THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 10, lssue of April 5, pp. 7469-7473,1993 Printed in U. S. A.

Expression Induced by Interleukin-6 of Tissue-type Transglutaminase in Human Hepatoblastoma HepG2 Cells*

(Received for publication, August 3, 1992)

Naoki Suto, Koji IkuraS, and Ryuzo Sasaki From the Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

We examined the effect of interleukin-6 (IL-6) on the expression of transglutaminase in human hepato- blastoma HepG2 cells. Treatment of cells with IL-6 increased their transglutaminase activity in a time- and dose-dependent way. Dexamethasone strength- ened the stimulation by IL-6. Half-maximum stimula- tion of transglutaminase activity in the cells occurred at a dose of 40 PM IL-6 regardless of the presence of dexamethasone. Based on its immunoreactivity, the transglutaminase induced was identified as tissue-type transglutaminase. Immunoblot analysis showed that the increase in transglutaminase activity was related to an increase in the amount of transglutaminase pro- tein. Northern blot analysis with a cDNA probe specific for human tissue-type transglutaminase showed that exposure of HepG2 cells to IL-6 increased the mRNA level of the enzyme, and the increase was detectable 3 h after IL-6 was added. Induction of the mRNA was maximum between 10 and 14 h. The increase in the mRNA level was not blocked by the presence of cyclo- heximide, suggesting that the increase was independ- ent of protein synthesis. Injections into mice of sub- stances that induce inflammation such as turpentine and lipopolysaccharides increased the liver transglu- taminase activity. These results indicated that trans- glutaminase may be involved in some biological proc- esses in hepatocytes regulated by IL-6 signaling.

Transglutaminases (protein-g1utamine:amine y-glutamyl- transferase, EC 2.3.2.13) are calcium-dependent acyltransfer- ases that catalyze the formation of an amide bond between the y-carboxamide group of peptide-bound glutamine residues and the primary amine group of various amines, including the t-amino group of lysine in certain proteins. These enzymes are widely distributed in tissues and fluids of animals and classified into at least four groups based on their biochemical properties: plasma, tissue, epidermal, and keratinocyte types. Several are involved in diverse biological functions in which they catalyze the formation of e(?-glutamyl) lysine cross- links with protein substrates (for reviews, see Refs. 1-4). These functions include stabilization of fibrin structure in blood clotting (5) and in endogenously occurring fibrinolysis (6), formation of a cornified envelope inside epidermal kera-

* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “oduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ TO whom correspondence should be addressed Dept. of Chem- istry and Materials Technology, Faculty of Engineering and Design, Kyoto Inst. of Technology, Matsugasaki, Kyoto 606, Japan. Tel.: 81- 75-724-7531: Fax: 81-75-724-7556.

tinocytes (7), stiffening of the erythrocyte membrane (81, and wound healing (9). Transglutaminases also seem to be in- volved in the regulation of cellular growth and differentiation

Liver transglutaminase is one of the most extensively stud- ied tissue-type transglutaminases, but its physiological role is not clearly understood. Hand et al. (14) reported a reduction of cytosolic transglutaminase activity in chemically induced liver carcinogenesis. A peritoneal injection of retinoic acid (15) increased the activity of this enzyme in rat liver. Liver transglutaminase may participate in the apoptosis of hepato- cytes (16, 17) and in the formation of cross-linked protein matrices at sites of cell-to-cell contact (18). cytosolic trans- glutaminase activity of guinea pig liver increases during the postnatal growing phase, and this change may be involved in the postnatal development of liver cells (19).

Interleukin-6 (IL-6)’ is a multifunctional cytokine that helps to regulate immune responses, hematopoiesis, and in- flammatory reactions (for review, see Ref. 20). It has also been called B cell stimulatory factor-2 (21), interferon 82 (22), hybridoma-plasmacytoma growth factor (23), myeloid blood cell differentiation-inducing protein (24), and hepatocyte- stimulating factor (25-27). The name “hepatocyte-stimulat- ing factor” was given because of its role as a regulator of acute-phase protein synthesis and its secretion in hepatocytes (27). In human primary hepatocytes (28,29), IL-6 induces the synthesis of fibrinogen, haptoglobin, serum amyloid A, C- reactive protein, and al-antichymotrypsin, and represses the synthesis of albumin and transferrin; in the rat, IL-6 regulates the transcription of acute-phase protein genes in liver (30). In rat hepatocytes, IL-6 stimulates the expression of the metallothionein (31) and T-kininogen (32) genes. IL-6 also represses DNA synthesis in rat primary hepatocytes (33). The distribution of labeled IL-6 after its injection into rats sug- gests that the liver is the major target organ of IL-6 (34). Rat hepatocytes have IL-6 receptors with a ligand-binding chain, which is structurally similar to that of human leukocytes (35).

