Role of glycogen synthase kinase-3 in TNF-alpha -induced NF-kappa B activation and apoptosis in hepatocytes

Robert F. Schwabe and David A. Brenner

Department of Medicine, Biochemistry, and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear factor-kappa B (NF-kappa B) prevents hepatocytes from undergoing apoptosis during development and liver regeneration. Mice with inactivated glycogen synthase kinase (GSK)-3beta die from hepatocyte apoptosis during development due to a defect in NF-kappa B activation (Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, and Woodgett JR. Nature 406: 86-90, 2000). In this study, we determined the role of GSK-3 in TNF-alpha -induced NF-kappa B activation and cell death in primary hepatocytes. LiCl, an established inhibitor of GSK-3, sensitized primary rat hepatocytes toward TNF-alpha -mediated apoptosis resulting in 90% cell death after 24 h. This was accompanied by increased caspase 8-like and 3-like activities, nuclear fragmentation and DNA laddering. LiCl treatment had no effect on Ikappa B-alpha degradation, Ikappa B kinase (IKK) activity, NF-kappa B binding activity, and p65 nuclear import and export, but decreased transcription of the NF-kappa B-dependent inducible nitric oxide synthase gene and a NF-kappa B-driven reporter gene. The p65 sequence revealed four potential GSK-3 phosphorylation sites within its COOH-terminal transactivation domains and recombinant GSK-3beta phosphorylated glutathione S-transferase (GST)-p65(354-551), but not GST-p65(1-305) in vitro. These results indicate that GSK-3 protects hepatocytes from TNF-alpha -induced apoptosis through p65 phosphorylation and upregulation of NF-kappa B transactivation.

p65 phosphorylation; p65 transactivation; Ikappa B kinase; tumor necrosis factor-alpha ; nuclear factor-kappa B


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR-alpha (TNF-alpha ) is a pleiotropic cytokine that may induce proliferation, apoptosis, or inflammatory reactions in target cells. These different cellular reactions toward TNF-alpha result from the activation of distinct cellular signaling pathways that interact with each other in a complex signaling network and allow a wide range of cellular responses (1, 6). Although TNF-alpha is a known activator of the apoptotic signaling cascade, TNF-alpha commonly does not induce apoptosis in target cells due to the parallel activation of protective signaling pathways that interfere with the onset of apoptosis. The nuclear factor-kappa B (NF-kappa B) pathway is strongly activated by TNF-alpha and prevents hepatocytes from undergoing TNF-alpha -induced apoptosis during development, liver regeneration, and other conditions associated with high levels of TNF-alpha (4, 20). Under normal circumstances, NF-kappa B is bound to its inhibitor Ikappa B and resides in an inactive state within the cytoplasm. When released from its inhibitor, NF-kappa B translocates to the nucleus where it activates the transcription of genes with kappa B sites. TNF-alpha controls this crucial step in the regulation of NF-kappa B activity through a kinase cascade that results in the phosphorylation of Ikappa B by the Ikappa B kinase (IKK) complex and its subsequent ubiquitination and degradation (23). Like many other transcription factors, the activity of NF-kappa B is believed to be additionally regulated by phosphorylation of its subunits containing transactivation domains (36, 37). This is supported by studies demonstrating that the NF-kappa B subunit p65 becomes phosphorylated on TNF-alpha or interleukin-1beta (IL-1beta ) stimulation (30, 34, 45) and that the transactivation potential of p65 is hereby increased (28, 45). Several kinases including IKK-alpha and -beta , casein kinase II, and calmodulin-dependent kinase 4 (22, 29, 34, 46, 47) have been demonstrated to phosphorylate p65. Recently, it was reported that mice with inactivated glycogen synthase kinase (GSK)-3beta die during development due to massive hepatocyte apoptosis, because of a defect in NF-kappa B activation independent of DNA binding activity, suggesting a role for GSK-3beta in the regulation of p65 transactivation (19).

