CCAAT Enhancer-binding Protein (cid:1) Is Required for Interleukin-6 Receptor (cid:1) Signaling in Newborn Hepatocytes*

The acute phase response is an evolutionarily conserved response of the liver to inflammatory stimuli, which aids the body in host defense and homeostasis. We have previously reported that CCAAT enhancer-binding protein (cid:1) (C/EBP (cid:1) ) is required for the induction of acute phase protein (APP) genes in newborn mice in response to lipopolysaccharide. In this paper, we describe a mechanism by which C/EBP (cid:1) knock-out mice are unable to induce APP gene expression in response to inflammatory stimuli. We demonstrate that the lack of acute phase response in C/EBP (cid:1) knock-out mice is because of a hepatocyte autonomous defect. C/EBP (cid:1) knock-out hepatocytes do not activate STAT3 in response to recombinant interleukin (IL)-6, indicating a defect in the IL-6 pathway. C/EBP (cid:1) knock-out hepatocytes also do not show activation of other IL-6 receptor (IL-6R)-mediated Janus kinase substrates, gp130, SHP-2, and Tyk2. Further examination of the IL-6 pathway demonstrated that C/EBP (cid:1) knock-out hepatocytes have decreased IL-6R (cid:1) protein levels caused, in part, by reduced protein stability. However, other components of the IL-6 pathway are intact, as demonstrated by rescue of STAT3 activation and APP gene induction with recombinant-soluble IL-6R

The acute phase response (APR) 1 is the rapid systemic response by the body to inflammatory stimuli such as tissue damage or infection. At the site of damage, macrophages and other surrounding cells detect injury and respond by secreting pro-inflammatory cytokines including IL-1, IL-6, and TNF-␣ into the blood stream to elicit a systemic response. The liver is highly responsive to these cytokines because of the high density of cytokine receptors on hepatocytes. These cytokines evoke changes in expression of acute phase protein (APP) genes, such as positively regulated APP genes like serum amyloid A (SAA), ␥-fibrinogen, and haptoglobin and negatively regulated APP genes like albumin and transferrin. These acute phase proteins are made by hepatocytes and then secreted into the bloodstream where they play a variety of roles in homeostasis, host defense, and minimizing tissue damage (see Refs. 1,2).
IL-1, IL-6, and TNF-␣ signal through known receptors to activate three major transcription factor families: NFB, C/EBPs, and STATs. These factors regulate APP gene expression at the transcriptional level. IL-1 and TNF-␣ activate NFB p50 and p65, which recognize the consensus sites in the promoters of many APP genes such the serum amyloid A gene family (3). IL-6 induces the tyrosine phosphorylation and DNA binding activity of STAT3, which bind fibrinogen and ␣ 2 -macroglobulin promoters among others (4,5). IL-1 and IL-6 induce C/EBP␤ and C/EBP␦ at both the transcriptional and protein activation levels, whereas C/EBP␣ is slightly down-regulated (6,7). This leads to the replacement of C/EBP␣ DNA binding at C/EBP sites in promoters of APP genes with C/EBP␤ and ␦. Previously, it was thought that C/EBP␣ played a passive role in the APR; however, our laboratory has shown that C/EBP␣ is required for the induction of many APP genes in mice (8). Neonatal C/EBP␣ null mice do not elevate APP genes but do induce NFB, C/EBP␤, and C/EBP␦ DNA binding in response to LPS injection. In contrast, C/EBP␣ knock-out (KO) mice do not activate STAT3 DNA binding in response to LPS, indicating a defect in the IL-6-STAT3 signaling pathway (8).
IL-6 is thought to be the main stimulator of the induction of APP genes during the hepatic acute phase response, whereas IL-1 and TNF-␣ influence the expression of subgroups of APP genes and induce IL-6 cytokine production (9). IL-6 knock-out mice induce APP genes in response to the systemic APR inducer bacterial LPS, whereas the response to a localized tissue damage by turpentine injection does not induce APP gene expression in these mice (10). Additionally, IL-6 null mice respond to LPS by activation of STAT3; however, STAT3 is not activated in response to turpentine treatment (11). The authors speculate that other STAT3 activating cytokines, such as leukemia inhibitory factor, IL-11, or Oncostatin M (OncM), may be induced in response to LPS but not to turpentine (10). STAT3 activation by an IL-6-like signaling pathway has recently been shown to be the most important regulator of APP gene expression in mice through tissue-specific inactivation of the STAT3 gene in the liver (12). In these tissue-specific STAT3 knock-out mice, most APP genes are not induced in response to LPS treatment. Interestingly, several genes found to be STAT3responsive do not have STAT3 binding sites identified in their promoters.
