Interleukin-1-induced Nuclear Factor-kappa B-Ikappa Balpha Autoregulatory Feedback Loop in Hepatocytes
A ROLE FOR PROTEIN KINASE Calpha IN POST-TRANSCRIPTIONAL REGULATION OF Ikappa Balpha RESYNTHESIS*

Youqi HanDagger , Tao MengDagger , Nicole R. Murray§, Alan P. Fields§, and Allan R. BrasierDagger parallel

From the Dagger  Department of Internal Medicine, § Sealy Center for Oncology and Hematology, and the  Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas 77555-1060

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The Ikappa B inhibitors regulate the activity of the potent transcription factor nuclear factor-kappa B (NF-kappa B). Following signal-induced Ikappa B proteolysis, NF-kappa B translocates into the nucleus to activate transcription of target genes, including Ikappa Balpha itself, initiating the "NF-kappa B-Ikappa Balpha autoregulatory feedback loop." Upon Ikappa Balpha resynthesis, NF-kappa B is subsequently inactivated and redistributed back into the cytoplasm. We have previously reported a robust NF-kappa B-Ikappa Balpha autoregulatory feedback loop in HepG2 hepatocytes. Sixty minutes after tumor necrosis factor (TNF-alpha ) stimulation, Ikappa Balpha is resynthesized to ~2-fold greater level than in control cells and completely inhibits NF-kappa B binding. Here we investigate the mechanism for Ikappa Balpha resynthesis comparing the effect of stimulation of TNF-alpha with that of interleukin-1 (IL-1alpha ). Although either TNF-alpha or IL-1alpha stimulation of protein kinase C (PKC)-down-regulated cells equivalently induces NF-kappa B translocation, the kinetics of Ikappa Balpha resynthesis is slowed. Moreover, pretreatment with selective calcium-dependent PKC inhibitors selectively slowed the kinetics of the IL-1alpha -induced overshoot without affecting that produced by TNF-alpha . Down-regulation of PKCalpha by antisense phosphorothioate oligonucleotides and expression vectors selectively blocked the IL-1alpha -induced Ikappa Balpha overshoot. In the absence of PKCalpha , although IL-1alpha induced similar amounts of Ikappa Balpha transcription and changes in steady-state mRNA, a greater component of Ikappa Balpha mRNA was retained in the nucleus. These data indicate a selective role for PKCalpha in IL-1alpha -induced Ikappa Balpha resynthesis, which is mediated, at least in part, by post-transcriptional control of mRNA export.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Multicellular organisms have evolved hormone-inducible signal transduction pathways for expression of homeostatic genes under conditions of stress. One prominent example, which is highly conserved in vertebrates, is the hepatic acute-phase response (APR),1 where infectious processes activate mononuclear cells to secrete the cytokines interleukin-1alpha (IL-1alpha ) or tumor necrosis factor-alpha (1, 2). After these cytokines enter the circulation, they bind to high affinity receptors on the hepatocyte plasma membrane, activating expression of genes required for blood pressure regulation (angiotensinogen (3)) and immune activation (interleukins-6 and -8 (1, 4)). A prominent mechanism for inducible gene expression during the APR is at the level of transcriptional initiation.

The transcription factor nuclear factor-kappa B (NF-kappa B) is an important signal transduction mediator of the APR. NF-kappa B is a family of related proteins that includes potent transactivator subunits (Rel A and c-Rel) and DNA-binding subunits (NF-kappa B1). In unstimulated hepatocytes, NF-kappa B is inactivated in the cytoplasm through reversible association with the inhibitory proteins, Ikappa Balpha , -beta , and -gamma (5). Following cytokine stimulation, Ikappa Balpha and -beta are inducibly phosphorylated by a ubiquitous multisubunit Ikappa B kinase and selectively degraded (Refs. 5 and 6 and references therein). The release of previously inactivated cytoplasmic NF-kappa B allows it to enter the nucleus where it binds to high affinity sites in the promoters of inducible genes and initiates transcription (3, 7, 8). NF-kappa B target genes include acute-phase responsive genes and, curiously, one of the NF-kappa B inhibitors, Ikappa Balpha . Ikappa Balpha resynthesis is directly activated by NF-kappa B through a mechanism that, at least in part, involves increased transcription (9-11).

Following its nuclear translocation, NF-kappa B remains only transiently in the nuclear compartment, redistributing back into the cytoplasm in its inactivated (non-DNA binding) form (5, 7). In contrast to its initial translocation, NF-kappa B inactivation requires protein synthesis (5, 11), indicating termination of NF-kappa B is an active event. Studies from our laboratory have shown that the timing of NF-kappa B inactivation coincides temporally with Ikappa Balpha resynthesis (5, 7). In unstimulated cells cytoplasmic NF-kappa B is complexed to many Ikappa B isoforms. In contrast, immediately following TNF-alpha stimulation, NF-kappa B is primarily associated only with Ikappa Balpha (5, 7). Because Ikappa Balpha can dissociate DNA-bound NF-kappa B, and capture it from the nucleus, Ikappa Balpha apparently plays a role as an initial terminator of NF-kappa B activity (12). The role of Ikappa Balpha in terminating nuclear NF-kappa B activity has been underscored in studies on ikappa balpha -deficient mice. Following TNF-alpha stimulation, NF-kappa B binding is sustained indefinitely in ikappa balpha -/- fibroblasts (13). Together, these data implicate the NF-kappa B-Ikappa Balpha autoregulatory feedback loop as one mechanism for terminating NF-kappa B activity.

In our previous studies on the mechanism for TNF-alpha -induced NF-kappa B activation in HepG2 hepatocytes, we observed the presence of a robust NF-kappa B-Ikappa Balpha autoregulatory feedback loop (5). Sixty minutes following TNF-alpha administration, nuclear NF-kappa B binding activity is completely but only transiently terminated due to Ikappa Balpha resynthesis and reassociation with Rel A. Termination of NF-kappa B binding is dependent on new protein synthesis and occurs temporally when Ikappa Balpha levels peak at 2-fold greater than that observed in control cells. Moreover, termination occurs concomitantly with reassociation of Ikappa Balpha with Rel A, before detectable resynthesis of any other Ikappa B isoform has occurred (5). Because the resynthesized Ikappa Balpha continues to be proteolyzed, NF-kappa B reappears in the nucleus (at a lower level) and persists until other Ikappa B isoforms are resynthesized (14).

