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Regulation of Milk Protein Gene Expression in Normal Mammary Epithelial Cells by Tumor Necrosis Factor¹

Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263

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

	Abstract

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Tumor necrosis factor- (TNF) is a physiologically significant regulator of mammary gland development, stimulating growth and branching morphogenesis of mammary epithelial cells (MEC) and modulating functional differentiation. The present studies were performed to determine the mechanism by which TNF modulated functional differentiation. In rat MEC in primary culture, TNF inhibited accumulation of whey acidic protein and ß-casein messenger RNAs in a time- and concentration-dependent manner. In contrast, levels of transferrin messenger RNA, the product of another milk protein gene, were not inhibited by TNF, suggesting selectivity. Using a nuclear run-on assay in the immortalized HC11 mammary epithelial cell line and the transcriptional inhibitor actinomycin D in MEC in primary culture, the effects of TNF were shown to be mediated by both a decrease in transcription and a decrease in the stability of the whey acidic protein and ß-casein transcripts. Additionally, TNF stimulated the binding of nuclear factor-B to a consensus B-oligonucleotide, increased the stability of matrix metalloproteinase-9 (MMP-9) transcripts, and increased MMP-9 activity. Together, these data suggest that TNF may exert its effects on milk protein gene expression either directly via nuclear factor-B modulation of transcription, or indirectly via MMP-9-induced remodeling of the architectural or hormonal environment surrounding the MEC.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
ALTHOUGH BEST KNOWN for its role in the immune system, tumor necrosis factor- (TNF) also plays a critical role in endocrine tissue, including the ovary, uterus, and placenta (1, 2). In addition, our laboratory has demonstrated that TNF plays a key role in the mammary gland (3, 4, 5, 6, 7). Specifically, TNF was shown to stimulate growth as well as induce extensive branching and alveolar morphogenesis of isolated rat mammary epithelial cells (MEC) in primary culture and under optimal medium conditions to inhibit the accumulation of casein proteins (3, 4, 5). Under suboptimal conditions, however, namely in the absence of epidermal growth factor (EGF), the effects of TNF on casein were biphasic, with low levels stimulating casein accumulation in parallel with a stimulatory effect on morphogenesis, and higher levels inhibiting casein protein levels. Furthermore, using agonistic antibodies specific to each of the receptors, we found that the p55 TNF receptor (TNFR) mediates the stimulatory effect of TNF on proliferation as well as the inhibitory effect on casein accumulation. In contrast, the p75 TNFR mediates an increase in casein accumulation (4).

Recent studies suggest that these effects of TNF on MEC are physiologically relevant. First, we found that secondary and tertiary branching of the mammary epithelium was inhibited in TNF null mice during puberty (7). Second, MEC were shown to express TNF as well as its two receptors, p55 and p75 TNFR, in a developmentally regulated manner (4). TNF messenger RNA (mRNA) increased markedly during pregnancy, then gradually decreased throughout lactation and involution; concomitant with this, the 26-kDa membrane form of TNF protein was first detected during pregnancy and was significantly elevated during lactation, but was not detected in pubescent rats or during involution. In contrast, p55 TNFR mRNA was elevated during pregnancy and early lactation, declining thereafter, and p75 TNFR mRNA increased during lactation and remained elevated through early involution. Taken together, these studies support the hypothesis that TNF, acting through the p55 TNFR, may play an important role in stimulating growth and morphogenesis during pregnancy; moreover, together with progesterone, TNF may inhibit the expression and secretion of milk proteins at this time. During lactation, however, the increased levels of p75 TNFR together with a significantly increased expression of the 26-kDa membrane form of TNF, which is thought to act selectively through the p75 TNFR (8), may stimulate functional differentiation, thus permitting the extensive synthesis and secretion of milk proteins seen at this developmental stage.

The regulation of milk proteins, the functional differentiation products of the mammary gland, has been studied by a number of investigators. In general, these studies have shown that several hormones act in concert to exert regulation at both transcriptional and posttranscriptional levels (9, 10). In vitro, PRL and a glucocorticoid, together with insulin are required for optimal transcription of the ß-casein and whey acidic protein (WAP) genes (10, 11), whereas progesterone is inhibitory (12, 13, 14, 15). No information is available on the mechanism by which TNF inhibits casein expression. Thus, the first objective of the work reported here was to determine whether the effect of TNF on casein accumulation was exerted at the RNA level. Once this was established, our second objective was to determine the mechanism by which steady state mRNA levels were inhibited by TNF, with focus on both mRNA stability as well as changes in transcription. As part of these investigations, we also determined whether casein was the only milk protein altered by TNF, as changes in at least one other protein would provide further support for a physiological role of TNF in the mammary gland. Finally, having determined that TNF inhibited the expression of both ß-casein and WAP by altering transcription as well as mRNA stability, the final objective was to carry out preliminary studies to provide leads as to how TNF might exert these effects. These latter studies focused on matrix metalloproteinase-9 (MMP-9), which we have recently found to play an important role in TNF-induced branching morphogenesis of MEC (6).