In this study, we assess the role of GSK-3 in the regulation of TNF-alpha -induced NF-kappa B activation in hepatocytes. We demonstrate that 1) LiCl, a pharmacological inhibitor of GSK-3, abolishes TNF-alpha -induced, NF-kappa B-dependent gene transcription in hepatocytes independent of NF-kappa B DNA binding activity and that 2) GSK-3beta phosphorylates the COOH terminus of the p65 unit of NF-kappa B in vitro. These results suggest a role for GSK-3 in the transactivation of NF-kappa B through p65 phosphorylation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatocyte isolation and culture. Hepatocytes were isolated from male Sprague-Dawley rats (200-225 g) by collagenase perfusion as previously described (18). Hepatocytes were plated onto collagen-coated cell culture dishes or collagen-coated coverslips in Waymouth medium containing 10% fetal bovine serum for 2 h. Cells were washed with PBS, and the medium was subsequently changed to hormonally defined medium as previously described (38). Before treatment with recombinant mouse TNF-alpha (R&D, Minneapolis, MN), hepatocytes were incubated with either LiCl (30 mM) or NaCl (30 mM) for 1 h in hormonally defined medium without insulin, if not indicated otherwise.

Western blot analysis. Whole cell extracts were prepared as previously described (38). For the analysis of Ikappa B-alpha degradation, 25 µg of whole cell extract were loaded onto a 10% SDS acrylamide gel and analyzed as previously described (38). For the detection of beta -catenin, 25 µg of whole cell extracts from LiCl- and NaCl-treated hepatocytes were loaded onto an 8% SDS acrylamide gel and transferred onto nitrocellulose membranes (Schleicher and Schull, Keene, NH). After blocking in 5% milk powder in Tris-buffered saline containing 0.5% Triton X-100, the membrane was incubated with antibodies to beta -catenin (Santa Cruz, Santa Cruz, CA) at a dilution of 1:500 for 6 h at room temperature. After extensive washing, the membrane was incubated with horseradish-peroxidase conjugated anti-goat secondary antibody at a dilution of 1:1,000 for 1 h at room temperature. After extensive washing, the bands were visualized by enhanced chemoluminescene (Amersham, Arlington Heights, IL) and exposure to Biomax film (Kodak, Rochester, NY). To demonstrate equal protein loading, membranes were incubated with alpha -tubulin antibody (Oncogene Science, Beverly, MA) for 1 h at a dilution of 1:1,000 and a horseradish-peroxidase conjugated anti-mouse secondary antibody (Santa Cruz) at a dilution of 1:1,000 for 1 h.

NF-kappa B reporter gene assay. Eight hours after isolation, 0.5 ×106 hepatocytes were infected with an adenovirus containing firefly luciferase linked to three kappa B sites at a multiplicity of infection (MOI) of 10 for 2 h (35). Cells were cultured for 12 h and then treated with TNF-alpha at a concentration of 30 ng/ml. After 6 h of TNF-alpha treatment, extracts were prepared and analyzed by luminometry. Luciferase activity was normalized to the protein content of the extracts.

RNA isolation and RT-PCR. Hepatocytes were pretreated with LiCl (30 mM) or NaCl (30 mM) for 1 h. After treatment with TNF-alpha (30 ng/ml) for 4 h, RNA was isolated by the TRIzol method. RT-PCR for inducible nitric oxide synthase (iNOS) or beta -actin was performed as previously described (38).

Electrophoretic mobility shift assay. Nuclear extracts were prepared as previously described (38). Five micrograms of nuclear extract were incubated with 1 ng of 32P-labeled probe containing the NF-kappa B consensus site and incubated at room temperature for 15 min as previously described. NF-kappa B DNA binding was determined by running the samples on a 5% acrylamide gel in 0.25× Tris-Borate-EDTA buffer and exposure to Biomax film.

Detection of apoptosis. After pretreatment with 30 mM LiCl or NaCl, cells were treated with TNF-alpha for the indicated times. For the detection of nuclear fragmentation, cells were fixed in 30% methanol/70% acetic acid and stained with propidium iodide and visualized under a fluorescent mircroscope (Olympus Melville, NY) using Spot digital imaging software (Diagnostic Instruments, Sterling Heights, MI). For the detection of caspase-like activity, cells were lysed in caspase lysis buffer and incubated in the presence of either IETD-7-amino-4-trifluoromethyl coumarin (AFC) or DEVD-AFC substrate as previously described (38). For the detection of DNA fragmentation, DNA was extracted as previously described (38) and analyzed on an 1.8% agarose gel.