In this report, we focus on understanding the detailed mechanism for the NF-kappa B-Ikappa Balpha feedback loop because this inhibitory pathway could potentially be modulated by anti-inflammatory therapeutics. We observe that the exaggerated NF-kappa B-Ikappa Balpha autoregulatory feedback loop also occurs with the cytokine IL-1alpha and PKC activator, phorbol 12-myristate 13-acetate (PMA). Surprisingly, we found that down-regulation of DAG-sensitive PKC isoforms blocked the rapid kinetics of IL-1alpha -induced Ikappa Balpha overshoot resynthesis, implicating a requirement for the phorbol ester-sensitive PKC isoforms PKCalpha , -beta II, or -delta . We further demonstrate that IL-1alpha requires PKCalpha to induce Ikappa Balpha overshoot resynthesis as follows: 1) IL-1alpha induces translocation and degradation of PKCalpha ; 2) the specific inhibitor of calcium-activated PKC (cPKC) isoforms, Gö 6976, blocks IL-1alpha -induced but not TNF-alpha -induced overshoot; and finally, 3) selective inhibition of PKCalpha expression by antisense oligonucleotide treatment blocks IL-1alpha -induced but not TNF-alpha -induced overshoot. The role of PKCalpha is independent of Ikappa Balpha transcription, as IL-1 induces equivalent Ikappa Balpha mRNA levels in the presence of PKC inhibitors. These observations indicate a role for activated PKCalpha in modulating IL-1alpha -induced NF-kappa B-Ikappa B autoregulatory feedback loop and identifies PKCalpha as a target for manipulating the inflammatory response in hepatocytes.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture and Treatment-- The human hepatoblastoma cell line HepG2 was obtained from ATCC (Rockville, MD) and grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and antibiotics (penicillin/streptomycin/fungizone) in a humidified atmosphere of 5% CO2. Recombinant human TNF-alpha (30 ng/ml), IL-1alpha (4 ng/ml), or PMA (1 µM) were added in serum-free culture medium, and cells were incubated for the indicated periods at 37 °C. For pretreatments, 3 µM bisindolylmaleimide I (Gö 6850), 3 µM Gö 6976, or 0.2 µM calphostin C were added in medium for 30 min, 30 min, or 6 h, respectively, prior to stimulation. The calcium-activated PKC (cPKC) and novel PKC (nPKC) isoforms in HepG2 cells were down-regulated by treatment with 0.5 µM PMA for 20 h at 37 °C, followed by a standard recovery period of 3 h in the normal growth medium for TNF receptor expression (15, 16). All above chemicals were obtained commercially (Calbiochem).

Plasmid Construction-- -346/+ 18 base pairs of the human Ikappa Balpha promoter driving luciferase reporter was constructed. Ikappa Balpha -specific primers, 5'-TCAGGATCCGACGACCCCAATTCAAATCG-3' and 5'-CGGAAGCTTGTGGGCTCTGCAGCGCCG-3' were used in the polymerase chain reaction with HepG2 genomic DNA as a template under standard conditions (17). Following purification of the 364-base pair product, the fragment was restricted with unique BamHI and HindIII sites (underlined) and ligated into the same sites in the promoterless luciferase reporter pOLUC (18). The sequence was confirmed by automated sequencing and purified using ion exchange chromatography prior to transfection. Rel A expression plasmid was constructed by ligating the BamHI/HindIII fragment of the human Rel A cDNA into the expression plasmid pEGFP-C1. Expression vectors for the full-length cDNAs encoding human PKCalpha and -beta II were cloned into the appropriate episomal expression vectors in the antisense orientation (19). Antisense PKCalpha was generated by excision of the full-length PKCalpha cDNA from pGEM4 with KpnI (5') and HindIII (3') and subsequent directional cloning into the KpnI and HindIII sites of pREP10. Antisense PKCbeta II was generated by excision of the full-length PKCbeta II cDNA in pBlueBac with BamHI and KpnI and cloned into the BamHI and KpnI sites of pMEP4 in the antisense orientation.

Transfections-- HepG2 cells were transiently transfected using Lipofectin (Life Technologies, Inc.) into triplicate 60-mm plates with 3 µg of Ikappa Balpha /Luc reporter, 1 µg of SV40 driven alkaline phosphatase internal control, and 4 µg of either antisense PKCalpha plasmid or pREP4 vector according to manufacturer's recommended protocol. After 48 h, cells were stimulated with ligand for 3 h and harvested for the measurement of luciferase and alkaline phosphatase activities. Luciferase activity was determined by subtracting machine background and normalizing each plate to alkaline phosphatase activity. Fold induction was calculated by dividing treatment values by control. For the transient expression of Rel A, antisense PKCalpha or -beta II in HepG2 cells, a mixture of 2.5 µg of DNA and 20 µl of Lipofectin was added to each 60-mm plate. Cells were harvested at 48 h for immunoblot analysis. Antisense PKCbeta II stable transfection was performed using 25 µg of DNA and 30 µl of LipofectAMINE for each 100-mm plate. After 42 h, cells were cultured in 200 µg/ml hygromycin-containing growth medium for 2 months with medium change twice each week. The pMEP4 vector was taken as control. For the antisense down-regulation of PKCalpha , phosphorothioate-modified PKCalpha antisense oligodeoxynucleotide (ODN, sequence 5'-GTTCTCGCTGGTGAGTTTCA -3') and scrambled version (5'-GGTTTTACCATCGGTTCTGG-3' obtained from Genemed Biotechnologies, South San Francisco, CA) were introduced into HepG2 cells as described (20). Where indicated, transiently transfected cells were isolated following cotransfection with 5 µg of plasmid CMV.IL2R encoding the IL-2 receptor. Transient transfectants in 100-mm plates (108 cells) were purified by adding anti-human CD25 (Caltag) and captured on magnetic beads conjugated to rabbit anti-mouse IgG (Dynabeads, Dynal Inc.) as described (21, 22).

Preparation of Subcellular Extracts-- For cytosol and particulate fractionation, HepG2 cells were incubated in hypotonic buffer (20 mM HEPES, pH 7.5, 10 mM potassium acetate, 1.5 mM magnesium acetate) for 5 min on ice. Lysis was completed in a Dounce homogenizer and verified by microscopic examination. Nuclei and unbroken cells were removed by low speed centrifugation, and supernatants were centrifuged at 100,000 × g for 1 h at 4 °C in a Beckman SW 55Ti rotor. The resultant supernatants were taken as cytosolic extract, and pellets were resuspended with the equal volume of 1% Nonidet P-40-containing hypotonic buffer as particulate extract. All extracts were normalized for protein amounts determined by Coomassie G-250 staining using bovine serum albumin as a standard (Bio-Rad).

Sucrose Density-purified Nuclear Extracts-- For the purification of nuclei, HepG2 cells were resuspended in Buffer A (50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml aprotinin, and 0.5% Nonidet P-40). After 10 min on ice, the lysates were centrifuged at 4,000 × g for 4 min at 4 °C. After discarding the supernatant, the nuclear pellet was resuspended in Buffer B (Buffer A with 1.7 M sucrose) and centrifuged at 15,000 × g for 30 min at 4 °C (5). The purified nuclear pellet was then incubated in Buffer C (10% glycerol, 50 mM HEPES, pH 7.4, 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 10 µg/ml aprotinin) with frequent vortexing for 30 min at 4 °C. After centrifugation at 15,000 × g for 5 min at 4 °C, the supernatant is saved for nuclear extract. Both of cytoplasmic and nuclear extracts were normalized for protein amounts determined by Coomassie G-250 staining.

