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Endocrinology Vol. 145, No. 6 2833-2844
Copyright © 2004 by The Endocrine Society

The Liver-Enriched Inhibitory Protein Isoform of CCAAT/Enhancer-Binding Protein ß, But Not Nuclear Factor-{kappa}B, Mediates the Transcriptional Inhibition of ß-Casein by Tumor Necrosis Factor-{alpha}

Haitao Zhang, Haiwei Zhang, Laura Lee and Margot M. Ip

Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263

Address all correspondence and requests for reprints to: Margot M. Ip, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263. E-mail: margot.ip{at}roswellpark.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TNF-{alpha} is a physiological regulator of mammary gland development that stimulates the growth of both normal and malignant mammary epithelial cells in primary culture and inhibits functional differentiation. To understand how TNF exerts its effects, the current study examined the mechanism by which TNF down-regulates expression of the ß-casein and whey acidic protein (WAP) genes. TNF treatment markedly decreased activity of the ß-casein and WAP promoters in transiently transfected HC11 mammary epithelial cells. Overexpression of the nuclear factor-{kappa}B (NF{kappa}B) p50 and/or p65 proteins increased the transcriptional activity of the ß-casein and WAP promoters in HC11 cells, suggesting that the inhibitory effect of TNF on transcription of these genes is not mediated by NF{kappa}B. This was further confirmed in experiments in which an NF{kappa}B super-repressor was overexpressed, and by deletion of an NF{kappa}B binding site in the ß-casein promoter. In contrast, we found that TNF induced both nuclear expression and the DNA-binding activity of liver-enriched inhibitory protein (LIP) isoform of CCAAT/enhancer-binding protein ß. Moreover, cotransfection of LIP and ß-casein expression vectors showed that LIP suppressed the transcriptional activity of the ß-casein promoter. Together, these results suggest that LIP plays a critical role in mediating TNF-induced down-regulation of the ß-casein gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH ORIGINALLY IDENTIFIED by its ability to induce necrosis of tumors, TNF-{alpha} is now widely regarded as a pleiotropic cytokine that affects the growth, differentiation, and function of many cell types (1, 2, 3, 4). In vivo and in vitro studies carried out in our laboratory have shown that TNF plays an important role in mammary gland development (5, 6, 7, 8, 9). Using a physiological three-dimensional culture model in which mammary epithelial cells (MEC) are cultured within a reconstituted basement membrane matrix in serum-free medium, our laboratory demonstrated that TNF stimulates the growth of MEC, in the presence or absence of epidermal growth factor (EGF) (5, 6, 7), an effect mediated by the p55 TNF receptor (6). TNF also induces extensive branching morphogenesis through a matrix metalloproteinase-9-dependent mechanism (5, 7, 8). The expression of TNF and its two receptors exhibits a highly regulated pattern during mammary gland development in vivo (6) (and unpublished data). TNF mRNA increases significantly during pregnancy and then gradually decreases during lactation and postweaning involution. TNF protein, measured by immunohistochemistry, is high in both stromal and epithelial cells during puberty, pregnancy, and involution but significantly reduced during lactation. Collectively, these results suggest that TNF is a physiological regulator of mammary gland development.

In addition to its effects on cell growth and morphogenesis of MEC, TNF also inhibits their functional differentiation. Recent studies in our laboratory have shown that TNF decreases the mRNA levels of both ß-casein and whey acidic protein (WAP) (9), and the protein level of ß-casein (5, 6, 7) in MEC in primary culture. The inhibitory effect of TNF on steady-state mRNA levels of these genes is exerted through decreased transcription as shown by nuclear run-on assay, as well as decreased mRNA stability as shown in actinomycin D treatment studies (9).

The tissue- and developmental stage-specific expression of the milk protein genes is tightly regulated by a number of hormones, growth factors, and components of the extracellular matrix (reviewed in Refs.10 and 11). Extensive efforts to understand the transcriptional regulation of these genes have revealed that this involves a network of interactions or cross-talk among an array of transcription factors, including CCAAT/enhancer-binding proteins (C/EBP), signal transducer and activator of transcription (STAT)5, glucocorticoid receptor, ying-yang 1 (YY-1), nuclear factor-{kappa}B (NF{kappa}B), and nuclear factor 1 (10, 11). Using an NF{kappa}B consensus oligo, our laboratory reported that TNF induces the DNA-binding activity of NF{kappa}B in normal and transformed MEC in primary culture (12), as well as in HC11 cells (9). Thus, we hypothesized that TNF regulates the transcription of ß-casein and WAP through activation of NF{kappa}B. To test this hypothesis and to delineate the signaling pathways activated by TNF in MEC, the current study was designed to examine the roles of NF{kappa}B and other transcription factors in mediating TNF-induced down-regulation of milk protein expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Fetal bovine serum, gentamicin, phenol red-free RPMI 1640, and the Lipofectamine PLUS transfection reagents were purchased from Life Technologies (Grand Island, NY). Insulin, hydrocortisone, phenylmethylsulfonylfluoride (PMSF), aprotinin, leupeptin, and benzamidine were purchased from Sigma (St. Louis, MO). Ovine prolactin (NIDDK-oPL-20) was provided by Dr. A. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program (Harbor-UCLA Medical Center), and mouse EGF was purchased from Upstate Biotechnology (Lake Placid, NY). Recombinant mouse TNF was obtained from Biosource International (Camarillo, CA). Immobilon polyvinylidene difluoride membrane was purchased from Millipore (Bedford, MA), and ECL Western blotting detection reagent was from Amersham Pharmacia Biotech (Arlington Heights, IL). pGL3 vectors, pRL vectors, and the luciferase assay kits were purchased from Promega (Madison, WI). Anti-YY1 (sc-7341X), anti-p65 (sc-109X), anti-C/EBPß (sc-150), and anti-STAT5 (sc-835) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Hsp70/Hsc70 (SPA-820) and anti-p50 (KAP-TF112) antibodies were purchased from Stressgen Biotechnologies Corp. (Victoria, British Columbia, Canada). Cloned pfu DNA polymerase was obtained from Stratagene (La Jolla, CA). The plasmids containing the 5'-flanking regions of the rat ß-casein and WAP genes were kindly provided by Dr. Jeffrey Rosen (Baylor College of Medicine, Houston, TX). The NF{kappa}B expression vectors, as well as the pFlag-I{kappa}B{alpha}-SR construct, were gifts from Drs. Albert S. Baldwin, Jr. (University of North Carolina, Chapel Hill, NC) and Warner Greene (University of California at San Francisco). The liver-enriched inhibitory protein (LIP) expression vector was generously provided by Dr. Pascal Gos (University of Geneva, Geneva, Switzerland). The 293 cells were generously provided by Dr. David Goodrich (Roswell Park Cancer Institute).