In vitro kinase assays. IKK activity was determined by immunocomplex kinase assay as previously described (38). To exclude a direct inhibitory effect of LiCl on IKK, which might be lost in the standard immunocomplex assay due to a decrease in LiCl concentrations after washing, we additionally added LiCl to the kinase reaction after washing. The GSK-3beta kinase assay was performed by incubating 100 units of recombinant GSK-3beta (New England Biolabs, Beverly, MA) in 25 µl kinase buffer containing 20 mM Tris (pH 7.5), 10 mM MgCl2, 5 mM DTT, and 50 µM ATP, and 0.5 µCi [32P]ATP with either glutathione S-transferase (GST)-p65 (1-305) or GST-p65 (354-551) as substrate for 30 min at 30°C.

Immunofluorescent staining. Hepatocytes (500,000) were plated on a glass coverslip and pretreated with LiCl (30 mM) or NaCl (30 mM) for 1 h. Cells were then treated with TNF-alpha for the indicated time and fixed with methanol for 5 min. Immunofluorescent staining of p65 was performed as previously described (38).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inhibition of GSK-3 by LiCl sensitizes hepatocytes toward TNF-alpha -mediated apoptosis. LiCl is an established inhibitor of GSK-3 (19, 24, 40). To demonstrate the inhibitory effects of LiCl on GSK-3beta activity in hepatocytes, we analyzed the levels of beta -catenin, a target of GSK-3beta , which is degraded more rapidly after phosphorylation by GSK-3beta (40). Two hours of LiCl treatment significantly enhanced beta -catenin expression, demonstrating an effective inhibition of GSK-3 by LiCl in hepatocytes (Fig. 1A). To investigate the potential role of GSK-3 in the TNF-alpha -induced apoptosis (19), primary hepatocytes were preincubated with LiCl followed by treatment with TNF-alpha . LiCl-pretreated hepatocytes displayed >90% cell death after 24 h of TNF-alpha treatment compared with only 9% cell death in hepatocytes pretreated with NaCl (Fig. 1B) and displayed typical morphological features of apoptosis including extensive nuclear fragmentation (Fig. 1C). Additionally, LiCl-pretreated hepatocytes displayed a DNA fragmentation pattern characteristic for apoptosis. (Fig. 1E). To further confirm that the occurring cell death was apoptotic, we measured caspase 8-like and caspase 3-like activity after TNF-alpha . Compared with NaCl-pretreated hepatocytes, LiCl-pretreated hepatocytes displayed a 16-fold induction of caspase 8-like and a 27-fold induction of caspase 3-like activity after TNF-alpha (Fig. 1D).


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Fig. 1.   Lithium stabilizes beta -catenin and sensitizes hepatocytes toward tumor necrosis factor-alpha (TNF-alpha )-mediated apoptosis. Hepatocytes were preincubated with LiCl (30 mM) or NaCl (30 mM) for 2 h. A: whole cell extracts were run on an 8% acylamide gel and analyzed for beta -catenin by Western blot analysis. To ensure equal protein loading, the blot was reprobed with antibodies specific for alpha -tubulin. B: hepatocytes were treated with TNF-alpha for 24 h. Viability was determined by trypan blue exclusion and expressed as number of viable cells per ×200 field. Shown is the average of 6 ×200 fields from 2 culture dishes. C: hepatocytes displayed typical morphological signs of apoptosis as demonstrated by phase contrast microscopy and propidium iodide staining of fixed hepatocytes. D: caspase 8-like and caspase 3-like activity were determined by 7-amino-4-trifluoromethyl coumarin (AFC) release assay after 10 h of TNF-alpha and are shown as AFC release (nmol) per µg protein. E: after 24 h of TNF-alpha treatment, DNA was isolated and analyzed on an 1.8% agarose gel.

