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[Cancer Research 60, 4085-4092, August 1, 2000]
© 2000 American Association for Cancer Research

Carcinogenesis

Nuclear Factor {kappa}B Subunits Induce Epithelial Cell Growth Arrest1

Cornelia S. Seitz2, Helen Deng2, Kaede Hinata, Qun Lin and Paul A. Khavari23

VA Palo Alto Health Care System and the Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear factor {kappa}B (NF-{kappa}B) gene-regulatory proteins play important roles in inflammation, neoplasia, and programmed cell death. Recently, blockade of NF-{kappa}B function has been shown to result in epithelial hyperplasia, suggesting a potential role for NF-{kappa}B in negative growth regulation. We expressed active NF-{kappa}B subunits in normal epithelial cells and found that NF-{kappa}B profoundly inhibits cell cycle progression. This growth inhibition is resistant to mitogenic stimuli and is accompanied by other features of irreversible growth arrest. NF-{kappa}B-triggered cell cycle arrest is also associated with selective induction of the cyclin-dependent kinase inhibitor p21Cip1, with overexpression of p21Cip1 alone inducing findings similar to those seen with NF-{kappa}B in vitro. An active NF-{kappa}B subunit expressed in the epidermis of p21Cip1-/- mice, however, displays only partial growth-inhibitory effects, suggesting that full NF-{kappa}B growth inhibition is only partially p21Cip1 dependent in this setting. These data indicate that NF-{kappa}B can trigger cell cycle arrest in epithelial cells in association with selective induction of a cell cycle inhibitor.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal somatic cells do not replicate indefinitely but ultimately undergo an irreversible withdrawal from the cell cycle that is resistant to mitogenic stimuli (1, 2, 3) . In stratified epithelium, this process occurs in association with commitment to terminal differentiation and outward migration (4 , 5) . Recent evidence suggests that neoplastic transformation in epithelia and other tissues may require mechanisms that, in addition to avoiding apoptosis, also bypass stimuli for such irreversible growth arrest (6, 7, 8) . Genes impacting cell cycle regulation, such as RB1 (9) , p53 (10, 11, 12) , p16INK4a (13) , and p21Cip1 (14) , play important roles in the induction of permanent growth arrest, as can signaling molecules of the Ras pathway (7) . Characterization of the molecular mechanisms controlling cellular growth arrest may offer insights into neoplasia and homeostasis of self-renewing tissues such as the epidermis, site of the most common cancers in the United States (15) .

Transcription factors can exert potent impacts in regulating cell proliferation, and their expression patterns may change with the induction of cellular growth arrest (16) . NF-{kappa}B/Rel gene-regulatory proteins influence important cell fate decisions and are activated in a range of conditions involving cellular stress and injury (17, 18, 19, 20) . NF-{kappa}B activity is controlled in several ways, important among these being its transition from an inactive cytoplasmic form to an active nuclear protein. I{kappa}B protein phosphorylation by I{kappa}B kinases in response to triggering stimuli leads to I{kappa}B degradation and nuclear translocation of NF-{kappa}B (21, 22, 23, 24, 25) . Additional forms of regulation exist via TPL-kinase, which regulates the proteolysis of the inhibitory precursors of p50 and p105 (26) . Recently, a role for NF-{kappa}B gene-regulatory proteins in regulating growth in stratified epithelium has been suggested (27) . In stratified epithelium, NF-{kappa}B subunits exist in the cytoplasm of the proliferative cells in the basal layer, then localize to the nuclei of the postmitotic suprabasal cells. Blockade of NF-{kappa}B function in the epidermis produces massive tissue hyperplasia whereas expression of constitutively active NF-{kappa}B subunits in transgenic mice produces epithelial hypoplasia, suggesting a possible role for NF-{kappa}B in negative cellular growth control (27) . The relevance of this observation to cutaneous carcinogenesis has recently been underscored by the demonstration that NF-{kappa}B-blockade can produce epidermal squamous cell carcinomas in transgenic mice over periods as short as 4 months (28) . In this study, we show that NF-{kappa}B induces epithelial cell cycle arrest in association with selective induction of the CKI4 p21Cip1.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of Mitotic Activity and Cell Cycle Distribution.
For analysis of cellular proliferation, cells were transduced in triplicate at densities of 104 cells/35-mm plate for each vector and time point, as described previously (29) . Following transduction, cells were harvested and counted in triplicate at 24-h intervals. Cell morphology was determined at each time point by phase contrast microscopy, and nuclear morphology was evaluated by fluorescence microscopy after staining with propidium iodide. Cell viability was determined by trypan blue exclusion. For mitogenic stimulation, cells were grown in the presence of EGF and KGF, both at concentrations of 10 ng/ml. For BrdUrd labeling in vitro, cells were grown on glass cover slides and incubated for 2 h with 10 µM BrdUrd (Boehringer Mannheim, Indianapolis, IN), then rinsed with PBS, fixed for 30 min in 70% ethanol, and air dried. After treatment with 0.07 N NaOH for 2 min the slides were thoroughly rinsed in PBS and stained with anti-BrdUrd monoclonal antibody (Becton Dickinson, San Jose, CA). Cells were then counterstained with propidium iodide (20 µg/ml) for 15 s to visualize all cells in a given field. For cell cycle analysis, cells were stained with propidium iodide 48 h after transduction then subjected to flow cytometry. Briefly, cells were trypsinized, washed in PBS, and incubated for 20 min in a solution containing 0.1% sodium citrate (pH 7.8), 0.1% Triton X-100, 50 µg/ml propidium iodide, and 1 mg/ml RNase. Then, an equal volume of a solution containing 0.375 M NaCl, 0.1% Triton X-100, 50 µg/ml propidium iodide was added and kept at 4°C until subjected to flow cytometry. Data were analyzed using ModFit software, as described previously (30) .