Cell culture
The mouse MEC line HC11 was obtained from Dr. Jeffrey Rosen (Baylor College of Medicine), with the permission of Dr. Bernd Groner (Institute for Experimental Cancer Research, Tumor Biology Center, Freiburg, Germany). Cells were plated and grown in basal medium (phenol red-free RPMI 1640 supplemented with 10% FBS, 1 ng/ml EGF, 2 mM glutamate, 5 µg/ml insulin, and 50 µg/ml gentamicin) for 4 d to reach confluency. The basal medium was then replaced with growth medium (phenol red-free RPMI 1640 supplemented with 10% FBS, 10 ng/ml EGF, 2 mM glutamate, 5 µg/ml insulin, and 50 µg/ml gentamicin). The cells were maintained in growth medium for 4 d before being switched to lactogenic medium (phenol red-free RPMI 1640 supplemented with 10% FBS, 5 µg/ml prolactin, 1 µg/ml hydrocortisone, 2 mM glutamate, 5 µg/ml insulin, and 50 µg/ml gentamicin). The cells were cultured in lactogenic medium for 5 d for maximal ß-casein induction and used for all the studies described below.

Transient transfection and reporter gene analysis
Supercoiled plasmid DNAs were prepared by the Qiagen column procedure (Qiagen, Valencia, CA). The day before transfection, HC11 cells were trypsinized and seeded in 100-mm culture dishes at 1 x 107 cells per dish in 10 ml lactogenic medium in the absence of gentamicin. Four micrograms of luciferase reporter construct were mixed with 750 µl RPMI 1640 and 20 µl PLUS reagent (Life Technologies, Rockville, MD) and incubated at room temperature for 15 min. Thirty microliters of LipoFECTAMINE reagent were diluted with 750 µl RPMI 1640 and mixed with DNA-PLUS complexes and incubated at room temperature for another 15 min. The HC11 culture medium was subsequently replaced with 5 ml RPMI 1640, and the DNA-PLUS-LipoFECTAMINE reagent complexes were overlaid onto the cells. After incubation at 37 C for 4 h in a 5% CO2 incubator, cells were trypsinized, resuspended with lactogenic medium, and equally divided into 6-well plates. Where indicated, TNF was then added to the treatment wells at a concentration of 40 ng/ml. Cells were lysed in 500 µl of 1x passive lysis buffer (Promega) 48 h after transfection and stored at –70 C until assay.

For cotransfection experiments, HC11 cells were seeded at 1 x 106 cells per well in 3 ml lactogenic medium minus gentamicin in a 6-well plate. One microgram of pGL3B-ßcas2.8 was mixed with various amounts of the expression vectors and 0.025 µg pRL-CMV, and diluted in 100 µl RPMI 1640 and 6 µl PLUS reagent. Four microliters of LipoFECTAMINE were mixed with 100 µl RPMI 1640 and added into the previous mixture. Similar steps were carried out as described above.

To determine the firefly luciferase activity, 20 µl cell lysate was mixed with 100 µl luciferase assay reagent (Promega) and analyzed using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). For dual-luciferase assay, the renilla luciferase activity was determined by addition of 100 µl of 1x stop-N-glow reagent (Promega). Total protein concentration of the lysate was measured by the Bradford method (13) using a protein assay reagent (Bio-Rad, Hercules, CA). The firefly luciferase activity was normalized against total protein concentration (TNF treatment experiments; LIP transfection experiment) or renilla luciferase activity and total protein concentration (all other experiments). Because LIP transfection inhibited both the control and ß-casein constructs, equal transfection efficiency in the LIP transfection experiments was verified by Southern analysis using a probe specific for the luciferase construct.

