Translational Control during the Acute Phase Response

Vol. 265, No. 24, Issue of August 25, pp. 1457%14578,199O Printed in U. S. A.

Translational FERRITIN

SYNTHESIS

Control during the Acute Phase Response IN RESPONSE

TO INTERLEUKIN-l* (Received

Jack T. Rogers$#ll, Philip E. AuronQ**,

Kenneth R. Bridges$, Gerard and Hamish N. Munro$

P. DurmowiczS,

Jonathan

for publication,

January

19, 1990)

Glass$II ,

From the $Divi.sion of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, SDepartment of Applied Biological Sciences and Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and the United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts, Boston, Massachusetts 02111

Interleukin1 (IL- lj3) increases the synthesis of both heavy and light (L)-ferritin subunits when added to human hepatoma cells (HepG2) grown in culture. RNase protection and Northern blot analysis with Lferritin probes revealed that no changes in L-ferritin mRNA levels occur after cytokine stimulation. However, the induction coincides with an increased association of the L-subunit mRNA with polyribosomes. Since the recruitment of stored ferritin mRNA onto polyribosomes is seen when iron enters the cell, the effect of IL- 16 on iron uptake was tested and was found to be unaffected by the lymphokine. Neither transferrin receptor mRNA levels nor the number of receptors displayed on the cell surface was affected by IL-l& However, the action of the cytokine on ferritin translation is inhibited by the action of the intracellular iron chelator deferoxamine. These data indicate that IL- 18 induces ferritin gene expression by translational control of its mRNA. The pathway of induction is different from iron-dependent ferritin gene expression whereas regulation requires the background presence of cellular iron.

Following infection, inflammation, or injury, an acute phase response (APR)’ occurs involving the synthesis and release from the liver of a series of proteins (acute phase reactants) such as al-acid glycoprotein, serum amyloid A, al-antitrypsin (alAT), and complement factor B (Baumann et al., 1987; Dente et al., 1985; Frisch and Ruley, 1987; Gehring et al., 1987; Geiger et al., 1987; Morrone et al., 1988; Perlmutter et al., 1986; Sipe et al., 1985). In contrast, the output of other liver-derived proteins such as albumin and transferrin diminishes (Ramadori et al., 1985; reviewed in Dinarello, 1988). The * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11To whom correspondence should be sent: Div. of Hematology, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5847. 11Present address: Hematology-Oncology, LSU Medical Center, Shreveport, LA 71130-3130. ** Present address: Lovett Research Group, Massachusetts General Hospital, Boston, MA 02114. ’ The abbreviations used are: APR, acute phase response; CQAT, oc,-antitrypsin; IL-l& interleukin-l@; H, heavy; L, light; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; Tf, transferrin; TfR, transferrin receptor; SDS, sodium dodecyl sulfate; RNP, ribonucleoprotein; MOPS, 4-morpholinepropanesulfonic acid; IRE, iron regulatory element.

the

altered hepatic transcription of these genes represents an adaptive response to minimize damage during the APR. Activated macrophages invade damaged tissues and release a number of factors into the bloodstream including IL-l& This 17.4-kDa lymphokine reproduces most acute phase changes when administered to rats (Auron et al., 1984; Ramadori et al., 1985). Some of these in uiuo responses are also reproduced by the administration of recombinant IL-l@ to hepatoma cells grown in vitro (Karin et al., 1985). However, purified cytokines do not induce the production and release of all the acute phase proteins from human hepatoma cells (Morrone et al., 1988). As an example, a1AT output is unchanged in hepatoma cells stimulated by IL-l@. Ferritin is a ubiquitous iron storage protein, the shell of which consists of a mixture of 24 heavy (H, M, 21,000) and light (L, M, 19,000) subunits (Theil, 1987). We studied the capacity of IL-l/3 to stimulate ferritin production by human hepatoma cells (HepG2) because plasma iron levels characteristically fall during the APR (Beissel, 1977). This reduction may result from an increase in liver ferritin synthesis as demonstrated in a rat model (Konijn and Hershko, 1977). Iron does increase the transcription of the L-subunit mRNA 2-3-fold in rat liver and in bullfrog red blood cells (White and Munro, 1987; Dickey et al., 1987). However, most of the ferritin induction seen in cells to which iron is administered occurs at the level of translation of both the H- and L-subunit mRNAs (Aziz and Munro, 1986; Rogers and Munro, 1987; Schull and Theil, 1982; Walden and Thach, 1986; Rouault et al., 1988). Since there is evidence that increased rat liver ferritin synthesis is also controlled at the level of translation during the APR (Konijn et al., 1981; Campbell et al., 1989), we chose HepGP cells to determine how human ferritin gene expression is regulated by the cytokine IL-lp. We find that IL-lp induces ferritin synthesis in HepG2 cells and that the translational efficiency of the L-subunit mRNA increases in the absence of changes in the steady-state levels of its mRNA. This occurs independently of any changes in iron uptake into the cell. An increase in the ferritin content of hepatocytes would increase the iron storage capacity of the liver, and the increase in iron retention within the organ may afford a protective response during the APR (see “Discussion”; Beissel, 1977; Konijn and Hershko, 1977). EXPERIMENTAL

