0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL. CHEMISTRY Vol. 269, No. 22, Issue of June 3, pp. 15925-15930, 1994

Printed in U.S.A.

Insulin and Interleukin-1 Differentially Regulate pp63, an Acute Phase Phosphoprotein in Hepatoma Cell Line*

(Received for publication, June 9, 1993, and in revised form, February 17, 1994)

Camklia AkhoundiSP, Martine Amiot+, Patrick Auberger, Alphonse Le Camn, and Bernard Rossill From the INSERM U-364 Faculte de Mkdecine, 06107 Nice Cedex 02, France and the llCentre CNRS-INSERM de Pharmacologie-Endocrinologie, 34094 Montpellier, France

We have reported previously that the phosphoprotein pp63, an acute phase protein, which has been recently identified as the rat fetuin, was capable of blocking the mitogenic effect of insulin on the rat Fao hepatoma cell line, without affecting metabolic effects of the hormone. Only the phosphorylated form of the protein has been shown to exhibit both anti-tyrosine kinase and growth inhibitory properties.

In this study, we used the FTO-2B rat hepatoma cell line to analyze the mechanisms involved in the control of synthesis and/or phosphorylation of pp63. For this purpose, we investigated the action of effectors known to modulate hepatic functions, such as cytokines (inter- leukin (IL)-1/3 and IL-6), which regulate the production of acute phase proteins, and insulin, which elicits pro- found effects on hepatocyte metabolism. Here, we dem- onstrate that IL-lp diminished markedly the pp63 pro- duction by affecting its mRNA transcription and that the cytokine was able to modify the N-glycosylation process of the protein. In contrast, insulin did not affect the biosynthesis of pp63 but dramatically decreased its extent of phosphorylation.

The phosphoglycoprotein pp63, secreted by ra t hepatocytes, is found in fetal plasma at concentrations ranging from 3 to 8 mg/liter (1-3). Recent studies based on the comparison of pro- tein sequences led to the conclusion that pp63 is in fact the rat fetuin (3). I t has also been established that several peptides generated from the bone sialoprotein synthesized by osteo- blasts are identical to corresponding regions of pp63 (4). Based on sequence homology, the human homologue of pp63 appears to be a2-HS’ glycoprotein (5-7). The rat phosphoglycoprotein purified by affinity chromatography or by the usual procedure used for purification of plasma acid-stable trypsin inhibitors blocks the insulin receptor tyrosine kinase activity and, con- comitantly, inhibits the growth promoting properties that in- sulin exerts on Fao, a rat hepatoma cell line (2,3). The protein pp63 is present in the plasma essentially in a dephosphorylated

*This research was supported by INSERM, University of Nice Sophia-Antipolis, and by Grants 6913 and 6285 from the Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must

U.S.C. Section 1734 solely to indicate this fact. therefore be hereby marked “aduertisement” in accordance with 18

$ The first two authors have contributed equally to this study. 8 Recipient of a fellowship from Wellcome France. 1 1 To whom correspondence should be addressed: INSERM U-364 Fac-

ulte de medecine 06107 Nice, Cedex 02 France. Tel.: 33-93-37-76-47;

’The abbreviations used are: a2-HS glycoprotein, a2 (Heremans-

APP, acute phase proteins; endo H, endo-p-N-acetylglucosaminidase H; Schmid) glycoprotein; IL, interleukin; TNFa, tumor necrosis factor a;

PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepro- panesulfonic acid.

Fax: 33-93-81-94-56.

form. It is noteworthy that only the phosphorylated form of the protein has been shown to be active (2). These data thus sup- port the assumption that a regulatory mechanism of phospho- rylation may modulate the inhibitory activity that pp63 elicits on the insulin receptor kinase activity and, by way of conse- quence, the mitogenic effects of the hormone.

During inflammatory processes, alterations in the concentra- tion of plasma acute phase proteins (APP) are observed (8). Previous studies have shown that in acutely inflamed rats, the production of pp63 by hepatocytes is severely impaired (9). Furthermore, in normal rats, treated with mediators of inflam- mation, it has been shown that the pp63 mRNA level is de- creased (10). These in vivo studies indicate that the rat fetuin is a negative APP. Consistent with results observed for the rat fetuin, the level of a2-HS glycoprotein expression in human plasma is also reduced during the inflammatory process (11). Over the last few years, several cytokines, including IL-6, IL- lp, TNFa, IL-11, leukemia inhibitory factor, and oncostatin M, which are implicated in the growth and differentiation control of hematopoietic cells, have been identified as modulators of APPs production (reviewed in Refs. 12 and 13). This family of proteins fall into two classes: type 1 proteins are regulated by IL-1p in combination with IL-6, whereas the type 2 proteins are maximally regulated in the sole presence of IL-6 (12). IL-6, IL-11, leukemia inhibitory factor, and oncostatin M seem to display similar qualitative regulatory properties, namely the stimulation of the expression of type 2 APPs (14-16). However, it is generally accepted that expression of each APP gene is in fact regulated by a specific combination of cytokines and hormones.