Western Analysis and Immunohistochemistry.
Whole cell extracts were prepared from cells grown in vitro and immunoblotted as described (29) after separation by SDS-PAGE on a 12% gel. Approximately 20 µg of protein, as determined by Bradford (Bio-Rad, Hercules, CA), were loaded per lane. Equal loading conditions were also confirmed by Coomassie Blue staining. In addition to antibodies to p21Cip1, p27Kip1, p57Kip2, p15INK4B, p16INK4A, p18INK4C, p19INK4D, p50, p65, and I{kappa}B{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA), blots were incubated simultaneously with polyclonal antiserum to BRG1 (31) , a constitutively expressed Mr 205,000 protein control for cell extract quality and protein transfer efficiency. Blots were visualized using the enhanced chemiluminescence-detection system (Amersham Corp., Arlington Heights, IL). Immunohistochemistry was performed as described (29 , 32) using antibodies to NF-{kappa}B subunits and to p21Cip1 (Santa Cruz Biotechnology), as well as to BrdUrd (Becton Dickinson). Prior to immunostaining, cells were rinsed with PBS, fixed for 10 min in acetone at room temperature, air-dried, and blocked with 5% normal goat serum. For staining, slides were incubated with primary antibodies for 30 min, followed by PBS washing and incubation with FITC-conjugated secondary antibodies (Sigma Chemical Co., St. Louis, MO), and mounted with Vectashield mounting media (Vector Laboratories Inc., Burlingame, CA). Where indicated, cells were counterstained 15 s with propidium iodide (20 µg/ml in PBS). For analysis of differentiation gene expression, the following antibodies were used for Western blotting and immunohistochemistry: keratin 1, keratin 10, involucrin, filaggrin, and loricrin (Babco, Richmond, CA). Differentiation gene immunostaining was performed by fixing transgenic cryosections in 100% acetone for 10 min., followed by blocking in 10% goat serum in PBS for 30 min. Following PBS rinse, primary antibodies were added for 1 h, then washed in PBS, and secondary antibodies were added for 30 min before PBS washing and counterstaining with Hoechst 33342 (2 µg/ml in PBS). Slides were then analyzed by fluorescence microscopy. For SA-ß-gal staining, cells were washed in PBS, fixed with 2% formaldehyde/0.2% glutaraldehyde for 5 min at room temperature, and stained for ß-gal at pH 6.0. (33) .

Cell Culture and Gene Transfer.
Normal human epithelial cells were isolated from human skin as described (34) . Cells were grown in a 1:1 mixture of serum-free medium (Life Technologies, Inc., Grand Island, NY) and 154 media (Cascade Biologicals, Portland, OR). The retroviral expression vectors for {Delta}SP, I{kappa}B{alpha}M, p50, and p65 were constructed as described (27) . For simultaneous expression of both p50 and p65, cells were transduced with both vectors. The p21Cip1 vector was produced by subcloning the full-length p21Cip1 cDNA into the EcoRI site of the LZRS backbone vector (35) after removal of the EcoRI fragment containing the lacZ gene. Amphotropic retrovirus production (32 , 35) and gene transfer with test and lacZ and GFP control vectors was performed as described previously (36) ; more than 98% gene transfer efficiency was confirmed for each vector by immunofluorescence staining with antibodies to NF-{kappa}B subunits, I{kappa}B, and p21Cip1. For analysis of differentiation gene expression in vitro, as a function of NF-{kappa}B activity, primary human keratinocytes were incubated in SFM/154 media with 1.5 mM calcium, as described previously (29) .