Western blotting
The nuclear extracts used here and in the following studies were prepared from HC11 cells as described previously (12). Specifically, cell pellets were resuspended in 400 µl ice-cold buffer A containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM PMSF, 1 µg/ml aprotinin, 20 µg/ml leupeptin, and 0.5 mg/ml benzamidine. After swelling on ice for 15 min, Nonidet P40 (Roche Molecular Biochemicals, Indianapolis, IN) was added to a final concentration of 0.6% (vol/vol), and the samples were sheared 10 times through a 22-gauge needle. Light microscopy and trypan blue were used to verify that nuclei remained intact while the plasma membrane was disrupted. Nuclei were collected by a 30-sec centrifugation at 7500 x g, washed once with 500 µl buffer A, and resuspended in 50 µl cold lysis buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 µg/ml aprotinin, and 20% glycerol. Samples were vigorously shaken for 20 min at 4 C and centrifuged at 12,000 x g for 10 min at 4 C. Supernatants were supplemented with 25 µg/ml leupeptin, aliquoted, frozen in liquid nitrogen, and stored at –80 C. Protein concentrations were determined by Bio-Rad protein assay. For Western blot, 30 µg nuclear extract was resolved on 4–20% gradient polyacrylamide-sodium dodecyl sulfate gels and transferred to polyvinylidene difluoride membrane. After blocking in Tris-buffered saline (150 mM NaCl; 10 mM Tris, pH 7.4) containing 5% nonfat milk powder overnight at 4 C, membranes were washed in Tris-buffered saline containing 0.1% Tween 20 and then incubated with a 1:1,000 dilution of rabbit polyclonal antiserum against mouse C/EBPß proteins or a 1:5,000 dilution of anti-YY1 mouse monoclonal antibody for 2 h at room temperature. Blots were washed, incubated with horseradish peroxidase-conjugated goat antirabbit secondary antibody (1:1,000 dilution) for 90 min at room temperature, and developed using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). Each blot was stripped and reprobed with a 1:20,000 dilution of anti-Hsp70/Hsc70 mouse monoclonal antibody. Immunoreactive bands were quantitated by volume densitometry using the ImageQuant 5.1 software (Molecular Dynamics, Sunnyvale, CA).

EMSA
Complementary strands of the probes were synthesized (Integrated DNA Technologies, Coralville, IA) and annealed by heating at 95 C for 10 min and cooling down gradually to room temperature. Each double-stranded probe was radiolabeled with [{gamma}-32P]dATP and T4 polynucleotide kinase and purified using a Sephadex G-25 spin column (Roche). Ten micrograms of nuclear extract were incubated with 25,000–50,000 cpm labeled probe for 30 min at room temperature in a 20-µl reaction vol containing 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol, and 1 µg poly (dI-dC). The reaction mixture was loaded onto a 5% polyacrylamide gel in 0.5% Tris-borate EDTA buffer. After electrophoresis at room temperature, the gel was dried and autoradiographed.

Sequence information of the probes can be found in Table 1Go.


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  		TABLE 1. Sequences of the oligonucleotides used in these experiments

 
Site-directed mutagenesis
Using sequential PCR, a mutated NF{kappa}B4 site (see below) was generated between the unique KpnI and PvuII restriction sites of a DNA segment of 1476 bp on pGL3B-ßcas2.8 (Fig. 1AGo) by a site-directed mutagenesis procedure (14). Briefly, primers no. 1–4 (see Table 1Go for sequence) were designed with primer no. 3 carrying a two-base mismatch to the template (refer to Table 1Go). Two separate PCR (PCR I and PCR II) reactions were performed to generate two halves of the desired segment using 10 ng pGL3B-ßcas2.8, 8 µl of 1.25 mM deoxynucleotide triphosphate, 1 µl each of 10 µM primers (primer no. 1 and primer no. 2 for PCR I; primer no. 3 and primer no. 4 for PCR II), 1 µl of 2.5 U/µl pfu DNA polymerase in 50 µl total vol. The PCR condition was 30 cycles of 94 C for 1 min, 55 C for 1 min, and 72 C for 1.5 min preceded by 94 C for 4 min. For PCR III, 20 ng each of the purified PCR I and PCR II products were combined with 2 µl each of 10 µM primer no. 1 and primer no. 4, 16 µl of 1.25 mM deoxynucleotide triphosphate, 2 µl of 2.5 U/µl pfu DNA polymerase in 100 µl total vol. The PCR condition was 30 cycles of 94 C for 1 min, 55 C for 1 min, and 72 C for 2 min preceded by 94 C for 4 min. The products of PCR III were purified and digested with KpnI and PvuII before being ligated with the 5.3-kb KpnI and PvuII restriction fragment from pGL3B-ßcas2.8. An aliquot of the ligation products was transformed into Escherichia coli DH5{alpha} cells, and the clones carrying the desired mutant plasmids were selected by sequencing.



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  		FIG. 1. TNF down-regulates ß-casein and WAP gene expression. A, Reporter gene constructs of the ß-casein and WAP promoters were generated by inserting the indicated regions into the pGL3-Basic vector. B, Reporter gene analysis. The reporter gene constructs were transiently transfected into mouse MEC HC11 cells. TNF was then added to the medium to a final concentration of 0 or 40 ng/ml. After 48 h, the cells were lysed and the luciferase activity was measured. Total protein concentration was also determined and used to normalize the results. Transfections were done in triplicate, and the results are shown as mean ± SEM. Three independent experiments were performed, and the result of a representative experiment is shown here.

 
DNA affinity immunoblotting (DAI)
The DAI assay was carried out as described (15). Briefly, the same double-stranded oligonucleotide used in the EMSAs was 5' end-labeled with biotin (Integrated DNA Technologies). Two hundred micrograms of nuclear extract were incubated with 10 pmol probe at 4 C for 30 min in a final vol of 400 µl containing 2 µg poly (dI-dC), 5 mM DTT, 100 mM NaCl in 1x DNA binding buffer (20 mM Tris-HCl, pH 7.2; 1 mM EDTA; 0.1% Triton X-100; and 4% glycerol). The DNA/protein complexes were captured by adding 0.1 mg magnetic streptavidin beads (Promega) to the above mixture and incubating at 4 C for 60 min. The beads were precipitated with a magnet and washed three times with 1x DNA binding buffer. The proteins were eluted from the beads by heating at 90 C for 5 min in 20 µl sample buffer (4% sodium dodecyl sulfate; 20% glycerol; 200 mM DTT; 120 mM Tris, pH 6.8; and 0.002% bromophenol blue). The eluted proteins were analyzed by Western blotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TNF on the transcription of the ß-casein and WAP genes
A portion of the rat ß-casein promoter region has previously been cloned and characterized (16). However, unlike its counterparts in other species, including human and ruminants, only the 0.8 kb immediately upstream of the transcriptional start site was sequenced. To investigate TNF regulation of the cis-regulatory elements in the rat ß-casein promoter further upstream, we sequenced an additional 1.5 kb upstream of the known region. The sequence was deposited in the DDBJ/EMBL/GenBank databases and was assigned an accession number AY19060.