The capacity of pp63 to behave as a negative APP during inflammatory processes prompted us to investigate where its expression could be regulated. To this end, we sought to deter- mine the level of biosynthesis and/or phosphorylation of pp63 under the action of cytokines and hormones involved in the modulation of hepatocyte functions and metabolism. The data presented here indicate that IL-lp and insulin both contribute to diminish the biological activity of pp63 by acting at different levels. IL-1p markedly decreased pp63 biosynthesis and al- tered its glycosylation processing, whereas insulin diminished only its degree of phosphorylation.

MATERIALS AND METHODS Reagents-Human recombinant insulin and dexamethasone were ob-

tained from Novo and Sigma, respectively. Human recombinant IL-lp and human recombinant IL-6 were obtained from Glaxo. Human re- combinant TNFa was purchased from Eurocetus. Neuraminidase, 0- glycosidase, N-glycosidase, endo H, and deoxyribonuclease I RNase-free were obtained from Boehringer Mannheim. [35SlCysteine and [32Plorthophosphate were from Amersham. [a-32P]UTP was obtained from Isotopchim (Ganagobie, France). The specific anti-pp63 serum used in this study contained antibodies against a p-galactosidase fusion protein containing a fragment of the 63-kDa phosphoglycoprotein which was obtained as described previously (2). Actin cDNA was kindly pro- vided by Dr. J. F. Peyron (INSERM U-364, Nice, France).

15925

15926 IL-1 and Insulin Differentially Regulate pp63

Cell Culture-The rat hepatoma FTO-2B cell line (17) kindly pro- vided by Dr. J. Auwerx (Facult4 des Sciences, Nice, France) was main- tained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics. The rat hepatoma Fao, a differentiated cell line derived from Reuber hepatoma (H-35) estab- lished and characterized by Deschatrette and Weiss (181, was main- tained in Dulbecco's modified Eagle's mediuMam's F-12 (Ill) supple- mented with 10% heat-inactivated fetal calf serum and antibiotics. The different cell lines were passaged once a week by trypsinization, and in most of the experiments, cells were plated a t a density of 25-50.103/well in 24-well Costar plates.

Isolation of Hepatocytes-Hepatocytes were prepared from livers of adult male Wistar rats (150-200 g) using the collagenase dissociation procedure. Briefly, after anesthesia, rat livers were perfused with col- lagenase, and the hepatocytes were dispersed in Krebs-Ringer buffer as described by Le Cam et al. (19). The viability of the cell suspension assessed by the trypan blue exclusion test was over 90%.

Biosynthetic Radiolabeling and Analysis of Secreted Proteins- FTO-2B cells were preincubated for 30 min in cysteine-free medium, and they were then biosynthetically labeled for 16 h a t 37 "C with [35Slcysteine (100 pCi/ml) in the absence or in the presence of different stimulating agents. Culture media were then collected and centrifuged to eliminate cell debris. A solution containing 0.2% Nonidet P-40, a mixture of protease inhibitors, and an excess of cold cysteine was added to the supernatants that were then used to immunoprecipitate pp63- related species with specific anti-pp63 polyclonal antibodies. The mix- tures were incubated for 2 h a t 4 "C, before adding protein A-Sepharose beads (Sigma) and continuing for an additional hour a t 4 "C. The beads were washed five times with phosphate saline buffer containing 0.2% Nonidet P-40 and protease inhibitors. 35S-Labeled immunoprecipitated proteins were analyzed on 10% SDS-PAGE under reducing conditions.

Cells treated for 36 h with different effectors or cytokines were washed twice with phosphate-free Krebs-Ringer buffer and incubated with 100 pCi/ml [32Plorthophosphate in phosphate-free Krebs-Ringer buffer for an additional 5 h at 37 "C. Culture media were then collected and centrifuged to discard cell debris. An equal volume of each super- natant was analyzed on 10% SDS-PAGE under reducing conditions, and 32P-incorporated proteins were revealed by autoradiography. When re- quired, immunoprecipitations from cell lysates or supernatants of 32P- labeled proteins were performed as described above and were then analyzed on 10% SDS-PAGE under reducing conditions.

