (35,36).

RESULTS

GM-CSF Induces Egr-1 mRNA in Murine Peritoneal Mac- rophages-To determine if GM-CSF induces Egr-1 mRNA, macrophages were treated with GM-CSF (25 units/ml) for 15-120 min and total cellular RNA was analyzed for Egr-1 mRNA at various time points by RNA blot using 32P-labeled murine Egr-1 cDNA. GM-CSF increased Egr-1 mRNA 12- 15-fold over baseline levels ( n = 11). As shown in a repre- sentative experiment in Fig. 1A, Egr-1 mRNA rapidly in- creases following stimulation with GM-CSF and reaches max- imal levels a t 30 min. In separate experiments (not shown), the level of Egr-1 mRNA was lower at 15 min than at 30 min after GM-CSF stimulation. The level of Egr-1 mRNA re- turned to baseline by 2 h. In some experiments, a very low level of Egr-1 mRNA was detectable in untreated cells. The amount of Egr-1 mRNA was consistently 12-15-fold higher following stimulation with GM-CSF, irrespective of the base- line level.

To determine whether induction of Egr-1 mRNA by GM- CSF requires de novo protein synthesis, macrophages were pretreated with cycloheximide (10 pg/ml) for 60 min prior to GM-CSF (25 units/ml). As shown in a representative exper- iment in Fig. 1B ( n = 2), GM-CSF induces Egr-l mRNA in the presence of cycloheximide. Thus, protein synthesis is not required for GM-CSF to increase Egr-1 mRNA in macro- phages. As described for other cell types, the basal level of Egr-I mRNA in macrophages is increased by cycloheximide

We sought to determine if the effect of GM-CSF on Egr-1 mRNA occurred in a concentration range similar to that a t which proliferation is induced. Macrophages were treated with varying doses of GM-CSF (1.25-50 units/ml) and assayed for Egr-1 mRNA by RNA blot after 30 min or for ['Hlthymidine incorporation after 72 h. As shown in Fig. 2a, Egr-1 mRNA

( 5 , 15-18).

Granulocyte-Macrophage CSF Increases Egr-1 5931

FIG. 1. Effect of GM-CSF on Egr-I mRNA. Recombinant GM-CSF (25 units/ml) was added to peritoneal macrophages and total RNA (30 pg/lane) was harvested at the time points indicated (panel A ) . RNA blot analysis using :'2P-labeled Egr-1 cDNA was conducted as described under "Experimental Procedures." The dried blot was exposed to Kodak XAR film in a double-sided intensifying screen for 1 day a t -70 "C. Equivalent amounts of RNA were present in each lane as determined by ethidium bromide staining and by probing for y-actin mRNA. Results similar t o those shown above were obtained in each of 11 separate experiments. To determine whether protein synthesis is required for GM-CSF to increase Egr-I mRNA, peritoneal macrophages were preincubated with cyclohexi- mide (IO pg/ml) for 60 min and then treated with recombinant GM- CSF (25 units/ml) or control buffer for 30 min (panel B ) . Egr-1 mRNA was measured by RNA blot analysis. Similar results were obtained in each of two separate experiments.

a 1 2 3 4 5

b

-1

0 I 2 5 1 7 5 2 5 5 0

GM-CSF unitslml

FIG. 2. Correlation between induction of Egr-I mRNA and proliferation by GM-CSF. Recombinant GM-CSF was incubated for 30 min a t various doses with peritoneal macrophages, and Egr-1 mRNA was measured by northern blot (panel A ) . The effect of GM- CSF at 0, 1.25, 12.5, 25, and 50 units/ml is shown in lanes 1-5, respectively. Hybridization with Egr-I mRNA is denoted by the arrow. In a parallel experiment (panel R ) , GM-CSF was added to macrophages and proliferation (["Hlthymidine incorporation) was measured after 72 h. The values shown represent the mean +- S.E. of triplicate determinations. Results similar to those shown were ob- tained in each of three separate experiments.

is induced in a dose-dependent manner beginning a t 12.5 units/ml of GM-CSF. The lowest concentration a t which proliferation is significantly induced by GM-CSF is also 12.5 units/ml (Fig. 2b). Therefore, the effects of GM-CSF on Egr- 1 and proliferation occur a t a similar concentration.