These findings indicated that IL-6 is a signaling factor for various functions of liver cells and led us to investigate whether transglutaminase may be involved in known and unknown biological processes in hepatocytes regulated by the action of the multifunctional cytokine IL-6. For these studies, we used HepG2 cells, a liver cell line derived from a human hepatoblastoma. This cell line expresses a variety of liver- specific metabolic functions, so it should be useful experimen- tally (36). HepG2 cells have two kinds of IL-6 binding sites with low and high affinity; when the cells are treated with IL- 6, the expression of acute-phase protein genes is stimulated (37). In this paper, we report that IL-6 induces the expression of tissue-type transglutaminase in HepG2 cells.

(10-13).

The abbreviations used are: IL-6, interleukin-6; kb, kilobase; kbp, kilobase pair.

7469

7470 Interleukin-6-induced Expression of Transglutaminase

EXPERIMENTAL PROCEDURES

Cell Culture-A human hepatoblastoma cell line, HepG2, was obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in minimum essential medium supple- mented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 units/ml penicillin, and 100 pg/ml streptomycin. Cultures were maintained at 37 "C in a humid atmosphere of 95% air and 5% Con. The effects of IL-6 (recombinant human IL-6, a gift from Ajinomoto Co., Inc., Japan) were tested by the addition of IL-6 to the medium after initial incubation for 24 h. Culture was started at the density of 2 X lo6 cells in dishes with 6-cm diameters and containing 5 ml of medium. Endotoxin was not detected in the medium used. This analysis was done by using an endotoxin assay kit, Toxicolor System (Seikagaku Corporation, Tokyo).

Animals-Male C3H/Hej mice (10 weeks old) were kept a t 24 "C in a cycle of 12 h of light and 12 h of darkness and fed ad libitum with a standard chow obtained commercially. The mice were injected intraperitoneally with lipopolysaccharide (10 pg dissolved in 300 p1 of phosphate-buffered saline (10 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl)) or subcutaneously with turpentine (50 pl). Twenty-four hours after the injection, the mice were killed and the liver was removed and assayed for transglutaminase.

Transglutaminme Assay-Cells grown in culture dishes were har- vested and washed in ice-cold phosphate-buffered saline containing 1 mM EDTA. The washed cells were suspended in 0.2 ml of lysis buffer (10 mM Tris-HC1, pH 7.5, 0.25 M sucrose, 1 mM EDTA, 1 mM (p-amidinopheny1)-methanesulfonyl fluoride, and 0.2 mM dithiothre- itol) and disrupted by five cycles of freezing and thawing. The cell lysate was fractionated into the cytosol and particulate fractions by centrifugation at 100,000 X g for 1 h. The supernatant was used as the cytosol fraction. The precipitate, after being washed twice by suspension in 2 ml of lysis buffer and centrifugation at 100,000 X g for 1 h each time, was resuspended in an appropriate volume of the lysis buffer and used as the particulate fraction. The cell lysates were assayed for transglutaminase, giving the total transglutaminase activ- ity, and the cytosol and particulate fractions were assayed, giving the activity of each fraction. Blood was removed from mouse livers by their perfusion with ice-cold 0.9% NaCl, after which the livers were cut into small pieces with scissors. The pieces of liver were made into 20% (w/v) homogenates in lysis buffer with a motor-driven Teflon homogenizer. The homogenate was assayed, giving the transgluta- minase activity of the liver. Transglutaminase activity was measured by the filter paper method (38) with acetyl a.l-casein (19) and [2,5- 3H]histamine as the substrates. The assay mixture contained, in a total volume of 100 pl, 50 mM Tris-HC1, pH 7.5, 5 mM CaCl,, 20 mM dithiothreitol, 4 m&ml acetyl aeI-casein, 1 mM [2,5-3H]histamine (4 &i), and an enzyme sample. The reaction was started by addition of the enzyme sample at 37 "C. Reaction mixtures (20 p1 each) were spotted at intervals onto filter paper discs (Whatman No. 3") treated with 5% trichloroacetic acid. The spotted filter paper discs were washed as described elsewhere (38), and the radioactivity on each disc was counted in 5 ml of toluene-based scintillation fluid by a liquid scintillation spectrometer (Packard model 3255). The count- ing efficiency of 13% was used to calculate the units of activity. One unit of enzyme activity is defined as the amount of activity that catalyzes the incorporation of 1 nmol of histamine into the protein substrate per h. The protein concentration of cell lysates was found by the Coomassie Brilliant Blue binding method of Bradford (39) with a Bio-Rad protein assay kit according to the manufacturer's protocol.