The computerized microscopic image processor Biocom 500 (Biocom, les Ulis, France) used for scanning comprises a PC/AT-compatible mi- crocomputer, a real time imaging processor, a control monitor, a color high definition monitor, and a Panasonic WV-CD5O camera.

Neuraminidase, 0-Glycosidase, N-Glycosidase, and endo H Di- gestions-Neuraminidase (50 milliunits), 0-glycosidase (4 milliunits), N-glycosidase (1.25 units), and endo H (10 milliunits) digestions were performed overnight a t 37 "C on protein Manti-pp63 immunoprecipi- tates resuspended in 50 pl of the appropriate enzyme buffer. For 0- glycosidase digestion, the samples were first incubated with neur- aminidase. The buffer used for N-glycosidase digestion was 50 mM so- dium acetate, pH 7, containing 10 mM EDTA, l% SDS, l% Triton X-100, 1% 2-mercaptoethanol, and for neuraminidase or/and 0-glycosidase di- gestions, the buffer was 50 mM sodium acetate, pH 5.5, containing 1% SDS and 1% Triton X-100. For endo H digestion, the buffer used was 100 llu~ sodium citrate, pH 5.5.

Analysis of pp63 mRNA Accumulation--Total cellular RNA were ex- tracted from FTO-2B cells incubated for 36 h with different effectors and purified using the RNAzolTM B kit (Bioprobe Systems, ZOL 1501- 100). Ten pg of total RNA were fractioned on a 1% agarose-MOPS- formaldehyde gel and transferred to a nylon membrane (Hybond N', Amersham). The blot was then hybridized overnight a t 65 "C in rapid RNA hybridization solution (Amersham) with a purified pp63 cDNA probe labeled by the random priming system (Megaprime DNA labeling system, Amersham). The membrane was then washed three times with 2 x SSC, 0.1% SDS for 5 min a t 65 "C and twice with 0.1 x SSC, 0.1% SDS for 15 and 30 min a t the same temperature and was exposed to X-AR (Kodak) films a t -80 "C.

Nuclei Isolation and Run-on Assay-Nuclei were isolated from FTO-2B cells according to the method described by Doglio et al. (20). Briefly, cells (50 x lo6) were resuspended in 10 m Tris-HC1 buffer, pH 7.5, containing 10 mM NaCl and 3 mM MgCI, and homogenized in a Dounce homogenizer. After addition of Nonidet P-40 (0.5%), nuclei were washed in the same buffer and resuspended in 50 mM Tris-HC1, pH 8.3, containing 5 mM MgCI,, 0.1 mM EDTA, and 40% glycerol. The transcrip- tion assay was initiated by adding to nuclei suspension (100 PI) an equal volume of 10 mM Tris-HC1, pH 8 ,5 mM MgCI,, 300 mM KCl, 0.5 mM each

Molecular . a b

45

31

C

two hepatoma cell lines (FTO-PB, Fao) and freshly isolated FIG. 1. Comparison of phosphorylated proteins secreted by

hepatocytes. After incubation of [32P]orthophosphate (100 pCi/ml) with hepatoma cells or freshly isolated hepatocytes for 5 h, total protein (1 pg) contained in culture supernatants were analyzed on 10% SDS- PAGE under reducing conditions: a , FTO-2B hepatoma cells (lane 1 ) and Fao hepatoma cells (lane 2). b, freshly isolated hepatocytes. c, immunoprecipitation pattern of phosphorylated 63 kDa from FTO-2B (lanes 1 and 2) and Fao (lane 3) culture supernatants using either a rabbit control serum (lane 1 ) or specific pp63 antibodies (lanes 2 and 3) coupled to protein A-Sepharose. Labeled immunoprecipitated proteins were revealed by autoradiography.

ofATP, CTP, and GTP and 100 pCi of [cY-~~PIUTP. After 50 min a t 30 "C, deoxyribonuclease I and proteinase K were added sequentially. After addition of tRNA (250 pg/ml) as carrier, nuclear RNAs were extracted with phenol and precipitated with propanol-2. Nylon membranes con- taining cDNA (5 pg) were hybridized for 48 h a t 65 "C with the 32P- labeled RNA(5.106 cpm) and washed four times with 2 x SSC, 0.1% SDS a t 65 "C for 2 h. Filters were exposed to X-AR films a t -80 "C.