PROCEDURES

Cell Culture-HepG2 cells were grown in Dulbecco’s modified Eagle’s medium (DEEM) supplemented with 10% fetal calf serum (FCS). L-elutamine, penicillin. and streptomycin (GIBCO). Cells were incubated with either (i) 0.2130 ng/& reiombinant human IL-l@ (Boehringer Mannheim); (ii) iron in the medium as diferric transferrin (0.4-5 pM FezTE Boehringer Mannheim); or (iii) with 400 pM

14572

Interleukin-1B

Induces Ferritin

deferoxamine, an iron-specific chelator (Ciba-Geigy). For studies involving labeled iron uptake, HepG2 cells were incubated with 0.4 /IM “gFe2Tf in DMEM containing only 1% FCS. Ferritirz Svnthesis-Cells were incubated first with IL-l/J, iron, or deferoxamine in DMEM supplemented with 10% or 1% FCS and then in methionine-free medium (RPM1 1640, GIBCO) supplemented with 25 clCi/ml lR5Slmethionine for the final 30 min. Equal numbers of cells (1 x 10’ cells/plate) were washed twice in cold phosphatebuffered saline at 4 “C and solubilized in a buffer containing 1% Triton X-100, 0.5% Nonidet P-40, 0.15 M NaCl, 10 mM Tris.HCl, pH 8, 5 mM EDTA (with 2 FM phenylmethylsulfonyl fluoride to prevent proteolysis). Total protein synthesis was measured by the amount of [““Slmethionine incorporated into trichloroacetic acidprecipitable material. Labeled ferritin and LU,AT were immunoprecipitated from HepG2 lysates as described previously (Rogers and Munro, 1987). The immunoprecipitated proteins were applied to 15% polyacrylamide gels containing 6 M urea, 0.1% SDS, 0.1 M sodium nhosnhate (DH 7.2) or onto 15% Laemmli SDS gels (Laemmli, 1970). The-two f&itin subunits (M, 19,000 and 21;OOO) and alAT (iVfr 55,000) were visualized by autofluorography of the gels after an overnight electrophoresis and impregnation with a fluorographic enhancer (Amplify, Amersham Corp.). Exposures were for l-2 days using Kodak XAR-5 film. Densitometric scanning of the autofluorographs was performed with a Bio-Rad model 620 videodensitometer. Hepatoma RNA Purification and Polyribosome Fractionation-Total cellular RNA was extracted using standard procedures (Chirgwin et al., 1979). After cell lysis for 4 h in guanidinium thiocyanate, the lysate was precipitated with 0.75 volume of ethanol and centrifuged at 10,000 rpm (Sorvall SA 600) for 10 min. The pellet was resuspended in l-2 ml of 7.5 M guanidium HCI and reprecipitated with 0.5 volume of ethanol. This selective precipitation of RNA from DNA was repeated twice, and the final pellet was phenol extracted and ethanol precipitated. For polyribosome gradients of liver lysates, approximately 5 X 106lo7 cells were harvested into 1 ml of polyribosome lysis buffer (0.15 M NaCl, 10 mM Tris.HCl (pH 7.2), 0.5% Nonidet P-40,10 mM MgC12, 50 pg of cycloheximide. ml-‘, 20 mM dithiothreitol, 100 units/ml RNasin). The postmitochondrial supernatant was layered onto 32 ml of a lo-50% sucrose gradient in a Beckman model SW 27 ultracentrifuge tube (25 X 89 mm) and spun for 4 h at 90,000 X g. The

