©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteolytic Processing of NF-B/IB in Human Monocytes
ATP-DEPENDENT INDUCTION BY PRO-INFLAMMATORY MEDIATORS (*)

(Received for publication, September 23, 1994; and in revised form, November 2, 1994)

Radhika Donald (1) Dean W. Ballard (2)(§) Jacek Hawiger (1)(¶)

From the  (1)Department of Microbiology and Immunology, Vanderbilt University School of Medicine, and the (2)Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, Tennessee 37232

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Proteolytic processing of select constituents of the nuclear factor kappaB (NF-kappaB)/inhibitor kappaBalpha (IkappaB) transcription factor system plays an important role in regulating the biological responses of monocytes to pro-inflammatory mediators. Nuclear translocation of NF-kappaB is preceded by the proteolytic degradation of IkappaBalpha, an ankyrin motif-rich inhibitor that traps NF-kappaB in the cytoplasm. In addition, formation of cytoplasmic NF-kappaB/IkappaBalpha complexes in quiescent cells requires constitutive proteolytic processing of p105, another ankyrin motif-rich inhibitory protein from which the p50 subunit of NF-kappaB is generated. We have demonstrated that, following stimulation of human monocytic cells with lipopolysaccharide or tumor necrosis factor-alpha, this critical p105 processing event is up-regulated in concert with the inactivation of IkappaBalpha. Moreover, the degradative loss of both p105 and IkappaBalpha is prevented in cells depleted of intracellular ATP. In activated monocytes, however, IkappaBalpha degradation occurs more rapidly than p105 processing to p50. Together these findings provide direct biochemical evidence that p105 and IkappaBalpha are differentially sensitive targets for inducible proteolysis via ATP-dependent degradative pathways.


INTRODUCTION

The NF-kappaB(^1)/Rel family of transcription factors participates in the induced expression of a diverse set of cellular and viral genes that are activated in response to immune and inflammatory signals. This set includes cellular genes that encode cytokines, cell adhesion molecules, and procoagulant factors. In addition, NF-kappaB is involved in the initiation of transcription from viral promoters that control the expression of human immunodeficiency virus 1 and cytomegalovirus (see (1) for review). Typically, NF-kappaB resides in the cytoplasm as a ternary complex composed of two DNA binding subunits, termed RelA (p65) and NF-kappaB1 (p50), bound to an ankyrin motif-rich inhibitor called IkappaBalpha (see (2) for review). This labile inhibitory protein is rapidly degraded in response to multiple cellular activation signals, thereby permitting nuclear expression of the functional NF-kappaB p50/RelA complex (see (3) for review).

An unusual feature of the NF-kappaB1 gene encoding p50 is its capacity to specify a larger precursor protein, termed p105(4, 5, 6, 7) . This precursor subunit has several key properties in common with IkappaBalpha. (i) p105 resides exclusively in the cytosolic compartment independent of the status of cellular activation(8, 9) ; (ii) p105 contains an ankyrin motif-rich carboxyl-terminal domain that inhibits the nuclear expression and DNA binding activity of its amino-terminal half(8, 9, 10, 11, 12) ; this IkappaBalpha-related domain is constitutively degraded in order to generate the p50 DNA binding subunit of NF-kappaB(13) ; (iii) p105, like IkappaBalpha, physically sequesters RelA in the cytoplasmic compartment (14) ; and (iv) the inducible genes encoding p105 and IkappaBalpha both contain functional NF-kappaB binding sites(15, 16, 17, 18) . These findings point to a dynamic relationship between p105/p50, IkappaBalpha, and RelA at the protein and DNA levels that define at least two distinct autoregulatory mechanisms for NF-kappaB-directed transcription(1, 3) .

Despite these structural and functional similarities of p105 and IkappaBalpha, the proteolytic mechanisms that control their inhibitory activities remain ill defined. Here we demonstrate that treatment of human monocytic cells with either bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF) stimulates p105 processing to p50 via an ATP-dependent pathway. Moreover, agonist-induced degradation of the structurally related IkappaBalpha protein, which occurs with more rapid kinetics relative to p105 processing, is also regulated by an ATP-dependent process. These findings suggest that proteolytic processing of p105 and IkappaBalpha in monocytic cells may be differentially induced during the course of an inflammatory reaction.


MATERIALS AND METHODS

Immunoblot and Gel Shift Analysis

THP-1 cells (American Type Culture Collection, Rockville, MD) were cultured and subjected to subcellular fractionation as described(19) . For immunoblotting studies, subcellular extracts were further fractionated on SDS-polyacrylamide gels (20) and transferred to nitrocellulose membranes(19) . Immunoreactive proteins were detected by enhanced chemiluminescence (ECL, Amersham Corp.) using either p105-specific (amino acids 947-969) or IkappaBalpha-specific (amino acids 289-317) antipeptide rabbit antibodies in combination with a secondary donkey anti-rabbit antibody linked to horseradish peroxidase (Amersham). For gel shift studies, DNA binding reactions containing a radiolabeled kappaB probe derived from the mouse immunoglobulin kappa enhancer were prepared and fractionated on native gels(19) .