Immunoblot Analysis-As described by Laemmli (40), cell lysates were made soluble in a sample buffer, and cell proteins were separated during SDS-9% polyacrylamide gel electrophoresis. Immunoblot analysis was done essentially by the method of Burnette (41), and details were described before (42). Human plasma transglutaminase (factor XIII) was purchased from Calbiochem. The reaction between antigen proteins and rabbit polyclonal antibodies to guinea pig liver transglutaminase was detected with peroxidase-labeled secondary antibodies to rabbit immunoglobulin G by a chemiluminescence re- action with an ECL-Western blotting detection system (Amersham International Plc., Amersham, UK) according to the manufacturer's protocol.

RNA Analysis-Total RNA was extracted form HepG2 cells by the guanidium isothiocyanate-CsC1 method (43). Northern blot analysis was done essentially by the method of Thomas (44). RNA was denatured with glyoxal and dimethyl sulfoxide and resolved by 1% agarose gel electrophoresis in 10 mM sodium phosphate buffer (pH

6.6). The RNA was transferred overnight to a nylon filter with 20 X SSC (0.015 M sodium citrate, pH 7.0, containing 0.15 M NaC1). The filter was dried at room temperature and then baked for 2 h at 80 'C. RNA blots were prehybridized overnight at 42 "C in a hybridization buffer (50% formamide, 5 X Denhardt's solution, 0.75 M NaC1, 0.06 M sodium phosphate, pH 7.4, 5 mM EDTA, 0.3% SDS, and 0.25 mg/ ml denatured salmon sperm DNA). Hybridization was done overnight a t 42 "C with a probe labeled with 32P in the hybridization buffer. The filter was washed four times at room temperature with 2 X SSC containing 0.1% SDS and twice at 42 "C with 0.1 X SSC containing 0.1% SDS before being dried and autoradiographed. The hybridiza- tion probes labeled with 32P for Northern blotting were prepared from cDNA fragments by use of [a-32P]dCTP and a multiprime DNA- labeling system (Amersham International) using the procedure de- scribed by the supplier. A 1.0-kbp fragment (nucleotide positions 600- 1600) of human tissue transglutaminase cDNA was prepared from a plasmid containing the cDNA of human endothelial cell transgluta- minase (45) (the gift of Drs. V. Gentile and P. J. A. Davies, University of Texas Medical School, Houston). A 1.1-kbp fragment of actin cDNA was obtained from a plasmid containing mouse actin cDNA (pAL41) (46) by PstI digestion. The size markers used were those in the RNA ladder kit (Life Technologies, Inc., Gaithersburg, MD).

RESULTS

Transglutaminase activity (8.7 units/mg of protein) was found in HepG2 cell lysates. The activity was inhibited com- pletely by the addition of 80 mM EDTA or 20 mM iodoace- toamide to the assay system, indicating that the transgluta- minase found in HepG2 cells is calcium-dependent and has SH group(s) essential for its catalytic function, like most animal transglutaminases examined (1, 2). The transgluta- minase activity in the HepG2 cell lysates was not affected by incubation of the lysates at 37 "C for 1 h or by treatment with thrombin (data not shown). Therefore, the transglutaminase activity detected seemed not to be a zymogenic transgluta- minase like blood coagulation factor XIII, which is activated through limited proteolysis by some proteases.

When the culture of HepG2 cells was treated with 0.8 nM IL-6 for 24 h, the total transglutaminase activity increased 3- fold (Fig. 1). The activity was increased rapidly after incuba-

'A 01 I I I I I

0 4 8 12 16 20 24 Time (hour)

FIG. 1. Stimulation of transglutaminase activity in HepG2 cells by IL-6. Cells were plated as described in the text and incu- bated in the presence of: 0, no additive; 0, 0.8 nM IL-6; A, 1 FM dexamethasone; or A, 0.8 nM IL-6 and 1 p~ dexamethasone. At different times after the addition, total transglutaminase activity in the cells was assayed. Activities were calculated as per milligram of total cellular protein and shown as relative to the activity of the untreated control cells (8.7 units/mg protein) at zero time of incu- bation.