gradients were separated into 12 fractions using an Isco fractionator (Lincoln, NE) with an absorbance monitor to trace the polyribosome profile of the gradients. Lysates were fractionated from the top to the bottom of sucrose gradients as postribosomal fractions (ribonucleoprotein, RNP), 40 and 60 S ribosomal subunits (monosomes, 80 S),

and polyribosomes (Ask and Munro, 1986; Rogers and Munro, 1987). RNA from each fraction was pooled, phenol extracted, and ethanol precipitated. RNA Blotting-Total RNA from the gradient (20 rg) was denatured in 50% formamide, 2.2 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 0.5 mM EDTA (pH 7.4) at 60 “C for 10 min and then fractionated by electrophoresis on 1.5% agarose-formaldehyde gels (Rave et al., 1979). RNA was transferred from gel to nylon bond membranes (Amersham) using standard procedures (Thomas, 1980). The RNA was filter immobilized by a 3-min exposure of the filters to ultraviolet light irradiation. Hybridization-Prehybridization of the filters was carried out for 3 h in a solution consisting of 50% formamide, 50 rg of denatured salmon sperm DNA per ml, 5 X SSC, 0.1% sodium dodecyl sulfate, and 5 x Denhardt’s solution. Overnight hybridization of the filters was carried out in 20 ml of the same buffer at 42 “C with the addition of 50 ng of randomly primed ‘*P-labeled probes. The filters were washed twice for a total of 1 h in 2 x SSC, 0.2% sodium dodecyl sulfate at room temperature followed by two washes for a total of 1 h in 0.5 X SSC, 0.1% sodium dodecyl sulfate at 55 “C. RNase Protection Analysis-Quantitation of L-ferritin and transferrin receptor (TfR) mRNA levels was performed as described elsewhere (Carrazana et al., 1988). A 630-b&e pair PstI fragment from an L-ferritin cDNA clone (Dorner et al.. 1985) and a 700-base vair PstI fragment from a transferrin receptor cDNA clone (McClelland et al., 1984) were subcloned into the PstI site of pBlueScript (Stratagene) as outlined in the maps in Figs. 2B and 6. A 480-base 32P.labeled L-ferritin cRNA probe (1 x lo6 cpm) and a 470.base TfR cRNA probe (1 X lo6 cpm) (Fig. 6) were transcribed with T7 and T3 polymerases, respectively, and each was hybridized to 20 pg of HepG2 RNA at 45 “C for 16 h followed by digestion with RNase A (40 Kg/ ml; Boehringer Mannheim) and RNase T, (2 fig/ml; Boehringer Mannheim). Protected cRNAs were separated by electrophoresis on

Synthesis in HepG2 Cells a 6% polyacrylamide gel containing 7 M urea. Hid -digested pBR322 DNA was kinase labeled and served for use as molecular weight markers to confirm the lengths of protected fragments. Iron Uptake-Labeling of transferrin with 59Fe has been described previously (Klausner et al., 1983). Triplicate wells each containing lo6 cells were incubated in DMEM, 1% FCS and 0.4 FM 59Fe2Tf for time intervals varying from 30 min to 6 h. One set of cells was incubated with 5 rig/ml IL-lo; a second set was pretreated with 1.25 ILM FelTf for 12 h: and a third set was left as control. Cells were washed in cold phosphate-buffered saline at the end of each incubation and solubilized with 1 ml of 0.5% SDS, 20 mM nitriloacetate (pH 6.4) (Davis and Czech 1986). The extracts were counted using a Beckman Compugamma counter to compare the rate of iron uptake by treated and untreated cells. Additional aliquots were assayed for protein content to ensure standardization of iron uptake rates between plates of equal numbers of cells. RESULTS

IL-ID Induces the Synthesis of Both Ferritin SubunitsDuplicate sets of HepG2 cells treated with 0.2 rig/ml IL-10 for 2 h, 6 h, and 14 h were labeled for 30 min to evaluate the synthesis of ferritin subunits in these cells compared with controls. Scanning of the autofluorogram shown in Fig. 1A showed that ferritin synthesis increased s-fold in cells exposed

for 2 and 6 h compared with an equal number (5 X 106) of control cells. Total protein synthesis increased about 25% after 6 h of IL-l@ stimulation as measured by trichloroacetic acid-precipitatable [35S]methionine within the lysates, suggesting that protein synthesis was in general only slightly affected in HepG2 cells within 6 h of IL-l@ exposure. Scan-