IL-6 cytokines signal by binding to an ␣-receptor subunit, IL-6R␣, which binds and signals through homodimerization of the common gp130 signaling molecule. The formation of these receptor complexes brings JAKs in close proximity to each other leading to cross-phosphorylation and activation of the JAKs, including Jak1, Jak2, and Tyk2. The JAKs activate the Ras-MAPK pathway through phosphorylation of the phosphatase, SHP-2, leading to downstream activation of C/EBP␤. JAKs also activate STAT3 by tyrosine phosphorylation, permitting dimerization and entry into the nucleus where STAT3 dimers bind to STAT sites in the promoters of many APP genes (13). Our laboratory has intensively investigated the roles of C/EBP␣ in mice. C/EBP␣ is a transcription factor highly expressed in liver and adipose tissue (14). C/EBP␣ null mice die shortly after birth because of hypoglycemia because C/EBP␣ is involved in regulating the expression of many glucose metabolism genes (15). We previously reported that neonatal C/EBP␣ null mice do not respond to LPS treatment by elevating APP genes and do not activate STAT3 DNA binding (8). However, it was not known whether the lack of APR in C/EBP␣ null mice was the result of a defect in the hepatocytes or in the extrahepatocellular signaling of the injury. In this paper, we report that C/EBP␣ null hepatocytes cultured in vitro are unable to respond to recombinant IL-6 to induce APP gene expression and STAT3 DNA binding. The IL-1 pathway is intact as the predominantly IL-1-responsive gene SAA3 (16) is induced upon IL-1 treatment of C/EBP␣ knock-out hepatocytes. Additionally, we show that STAT3 is not activated because of defects in the IL-6R signaling pathway. Specifically, IL-6R␣ protein levels in C/EBP␣ null hepatocytes are decreased, which is attributed in part to decreased protein stability of the receptor. Finally, the addition of the IL-6-like cytokine Oncostatin M or the soluble IL-6R␣ linked to IL-6, termed Hyper-IL-6 (17), is capable of activating STAT3 DNA binding and synergistic induction of the acute phase protein gene, SAA1, in primary hepatocytes derived from C/EBP␣ null mice. We propose that the dramatically reduced expression of the IL-6 receptor is responsible for the failure of C/EBP␣ knock-out mice to respond to inflammatory stimuli.

EXPERIMENTAL PROCEDURES
APR Induction in Mice-Newborn mice were injected with 5 mg/kg body weight of LPS (Sigma) intraperitoneally to induce a generalized inflammatory response, and livers were harvested at time points after LPS injection. All of the neonatal mice were periodically injected with 10% glucose subcutaneously to counteract the perinatal hypoglycemia seen in C/EBP␣ knock-out pups.
Primary Hepatocyte Isolation and Cell Culture-Livers from newborn C/EBP␣ KO and WT mice were harvested and were mechanically dissociated by scalpel. Hepatocytes were further dissociated from the liver pieces by shaking at 37°C in EDTA-Earl's balanced salt solution (Invitrogen) for 10 min followed by a brief wash with M/M medium (three-parts Eagle's minimal essential medium and one-part Waymouth MAB 83/7 (Invitrogen) with 10% fetal calf serum). Hepatocytes were then incubated in M/M medium containing 0.4 mg/ml collagenase P (Roche Applied Science) and 0.6 units/ml Dispase (Invitrogen) for 10 min at 37°C followed by trituration. Isolated hepatocytes were filtered through a 70-m nylon cell strainer (BD Falcon) to remove cellular aggregates and tissue debris. Hepatocytes were plated in M/M medium and allowed to attach for 2 h, and then cells were washed in M/M medium to remove non-adherent hematopoietic cells. Hepatocytes were finally plated in M/M medium containing dexamethasone (10 Ϫ6 ) (Sigma), epidermal growth factor (50 ng/ml) (Invitrogen), and insulin (5 g/ml) (Sigma). Cells were fed every other day and split at 80 -90% confluency. Experiments were performed on days 7-10 post-isolation of primary hepatocytes.
Cytokine Treatment-Primary hepatocytes were treated with all of the cytokines used at a concentration of 50 ng/ml in fresh medium for the time indicated in the figures. IL-1 was obtained from PeproTech (Rocky Hill, NJ). Oncostatin M and IL-6 were obtained from Sigma.
Hyper-IL-6 was a generous gift from Dr. Rose-John at Christian-Albrechts Universitat (Kiel, Germany).
Northern Analysis-Northern blot analysis was performed on liver RNA harvested 4 h after LPS injection and on primary hepatocyte RNA after 20 h of cytokine treatment. Total RNA was isolated using RNA STAT60 according to manufacturer's directions (Tel-Test, Friendswood, TX). Northern blot was performed as described previously (8).
Protein Extraction-Preparation of nuclear extracts was performed as described previously (18). Liver tissue or primary hepatocytes were homogenized, and nuclei were pelleted. Nuclei were lysed in a high salt extraction buffer on ice for 20 min and centrifuged to remove debris. RIPA whole cell extracts were used for membrane-bound proteins. Liver tissue or hepatocytes were lysed in a modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM Na 3 Vo 4 , 1 mM NaF, and 1ϫ complete protease inhibitors (Roche Applied Science)) by Dounce homogenization on ice for 20 min. Lysate was centrifuged for 10 min to remove cellular debris.
Immunoprecipitation of Tyrosine-phosphorylated Proteins-1 mg of RIPA liver extract was pre-cleared with 100 l of BioMag protein A/G beads (Qiagen) rocking at 4°C for 30 min. Tyrosine-phosphorylated proteins were immunoprecipitated using a mixture of several antibodies: PY20 (3 g, BD Biosciences); PY69 (3 g, BD Biosciences); and rabbit immunoaffinity-purified IgG anti-phosphotyrosine (3 g, Upstate Biotechnology, Lake Placid, NY) for 2 h of rocking at 4°C. 100 l of BioMag protein A/G beads were added to precipitate the proteinantibody complexes for 1 h. Immunocomplexes were isolated using magnetic separators (Qiagen), and the non-precipitated supernatant was saved for Western blot analysis. The immune complexes were washed six times with modified RIPA buffer and boiled in SDS loading buffer. Pre-cleared extracts, immunoprecipitated proteins, and the supernatant were electrophoresed on 1% SDS-10% polyacrylamide gels for Western blot analysis.
