©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytokine Induction of Nuclear Factor B in Cycling and Growth-arrested Cells
EVIDENCE FOR CELL CYCLE-INDEPENDENT ACTIVATION (*)

(Received for publication, February 1, 1995; and in revised form, June 9, 1995)

Colin S. Duckett (§) Neil D. Perkins (¶) Kwanyee Leung Adam B. Agranoff Gary J. Nabel (**)

From theHoward Hughes Medical Institute and the Departments of Internal Medicine and Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0650

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nuclear factor kappaB (NF-kappaB) is a pleiotropic transcription factor which regulates the expression of a large number of cellular and viral genes. Induction of NF-kappaB has been shown previously to occur during cell cycle transition from G(0) to G(1), but the relationship of cytokine induction of this transcription factor to cell cycling has not been directly addressed. Here we examine the induction of NF-kappaB in serum-deprived and cycling cells in response to tumor necrosis factor-alpha (TNF-alpha). In 3T3 fibroblasts deprived of serum, and in the temperature-sensitive G(2) phase mutant carcinoma line FT210, we find that NF-kappaB DNA binding activity is rapidly induced upon addition of TNF-alpha. In addition, NF-kappaB induction in cycling cells occurs without a significant change in cell cycle distribution. These data reveal that NF-kappaB is rapidly induced by TNF-alpha in both proliferating and arrested cells and suggest that distinct activation pathways can lead to cell cycle-dependent or -independent induction of NF-kappaB.


INTRODUCTION

Nuclear factor kappaB (NF-kappaB) (^1)is comprised of a family of at least five transcription factors(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) related to and including the Rel oncoprotein(12, 13, 14, 15) . First identified as an activity in B cells which binds to a 10-base pair conserved element in the immunoglobulin light chain enhancer(16) , NF-kappaB is now known to be widely expressed in mammalian cells. The best characterized form of NF-kappaB is a heterodimer composed of 50- and 65-kDa subunits (15) ; however, both homodimeric and heterodimeric forms of NF-kappaB/Rel in most combinations are able to bind, albeit with different affinities, to kappaB-like regulatory elements found in a diverse array of cellular and viral genes(9, 10, 17, 18, 19) . In most unstimulated cells NF-kappaB is thought to reside in the cytoplasm bound to an inhibitor protein, designated IkappaB(20, 21, 22) ; upon stimulation or activation of the cell by cytokines(23, 24, 25, 26) , phorbol esters(27) , and viral trans-activators(28, 29) , NF-kappaB is able to rapidly dissociate from IkappaB and translocate into the nucleus, an event which involves the phosphorylation of IkappaB(30, 31) .

In order for most nontransformed cells to progress through the cell cycle, appropriate extracellular signals are required(32) . In cultured cells, these signals are usually provided by the presence of growth factors contained within serum added to the media(33, 34, 35, 36) . Deprivation of these factors has been shown to result in the cessation of both cell growth and nuclear division, and the cells become arrested in a quiescent state known as G(0)(36) . Cells arrested in this manner may remain in G(0) for days or weeks without a significant decrease in viability. The subsequent addition of serum to such cells allows re-entry into G(1), which is followed by continued growth and cell division(32) . Many of the growth factors contained within serum have been characterized, as well as the cellular factors which they induce(37, 38, 39, 40) . In many cases, the addition of serum results in the induction of mRNAs encoding transcription factors, such as c-myc, c-fos, and c-jun(41, 42, 43, 44, 45, 46, 47, 48) . Activation of these factors is probably necessary to induce the expression of sets of genes whose products are essential for progression through the cell cycle.

Previous studies have revealed that NF-kappaB also fits into this category of serum-induced or immediate-early genes(49) ; the addition of serum to G(0)-arrested 3T3 cells has been shown to induce both nuclear translocation of NF-kappaB and kappaB-directed transcriptional activation. However, while NF-kappaB is induced at the G(0) to G(1) transition, it is unknown whether this is the only stage of the cell cycle at which NF-kappaB induction can occur. In addition, it is unclear whether induction of NF-kappaB is associated with changes in the distribution of cells at different points of the cell cycle. Here we show, using cell cycle analysis of 3T3 cells and the G(2) mutant mouse mammary carcinoma line, FT210(50) , that NF-kappaB may be activated with equivalent efficiency in both proliferating and arrested cells. Furthermore, we show that activation of NF-kappaB has no effect on the rate of progression or checkpoint arrest throughout cell cycle. We conclude that NF-kappaB may be induced in both a cell cycle-dependent and -independent manner and suggest a model in which distinct signaling mechanisms can lead to NF-kappaB activation.


