Tumor necrosis factor alpha promotes nuclear localization of cytokine-inducible CCAAT/enhancer binding protein isoforms in hepatocytes.

Hepatocytes were cultured in the presence of recombinant tumor necrosis factor (TNF) α or mutated TNF α peptides that specifically activate either p55 or p75 TNF receptors to determine if TNF α can activate cytokine-inducible CCAAT/enhancer binding protein (C/EBP) isoforms by post-transcriptional mechanisms that are initiated by TNF receptors. Within 5-10 min after treatment with any of these agents, nuclear concentrations of C/EBP β and C/EBP δ double and remain 2-4-fold greater than control cultures for 30 min (p < 0.01). Consistent with these results, gel mobility shift assays demonstrate 3-fold increased nuclear C/EBP β- and C/EBP δ-DNA binding activity in TNF α-treated cells, and immunocytochemistry confirms rapid redistribution of these C/EBP isoforms into the nucleus. In contrast, mRNA and whole cell protein concentrations of C/EBP β and δ are not altered by TNF α exposure, and nuclear concentrations of another C/EBP isoform, C/EBP α, are decreased by 80%. This novel evidence that TNF α initiates post-transcriptional activation of cytokine-inducible C/EBP isoforms identifies a mechanism that enables hepatocytes to respond immediately to inflammatory stress.

TNF 1 ␣ is a pleotropic cytokine. Diverse phenotypic responses to TNF ␣ reflect, at least in part, its ability to modulate the activity of both ubiquitous and tissue-specific transcription factors. For example, in many cells, TNF ␣ initiates mitogenactivated protein kinase cascades, which modulate the transcriptional and/or DNA binding activity of AP-1 components. In macrophages, transcription of c-fos is induced by TNF ␣-initiated signals that activate microtubule-activated protein early response kinase, microtubule-activated protein early response kinase kinase, and early response kinase, leading to phosphorylation of ELK-1, which, in turn, increases the transcription of c-fos (1). In fibroblasts, TNF ␣ induces Jun nuclear kinase, which phosphorylates and transcriptionally activates c-Jun, promoting increased expression of this transcription factor (2). TNF ␣ can also induce NF-␤ activity by triggering events that favor redistribution of this transcription factor from the cytosol to the nucleus (3)(4)(5). Taken together, these data demonstrate that TNF ␣ employs several post-transcriptional mechanisms to activate an array of ubiquitous transcription factors, which, in turn, influence the expression of diverse target genes.
Evaluation of the regenerating liver after partial hepatectomy (PH) suggests that TNF ␣ may also operate post-transcriptionally to activate members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. Nuclear concentrations of C/EBP ␤ and C/EBP ␦ proteins increase early during the prereplicative period after PH (6 -10). These increases are inhibited in animals pretreated with neutralizing anti-TNF antibodies, although there is no difference in post-PH induction of their respective mRNAs (7). Because the C/EBPs regulate tissue-specific gene expression in hepatocytes, TNFmediated alterations in C/EBP activity are likely to modulate liver-specific functions during liver regeneration. Indeed, during systemic inflammation, another situation that increases TNF ␣, the profile of hepatocyte gene expression is altered radically (11). In that setting, TNF ␣ is thought to regulate hepatocyte gene expression indirectly by inducing other cytokines (e.g., interleukins 1 and 6) that can interact directly with hepatocytes to activate C/EBP ␤ and C/EBP ␦ (11)(12)(13)(14). Because IL-1 and IL-6 are also induced after PH (15,16), it is unclear if TNF ␣ or other cytokines are responsible for the observed variations in C/EBP expression that occur in the regenerating liver. The purpose of this study was to evaluate the effect of human recombinant TNF ␣ and synthetic TNF peptides that specifically activate either class 1 or class 2 TNF receptors on nuclear concentrations of C/EBP ␤ and C/EBP ␦ in cultured hepatocytes and to determine if TNF-induced changes in the nuclear concentrations of these proteins require altered expression of their mRNAs. Our results indicate that activation of both classes of TNF receptors rapidly induce a transient increase in the nuclear concentrations of these C/EBP family members without altering the expression of their mRNAs or increasing total cellular concentrations of these C/EBP isoforms. Immunocytochemistry suggests that TNF-initiated changes in the subcellular distribution of C/EBP ␤ and C/EBP ␦ are involved in post-transcriptional regulation of C/EBP activity. Baltimore, MD) (19). Antibodies were prepared by immunizing rabbits with peptides corresponding to an internal amino acid sequence of C/EBP ␣ (present in both p42 C/EBP ␣ and p30 C/EBP ␣ ) or to amino acids 278 -295 (LRNLFKQLPEPLLASAGH) of C/EBP ␤ or 115-130 (ARG-PLKREPDWGDGDA) of C/EBP ␦. As previously reported, affinity-purified antiserum used for Western blots or supershifting was specific for each C/EBP isoform, and interactions with other C/EBP isoforms were not observed (19,20). All the other chemicals were from Sigma or J. T. Baker Inc. (Phillipsburg, NJ).