to IL-l/3

ning also revealed

a 20-fold

increase

in ferritin

subunit

syn-

thesis in HepG2 cells responding to overnight exposure to ILl/3. These values were determined by combining the readings from the data displayed in Fig. IA with those from a duplicate set of labelings and immunoprecipitations. In a separate experiment, duplicate flasks of HepG2 cells were exposed to iron in the form of 1.25 pM Fe2Tf for 2.5 h. Fig. 1B shows a IO-fold increase in ferritin synthesis in ironexposed cells relative to control. No ferritin was immunoprecipitated from lysates of iron-treated HepG2 cells incubated with preimmune rabbit serum (Fig. lB), reflecting the specificity of the antiferritin antibody immunoprecipitations. L-ferritin mRNA Levels Are Unchanged by IL-10 Stimulation of HepGZ Cells-Total RNA was extracted from control HepGP cells, from cells treated for 2, 6, and 14 h with 0.2 ng/ ml IL-lp, and from cells treated for 2 h with 2.5 PM human FelTf as an iron source. Equal aliquots of these mRNA samples (20 pg/slot) were used in a Northern blot analysis of L-ferritin mRNA by hybridization with a human L-ferritin cDNA insert probe (Fig. 2) (Dorner et al., 1985). Subsequently the filter was rehybridized with a mouse @-actin cDNA probe, and the data in Fig. 2A represent different exposures of the same filter hybridized with each probe separately. Scanning of these autofluorographs showed that the ratio of L-ferritin mRNA to actin mRNA levels within HepGP was not increased after stimulation with either iron or IL-l@ RNase protection of a predicted 380-base L-ferritin cRNA fragment by the same RNA preparations is shown in Fig. 2B. Experiments were performed with a calculated loo-fold excess of cRNA probe to the estimated levels of ferritin mRNA in a total RNA preparation. These data confirm that there is no increase in L-ferritin mRNA levels in HepG2 cells stimulated with IL1P. Effect of IL-ID and Iron on the Polyribosome Distribution of Ferritin mRNAs-Iron increases polyribosomal association of stored cytoplasmic H- and L-ferritin mRNAs (Aziz and Munro, 1986; Rogers and Munro, 1987). In order to investigate possible changes by IL-lfi of the polyribosome distribution of ferritin mRNAs, equal numbers of HepG2 cells were exposed

14574

Interleukin-l@

Induces Ferritin

Synthesis in HepG2 Cells IL-0

11-lB

Con 2h

6h

kc 5-i-A 14h -28s ACTIN

-18s

-r: r) a

L-FE??

IL-tp U

4 ._

ANT/

6 M

CON

CON

-

FE/3

Fe

C

2

6

I 14hM

4

bp

-1631

NRS

Fe

Fe

Fe

B H L

FIG. 1. A, the effect of IL-l[j on the biosynthesis of ferritin in human hepatoma cells. IL-l/j at a concentration of 0.2 rig/ml was added to duplicate sets of HepG2 cells for the indicated times. Protein synthesis was measured by adding [““S]methionine for 30 min after each incubation. Labeled ferritin was immunoprecipitated and separated by electrophoresis in a 15% polyacrylamide gel containing 0.1% SDS, 6 M urea, 0.1 M sodium phosphate buffer, pH 7.0. The data displayed show the inductions from one of the duplicate sets of cells. The amount of ferritin induction reported in the results was calculated from scanning based on four separate experiments. Con, control. H, the action of iron on ferritin synthesis in HepG2 cells. Duplicate sets of control and iron-treated cells were labeled with [““Slmethionine after exposure of cells to iron in the form of 1.25 JLM FezTf for 2.5 h. Control (Con) and iron-induced (Fe) lysates from these cells were immunoprecipitated and gel fractionated as above. The control and iron-induced lysates were immunoprecipitated with antiferritin antibody (anti-Fer) whereas equal aliquots of iron-treated lysate were immunoprecipitated with preimmune rabbit serum. NRS, normal rabbit serum.