To study the effect of TNF on the transcription of the milk protein genes, a series of reporter gene constructs was generated by inserting various DNA fragments derived from the 5'-flanking region of the ß-casein and WAP genes upstream of a promoterless firefly luciferase reporter gene (Fig. 1AGo). These constructs were transiently transfected into HC11 cells, and luciferase activity was measured in the presence or absence of TNF treatment. As shown in Fig. 1BGo, TNF decreased the activity of both the ß-casein and WAP promoters, indicating that TNF exerts its role in down-regulating the expression of these genes at the transcriptional level. This is consistent with our previous data obtained using the nuclear run-on assay (9) and validates the transfection approach for the studies described below. Furthermore, the shortest ß-casein reporter construct, pGL3B-ßcas0.7, which contains –179 to +490 of the ß-casein promoter, showed a similar fold of inhibition by TNF compared with the other ß-casein reporter constructs, suggesting the presence of TNF-responsive element(s) in this proximal ß-casein promoter region, as well as upstream.

NF{kappa}B does not mediate the inhibitory effect of TNF on ß-casein expression
Many functions of TNF are mediated through the activation of NF{kappa}B (17). Using an NF{kappa}B consensus oligonucleotide, previous studies in our laboratory demonstrated that TNF induces the DNA-binding activity of the p50 homodimer of NF{kappa}B in both normal and transformed MEC in primary culture (12) and the p50/p65 heterodimer of NF{kappa}B in the HC11 MEC line (9). By sequence analysis using the MatInspector program and the TRANSFAC 5.0 database, we identified several potential NF{kappa}B binding sites in the ß-casein promoter (Fig. 2Go and Table 1Go). To test whether NF{kappa}B mediates TNF-induced down-regulation of ß-casein expression, various amounts of the expression vectors encoding NF{kappa}B p50 or p65 were cotransfected into HC11 cells with the ß-casein reporter construct, pGL3B-ßcas2.8. As shown in Fig. 3AGo, both p50 and p65 induced an increase in ß-casein promoter activity in a concentration-dependent manner, although p65 was much more potent than p50. These observations suggest that NF{kappa}B is a positive regulator of ß-casein expression, in contrast to our original hypothesis of the role of NF{kappa}B in mediating the inhibitory effect of TNF.



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  		FIG. 2. Schematic diagram of the promoter region of the rat ß-casein gene. This figure shows the previously identified composite response elements, as well as the four putative NF{kappa}B binding sites revealed by sequence analysis; adapted from Rosen et al. (10 ).

 


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  		FIG. 3. NF{kappa}B is a positive regulator of ß-casein expression. A, Overexpression of NF{kappa}B increases the transcription of the ß-casein gene. Previous studies have shown that TNF induced the DNA binding activity of the NF{kappa}B p65/p50 heterodimer in HC11 cells. To study the effect of NF{kappa}B on the promoter activity of ß-casein, 1 µg pGL3B-ßcas2.8 was cotransfected into HC11 cells with various amounts of the NF{kappa}B expression vectors, and the luciferase activity was determined. B, The NF{kappa}B super-repressor I{kappa}B{alpha}-SR decreases ß-casein promoter activity. I{kappa}B{alpha}-SR inhibits the function of NF{kappa}B by binding to and preventing its nuclear translocation. One microgram of pGL3B-ßcas2.8 was cotransfected into HC11 cells with various amounts of the pFlag-I{kappa}B{alpha}-SR expression vector, and the luciferase activity was determined. In both A and B, pRL-CMV was also cotransfected and used to normalize for transfection efficiency. All the transfections shown in the figures were done in triplicate, and the results are shown as mean ± SEM. Three independent experiments were performed, and the result of a representative experiment is shown here.

 
To further confirm the above findings, we performed a cotransfection experiment using an expression vector encoding the I{kappa}B{alpha} super-repressor, I{kappa}B{alpha}-SR. It has been shown that I{kappa}B{alpha}-SR inhibits the function of NF{kappa}B by binding to p65 and preventing the nuclear translocation of the p65/p50 heterodimer (18). When cotransfected into HC11 cells with pGL3B-ßcas2.8, I{kappa}B{alpha}-SR caused a decrease in the promoter activity of ß-casein (Fig. 3BGo). This observation agrees with the p50/p65 cotransfection results in establishing a positive role of NF{kappa}B in regulating ß-casein expression. Thus, both these studies suggest that TNF-induced inhibition of ß-casein expression is not mediated directly through the activation of NF{kappa}B.