Ribonuclease Protection Assay-Ribonuclease protection assay was performed using the RiboQuant Multi-Probe RNase protection assay system (BD Biosciences) according to manufacturer's instructions using 10 g of RNA. mCR-4 multi-probe template set (BD Biosciences) was used to examine IL-6R␣, gp130, and GAPDH RNA levels in primary hepatocytes. Quantitation was performed by phosphorimaging. Statistical significance was determined by paired Student's t test.
Pulse-Chase Stability Analysis-Pulse-labeling experiment was performed as below in pulse-chase experiments with the time points indicated for the pulse with a 1-h pretreatment of the cells with 200 M chloroquine and 50 M MG132. C/EBP␣ WT and KO hepatocytes were washed twice with methionine-cysteine-free minimum Eagle's medium and starved for 20 min in methionine-cysteine-free minimum Eagle's medium. The cells were then pulsed with 70 Ci/ml Easytag Express protein-labeling mixture of [ 35 S]methionine and cysteine (PerkinElmer Life Sciences) for 45 min. Cells were then washed three times with chase medium (M/M, 10% fetal calf serum, 15 g/ml cold methionine, 31.3 g/ml cold cysteine) and then incubated in 3 ml of chase medium for the time points. RIPA cell lysates were made as described earlier. 400 g of extract was pre-cleared with protein A-agarose (Sigma) for 1 h. IL-6R␣ was immunoprecipitated with 6 g of polyclonal IL-6R␣ (M-20, Santa Cruz Biotechnology) overnight at 4°C. 75 l of protein A-agarose was added for 2 h to precipitate the protein antibody complex. Immune complexes were washed 4ϫ with modified RIPA buffer, boiled in SDS-loading buffer, and run on 8% SDS-polyacrylamide gels. Graph represents the averages of two individual experiments.
Cycloheximide Protein Stability Analysis-C/EBP␣ WT and KO hepatocytes were treated with 20 g/ml cycloheximide for 0 -4 h. RIPA extracts were made at 0-, 0.5-, 1-, 2-, and 4-h time points. IL-6R␣ protein levels were analyzed by Western blot analysis and densitometry using a Molecular Dynamics personal densitometer, and ImageQuant software was performed to quantitate protein levels normalized to ␤-actin loading. Graph represents the averages of three experiments. C/EBP␣ WT and KO hepatocytes were also pre-treated with 200 M chloroquine or 50 M MG132 for 2 h prior to cycloheximide treatment for 0 and 4 h.
Real-time RT-PCR--Primary hepatocyte RNA was isolated using the Qiagen RNeasy Mini-kit with the RNase-free DNase kit in accordance with manufacturer's protocols. 2 g of total RNA was used for reverse transcription using oligo(dT) and Superscript II RNase H Ϫ reverse transcriptase (Invitrogen). The RT reactions were diluted to 1 ng/l by spectrophotometer 280/260 reading, and 3 l was used for real-time PCR. Real-time PCR was performed using the SYBR Green PCR Master mixture (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 sequence detection system (Applied Biosystems). Primers used for SAA1 were: forward 5Ј-GGAGTCTGCCATGGAGGGTT-3Ј and reverse 5Ј-CCCTTGGAAAGCCTCGTGA-3Ј. ␤-actin-loading control primers were: forward 5Ј-CTGCTCTGGCTCCTAGCACC-3Ј and reverse 5Ј-CGCTCAGGAGGAGCAATGA-3Ј. Graphic calculations were performed using the comparative C T method with data scaled to the control samples of each genotype.

Lack of IL-6-induced APP Gene Induction during the APR Is a Hepatocyte Autonomous Defect in C/EBP␣ Null Mice-New-
born C/EBP␣ null mice are unable to respond to the bacterial endotoxin LPS and do not induce APP genes, such as ␥-fibrinogen and SAA1 (Fig. 1A). However, we have observed that IL-1, TNF-␣, and IL-6 mRNA levels are induced in both C/EBP␣ KO and WT livers in response to LPS treatment (data not shown). Therefore, to determine whether the defect in APP gene induction was because of a systemic defect in the C/EBP␣ knock-out mice or a defect in the hepatocytes themselves, primary hepatocytes derived from newborn C/EBP␣ knock-out and wild type mice were cultured for 7-10 days and treated with recombinant cytokines IL-1 and IL-6 to simulate the acute phase response or directly with LPS. APP genes in WT primary hepatocytes were elevated in response to the recombinant cytokine mixture of IL-1 and IL-6 and LPS, whereas C/EBP␣ KO primary hepatocytes showed no increase in APP expression (Fig. 1, B and C). Basal levels of ␥-fibrinogen are decreased in C/EBP␣ KO hepatocytes ( Fig. 1B) but detectable in longer exposures (data not shown). WT primary hepatocytes showed increased expression (ϳ2-fold) of ␥-fibrinogen over basal levels and a large induction of SAA1 from the undetectable basal expression, whereas C/EBP␣ KO hepatocytes do not induce these genes. However, the SAA3 gene, which is predominantly IL-1-responsive (16), is induced in both WT and KO hepatocytes when treated with recombinant IL-1 and IL-6 mixture (Fig. 1B) and with IL-1 alone (data not shown), indicating that the IL-1 pathway is intact in hepatocytes lacking C/EBP␣. The fact that the C/EBP␣ KO hepatocytes do not respond to the addition of recombinant IL-6 to elevate most APP genes indicates that the hepatocytes lack some component of this pro-inflammatory signaling pathway.