MATERIALS AND METHODS

Plasmids

The four-copy kappaB CAT plasmid and the kappaB mutant CAT control plasmid have been described previously(18, 28) . The RSV-beta-galactosidase plasmid used to standardize transformation efficiency was a kind gift of Dr. B.-Y. Wu.

Cells

NIH 3T3 cells were maintained in Dulbecco's modified Eagle medium with 10% fetal calf serum, penicillin, and streptomycin, and serum starvation conditions were established as described previously(49) . FT210 and FM3A cells were maintained in RPMI 1640 medium with 10% fetal calf serum, penicillin, and streptomycin at 33 °C as described previously(50) . FT210 cells were synchronized at G(2)/M by incubation at 39 °C for 48 h.

Cell Cycle Analysis

3T3 cells (10^6) were pelleted by centrifugation at 300 g for 5 min at room temperature and washed twice with 5 ml of phosphate-buffered saline. Cells were resuspended in 1 ml of phosphate-buffered saline and pelleted as described above. Pellets were taken up in 0.5 ml of nuclear staining solution (0.05 mg/ml propidium iodide, made as a 10 stock in 1.12% sodium citrate, 0.3% Triton X-100) and incubated at room temperature for 15 min. RNase A (0.5 ml, 6.5 Kunitz units/ml) was added, and cells were analyzed by flow cytometry using a FACScan (Becton Dickinson). Fluorescence measurements were accumulated to form a distribution curve of DNA content. Fluorescence events due to debris were substracted before analysis. Areas under the curve representing the G(0)/G(1), S, and G(2)+M phases of the cell cycle were then calculated.

Transfections and CAT Assays

3T3 cells were cotransfected with 10 µg of kappaB reporter plasmid or mutant kappaB-CAT plasmid and 10 µg of RSV-betagal by the calcium phosphate procedure as described previously (49) with the following modifications: cells were plated to 25% confluence 20 h prior to transfection. The culture medium was replaced 12 h after transfection with medium containing 0.5% serum or, as a control, 10% serum. Cells were maintained for a total of 48 h from the time of transfection to the time of harvest for CAT assays. Where indicated, media was replaced with fresh media containing 20% serum or 200 units/ml of TNF-alpha 12 h prior to harvest. FT210 and FM3A cells were transfected with 10 µg of kappaB reporter plasmid or control plasmid by the DEAE-dextran method(7) . For temperature shift experiments, FT210 or FM3A cells were transferred to a 39 °C incubator 4 h after transfection and incubated further for 44 h. Cellular extracts were prepared as described previously(7) . Protein concentrations were determined by the method of Bradford(51) . 3T3 extracts were standardized by cotransfection of beta-galactosidase expression vectors prior to CAT assays. FT210 and FM3A cells were standardized by protein concentration. CAT assays were performed as described previously(52) .

Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSA)

Nuclear extracts were prepared by the method of Osborn et al.(23) . EMSA analysis was performed essentially as described previously(19) . Double-stranded oligonucleotide probes encompassing the HIV kappaB site were labeled by the Klenow enzyme with [P]dCTP (ICN). Nuclear extracts (4 µg) were incubated in a 20-µl reaction containing 2 10^4 counts/min radiolabeled probe and 1 µg of poly d[IbulletC]-poly d[IbulletC] at room temperature for 10 min and loaded without dye onto a 0.25 Tris borate-EDTA (TBE) polyacrylamide gel. Electrophoresis was performed at 10 V/cm at room temperature for 3 h. Gels were dried and exposed to Kodak AR-5 film at -70 °C using intensifying screens.


RESULTS

NF-kappaB-mediated Transcription Is Activated in Serum-deprived Cells by TNF-alpha

To determine whether activation of NF-kappaB required active cell cycling, 3T3 cells were transfected with either a HIV kappaB-responsive reporter plasmid or a negative control containing mutated versions of the kappaB site, deprived of serum for 24 h, treated with either serum or TNF-alpha, and maintained for a further 12 h before harvest. The addition of serum to growth-arrested cells was found to potently activate kappaB-mediated transcription (Fig.1), consistent with earlier studies(49) . Interestingly, the addition of TNF-alpha to either serum-deprived or cycling cells also stimulated equivalent levels of kappaB-dependent gene expression (Fig.1). A similar result was observed following stimulation with interleukin-1 (data not shown).