FIG. 2. A comparison of total levels of L-ferritin (L-Fer) and /3-actin mRNAs isolated from human hepatoma cells grown in the presence of IL-lb and iron. A, gel-fractionated RNA (5 pg/well) was Northern blotted. First lane from left, RNA from 2.5 pM FerTf-treated cells; second lane, RNA from control cells (C); right three lanes, RNA from cells treated with 0.2 ng of IL-lb for 2, 6, and 14 h. B, RNA (20 /*g/reaction) was hybridized with lo6 cpm of a 480-base L-subunit cRNA, and a 380-base RNase Tl/RNase Aresistant fragment was fractionated on 6% acrylamide; U is undigested cRNA; C is control RNA; middle three lanes are protected RNA from cells treated with IL-18 for 2, 6, and 14 h. M, marker: kb, kilobase; bp, base pairs. IL-l@ for 7 and 14 h or to iron as 2.5 PM Fe,Tf for 4 h. The lysates from these cells were separated through 15-50% sucrose gradients. Total polyribosome profiles, analyzed by UV absorbance, were unaffected by IL-lb stimulation of HepG2 cells. Fractions at the top of each gradient were devoid of ribosomes and were designated as RNP; those containing only 18 S ribosome subunits, as the 40 S peak (Aziz and Munro, 1986); monosome fractions, as the 80 S peak; and polyribosome fractions were at the bottom of each gradient. Northern blots were used to assess the L-ferritin mRNA distribution among the fractions. The autoradiographs were scanned, and the ferritin mRNA distributions across the gradients are shown in Fig. 3. L-ferritin mRNA was present in all four regions of the

to 0.2 rig/ml

Interleukin-1B

RNP

40 s

80

RNP

s POLYSOME

Induces Ferritin

40

s

so

s FQLYSO)rlE

FRACTION

FRACTION

Synthesis in HepG2 Cells was determined in triplicate over a 6-h time course (Fig. 4). The rate of uptake of radiolabeled iron into cells is unaltered by stimulation of HepG2 cells for up to 6 h with IL-W This indicates that IL-l/3 does not change transferrin receptor activity. To exemplify the effect of a significant influx of iron on receptor activity, HepG2 cells were pretreated overnight with 1.25 PM FezTf. The rate of labeled iron uptake into these cells was similar to control and IL-I@-treated cells. However, the absolute levels of labeled iron accumulation into iron-treated cells was significantly reduced compared with control or ILl&treated cells, suggesting that the influx of iron from Fe2Tf reduced the number of transferrin receptors. To confirm this, transferrin binding to the cell surface at

4 “C was determined. There was no increase in transferrin receptors on the cell surface in HepG2 cells stimulated with IL-l@ RNP

40

s

90

s

POLYSOME

RNP

FRACTION

40

80

9

S POLYSOME

FR,%CTION

for 6 h compared

with control

cells. Untreated

HepG2

cells possess 35,000 receptors/cell whereas after 6 h of IL-10 stimulation the same cells express 30,000 receptors/cell. As expected, the influx of iron into the cells treated with 5 PM FezTf decreased the number to 22,900 receptors/cell. Intracellular Iron Chelation Prevents IL-lb-induced Ferritin Synthesis-The chelator deferoxamine binds intracellular

iron, making it completely unavailable for metabolic use. Fig. 5 shows that deferoxamine crease in ferritin synthesis ” RNP

40

s

80

s POLYSOME

mRNAs stimulated

from

control, HepG2 cells.

40

80

s POLYSOME

and 14-h

&actin IL-lj3-

s

FRACTION

FRACTION

FIG. 3. Polyribosomal

”

RNP

distribution iron-induced,

of L-ferritin and 7-h and

Cytoplasmic extracts were fractionated on 15-50% sucrose gradients, and the total RNA was isolated from different fractions of the gradient by phenol-chloroform extraction. RNA from polyribosomes, monosomes (80 S), small ribosomal subunit (40 S), and fractions free of ribosomal subunits (RNP) were identified by the UV absorbance and the rRNA pattern after gel fractionation (Aziz and Munro, 1986). The samples were pooled and Northern blotted onto nylon membranes. The polyribosome distribution of both the L-subunit and @actin mRNAs was determined by hybridization of the filters with labeled probes and densitometry of the resulting autofluorograms. The mRNA detected in each fraction is expressed as a percentage of the total amount of the same mRNA present in all the fractions of the gradient. Polyribosome gradients separate from those described have been presented in abstract form (Rogers et al., 1989). control gradient with about 15% as RNP, about 30% in the 40 S peak fractions, 30% in the SO S peak fractions, and 17% of L-ferritin mRNA associated with the polyribosomes. The L-ferritin mRNA distribution from either 7-h or 14-h IL-l& stimulated HepG2 lysates was shifted toward the polyribosome fractions. Fig. 3 shows that 50% of L-ferritin mRNA becomes associated with 80 S fractions while 30% is present in polyribosome fractions. Iron also induces increased polyribosome association. Reprobing of the same blots with labeled H-ferritin cDNA demonstrated a distribution of heavy subunit mRNA within polyribosome gradients similar to that of the L-chain mRNA (data not shown). In contrast, actin and cvlAT (not shown) mRNAs were exclusively associated with the polyribosomes irrespective of growth conditions. These data show that IL-l/3 increases the translational efficiency of ferritin mRNAs without increasing mRNA levels. The Rate of Iron Uptake Is Unaffected by the Presence of Inter&kin-l&-An increase in iron uptake from transferrin would indirectly induce ferritin synthesis by elevating intracellular iron levels. Therefore, the effect of IL-lp on the uptake of labeled iron from 5gFezTf into HepG2 cells at 37 “C