Interleukin-6-induced Expression of Transglutaminase 7471

tion for 8 h and reached near its maximum by 16 h. Activity remained high for at least 8 h. Dexamethasone at the concen- tration of 1 p M increased the stimulatory effect of IL-6 almost 2-fold. Little change in the transglutaminase activity was found in the cells treated with 1 p~ dexamethasone alone or in untreated control cells incubated for 24 h. The increase in transglutaminase activity depended on the concentration of IL-6 added in both the presence and the absence of dexa- methasone (Fig. 2). Regardless of the presence of dexameth- asone, half-maximum stimulation occurred when IL-6 was present at the concentration of about 40 pM, and this concen- tration was on the order of the dissociation constant, 15 pM, for the high affinity IL-6 binding sites of HepG2 cells (37). Stimulation was maximum between 0.6 and 1.5 nM IL-6.

Immunoblot analysis was done of lysates of HepG2 cells treated with IL-6 to identify any changes in the amount of transglutaminase during culture. Polyclonal antibodies to guinea pig liver transglutaminase that did not form antibody- antigen complexes with human factor XI11 showed that HepG2 cells contained tissue-type transglutaminase with a molecular mass of 80 kDa (Fig. 3, lanes 1-3). The amount of 80-kDa transglutaminase protein increased with the time of IL-6 treatment (Fig. 3, lanes 3-6), but the amount in untreated control cells was unchanged after 18 h of incubation (Fig. 3, lane 7). Dexamethasone strengthened the induction by IL-6 (Fig. 3, lane 10) by about the same extent as it increased enzyme activity.

In HepG2 cells, about 90% of the total transglutaminase activity was in the cytosol fraction and the other 10% of the activity was in the particulate fraction. The treatment of HepG2 cells with IL-6 stimulated the increase in transgluta- minase activity in both fractions at a similar rate (Table I).

The kinetics of the accumulation of tissue-type transglu- taminase mRNA in HepG2 cells treated with IL-6 is shown in Fig. 4. A single 4.0-kb band (tissue-type transglutaminase mRNA) had increased significantly 3 h after the addition of IL-6, and a maximum was reached at about 14 h. Dexameth- asone strengthened the induction by IL-6 of transglutaminase mRNA. In samples from untreated cells and cells treated with dexamethasone alone, the band of this mRNA was faint. To

6 -

5 -

.- 0 4 -

%

d 2 -

.- > L

a) 3 - .- L > m -

1 -

O: t

10 100 lo00 11-6 0

FIG. 2. Dose response to IL-6 by change in transglutamin- ase activity in HepG2 cells. Cells were plated as described in the text and treated with IL-6 (0) or IL-6 plus 1 p M dexamethasone (0) a t the concentrations indicated for 18 h. Total transglutaminase activity in the cells was assayed. Activities were calculated as per milligram of total protein and shown as relative to the activity of the control cells not treated with IL-6 (8.6 units/mg protein).

1 2 3 4 5 6 7 8 9 1 0

(kDa)

94- TGase c

67-

43-

30-

FIG. 3. Immunoblot analysis of transglutaminase (muse) in HepG2 cells treated with IL-6. Lanes 1 and 2,0.2 and 1.0 pg of human plasma factor XIII, respectively; lanes 3-6, lysates of HepG2 cells cultured in the presence of 0.8 nM IL-6 for 0, 5, 10, or 18 h, respectively; lane 7, lysate of HepG2 cells cultured for 18 h in the absence of IL-6; and lanes 8-10, lysates of HepG2 cells cultured in the presence of 0.8 nM IL-6 plus 1 p~ dexamethasone for 5, 10, or 18 h, respectively. Two microliters of a cell lysate was used per lane. The arrowhead indicates the position of tissue-type transglutaminase. Relative densities of bands representing tissue-type transglutaminase were: lane 1,O; lane 2,O; lane 3, 1.0; lane 4, l . l ; lane 5,2.3; lane 6,3.O; lane 7, 0.9; lane 8, 1.0; lane 9, 2.5; and lane IO, 4.6. Numbers on the left-hand side show the positions of molecular mass marker proteins (from top: phosphorylase b, bovine serum albumin, ovalbumin, and carbonic anhydrase).