Mutation of a NF{kappa}B binding site in the ß-casein promoter has no effect on the response to TNF
The above data suggested that the putative NF{kappa}B sites in the ß-casein promoter (Fig. 2Go) might not bind NF{kappa}B proteins, and/or that the sites were not functionally significant. To address this, we used oligonucleotide probes encompassing these sites to investigate whether NF{kappa}B proteins bound to these putative elements. No NF{kappa}B binding was detected when NF{kappa}B1, NF{kappa}B2, and NF{kappa}B3 oligonucleotides were used (data not shown); further studies on these sites were therefore not undertaken. However, when NF{kappa}B4 was used as the probe, we observed three specific binding complexes, two of which were induced by TNF treatment (Fig. 4AGo, lanes 1–3). These complexes appear to consist of the p65/p50 heterodimer, and the p50/p50 homodimer, as suggested by supershift studies using p50 and p65 antibodies, respectively (Fig. 4AGo, lanes 4 and 6). The results were confirmed by DAI assays using biotinylated NF{kappa}B4, where both p50 and p65 were found to be present in the DNA-binding complex eluted from the probe (data not shown).



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  		FIG. 4. Mutation of the NF{kappa}B4 site has no effect on the response of the ß-casein promoter to TNF treatment. A, Characterization of the NF{kappa}B4 binding site (Table 1Go, and below) in the ß-casein promoter. Nuclear extracts were prepared from HC11 cells treated with TNF (0 or 40 ng/ml) for 72 h and incubated with radiolabeled NF{kappa}B4 probe. Using a 100-fold excess of unlabeled probe (lane 3), three specific binding complexes, indicated by the arrows on the left side, were identified. TNF induced the formation of the two upper complexes. Antibodies against p50, p52, p65, c-Rel, and RelB were included in the binding reactions (lanes 4–8) to reveal the composition of the complexes. The supershift bands (lanes 4 and 6) are indicated by the arrowheads. B, Mutation was introduced into the NF{kappa}B4 site by replacing the two highly conserved bases (TC) with nonrelevant sequence (GA), as indicated: NF{kappa}B4, 5'-GGTTCAGGCCTTTCCCTAAT-3'; NF{kappa}B4-Mut, 5'-GGTTCAGGCCTTGACCTAAT-3'. This mutation abolished the binding of NF{kappa}B proteins to the NF{kappa}B4 site. Arrows indicate the p50/p65 and p50/p50 DNA-binding complexes, respectively. C. Wild-type or mutant pGL3B-ßcas2.8 were transfected into HC11 cells, and TNF was then added to the medium to a final concentration of 0 or 40 ng/ml. After 48 h, the cells were lysed, and the luciferase activity was measured. Total protein concentration was also determined and used to normalize the results. Transfections were done in triplicate, and the results are shown as mean ± SEM. The results shown in A–C are representative of at least three independent experiments.

 
To further confirm the specificity of the binding of the NF{kappa}B proteins, 2 bp near the 3' end of NF{kappa}B4 that are highly conserved among NF{kappa}B binding sites (19) were modified as indicated in the legend to Fig. 4Go. As shown in Fig. 4BGo, this modification totally abolished the binding of NF{kappa}B proteins to this site, suggesting that the binding is highly sequencespecific.

Although the results from Fig. 1BGo suggest the presence of TNF-responsive element(s) in the region from –179 to +490 of the ß-casein promoter, it does not exclude the possibility that redundant TNF-responsive element(s) also exists in the distal promoter region. To study the functional relevance of the NF{kappa}B4 site in the context of the ß-casein promoter, the same 2-bp mutation was introduced into pGL3B-ßcas2.8 by site-directed mutagenesis. Both the wild-type and mutant pGL3B-ßcas2.8 were (separately) transfected into HC11 cells and the cells lysed 48 h after addition of TNF. As shown in Fig. 4CGo, TNF inhibited the transcriptional activity of both constructs to a similar extent, suggesting that the mutation that abolished the binding of NF{kappa}B proteins to the NF{kappa}B4 site has no effect on the TNF response. This result suggests that the suppression of gene transcription by TNF is not exerted through inducing the binding of NF{kappa}B to its target site in the ß-casein promoter. We therefore performed additional studies to determine how the TNF effect might be mediated.

TNF does not affect either the nuclear abundance or the DNA-binding activity of YY-1
Other investigators have demonstrated that YY-1 plays an important role in suppressing the expression of ß-casein during mammary development (20, 21). If TNF exerts its inhibitory effect on ß-casein expression by acting on YY-1, it would be expected that the nuclear abundance and/or the DNA-binding activity of YY-1 would be elevated by TNF. To test this possibility, Western blotting analysis and EMSAs were performed using nuclear extracts prepared from HC11 cells treated with TNF for the indicated times (Fig. 5Go). TNF did not change nuclear YY-1 levels at any of the time points examined (24, 48, 72, or 96 h of TNF treatment) (Fig. 5AGo). For EMSA, we used an oligonucleotide probe derived from –129 to –98 of the ß-casein promoter, which contains a previously characterized YY-1 binding site (20). No consistent changes in the DNA-binding activity of YY-1 were found after TNF treatment (Fig. 5BGo). Collectively, these results suggest that YY-1 is not involved in mediating TNF action on ß-casein expression.



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  		FIG. 5. TNF does not change either the nuclear level (A) or the DNA-binding activity (B) of YY-1. Nuclear extracts were prepared from HC11 cells after TNF treatment for the indicated times, as described in Materials and Methods. A, Western blotting analysis using a YY-1-specific antibody. The same membrane was stripped and reblotted with an Hsc70-specific antibody to demonstrate the equality of sample loading. B, EMSA using a DNA probe containing the YY-1 binding site in the ß-casein promoter. At least three independent experiments were performed, and the result of a representative experiment is shown here.