blocks the IL-lp-mediated inin HepG2 cells. Densitometric

scans of lanes 2 and 4 showed that IL-l@ induced a 3-fold increase in the amount of [35S]methionine incorporated into both H- and L-ferritin subunits by pulse labeling. Under these conditions, [35S]methionine incorporation into (Y~AT (Mr 55,000) was unchanged (lanes 1 and 3). IL-p-induced ferritin synthesis is absent from cells grown in the presence of 100 FM deferoxamine (lane 6), whereas oiAT production is unchanged (lane 5). The synthesis of olAT was determined by immunoprecipitation from the same lysates as ferritin, but unlike ferritin it was not affected by exposure of the cells to deferoxamine, IL-l& or iron over several experiments. IL-l@ Does Not Affect Transferrin

Receptor mRNA

Levels-

Previous reports have suggested that the stability of transferrin receptor mRNA would diminish if IL-l/3 would act to 30000

z 8

,

/ q

arrmoc

l

IL-l

XFCN

20000

/

/

/

/

It! 08 In

0

100

200 TIME

300

400

(MN)

FIG. 4. Uptake of “Fe (from FezTf) into HepG2 cells; the effect of co-incubation with IL-16 compared with pretreatment with Fe’Tf. Cells (106) were incubated for the indicated times

in triplicate with 0.4 pM 59FeZTf in DMEM containing 1% FCS. Cells were either left as control, coincubated with 5 ng of IL-l& or pretreated with 1.25 pM FezTf. The wells were washed three times in phosphate-buffered saline and solubilized in 0.5% SDS, 20 mM nitriloacetate. The intracellular 5gFe incorporated was then counted using a y-counter, and the iron uptake for each time period was calculated from the average of each of the triplicates. Two independent experiments provide similar estimates of iron influx.

14576

Interleukin-1B

I

Con 12

I

Induces Ferritin

Synthesis in HepG2 Cells

-Df +Df III 3456 -qAT b

C2

614hrs

C FeDf

M bp -1631 1;;:

-H -L I ! E FIG. 5. The effect of deferoxamine on IL-l@-induced ferritin synthesis. Cells were labeled with [““Slmethionine for 30 min after prior incubation with IL-1B and/or deferoxamine (Ofi. Equal aliquots from each lysate were immunoprecipitated with either antiferritin antibody or with antibody to cu,AT. The immunoprecipitates were separated using a 15% acrylamide gel (Laemmli, 1970), and autofluorography was performed. Lane I, (u,AT from control (con) cells; (ana 2, ferritin from the same labeling as lane 1; lane 3, n,AT from cells treated with and 30 rig/ml IL-10 for 6 h; lane 4, ferritin from the same labeling as lane 3; lane 5, (u,AT from cells treated with 30 rig/ml IL-la and 100 pM deferoxamine for 6 h; lane 6, ferritin from the same labeling as lane 5.