LiCl inhibits NF-kB dependent gene transcription. NF-kappa B is a critical factor in protecting hepatocytes from apoptosis. Previous studies (19), as well as our findings that LiCl sensitizes hepatocytes to TNF-alpha -mediated apoptosis, suggest that GSK-3 is involved in the regulation of NF-kappa B activation. To test this hypothesis, we evaluated the effect of GSK-3 inhibition on the NF-kappa B-dependent iNOS gene that is one of several NF-kappa B-dependent factors protecting hepatocytes from apoptosis (17). After 4 h of TNF-alpha treatment, the mRNA coding for iNOS was significantly induced in NaCl pretreated hepatocytes (Fig. 2A). In contrast, no iNOS mRNA was detectable after 4 h of TNF-alpha treatment in LiCl-pretreated hepatocytes. There was no difference in beta -actin expression between LiCl and NaCl treated hepatocytes (Fig. 2A). To demonstrate that the effects of LiCl on gene transcription were solely NF-kappa B-dependent, we transduced hepatocytes with a reporter gene in which luciferase transcription is driven by three NF-kappa B consensus sites. After 6 h of TNF-alpha , NF-kappa B-dependent transcriptional activity was highly upregulated in NaCl-pretreated hepatocytes (Fig. 2B). LiCl decreased the TNF-alpha -induced reporter gene transcription by 85% confirming that the effects of LiCl on gene transcription are mediated by its effect on the NF-kappa B pathway.


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Fig. 2.   Lithium inhibits inducible NO synthase (iNOS) mRNA expression and nuclear factor-kappa B (NF-kappa B)-dependent gene transcription in hepatocytes. Hepatocytes were preincubated with LiCl (30 mM) or NaCl (30 mM) for 2 h. A: hepatocytes were treated with TNF-alpha for 4 h. RNA was extracted, and RT-PCR for iNOS or beta -actin was performed as described in MATERIAL AND METHODS. B: hepatocytes were infected with an adenovirus containing a NF-kappa B-luceriferase reporter gene at a multiplicity of infection of 10. After treatment with TNF-alpha for 6 h, luciferase activity was measured and adjusted to the protein content of the extracts. RLU, relative light units.

LiCl does not inhibit IKK activity, Ikappa B degradation, and p65 nuclear import or export. To determine at what level GSK-3 influences NF-kappa B activation, we assessed the effects of LiCl at different levels of the NF-kappa B signaling cascade after TNF-alpha stimulation. Activation of the IKK complex has been demonstrated to be critical for NF-kappa B activation and protection from apoptosis after TNF-alpha as apparent by massive hepatocyte apoptosis of IKK-beta -/- mice during development and sensitization of hepatocytes transduced with dominant negative IKK-beta (25, 26, 38, 41). Therefore, we performed an immunocomplex kinase assay to assess the potential effect of LiCl on IKK activation. LiCl-treated hepatocytes displayed a robust activation of IKK after 10 min of TNF-alpha treatment that did not differ from NaCl-treated hepatocytes (Fig. 3A, lanes 2 and 4, respectively). Because LiCl acts as a competitive inhibitor of GSK-3 (33) and its inhibitory effects may be lost during the immunoprecipitation and washing, we added LiCl directly to the kinase reaction. We did not detect a significant direct influence of LiCl on IKK activity (Fig. 4A, lanes 5-8). After the phosphorylation of Ikappa B-alpha by the IKK complex, Ikappa B-alpha is degraded and releases NF-kappa B, which translocates to the nucleus. Therefore, we next analyzed the effects of LiCl on Ikappa B-alpha degradation by Western blot analysis. In NaCl-pretreated hepatocytes, Ikappa B-alpha was degraded 15 min after TNF-alpha and was resynthesized after 60 min (Fig. 3B). In LiCl-treated hepatocytes, Ikappa B-alpha degradation occurred in a similar manner, but the resynthesis of Ikappa B-alpha was strongly inhibited. Because the resynthesis of Ikappa B-alpha is a NF-kappa B-dependent event due to the kappa B site in the Ikappa B-alpha promoter, the inhibition of Ikappa B-alpha resynthesis confirms our initial finding that LiCl inhibits NF-kappa B activation. Electrophoretic mobility shift assay revealed a significant increase in NF-kappa B DNA binding activity in LiCl-pretreated hepatocytes after TNF-alpha with no significant difference compared with NaCl-pretreated hepatocytes (Fig. 3C). It has been demonstrated that GSK-3 influences nuclear factor of activated T cells (NFAT) and cyclin D1 by upregulating their nuclear export and degradation (2, 11). To assess whether nuclear NF-kappa B import and export differed between LiCl- and NaCl-pretreated hepatocytes, we performed immunofluorescent staining for p65 at different times after TNF-alpha stimulation. In both LiCl- and NaCl-pretreated hepatocytes, p65 was imported into the nucleus within the first 30 min after TNF-alpha (Fig. 3D). One hour after TNF-alpha , p65 was still predominantly located in the nucleus and redistributed to the cytoplasm over the next 2 h in both LiCl- and NaCl-pretreated hepatocytes.