C/EBP␣ Null Primary Hepatocytes Are Unable to Activate STAT3 in Response to IL-6 Treatment-The IL-6 signaling pathway activates STAT3 DNA binding and tyrosine phosphorylation of STAT3. We then asked whether STAT3, the main transcription factor activated by IL-6 and involved in the induction of APP genes, can be activated by LPS or the IL-1/IL-6 cytokine mixture in C/EBP␣ knock-out mice. STAT3 is activated in liver in response to 4 h of LPS treatment in WT mice ( Fig. 2A) as demonstrated by neutralization of most of the binding with STAT3 antibodies (S3). However, STAT3 DNA binding is not induced in C/EBP␣ knock-out liver in response to LPS treatment ( Fig. 2A). In WT primary hepatocytes, STAT3 DNA binding is induced at both 1 and 4 h after IL-1/IL-6 treatment, whereas C/EBP␣ null primary hepatocytes induce very little or no STAT3 DNA binding at any time point after IL-1/IL-6 treatment (Fig. 2B). The addition of STAT3 antiserum to the binding reaction causes neutralization of STAT3 DNA binding. The specificity of STAT3 binding to a STAT3 consensus-binding oligomer (m67SIE) is shown by competition with cold m67SIE competimer (CC). Western blot analysis of liver nuclear extracts using phospho-tyrosine 705-specific STAT3 antibodies show induction of STAT3 tyrosine phosphorylation in WT mice in response to LPS treatment, whereas C/EBP␣ KO mouse liver shows very little phosphorylated STAT3 induced by LPS (Fig. 2C). In addition, cultured WT hepatocytes induce STAT3 phosphorylation in response to the IL-1 and IL-6 mixture (Fig. 2D). However, C/EBP␣ KO primary hepatocytes showed no STAT3 tyrosine phosphorylation in response to the IL-1/IL-6 treatment (Fig. 2D). Because the IL-6-STAT3 pathway is the major inducer of the hepatic acute phase response, it is probable that the lack of APP gene induction in C/EBP␣ KO mice and hepatocytes is because of a defect in the IL-6-STAT3 signaling cascade.
JAK-associated Tyrosine Phosphorylation Is Reduced in C/EBP␣ Knock-out Mice-Because JAKs are the family of kinases responsible for the tyrosine phosphorylation of STAT3 in the IL-6 signaling pathway (13), we then asked whether JAK kinases were activated in response to LPS in C/EBP␣ KO mice. We analyzed the activation of JAK by examining the phosphorylation status of targets of JAK kinases, gp130 and SHP-2, and a member of the JAK family of kinases itself, Tyk2. Immunoprecipitation of proteins containing phosphorylated tyrosine residues and Western blot analysis of gp130, SHP-2, and Tyk2, downstream targets of IL-6 receptor signaling, demonstrated reduced phosphorylation of these proteins in C/EBP␣ KO liver compared with WT liver following LPS treatment (Fig.  3A). Equal levels of gp130 and SHP-2 in the WT and KO extracts are shown in Fig. 3B, whereas Tyk2 is not detectable at 0 h but induced in both WT and KO liver following LPS treatment. The lack of tyrosine phosphorylation of four substrates (STAT3, gp130, SHP-2, and Tyk2) of JAK kinases in C/EBP␣ KO liver indicates that the entire IL-6 pathway is not activated in response to recombinant IL-6 or LPS.

C/EBP␣ KO Liver and Hepatocytes
Have Decreased Levels of the IL-6R␣-Because C/EBP␣ KO hepatocytes do not respond to IL-6 by elevating APP gene expression, activating JAKs, or activating STAT3, expression levels of the IL-6 receptor signaling components were examined. Ribonuclease protection assay analysis of IL-6R␣ and gp130 mRNA levels were performed on three independent sets of primary hepatocytes derived from C/EBP␣ KO and WT mouse livers. The graphs in Fig. 4, A and  B, show the phosphorimage quantitation of the mRNA levels of IL-6R␣ and gp130 as a ratio to a GAPDH-loading control. IL-6R␣ mRNA levels are not statistically different (p ϭ 0.40, 0.25, and 0.62, respectively, for 0, 1, and 4 h) between the genotypes in control or IL-1 and IL-6 cytokine-treated primary hepatocytes. C/EBP KO and WT hepatocytes also express gp130 mRNA levels (Fig. 4B) that are not statistically significant different (p ϭ 0.78, 0.49, and 0.19, respectively, for 0, 1, and 4 h).