Cell Culture-The SV40 tsA255 virus-transformed rat adult liver cell line RALA255-10G (17) was initially grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 4% fetal bovine serum for 3-5 days at 33°C. When cells reached about 70% confluence (time 0), cultures were treated with human recombinant TNF ␣ or TNF mutant polypeptides for variable periods of time, ranging from 5 min to 24 h.
Northern Blot Analysis-Total RNA was extracted from cultured cells by the method of Chomczynski and Sacchi (21). 20 g/lane of RNA samples was fractionated on denaturing agarose gels and transferred to GeneScreen membranes (NEN Research Products, Boston, MA). Membranes were stained with 0.04% methylene blue to confirm the lanelane equivalency of RNA loading/transfer and then hybridized with cDNA probes for C/EBP ␤ and C/EBP ␦ as described previously (7). After washing under stringent conditions, membranes were exposed to Kodak XAR film with intensifying screens. Autoradiograms from three to four unique experiments were analyzed by scanning laser densitometry (Molecular Dynamics, Sunnyvale, CA).
Protein Extraction and Western Blot Analysis-Parallel plates of similarly confluent cultures were used to isolated nuclear and whole cell protein. Nuclear protein was isolated by NUN buffer as described (22) with some modification. Briefly, cells were lysed in 10 mM Hepes, pH 7.5, 0.5 mM spermidine, 0.15 mM spermine, 5 mM EDTA, 1 mM dithiothreitol, 0.35 mM sucrose, and 0.5% Nonidet P-40. Nuclei were pelletted by centrifugation at 12000 ϫ g and resuspended in NUN buffer containing 25 mM Hepes, pH 7.5, 300 mM NaCl, 1 M urea, 1% Nonidet P-40, and 1 mM dithiothreitol. Chromatin was removed by centrifugation, and the nuclear protein extract was aliquoted and stored at Ϫ70°C. To isolate the whole cell protein, cells were washed with cold phosphate-buffered saline and lysed on ice in RIPA buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10 mM phenylmethylsulfonyl fluoride) followed by centrifugation. The soluble protein portion was stored at Ϫ70°C.
Treatment-related variations in C/EBP proteins were analyzed by Western blot, as described previously (7). In brief, proteins were separated on 12% SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes (Millipore Co. Bedford, MA). The blots were probed with affinity-purified polyclonal anti-C/EBP ␣, anti-C/EBP ␤, or anti-C/EBP ␦ antibodies, and proteins were visualized by the Enhanced Chemiluminescence detection system (Amersham Corp.). Blots obtained from three to four unique experiments were evaluated by scanning laser densitometry (Molecular Dynamics, Sunnyvale, CA).
Immunocytochemical Analysis-Subcellular localization of C/EBPs was examined by fluorescent immunocytochemical staining of RALA cells according to Tse et al. (23). Cells were cultured on glass slides and fixed in 2% paraformaldehyde, 0.1 M lysine, 0.01 M sodium periodate, and 0.05 M phosphate buffer. After quenching with 0.25% NH 4 Cl and permeabilizing with 0.06% digitonin, slides were blocked with 0.2% gelatin, immunostained with primary antibodies to C/EBP ␤ or C/EBP ␦, and visualized by staining with the fluorescein isothiocyanate-conjugated secondary antibody to rabbit IgG (Kirkgaard & Perry Lab. Inc., Gaithersburg, MD). Slides were examined under Zeiss Axioplan fluorescence microscope by two independent observers, and the number of cells with predominately nuclear or predominately cytoplasmic staining were quantitated in 10 high power (400ϫ) fields/slide. Interobserver variation was Ͻ2%.