Transgenic Mice.
p50/p21Cip1-/- mice were generated by crossing K14-p50 mice with p21Cip1-/- mice, then performing the appropriate back crosses to reobtain homozygous null p21 alleles in the setting of epidermal p50 expression. p21Cip1-/- mice (37) were a generous gift of P. Leder (Howard Hughes Medical Institute, Boston, MA). K14-p50 transgenic C57/BL6 mice were generated as described previously (27) , however, the K14-p50 mice used for these studies were derived from two founders that were mildly affected, displaying less severe epidermal hypoplasia and surviving to reproductive age. Skin biopsy specimens were obtained from site-matched mid-back skin of genetically matched normal control as well as p50, p21Cip1-/-, and p50/p21Cip1-/- mice at 4 weeks of age. Data represent the average epidermal thickness measured on three separate histological sections per mouse; four independent mice were evaluated in each group.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NF-{kappa}B Subunits Induce Cell Morphological Changes.
To examine a potential growth-regulatory role for NF-{kappa}B, we transduced normal epithelial cells (passage 1) with amphotropic retroviral expression vectors for proteins with impacts on NF-{kappa}B function. These include constitutively active p50 (38) and p65/RelA (39) as well as molecules dominant-negative for NF-{kappa}B function, including I{kappa}B{alpha}M (40) and the transcriptionally inactive {Delta}SP p50 internal deletion mutant (41) . Retroviral expression vectors for lacZ (35) and GFP (42) marker genes as well as mock transduction served as controls. Using recently refined approaches (36) , gene transfer was effected with these vectors at efficiencies of >98%, as judged by immunohistochemical staining (data not shown); these vectors effectively express full-length mutant proteins in these cells. In this setting, we have recently confirmed that p50 and p65, both separately and together, produce strong constitutive activation of NF-{kappa}B-directed gene expression whereas {Delta}SP p50 and I{kappa}B{alpha}M inhibit phorbol ester-induced NF-{kappa}B-driven reporter gene activity (27) . Control-transduced cells and cells expressing molecules that are dominant-negative for NF-{kappa}B function, including I{kappa}B{alpha}M and {Delta}SP, exhibit the normal polygonal shaped cell morphology and colony growth pattern (Fig. 1, d, and eCitation ). In contrast, NF-{kappa}B subunits produce cell morphological changes as early as 24 h after gene transfer (Fig. 1 and cCitation ). Cells enlarge, flatten, and lose the pattern of growth in colonies, displaying the morphological changes consistent with epithelial cells undergoing permanent cell cycle arrest (43) . Similar effects are seen with coexpression of p50 and p65 subunits, and these NF-{kappa}B subunits fail to induce epidermal differentiation markers involucrin, keratin 10, and filaggrin, either in vivo (27) or in vitro (data not shown).



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  		Fig. 1. NF-{kappa}B induces cell morphological changes. Morphological changes of lacZ control (a), p50[+] (b), p65[+] (c), {Delta}SP (d), and I{kappa}B{alpha}M (e) cells 24 h after retroviral gene transfer; magnification, x20.

 
NF-{kappa}B Subunits Inhibit Cell Cycle Progression.
In addition to undergoing cell morphological changes, p50- and p65-expressing cells fail to proliferate normally (Fig. 2aCitation ). Nuclear stains fail to reveal morphological changes characteristic of apoptosis, and nonviable cells comprise <5% of cells in each vector group at all time points, indicating that this finding is not due to increased cell death (data not shown). NF-{kappa}B blockade in I{kappa}B{alpha}M-expressing cells, in contrast, demonstrate increased proliferation after 1 week in culture (Fig. 2aCitation ); this finding is most pronounced at later time points when cells have reached confluence. We next examined cell cycle distribution at 2 days after transduction with retroviral expression vectors for p50 and p65. Within 48 h after gene transfer, NF-{kappa}B subunit expression decreases the percentage of epithelial cells in S phase by >50% concomitant with an increase in the proportion of cells in G0-G1; BrdUrd incorporation is, likewise, decreased by >50% (Fig. 2bCitation and data not shown).