The protein levels of IL-6R␣ and gp130 were also analyzed by Western blot analysis. As shown in Fig. 4C, the protein levels of IL-6R␣ are greatly reduced in C/EBP␣ KO primary hepatocytes. Densitometric quantitation of the ratio of IL-6R␣ to ␤-actin protein levels is shown below the panel. This quantitation shows IL-6R␣ protein levels to be 4 -5-fold lower in C/EBP␣ KO hepatocytes compared with WT hepatocytes, whereas gp130 protein levels are approximately equal between WT and KO primary hepatocytes with ␤-actin shown as a loading control. Additionally, we examined the protein levels of IL-6R␣ in C/EBP␣ KO and WT mice in C/EBP␣ expressing tissues, liver, lung, and brown adipose tissue to determine whether C/EBP␣ regulates IL-6R␣ in all of the C/EBP␣-expressing tissues. The IL-6R␣ protein levels are reduced in C/EBP␣ KO liver but are not different between other C/EBP␣ WT and KO tissues, lung and brown fat (Fig. 4D). This indicates that decreased IL-6R␣ protein levels in C/EBP␣ KO liver is a hepatocyte-specific defect. The discrepancy between IL-6R␣ mRNA and protein levels in C/EBP␣ KO and WT primary hepatocytes suggests that the protein is differentially regulated between the genotypes post-transcriptionally, possibly at the level of protein stability.
Decreased Protein Stability of IL-6R␣ in C/EBP␣ KO-derived Primary Hepatocytes-We asked whether the rate of translation of IL-6R␣ was changed between C/EBP␣ KO and WT hepatocytes. Primary hepatocytes were metabolically labeled with [ 35 S]methionine and cysteine in the presence of protease inhibitors, MG132 and chloroquine, over several time points. The rate of [ 35 S] incorporation into IL-6R␣ protein is not statistically different between C/EBP␣ WT and KO hepatocytes with p ϭ 0.26 and p ϭ 0.21 for the 30-and 60-min time points, respectively (Fig. 5A). We then asked whether decreased protein stability is responsible for the reduced protein levels in C/EBP␣ KO hepatocytes. To measure the half-life of IL-6R␣ protein in primary hepatocytes, we performed a pulse-chase experiment. WT and C/EBP␣ KO hepatocytes were metabolically labeled with [ 35 S]methionine and [ 35 S]cysteine for 45 min and chased with excess unlabeled methionine and cysteine for the times indicated in Fig. 5, B and C. IL-6R␣ was immunoprecipitated and resolved by SDS-PAGE. Pulse-chase analysis of IL-6R␣ shows the half-life to be approximately two times longer in C/EBP␣ WT hepatocytes compared with KO hepatocytes. Confirming that C/EBP␣ WT hepatocytes have a longer IL-6R␣ protein half-life, cycloheximide treatment of the hepatocytes was employed to block new protein synthesis. The rate

FIG. 2. Reduced STAT3 DNA binding and STAT3 phosphorylation in C/EBP␣ KO livers and primary hepatocytes in response to
LPS and IL-1/IL-6 treatments, respectively. EMSA of STAT3 DNA binding activity to a STAT consensus oligomer (M67-SIE) using C/EBP␣ WT and KO liver nuclear extracts (A) and C/EBP␣ WT and KO derived primary hepatocyte nuclear extracts (B) is shown. Preincubation with either no antiserum (Ϫ), antiserum to STAT3 (S3), or antiserum to STAT1 (S1) was used to identify proteins in the complexes. The addition of cold competimer (CC) oligomer is used to demonstrate the specificity of the binding. Western blot analysis of nuclear extracts from C/EBP␣ KO and WT mice liver (C) and C/EBP␣ WT and KO primary hepatocytes (D) probed with antibodies to phospho-tyrosine 705 STAT3 and total STAT3 is shown. Representative blots for at least three WT and KO animals are shown.

FIG. 3. Decreased JAK-associated tyrosine phosphorylation of gp130 and Shp2 in C/EBP␣ KO mouse livers.
A, RIPA extracts from WT and KO liver were immunoprecipitated with antisera to phosphotyrosine, and Western blot analysis was performed using antibodies to gp130, SHP-2, and Tyk2. IP:P-Y, phospho-tyrosine-immunoprecipitated proteins; Sup, the unprecipitated proteins in the supernatant. B, the side panel shows total proteins levels of gp130, Shp2, and Tyk2 in the extracts. Representative blot for three experiments is shown. of protein degradation of IL-6R␣ was determined over a 4-h treatment period. Basal levels of IL-6R␣ protein are decreased in C/EBP␣ KO hepatocytes; therefore, two to three times more protein was loaded from the C/EBP␣ KO hepatocytes to detect IL-6R␣. IL-6R␣ levels were quantitated by densitometry and graphed as a percentage of the starting levels to show the difference in protein degradation rates in four independent experiments (Fig. 5D). The half-life of IL-6R␣ in C/EBP␣ WT hepatocytes is ϳ2-fold longer than in C/EBP␣ KO hepatocytes. A representative Western blot of IL-6R␣ protein levels during the cycloheximide treatment is shown in Fig. 5E. Although it is noted that rates of degradation are slightly longer in the pulsechase experiment compared with the cycloheximide experiment, degradation differences are probably due to differences in the two techniques because cycloheximide blocks all protein synthesis and can cause alterations in the hepatocytes. To determine the contribution of different degradation pathways on IL-6R␣ stability, we pre-treated the hepatocytes with inhibitors of the main pathways of protein degradation, proteosomal and lysosomal. Both chloroquine, an inhibitor of the lysosomal degradation pathway, and MG132, a proteosome inhibitor, partially stabilized IL-6R␣ protein degradation during cycloheximide treatment in C/EBP␣ WT and KO hepatocytes (Fig. 5F). These data indicate that decreased IL-6R␣ protein stability contributes to the decreased IL-6R␣ protein seen in C/EBP␣ KO hepatocytes and that both the lysosomal and proteosomal pathways of protein degradation are involved in IL-6R␣ protein degradation in hepatocytes.