Electrophoretic Mobility Shift Assays (EMSAs)-- EMSAs were performed as described previously with minor modifications (5). Nuclear extracts (10 µg) were incubated with 40,000 cpm of 32P-labeled APRE WT duplex oligonucleotide probe and 2 µg of poly(dA-dT) in a buffer containing 8% glycerol, 100 mM NaCl, 5 mM MgCl2, 5 mM dithiothreitol, and 0.1 µg/ml phenylmethylsulfonyl fluoride in a final volume of 20 µl, for 15 min at room temperature. The complexes were fractionated on 6% native polyacrylamide gels run in 1× TBE Buffer (89 mM Tris, 89 mM boric acid, and 2.0 mM EDTA), dried, and exposed to Kodak X-AR film at -70 °C. Competition was performed by the addition of 100-fold molar excess nonradioactive double-stranded oligonucleotide competitor at the time of addition of radioactive probe. The sequences of the APRE double-stranded oligonucleotides are shown in Oligonucleotide APRE WT, M2, and M6.
<AR><R><C><UP>APRE  WT,</UP></C><C><UP>GATCCACCACAGTTGGGATTTCCCAACCTGACCA</UP></C></R><R><C></C><C><UP>            GTGGTGTCAACCCTAAAGGGTTGGACTGGTCTAG</UP></C></R><R><C><UP>APRE  M2,</UP></C><C><UP>GATCCACCACAGTTGTGATTTCACAACCTGACCA</UP></C></R><R><C></C><C><UP>            GTGGTGTCAACACTAAAGTGTTGGACTGGTCTAG</UP></C></R><R><C><UP>APRE  M6,</UP></C><C><UP>GATCCACCACATGTTGGATTTCCGATACTGACCA</UP></C></R><R><C></C><C><UP>            GTGGTGTACAACCTAAAGGCTATGACTGGTCTAG</UP></C></R></AR>
<UP><SC>Oligonucleotide</SC> APRE  WT, M2, and M6</UP>

Western Immunoblots-- For immunoblot analysis, a constant amount of indicated cellular extracts (200-300 µg as indicated) were boiled in Laemmli Buffer, separated on 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked in 8% milk and immunoblotted with the affinity purified rabbit polyclonal antibodies (Santa Cruz Biotechnology) for either Ikappa Balpha (reactive with amino acids 297-317), Rel A (reactive with amino acids 3-19), NF-kappa B1 (reactive with amino acids 350-363), PKCalpha (reactive with amino acids 651-672), PKCbeta II (reactive with amino acids 657-673), PKCdelta (reactive with amino acids 554-673), PKCepsilon (reactive with amino acids 723-737), or PKCzeta (reactive with amino acids 557-592). Anti-PKCdelta and -zeta antibodies were purchased from Upstate Biotechnology Inc., and all others were from Santa Cruz Biotechnology. Immune complexes were detected by binding donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce) followed by reaction in the enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech) according to the manufacturer's recommendations.

Northern Blots-- 50 µg of total cellular RNA was extracted from HepG2 cells using acid guanidinium/phenol extraction (RNazol B, Tel-Test Inc., Friendswood, TX), fractionated by electrophoresis on a 1% agarose-formaldehyde gel, capillary-transferred to a nylon membrane (Hybond, Amersham Pharmacia Biotech, United Kingdom), and prehybridized for 4 h in hybridization buffer (0.5 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, 1% bovine serum albumin) at 65 °C. The membrane was hybridized with 1 × 106 cpm 32P-Ikappa Balpha cDNA probe per ml overnight in the same buffer, washed three times (20 min each) with 1% SDS-containing phosphate buffer at 65 °C, and exposed to X-AR film for 24-48 h. For the 18 S RNA hybridization, the same membrane was reprobed with 32P-labeled human 18 S cDNA and briefly exposed. Bands in the autoradiogram were quantitated by exposure to Molecular Dynamics PhosphorImager cassette.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Ligand Independence of the Ikappa Balpha Overshoot Resynthesis-- A time course of nuclear NF-kappa B binding activity following TNF-alpha (30 ng/ml) stimulation in HepG2 cells is displayed in Fig. 1A to demonstrate salient features of the NF-kappa B-Ikappa Balpha autoregulatory feedback loop. Three major complexes that bind to the angiotensinogen APRE are seen by EMSA. Of relevance here, the TNF-alpha -inducible C2 complex is rapidly induced within 15 min of stimulation; we previously reported that C2 binds with NF-kappa B binding specificity and is supershifted by the addition of Rel A or NF-kappa B1 antibodies, indicating C2 is a Rel A·NF-kappa B1 heterodimer (5). Thirty minutes after TNF-alpha stimulation, C2 binding begins to diminish, and by 60 min, it is completely abolished (at subsequent times C2 binding reappears at a lower level as a result of ongoing Ikappa Balpha proteolysis before the resynthesis of Ikappa Bbeta ). Also in Fig. 1A (bottom), cytoplasmic Ikappa Balpha abundance is determined by Western immunoblot under conditions where we have shown that the Ikappa Balpha signal is linear to input protein (23). Ikappa Balpha , present in unstimulated cell cytoplasm, completely disappears 15 min following TNF-alpha administration. At 30 min following TNF-alpha stimulation, Ikappa Balpha begins to be resynthesized, and resynthesis peaks after 60 min to levels ~2-fold greater than that observed in control cells. In previous studies, at 60 min, all of the resynthesized Ikappa Balpha is associated with Rel A as determined by nondenaturing coimmunoprecipitation assay, and inhibition of its resynthesis blocks the 60 min termination of C2 binding (5).


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Fig. 1.   A, time course of TNF-alpha -induced NF-kappa B binding and Ikappa Balpha overshoot resynthesis. Top, autoradiogram of EMSA using 10 µg of nuclear protein prepared from cultured HepG2 cells stimulated for the various times (minutes) with 30 ng/ml TNF-alpha binding to the radiolabeled angiotensinogen NF-kappa B-binding site (APRE WT, see "Experimental Procedures"). The bound complexes are shown. C2, the Rel A·NF-kappa B1 complex (5), is rapidly induced with biphasic kinetics, the first peak at 15 and 30 min, and by 60-min nadirs. Relative to control values, C2 binding increases 5.1- (15 min), 4.9- (30 min), 0.6- (60 min), 4- (120 min), and 3-fold (360 min). Bottom, Western immunoblot of 37-kDa cytoplasmic Ikappa Balpha from the same samples (Ikappa Balpha is not detectable in the nuclear fraction (5)). Ikappa Balpha is rapidly degraded at 15 min and subsequently is resynthesized, peaking at 60 min (corresponding to the nadir in NF-kB binding). Relative to control values, Ikappa Balpha is 0.008- (15 min), 0.7- (30 min), 2.2- (60 min), 1.6- (120 min), and 1-fold (360 min). B, time course of IL-1alpha -induced NF-kappa B binding and Ikappa Balpha overshoot resynthesis. Cells were stimulated with 4 ng/ml IL-1alpha for indicated times. Top, autoradiogram of EMSA (as described in A). Relative to control values, C2 binding increases 42- (15 min), 38- (30 min), 1.3- (60 min), 28- (120 min), and 20-fold (360 min). Bottom, Western immunoblot of cytoplasmic Ikappa Balpha from same samples. Ikappa Balpha is rapidly degraded at 15 min and resynthesized thereafter, concomitantly with the appearance of a phosphorylated isoform migrating ~3 kDa slower seen at the 30- and 60-min time points. Relative to control values, Ikappa Balpha is 0.6- (30 min), 1.7- (60 min), 0.9- (120 min), and 1.2-fold (360 min). C, time course of PMA-induced NF-kappa B binding and Ikappa Balpha overshoot resynthesis. Cells were stimulated with 1 µM PMA for indicated times. Top, autoradiogram of EMSA (A). Relative to control values, C2 binding increases 12- (15 min), 12- (30 min), 9- (120 min), and 9-fold (360 min). The 60-min point is undetectable. Bottom, Western immunoblot of cytoplasmic Ikappa Balpha from same samples. Relative to control values, Ikappa Balpha is 0.2- (15 min), 0.3- (30 min), 1.2- (60 min), 0.6- (120 min), and 0.8-fold (360 min).