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Fig. 3.   Lithium inhibits NF-kappa B downstream of the Ikappa B kinase (IKK) complex. To analyze at which level LiCl inhibited NF-kappa B, different levels of NF-kappa B activation cascade were analyzed. A: IKK activation was analyzed by kinase assay. After 10 min of TNF-alpha (30 ng/ml), extracts were prepared, and the kinase assay was performed (lanes 1-4). To account for the possibility of losing LiCl during the washing procedures, LiCl was added to some samples of the kinase reaction (lanes 5-8). B: Ikappa B-alpha degradation was analyzed by Western blot analysis after various times of TNF-alpha stimulation. C: NF-kappa B binding activity was analyzed by electrophoretic mobility shift assay after 30 min of TNF-alpha stimulation. Supershift analysis revealed p50 and p65 (lanes 5 and 6) as main components of the NF-kappa B complex. D: NF-kappa B localization was analyzed by p65 immunofluorescent staining after various lengths of TNF-alpha stimulation. GST, glutathione S-transferase; ns, nonspecific.



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Fig. 4.   p65 Contains GSK-3 sites and is phosphorylated by GSK-3beta in its COOH-terminal domain. A: p65 sequence was analyzed for GSK-3 sites. Shown are the two transactivation domains (TAD) of p65, each containing a pair of adjacent GSK-3 sites. B: recombinant GSK-3beta was incubated with either GST-p65 (1-305) or GST-p65 (354-551) in an in vitro kinase reaction for 30 min at 30°C. Left, presence of the substrates in the kinase reaction is shown by Coomassie staining; right, phosphorylation of the substrates and autophosphorylation of GSK-3beta is shown.

GSK-3beta phosphorylates p65. Because LiCl exerted its inhibitory effects on NF-kappa B activation independent of IKK activation, Ikappa B-alpha degradation and NF-kappa B DNA binding activity, we determined whether GSK-3beta was able to phosphorylate p65. Phosphorylation of p65 has been proposed as the critical modulator of NF-kappa B transactivation and may enhance the binding of transcription factor IIB, TATA-binding protein and cofactors (37, 45). Analysis of the p65 sequence revealed four GSK-3 sites within the COOH-terminal transactivation domain of p65 (Fig. 4A). We analyzed the capacity of GSK-3 to phosphorylate p65 by incubating recombinant GSK-3beta with different p65 substrates in an in vitro kinase assay. We did not observe phosphorylation of a GST-p65 (1-305) substrate in this kinase reaction, but the GST-p65 (354-551) substrate was strongly phosphorylated by GSK-3beta (Fig. 4B, right). The presence of the GST-p65 substrates was confirmed by Comassie blue staining (Fig. 4B, left). GSK-3beta activity was demonstrated by a significant amount of autophosphorylation in each kinase reaction (Fig. 4B, right). The lack of phosphorylation of GST-p65 (1-305) showed that the phosphorylation of the GST-p65 (354-551) substrate did not occur within the GST sequence. Importantly, phosphorylation occurred only in the substrate containing the COOH-terminal domain with the two transactivation domains. The presence of several bands in the gel indicates multiple phosphorylation of the GST-p65 (354-551) substrate. Pairwise organization of the GSK sites (Fig. 4A) may facilitate the observed multiple phosphorylation, because GSK-3 is believed to act as its own priming kinase in adjacent sites (16). Similar to some of the GSK-3 substrates including beta -catenin and axin (21), GST-p65 did not require priming of the substrate by another kinase to be phosphorylated by GSK-3beta .