 
TNF activates C/EBPß
We next considered whether C/EBPß might mediate the effect of TNF. C/EBPß is required for expression of ß-casein (22, 23), and both the full-length liver-enriched activating protein (LAP) isoform, as well as the N-terminally truncated LIP isoform are developmentally regulated (22, 24) in the mammary gland. LIP, which contains an intact basic zipper domain and a truncated trans-activation domain, is capable of binding to C/EBP sites and inhibiting the trans-activating activity of LAP in a dominant negative fashion. To determine the effect of TNF on the nuclear expression of C/EBPß, Western analysis was performed using nuclear extracts from HC11 cells treated with TNF (Fig. 6AGo). TNF increased the nuclear levels of both the LIP and LAP C/EBPß isoforms (Fig. 6AGo). The induction could be detected as early as 12 h and persisted over a period of 72 h. Note that the C/EBPß antibody has previously been shown to cross-react nonspecifically with a 45-kDa protein (22), and we confirmed this using a cell lysate from 293 cells that do not express C/EBPß (Fig. 6BGo). Specific LAP and LIP bands are at 35 and 21 kDa, respectively. The band at approximately 38 kDa most likely represents a posttranslationally modified form of LAP (24). Densitometric quantitation from three independent experiments (Fig. 6CGo) revealed that, at 12 h, levels of LAP were increased by 3.3-fold, whereas LIP expression was up-regulated to a greater extent at 5.9-fold; this increase in LIP, relative to LAP, was maintained for up to 72 h. Thus, the ratio of LAP/LIP in the nucleus was decreased by TNF. These data would be consistent with an inhibitory effect of TNF on expression of the ß-casein gene.



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  		FIG. 6. TNF induces the nuclear expression of the C/EBPß proteins. A, Western blotting analysis using an antibody against C/EBPß. Thirty micrograms of nuclear extracts prepared from HC11 cells after different durations of TNF treatment were analyzed. The hsc70 band was used as a loading control. B, Western blot of a nuclear extract of HC11 cells, and a whole-cell lysate from 293 cells. This figure demonstrates that the 45-kDa band seen in nuclear extracts of HC11 cells with the C/EBPß antibody is nonspecific (ns), because 293 cells do not express C/EBPß. C, Quantitation by scanning densitometry of the LIP/hsc70 and LAP (35 kDa)/hsc70 ratios on Western blots from three independent experiments. This figure shows the fold induction by TNF at each time point (mean ± SEM). Note that, to ensure linearity, the quantitation of the LAP bands was done from films with a shorter exposure time than those of the LIP bands. Because of the difficulties inherent in summarizing quantitative data from Western blots performed at different times (i.e. the three separate experiments reported here), statistical significance (P = 0.05, t test) was observed only at the 24-h time point. However, it should be noted that, at each of the data points in each experiment, LIP was stimulated by TNF to a greater extent than LAP.

 
EMSAs were carried out using three oligonucleotides, each representing a previously characterized C/EBPß binding motif derived from the ß-casein promoter (25) (Table 1Go). Low DNA-protein interaction was detected with C/EBPß-1 and C/EBPß-2, and no TNF effect was observed (data not shown). In contrast, three DNA/protein complexes were detected when C/EBPß-3 (Table 1Go) was used, as indicated by arrows in Fig. 7AGo. Competition with unlabeled probe revealed the specificity of all the complexes (Fig. 7AGo). The intensity of complex 2 did not seem to be affected by TNF, whereas those of complexes 1 and 3 were increased by TNF (Fig. 7AGo). The induction could be observed as early as 12 h after TNF treatment and persisted over a period of 72 h (data not shown). Both complexes 1 and 2 contain C/EBPß proteins, because the intensity of both bands decreased when the antibody against C/EBPß was used in the supershift assay (lanes 3 and 7, Fig. 7AGo). Because the probe C/EBPß-3 also contains a STAT5 binding site (26), we examined the presence of STAT5 protein in these complexes. The intensity of both complexes 1 and 2 was decreased by the anti-STAT5 antibody, suggesting that STAT5 is also present in these complexes. This was confirmed in the DAI assay, which demonstrated the presence of STAT5 in the protein complex eluted from the C/EBP oligonucleotide (data not shown). Complex 3 was not supershifted by either antibody. Due to our inability to obtain antibodies that could differentiate the LAP and LIP isoforms of C/EBPß, we employed the DAI assay to further confirm the presence of C/EBPß and identify which isoform(s) was present in these complexes. The same C/EBPß oligonucleotide used in the EMSAs was biotinylated, and the DNA/protein complexes in the nuclear lysates shown in Fig. 6AGo were captured by streptavidin-conjugated magnetic beads. After elution, the proteins were subjected to Western analysis. As shown in Fig. 7BGo, LAP and its putative posttranslationally modified 38-kDa variant, as well as LIP, were present, and their levels were elevated by TNF. To control for the specificity of the DAI assay, the same blot was probed with an antibody for hsc70, and no hsc70 was detected (data not shown). The absence of the nonspecific 45-kDa band in the proteins eluted from the NF{kappa}B oligo/protein complex also demonstrates the specificity of the assay. Together, the above studies demonstrate that TNF enhances the binding of C/EBPß to the ß-casein promoter.