In comparison to that produced by TNF-alpha , we next determined whether the phenomenon of Ikappa Balpha overshoot resynthesis occurs following treatment with IL-1alpha (4 ng/ml, Fig. 1B). IL-1alpha is a more potent inducer of C2 complex; however, the 60-min nadir in the Rel A·NF-kappa B1 binding is produced concomitantly with Ikappa Balpha overshoot resynthesis. The same phenomenon occurs with the PKC activator, PMA (1.0 µM, Fig. 1C), where a nadir in Rel A·NF-kappa B1 binding is also seen concomitantly with the Ikappa Balpha overshoot resynthesis at 60 min. Together, these data indicate that the overshoot in Ikappa Balpha resynthesis is ligand-independent.

The mechanism underlying the overshoot in Ikappa Balpha resynthesis was investigated by measurement of relative changes in steady-state Ikappa Balpha mRNA abundance by Northern blot analysis (Fig. 2). In control cells, Ikappa Balpha mRNA is detectable as a 1.8-kilobase pair transcript. For each treatment condition, TNF-alpha (Fig. 2A) and IL-1alpha (Fig. 2B), or PMA (Fig. 2C), Ikappa Balpha mRNA was detectable in control cells and rapidly depleted at 15 min, perhaps indicating the presence of translation-coupled degradation (24). However, Ikappa Balpha mRNA was robustly increased at 60 min, a time point when the peak in Ikappa Balpha resynthesis occurs, and gradually decays over the next 5 h. These data indicate that the Ikappa Balpha overshoot resynthesis is dependent on the enhanced expression of Ikappa Balpha mRNA.


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Fig. 2.   Mechanism for Ikappa Balpha overshoot resynthesis. A, Northern blot analysis of 50 µg of total cellular RNA isolated from HepG2 cells stimulated for the indicated times with 30 ng/ml TNF-alpha . Top, autoradiogram following hybridization for Ikappa Balpha . The 1.8-kilobase pair mRNA is shown. Bottom, autoradiogram following rehybridization of the same membrane for 18 S ribosomal RNA (18 S). For each time point, the Ikappa Balpha /18 S hybridization ratio was determined and expressed relative to control values. Ikappa Balpha /18 S increases 1.2- (15 min), 2.9- (30 min), 3-(60 min), 1.5- (120 min), and 2.3-fold (360 min). B, Northern blot analysis following stimulation with 4 ng/ml IL-1alpha . Experiment shown is as described in A. Relative to control values, Ikappa Balpha /18 S increases 0.2- (15 min), 0.9- (30 min), 3.4- (60 min), 1.8- (120 min), and 0.8-fold (360 min). C, Northern blot analysis following stimulation with 1 µM PMA. Experiment shown is as described in A. Relative to control values, Ikappa Balpha /18 S increases 0.4- (15 min), 0.4- (30 min), 1.7- (60 min), 0.8- (120 min), and 0.9-fold (360 min). D, Rel A activates Ikappa Balpha expression. HepG2 cells were transfected with IL-2 receptor expression plasmid in the absence (-) or presence of Rel A expression plasmid (Rel A). Transient transfectants were subsequently enriched using anti-IL-2 receptor antibody captured on magnetic beads (see "Experimental Procedures"). Shown is a Western immunoblot for Ikappa Balpha and as control NF-kappa B1. Relative to empty expression plasmid, Rel A activates Ikappa Balpha protein abundance 8.5-fold.

Other studies have shown that cytokines are potent activators of Ikappa Balpha expression through a mechanism dependent on NF-kappa B translocation (9, 11, 25). To confirm this effect in HepG2 cells, the effect of transactivator subunit (Rel A) expression on Ikappa Balpha abundance was determined in transient overexpression assays. We observed that Rel A expression strongly induced Ikappa Balpha protein abundance (Fig. 2D), indicating that NF-kappa B is sufficient for activation of the autoregulatory feedback loop.

Phorbol Ester-sensitive PKC Isoforms Are Involved in the Kinetics of Ikappa Balpha Overshoot Resynthesis-- Because both TNF-alpha and IL-1alpha cytokines have been shown to activate PKC activity (16), we explored the PKC dependence for Ikappa Balpha overshoot resynthesis. For this, DAG-sensitive PKC isoforms were down-regulated by chronic exposure to PMA prior to cytokine stimulation (Fig. 3). Following 15 min stimulation with either TNF-alpha or IL-1alpha , Rel A·NF-kappa B1 binding was strongly induced and Ikappa Balpha proteolyzed, indicating signals necessary for initial Rel A·NF-kappa B1 translocation are PKC-independent (16). Surprisingly, however, we observed the 60-min nadir in Rel A·NF-kappa B1 was eliminated by PKC down-regulation for either cytokine. Moreover, in PKC down-regulated cells, although Ikappa Balpha resynthesized, its reaccumulation was gradual, without exceeding control levels at the 60-min time point. As expected, acute exposure of PKC down-regulated cells to PMA produced no induction of Rel A·NF-kappa B1 binding activity or Ikappa Balpha proteolysis (Fig. 3C). These data indicate a requirement for PMA-sensitive PKC isoforms for the rapid kinetics of TNF-alpha and IL-1alpha -induced Ikappa Balpha resynthesis.


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Fig. 3.   Phorbol ester-sensitive PKC isoforms are required for cytokine-induced Ikappa Balpha overshoot resynthesis. DAG-sensitive PKC isoforms were down-regulated by chronic agonist exposure prior to stimulation. A, time course of TNF-alpha stimulation. Top, autoradiogram of EMSA following 30 ng/ml TNF-alpha (described in Fig. 1A). C2, the Rel A·NF-kappa B1 complex (5), is rapidly induced and declines with a monophasic profile, peaking at 15 and 30 min and declining thereafter without a detectable nadir. Relative to control values, C2 binding increases 7.2- (15 min), 6.6- (30 min), 4.4- (60 min), 3.5- (120 min), and 3-fold (360 min). The nadir in C2 binding, previously seen at 60 min, is absent. Bottom, Western immunoblot of cytoplasmic Ikappa Balpha from the same samples. The overshoot resynthesis of Ikappa Balpha , previously seen at 60 min, is absent. Relative to controls, Ikappa Balpha abundance decreases 0.01- (15 min), 0.8- (30 min), 1.3- (60 min), 1.4- (120 min), and 1.5-fold (360 min). B, time course of IL-1alpha stimulation. Top, autoradiogram of EMSA following 4 ng/ml IL-1alpha (described in Fig. 1A). Rel A-NF-kappa B1 binding (C2) similarly follows a monophasic profile, peaking at 15 min and declining thereafter without a detectable nadir. Relative to control values, C2 binding increases 6- (15 min), 5.3- (30 min), 4- (60 min), 1.5- (120 min), and 1.7-fold (360 min). The 60-min nadir in C2 binding is similarly absent. Bottom, Western immunoblot of cytoplasmic Ikappa Balpha from the same samples. The overshoot resynthesis of Ikappa Balpha is absent. Relative to controls, Ikappa Balpha abundance decreases 0.01- (15 min), 0.07- (30 min), 0.8- (60 min), 1- (120 min), and 1.3-fold (360 min). C, time course of PMA stimulation. Top, autoradiogram of EMSA following 1 µM PMA (as described in Fig. 1A). Bottom, Western immunoblot of cytoplasmic Ikappa Balpha from the same samples. No significant induction of C2 binding or change in Ikappa Balpha abundance was detected, indicating that DAG-activated PKC isoforms were completely hydrolyzed and required for NF-kappa B activation.