Pulse-Chase Experiments

THP-1 cells (10^6 cells/ml) were cultured in methionine and cysteine-free medium for 1 h and then pulse-labeled for 3 h with 100 µCi/ml [S]methionine/[S]cysteine (TranS-label; DuPont NEN). Radiolabeled cells were washed and resuspended in medium containing either LPS (10 µg/ml Escherichia coli LPS 0127:B8; Difco), TNF (100 units/ml; Mallinckrodt), or no agonist. Cytosolic extracts were prepared at the times indicated, diluted with RIPA buffer (21) containing 1% deoxycholate, and centrifuged at 100,000 times g (1 h at 4 °C) to remove particulate matter. Lysates were initially precleared with normal rabbit serum in combination with protein A-Sepharose CL-4B beads (Sigma) and then immunoprecipitated with p105/p50-specific (amino acids 1-21) antipeptide rabbit antibody in combination with protein A-Sepharose CL-4B beads. Immunoprecipitates were washed in RIPA buffer and proteins were eluted by boiling in the presence of 1% SDS and specific peptide (1 mg/ml). Supernatants were diluted to 0.1% SDS with RIPA buffer and immunoprecipitated with p105-specific antiserum (amino acids 947-969). Immunoprecipitates were washed in RIPA buffer, boiled in the presence of 4% SDS and 10% beta-mercaptoethanol, and subjected to electrophoresis on SDS-polyacrylamide gels. Radiolabeled polypeptides were visualized by fluorography and quantitated by phosphorimager analysis (Molecular Dynamics model 425E).

Intracellular ATP Depletion Studies

THP-1 cell cultures (10^6 cells/ml) were treated with antimycin A (4 µg/ml) and 2-deoxy-D-glucose (6.5 mM; Sigma) for 2 h (22) , followed by treatment with either LPS (10 µg/ml) or TNF (100 units/ml) for the times indicated. Cell extracts were prepared as described above and subjected to immunoblot and electrophoretic mobility shift analyses. To assess the efficiency of ATP depletion, stimulated cultures were lysed in 2% trichloroacetic acid, neutralized by addition of 0.1 M Tris, pH 9 (1:1), and analyzed for ATP levels using a luciferase enzyme assay in conjunction with calibration standards relating ATP concentrations to bioluminescence (ATP bioluminescence kit; Sigma).


RESULTS

Induction of p105 Proteolysis in Monocytes by Pro-inflammatory Mediators

The NF-kappaB signaling pathway is activated in cells of the monocyte/macrophage lineage by several pro-inflammatory agents, including LPS and TNF(19, 23) . Despite the induction of NF-kappaB1 mRNA in response to LPS(19) , cellular stimulation led to significant loss of p105 protein within 2 h (Fig. 1, middlepanel, lanes1 and 4). The reappearance of p105 protein at 3 h following LPS stimulation (Fig. 1, middlepanel, lane5) is attributed to the 15-fold induction of NF-kB1 mRNA in response to LPS(19) . Of note, two electrophoretically distinct forms of p105 were detected in these experiments, both of which diminished during cellular stimulation (indicated by arrows in Fig. 1, middle and lowerpanels). Proteolysis of p105 was even more pronounced when cells were pretreated with the translation inhibitor cycloheximide (CHX) to preclude de novo synthesis of p105 (Fig. 1, lowerpanel, lanes3-5), which eliminated the increase in p105 protein observed at later time points (Fig. 1, middlepanel, lane5). NF-kappaB-specific DNA binding activity was readily detected in nuclear extracts prepared from the same LPS-stimulated cells (Fig. 1, middlepanel, lanes 7-10). Using competition and supershift assays, we have previously demonstrated that these gel shift activities correspond to p50/RelA and p50/c-Rel complexes(19) . Comparative experiments performed with TNF as the NF-kappaB inducing agent indicated that TNF also stimulates p105 degradation, albeit with lower efficiency (see below).


Figure 1: Effect of LPS activation on p105 and nuclear NF-kappaB expression in human monocytic cells. THP-1 cells were treated with CHX (10 µg/ml), LPS (10 µg/ml), or a combination of CHX and LPS at these concentrations for the times indicated. In CHX + LPS treatment, CHX was added 30 min prior to LPS. Cytosolic extracts were subjected to immunoblot analyses using p105-specific (amino acids 947-969) antipeptide antibody. Arrows indicate the positions of the major forms of p105-specific polypeptides. The minor immunoreactive species migrating below the major p105-specific polypeptides is also eliminated in blocking studies with p105-specific (amino acids 947-969) peptides. Nuclear extracts from the same cells were assayed for NF-kappaB DNA binding activity by gel shift analyses. A composite of the resultant kappaB-specific nucleoprotein complexes are shown.