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  		Fig. 2. NF-{kappa}B inhibits cell cycle progression. a, growth curves. Cells were transduced in triplicate at densities of 104 cells/35-mm plate for each vector and counted at the indicated time points following transduction with retroviral expression vectors for p50, p65, I{kappa}B{alpha}M, and lacZ control. Each experiment was performed three times, and each time point was determined in triplicate independent transductions. p50/p65 denotes cells expressing both subunits. b, cell cycle analysis of NF-{kappa}B-transduced cells versus control. Cells were transduced in triplicate with retroviral expression vectors for p65 and lacZ control, then stained with propidium iodide, and cell cycle distribution was determined via flow cytometry, with data analyzed using Modfit software.

 
NF-{kappa}B Subunits Induce Features of Irreversible Growth Arrest.
In stratified epithelium, NF-{kappa}B subunits redistribute from cytoplasm to nucleus in cell compartments undergoing cell cycle arrest, outward migration, and commitment to terminal differentiation (27) , raising the possibility that NF-{kappa}B could induce the growth arrest associated with this process. To analyze NF-{kappa}B growth-inhibitory effects further, we passaged NF-{kappa}B subunit-expressing cells. On repeated passaging, these cells fail to proliferate exponentially in contrast to lacZ controls. Additionally, these cells can be maintained for up to 4 weeks, displaying the same morphology and apparent growth arrest (data not shown). Combined with the morphological changes noted above, these findings raised the possibility that NF-{kappa}B subunits trigger an irreversible cell cycle arrest in epithelial cells.

In addition to cell morphological changes, such permanent arrest has been associated with resistance to growth factor stimulation (2 , 44) . To examine whether NF-{kappa}B-induced growth arrest confers such resistance to mitogenic stimuli, NF-{kappa}B subunit-transduced cells were grown either in minimal media lacking growth factors or in media containing both EGF and KGF. Under appropriate conditions, these factors can serve as epithelial cell mitogens in vitro (34 , 45) . lacZ-transduced controls in minimal media grow slowly (Fig. 3aCitation ). As expected, lacZ control cells begin to proliferate exponentially in the presence of growth factors (Fig. 3aCitation ). Growth factors, however, did not overcome the growth arrest seen in both NF-{kappa}B p50 and p65 subunit-expressing cells (Fig. 3aCitation and data not shown), suggesting that NF-{kappa}B rendered these cells resistant to these mitogenic stimuli. An additional feature that can be seen in permanently arrested epithelial cells is the induction of a SA-ß-gal that can be specifically detected in vitro at pH 6.0 (33) . Although absent early, SA-ß-gal is observed in a significant proportion of NF-{kappa}B-expressing cells by 3 days after gene transfer, and the percentage of SA-ß-gal-positive cells consistently increases over the following 4 days (Fig. 3 and cCitation ). Normal cells are almost devoid of SA-ß-gal staining under these conditions, as are cells expressing the transcriptionally inactive {Delta}SP p50 mutant and the additional NF-{kappa}B inhibitor I{kappa}B{alpha}M (Fig. 3cCitation ).



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  		Fig. 3. NF-{kappa}B induces features of irreversible growth arrest. a, NF-{kappa}B-induced growth arrest is not reversible by the addition of growth factors. p50 and lacZ control-transduced cells were plated at low density, then grown in media supplemented with either [+] or without [-] a combination of EGF and KGF. Cells were harvested and counted at indicated times after plating. b, SA-ß-gal staining. SA-ß-gal staining was performed on p50[+], p65[+], and GFP[+] control cells 5 days after gene transfer. c, proportion of cells demonstrating SA-ß-gal staining. At least 500 cells were counted for each plate; all time points were performed in duplicate. d, differentiation gene expression as a function of NF-{kappa}B activity. Murine skin transgenic for NF-{kappa}B activation (p50, p65) or blockade of function (I{kappa}B{alpha}M) along with normal littermate controls was immunostained using antibodies to filaggrin and loricrin. Note the correctly localized expression in the outer epidermis in all cases and the lack of significant alterations in expression levels as a function of NF-{kappa}B activity. Shown also is skin stained with Hoechst 33342 to highlight cellular nuclei (Hoechst) and background staining obtained with secondary antibody alone (Control). Bars, 30 µM.