Figure 1: Effects of serum addition and TNF-alpha treatment on cycling versus growth-arrested 3T3 cells. 3T3 cells were transfected with multimerized kappaB reporter plasmid (A) or a kappaB mutant control (B), as described under ``Materials and Methods.'' Cells were either serum starved or mock treated 12 h after transfection. Serum or TNF-alpha addition was performed 36 h post-transfection, and cells were harvested 48 h after transfection. Transfected cells were harvested, protein extracts made, and CAT assays performed as described under ``Materials and Methods.'' The results shown are representative of at least two independent experiments.



TNF-alpha Induces NF-kappaB DNA Binding Activity in Growth-arrested Cells

To confirm that the activation of kappaB-directed gene expression by TNF-alpha in serum-starved cells correlated with the induction of a nuclear, kappaB-specific DNA binding activity, EMSA analysis was performed. Nuclear extracts were prepared from cycling or growth arrested cells which had been either mock treated or stimulated with TNF-alpha and incubated with a P-labeled oligonucleotide probe containing a single copy of the HIV kappaB site. An NF-kappaB-like binding activity was induced by treatment with TNF-alpha in both cycling and growth arrested cells (Fig.2). Competition analysis with unlabeled wild type and mutant kappaB sites confirmed the specificity of this activity (lanes 6 and 7). The NF-kappaB proteins induced by TNF-alpha treatment have previously been shown to be composed of p50 and p65 subunits; supershift analysis of NF-kappaB induced in cycling versus serum-deprived cells revealed their subunit composition to be identical (data not shown). These findings suggest that NF-kappaB can be induced by TNF-alpha irrespective of the cycling state of the cell.


Figure 2: Evidence that TNF-alpha can induce nuclear NF-kappaB activity in both cycling and G(o)-arrested cells. 3T3 cells were seeded at 50-70% confluence and maintained for 24 h in medium containing 10% serum (lanes 1 and 2) or 0.5% serum (lanes 3-7), followed by the addition of TNF-alpha (lanes 2, 4-7) for 2 h. Nuclear extracts were prepared and analyzed by EMSA as described under ``Materials and Methods.'' Lanes 4-6 show the results of a competition experiment in which reactions additionally contained 10 ng of unlabeled double-stranded oligonucleotide encompassing either the wild type kappaB site (lane 6) or a mutated kappaB control (lane 7).



Cell Cycle Progression Is Unaffected following NF-kappaB Induction by TNF-alpha

The previous experiments revealed that NF-kappaB induction by TNF-alpha occurs at equivalent levels in both cycling and serum-deprived 3T3 cells. One possible explanation for this finding is that the process of NF-kappaB activation can itself release the cells from the G(0) block. To test this possibility, 3T3 cells which had been subjected to different degrees of serum deprivation were stimulated with TNF-alpha, treated with propidium iodide, and analyzed for DNA content by flow cytometry and FACScan analysis. The addition of TNF-alpha to cycling cells had no observable effect on the distribution of cells at various stages of the cell cycle (Fig.3C, Table 1). Furthermore, while serum deprivation was found to arrest greater than 70% of cells in G(0)/G(1) (Fig.3D, Table 1), the cell cycle distribution following addition of TNF-alpha to serum-deprived cells was essentially unchanged (Fig.3F, Table 1). This finding strongly suggests that, in contrast to the induction of NF-kappaB by the addition of serum to serum-deprived cells, NF-kappaB activation by TNF-alpha occurs in a manner which is independent of the cycling state of the cell. In addition, these data suggest that the process of NF-kappaB activation does not immediately alter cell cycle progression.


Figure 3: Cell cycle analysis of 3T3 cells. Aliquots of the transfected 3T3 cells were taken at the same time as the harvest for CAT assay as described in the legend to Fig.1, washed with phosphate-buffered saline, resuspended in nuclear staining solution, and analyzed by flow cytometry as described under ``Materials and Methods.'' Representative samples are shown depicting cycling cells, mock treated with 10% serum (a), cycling cells treated with 20% serum for 24 h (b), cycling cells treated with TNF-alpha for 12 h (c), serum-starved cells, mock treated with 0.5% serum for 12 h (d), serum-starved cells treated with 20% serum for 12 h (e), and serum-starved cells treated with TNF-alpha for 12 h (f). The regions between the vertical lines from left to right represent cells in G(0)/G(1), S, and G(2)/M, respectively.