RESULTS FTO-2B and Fa0 Cells Both Secrete the pp63 Phospho-

glycoprotein-The two hepatoma cell lines FTO-2B and Fao were tested for their capacity to produce secreted phosphopro- teins. After the ATP pool was labeled by incubating cells with [32P]orthophosphate for 5 h, cell culture supernatants were analyzed. The results presented in Fig. l a show that FTO-2B (lane I ) and Fao (lane 2 ) cells secreted only two major phos- phorylated proteins of 63 and 38 kDa. "he pattern of secreted phosphoproteins displayed by hepatoma cell lines or freshly isolated hepatocytes (Fig. l b ) supernatant were very similar with only two bands migrating with an apparent molecular mass of 63 and 38 kDa, respectively. Immunoprecipitation ex- periments, using specific anti-pp63 antibodies, confirmed that the 63-kDa band found in FTO-2B and Fao cell culture super- natants did correspond to the phosphorylated 63-kDa molecule secreted by rat hepatocytes (Fig. IC, lanes 2 and 3). The identity of the phophorylated 38-kDa protein has not been yet deter- mined. Although the two hepatoma cell lines secreted the 63- kDa molecule, the FTO-2B cells produced a much higher

IL-1 and Insulin Differentially Regulate pp63 15927

Molmlnr a b mass(kDa) I 2 3 4 S 6 7 8 1 2 3 4

"i 45

I I 1 I

Molecular mass (kDn) C

97

671"" "I 4 5 1 31

1 I

FIG. 2. Down-regulation of secreted phosphorylated 63-kDa molecule by IL-lp and/or IJL-6 in combination with dexametha- sone. a, FTO-2B cells were treated for 36 h without effector (lane 1) or with 0.1 p~ dexamethasone (lane 2) , 5 ng/ml IL-1p (lanes 3 and 4) , 500 unitdm1 IL6 (lanes 5 and 6 ) or with IL-1p + IL-6 (lanes 7 and 8) in combination with 0.1 PM dexamethasone (lanes 4, 6, and 8). Cells were then labeled for 5 h with [32Plorthophosphate, and culture media were analyzed on 10% SDS-PAGE under reducing conditions. b, qualitative analysis of [32Plorthophosphate-labeled and immunoprecipitated pp63 by FTO-2B cells. Before labeling, cells were treated without (lanes I and 4 ) or with 5 ng/ml I L l p (lanes 2 and 3) for 36 h. The cytokine was omitted (lane 6') or added (lane 3 ) during the 5-h 32P-labeling incuba- tion. c, Autoradiogram of [35Slcysteine-labeled proteins secreted by FTO-2B cells. Immunoprecipitation were performed with specific pp63 antibodies (lanes 1-4) or a polyclonal anti-hSAserum as a control (lanes 5-8). Before labeling, cells were treated for 36 h with 5 ng/ml of IL-1p alone or in combination with 500 units/ml of IL6 as follows: no effector (lanes I and 5); lanes 2 and 6, cells treated with I L l p alone, lanes 3 and 7, cells treated with IL-6 alone, lanes 4 and 8, cells treated with I L l p and IL6.

amount than Fao cells, at an extent which matched that pro- duced by freshly isolated rat hepatocytes when comparable amounts of protein were analyzed.

IL- lp Is the Major Cytokine Regulating the Production of pp63-We used the FTOdB cell line, which appears to be the most appropriate, to analyze the effects of individual cytokines on the production of 63-kDa phosphorylated protein. FTO-2B cells were treated with optimal concentrations of IL-6 (500 unitdml) or IL-1p (5 ng/ml) for 36 h before labeling with [32Plorthophosphate for 5 h, in a serum-free and phosphate-free medium. Analysis of total phosphorylated secreted proteins pre- sented in Fig. 2a indicates that, in the presence of IGlp, the amount of 63-kDa phosphoprotein was diminished by 65%, as assessed by densitometric scanning (lane 31, whereas IL-6 had no effect on the level of pp63 phosphorylation (lane 5). Albeit IL-6 had no effect per se, it potentiated IL-1p action, the simul- taneous addition of the two effectors decreasing the extent of pp63 phosphorylation by 80% (lane 7). As expected, IL-1p can be replaced qualitatively by TNFa (result not shown).