Effect of TNF-alpha and IL-1alpha on Particulate-Cytosol Redistribution of PKC Isoforms-- Following PKC activation, cytosol-particulate (membrane) redistribution and degradation occurs. To tentatively identify PKC isoforms involved in Ikappa Balpha resynthesis, the effect of cytokines on subcellular distribution of various PKC isoforms was determined. At various times following cytokine stimulation, HepG2 cells were fractionated into cytosolic and particulate fractions by ultracentrifugation (see "Experimental Procedures"), and PKC isoform abundance was determined by Western immunoblot (Fig. 4A). Unstimulated HepG2 cells express cPKC isoforms, PKCalpha and PKCbeta II, the nPKC isoforms, PKCdelta and PKCepsilon , and the atypical PKC (aPKC) isoform PKCzeta (Fig. 4A) (26). All of these isoforms were distributed in both the cytosolic and particulate fractions, except PKCbeta II (which was primarily distributed in the particulate fraction).


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Fig. 4.   PKC isoforms activated by cytokines and PMA. A, Western immunoblot for PKC isoforms distribution in subcellular fractions. HepG2 cells were stimulated with TNF-alpha or IL-1alpha and fractionated into cytosolic (Cytosol) or particulate (Particulate) fractions at various times (indicated at top). Samples (200 µg) were fractionated, and changes in PKC isoform abundance were detected by immunoblot using indicated primary antibody (at left). In unstimulated cells, 78-kDa PKCbeta II (beta ) is primarily detected in the particulate fraction. Distributed in both cytosol and particulate fractions are 80-kDa PKCalpha (alpha ), 70-kDa PKCdelta (delta ), 72-kDa PKCepsilon (epsilon ), and 76-kDa PKCzeta (zeta ). IL-1alpha induced PKCalpha degradation in the cytosol fraction (relative to control, PKCalpha declined to 0.6 (5 min), 0.3 (15 min), and 0.3 (45 min)). Both TNF-alpha and IL-1alpha induced a rapid PKCalpha translocation to the particulate fraction at 5 min of 6- and 13-fold (respectively). IL-1alpha also induced a later degradation of PKCalpha in the particulate fraction where PKCalpha abundance declined to 9.5-fold (15 min) and 5.4-fold (45 min). Because IL-1alpha induces initial PKCalpha cytosol-particulate redistribution and degradation in both compartments, these data indicate IL-1alpha activates PKCalpha in HepG2 cells. B, effect of agonist exposure on PKC isoform abundance. Western immunoblot of cytosol and particulate fractions (200 µg) from control and PMA down-regulated HepG2 cells. PMA down-regulates cytosolic and particulate PKCalpha , particulate PKCbeta II, and cytosolic PKCdelta . No significant effects were seen on the PKCepsilon or PKCzeta .

Cytokine-induced changes were observed for PKC isoforms alpha , delta , and zeta . Although TNF-alpha had no consistent effect on cytosolic PKCalpha , IL-1alpha induced rapid cytosolic PKCalpha degradation, initially detectable after 15 min stimulation. Treatment with either TNF-alpha or IL-1alpha induced translocation of PKCalpha to the particulate fraction within 5 min after stimulation. In contrast to TNF-alpha , IL-1alpha also induced particulate PKCalpha degradation after 15 min of stimulation. Taken together, these data indirectly indicate that IL-1alpha is a potent activator of PKCalpha . No significant effect by either cytokine was seen on particulate PKCbeta II. In contrast, following either TNF-alpha or IL-1alpha stimulation, PKCdelta also was degraded primarily in the cytosolic fraction and weakly accumulated in the particulate fraction. In addition, following IL-1alpha treatment, PKCzeta was degraded only in the particulate fraction.

A similar analysis was performed to determine the effect of PKC isoforms hydrolyzed by chronic PMA exposure (Fig. 4B). Chronic exposure to PMA significantly hydrolyzed PKCalpha (both in cytosolic and particulate fractions), PKCbeta II, and PKCdelta (in the cytosolic fraction). Together, these data indicate PKCalpha , -beta II, and -delta as candidate isoforms mediating the cytokine-induced Ikappa Balpha overshoot resynthesis.

cPKC Inhibitors Block IL-1alpha -induced Ikappa Balpha Overshoot-- As additional evidence for the role of PKC isoforms, the potent and selective PKC inhibitors bisindolylmaleimide I (Gö 6850) and Gö 6976 were tested for their effects on Ikappa Balpha overshoot resynthesis at concentrations that inhibit cPKC isoforms (27). These agents are highly selective inhibitors for PKC by competitive inhibition of the ATP-binding site, without effect on tyrosine, cAMP-dependent protein, myosin, or phosphorylase kinases (27). Gö 6850 inhibits cPKC and, at higher concentrations, nPKC isoforms (27). In these experiments, Gö 6850 was used in concentrations that selectively inhibit the cPKC isoforms. Gö 6850 had no effect on TNF-alpha -induced Ikappa Balpha overshoot but completely blocked the effects of IL-1alpha (Fig. 5A). As a control for its inhibitory effect, Gö 6850 blocked the ability of PMA to induce C2 binding and Ikappa Balpha proteolysis (Fig. 5A). Gö 6976 is a selective inhibitor of cPKC isoforms (28). Similarly, Gö 6976 interfered with the IL-1alpha -induced Ikappa Balpha overshoot but not that produced by TNF-alpha (Fig. 5B). Calphostin C, an inhibitor of the aPKC isoforms by binding to the unique regulatory domain (without effect on cPKC isoforms (29)) was next tested to determine whether aPKC plays a role in the autoregulatory feedback loop. Calphostin C had no effect on either NF-kappa B activation or Ikappa Balpha overshoot resynthesis. Together, these data indicate that cPKC isoforms (PKCalpha or beta II) influence the kinetics of IL-1alpha -induced Ikappa Balpha resynthesis (and transient NF-kappa B termination) in hepatocytes.