	Materials and Methods

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Materials
[-³²P]Deoxy-CTP, [-³²P]deoxy-ATP, and [-³²P]UTP were purchased from NEN Life Science Products (Boston, MA). Insulin, progesterone, hydrocortisone, transferrin, ascorbic acid, fatty acid-free BSA, phenylmethylsulfonylfluoride, actinomycin D, and phenol red-free DMEM/Ham’s F-12 (1:1) tissue culture medium containing 15 mM HEPES were products of Sigma (St. Louis, MO). RPMI 1640, gentamicin, and TRIzol were purchased from Life Technologies, Inc. (Grand Island, NY). Collagenase class III was obtained from Worthington Biochemical Corp. (Freehold, NJ). Grade II dispase, leupeptin, and the RNA labeling kit (SP6) were obtained from Roche Molecular Biochemicals (Indianapolis, IN). FBS and normal calf serum (NCS) were purchased from HyClone Laboratories, Inc. (Logan, UT). The Multiprime DNA Labeling Kit, Hybond N nylon membrane, and the enhanced chemiluminescence Western blotting detection reagents were products of Amersham Pharmacia Biotech (Arlington Heights, IL). Mouse EGF and liquid dispase (50 caseinolytic units/ml) were products of Collaborative Research (Bedford, MA). Ovine PRL (NIDDK oPRL-19) was a gift from Dr. A. Parlow at the National Hormone and Pituitary Program, NIDDK. Donkey antirabbit peroxidase-conjugated IgG was a product of Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). The complementary DNA (cDNA) probe for recombinant human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The cDNA probes for rat WAP and rat ß-casein were gifts from Dr. J. Rosen (Baylor University, Houston, TX), the cDNA probe for rat 92-kDa gelatinase B (MMP-9) was a gift from Dr. L. Matrisian (Vanderbilt University, Nashville, TN), and the riboprobe for rat transferrin was a gift from Drs. M. Griswold and S. Sylvester (Washington State University, Pullman, WA). The 220-bp mouse ß-casein probe was generated by PCR using forward (positions 5921–5944) and reverse (6996–7017) primers from the mouse ß-casein gene. Recombinant human TNF (2.5 x 10⁶ U/mg), a gift from Asahi Chemical Industry Co. (Fuji, Shizuoka, Japan), was used in all studies involving primary MEC. Recombinant mouse TNF (1 x 10⁷ U/mg), purchased from Biosource International (Camarillo, CA), was used for experiments with the mouse mammary HC11 cell line. The nuclear factor-B (NFB) antibodies used in the supershift study, p65 (sc-109X), p50 (sc-114X), p52 (sc-298X), and c-Rel (sc-070X), were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Animals
Virgin, 50- to 55-day-old female Sprague Dawley CD rats (Crl:CD BR), purchased from Charles River Laboratories, Inc. (Wilmington, MA) were used as the source of mammary glands in the time- and dose-curve experiments, whereas Sprague Dawley [Tac:N(SD)fBR] rats from Taconic Farms, Inc. (Germantown, NY), were used for all other experiments reported herein. Female CD2F₁ mice purchased from NCI-Frederick Cancer Research Facility, Biological Testing Branch (Frederick, MD) were used to carry the Engelbreth-Holm-Swarm sarcoma. Animals were fed chow diets (Teklad, Madison, WI) ad libitum and had free access to water. All animal work was conducted using approved protocols from the institute animal care and use committee, and met the highest standards of humane animal care.

Preparation of reconstituted basement membrane
The reconstituted basement membrane (RBM) matrix was extracted from the Engelbreth-Holm-Swarm sarcoma as previously described (16). Final dialysis was carried out using a dialysis membrane with a 14-kDa cut-off.