enhance the translational efficiency of ferritin mRNAs by changing cellular iron levels (Mullner and Kuhn, 1988; Mullner et al., 1989). However, RNase protection analysis with a vast excess of cRNA probe shows that IL-l/3 does not influence receptor mRNA levels compared with control cells within the same 14-h time period that ferritin translation is induced. The amount of RNase protection of a predicted 420-base and a 300-base TfR fragment is unchanged by RNA isolated from control or IL-l/3-stimulated HepG2 cells (Fig. 6). As expected, TfR mRNA is more abundant after chelation of intracellular iron from HepG2 cells with deferoxamine whereas irontreated cells exhibit TfR levels diminished to a half that seen in controls (Fig. 6B). The unexpected smaller 300-base TfR cRNA is probably the result of a polymorphism between HepG2 TfR mRNA and the cloned TfR (McClelland et al., 1984), which permits RNase digestion at mismatched base(s). Northern blot analysis also shows that TfR mRNA levels are unchanged in hepatoma cells after IL-l@ stimulation, and actin mRNA levels remain unchanged under the same conditions (data not shown). DISCUSSION

Ferritin gene expression is regulated by iron through a well described translational control mechanism. Our results demonstrate that interleukin-l& a major mediator of inflammation and the APR, stimulates the synthesis of both H- and Lferritin subunits. Detailed investigation of L-subunit mRNA expression revealed that translational control mechanisms regulate L-subunit synthesis in response to IL-l@ in human hepatoma cells. Northern blot and RNase protection analyses show that L-ferritin mRNA levels in HepG2 cells are unaffected by IL-l/3 treatment. The response of ferritin synthesis to IL-lp is accompanied by a redistribution of L-ferritin mRNA toward the polyribosomes consistent with an increase in translational efficiency. This occurs within 2 h of cytokine administration and persists for at least 14 h. Previous studies are consistent with our data. Rat liver and

w-4

-396 -344 -298

v-154 FIG. 6. The effect of IL-lb and iron perturbation on transferrin receptor mRNA levels. A, RNA (20 pg) isolated from control or IL-M-stimulated HeuG2 cells was used to RNase protect 10’ cpm of a ‘470-base TfR ERNA fragment. 420- and 360-base protected TfR cRNA fragments were scanned to estimate TfR mRNA levels in HepGP cells. U is undigested cRNA; C is control RNA; right three lanes are IL-1B 2-h, 6-h, and 14-h RNAs. B, protections of TfR cRNA with RNA isolated from control (C), iron-treated (Fe), and deferoxamine (@-)-treated HepG2 cells. M, markers; bp, base pairs.

spleen ferritin synthesis is elevated 3-4-fold 6 h after the onset of an experimentally induced inflammatory response (Konijn and Hershko, 1977; Campbell et al., 1989). Konijn et al. (1981) suggested that increased ferritin synthesis occurs as the result of translational mechanisms since cytoplasmic extracts taken from rat liver reproduced this induction in the absence of nucleii in vitro. More recently L-subunit mRNA was shown to be recruited from mRNPs to polyribosomes in rat liver and spleen cells 12 h after a turpentine-induced inflammation (Campbell et al., 1989). The mRNAs for both H- and L-ferritins are translationally activated within the first 2 h of administering iron to human and rat hepatoma cells (Rogers and Munro, 1987), human erythroleukemia cells (K562) (Rouault et al., 1988), and mouse fibroblast cell lines (Walden and Thach, 1986). In intact animals, a similar induction by iron results in a lo-20-fold increase in liver ferritin synthesis (Aziz and Munro, 1986; Schull and Theil, 1982; White and Munro, 1988). Since translation of ferritin mRNA is so sensitive to changes in intracellular iron levels we sought to exclude the possibility that IL-l@ acts to stimulate transferrin receptormediated iron uptake into cells. Under such circumstances IL-l@ regulation of ferritin synthesis would be indirect. However, the rate and levels of labeled iron uptake from transferrin into HepG2 cells were not increased by the presence of IL-lp. In contrast, transferrin receptor number and iron uptake were down-regulated in cells preloaded with iron (Fig. 4). Therefore, IL-lp appears not to stimulate ferritin translation by increasing the influx of exogenous iron through either an increase in transferrin receptor number or receptor cycling rate. Transferrin receptor mRNA levels are mediated by the same iron-sensitive trans-acting factor that controls ferritin