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Fig. 5.   Effect of selective PKC inhibitors on cytokine-induced NF-kappa B binding and Ikappa Balpha overshoot resynthesis. Top, autoradiogram of EMSA. Bottom, Western immunoblot of Ikappa Balpha from same samples. A, effect of cPKC and nPKC inhibitor, bisindolylmaleimide I (Gö 6850). Cells were preincubated with 3 µM Gö 6850 prior to stimulation for various times with indicated ligands (30 ng/ml TNF-alpha , 4 ng/ml IL-1alpha , and 1 µM PMA). C2 binding (relative to control) following TNF-alpha stimulation is increased 2.4- (15 min) and 1.2-fold (60 min); following IL-1alpha stimulation is increased 2.6- (15 min) and 2.0-fold (60 min); following PMA stimulation is increased 0.6- (15 min) and 0.6-fold (60 min). Ikappa Balpha abundance (at 60 min relative to control) following TNF-alpha stimulation is increased 1.6-fold, following IL-1alpha stimulation is 1-fold, and following PMA is increased 0.8-fold. Gö 6850 blocks IL-1alpha -induced Ikappa Balpha overshoot resynthesis but not that induced by TNF-alpha . B, effect of cPKC inhibitor, Gö 6976. Cells were preincubated with 3 µM Gö 6976 prior to stimulation for various times with indicated ligands (30 ng/ml TNF-alpha , 4 ng/ml IL-1alpha , and 1 µM PMA). C2 binding (relative to control) following TNF-alpha stimulation is 4.6- (15 min) and 1.5-fold (60 min), following IL-1alpha stimulation is 5.2- (15 min) and 5-fold (60 min), and following PMA stimulation is 3.2- (15 min) and 3.4-fold (60 min). Ikappa Balpha abundance (at 60 min relative to control) following TNF-alpha stimulation is 1.1-fold, following IL-1alpha stimulation is 0.3-fold, and following PMA is 0.7-fold. Gö 6976 partially blocks PMA-induced NF-kappa B activation and Ikappa Balpha proteolysis. In addition, Gö 6976 blocks IL-1alpha -induced Ikappa Balpha overshoot resynthesis but not that induced by TNF-alpha . C, effect of aPKC inhibitor, calphostin C. Cells were preincubated with 0.2 µM calphostin C prior to stimulation for various times with indicated ligands (30 ng/ml TNF-alpha , 4 ng/ml IL-1alpha , and 1 µM PMA). Calphostin C has no effect on NF-kappa B activation, Ikappa Balpha proteolysis, or Ikappa Balpha overshoot resynthesis by any ligand, making a contribution of atypical PKC isoforms unlikely.

Effect of PKCalpha Down-regulation on IL-1alpha -induced Ikappa Balpha Overshoot Resynthesis-- To isolate selectively the contribution of PKCalpha in IL-1alpha signaling, we initially sought to make stable HepG2 cells with a down-regulated PKCalpha isoform by constitutive expression of a PKCalpha antisense expression plasmid. However, we were unable to isolate PKCalpha -deficient cells in numerous attempts, even though we were successful in down-regulating the PKCbeta II isoform, perhaps indicating an important role for PKCalpha in permitting cell proliferation. As an alternative approach, we transiently down-regulated PKCalpha by introducing phosphorothioate-modified antisense oligodeoxynucleotides (ODN) using liposome-mediated transfection (20). In preliminary experiments, a time course for optimal PKCalpha down-regulation following introduction of the antisense PKCalpha ODN was performed. Peak inhibition of PKCalpha was observed at 24 h (Fig. 6A). At this time point, the antisense ODN inhibited expression of PKCalpha by 81%, whereas the control scrambled ODN affected PKCalpha abundance by less than 11%. Following PKCalpha down-regulation, the effect of TNF-alpha and IL-1alpha on the Ikappa Balpha overshoot resynthesis was determined (Fig. 6B). PKCalpha was required for IL-1alpha - induced but not the TNF-alpha -induced Ikappa Balpha overshoot. To exclude an additional role of PKCbeta II, HepG2 cells deficient in PKCbeta II were generated (Fig. 6C). Down-regulation of PKCbeta II had no effect on either the TNF-alpha - or IL-1alpha -induced overshoot (Fig. 6D). Together, these data strongly implicate a requirement for PKCalpha in mediating Ikappa Balpha overshoot resynthesis selectively for IL-1alpha .


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Fig. 6.   Effects of down-regulating PKC isoforms on cytokine-induced Ikappa Balpha overshoot resynthesis. A, changes in steady-state PKCalpha abundance following antisense phosphorothioate ODN treatment. HepG2 cells were treated with antisense or scrambled phosphorothioate ODN (see "Experimental Procedures"). Whole cell lysates were prepared and analyzed by Western immunoblot for PKCalpha . PKCalpha abundance was 0.2-fold (antisense) and 0.9-fold (scrambled) that seen in control HepG2 cells. B, effect of PKCalpha down-regulation on cytokine-induced Ikappa Balpha overshoot resynthesis. HepG2 cells were treated with antisense or scrambled ODN (as in A) and stimulated with indicated cytokine for various times (top). Western immunoblot is shown. Top, Ikappa Balpha antibody. Bottom, internal control Rel B. For antisense-treated cells stimulated with TNF-alpha , Ikappa Balpha abundance increased 3-fold at 60 min, whereas following IL-1alpha treatment, Ikappa Balpha resynthesis was blocked (0.3-fold at 60 min). In the scrambled ODN-treated cells, Ikappa Balpha increased to 4.7- and 5.9-fold at 60 min for both TNF-alpha and IL-1alpha , respectively. These data indicate PKCalpha is required for IL-1alpha -induced Ikappa Balpha overshoot resynthesis. C, down-regulation of PKCbeta II. HepG2 cells were transfected with PKCbeta II antisense expression plasmid (AS PKCbeta II) or empty expression plasmid (pMEP4) and stably selected with hygromycin. Western immunoblot of total cytoplasmic lysate using primary anti-PKCbeta II antibody (top) or internal control Rel B (bottom). D, effect of PKCbeta II down-regulation on cytokine-induced Ikappa Balpha overshoot resynthesis. Stably transfected HepG2 cells (shown in C) were stimulated with TNF-alpha (top) or IL-1alpha (bottom) for various times (minutes, indicated at top) prior to extraction of cytoplasmic protein. Shown is a Western immunoblot for Ikappa Balpha . Down-regulation of PKCbeta II does not interfere with TNF-alpha - or IL-1alpha -induced Ikappa Balpha overshoot resynthesis.

TNF-alpha and IL-1alpha Activate Ikappa Balpha Transcription in a PKCalpha -independent Manner-- The effect of PKCalpha in blocking the Ikappa Balpha overshoot could be the result of inhibition of Ikappa Balpha gene expression. As shown earlier (Fig. 2D), Ikappa Balpha expression is dependent on Rel A translocation. To exclude the trivial possibility that IL-1alpha -induced Rel A translocation is dependent on PKCalpha , a Western immunoblot for changes in the Rel A isoform was determined under conditions where the signal is proportional to input protein (5). Following addition of IL-1alpha in the absence or presence of PKC inhibitors (Gö 6976 or Gö 6850) or following PKC down-regulation, equivalent amounts of Rel A translocated into the nucleus (data not shown). To demonstrate additionally that TNF-alpha or IL-1alpha affect Ikappa Balpha promoter expression independent of PKCalpha activity, transient transfections of the Ikappa Balpha promoter linked to luciferase reporter gene were performed in the presence of PKCalpha antisense expression plasmid. TNF-alpha induced a 3.8-fold and IL-1alpha induced a 7.5-fold increase in Ikappa Balpha promoter activity (Fig. 7A). The level of stimulation was not affected by transient PKCalpha down-regulation. Demonstration that PKCalpha was down-regulated is presented in Fig. 7B.