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  		FIG. 7. TNF down-regulates ß-casein expression through the action of LIP. A, TNF induces DNA/protein interactions at a C/EBPß binding site in the ß-casein promoter region (EMSA). An oligonucleotide probe, C/EBPß3, containing a previously characterized C/EBPß binding site, was incubated with 10 µg nuclear extract purified from HC11 cells after 24 h of TNF treatment. Three binding complexes were identified, as indicated by the arrows on the left side. Unlabeled C/EBPß probe (cold probe) was added at 100-fold excess to show the specificity of the binding (lanes 4 and 8). Antibodies directed against C/EBPß (lanes 3 and 7) and STAT5 (lanes 2 and 6) were used to reveal the composition of the complexes. B, DAI assay identified the presence of LIP and LAPs in the DNA-protein complexes. The same probe used in EMSA was biotinylated and incubated with 200 µg nuclear extracts from HC11 cells treated with TNF for the indicated times. The DNA/protein complexes were captured by magnetic streptavidin beads. After elution, the proteins were analyzed by Western blotting using the C/EBPß antibody. To control for the specificity of the DAI assay, the blot was probed for hsc70, and the result was negative (i.e. no hsc70 was detected). The hsc70 content of the nuclear extract used for the DAI assay is shown as INPUT hsc70 and demonstrates equivalent input in each of the control and TNF treatment groups. C, Overexpression of LIP results in decreased transcription of the ß-casein promoter. Various amounts of the pCMV-LIP expression vector were cotransfected into HC11 cells with 1 µg pGL3B-ßcas2.8. Total protein concentration was measured and used to normalize for experimental variability. Transfections were done in triplicate, and the results are shown as mean ± SEM. Three independent experiments were performed, and the result of a representative experiment is shown here. RLU, Raw luciferase units.

 
Overexpression of LIP results in decreased activity of the ß-casein promoter
To study the effect of up-regulation of LIP expression on the transcription of the ß-casein gene, a LIP expression vector was cotransfected into HC11 cells with pGL3B-ßcas2.8. Compared with the vector control, overexpression of LIP inhibited the reporter gene activity in a concentration-dependent manner (Fig. 7CGo). Total protein concentration was used to normalize for experimental variability. Transfection efficiency was monitored by Southern analysis of the cell lysates using a probe specific for the ß-casein-luciferase construct. Similar transfection efficiencies were observed among different transfection groups (data not shown); thus the observed LIP-mediated reduction in ß-casein promoter activity was not due to differences in transfection efficiency.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study agrees with our previous experiments demonstrating that TNF inhibits the expression of the ß-casein and WAP genes at the transcriptional level (9). Our data also confirm the report of Geymayer and Doppler (27) that TNF inhibits ß-casein reporter activity in HC11 cells transfected with a construct containing the –300 to –1 region of the promoter, and extend their data to demonstrate that TNF significantly inhibited luciferase reporter activity of both longer (–2325 to +490) and shorter (–179 to +490) promoter constructs. Because the expression of the ß-casein and WAP genes is regulated similarly by TNF, we used ß-casein as a model to investigate the mechanism by which TNF inhibits the transcription of these genes. Contrary to our original hypothesis, we found that NF{kappa}B does not seem to be directly involved in mediating the TNF effect. Instead, our results suggest a potential role for the LIP isoform of C/EBPß as a downstream mediator of TNF-induced transcriptional repression of ß-casein expression.

The involvement of C/EBPß in mammary gland development has been well studied. C/EBPß expression is induced during pregnancy and involution, and decreased during lactation (24, 28), a pattern that correlates very well with that of TNF (6). C/EBPß knockout mice exhibit abnormal mammary ductal morphogenesis, limited lobuloalveolar development, and diminished expression of the ß-casein and WAP genes (22). The ratio of LAP/LIP is tightly regulated during mammary development and is correlated with ß-casein expression (24). Overexpression studies have previously demonstrated that LIP inhibits casein expression (29, 30), an observation that we have confirmed here. However, despite its developmental regulation in the mammary gland and its specific appearance during liver regeneration (31), the physiological significance of the LIP isoform has been controversial, with its generation being attributed to an alternative internal translation start site (31, 32, 33, 34, 35), regulated proteolytic cleavage (31, 36), or artifactual proteolytic cleavage (30, 37). The studies described herein demonstrate that TNF increases the nuclear level of LIP and decreases the LAP/LIP ratio, leading to the suppression of ß-casein expression. The observation that LIP increased to a greater extent than LAP would tend to argue against LIP being a proteolytic artifact, in agreement with the data of Hirai et al. (38). Of special interest is that elevation of LIP levels has been reported to be associated with increased MEC proliferation (39). This is consistent with the previous findings in our laboratory that TNF stimulates the growth of both normal and malignant MEC (5, 6, 7, 12). Overexpression of LIP was also found in human breast cancers (40). Based on these findings, it is appropriate to propose that TNF regulation of C/EBPß is a physiologically important event during the course of normal mammary gland differentiation. Aberrant expression of TNF could lead to increased LIP expression, which in turn could contribute to mammary tumorigenesis.

The role of C/EBPß in conveying the TNF signal has been described in other cell types. In human keratinocytes and HeLa cells, C/EBPß mediates TNF-induced activation of the K6b promoter (41). TNF activates C/EBPß through a posttranscriptional mechanism in an adult rat hepatocyte line (42) and induces C/EBPß expression in mouse primary astrocytes (43). Binding of C/EBPß to the GLUT4 promoter is induced by TNF in 3T3-L1 adipocytes (44). More interestingly, Iraburu et al. (45) found that, in hepatic stellate cells, TNF down-regulated the expression of {alpha}1(I) collagen through LIP and C/EBP{delta}. In a similar study, Greenwel et al. (46) found that TNF inhibition of the expression of {alpha}2(I) collagen was mediated by C/EBPß and C/EBP{delta} in cultured fibroblasts. These findings are analogous to what we have found in MEC, suggesting that C/EBPß-mediated TNF regulation of gene expression is a common mechanism that is not unique to the mammary gland. Whether C/EBP{delta} is involved in the down-regulation of ß-casein in our system remains to be determined.