Primary mammary epithelial organoid isolation, cell lines, and culture conditions
Procedures for isolation of primary mammary epithelial organoids have been described previously (16, 17, 18). In brief, excised mammary glands from 12–15 rats/experiment were minced finely, placed in digestion solution (10 ml/g wet wt) consisting of 0.2% (wt/vol) collagenase type III and 0.2% (wt/vol) dispase grade II in phenol red-free RPMI 1640 containing 5% (vol/vol) NCS and 50 µg/ml gentamicin, and incubated at 37 C for approximately 13 h. The digested tissue was then pelleted, washed twice with RPMI 1640, resuspended in RPMI 1640, and filtered initially through a 530-µm Nitex filter (Tetko, Depew, NY) and then through a 60-µm Nitex filter to trap the epithelial organoids (which were saved) but allow passage of small cell clusters and single cells (which were discarded). The organoids were washed off the Nitex filter with a 1:1 mixture of DMEM/Ham’s F-12 (phenol red-free), 5% NCS, and 50 µg/ml gentamicin; placed in a plastic tissue culture flask; and incubated for 4 h at 37 C to facilitate the attachment and subsequent removal of stromal contaminants. The cells within the nonadherent mammary organoids were enumerated by isolation and counting of nuclei after dilution into 0.1 M citric acid as described previously (16), pelleted by centrifugation at 500 x g for 10 min, and resuspended in ice-cold RBM matrix at a concentration of 1.5 x 10⁶ cells/ml matrix. For each experiment, 200 µl of this cell-RBM suspension were plated on top of 200 µl solidified cell-free RBM in 24-well tissue culture plates and incubated at 37 C for 3 h. After gelation of the cell-RBM suspension, 1 ml serum-free medium was added to each well. Alternatively, for the RNA studies, the mammary organoids were resuspended in ice-cold RBM matrix at a concentration of 4 x 10⁶ cells/ml, and 2.5 ml of this suspension were plated on top of 2.5 ml solidified RBM in petri dishes and incubated at 37 C for 4 h. After gelation, 12 ml serum-free medium were added to each dish.

The serum-free medium used in these studies consisted of phenol red-free DMEM/F-12 (1:1) containing 10 µg/ml insulin, 1 µg/ml progesterone, 1 µg/ml hydrocortisone, 10 ng/ml EGF, 1 µg/ml PRL, 5 µg/ml transferrin, 5 µM ascorbic acid, 1 mg/ml fatty acid-free BSA, and 50 µg/ml gentamicin. MEC were cultured for 7–10 days in complete serum-free medium or in medium lacking hydrocortisone, as noted. Vehicle (PBS) or TNF (0.4–40 ng/ml) was added either at time zero (continuously present) or as noted in the text. Cells were refed with fresh medium twice per week.

The mouse HC11 mammary epithelial cell line obtained from Dr. J. Rosen (Baylor University, Houston, TX) with the permission of Dr. B. Groner (Institute for Biomedical Research, Frankfurt/Main, Germany) was grown to confluence and maintained for 4 days in RPMI 1640 medium supplemented with 10% FBS containing 10 ng/ml EGF, 2 mM glutamine, 5 µg/ml insulin, and 50 µg/ml gentamicin (growth medium). The growth medium was then removed, and the cells were switched to lactogenic medium (RPMI 1640 supplemented with 10% FBS containing 5 µg/ml insulin, 5 µg/ml PRL, 1 µg/ml hydrocortisone, 2 mM glutamine, and 50 µg/ml gentamicin). For the nuclear run-on studies, TNF (40 ng/ml) or vehicle (PBS) was added on day 3 of lactogenic medium (48 h point) or day 5 (2 and 4 h points), and all cells were harvested on day 5 of culture in lactogenic medium.

Cell number
Viable cell number was determined for MEC using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay as previously described (5). For HC11 cells, viable cell number was quantitated by counting an aliquot of harvested cells using a hemocytometer and trypan blue.