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Fig. 7.   IL-1alpha activates Ikappa Balpha transcription independently of PKCalpha . A, PKCalpha -independent activation of the Ikappa Balpha promoter. HepG2 cells were transfected with Ikappa Balpha promoter/luciferase reporter in the absence (pREP4) or presence of antisense coding sequences for PKCalpha (AS PKCalpha ) and stimulated with nothing (con), 30 ng/ml TNF-alpha , 4 ng/ml IL-1alpha , or 1 µM PMA as indicated. Cells were harvested after 6 h, and reporter activity was normalized to control values. All three agonists are inducers of Ikappa Balpha transcription independently of PKCalpha . B, transient down-regulation of PKCalpha by antisense expression vector. HepG2 cells were transiently transfected with IL-2 receptor expression plasmid and empty expression vector (pREP4), antisense PKCalpha (AS PKCalpha ), or antisense PKCbeta II (AS PKCbeta II). Transient transfectants were isolated by affinity purification using anti-IL-2 receptor antibody (see "Experimental Procedures"). Shown is a Western immunoblot for steady-state PKCalpha (top) or internal control Rel B (bottom). C, cytokines induce equivalent changes in Ikappa Balpha mRNA in the presence of PKC inhibitors. HepG2 cells were stimulated with TNF-alpha or IL-1alpha pretreated in the absence (-) or presence of chronic agonist stimulation (PMA-20 h) or the PKC inhibitors Gö 6850 or Gö 6976 as indicated at top. Shown is autoradiogram of Northern blot hybridization of Ikappa Balpha mRNA (top) or 18 S ribosomal RNA (bottom). Relative to control values, TNF-alpha increased Ikappa Balpha mRNA levels by 2.4- (no inhibitor), 2.1- (PMA-20 h), 2.2- (Gö 6850), and 3.4-fold (Gö 6976). IL-1alpha increased Ikappa Balpha mRNA levels by 5.9- (no inhibitor), 6.5- (PMA-20 h), 6.2- (Gö 6850), and 6.0-fold (Gö 6976). Stimulation in the presence of PKC inhibitor has no effect on the magnitude of Ikappa Balpha mRNA induction.

Similarly, Northern blot analysis was done to determine the effect of PKC inhibition on cytokine-induced steady-state Ikappa Balpha mRNA expression. Following TNF-alpha administration, Ikappa Balpha mRNA increased by 2.5-fold (Fig. 7C). The induction by TNF-alpha was not altered by presence of PKC inhibitors (Gö 6976 or Gö 6850) or following PKC down-regulation. Similarly, IL-1alpha induced a 6-fold increase in steady-state Ikappa Balpha mRNA that was not affected by PKC inhibitors or down-regulation. Together, these data indicate IL-1alpha produced similar levels of Rel A translocation and Ikappa Balpha gene expression in the absence or presence of PKCalpha .

Post-transcriptional Role of PKC Is, in Part, through Facilitation of Ikappa Balpha mRNA Nuclear Export-- Because equivalent amounts of steady-state Ikappa Balpha mRNA were produced following IL-1alpha stimulation in the absence or presence of PKCalpha , we concluded that PKCalpha apparently plays a role in post-transcriptional control of Ikappa Balpha resynthesis. One potential mechanism, previously described to control Ikappa Balpha resynthesis in monocytes, was at the level of mRNA decay in the nucleus (24). To determine the relative distribution of Ikappa Balpha RNA, RNA from cytoplasmic and nuclear fractions from control and IL-1alpha -stimulated HepG2 cells were analyzed for changes in Ikappa Balpha abundance by Northern blot (Fig. 8). Following IL-1alpha stimulation, Ikappa Balpha mRNA primarily increased in the cytoplasmic fraction with 15% of the total signal detected in the nuclear compartment. However, IL-1alpha stimulation in cells following PKC down-regulation resulted in detectable accumulation of nuclear Ikappa Balpha RNA. In this case, 32% of the total signal was nuclear. These data indicate a potential role for DAG-sensitive PKC isoforms in controlling Ikappa Balpha RNA export.


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Fig. 8.   Post-transcriptional mechanism for Ikappa Balpha overshoot resynthesis. Effect on Ikappa Balpha mRNA nuclear-cytoplasmic distribution. HepG2 cells were stimulated with IL-1alpha (lanes 3-6) pretreated in the absence (lanes 3 and 4) or presence of chronic agonist stimulation (lanes 5 and 6). Lanes 1 and 2 are control cells. Cells were fractionated into nuclear (lanes 2, 4, and 6) and cytoplasmic (lanes 1, 3, and 5) fractions. Equivalent cell numbers were analyzed for Ikappa Balpha mRNA abundance by Northern blot hybridization. Shown is autoradiogram following hybridization to Ikappa Balpha mRNA probe (top) or 18 S ribosomal RNA probe (bottom). For each time point, nuclear 18 S RNA signal is ~50% that of the cytoplasmic signal. Following IL-1alpha treatment, a strong 6-fold increase in cytoplasmic Ikappa B abundance is seen. Following normalization to 18 S signal, nuclear Ikappa Balpha RNA abundance increases 7.2-fold in IL-1alpha treated cells and 17.7-fold in IL-1alpha treated/PKC down-regulated cells.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

IL-1alpha is a proinflammatory cytokine that plays an important role in initiating the cytokine cascade in sepsis and inflammation. This peptide exerts its biological effects by binding a single transmembrane spanning receptor on diverse cell types (30). Following activation of the IL-1 receptor, cytoplasmic proteins associate with the cytoplasmic tail, generating second messengers and activating intracellular kinases (31-33). An important intracellular target for NF-kappa B-activating agents is the Ikappa B signalsome, a multisubunit complex of proteins responsible for the phosphorylation of Ikappa B. IL-1alpha - and TNF-alpha -induced signals converge on the NF-kappa B-inducing kinase, a protein complexed with the Ikappa B kinase IKKalpha (Refs. 6 and 31 and references therein). Following Ikappa B phosphorylation the inhibitor is targeted for proteolytic degradation. In hepatocytes, we have shown that IL-1alpha and TNF-alpha activate the NF-kappa B transcription factor in a process involving its nuclear translocation coincidentally with Ikappa B proteolysis (5, 34, 35). Once translocated into the nucleus, NF-kappa B-responsive (APR and cytokine) genes are inducibly transcribed.

Following NF-kappa B translocation, the ikappa balpha gene is also expressed, resulting in its rapid resynthesis. Resynthesized Ikappa Balpha captures nuclear NF-kappa B protein and redistributes it back into the cytoplasm to restore cellular homeostasis, resulting in what is called the NF-kappa B-Ikappa Balpha autoregulatory pathway. Studies by others (9-11) have shown that an important component of this autoregulatory pathway depends on transcriptional activation of the Ikappa Balpha promoter. The marked induction of Ikappa Balpha promoter activity following cytokine stimulation dependent on NF-kappa B-binding sites and enhanced Ikappa Balpha expression following Rel A overexpression are in support of this transcriptional component (10, 11). However, post-transcriptional control mechanisms of Ikappa Balpha expression are also important. Here we report evidence for a multistep mechanism for NF-kappa B-Ikappa Balpha autoregulatory pathway induced by IL-1alpha in hepatocytes. We demonstrate the unanticipated findings that IL-1alpha -induced Ikappa Balpha resynthesis also requires the participation of activated PKCalpha . IL-1alpha stimulation following selective antagonists of PKCalpha results in nuclear retention of a fraction of Ikappa Balpha mRNA.