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrate that 1) inhibition of GSK-3 downregulates NF-kappa B-dependent gene transcription induced by TNF-alpha and sensitizes hepatocytes to TNF-alpha -mediated apoptosis, and 2) GSK-3beta phosphorylates the COOH terminus of p65. A recent study has demonstrated that GSK-3beta -/- mice die of massive hepatocyte apoptosis during development (19) similar to animals with defects in the NF-kappa B pathway (4, 25, 26, 32), suggesting a role for GSK-3 in regulating NF-kappa B activity. Although GSK3beta -/- mouse embryonic fibroblasts (MEFs) had no defect in NF-kappa B DNA binding activity, they displayed a profound reduction in the transcription of a NF-kappa B-dependent reporter gene, suggesting that hepatocytes in these animals underwent apoptosis during development due to a similar defect in NF-kappa B activation (19). Embryological lethality of the GSK3beta -/- mice prevents studying the effect of GSK3-beta in hepatocytes of these animals. Therefore, we chose to inhibit GSK-3 pharmacologically by LiCl, an established inhibitor of GSK-3, which downregulated GSK-3 activity in hepatocytes as demonstrated by an upregulation of its target beta -catenin. Our results corroborate the data obtained by Hoeflich et al. (19) in GSK-3beta -/- MEFs in that NF-kappa B activity was impaired only at the transcriptional level in LiCl treated hepatocytes. Other steps in the NF-kappa B pathways including the activation of IKK, Ikappa B degradation, and NF-kappa B DNA binding activity remained intact in LiCl-treated hepatocytes. The mechanism by which GSK-3beta might have such a profound influence on NF-kappa B-dependent gene transcription was not addressed in previous studies. In this study, we tried to determine how GSK-3beta might affect NF-kappa B-dependent transcription in hepatocytes. It has been shown that besides its role in the Wnt pathway and the regulation of glycogen homeostasis, GSK-3beta phosphorylates several transcription factors including c-Jun, NFAT, CCAAT/enhancer binding proteins (C/EBP-alpha ), and the NF-kappa B family member p105 (2, 5, 10, 31), as well as the cell cycle regulator cyclin D1 (11). GSK-3beta -mediated phosphorylation strikingly alters the function of these transcription factors as seen by the enhanced transactivation of C/EBP-alpha (31), the decreased transactivation of c-Jun (5) and the increased nuclear export of NFAT (2). GSK-3beta -mediated phosphorylation of cyclin D1 decreases its stability and enhances its redistribution to the cytoplasm and consequent degradation (11). To assess the potential effect of GSK-3 inhibition on the subcellular localization of NF-kappa B and its stability, we stained hepatocytes for p65 after various lengths of TNF-alpha stimulation. Previous studies (38) have shown that the p50-p65 heterodimer is the main component of NF-kappa B binding activity after TNF-alpha in hepatocytes and that p65 staining accurately reflects the localization of the majority of biologically relevant NF-kappa B. LiCl did not influence TNF-alpha -induced p65 translocation or cytoplasmic redistribution and did not decrease the intensity of p65 staining compared with NaCl. Accordingly, a recent study has demonstrated that acetylation is a key mechanism to regulate the duration of the NF-kappa B response and the cellular localization of NF-kappa B (7).