We next studied the effect of different combinations of cyto- kines in the presence or the absence of dexamethasone. The results presented in Fig. 2a demonstrate that dexamethasone

M) moderately decreased (40%) the level of pp63 (lane 2) , whereas it induced a strong stimulation in the degree of phos- phorylation of the 38-kDa protein. Interestingly, dexametha- sone synergized the IL-1p inhibitory effect on the 63-kDa pro- tein production which decreased by 90% (lane 4) , although these two effectors behave antagonistically on the inflamma-

tory process. As shown in lane 7, IL-6 and IL-1p also exerted synergistic effects, since the pp63 level was decreased by 80%. However, only the combination of IL-lp, IL-6, and dexametha- sone resulted in an almost complete inhibition (98%) of the production of the phosphorylated form of the 63-kDa molecule (lane 8). Finally, we found that the two secreted phosphopro- teins demonstrated distinct 32P-labeling behavior in response to the different combinations of mediators (lanes 2-8).

IL-10 Decreases pp63 Synthesis at the I).anscriptional Level-Changes in the level of the phosphorylated form of rat fetuin may result from either a change in the protein biosyn- thesis or a decreased level of phosphorylation. To discriminate between these two possibilities, we investigated, the synthesis of pp63 by labeling the cells with [35Slcysteine followed by im- munoprecipitation with specific anti-pp63 antibodies. In all ex- periments, a second cycle of immunoprecipitation was per- formed to ensure that all the protein was immunoprecipitated. The results presented in Fig. 2c (lane 2 ) indicate that IL-1p down-regulated the level of pp63 synthesis by 33%, as assessed by densitometric scanning. On the other hand, IL-6 alone had no significant effect on the production of the 63-kDa molecule when used alone (lane 31, albeit it potentiated the effect of IL-1p when used in combination (lane 4) . Interestingly, the apparent molecular mass of pp63 was found to be slightly de- creased under IL-1p treatment (Fig. 2, b and c, lanes 3 and 2, respectively). A similar molecular mass modification was also observed with TNFa (result not shown). The level of production of albumin, a well known negative AF'P, was also reduced in the presence of IL-1p or a combination of IL-1p plus IL-6; however, no modification of the molecular mass of albumin was observed (Fig. 2c, lanes 5-8).

Since pp63 processing was affected under IL-1p treatment, the question raised to know whether the decreased level of pp63 corresponded to a reduction of the transcription or the translation step. The data presented in Fig. 3 demonstrate that IL-1p alone decreased the level of pp63 transcripts. As shown in Fig. 3, lane 4, IL-6 acted in synergy with IL-1p to decrease the level of pp63 transcripts, in accord with the effects observed on the protein (Fig. 2c). In an attempt to better understand the effect of IL-1p on the transcription, we performed run-on ex- periments. As shown in Fig. 4, IL-lp decreased pp63 mRNA synthesis (Fig. 4a, lane 2) compared with the control or IL-6 conditions (Fig. 4a, lanes 1 and 3). Dexamethasone also de- creased pp63 mRNA transcription, in accord with the decrease observed in the level of phosphorylated pp63 (Fig. k). It has to be noticed that neither IL-10 nor IL-6 modified the level of actin mRNA which was used as an internal standard. These data supported the idea that the decrease in the level of pp63 protein induced by this cytokine mainly stems from a regula- tion at the transcriptional level.

IL-lp Affects pp63 N-Glycosylation Processing-In an at- tempt to better understand the mechanism underlying the de- crease in pp63 molecular mass observed in hepatoma cells ex- posed to IL-lp, we sought to examine for possible modifications occurring in the glycosylation steps involved in pp63 matura- tion. Proteins of the fetuin family undergo glycosylation at oxygen and nitrogen residues (21,22). Sequential treatments of newly synthesized pp63 with N-glycosidase, 0-glycosidase, and neuraminidase resulted in a graduated loss in molecular mass, indicating that sialic acid and 0- and N-linked sugar moieties were present on this protein. Removal of all types of sugar residues by using a combination of neuraminidase and N- and 0-glycanases (Fig. 5, lanes 7 and 8) abolished the change in molecular mass that IL-lp induced in neosynthesized pp63 (lanes 1 and 2) . Under those conditions, the apparent molecu- lar mass was around 43 kDa, a size which corresponds to the protein core as estimated from the nucleotide sequence (2).