GM-CSF Induces Transcriptional Activation of Egr-1 mRNA-To determine if the increased level of Egr-1 mRNA in GM-CSF-stimulated macrophages is due to an increase in the rate of transcription, nuclear runoff analysis was con- ducted. Nuclei from control and GM-CSF (50 units/ml)- treated macrophages were harvested at various time points,

and the relative amount of nascent mRNA was quantitated by nuclear runoff transcription as described above. Since c- fos has been shown to be often co-regulated with Egr-1 in other cells (5), we also analyzed the transcription rate of c-fos in GM-CSF-treated macrophages in parallel. An equal amount of trichloroacetic acid-precipitable '"P-labeled mRNA from each sample was used in the hybridization. The levels of three control genes (murine cyclophilin, human y-actin, human HLA class I) were measured to further confirm that equal amounts of labeled mRNA were used a t each time point. As shown in a representative experiment in Fig. 3A ( n = 3), GM-CSF induces a 10-fold increase in the rate of transcrip- tion of Egr-1 by 10 min. The effect of GM-CSF is maximal at 25 min (13-fold). By 45 min, the effect of GM-CSF in Egr- 1 transcription is reduced to a level near that in unstimulated cells. The effect of GM-CSF on the transcription rate of Egr- 1 mRNA was similar when the Egr-1 signal was corrected to that of the control genes (e.g. y-actin, Fig. 3B). The increase in the relative transcription rate of Egr-1 induced by GM- CSF is similar to the magnitude of its effect on Egr-1 mRNA levels in the RNA blot experiments.

Egr-1 mRNA Is Rapidly Degraded in GM-CSF-treated Mac- rophages-To further characterize the mechanism whereby GM-CSF increases Egr-1 mRNA, the effect of GM-CSF on the stability of Egr-1 mRNA was measured. The rate of decay of Egr-1 mRNA in control and GM-CSF-treated macrophages was determined after inhibiting transcription with actino- mycin D (10 pg/ml). The half-life of Egr-1 mRNA in GM- CSF-stimulated macrophages was 13.3 and 21.2 min in two separate experiments (data not shown). Despite using poly(A+)-selected mRNA (37), the amount of Egr-1 mRNA in control cells was insufficient to conduct reliable densito- metric analysis for the purpose of determining the half-life in unstimulated cells. Moreover, the very short half-life of Egr- 1 mRNA makes it technically difficult to detect stabilization of Egr-1 mRNA by GM-CSF.

Egr-1 Induction by GM-CSF Is through a Protein Kinase

A GM - CSF

Control 5 7 T T Z ' Epr-1

c-bas HLA

Cyclophilin - - y-Adin L I* 1, - P -CAT

A B C D E

.t .

B

FIG. 3. Effect of GM-CSF on the transcription rate of Egr- 1. A, at the various time points indicated after addition of GM-CSF (50 units/ml), nuclei were harvested and nuclear runoff transcription was conducted as described under "Experimental Procedures." Equal amounts of labeled RNA from each time point were hybridized with 1 pg of each of the indicated cDNA probes immobilized on nitrocel- lulose. P-CAT is a negative control plasmid comprised of the chlor- amphenicol acetyltransferase gene. Similar results were obtained in each of three separate experiments. R, the autoradiograph shown in panel A was analyzed by computerized densitometry. The relative transcription rate was calculated as described under "Experimental Procedures."

5932 Granulocyte-Macrophage CSF Increases Egr-l

C-independent Pathway-Studies in other cells have shown that TPA induces Egr-1 mRNA levels (17,19,29). The effect of TPA may occur through TPA response elements identified in the 5’-flanking sequence of Egr-1 (17,24). Moreover, Egr- 1 induction has been shown to be both protein kinase C- dependent and -independent (17,38). In view of the potential role of protein kinase C in inducing Egr-1, we undertook a series of experiments to determine whether the effect of GM- CSF on Egr-1 is mediated through protein kinase C. As described in other cell types, TPA induced Egr-1 mRNA in macrophages in a dose-dependent manner. The maximal ef- fect of TPA was observed at 40 rig/ml (data not shown). We then asked if GM-CSF could increase Egr-1 mRNA in the presence of maximal stimulation with TPA. As shown in Fig. 44, GM-CSF (50 units/ml) plus TPA (100 rig/ml) increased Egr-1 mRNA above that observed for either agent alone. These data suggest that GM-CSF does not utilize a protein kinase C-dependent pathway.