Interleukin-10

Induces Ferritin

translation (Mullner and Kuhn, 1988; Mullner et al., 1989). This protein binds to a conserved 28-base sequence in the 5’untranslated region of ferritin mRNAs (iron regulatory elements, IREs (Aziz and Munro, 1987; Hentze et al., 1987; Leibold and Munro, 1988; Rouault et al., 1988) and to similar regions in the 3’-untranslated region of transferrin receptor mRNA in such a way as to regulate both ferritin translation and transferrin receptor stability in an iron-dependent fashion (Bridges and Cudkowicz, 1984; Klausner and Harford, 1989; Mattia et al., 1984; Mullner and Kuhn, 1988; Rao et al., 1986; Rudolf et al., 1985). The exact mechanism by which iron regulates the binding of this trans-acting factor to IREs is at present undetermined. Iron may mediate direct conformational changes to the IRE-binding protein (Klausner and Harford, 1989). Alternatively, it may increase hemin synthesis, which has been shown to derepress ferritin translation in vitro (Lin et al., 1990). A third possibility is that other factors may be stimulated by iron, which serves to modulate binding of the repressor to IREs. The absence of any changes in TfR mRNA with IL-lp levels suggests that the cytokine does not redistribute intracellular iron pools in order to stimulate ferritin synthesis (Fig. 6). Such events would change transferrin receptor mRNA levels by mechanisms associated with the binding of the ironregulated factor to receptor mRNA. These data, therefore, indicate that the cytokine acts to stimulate L-ferritin translation in a manner different from iron-induced translation. The action of IL-lfi does depend on the baseline availability of iron since deferoxamine inhibits the action of the lymphokine in HepG2 cells (Fig. 5). The complete absence of iron within cells treated with chelator may serve to “lock” the iron-dependent repressor to the IRE present in the 5’-untranslated region of all ferritin mRNAs. This would prevent a ribosomal association of ferritin mRNAs in any circumstance. It remains to be seen whether IL-@ induction of Lferritin mRNA translation is triggered by intermediate signals that permit altered binding of either the IRE-binding factor or other factors to L-subunit mRNPs. In this regard IL-P may enhance expression of trans-acting factors that modulate binding of repressor to the IRE of ferritin mRNAs but not to TfR mRNA IREs or indeed binding to alternative sites on the ferritin mRNA. The steady-state levels of H-subunit mRNAs are also unchanged, and its translational efficiency is altered in response to IL-l@.’ These observations are consistent with the movement of iron between the serum and major tissue storage sites reported by several other studies (Konijn and Hershko, 1977; Konijn et al., 1981; Campbell et al., 1989). Infections associated with fever cause a depression in serum iron levels of human subjects (Beissel, 1977) whereas both endotoxin and IL-l/3 also cause a depression of serum iron levels in chickens (Klasing, 1984). In addition, the depletion of iron from the serum of rats in which inflammation is induced by turpentine correlates with an increased ferritin content in the liver and spleen (Konijn and Hershko, 1977; Konijn et al., 1981; Campbell et al., 1989). Several groups have proposed that such alterations might serve to divert labile intracellular iron to storage sites thereby reducing its availability for release from tissues into the serum (Konijn and Hershko, 1977; Weinberg, 1978,1985). The liver is the major iron storage tissue although a marked increase in the translation of L-ferritin mRNA also occurs in rat spleen macrophages during inflammation (Campbell et al., 1989). Reduction in serum iron during the acute phase response *J.

Rogers,

K. R. Bridges,

G. P. Durmowicz,

and H. N. Munro, unpublished

observations.

J. Glass,

P. E. Auron,

14577

Synthesis in HepG2 Cells

may serve a protective role by withholding iron from the siderophores of opportunistic bacteria. A reduction in the bioavailability of iron may also provide protection against cell injury by hydroxyl radicals that are generated from macrophage-derived superoxide in the presence of serum iron (Babior, 1984; Thomas et al., 1985). Human hepatoma cells do exhibit a marked increase in the steady-state levels of ferritin shells as measured by protein staining on nondenaturing gels.’ These data suggest that ferritin protein accumulates in these cells rather than being degraded at an increased rate when stimulated by IL-l@. Tumor necrosis factor, a peptide released from mature macrophages, increases the level of H-ferritin mRNA 4-6fold after a 48-h exposure of mouse adipocytes and human muscle cells (Torti et al., 1988). This effect probably results from of enhanced transcription of the ferritin H gene. Our experiments focused on the early responses to IL-l@ and have not ruled out subsequent transcriptional responses of ferritin genes to the cytokine. These observations support our conclusions that translational control is exerted on L-ferritin synthesis early in the acute phase response. Acknowledgments-We greatly of Carol McKinlev, Massachusetts ture Center, and-the suggestions Bunn.

appreciate the technical assistance Institute of Technoloev Cell Culof Dr. L. LaCroix and-Dr. H. F.

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