PKC are a family of multifunctional serine-threonine lipid-dependent kinases grouped broadly into three families based on structural and functional criteria. The calcium-dependent or classic cPKC isoforms (alpha , beta , gamma ), novel PKC isoforms (delta , epsilon , eta , theta ), and atypical PKC isoforms (zeta , iota , lambda ) fulfill distinct, nonredundant functions in a cell involved in intracellular signaling and cell proliferation (19). We have shown that HepG2 cells express the cPKC (alpha , beta II), nPKC (delta , epsilon ), and aPKC (zeta ) isoforms, confirming observations of others (26). Our data extend these observations to show that several of these PKC isoforms are responsive to cytokine stimulation. PKCalpha is apparently activated by IL-1alpha because IL-1alpha induces cytosol-to-particulate translocation of PKCalpha and, later, its proteolysis. PKCdelta is also apparently activated by either TNF-alpha or IL-1alpha , as the protein is rapidly degraded in the cytoplasm.

Here we report that selective down-regulation of PKCalpha affects intracellular signaling produced by IL-1alpha but not TNF-alpha . However, the Ikappa B overshoot produced by TNF-alpha is influenced by PKC down-regulation, probably indicating that a different PMA-sensitive PKC isoform, perhaps PKCdelta , subserves the role that PKCalpha plays in IL-1 signaling. The role of PKCdelta in TNF-alpha signaling will require additional investigation. In a variety of cell types, IL-1alpha induces generation of a variety of lipid-derived second messenger including ceramide, DAG, and arachidonic acid (33). In IL-1alpha -stimulated T-lymphocytes, DAG is rapidly produced as a consequence of phosphatidylcholine hydrolysis, peaking 1 min after addition of ligand (33). Of the known second messengers that activate PKCalpha , DAG is likely to be an important mediator because IL-1alpha does not induce measurable changes in bulk intracellular calcium concentrations (33). The second messenger(s) responsible for IL-1alpha activation of PKCalpha in hepatocytes will require further study.

In other studies, nuclear processing of Ikappa Balpha mRNA was proposed to be important in regulating Ikappa Balpha protein levels following monocyte adherence (24). In these cells, Ikappa Balpha mRNA is predominantly nuclear. This contrasts with our observations in hepatocytes where in both unstimulated and cytokine-stimulated hepatocytes, Ikappa Balpha mRNA is predominantly cytoplasmic. However, in PKC down-regulated cells, retention of newly synthesized nuclear Ikappa Balpha mRNA is observed. By its indistinguishable size with cytoplasmic mRNA, the nuclear Ikappa Balpha RNA is apparently spliced and polyadenylated, but this will require direct demonstration. Together these observations indicate that Ikappa Balpha nuclear processing may be a general mechanism for control of its cytoplasmic abundance. The role for PKCalpha in IL-1alpha -induced Ikappa Balpha resynthesis is apparently at a post-transcriptional level, mediated in part through facilitating rapid kinetics of mRNA export. Whether other potential mechanisms play a role in Ikappa Balpha resynthesis, such as efficiency of translational initiation, have not been directly tested in these studies, and their additional contribution(s) cannot be excluded. In eukaryotic cells, nuclear export of mRNA is dependent on the association with RNA-binding proteins. Apparently there are requirements for helicases (to disassociate the nascent RNA from spliceosome complex) and "zipcode" RNA-binding proteins (to form suitable ribonucleoprotein complexes) for mRNA to exit the nuclear pore (36). Presumably some component of this pathway is influenced by PKCalpha activation. Both functional (16) and immunoblot data (presented here, see Fig. 5) indicate that, following cytokine stimulation, PKCalpha translocates to the particulate (membrane) fraction. In this compartment, membrane PKC activity may regulate the proteins or motors controlling nuclear mRNA export.

Although our study focuses on Ikappa Balpha expression as a model for understanding intracellular mechanisms in IL-1alpha signaling, other pathways are required for complete termination of NF-kappa B activity. Our data indicate that activation of the Ikappa Balpha autoregulatory feedback loop only transiently terminates NF-kappa B signaling. Following its overshoot resynthesis, Ikappa Balpha undergoes a second wave of proteolysis allowing liberation of NF-kappa B and its subsequent re-entry into the nucleus after 2 h, probably indicating that the inducible proteolytic pathway is still active at this time. However, we note that NF-kappa B binding activity observed following Ikappa Balpha resynthesis is significantly reduced from that observed immediately after Ikappa Balpha proteolysis (cf. Fig. 1, A-C) indicating that resynthesized Ikappa Balpha sequesters and removes a significant component of NF-kappa B from the nuclear compartment. This is in apparent contradiction to the absolute requirement of Ikappa Balpha in termination of NF-kappa B activity in ikappa balpha -/- fibroblasts (13) and may be a consequence of differences in cell type as the spectrum and regulation of the Ikappa B inhibitors is cell type-dependent (14, 23). Additional studies will be required to determine the later mechanism for NF-kappa B termination in hepatocytes.

NF-kappa B is a signaling molecule important in mediating cytokine and acute phase reactant gene expression in a variety of inflammatory pathophysiological conditions. Understanding the mechanisms involved in termination of NF-kappa B activity may identify novel target for anti-inflammatory therapeutics. Here we describe a ligand-dependent role for PKCalpha in controlling subcellular distribution of Ikappa Balpha mRNA during the NF-kappa B-Ikappa Balpha autoregulatory feedback loop. We hypothesize that novel anti-inflammatory therapeutics could be designed that modulate the Ikappa Balpha mRNA nuclear export pathway.

    ACKNOWLEDGEMENTS

Plasmid CMV.IL2R was a generous gift of B. Howard and R. Padmanabhan, NICHD, National Institutes of Health.

    FOOTNOTES

* This work was supported by Grant 1R01 55630-01A2 from NHLBI, National Institutes of Health (to A. R. B.), Grant 4017 from the Council for Tobacco Research (to A. R. B), and Grant P30 ES06676 from NIEHS, National Institutes of Health (to R. S. Lloyd, University of Texas Medical Branch).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Established Investigator of the American Heart Association. To whom correspondence should be addressed: University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1060. Tel.: 409-772-2824; Fax: 409-772-8709; E-mail: arbrasie{at}utmb.edu.

The abbreviations used are: APR, acute-phase response; aPKC, atypical PKC isoform; APRE, acute-phase response element; cPKC, calcium-regulated PKC isoform; DAG, diacylglycerol; EMSA, electrophoretic mobility shift assay; Ikappa B, inhibitor of NF-kappa B; IL-1alpha , interleukin-1alpha ; NF-kappa B1, nuclear factor-kappa B 50-kDa subunit; nPKC, novel PKC isoform; PKC, protein kinase C; Rel A, NF-kappa B 65-kDa subunit; TNF-alpha , tumor necrosis factor alpha ; PMA, phorbol 12-myristate 13-acetate; WT, wild type; ODN, oligodeoxynucleotides.
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Abstract
Introduction
Procedures
Results
Discussion
References

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