To further examine the role of protein kinase C in the induction of Egr-1 mRNA by GM-CSF, macrophages were treated with TPA (100 rig/ml) for 24 h to down-regulate endogenous protein kinase C (39). The down-regulation of protein kinase C under these experimental conditions was confirmed by the finding that TPA-induced translocation of protein kinase C activity from the cytosol to the membrane (40) was blocked by chronic TPA-treatment for 24 h. Mac- rophages were treated with TPA (100 rig/ml) or diluent buffer for 24 h and then with GM-CSF (25 units/ml) or TPA (100 rig/ml) for 30 or 60 min prior to harvesting total RNA. As shown in Fig. 4B (lanes 1-4 and 7), both TPA and GM-CSF increased Egr-1 mRNA in macrophages pretreated with buffer alone. In macrophages pretreated with TPA, however, the effects of GM-CSF and TPA diverged. As expected, down- regulation of protein kinase C by chronic TPA pretreatment eliminated the effect of TPA on Egr-1 expression (Fig. 4B,

A

B 123456789

28s

FIG. 4. A, effect of TPA and GM-CSF on Egr-I mRNA. Peritoneal macrophages were treated with diluent alone (lane I), TPA (100 ng/ ml, lane 21, TPA plus GM-CSF (lane 3), or GM-CSF (25 units/ml, lane 4) for 30 min prior to measuring Egr-1 mRNA by RNA blot analysis. The autoradiograph density of Egr-1 mRNA for each con- dition was measured using a computerized densitometer. Control (1,356 density units), TPA (57,807 density units), TPA plus GM-CSF (115.295 densitv units). GM-CSF (71.064 densitv units). B, effect of down-regulation of protein kinase 6 on the induction of kgr:l mRNA by GM-CSF. Induction of Egr-2 mRNA by GM-CSF (25 units/ml) or by TPA (100 rig/ml) was compared in peritoneal macrophages pretreated with buffer alone or TPA (100 rig/ml) for 24 h to down- regulate protein kinase C. Total RNA was harvested and Northern blot analysis of &r-l mRNA was conducted. Lane 1, control; lane 2, TPA for 24 h; lanes 3 and 4, TPA for 30 or 60 min following pretreatment with buffer alone for 24 h; lanes 5 and 6, TPA for 30 or 60 min followina nretreatment with TPA for 24 h: lane 7. GM-CSF for 60 min follo%ng pretreatment with buffer alone; lank 8 and 9, GM-CSF for 30 or 60 min following pretreatment with TPA for 24 h. Equivalent amounts of RNA were present in each lane as determined by reprobing the RNA blots for y-actin. Similar results to those shown above were obtained in each of three separate experiments.

hnes 5 and 6). In contrast, however, the enhancement of Egr- 1 mRNA by GM-CSF was preserved in protein kinase C down-regulated macrophages (Fig. 4B, lanes 8 and 9). In concert with the lack of effect of GM-CSF on protein kinase C observed previously (40), these data suggest that GM-CSF induces Egr-1 mRNA through a pathway independent of protein kinase C.

DISCUSSION

These data show that GM-CSF induces a marked increase in the transcription rate of Egr-1 within 10 min in murine macrophages. The increased transcription is followed by a 12-E-fold increase in Egr-1 mRNA within 30 min. The effect on Egr-1 mRNA is dose-dependent and does not require de nova protein synthesis. These data provide the first evidence that Egr-1 is regulated at the transcriptional level in primary cells. Moreover, these data demonstrate that hematopoietic growth factors such as GM-CSF increase transcription of Egr-1. The rapid and dramatic increase in Egr-1 mRNA are consistent with the possibility that the Egr-1 gene product may be a proximal component of the intracellular signals triggered by GM-CSF.

The kinetics of GM-CSF’s effect on Egr-1 mRNA in mac- rophages is very similar to that observed in neutrophils and 32D cells (29). A similar pattern of induction has also been observed in 3T3 cells following stimulation with serum or platelet-derived growth factor (16). The reason for the fluc- tuation in the basal level of Egr-1 mRNA in macrophages is unclear. Data from other cell systems have also revealed a fluctuation in the basal level of Egr-1 mRNA and protein (5, 20, 23,41).