TNF-alpha Induces NF-kappaB in the G(2) Phase Mutant Cell Line FT210

To verify that NF-kappaB may be activated by cytokines regardless of their point in the cell cycle, NF-kappaB induction was examined in FT210 murine mammary carcinoma cells, which can be readily growth arrested at a different point in the cell cycle. The FT210 cell line is a temperature-sensitive G(2) phase mutant, resulting from a mutation in the cdc2 kinase gene(50) . While cell growth and division is unimpaired at the permissive temperature of 33 °C, cells incubated at the non-permissive temperature of 39 °C rapidly arrest at the G(2)/M boundary. To evaluate the properties of NF-kappaB induction at G(2)/M, FT210 cells or the wild type parental line FM3A were transfected in parallel with the kappaB reporter plasmid, and responsiveness to TNF-alpha was determined at both permissive and non-permissive temperatures. TNF-alpha was able to induce CAT expression, specifically through the kappaB sites, at both 33 and 39 °C (Fig.4). As shown previously(50) , FT210 cells maintained at the non-permissive temperature of 39 °C were completely arrested at G(2)/M (Fig.4C). To confirm that kappaB-directed expression correlated with the activation of NF-kappaB, nuclear extracts from both resting and stimulated FT210 cells were compared by EMSA. Analysis of extracts prepared from TNF-alpha-treated cells revealed the presence of a kappaB-specific binding activity (Fig.5). This nucleoprotein complex was observed in extracts which had been grown at both 33 and 39 °C but only following TNF-alpha stimulation. Interestingly, no activation of NF-kappaB was observed using a common inducing agent, phorbol 12-myristate 13-acetate (PMA), in either FM3A or FT210 cells (Fig.5; data not shown), suggesting that additional activation pathways may also exist which are defective in this cell line. Taken together, the experiments performed in FT210 cells suggest that NF-kappaB is also induced at the G(2)/M boundary by TNF-alpha.


Figure 4: Activation of kappaB-directed CAT expression in G(2)/M-arrested FT210 cells. FT210 cells (A) and the parental line, FM3A (B) maintained at 33 °C, were transfected with either the wild type (wt) or mutant kappaB reporter plasmids as described under ``Materials and Methods.'' Each sample was split equally between two flasks 4 h after transfection; one sample was maintained at 33 °C while the other was incubated at 39 °C, as indicated. Preparation of cellular extracts and CAT analyses were performed as described under ``Materials and Methods.'' TNF-alpha addition was performed 24 h after transfection, and cells were harvested 48 h post-transfection. C, cell cycle analysis of FM3A and FT210 cells. FT210 and FM3A cells were incubated at 33 or 39 °C as indicated, for 24 h, and analyzed by flow cytometry as described under ``Materials and Methods.''




Figure 5: Induction of NF-kappaB activity by TNF-alpha in G(2)/M-arrested FT210 cells. FT210 cells were incubated for 24 h at 39 °C and stimulated with 10 ng/ml PMA or 200 units/ml TNF-alpha for 2 h. Preparation of nuclear extracts and EMSA were performed as described under ``Materials and Methods.'' The arrow indicates the position of the inducible, kappaB-specific nucleoprotein complex.




DISCUSSION

Cellular growth and differentiation is regulated by a combination of growth factors which are present in serum at nanomolar to picomolar concentrations. Addition of serum growth factors to G(0)-arrested cells induces a set of genes, often encoding transcription factors, whose products are required for progression into G(1) and continued cell growth. Previous studies have demonstrated that NF-kappaB represents an immediate-early response gene (49) . Like other immediate-early response genes, induction of NF-kappaB occurs in the presence of protein synthesis inhibitors(49, 53) presumably due to its presence as a covert, cytoplasmic complex with IkappaB. As shown previously(49) , we find that NF-kappaB is inducible following serum addition to serum-deprived cells. However, we also find that NF-kappaB induction in response to TNF-alpha occurs in a cell-cycle independent fashion.