15928 IL-1 and Insulin Differentially Regulate pp63

1 2 3 4 a

28s

- 18s

Fro. 3. Northern blot analysis of pp63 mRNA accumulation. Total cellular RNA were extracted from untreated FT0-2B hepatoma cells (lane 1 ) or from cells treated with 5 ng/ml of I L l p (lane 2), with 500 units/ml of IL-6 (lane 3 ) or with a combination of the two cytokines (lane 4) . They were then separated on a 1% agarose-MOPS gel and transferred to a nylon membrane as indicated under "Materials and Methods." a, autoradiography of total FTO-2B cells RNA hybridized with a 3ZP-labeled pp63 probe. b, ethidium bromide staining of the transferred 28 S RNA.

Secreted pp63 has been described to be extensively sialylated. However, a neuraminidase treatment (Fig. 5, lanes 3 and 4 ) did not reduce the difference in molecular mass between the spe- cies obtained in the absence or in the presence of IL-1p. Along the same line, removal of both sialic acid and 0-glycosylated residues did not eliminate the difference in the migration of the two species (lanes 5 and 6). Altogether, these data suggest that the impairment of the N-glycosylation step is likely to be the cause of the reduction in the pp63 molecular mass observed under IL-1p action.

To better predict where the N-glycosylation modification oc- curred, we analyzed the effect of endo H treatment. This en- zyme is known to cleave high mannose sugar motifs before their processing by the mannosidase I1 activity. We found that the constitutive secreted pp63 was endo H-resistant (Fig. 6, lane 2) , whereas the pp63 secreted in presence of IL-1p was sensitive to endo H treatment (Fig. 6, lane 4) . The three bands observed after endo H digestion likely correspond to different degrees in high mannose motif incorporation, according to the presence of three putative N-glycosylation sites. These data demonstrate that IL-1p induced an alteration in the matura- tion process of the N-linked oligosaccharidic chains, a t a step occurring before the action of the Golgian mannosidase 11. In consequence, the presence of sialic residues observed on the secreted form of pp63 in the presence of IL-1p (Fig. 5, lanes 3

1 a

I * I I

Fro. 4. Nuclear run-on assay and analysis of pp63 and actin gene transcription rates. FTO-2B cells were treated without (lanes I ) or with IL-1p (lanes 2). IL-6 (lanes 3) , or with dexamethasone (lanes 4 ) for 36 h. The nuclei were then isolated as described under "Materials and Methods," and transcription was performed in the presence of [O~-~~P]UTP. After extraction of the 32P-labeled RNAs (see "Materials and Methods"), they were then hybridized to different cDNAs (5 pg) for 48 h a t 65 "C: a, pp63 cDNA; b, actin cDNA.

Molecular mas(kDa) I 2 3 4 5 6 7 8

Fro. 5. IL-lp effect on pp63 glycosylation. FTO-2B cells were incubated without (lanes I , 3,5, and 7) or with 5 ng/ml of IL-lp (lanes 2, 4, 6, and 8) for 36 h, and the culture supernatants, containing 35S- labeled secreted proteins were used for immunoprecipitation with pp63 antibodies. "he samples were incubated without (lanes 1 and 2) or with neuraminidase (lanes 3 and 4), neuraminidase plus 0-glycosidase (lanes 5 and 6), or treated sequentially with neuraminidase, O-glycosi- dase, and N-glycosidase (lanes 7 and 8) and analyzed on 10% SDS- PAGE under reducing conditions.

~ _ _ _ ~ ~

and 4 ) could be attributed to the sialylation of the 0-linked sugar chains whose processing is apparently not modified by the action of the cytokine.

Insulin Down-regulates pp63 Phosphorylation Level without Affecting Its Biosynthesis-Because insulin acts as a growth- promoting agent in hepatoma cells, an effect which can be strongly inhibited by pp63 (2), it was interesting to explore the possibility that insulin might regulate pp63 production, thus creating a putative negative feedback loop. The long term effect of insulin was investigated in the same conditions described above for cytokines. After a 36-h incubation in the presence of insulin followed by a biosynthetic labeling with [3sS]cysteine for 16 h, the production of pp63 was assessed by immunoprecipi- tation with anti-pp63. No modification in pp63 synthesis was detected in the presence of concentration of insulin of 5 and 50 rm (Fig. 7, lanes 2 and 3) compared with basal conditions (Fig. 7, lane 1). In contrast, insulin (5 m) dramatically decreased the 32P incorporation into both pp63 (Fig. 8, a and b, lanes 2)

IL-1 and Insulin Differentially Regulate pp63 15929

Molecular mass W a ) 1 2 3 4

116- 97 - 67-

o .) _ _ _ 45 -

31 -

Endo H - + - FIG. 6. &l/3 affects the pp63 N-glycosylation. FT0-2B cells were

incubated without (lanes 1 and 2) or with I L l p (lanes 3 and 4) . After incubation, 35S-labeled supernatants were used for immunoprecipita- tion with anti-pp63 serum. pp63 pellets were treated without (lanes 1 and 3) or with endo H (lanes 2 and 4 ) as described under "Materials and Methods."