TABLE I Transglutaminase activity in cytosol and particulate fractions

HepG2 cells were cultured in the presence and the absence of 0.8 nM IL-6 for 18 h. Transglutaminase activity was assayed as described in the text.

Transglutaminase activity

Total Cytosol Particulate unitslmg total cellularprotein

Cells

Control 6.75 5.87 0.63 Treated with IL-6 17.62 16.61 2.14

examine whether the IL-6-induced increase in tissue-type mRNA was dependent on protein synthesis, we tested the effect of cycloheximide on this induction (Fig. 5). Under the conditions used, cycloheximide decreased the protein synthe- sis of HepG2 cells by more than 90% (data not shown). In both control cells and cells treated with IL-6, cycloheximide seemed to increase the accumulation of transglutaminase mRNA (see “Discussion”), indicating that the IL-6-induced increase in the mRNA was not dependent on protein synthe- sis.

Injections of turpentine or lipopolysaccharide, often used to induce the acute-phase response in experimental animals, increased the liver transglutaminase activity in mice (Table 11). Induction of the acute-phase response by turpentine or lipopolysaccharide was confirmed by immunochemical detec- tion of an increase in the amount of haptoglobin, a typical acute-phase protein, in plasma from the mice (data not shown). IL-6 (0.8 nM) increased the transglutaminase activity of human hepatoma Hep3B cells 6-fold during culture for 18 h.

DISCUSSION

Our results as reported here suggested a relationship be- tween IL-6 signaling and the induction of transglutaminase in the hepatocytes of mammals. Perhaps tissue-type transglu- taminase is involved in the acute-phase response of hepato-

7472 Interleukin-6-induced Expression of Transglutaminase IL-6 + - + - DEX -

”-

+ hours

9.5 - 7.5 - 4.4 - 4 T G a s e

2.4 -

1.3-

FIG. 4. Changes in tissue-type transglutaminase mRNA level in HepG2 cells during IL-6 treatment. Cells were plated as described in the text and cultured in the presence of both 0.8 nM IL-6 and 1 p~ dexamethasone (DEX), one of these, or neither. A t the incubation times indicated after the addition of a factor(s), total RNA was isolated from the cells and analyzed (20 pgllane) by Northern blotting with a 32P-labeled probe prepared from cDNA of human tissue-type transglutaminase. After stripping off the probes, the same blot was probed again with a 32P-labeled probe prepared from cDNA of mouse actin. Transglutaminase ( TGase) shows the 4.0- kb band of human tissue-type transglutaminase mRNA. Numbers on the left-hand side show the positions of RNA size markers.

1 2 3 4

9.5- 7.5-

4.4- TGase

2.4-

1.3-

(kb)

FIG. 5. Effect of cycloheximide on the E-6-induced accu- mulation of tissue-type transglutaminase mRNA. HepG2 cells were cultured for 1.5 h after the addition of 0.8 nM IL-6, 10 pglml cycloheximide, or both, and total RNA was isolated and analyzed by Northern blotting (20 pgllane). Lane 1, control cells, with no addi- tions; lane 2, cells treated with IL-6; lane 3, cells treated with cyclo- heximide; lane 4, cells treated with IL-6 and cycloheximide. mRNAs of transglutaminase and actin on the blot were probed with 32P- labeled probes as described in the legend for Fig. 4. Transglutaminase

mRNA. (TGase) shows the 4.0-kb of human tissue-type transglutaminase

cytes. Bungay et al. (47) have reported that islet transgluta- minase may participate in the membrane-mediated events necessary for glucose-stimulated insulin release by the pan- creatic beta cells. Hepatocyte transglutaminase might be cru- cial in the secretion of acute-phase proteins.

TABLE I1 Transglutaminase activity in an acute-phase response in mouse livers

An acute-phase response was induced in mice by an injection of turpentine (50 pl) or lipopolysaccharide (10 pg). At 24 h after the injection, the mice were killed, the liver was removed and homoge- nized, and transglutaminase activity in the homogenate was assayed as described in the text. Means k standard deviation are shown ( n = 6).