The effect of GM-CSF on Egr-1 mRNA in macrophages was observed under serum-free conditions. The cells were cultured in the absence of serum to reduce the possibility of carry-over of a serum factor that might be required for GM- CSF to exert an effect. Moreover, the recombinant GM-CSF preparation did not contain detectable endotoxin that may confound the results. Therefore, GM-CSF alone is sufficient to increase Egr-1 mRNA.

The concentration of GM-CSF required to increase Egr-1 mRNA is similar to that required to increase 13H]thymidine uptake in the present study (Fig. 2b) and both oxidative metabolism and Fc-dependent phagocytosis in a previous study (31). These data are consistent with a possible role for Egr-1 in mediating the intracellular events that lead to alter- ations in macrophage growth and/or function. The diversity of signals that induce Egr-1 in both proliferative and post- mitotic cell types further supports its potential involvement in a diverse array of cell functions (5, 15-18, 37,41).

The lack of a requirement for protein synthesis for GM- CSF to increase Egr-1 is common to the entire group of immediate early response genes (4,5,15-18). The increase in Egr-I (Zif 268) mRNA induced by cycloheximide has been previously shown in fibioblasts to be due to both increased transcription and stabilization of mRNA (42).

The kinetic pattern and magnitude of increased transcrip- tion of Egr-1 are sufficient to account for the effect of GM- CSF on Egr-1 mRNA. However, we cannot exclude an effect of GM-CSF on Egr-1 at the post-transcriptional level. The short half-life observed in the present report is similar to that reported for Egr-1 (Zif268) in 3T3 cells (42). The short half- life of Egr-1 mRNA may be conferred by AU-rich sequences in the 3’-untranslated end of Egr-1 (5, 25). Similar AU-rich sequences have been identified in a number of short-lived mRNAs (reviewed in Ref. 44).

The c&-acting regulatory elements that mediate the effect

Granulocyte-Macrophage CSF Increases Egr-1 5933

of GM-CSF on Egr-1 transcription are uncertain. Of the putative transcriptional regulatory sites, only the serum re- sponse elements in the 5' region have been shown to be functionally active (45). Since blockade of the protein kinase C cascade with chronic TPA treatment did not inhibit the effect of GM-CSF, it is unlikely that the TPA response elements identified at -610 and -867 mediate the effect of GM-CSF on Egr-1.

Egr-1 has been shown to be inducible by cAMP in some cell types (18, 24). We have previously demonstrated that GM-CSF increases adenylate cyclase activity and intracellu- lar cAMP within 5 min in peritoneal macrophages (40). In subsequent experiments, however, we have been unable to detect an effect of GM-CSF on cyclic AMP-dependent protein kinase? Morever, Egr-1 mRNA is not inducible in macro- phages with diffusible cAMP analogue^.^ Therefore, it seems unlikely that the effect of GM-CSF on Egr-I in macrophages is mediated through the putative cyclic AMP response ele- ments. Experiments are presently underway to more rigor- ously characterize the cis-acting elements that mediate the effect of GM-CSF on Egr-1 transcription.

The common intracellular pathway triggered in macro- phages by activation signals such as GM-CSF is uncertain (46). The prototypic macrophage-activating factor, y-inter- feron, has been previously shown to activate protein kinase C in peritoneal macrophages (47). GM-CSF has no effect, how- ever, on protein kinase C in neutrophils (48) or macrophages (40). Together with our data showing protein kinase C-inde- pendent induction of Egr-1 by GM-CSF, it is unlikely that protein kinase C is involved in the early genetic response to GM-CSF. Therefore, macrophage activation may be triggered by multiple intracellular pathways.

The rapid transcriptional activation of Egr-1 by GM-CSF in murine macrophages provides an important model to char- acterize the intracellular signals that lead to macrophage activation and growth. Characterization of the trans-acting factors that mediate the effect of GM-CSF on Egr-1 will provide clues to the proximal components of the signaling cascade initiated by GM-CSF. Furthermore, characterization of the genes whose transcription is regulated by Egr-1 protein will provide insight into the sequence of genetic changes that lead to alterations in macrophage phenotype.

Acknowledgments-We wish to express our gratitude to Ann Bar- tiss for expert technical assistance, to Georgi Danley and Jennie Annatone for preparation of the manuscript, and to Anneli Rupprecht and Jeff Chodakewitz for their careful review of the manuscript.

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