 
Because induction of terminal cell cycle arrest is associated with terminal differentiation in the epidermis, we examined NF-{kappa}B impact on expression of a panel of terminal differentiation markers, including keratin 1, keratin 10, filaggrin, and loricrin. Epidermis transgenic for NF-{kappa}B activation (p50, p65) or blockade (I{kappa}B{alpha}M) displays normally localized expression of terminal differentiation marker gene expression (Fig. 3dCitation and data not shown), consistent with earlier observations with the differentiation marker involucrin (27) . In addition, human keratinocytes transduced at high efficiency with retroviral expression vectors for gain (p50, p65) and loss (I{kappa}B{alpha}M and {Delta}SP) of NF-{kappa}B function, as well as GFP control, then differentiated in vitro with elevated media calcium to 1.5 mM (29) display no differences in differentiation gene induction (data not shown), arguing against a primary role for NF-{kappa}B in regulating differentiation.

NF-{kappa}B Subunits Induce p21Cip1.
To investigate the basis for NF-{kappa}B inhibition of cellular growth, we studied NF-{kappa}B effects on expression of p53 and CKIs of the Cip/Kip and INK4 families. NF-{kappa}B subunit-expressing cells induce p21Cip1 protein and mRNA expression (Fig. 4 and bCitation ); lower levels of p21Cip1 expression could be detected in controls (Fig. 4 and bCitation ). Both p50 and p65 alone or both subunits expressed together produce this induction (Fig. 4 and bCitation ). Such induction is not observed in cells transduced with the transcriptionally inactive {Delta}SP p50 deletion mutant or I{kappa}B{alpha}M (Fig. 4aCitation and data not shown). To analyze this effect at the level of individual cells, immunofluorescence staining was then performed with antibodies to p21Cip1 with cells expressing p50, p65, I{kappa}B{alpha}M, or lacZ control (Fig. 4 and dCitation ). Expression of active NF-{kappa}B subunits was associated with an augmented proportion of cells with nuclear p21Cip1 (Fig. 4 and dCitation ). NF-{kappa}B induction of p21Cip1 seems selective in that it is not accompanied by significant changes in the levels of other CKIs, including p27Kip1 and p57Kip2, or the INK4 family proteins p15INK4B, p16INK4A, p18INK4C, and p19INK4D (Fig. 4eCitation and data not shown). In addition, this p21Cip1 induction occurs without an increase in p53 expression (Fig. 4eCitation ). Normal mice treated topically with the NF-{kappa}B inhibitor PDTC (27) demonstrate a decrease in p21Cip1 protein detectable by immunostaining in the epidermis to levels approaching p21Cip1-/--negative control skin (data not shown), consistent with NF-{kappa}B regulation of p21Cip1 in vivo.



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  		Fig. 4. NF-{kappa}B induces p21Cip1. a, induction of p21Cip1 protein expression by NF-{kappa}B subunits. Nine (Lanes 1, 3, 5, 7, and 9) and 18 (Lanes 2, 4, 6, 8, and 10) h after retroviral transduction with expression vectors for p50, p65, I{kappa}B{alpha}M, and lacZ control, cellular extracts were prepared. Immunoblotting was performed with antibodies to p21Cip1. b, NF-{kappa}B impact on p21Cip1 mRNA expression. Nine h after retroviral transduction, RNA was harvested and subjected to Northern analysis using a human p21Cip1 cDNA probe; ß-actin control for RNA sample loading and quality is shown below the p21Cip1 Northern blot. Note the increase in p21Cip1 mRNA expression by p50/p65 over control levels and, conversely, the decrease in expression with I{kappa}B{alpha}M. c, induction of nuclear p21Cip1 expression by NF-{kappa}B. Eighteen h after gene transfer, cells were stained with antibody to p21Cip1. Top, p21Cip1 expression (green); bottom, the same fields counterstained with propidium iodide to show all of the cells present in the same field. Shown are fields representative of six independent transductions for each vector group. d, proportion of cells expressing nuclear p21Cip1. Three independent transductions were evaluated for each vector; at least 250 cells were counted for each transduction. Data are expressed as cells expressing nuclear p21Cip1 as a percentage of total. e, NF-{kappa}B does not alter expression of p53 or the CKIs p27Kip1, p15INK4B, and p16INK4A. Eighteen h after retroviral transduction with expression vectors for p50, p65, and the I{kappa}B{alpha}M mutant trans-dominant for NF-{kappa}B function along with lacZ control, cellular extracts were prepared. Immunoblotting was performed with antibodies to p53, p27Kip1, p15INK4B, and p16INK4A. Blots were simultaneously incubated with antibodies to BRG1, a Mr 205,000 constitutively expressed control for extract quality and protein transfer efficiency.