Injection Transglutaminase activity unitslmg protein

None 5.9 k 0.4 Turpentine 7.4 f 1.1 Lipopolysaccharide 13.0 -C 1.1

IL-6 induces the differentiation of B and T cells, macro- phages, and neural cells and also the growth of hematopoietic stem cells and mesangial cells. IL-6 increased 7-fold the transglutaminase activity of cultured mouse myeloid leukemia M1 cells: which are made to differentiate into mature mac- rophage-like cells by IL-6 (48). I t is not known if there is a relationship between IL-6 signaling and the differentiation of liver cells. Perhaps transglutaminase participates in the dif- ferentiation induced by IL-6 of several kinds of cells.

The increase in transglutaminase activity in HepG2 cells treated with IL-6 seemed to be related to the increased syn- thesis of this enzyme and to the increased level of the mRNA for tissue-type transglutaminase. The IL-6-induced increase in the mRNA level seemed to be independent of protein synthesis, but it is not clear whether IL-6 increased the stability of the mRNA or increased the relative transcription rate of the transglutaminase gene. Rat and human genes of various acute-phase proteins, the synthesis and secretion of which are induced by IL-6, contain IL-6-responsive elements in their 5”upstream regions, and the induction by IL-6 of these proteins is probably caused by an increase in the tran- scription rate of their genes (20, 27). The IL-6-responsive elements of several acute-phase genes such as those coding for hemopexin, haptoglobin, C-reactive protein, and al-acid glycoprotein have a consensus sequence recognized by the transcription-controlling factor NF-IL-6, a nuclear factor for IL-6 expression (49). The IL-6-induced transcription of these genes seems to be regulated a t least in part by this factor. Preliminary studies3 indicated that the 5”upstream region of the guinea pig gene for liver transglutaminase contains the consensus sequence of the NF-IL-6 recognition sites. If these findings are taken into account, the IL-6-induced increase in the transglutaminase mRNA level in HepG2 cells is likely to be caused through an increase in the relative transcription rate of the corresponding gene.

The increase by cycloheximide of the level of transgluta- minase mRNA might occur because this mRNA is subject to transcriptional regulation that is under the control of labile repressor proteins. Alternatively, cycloheximide might pro- long the biological half-life of transglutaminase mRNA as the mRNAs of histones H3 and H4 are stabilized by protein synthesis inhibitors (50). Superinduction of transglutaminase mRNA in the presence of cycloheximide has been observed in cultured human erythroleukemia cells (51). Cycloheximide had no effect on the level of tissue-type transglutaminase mRNA in mouse resident peritoneal macrophages (52). These findings suggest that the expression of tissue-type transglu- taminase genes in various cells may or may not be dependent on the protein synthesis, depending on the kind of cells.

* N. Suto, K. Ikura, and R. Sasaki, unpublished observations. N. Suto, K. Ikura, R. Shinagawa, and R. Sasaki, unpublished

observations.

Interleukin-6-induced Expression of Transglutaminme 7473

Treatment of HepG2 cells with the synthetic glucocorticoid dexamethasone leads to up-regulation of their IL-6 receptors (53). This probably explains our finding that dexamethasone treatment alone did not increase the mRNA and protein levels of transglutaminase in HepG2 cells but that dexamethasone strengthened their induction by IL-6. Monokines such as IL- 1, IL-6, and tumor necrosis factor that are secreted from monocytes and macrophages during inflammation stimulate the release of adrenocorticotropic hormone from pituitary cells, increasing the secretion of glucocorticoids by adrenocor- tical cells. The glucocorticoids may help to regulate the syn- thesis of tissue-type transglutaminase as well as the synthesis of acute-phase proteins in hepatocytes.

To our knowledge, this is the first report describing the induction of tissue-type transglutaminase by IL-6. Several other factors induce tissue-type transglutaminase in different cell systems. Retinoic acid increases the level of tissue-type transglutaminase in mouse macrophages (52) and human erythroleukemia cells (51) by increasing the rate of gene transcription. Morphological changes caused by retinoids in vascular endothelial cells are associated with an increase in tissue-type transglutaminase and its mRNA level (54, 55). Transforming growth factor-@ induces tissue-type transglu- taminase in undifferentiated normal human epidermal cells but does not induce their terminal differentiation (56). So- dium butyrate can also induce the activity and expression of tissue-type transglutaminase in cultured rat pheochromocy- toma PC12 cells (57) and virus-transformed human embry- onic fibroblasts (58). These findings suggest that as with other eucaryotic genes, the expression of tissue-type transglutamin- ase genes can be controlled by various signaling factors so that the enzyme works effectively and when needed in differ- ent tissues.

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16. 17.

18.