Gel Mobility Shift Assay-Gel mobility shift assays were performed as described by Garner and Revzin (24) and Fried and Crothers (25). Each 20-l reaction mixture contained 8 -10 g of nuclear protein plus a ␥-32 P-labeled 25-base pair oligonucleotide probe containing the C/EBP binding site in the c-fos promoter (26) in binding buffer (10 mM Hepes, pH 7.5, 0.5 mM spermidine, 0.15 mM spermine, 5 mM EDTA, 10 mM dithiothreitol, 0.35 mM sucrose). The reaction mixture was incubated at room temperature for 15 min and loaded directly onto a 6.5% polyacrylamide (49:0.6 acrylamide/bisacrylamide) gel in a buffer of 25 mM Tris borate (pH 8.0), 0.25 mM EDTA. In some experiments, antisera specific for unique C/EBP isoforms or preimmune sera were added to reaction mixtures to determine the composition of protein-probe complexes. For these "supershift" assays, extracts were incubated with 1 l of preimmune sera or an equal volume of anti-C/EBP ␣, anti-C/EBP ␤, and/or anti-C/EBP ␦ antisera at 4°C for 30 min prior to addition of ␥-32 Plabeled probe. In all experiments, proteins were separated by electrophoresis at 200 V for 2 h at room temperature. Gels were dried and exposed to Kodak XAR film with intensifying screens. Assays were

RESULTS
To determine if TNF ␣ acts directly on hepatocytes to alter nuclear concentrations of C/EBP ␤ and/or C/EBP ␦, human recombinant TNF ␣ was added to the SV40 virus conditionally transformed adult rat hepatocyte line (RALA255) during culture at the permissive temperature (33°C). As shown in Fig. 1, nuclear concentrations of C/EBP ␤ and C/EBP ␦ double within 5 min of TNF treatment and remain 2-4-fold greater than untreated cultures for 30 min. Increased nuclear concentrations of the C/EBPs are not likely to be mediated by increased synthesis of these proteins because mRNA and whole cell protein concentrations of C/EBP ␤ (Fig. 2) and C/EBP ␦ (data not shown) remain relatively constant during this time. Immunocytochemistry indicates that under these conditions, TNF causes a rapid redistribution of C/EBP ␤ (Fig. 3) and C/EBP ␦ (Fig. 4) into the nucleus. To determine if these effects of human recombinant TNF ␣, which predominately activates class 1 TNF receptors, can also result from TNF receptor 2-initiated signals, experiments were repeated with synthetic peptide ligands that have been shown to selectively activate either class 1 (p55) or class 2 (p75) TNF receptors (18). As shown in Fig. 5, nuclear concentrations of C/EBP ␦ increase within 5 min and remain elevated for 30 min after treatment with either ligand. Increases in nuclear C/EBP ␤ are also short-lived but appear to occur sooner after the addition of the p75 agonist than after addition of the p55 agonist. Thus, both ligands cause a rapid, albeit transient, increase in the nuclear concentrations of C/EBP ␤ and C/EBP ␦. In contrast, nuclear concentrations of a closely related transcription factor, C/EBP ␣, transiently decrease after treatment with either agent (Fig. 5). TNF ␣-dependent increases in the nuclear concentrations of C/EBP ␤ and C/EBP ␦ are accompanied by increased binding activity of these C/EBP isoforms. As shown in Fig. 6, complex formation between nuclear extracts and the oligonucleotide probe is increased 3-fold in cells treated with either the p55 or p75 mutein FIG. 3. Subcellular localization of C/EBP ␤ protein before and after TNF ␣ treatment. RALA255-10G cells were cultured at 33°C; human recombinant TNF ␣ (60 ng/ml) was added to the plates for 30 min, and then the plates were washed extensively and processed for immunocytochemistry (see "Experimental Procedures") to detect treatment-related differences in the compartmentalization of C/EBP ␤ protein. In all experiments, parallel cultures treated with preimmune sera or primary anti-C/EBP ␤ antisera that had been preincubated with in vitro translated C/EBP ␤ protein demonstrated no fluorescence after treatment with the secondary fluorescein isothiocyanate-conjugated antibody (data not shown). A, representative photomicrographs of anti-C/ EBP ␤ stained cells before and after TNF ␣ treatment. B, graphical summary of results obtained in four separate experiments. Over 100 positively stained cells were counted in each experiment and ranked for either predominately nuclear or predominately cytoplasmic staining by two independent observers who were unaware whether