Hyper-IL-6 or Oncostatin M Can Rescue STAT3 Activation and SAA1-synergystic Induction in C/EBP␣ Null Hepatocytes-Other gp130 signaling cytokines besides IL-6 have been reported to activate STAT3 DNA binding and induce APP gene expression (19). We then asked whether gp130 and the other components of the IL-6 pathway are functional in C/EBP␣ KO hepatocytes, excluding the IL-6R␣. We examined the response of C/EBP␣ KO and WT primary hepatocytes to Oncostatin M and Hyper-IL-6. OncM is an IL-6-like cytokine that signals through a dimer of OncMR and gp130 (20). Hyper-IL-6 (HYP) is a recombinant protein designed to have the soluble IL-6R␣ peptide linked to the IL-6 cytokine, which signals through gp130 in a constitutive manner (17). WT primary hepatocytes respond to IL-6, HYP, and OncM by inducing STAT3 DNA binding (Fig. 6A). C/EBP␣ KO hepatocytes do not induce STAT3 DNA binding in response to IL-6 treatment as described earlier in this paper (Figs. 2B and 6A). However, C/EBP␣ KO hepatocytes induce STAT3 DNA binding in response to Hyper-IL-6 and Oncostatin M. Interestingly, hepatocytes of both genotypes induce stronger STAT3 DNA binding in cells treated with Oncostatin M than with IL-6 or Hyper-IL-6. This may reflect differences in the receptor affinities of OncMR and IL-6R␣ for the gp130 signaling protein and their ligands. SAA1 has previously been shown to be induced by IL-1 alone and have synergistic induction with IL-1 and IL-6 together, whereas IL-6 alone is not a very good inducer of SAA1 in hepatocytes (21). The cytokine induction of SAA1 mRNA is a useful response to measure because of its low basal level and very high transcriptional induction in response to cytokines. We examined SAA1 mRNA induction by real-time RT-PCR in WT and C/EBP␣ KO hepatocytes in response to several combinations of cytokines. WT hepatocytes responded, in agreement with previously published data (21), with very little induction of SAA1 in response to IL-6, ϳ20-fold induction with IL-1, and ϳ60-fold induction in cells treated with IL-1 and IL-6 together, displaying a synergistic induction of SAA1 mRNA to both cytokines (Fig. 6B). KO hepatocytes showed very little induction in response to IL-6 and showed a similar fold induction as WT cells in response to IL-1, ϳ20-fold (Fig. 6B). However, C/EBP␣ KO cells responded to IL-1 and IL-6 treatment with only the same level of induction as seen with IL-1 alone, indicating that the SAA1 gene was not synergistically induced in response to both cytokines. This finding confirms our previous data that the IL-6 pathway is not functional in C/EBP␣ KO primary hepatocytes. To determine whether other gp130 signaling cytokines were able to rescue the synergistic induction of SAA1 mRNA, we treated hepatocytes with Oncostatin M or Hyper-IL-6, either alone or with IL-1. Both WT and C/EBP␣ KO FIG. 4. IL-6R␣ and gp130 mRNA levels are not changed in C/EBP␣ KO primary hepatocytes, but IL-6R␣ protein levels are reduced in C/EBP␣ KO primary hepatocytes and liver but not in other C/EBP␣-expressing tissues. Ribonuclease protection assay was performed on 10 g of total RNA from primary hepatocytes with IL-6R␣, gp130, and GAPDH-specific probes. Quantitation by phosphorimaging analysis was performed on three independent experiments, and mRNA levels of the receptors were compared with GAPDH for loading control. A and B, the quantitation of IL-6R␣ and gp130 mRNA levels of primary hepatocytes, respectively. Western blot analysis of the protein levels of IL-6R␣, gp130, and ␤-actin in C/EBP␣ WT and KO-derived primary hepatocytes treated with IL-1 and IL-6 mixture (C) and in liver, lung, and brown fat of C/EBP␣ WT and KO mice is shown (D). The numbers below the IL-6R␣ panel are the densitometric quantitation of the ratio of IL-6R␣ to ␤-actin levels.