Pulse-Chase Analysis

To determine the half-life of p105 under steady-state conditions reflecting constitutive versus signal-dependent proteolysis, THP-1 cells were pulse-labeled for 3 h and chased for up to 8 h in media containing LPS, TNF, or no agonist (Fig. 2a). Based on five independent experiments, the half-life (t) values for p105 were derived from best-fit curves encompassing all data points. The t of p105 in unstimulated cells was greater than 8 h, whereas the corresponding t values in LPS- and TNF-stimulated cells were approximately 70 min and 300 min, respectively (Fig. 2b).


Figure 2: Pulse-chase analysis of p105 in unstimulated and LPS- or TNF-stimulated THP-1 cells. a, a representative pulse-chase experiment performed as described under ``Materials and Methods.'' ^14C-Labeled molecular weight standards (Amersham Corp.) are indicated in kilodaltons (kDa). b, quantitative phosphorimager analysis of pulse-chase data from five independent experiments. Each point represents the mean (± S.E.) of radiolabeled p105 relative to that detected in pulse-labeled cells (arbitrarily set as 100%) at time = 0.



Precursor/Product Relationship

The functional significance of LPS-regulated proteolytic processing of p105 was explored by monitoring the appearance of p50 during cellular activation. For these experiments, the pulse-labeling time was reduced to 2 h to minimize the incorporation of radiolabeled amino acids into p50. Radiolabeled proteins were detected in whole cell extracts by immunoprecipitation with p105/p50-specific antipeptide antibody and elution with the corresponding antibody-specific peptide. In addition to the radiolabeled pools of p105 and p50, radiolabeled IkappaBalpha was also detected due to its tight association with NF-kappaB complexes containing p50 (Fig. 3a). As shown in Fig. 3b, the loss of p105 in LPS-stimulated cells was associated with a stoichiometric increase in the amount of p50. These results strongly imply a direct precursor/product relationship between p105 and p50 in activated monocytic cells, a finding that is fully consistent with that described for the basal processing of ectopic p105 in transfected monkey COS cells(13) .


Figure 3: Pulse-chase analysis of p50, p105, and IkappaBalpha. a, THP-1 cells were pulse-labeled (lanes 1 and 5) with [S]methionine/[S]cysteine and chased either in the absence (lanes 2-4) or presence (lanes 6-8) of LPS (10 µg/ml) for the indicated times. Whole cell extracts were immunoprecipitated with p105/p50-specific (amino acids 1-21) antipeptide antibody, fractionated on SDS-polyacrylamide gels, and visualized by fluorography. ^14C-Labeled molecular weight standards are indicated in kilodaltons (kDa). p105, p50, and IkappaBalpha proteins are indicated by arrows. b, quantitative phosphorimager analysis of pulse-chase data from three independent experiments. Each point represents the mean (± S.E.) of each radiolabeled protein relative to that detected for that protein in pulse-labeled cells (arbitrarily set as 0% for p50 and as 100% for p105 and IkappaBalpha) at time = 0.



Intracellular ATP Depletion Studies

Fan and Maniatis (13) have reported that processing of p105 in vitro occurs via an ATP-dependent mechanism. We extended these observations to the in vivo processing of p105 induced in human monocytic cells by LPS. THP-1 cell cultures were depleted of intracellular ATP prior to stimulation. Based on bioluminescence assays (see ``Materials and Methods''), the level of ATP in these THP-1 cells was reduced by 93-97% of that measured in untreated cells. In immunoblotting studies, the degradation of p105 was completely inhibited in ATP-depleted THP-1 cells (Fig. 4, lower panel, lanes 1-5). Furthermore, gel shift analyses of nuclear extracts from these cells showed that the depletion of intracellular ATP also prevented LPS-induced kappaB DNA binding activity (Fig. 4, lower panel, lanes 6-10). These in vivo findings strongly suggest that p105 processing involves a proteolytic mechanism that requires ATP utilization.


Figure 4: Effect of intracellular ATP depletion on p105 proteolysis and nuclear NF-kappaB expression in LPS-stimulated monocytes. THP-1 cells were depleted of intracellular ATP for 2 h (see ``Materials and Methods'') and then cultured either in the presence (lower panels) or absence (upper panels) of LPS (10 µg/ml). As a control, cells replete with ATP were stimulated with LPS (10 µg/ml; middle panels). Cytosolic extracts were subjected to immunoblot analyses using p105-specific antipeptide antibody. Nuclear extracts from the same cells were assayed for NF-kappaB DNA binding activity by gel shift analyses. A composite of the resultant kappaB-specific nucleoprotein complexes is shown.