Molecular

FIG. 7. Insulin does not affect the pp63 synthesis. pp63 (lanes 1-3) or albumin (lanes 4-6) were immunoprecipitated from culture media containing 35S-labeled secreted proteins and analyzed on 10% SDS-PAGE. The FTO-2B cells were incubated for 36 h without (lanes 1 and 4 ) or with 5 n~ (lanes 2 and 5) or 50 nM insulin (lanes 3 and 6).

rnolrcular a b

FIG. 8. Insulin decreases the pp63 phosphorylation level. a, analysis of 32P-labeled proteins present in FTO-2B culture supernatants of cells incubated without (lane 1) or with (lane 2) insulin. 6 , analysis of 32P-labeled secreted (lanes 1 and 2) or intracellular (lanes 3 and 4 ) proteins immunoprecipitated with anti-pp63 serum. The FTO-2B cells were incubated for 36 h without (lanes 1 and 3) or with (lanes 2 and 4 ) 5 rm insulin.

and the 38-kDa protein, suggesting an overall effect on the phosphorylation balance. Insulin effect was dose-dependent from 0.5 to 5 IIM, which corresponds to the physiologic range of concentration (data not shown).

To define the step where the decrease in pp63 phosphoryla-

I I

proteins from supernatants of FTO-2B cells incubated without (lane 1) FIG. 9. Kinetics study of insulin action. Analysis of 32P-labeled

or with 5 nM of insulin for 3 (lane 2), 6 (lane 3), or 20 h (lane 4) .

tion occurred, we performed immunoprecipitation on 32P-la- beled cell lysates. The results presented in Fig. 8 indicated that insulin produced a dramatic decrease in pp63 phosphorylation (Fig. 8b, lanes 3 and 4 ) in the intracytoplasmic compartment. These data suggest that insulin regulation occurred before se- cretion and was due to an intracellular process.

To get insight into the mechanism whereby insulin impaired the degree of phosphorylation of the two secreted proteins, kinetic studies were performed. Only 3 h of incubation of FTO-2B cells in the presence of insulin were sufficient to sig- nificantly decrease the production of the 38-kDa phosphopro- tein (Fig. 9, lane 2); whereas, 20 h were required to affect the phosphorylation level of pp63 (Fig. 9, lane 4).

DISCUSSION The aim of the present study was to identify the effectors

controlling both the concentration and phosphorylation level of the phosphoprotein pp63 to gain information on the mechanism underlying regulation of its biological activity. In this purpose, we used the FTO-2B hepatoma cells for their ability to secrete the phosphorylated form of pp63 as described in normal adult rat hepatocytes (23). Since pp63 (rat fetuin) belongs to the negative acute phase protein family, we focused our attention first on cytokines that are known to be implicated in the set-up of the inflammation, namely IL-1p and IL-6, and second on insulin, which is a major regulatory effector of hepatic func- tions. Our observations indicate that IL-lp displayed a major effect on the negative modulation of pp63 synthesis as meas- ured by [35Slcysteine incorporation which resulted mainly from a marked decrease in the mRNA transcription process as as- sessed by run-on experiments. Interestingly, IL6, which also plays a pivotal role in liver response during inflammation, had no detectable effect on pp63 synthesis. As expected, TNFa could substitute qualitatively for IL-lp, an observation that is con- sistent with the fact that IL-1p and "NFa share many biologi- cal features (24). The IL-lp inhibitory effect on pp63 synthesis was elicited independently of the presence of glucocorticoids but addition of dexamethasone synergized with the effect of the interleukin. These data confirm that pp63 regulation, as ob- served for other APP, is controlled by a combination of various cytokines and suggest that the capacity exists for a fine and specific regulation for each APP (25). IL-lp-mediated inhibition of pp63 synthesis occurred concomitantly with a reduction of the apparent molecular mass of the secreted pp63 protein. It has been shown previously that an increase in the level of APP is often accompanied by changes at the post-translational gly- cosylation steps occurring during maturation of these proteins (26-28). In an attempt to characterize the mechanism govern- ing the downward shift in molecular mass of pp63, we investi- gated the possibility that the cytokine modified the glycosyla- tion process of pp63, as suggested previously for other proteins in cultured hepatocytes (29, 30). By the use of different glyco- sidases, we were able to determine that removal of sialic acid or