hepatocytes showed little induction in response to the gp130 signaling cytokines alone. However, both Oncostatin M and Hyper-IL-6 in conjunction with IL-1 synergistically induced SAA1 mRNA in both C/EBP␣ KO and WT hepatocytes (Fig.  6B). Hyper-IL-6 with IL-1 induced both WT and KO hepatocytes to similar levels induced by WT hepatocytes treated with IL-1 and IL-6 (ϳ60-fold), whereas Oncostatin M in combination with IL-1 induced SAA1 to an even greater extent (ϳ100 -150fold) than IL-1/IL-6 or Hyper-IL-6 with IL-1. Additionally, KO hepatocytes responded more strongly to Oncostatin M and IL-1 than WT cells, which may indicate a difference in OncMRs between the genotypes. Together these data demonstrate that the extent of the defect in the IL-6 pathway is limited to the IL-6R␣ because other gp130 signaling molecules can activate STAT3 DNA binding and synergistically induce SAA1 mRNA in C/EBP␣ KO hepatocytes. DISCUSSION The acute phase response is a highly regulated response by the liver to inflammatory stimuli. Our laboratory previously reported that C/EBP␣ null mice were unable to mount an APR in response to bacterial LPS (8). This study examines the mechanism of the defect observed in C/EBP␣ knock-out mice with primary hepatocytes in culture during the APR. We demonstrate that C/EBP␣ is required specifically in hepatocytes for proper elevation of acute phase protein gene expression in response to inflammatory stimuli such as LPS or recombinant IL-1 and IL-6 cytokines. Primary hepatocytes derived from C/EBP␣ null livers are unable to activate the IL-6-JAK-STAT3 signaling pathway. We have shown that the lack of IL-6 signaling is due to decreased protein levels of IL-6R␣. The decreased IL-6R␣ protein levels are due to decreased protein stability in C/EBP␣ null primary hepatocytes. However, gp130 and the other components of the IL-6 pathway are intact in C/EBP␣ null hepatocytes as demonstrated by the rescue of STAT3 activation and synergistic SAA1 gene induction by treatment with other gp130 signaling cytokines, such as recombinant Hyper-IL-6 or Oncostatin M in combination with IL-1.
The data presented here show the importance of IL-6-STAT3 signaling in the acute phase response in newborn mice. Currently, IL-6R␣ knock-out mice have not been generated for study. However, studies of the acute phase response in adult IL-6 knock-out mice have shown that STAT3 and most APP genes are induced in response to the systemic inflammatory stimulus, LPS (10). However, the authors have suggested that other gp130 signaling cytokines may be responsible for this response in the IL-6 null mice. The differences observed between IL-6 knock-out mice and our C/EBP␣ knock-out mice with low IL-6R␣ levels in response to LPS may be reflective of the differences in the ages of mice studied. Newborn mice, as used in this study, may not be able to induce these compensatory gp130 signaling cytokines in response to LPS as adult mice, although gp130 itself appears to be functional in newborns. However, the critical importance of gp130 signaling in the acute phase response is exemplified by the lack of APP gene induction in gp130 conditional knock-out mice (22). A broad comparison of cytokine induction between newborn and adult mice would aid in the clarification of this issue. Additionally, it is possible that C/EBP␣ null mice may have other defects in addition to low IL-6R␣ levels that contribute to their defective APR.
In C/EBP␣ knock-out hepatocytes, STAT3 was not activated in response to IL-6; however, we were able to induce STAT3 DNA binding and APP gene expression in response to Oncostatin M and Hyper-IL-6. Our observation that rescue of STAT3 activation correlates with APP gene induction is in agreement with previously published work (10 -12). It was noted in IL-6 KO mice that STAT3 was activated and APP genes induced with LPS as the stimulus, whereas in response to turpentine oil, the IL-6 KO mice did not activate STAT3 and were unable to induce APP gene expression (10,11). Turpentine oil induces a localized inflammatory response, whereas LPS induces a systemic response, which may use a different pathway and elevate other gp130 signaling cytokines such as Oncostatin M or IL-11. Additionally, conditional inactivation of STAT3 in adult mouse liver has conclusively shown STAT3 to be a critical transcription factor required for APP gene induction in response to LPS treatment (12).
IL-6R␣ is required for IL-6 cytokine binding leukemia inhibitory factor leading to increased affinity for the gp130 signaling subunits of the receptor complex. C/EBP␣ has been shown to be involved in granulocyte differentiation, and C/EBP␣ KO mice do not make fully differentiated neutrophils (23). Zhang et al. (24) have shown that mRNA and functional protein levels of the IL-6R␣ are highly reduced in day 19 embryo liver and cultured hematopoietic cells from C/EBP␣ KO mice. Our data show that in addition to the previously observed reduction of IL-6R␣ levels in hematopoietic cells from C/EBP␣ KO mice, C/EBP␣ KO hepatocytes also have decreased IL-6R␣ levels. However, the mechanism of IL-6R␣ regulation by C/EBP␣ appears to be different between hematopoietic cells at the mRNA level and hepatocytes at the protein level.
We have observed 4-fold lower steady-state levels of IL-6R␣ protein in C/EBP␣ null hepatocytes compared with WT hepatocytes. Cycloheximide and pulse-chase studies showed the half-life of IL-6R␣ protein in WT hepatocytes to be approximately twice as long as in the KO hepatocytes. Previous studies have shown the protein half-life of IL-6R␣ to be 2-3 h in several types of cell lines, similar to our results (25). Protein steadystate levels are determined by a variety of factors including mRNA levels, the rate of translation, the rate of degradation, and the developmental stage at which IL-6R␣ begins expression. In our study, we found larger differences in steady-state levels of IL-6R␣ than differences in protein half-life. We ruled out differences in mRNA levels and protein translation as being the cause of the difference in steady-state IL-6R␣ protein levels. In addition, we found the rate of IL-6R␣ protein degradation to be significantly different between WT and C/EBP␣ KO hepatocytes, contributing to the differences in steady-state IL-6R␣ protein levels.