REFERENCES Folk, J. E. (1980) Annu. Reu. Biochem. 49,517-531 Lorand, L., and Conrad, S. M. (1984) Mol. Cell. Biochem. 68,9-35

Greenberg, C. S., Birckbichler, P. J., and Rice, R. H. (1991) FASEB J. 6, Fesus, L., and Thomazy, V. (1988) Adu. Exp. Med. Biol. 2 3 1 , 119-134

Lorand, L. (1972) Ann. N. Y. Acad. Sci. 202,6-30

Rice, R. H., and Green, H. (1978) J. Cell Biol. 76,705-711 Sakata, Y., and Aoki, N. (1982) J. Clin. Inuest. 69,536-542

Siefring, G. E., Jr., Apostel, A. B., Velasco, P. T., and Lorand, L. (1978)

Mosher, D. F., and Schad, P. E. (1979) J. Clin. Inuest. 6 4 , 781-787 Birckbichler, P. J., and Patterson, M. K., Jr. (1978) Ann. N. Y. Acad. Sci.

Birckbichler, P. J., Orr, G. R., Patterson, M. K., Jr., Conway, E., and Carter,

Kannaa, R., Teshigawara, K., Noro, N., and Masuda, T. (1982) Biochem.

Murtaugh, M. P., Arend, W. P., and Davies, P. J. A. (1984) J. Exp. Med.

Hand, D., Elliott, B. M., and Griffin, M. (1988) Biochem. Biophys. Acta

3071-3077

Biochemistry 17,2598-2604

312,354-365

H. A. j1981) Proc. Natl. Acad. Sci. U. S. A. 7 8 , 5005-5008

Biophys. Res. Commun. 106,164-171

169 , 114-125

970.137-145 Piacentini, M., Fesus, L., Sartori, C., and Ceru, M. P. (1988) Biochem. J . 263.33-38

20. Akira, S., Hirano, T., Taga, T., and Kishimoto, T. (1990) FASEB J. 4 , 19. Ikura, K., Suto, N., and Sasaki, R. (1990) FEBS Lett. 2 6 8 , 203-205

21. Hirano, T., Yasukawa, K., Harada, H., Taga, T., Watanabe, Y., Matsuda, 2860-2867

T., Kashiwamura, S., Nakajima, K., Kayama, K., Iwanatsu, A., Tsuna- sawa, S., Sakiyama, F., Matsui, H., Takahara, Y., Taniguchi, Y., and

22. Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, Kishimoto, T. (1986) Nature 324,73-76

Natl. Acad. Sci. U. S. A. 79,2768-2772 U., Wallach, D., Perricaudet, M., Tiollais, P., and Revel, M. (1988) Proc.

23. Van Damme, J., Opdenakker, G., Sim son, R. J., Rubira, M. R., Cayphas, S., Vink, A., Billiau, A,, and Van &nick, J. (1987) J. Exp. Med. 166 , 914-919

24. Shabo, Y., Lotem, J., Rubinstein, M., Revel, M., Clark, S. C., Wolf, S. F.,

25. Andus, T., Geiger, T., Hirano, T., Northoff, H., Ganter, U., Bauer, J., Kamen, R., and Sachs, L. (1988) Blood 72,2070-2073

26. Gauldie, J., Richards, C., Harnish, D., Lansdorp, P., and Baumann, H. Kishimoto, T., and Heinrich, P. C. (1987) FEBS Lett. 2 2 1 , 18-22

27. Heinrich, P. C., Castell, J. V., and Andus, T. (1990) Bmchem. J. 266,621- (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 7251-7255

28. Castell, J. V., Gomez-Lechon, M. J., David, M., Hirano, T., Kishimoto, T., 636

29. Castell, J. V., Gomez-Lechon, M. J., David, M., Andus, T., Geiger, T., and Heinrich, P. C. (1988) FEBS Lett. 232,347-350

Trullenque, R., Fabra, R., and Heinrich, P. C. (1989) FEBS Lett. 2 4 2 , 797-7'10

30.

31.

32.

33.

34.

GeFT, T., Andus, T., Klapproth, J., Hirano, T., Kishimoto, T., and

Schroeder, J. J., and Cousins, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A.