or not the slides were obtained before or after TNF treatment. Data demonstrate the means Ϯ S.E. percentage of cells with either nuclear or cytoplasmic staining for each condition. for 10 min. Supershift experiments confirm that this is predominately due to increased C/EBP ␤ and C/EBP ␦ binding activity (Fig. 6). DISCUSSION The finding that TNF ␣ receptor activation directly stimulates nuclear accumulation of C/EBP ␤ and C/EBP ␦ in hepatocytes is novel. Published work in animal models suggests that during acute inflammatory responses, TNF ␣ modulates the hepatocyte phenotype indirectly, by inducing other cytokines such as IL-1 and IL-6 (11). Although recombinant TNF ␣ can down-regulate C/EBP ␣ expression in several cell lines (27), an indirect role for TNF ␣ in C/EBP ␤ and C/EBP ␦ induction has been presumed because, in vitro, recombinant IL-1 or IL-6 can reproduce most of changes in C/EBP-regulated gene expression that occur in vivo during an acute inflammatory response. In particular, IL-1 and IL-6 have been shown to increase the respective DNA binding activities of C/EBP ␤ and C/EBP ␦, important transcriptional regulators of hepatic acute phase response genes (11)(12)(13)(14). The present results demonstrate that at least in this conditionally transformed adult hepatocyte line, TNF ␣ interacts directly with its receptors to rapidly increase nuclear concentrations of these C/EBP isoforms. Indeed, this in vitro response closely mimics the kinetics of C/EBP ␤ and ␦ induction, which occurs during liver regeneration after PH in intact rats (6 -10). This observation, coupled with the fact that PH induction of these C/EBP isoforms is inhibited by pretreatment with neutralizing anti-TNF antibodies (7), suggests that TNF ␣ may also directly mediate C/EBP ␤ and C/EBP ␦ induction in vivo.
Although it is apparent that several pro-inflammatory cytokines can increase the activity of C/EBP ␤ and C/EBP ␦ in hepatocytes, the mechanisms involved have not been identified. To our knowledge, altered compartmentalization of cytokine-inducible C/EBP isoforms have not been reported after treatment with TNF ␣ or TNF-inducible cytokines. Perhaps this is because technical problems are likely to confound efforts to identify variations in the subcellular localization of these C/EBPs in hepatocytes. Commercially available anti-C/EBP ␤ and anti-C/EBP ␦ antibodies recognize several cross-reacting antigens on immunoblots of liver extracts (28), and hence, immunocytochemical variations in antigen expression may be nonspecific. Furthermore, hepatocyte isolation and culture conditions often induce C/EBP ␤ and C/EBP ␦ (9, 10), making it difficult to detect further cytokine-mediated induction of these isoforms.
The present experiments were designed to minimize these obstacles. The anti-C/EBP ␤ and anti-C/EBP ␦ antibodies employed were raised against specific internal domains of each C/EBP peptide (19,20) and affinity-purified, and antibody specificity was validated by immunoblot analysis before proceeding to immunocytochemistry. All immunocytochemical data were also confirmed by immunoblot and gel mobility shift analyses of nuclear extracts prepared from parallel cultures. Studies were done in RALA255 cells, a conditionally transformed adult hepatocyte line that was selected because when cultured at the permissive temperature, this cell line expresses trivial levels of C/EBP ␤ and C/EBP ␦ proteins in the nucleus (29,30). In this regard, RALA255 cells cultured at 33°C more closely approximate mature hepatocytes in the healthy liver  1-4) or with p55 TNF muteins (lanes 5-8) or p75 TNF muteins (lanes 9 -12) as described under "Experimental Procedures." The DNA-protein complexes were separated from free probe by polyacrymide gel electrophoresis. In some reactions (lanes 2-4, 6 -8, and 10 -12), specific antibodies against C/EBPs were added to the binding reaction to distinguish the binding specificity. Lanes 2, 6, and 10 include antisera to C/EBP ␣; lanes 3, 7, and 11 include antisera to C/EBP ␤; and lanes 4, 8, and 12 include antisera to C/EBP ␦. Supershifting of protein-probe complexes is apparent only in lanes that include antisera to C/EBP ␤ or C/EBP ␦. B, control experiments confirm specificity of binding reactions with this oligonucleotide. Comparison of lanes 1 (no protein), 2 (10 g of protein), and 4 (2 g of protein) demonstrates