Interestingly, the 4-fold lower levels of IL-6R␣ in C/EBP␣ KO hepatocytes results in almost no IL-6-STAT3 signaling. This finding suggests that the IL-6R␣ levels are below a critical threshold required for IL-6R␣-gp130 signaling. The fact that C/EBP␣ is a transcription factor localized to the nucleus suggests that C/EBP␣ does not directly regulate the degradation of IL-6R␣ protein at the cell membrane. The indirect regulation of IL-6R␣ protein by C/EBP␣ in liver is further substantiated by the fact that other C/EBP␣-expressing tissues such as brown fat and lung have normal levels of IL-6R␣. It is probable that C/EBP␣ transcriptionally regulates a gene whose protein product influences the degradation of IL-6R␣ specifically in hepatocytes.
Down-regulation of IL-6R␣ by internalization and degradation has been studied in response to IL-6 ligand stimulus; however, little is known regarding the basal regulation of IL-6R␣ protein. Upon IL-6 binding, IL-6R␣ is rapidly endocytosed, resulting in complete removal of IL-6 binding sites from the cell surface by 30 -60 min (26,27). The ligand-induced endocytosis of IL-6/IL-6R␣ has been shown to be mediated by gp130 through a dileucine motif in the cytoplasmic domain of gp130 (28). Additionally, a soluble form of IL-6R␣ can be generated by proteolytic cleavage by a metalloproteinase or by alternative RNA splicing (29,30). However, generation of soluble IL-6R␣ does not inhibit IL-6 signaling but rather increases the plasma half-life of IL-6 and activates cells expressing gp130 that might not normally be responsive to IL-6 (31). It is not clear in FIG. 6. Oncostatin M and Hyper-IL-6 can rescue STAT3 activation and synergistic induction of SAA1 mRNA in C/EBP␣ KO hepatocytes. A, EMSA of nuclear protein binding to a STAT3 consensus site (M67-SIE) oligomer in C/EBP␣ WT and KO primary hepatocytes treated with either control medium or 50 ng/ml IL-6, Hyper-IL-6 (HYP), or OncM for 1 h of treatment. Specificity of STAT3 DNA binding confirmed by neutralization with STAT3 antibodies (Stat3Ab) and coldcompetition (C.C.). B, C/EBP␣ WT and KO hepatocytes were treated with 50 ng/ml each of IL-6; IL-1; IL-1 and IL-6; OncM; OncM and IL-1; HYP; or HYP and IL-1 for 20 h. Real-time RT-PCR was performed on total RNA for SAA1 and ␤-actin. The bar graphs show fold induction of SAA1 mRNA compared with the control (Cont) SAA1 levels for each genotype for three individual experiments. C/EBP␣ KO hepatocytes whether IL-6R␣ is being constitutively endocytosed from the cell membrane or degraded in the cytoplasm by an alternative mechanism. It is unlikely that soluble IL-6R␣ is being generated in C/EBP␣ KO hepatocytes because the hepatocytes would still respond to IL-6 cytokine. Further studies of IL-6R␣ protein regulation in C/EBP␣ KO hepatocytes will help elucidate basal mechanisms of IL-6R␣ protein turnover.
In this study, we have shown that reduced IL-6R␣ levels are responsible for the lack of IL-6 signaling in C/EBP␣ KO hepatocytes by rescuing IL-6-like signaling with other gp130 signaling cytokines. Hyper-IL-6 is a recombinant molecule engineered to take advantage of the functional design of the IL-6 receptor, which does not require a cytoplasmic domain to signal in cells. Hyper-IL-6 contains the soluble IL-6 receptor peptide linked to IL-6 (17). When added to cell culture medium, Hyper-IL-6 can signal through gp130 to activate STAT3 (17), whereas the IL-6-like cytokine, Oncostatin M, forms a high affinity dimeric structure containing one OncMR and one gp130 signaling protein (32). Previous studies have shown that Oncostatin M and Hyper-IL-6 are capable of stimulating APP gene induction similar to IL-6 (17,19). We show that Oncostatin M and Hyper-IL-6 activate STAT3 DNA binding and synergistically induce SAA1 mRNA in C/EBP␣ KO hepatocytes with IL-1. The mouse SAA1 promoter has not been characterized; however, Alonzi et al. (12) identified two conserved potential STAT binding sites by computer-assisted alignment, which correspond with the profoundly defective activation of SAA1 in the absence of STAT3 in the liver. We also observed that Oncostatin M is a stronger activator of STAT3 and SAA1 than IL-6 or Hyper-IL-6. This may be related to gp130 levels because OncMR only requires one gp130 signaling protein compared with two gp130 proteins needed for IL-6R signaling (20). In this report, we show that the IL-6R␣ is the only deficient component of the IL-6 pathway in C/EBP␣ null hepatocytes by using Oncostatin M and Hyper-IL-6 to activate STAT3 DNA binding and induce SAA1 mRNA.
C/EBP␣ is a transcription factor highly expressed in hepatocytes. C/EBP␣ has been shown to play critical roles in glucose metabolism and growth arrest in the liver (15,18). In this study, we have shown that C/EBP␣ is required for IL-6 signaling in newborn hepatocytes. Premature infants have been observed to have neonatal hypoglycemia and abnormal lipid metabolism similar to the phenotype of C/EBP␣ knock-out mice (33). Therefore, it has been proposed that C/EBP␣ null mice may serve as a good model system for better understanding liver complications and the inflammatory responses of premature infants.