The data reveal that NF-kappaB can be induced in serum-deprived cells by cytokines ( Fig.1and Fig. 2). At least two possibilities existed to account for these effects. First, the activation of NF-kappaB might itself be sufficient to induce checkpoint release, leading to re-entry into the cell cycle. Second, the activation of NF-kappaB by cytokines might occur independently of cell cycle, predicting that NF-kappaB induction alone would not change the cell cycle profile. The data presented in Fig.3appear to fit the second model; induction of NF-kappaB in G(0)-arrested cells by TNF-alpha did not dramatically affect the cell cycle profile, while in the same experiment the addition of serum almost completely relieved the G(0) arrest. Under some conditions, TNF-alpha stimulation has been shown to shorten the duration of S phase(54) , but this finding was not observed in our experimental system, using a different cell type and alternative conditions of growth arrest (Fig.3, Table 1). The addition of TNF-alpha to G(2)/M-arrested FT210 cells induced NF-kappaB ( Fig.4and Fig. 5), providing further support for this model.

Interestingly, in both 3T3 and FT210 cells, a significant degree of cell death was observed after TNF-alpha addition to arrested cells, starting 2-3 h following addition of cytokine. (^2)This effect was not observed with IL-1 which, at least in 3T3 cells, induced NF-kappaB to the same extent as TNF-alpha (data not shown). This result suggests that the signaling events leading to cell death by TNF-alpha are mediated by the Fas-related domains (55) and is therefore a consequence of apoptosis rather than induction of NF-kappaB per se.

Considering the rapid kinetics of induction (seconds to minutes), as well as its ability to be induced in the presence of protein synthesis inhibitors(53, 56) , NF-kappaB is a good candidate as both an immediate-early, cell cycle-responsive factor, and a cytokine-induced, cell cycle-independent factor. It is most likely that alternative signal transduction pathways are utilized in these two different events. In support of this hypothesis, much evidence has accumulated suggesting the involvement of serine-threonine kinases, including protein kinase C, in the cytokine-induced (cell cycle-independent) activation of NF-kappaB(30, 57, 58) . However, the observation that PMA was unable to induce NF-kappaB in FT210 cells suggests that protein kinase C-independent pathways of NF-kappaB activation might also exist, as described previously in another cell type(23) . Analysis of cytoplasmic extracts following cellular stimulation has revealed that only a fraction of NF-kappaB relocates to the nucleus.^2 This suggests that there may be distinct cytoplasmic stores of NF-kappaB, perhaps associated with different isoforms of IkappaB. These forms could be subject to modification by distinct kinases and phosphatases, allowing for regulation by distinct signaling pathways. Furthermore, several studies have raised the possibility of distinct pathways being involved in the cell cycle-dependent induction of NF-kappaB. The first of these is the implication that the addition of platelet-derived growth factor, an event which activates the intrinsic tyrosine kinase activity of the platelet-derived growth factor receptor, to serum-starved 3T3 cells can induce NF-kappaB(49) . Also, stimulation of the T cell costimulatory molecule, CD28, has been shown to induce NF-kappaB(59) . Finally, induction of NF-kappaB by some stimuli can be blocked by the tyrosine kinase inhibitor, herbimycin A, but this reagent is unable to inhibit NF-kappaB induction by TNF-alpha(60) . We therefore speculate that independent signaling pathways converge to activate similar NF-kappaB family members. It remains to be determined whether this convergence occurs upstream of IkappaB, for example, by regulation of a kinase of IkappaB(61) , downstream by modification of the NF-kappaB family members themselves (62) or at the level of IkappaB itself, perhaps by differential phosphorylation or degradation. The dissection of these signaling events, as well as the levels at which they converge, will provide insight into the cell cycle-dependent and -independent mechanisms of cellular and viral gene regulation.


FOOTNOTES

*
This work was supported by Grant AI29179 from the National Institutes of Health. 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.

§
Present address: Howard Hughes Medical Institute, The University of Chicago, 924 E. 57th St., Chicago, IL 60637.

Recipient of a Scholar Award from the American Foundation for AIDS Research (AmFAR) made in memory of James Hoese.

**
To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Michigan Medical Center, 1150 W. Medical Center Dr., 4520 MRSB I, Ann Arbor, MI 48109-0650. Tel.: 313-747-4798; Fax: 313-747-4730.

^1
The abbreviations used are: NF-kappaB, nuclear factor kappaB; CAT, chloramphenicol acetyltransferase; TNF-alpha tumor necrosis factor alpha; EMSA, electrophoretic mobility shift assay; PMA, phorbol 12-myristate 13-acetate.

^2
C. S. Duckett and G. J. Nabel, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Patrick Brown for providing FM3A and FT210 cells, Donna Gschwend for secretarial assistance, Karen Carter for computer graphics, and other members of the Nabel laboratory for their helpful advice and comments.


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