 
p21Cip1 Triggers the Growth Arrest Features Induced by NF-{kappa}B.
In addition to a recently appreciated role in regulating the complex process of epidermal differentiation (5) , p21Cip1 has recently been implicated in negative growth regulation in epithelium (30) . In light of the fact that NF-{kappa}B induces p21Cip1, we wished to determine whether p21Cip1 is sufficient to induce the similar cell cycle distribution and growth arrest features induced by NF-{kappa}B in epithelial cells. To do this, we produced an amphotropic retroviral vector for constitutive p21Cip1 expression. After confirming expression of full-length p21Cip1 protein (Fig. 5aCitation ) and >98% gene transfer efficiency by immunofluorescence analysis of transduced cells using antibody to p21Cip1 (data not shown), we analyzed cells for morphological changes, proliferation kinetics, and appearance of SA-ß-gal. Similar to NF-{kappa}B, p21Cip1-expressing cells demonstrate morphological alterations and induction of SA-ß-gal (Fig. 5 and cCitation ). In addition, p21Cip1 caused cell cycle arrest, with cell cycle distribution similar to that induced by NF-{kappa}B subunits (Fig. 5dCitation ).



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  		Fig. 5. p21Cip1 inhibits epithelial growth. a, expression of p21Cip1. Cells were transduced with a retroviral expression vector for p21Cip1 along with GFP control. Cell extracts were prepared before transduction and 9 h after gene transfer. Western blotting was performed using antibodies to p21Cip1 and BRG1 control. b, SA-ß-gal is induced by p21Cip1. ß-gal staining was performed 5 days after gene transfer (arrows indicate SA-ß-gal[+] cells). c, quantitation of percentage of cells demonstrating SA-ß-gal expression. Each transduction was performed in triplicate; at least 500 cells were counted for each transduction. d, p21Cip1 expression produces cell cycle distribution changes similar to that seen with NF-{kappa}B activation. Forty-eight h after transduction with the retroviral expression vector for p21Cip1, cell cycle distribution was analyzed by flow cytometry in triplicate independent transductions for the p21Cip1 vector and GFP control.

 
p50 Partially Inhibits Epidermal Hyperplasia in p21Cip1-/- Mice.
The selective induction of p21Cip1 noted above and the fact that p21Cip1 alone seems to replicate NF-{kappa}B growth-inhibitory effects raised the question of whether NF-{kappa}B growth inhibition is entirely dependent on p21Cip1 or if it can be fully mediated by other factors. To address this issue, we expressed p50, previously shown to induce epidermal growth inhibition and hypoplasia in transgenic mice (27) , in a p21Cip1-/- background (37) . Mildly affected mice expressing p50 targeted to epithelium via the K14 promoter were mated with p21Cip1-/- mice to generate p50/p21Cip1-/- mice, and epidermal thickness was analyzed. NF-{kappa}B subunits induce epidermal hypoplasia and growth inhibition, as measured by BrdUrd incorporation in vivo (27) ; consistent with this, p50 epidermis was relatively hypoplastic compared with control (Fig. 6Citation ). p21Cip1-/- mice, in contrast, are known to exhibit hyperplasia in various tissues (37) and display epidermal hyperplasia (Fig. 6 and bCitation ). p50 expression only partially reverses this hyperplasia (Fig. 6 and bCitation ), suggesting that although p21Cip1 may, in part, mediate NF-{kappa}B subunit growth inhibition in the epidermis, additional factors may be required for full NF-{kappa}B subunit-induced hypoplasia.