STAT5, previously known as mammary gland factor, is the primary transcription factor that mediates prolactin-induced activation of the milk protein genes in the mammary gland (47). There are two STAT5 sites in the composite response element of the ß-casein promoter (26), which play an essential role in regulating milk protein gene expression (10, 11). Wyszomierski and Rosen (29) have shown that STAT5 and C/EBPß cooperatively transactivate ß-casein expression in the presence of the glucocorticoid receptor. The N-terminal transactivation domain of LAP is essential for such an activity, because substituting LIP for LAP resulted in the abolishment of the transcriptional cooperation between C/EBPß and STAT5. The authors suggested that it is possible that LIP inhibits ß-casein expression by binding to the neighboring C/EBPß3 site and interfering with the function of STAT5 (29). Our EMSA and DAI results, demonstrating: 1) that C/EBPß and STAT5 coexist in two DNA/protein complexes, and 2) that TNF treatment results in increased binding of LIP to the C/EBPß3 site, lend support to such a model. Together with the previously reported ability of TNF to inhibit STAT5 phosphorylation in HC11 cells (27), these observations suggest an important mechanism by which TNF inhibits ß-casein transcription.

Our data suggest that NF{kappa}B does not directly mediate the inhibitory effect of TNF on ß-casein gene transcription. Using EMSA analysis, we detected specific binding of nuclear extract proteins to the NF{kappa}B1, NF{kappa}B3, and NF{kappa}B4 sites; however, antibodies to p50, p52, p65, c-rel, or RelB did not supershift or decrease the intensity of the NF{kappa}B1 or NF{kappa}B3 complexes, suggesting that NF{kappa}B proteins did not bind to these two sites. This is consistent with the report of Geymayer and Doppler (27) that mutation of the –84 to –75 region of the NF{kappa}B site on the ß-casein promoter did not inhibit NF{kappa}B repression of STAT5. Based on the TNF inducibility of the p65/p50 heterodimer and the p50/p50 homodimer NF{kappa}B complexes on the NF{kappa}B4 oligo, and the loss of DNA-binding activity upon mutation of the oligo, we expected that the NF{kappa}B4 site would be functional. However, transfection of a ß-casein construct with a mutated NF{kappa}B4 site did not affect the ability of TNF to inhibit ß-casein transcription, suggesting that the inhibitory effect of TNF is not mediated by binding of NF{kappa}B to the promoter.

Unexpectedly, our data suggest that NF{kappa}B alone acts as an activator of ß-casein transcription in HC11 cells. NF{kappa}B/p65 or the p65/p50 heterodimer were most active in this regard, probably because bcl3, which is a critical coactivator of NF{kappa}B/p50 (which lacks a transcriptional activation domain), is not present in HC11 cells (L. Lee and M. Ip, unpublished data). In a study using 293 cells, a human kidney epithelial cell line, Geymayer and Doppler (27) reported that the p65/p50 heterodimer of NF{kappa}B inhibited ß-casein expression. This apparent discrepancy may be due to the difference in signaling pathways in different cell types, especially in view of the fact that the full complement of transcription factors and coactivators necessary for ß-casein transcription are present within HC11 cells but not within 293 cells. Alternatively, it could be attributed to different experimental designs. In Geymayer and Doppler’s study, NF{kappa}B was cotransfected with STAT5 and a ß-casein promoter construct, and it was found that NF{kappa}B inhibited ß-casein expression indirectly by blocking prolactin-induced activation of STAT5. In the absence of prolactin, however, NF{kappa}B stimulated ß-casein transcription (27). Our study aimed to investigate the direct involvement of NF{kappa}B in mediating TNF-induced down-regulation of milk protein genes by cotransfecting NF{kappa}B with the ß-casein promoter construct into lactogenic-competent mouse MEC HC11. The results showed that NF{kappa}B is unlikely to be directly involved in repressing ß-casein expression, but we could not rule out the possibility that NF{kappa}B may play an indirect role by activating other genes that may indirectly alter ß-casein transcription or by interacting with other transcription factor(s) activated by TNF. One such candidate is C/EBPß. In the DAI assay, we detected the presence of NF{kappa}B p50 and p65 in the DNA/protein complexes formed by incubating the C/EBP probe with nuclear extracts from HC11 cells (data not shown). It has yet to be determined to which isoform of C/EBPß NF{kappa}B binds. However, it could be speculated that a direct binding of NF{kappa}B to C/EBP-LAP might interfere with the transcriptional activation activity of LAP on the ß-casein promoter. Further studies along this line are being conducted in our laboratory.

In summary, the work described herein demonstrates that C/EBPß LIP plays a critical role in mediating the effect of TNF on the expression of ß-casein. Current studies in our laboratory are focused on identifying other transcriptional regulators that are involved, as well as the cross-talk among these factors. A detailed knowledge of the molecular events initiated by TNF in MEC will not only provide the basis for fully understanding the role of TNF in mammary differentiation and tumorigenesis but may also help identify potential therapeutic targets for breast cancer.


   Acknowledgments
 
We are grateful to Drs. Molly Kulesz-Martin and Chad Knights for their assistance in setting up the DAI assay in our laboratory and to Dr. Fengzhi Li for helpful discussions.


   Footnotes
 
This work was supported by National Institutes of Health Grant CA 77656 and by the shared resources of the National Cancer Institute Cancer Center Support Grant CA 16056.

Abbreviations: C/EBP, CCAAT/enhancer-binding protein; DAI, DNA affinity immunoblotting; DTT, dithiothreitol; EGF, epidermal growth factor; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitory protein; MEC, mammary epithelial cell(s); NF{kappa}B, nuclear factor-{kappa}B; PMSF, phenylmethylsulfonylfluoride; STAT, signal transducer and activator of transcription; WAP, whey acidic protein; YY-1, ying-yang 1.

Received December 22, 2003.

Accepted for publication February 10, 2004.


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 Introduction
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 Results
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