Because the effects of GSK-3 inhibition by LiCl seemed to be independent of NF-kappa B localization and DNA binding activity, we tested whether NF-kappa B might be a substrate of GSK-3beta . Phosphorylation of the COOH-terminal transactivation domains of p65 is believed to enhance its transcriptional activity, possibly through enhanced binding of basal transcription factors, such as transcription factor IIB, TATA-binding protein and cofactors (37). After TNF-alpha , IL-1beta , or CD40 stimulation (34, 38, 39, 45), p65 is phosphorylated, and this phosphorylation upregulates p65 transcriptional activity (28, 45). Our study demonstrates that GSK-3beta phosphorylates a COOH-terminal p65 peptide but not an NH2-terminal p65 peptide. GSK-3beta -mediated phosphorylation often requires the priming by another kinase, such as by casein kinase II or cAMP-dependent protein kinase (8, 12, 13). After initial priming by other kinases, GSK-3 may act as its own priming kinase and thus phosphorylate multiple adjacent GSK-3 sites with high efficiency leading to hyperphosphorylation of certain substrates (16). GSK-3-mediated phosphorylation may also occur independently of priming as demonstrated for substrates, such as axin and beta -catenin (21). Recently, distinct residues have been described to be critical for the phosphorylation of primed and nonprimed GSK-3 substrates (14) indicating that the phosphorylation of primed and nonprimed substrates may rely on two different molecular mechanisms. In some substrates, certain residues are phosphorylated weakly in the absence of a priming kinase but are phosphorylated much more efficiently after priming (15). Our data demonstrate that phosphorylation of p65 by GSK-3beta does not require priming in vitro. However, the four GSK-3 sites within the transactivation domains of p65 are organized in pairs so that phosphorylation of the second site by GSK-3 may be facilitated through a priming effect after phosphorylation of the first site. This phenomenon might explain the multiple phosphorylation we observed with the GST-p65 (354-55) substrate. Combined with our findings that inhibition of GSK-3 downregulates NF-kappa B-dependent gene transcription it is very likely that phosphorylation of p65 by GSK-3beta enhances its transactivation. GSK-3beta might be a highly suitable kinase for this purpose, because multiple phosphorylation of p65 appears to be advantageous for its transactivation (37). Future studies are needed to further define the functional importance of GSK-3-mediated p65 phosphorylation for NF-kappa B transactivation.

Our study did not assess whether GSK-3 inhibition by LiCl affects other GSK-3 targets that might explain the observed sensitization toward TNF-alpha . c-Jun is a target whose function is downregulated by GSK-3. Observations from our laboratory (R. F. Schwabe, unpublished observations) and a study from another group (27) show that the c-Jun NH2-terminal kinase (JNK)/c-Jun pathway strongly enhances TNF-alpha -mediated apoptosis in rat hepatocytes. However, proapoptotic effects of JNK/c-Jun require the inhibition of NF-kappa B and make it unlikely that c-Jun is the main target of GSK-3 in our experimental model (9, 27, 42). We also cannot completely exclude that LiCl affects other systems besides GSK-3 in hepatocytes through which it may possibly enhance the sensitivity of hepatocytes toward TNF-alpha . One such target of LiCl is inositol monophosphatase 1, but to the best of our knowledge, there is no link between inositol monophosphatase 1 activity and apoptosis. Considering the critical role of NF-kappa B in the protection from TNF-alpha -mediated apoptosis (3, 43, 44) and the necessity of GSK-3beta for the protection from hepatocyte apoptosis during development (19), it is most likely that the LiCl inhibits GSK-3-dependent NF-kappa B activation and thereby, sensitizes hepatocytes to TNF-alpha -mediated apoptosis.


    ACKNOWLEDGEMENTS

The authors thank Dr. Hiroaki Sakurai (Tanabe Seiyaku, Osaka, Japan) for providing GST-p65 plasmids, Dr. John Engelhard (University of Iowa, Iowa City, IA) for providing nuclear factor-kappa B-luciferase adenovirus, and the laboratory of Dr. John Lemasters (University of North Carolina, Chapel Hill, NC) for providing primary rat hepatocytes. We thank Carrie Purbeck for excellent technical assistance.


    FOOTNOTES

This study was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34987 and National Institute of General Medical Sciences Grant GM-41804.

Address for reprint requests and other correspondence: D. A. Brenner, Univ. of North Carolina, Dept. of Medicine, CB #7038, Chapel Hill, NC 27599 (E-mail: dab{at}med.unc.edu).

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.

First published February 20, 2002;10.1152/ajpgi.00016.2002

Received 14 January 2002; accepted in final form 7 February 2002.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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