Comparative Analysis of IkappaBalpha

In comparison to p105, pulse-chase analysis demonstrated that LPS-stimulation led to the rapid degradation of IkappaBalpha as evidenced by the loss of over 70% of radiolabeled IkappaBalpha within 30 min (Fig. 3b). Consistent with these results, immunoblot analyses using IkappaBalpha-specific antisera demonstrated that IkappaBalpha is degraded within 30 min following LPS or TNF stimulation (Fig. 5, a and b, middlepanels, lane2). In addition, newly synthesized IkappaBalpha reappeared between 1 and 2 h after either TNF or LPS stimulation (Fig. 5b, middlepanel, lanes3 and 4). The rapid kinetics of induced IkappaBalpha degradation was in sharp contrast to the relatively slow kinetics of p105 processing but was fully consistent with the pattern of nuclear NF-kappaB expression in LPS-stimulated cells (Fig. 1, middle panel, lanes 7-10). This finding strongly suggests that the rapid mobilization of NF-kappaB during the initial phases of monocyte activation is primarily due to the degradation of IkappaBalpha. Like p105, proteolysis of IkappaBalpha induced by LPS and TNF appears to occur through an ATP-dependent mechanism(s) since this degradative pathway was also blocked in cells depleted of ATP (Fig. 5, c and d, lower panels).


Figure 5: IkappaBalpha proteolysis in stimulated and ATP-depleted monocytes. a and b, immunoblot analyses using IkappaBalpha-specific (amino acids 289-317) antipeptide antibody were performed on cytosolic extracts from THP-1 cells treated with cycloheximide (10 µg/ml), lipopolysaccharide (10 µg/ml), tumor necrosis factor-alpha (100 units/ml), or the indicated combinations of these reagents. CHX was added 30 min prior to the addition of agonists. c and d, immunoblot analyses using IkappaBalpha-specific antipeptide antibody were performed on cytosolic extracts from THP-1 cells that were depleted of intracellular ATP and cultured either in the absence (-ATP; upper panels) or presence (lower panels) of either LPS (10 µg/ml) or TNF (100 units/ml) for the indicated times. In control experiments, cells replete with ATP were stimulated with either LPS (10 µg/ml) or TNF (100 units/ml) (middle panels).




DISCUSSION

These studies with human monocytic cells demonstrate that proteolytic processing of p105 is stimulated significantly in response to the pro-inflammatory agents LPS and TNF. As such, inducible proteolysis regulates not only the level of IkappaBalpha in activated monocytes(19, 23, 24) , but also the levels of p105 and p50. These findings are in accord with some (25, 26) but not all (27) reports regarding the regulation of p105 in other experimental systems. The rate of p105 processing may therefore be influenced in an agonist- and/or cell type-dependent manner. We have also shown that the in vivo processing of p105 in human monocytic cells is ATP-dependent, thus extending the in vitro findings of Fan and Maniatis (13) . In this regard, evidence is emerging that p105 and IkappaBalpha are both phosphorylated during cellular activation(19, 26, 28) . However, the relationship between phosphorylation and degradation remains unknown. Notwithstanding this uncertainty, the critical requirement for ATP in p105 processing documented in our studies is fully consistent with recent findings that the 26S proteasome specifically mediates this p105 processing event(29) .

Our results provide further evidence that the NF-kappaB transcription factor system can be regulated at multiple levels through the induced proteolysis of IkappaB inhibitory subunits and NF-kappaB precursor proteins. The different rates observed for induced IkappaBalpha and p105 proteolysis in these studies imply a bimodal function for proteolysis, namely to permit nuclear translocation of NF-kappaB and to supply a second wave of p50 subunits. This second wave could include p50 homodimers, which have been reported to repress gene transcription in a LPS-tolerant monocytic cell line(30) . In addition, the dilatory generation of p50 in association with RelA could provide a mechanism to replenish the cytoplasmic pool of preformed NF-kappaB that is released from IkappaBalpha and rapidly mobilized to the nucleus.


FOOTNOTES

*
This study was supported in part by National Institutes of Health Grants HL 30647 and HL 45994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Howard Hughes Medical Institute and Grant 1 RO1 AI33839 from NIAID, National Institutes of Health.

To whom correspondence should be addressed. Tel.: 615-322-2087; Fax: 615-343-7392.

(^1)
Abbreviations used: NF-kappaB, nuclear factor kappaB; IkappaBalpha, inhibitor kappaBalpha; LPS, lipopolysaccharide; TNF, tumor necrosis factor-alpha; CHX, cycloheximide.


ACKNOWLEDGEMENTS

We thank Tom Maniatis for sharing data prior to publication and Michael Karin for advice concerning pulse-chase experiments. We also thank Emily Vance and Carol Walter for assistance in the preparation of this manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.