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  		Fig. 6. p50 partially inhibits epidermal hyperplasia in p21Cip1-/- mice. a, histology of normal control (NL), K14-p50 (p50), p21Cip1-/-, and p50/p21Cip1-/- mice. b, epidermal thickness of normal control (NL), K14-p50 (p50), p21Cip1-/-, and p50/p21Cip1-/- mice. *, epidermal thickness of p50/p21Cip1-/- mice is significantly different from both p21Cip1-/- mice and normal control (P < 0.01). Data represents the average epidermal thickness measured on three separate histological sections per mouse ± SD; four independent mice were evaluated in each group.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we show that NF-{kappa}B inhibits epithelial cell cycle progression. Whereas cell cycle-regulatory proteins and cell cycle entry have been shown to affect NF-{kappa}B function in some settings (17 , 46) , NF-{kappa}B-triggered inhibition of cellular proliferation affecting cell cycle-regulatory proteins such as CKIs has not been previously reported in epithelial cells. Many prior studies have associated NF-{kappa}B with the promotion of cellular proliferation. In this regard, an important role for NF-{kappa}B proteins in the outgrowth of the vertebrate limb bud has recently been appreciated (47 , 48) . Induction of NF-{kappa}B DNA-binding activity is also associated with the G0 to G1 transition in murine fibroblasts in vitro (49) . Furthermore, in Ras-transformed NIH 3T3 fibroblasts, antisense inhibition of p65 expression decreased tumor cell growth in vivo (50) . Consistent with this, in another study with 3T3 fibroblasts, I{kappa}B{alpha} blockade increased neoplastic growth in vivo (51) . A similar NF-{kappa}B growth-promoting role has been suggested in lymphocytes (52) and in HTLV-1-transformed lymphoid cells (53) . These studies suggesting a growth-promoting role for NF-{kappa}B seem to be in contradiction with our current findings in normal epithelial cells.

This discrepancy may stem from the fact that NF-{kappa}B effects may differ depending on tissue and cell-intrinsic differences, including developmental state. In developmentally mature nontransformed cells, for example, proto-oncogene function can be entirely opposite to that seen in immortalized cell lines. This has been described in the case of Ras induction of cellular growth arrest and senescence (7) , and this disparity may also apply in the case of NF-{kappa}B. In further support of a growth-inhibitory role for NF-{kappa}B in the epidermis are recent data in mice with blocked NF-{kappa}B function via inhibitor of {kappa}B kinase {alpha} (IKK{alpha}/IKK1) gene disruption (54 , 55) . The mechanistic basis for the potent NF-{kappa}B growth effects observed in IKK knock-out (54 , 55) and prior transgenic mouse models (27) , however, has been unclear.

Here, we report that NF-{kappa}B can induce p21Cip1 and that this CKI alone is sufficient to induce epithelial cell growth arrest. Consistent with this is the observation that c-rel can promote G1 arrest and increased p21Cip1 expression in the transformed HeLa cell line (56) . p21Cip1 expression is induced concomitantly with cell cycle withdrawal in G1 prior to differentiation in a variety of tissues (57) and has been implicated as an important factor in this process (58, 59, 60) . In stratified epithelia, p21Cip1 seems to play an important role in growth control (30) . p21Cip1, similar to activated NF-{kappa}B, is expressed in a pattern restricted to growth-arrested cells; this expression normally occurs in the absence of detectable p53 (61 , 62) . p21Cip1 expression in this setting, consistent with recent findings for NF-{kappa}B (27) , does not induce expression of terminal differentiation genes (5) . Instead, p21Cip1 seems to exert an inhibitory effect on their expression, suggesting additional mechanisms of p21Cip1 regulation of growth and differentiation in stratified epithelium (5) . These findings and our current work indicate that permanent cell cycle arrest and subsequent induction of differentiation are not obligately linked in stratified epithelium, suggesting that NF-{kappa}B and p21Cip1 function to induce cell cycle arrest in a step discrete from terminal differentiation. These data indicate that NF-{kappa}B may, thus, function more as a pure growth inhibitor. Of interest, these data, when combined with the recently published IKK1/IKK{alpha} knock-out phenotypes (54 , 55 , 63) , implicate IKK1/IKK{alpha} as activating NF-{kappa}B to arrest epithelial growth as well as triggering a separate NF-{kappa}B-independent pathway that impacts differentiation, a possibility partially consistent with a recently proposed model (64) .

Prior studies showed NF-{kappa}B induction within one h after UV-exposure in human skin (65) . Other studies primarily examining p21Cip1 expression have shown that p21Cip1 is markedly increased over baseline levels within 8–12 h following UV injury (61 , 62) . These separate lines of data raise the possibility that NF-{kappa}B activation, followed by induction of p21Cip1 and irreversible growth arrest, might be an important safeguard to remove cells with DNA-damage induced by UV injury, in a manner that may complement p53 effects. Consistent with this, we have observed that UV-induction of p21Cip1 mRNA in primary human epithelial cells is blocked in the presence of I{kappa}B{alpha}M.5 Cell cycle control by the CKI p21Cip1, then, seems to be important in normal negative growth regulation in stratified epithelium, and the induction of p21Cip1 by NF-{kappa}B is consistent with the NF-{kappa}B-mediated growth inhibition we have observed in this setting.