Chen, H.-M., Considine, K. B., and Liao, W. S. L. (1991) J. Biol. Chem. 87,3137-3141

Nakamura, T., Arakaki, R., and Ichihara, A. (1988) Exp. Cell Res. 179 , 266,2946-2952

Castell, J. V., Geiger, T., Gross, V. Andus, T., Walter E. Hirano, T., 488-497

Kishimoto, T., and Heinrich, P. d. (1988) Eur. J. B&heh. 177 , 357- a m

", ""

emnch, P. C. (1988) Eur. J . Immunol. 18 , 717-721

35. Baumann, M., Baumann, H., and Fey, G. H. (1990) J. Biol. Chem. 266 ,

36. Javitt, N. B. (1990) FASEB J. 4,161-168 37. Baumann, H., Isseroff, H., Latimer, J. J., and Jahreis, G. P. (1988) J. Biol.

38. Lorand, L., Campbell-Wilkes, L. K., and Cooperstein, L. (1972) A d .

39. Bradford, M. (1976) Anal. Biochem. 72,248-254 40. Laemmli, U. K. (1970) Nature 227,680-685 41. Burnette, W. N. (1981) A d . Biochem. 112,195-203 42. Ikura, K., Tsuchiya, Y., Sasaki, R., and Chiba, H. (1990) Eur. J . Biochem.

19853-19862

Chem. 283,17390-17396

Biochem. 62,623-631

187 . 705-71 1 43. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J.

44. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 7 7 , 5201-5205 (1979) Biochemistry 18,5294-5299

45. Gentile, V., Saydak, M., Chiocca, E. A., Akande, O., Birckbichler, P. J., Lee, K. N., Stein, J. P., and Davies, P. J. A. (1991) J. Biol. Chem. 2 6 6 ,

~ , . . . . - -

478-4U2 46. Minty, A. J., Alonso, S., Guenet, J.-L., and Buckingham, M. E. (1983) J.

47. Bungay, P. J., Owen, R. A,, Coutts, I. C., and Griffin, M. (1986) Biochem.

48. Miyaura, C., Onozaki, K., Akiyama, Y., Taniyama, T. Hirano, T., Kishi- J. 236,269-278

49. Akira, S. Isshiki, H., Su 'ta, T. Tanabe, O., Kinoshita, S. Nishino, Y., moto, T., and Suda, T. (1988) FEBS Lett. 234 ,17- i l

Nakajima, T., Hirano, ?., and'Kishimoto, T. (1990) EMBb J. 9 , 1897- 1906

"- Mol. Bid. 167 , 77-101

50. Sive,-H. L., Heintz, N., and Roeder, R. G. (1984) Mol. Cell. Biol. 4 , 2723- 2734

51. Suedoff, T., Birckbichler, P. J., Lee, K. N., Conway, E., and Patterson, M.

52. Chiocca, E. A,, Davies, P. J. A., and Stein, J. P. (1988) J. Biol. Chem. 263 , K., Jr. (1990) Cancer Res. 60,7830-7834

1 1 584-1 1589 53. Rose-John, S., Schooltink H., Lenz, D., Hipp, E., Dufhues, G., Schmitz,

J. Biochem. 1 9 0 , 7 9 4 3 H., Schiel, X., Hirano, T:, Kishimoto, T., and Heinrich, P. C. (1990) Eur.

54. Nara, K., Nakanishi, K., Hagiwara, H., Wakita, K., Kojima, S., and Hirose, S. (1989) J. Biol. Chem. 264,19308-19312

55. Nakanishi. K.. Nara. K.. Haeiwara. H.. Aovama. Y.. Ueno. H.. and Hirose.

"" - "_"

- -- .-- " S. ( m 1 j E&. J. Bkhem~202, '15-21 ~ ' ' I ,

Fesw L., T h o m w , v., and Falus, A. (1987) RVLS Lett. 2 2 4 , 104-108 56. George, M. D., Vollberg, T. M. Floyd E. E., Stein, J. P., and Jetten, A. M. Fesus, L., Thomazy, V., Autuori, F., Ceru, M. P., Tarcsa, E., and Piacentini, (1990) J. Biol. Chem. 266, i1098-il l04

Slife, c. W., Dorsett, M. D., and Tillotson, M. L. (1986) J. Biol. Chem. 58. Lee, K. N., Birckbichler, P. J., Patterson, M. K., Jr., Conway, E., and M. (1989) FEBS Lett. 2 4 6 , 150-154

261,3451-3456 Maxwell, M. (1987) Biochem. Biophys. Acta 928,22-28

57. Byrd, J. C., and Lichti, U. (1987) J. Biol. Chem. 262,11699-11705