15930 IL-1 and Insulin Differentially Regulate pp63

0-linked sugars did not attenuate the difference in molecular mass between the basal and IL-lp-modulated forms of the pro- tein. Nevertheless, removal of the entire sugar moieties by sequential treatment with different enzymes gave rise to two molecular species migrating with an identical molecular mass of 43 kDa, corresponding to that expected for the protein core as deduced from sequence analysis (2). These data rule out the possibility that IL-1p might induce a proteolytic degradation of the protein, but rather suggest that the difference in migration observed between the two forms of the protein likely arose from a modification in the enzymatic activities involved in the elabo- ration of the nitrogen-bound sugar motif, since neither sialic acid nor 0-linked sugars content seems to be altered by expo- sure to IL-16. To gain information on this point, we used endo H, which cleaves only high mannose-bearing motifs before they are processed by mannosidase 11. Only the species of pp63 secreted from IL-18-treated cells was reactive to this enzyme, indicating that IL-lP impaired an early step of the maturation of the N-linked sugars, that intervenes in the endoplasmic re- ticulum or &-Golgi before the action of mannosidase 11. The question remains as to whether the differential glycosylation of pp63 is of biological importance.

Phosphorylated rat fetuin acts as an insulin receptor anti- tyrosine kinase capable of selectively blocking the mitogenic effect of insulin, whereas it preserves the metabolic action of the hormone on glucose metabolism andor amino acid uptake in Fao cells. In this respect, it has been demonstrated previ- ously that the phosphorylation status of pp63 is of critical im- portance for the insulin receptor tyrosine kinase inhibitory ac- tivity of this protein (2). In fact, contrary to IL-lp, insulin produced a dramatic and specific decrease in the level of phos- phorylation without affecting the biosynthesis of pp63. In con- trast, IL-6 had no or slight effect on the pp63 extent of phos- phorylation. IL-10 diminished to the same extent the level of phosphorylation and protein biosynthesis of pp63, suggesting that the phosphorylation status of pp63 remained unaltered. Insulin strongly diminished the level of phosphorylation of pp63 without any effect on its synthesis. Furthermore, combi- nation of insulin with IL-lp, IL-6, or dexamethasone did not modify the effect observed for each effector when used alone. We could demonstrate by the use of immunoprecipitation ex- periments that insulin induced dephosphorylation of the intra- cellular form of pp63, suggesting that impairment of the pp63 phosphorylation process occurred prior to secretion. It is known that insulin produced an overall effect on the intracytoplasmic phosphorylatioddephosphorylation balance that could be due to either an inhibition of a protein kinase or the activation of a protein phosphatase activity. Although insulin's major effect concerns protein kinases activation, it is well established that insulin induces dephosphorylation on several substrates (31). For example, archetypical glycogen synthase kinase has been shown to be activated once dephosphorylated on serine resi- dues under insulin stimulation. These data provide interesting paths to follow in the search of the mechanism by which insulin regulates the phosphorylation level of pp63.

Fetuin has been proposed to be involved in several biological processes, but most of them remain elusive (32,33). It has been proposed that fetuin can play a role in the control of growth on the basis of its high circulating concentration during fetal de- velopment (34,35). On the physiological viewpoint the question arises thus, as fetuin exerted a growth inhibitory effect on the FTO-2B hepatoma cell line because of a possible dedifferenti-

ated status, rendering it similar to fetal hepatic cells. However, this hypothesis can be ruled out on the basis that FTO-2B cells have been reported to express markers of highly differentiated hepatic cells, like tyrosine aminotransferase and alanine ami- notransferase whose expression is switched on at birth or sev- eral days after birth, respectively (17, 18). Owing to the close relationship existing between the phosphorylation status of pp63 and its inhibitory activity (21, it is tempting to imagine that the action of insulin might create a negative feedback loop that tends to relieve the inhibitory effect that pp63 exerts on the insulin mitogenic effects.

Acknowledgments-We are grateful to B. Ferrua for providing the cytokines, and we thank R. Mescatulo and C. Minghelli for their excel- lent illustration work.

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4.

5.

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7.

8.

10. 9.

11.

12. 13.

14. 15. 16.

17. 18.

20. 19.

21. 22.

23.

24. 25.

26.

27. 28.

29.

30.

31.

32.

33. 34.

35.

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