Whereas p53 is perhaps the best recognized transcriptional activator of p21Cip1 expression (66) , a number of sequence-specific DNA binding proteins have been implicated as capable of activating p21Cip1 expression in different cell types under various conditions (67 , 68) . Among such proteins are AP2 (69) , the ets oncogene family transcription factor E1AF (70) , the glucocorticoid receptor (71) , the EBV immediate early transactivator Zta (72 , 73) , STAT1 (74) , MyoD (58 , 59) , Sp1/Sp3 (75) , and C/EBP{alpha} (76) . Here, we demonstrate that NF-{kappa}B induction of p21Cip1 may occur, at least in part, via regulation of p21Cip1 mRNA expression. Consistent with this are our observations that NF-{kappa}B subunits induce p21Cip1 mRNA expression and that p21Cip1 induction is dependent on an intact transactivation domain in the case of p50 (data not shown). Further supporting this possibility is the observation that NF-{kappa}B blockade in these epithelial cells reduces p21Cip1 mRNA expression and the fact that multiple consensus GGGRNNYYYC NF-{kappa}B DNA-binding site motifs (77) can be found in the published sequence of the human p21Cip1 promoter (62) , including at positions -302 and -2006. p21Cip1 regulation by the transcription factors noted above, however, includes mechanisms acting at levels other than mRNA expression, including enhancement of p21Cip1 protein stability (76) , and additional mechanisms of p21Cip1 regulation by NF-{kappa}B cannot currently be excluded. Why so many individual transcription factors seem capable of activating p21Cip1 and whether their effects are direct or indirect is, thus, still unclear in most cases. Specific p21Cip1 inducers, however, may be of relevance in only specific cell types and tissue settings.

Many transcription-regulatory proteins are important in triggering growth arrest either prior to terminal differentiation or in situations where continued cell division is undesirable, such as following genotoxic injury as well as during infection and inflammation. Such a theme seems to apply to NF-{kappa}B activation in stratified epithelium. NF-{kappa}B activation, as judged by nuclear translocation (27) , occurs in epithelial cells undergoing irreversible cell cycle arrest, a similar p21Cip1-inductive setting to that seen with MyoD in terminal muscle differentiation. If p21Cip1 is the sole mediator of NF-{kappa}B inhibition, we would expect no NF-{kappa}B subunit effects on the epidermal hyperplasia seen in p21Cip1-/- mice. On the other hand, if NF-{kappa}B-induction of p21Cip1 is redundant or unnecessary for its growth inhibition, we would expect to see epidermal hypoplasia in p50/p21Cip1-/- mice comparable with p50 control. The fact that we have observed an intermediate situation, in which p50 partially inhibits epidermal hyperplasia, suggests that although p21Cip1 may mediate a portion of NF-{kappa}B growth-inhibitory effects, there are additional factors required. The potential existence of such partially redundant effectors of growth inhibition downstream of NF-{kappa}B may be important in a self-renewing tissue prone to neoplasia, such as the stratified epithelium of the epidermis. Our findings support a new growth-inhibitory role for NF-{kappa}B in stratified epithelia and identify induction of the CKI p21Cip1 as a potential mechanism contributing to this growth regulation.


   ACKNOWLEDGMENTS
 
We thank P. Jackson for helpful early discussions and advice and J. Healy for helpful comments. We thank A. Israel for the XbaI p105 plasmid and p50 {Delta}SP mutant, P. Leder for p21Cip1-/- mice, I. Verma for I{kappa}B{alpha}M, P. Jackson for the p21Cip1 cDNA, E. Fuchs for the K14 promoter, and N. Griffiths and P. Bernstein for administrative support.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Office of Research and Development, Department of Veterans Affairs; a Veterans Affairs Merit Review Award (to P. A. K.); and NIH Grants AR43799, AR45192, and AR44012 (to P. A. K.). C. S. S. is the recipient of a postdoctoral fellowship award from Deutsche Forschungsgemeinschaft. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Stanford University School of Medicine, 269 Campus Drive, Room 2145, Stanford, CA 94305. Phone: (650) 725-5266; Fax: (650) 723-8762; E-mail: khavari{at}CMGM.stanford.edu Back

4 The abbreviations used are: CKI, cyclin-dependent kinase inhibitor; BrdUrd, bromodeoxyuridine; EGF, epidermal growth factor; KGF, keratinocyte growth factor; SA-ß-gal, senescence-associated ß-galactosidase. Back

5 K. Hinata et al., manuscript in preparation. Back

Received 9/15/99. Accepted 5/25/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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