that binding activity is dependent on protein concentration. The addition of antisera to C/EBP ␣, C/EBP ␤, and C/EBP ␦ (lane 3) or 20-fold molar excess unlabeled C/EBP oligonucleotide (lane 5) significantly reduces binding activity; 20-fold molar excess unlabeled AP-1 oligonucleotide does not (lane 6). than do hepatocytes in primary culture, because hepatocytes in the healthy adult liver constitutively express relatively little C/EBP ␤ and C/EBP ␦ (6 -9). Low "background" nuclear expression of these C/EBP isoforms in RALA255 hepatocytes facilitates identification of TNF ␣-mediated increases in nuclear C/EBP ␤ and C/EBP ␦ concentrations. TNF ␣ is recognized by a binary system of receptors with apparent molecular masses of 55 (p55) and 75 kDa (p75), which belong to the TNF/nerve growth factor receptor family. Binding of TNF ␣ to either receptor provokes receptor oligomerization and initiates signal transduction. Most cell lines and primary tissues coexpress both receptor types, although distinct mechanisms control expression of p55 and p75. Typically, p55 is constitutively expressed at relatively low levels, whereas p75 expression is induced by external stimuli (31,32). Different cellular proteins associate with the two receptors, suggesting that they may independently regulate separate cellular responses (33,34). For example, specific activation of p55 typically induces cytotoxicity (35)(36)(37), whereas stimulation of p75 often induces proliferation (37)(38)(39), although cooperative interactions between the two classes of receptors have also been documented (40,41). Recent evidence suggests that the two receptors may also differ in their sensitivity to different forms of TNF ␣. In several cell lines, p55 is activated preferentially by soluble TNF ␣, whereas membrane-associated TNF ␣ is a better activator of p75 (14). The present results indicate that in rat hepatocytes, both TNF receptors can initiate signals that result in nuclear localization of C/EBP ␤ and C/EBP ␦. Indeed, given the relatively low affinity of rodent p75 receptors for the p75 mutants (18), similarities in the responses evoked by the two TNF mutants suggest that p75 may play a major role in transducing this TNF ␣-initiated signal in vivo, particularly after PH when hepatic TNF ␣ expression is relatively modest (42).
TNF activation of its receptors initiates signals that regulate gene expression at multiple levels. Post-transcriptional mechanisms appear to play particularly important roles in TNF ␣ regulation of transcription factors. For example, TNF ␣ induces Jun nuclear kinase, which phosphorylates c-Jun, increasing the transcriptional activity of AP-1 (2). TNF ␣ also leads to hyperphosphorylation of IB␣, which permits NF-B to move into the nucleus and activate the transcription of B-regulated genes (3)(4)(5). Post-transcriptional events have been incriminated in cytokine-dependent induction of C/EBP ␤ and ␦ because treatment with agents (e.g., bacterial lipopolysaccharide) that increase TNF ␣ and TNF-inducible cytokines stimulates dramatic increases in the DNA binding activities of C/EBP ␤ and C/EBP ␦ but induces relatively small increases in the transcription of these C/EBPs (12). C/EBP ␤ can be a target for post-transcriptional regulation. Both protein kinase A (43) and calcium calmodulin-dependent kinase (44) have been shown to phosphorylate C/EBP ␤. Phosphorylation modulates the DNA binding and transcriptional activity of C/EBP ␤, but it is unknown if changes in phosphorylation influence subcellular compartmentalization of this protein. However, in one report, treatments that increased cAMP in PC-12 cells led to a redistribution of C/EBP ␤ from the cytosol to the nucleus (26). The present results demonstrate that TNF ␣ induces nuclear localization of C/EBP ␤. This is unlikely to be mediated by cAMPdependent mechanisms because TNF ␣ inhibits activation of adenylyl cyclase and reduces intracellular cAMP concentrations (45). Further investigation of the mechanisms responsible for TNF ␣-initiated redistribution of cytokine-inducible C/EBP isoforms will be required to clarify the molecular basis for this response. The process has important physiological implications because it permits TNF ␣ to affect rapid changes in hepatocyte gene transcription, enabling these cells to respond immediately to acute inflammatory stress.