RNA isolation and Northern blot analysis
For MEC in primary culture, medium was removed from the culture dishes, and 10 ml dispase (5 caseinolytic units/ml) in PBS were added. The RBM was dissociated with a cell scraper, followed by gentle up and down pipetting, and was digested by incubation at 37 C for 30 min with gentle stirring. The MEC were then collected by centrifugation for 10 min at 500 x g at 25 C. For studies using HC11 cells, cells were harvested from T175 flasks by trypsinization. After harvesting MEC and HC11 cells, 1 ml TRIzol reagent was added per 10⁷ cells, and total RNA was isolated according to the manufacturer’s protocol. Denatured RNA (20–30 µg) was separated by electrophoresis on a 1% (wt/vol) agarose gel containing 1 x 3-[N-morpholino]propanesulfonic acid (MOPS) and 2.2 M formaldehyde and transferred by capillary action overnight to Hybond N nylon membrane. The RNA was then cross-linked to the membranes by UV irradiation (UV Stratalinker 1800, Stratagene, La Jolla, CA). Membranes were prehybridized in a buffer containing 5 x SSC (standard saline citrate), 20 mM sodium phosphate (pH 6.5), 0.2% (wt/vol) SDS, 5 x Denhardt’s solution, 250 µg/ml salmon sperm DNA, and 50% formamide, using a modification of the standard method (19), for 4 h at 52 C. Hybridization with 2 x 10⁶ cpm/ml [-³²P]deoxy-CTP multiprimed cDNA probes or 2 x 10⁶ cpm/ml [-³²P]UTP-labeled RNA probes was performed in prehybridization buffer containing 10% dextran sulfate for 16 h at 52 C. The membranes were washed in 6 x SSC and 0.1% SDS, and autoradiography was performed at -80 C with Kodak X-Omat AR film using DuPont Cronex cassettes (Wilmington, DE) and intensifying screens. The bands were scanned using a Molecular Dynamics, Inc. (Sunnyvale, CA) laser densitometer, and quantitated using ImageQuant software.

Nuclear run-on assay
Nuclei were prepared by a slight modification of the method of Lamers et al. (20). Confluent HC11 cells were harvested by trypsinization after 5 days in lactogenic medium (in the presence or absence of 40 ng/ml TNF for the indicated times) and gently resuspended in lysis buffer (20 mM Tris HCl (pH 7.5), 2 mM MgCl₂, and 10 mM NaCl). All procedures were performed at 4 C unless otherwise noted. Nonidet P-40 (0.5% final concentration) was added to each sample, and the nuclei were gently pipetted up and down on ice for 5–15 min with periodic examination under the microscope to check on the progress of cellular lysis. When more than 60% of the cells were lysed, the samples were centrifuged at 500 x g; nuclei were washed twice and resuspended in a buffer containing 40% glycerol, 5 mM MgCl₂, and 50 mM Tris-HCl (pH 7.5) at 4 C and 0.1 mM EDTA; and aliquots (5 x 10⁷ nuclei) were frozen in liquid nitrogen and stored at -80 C until use.

The transcriptional activity of the nuclei was measured by determining the incorporation of 250 µCi [-³²P]UTP into RNA transcripts elongated in vitro. For hybridization, a slot blot was prepared that contained 2.5 µg linearized DNA (pGEM vector and transferrin), 50 ng purified cDNA insert (GAPDH), 100 ng purified cDNA insert (WAP) or 100 ng PCR-generated DNA (ß-casein). Immobilization of DNA probes on nylon membrane, hybridization, and washes were performed as previously described (21). Autoradiography was performed at -80 C with Kodak X-Omat AR film using DuPont Cronix cassettes and intensifying screens. The bands were scanned using a Molecular Dynamics, Inc. laser densitometer, and quantitated using ImageQuant software.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from control and TNF-treated HC11 cells were prepared according to the method of Olnes and Kurl (22). For EMSA, 20 µg nuclear protein and 1 µg poly(dI-dC) were incubated for 20 min at 25 C in EMSA buffer [10 mM Tris HCl (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol]. Seventy-five femtomoles of the NFB consensus oligonucleotide (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), labeled with [-³²P]ATP using T4 polynucleotide kinase (Promega Corp., Madison, WI), were added to the nuclear protein samples, and the incubation was continued for an additional 13 min before applying the samples to the gel. For the competition study, nuclear extracts were incubated with a 50-fold excess of unlabeled NFB consensus oligonucleotide for 20 min at room temperature before addition of the ³²P-labeled NFB oligonucleotide. To determine the protein composition of the NFB-DNA complexes, nuclear extracts were incubated with ³²P-labeled NFB oligonucleotide for 15 min at room temperature, then 4 µg antibody against p65, c-Rel, p52, or p50 were added. The samples were incubated overnight at 4 C before loading on the gel. The samples were run on 4% or 5% native polyacrylamide gels in 0.5 x TBE running buffer [50 mM Tris borate (pH 8.0) and 1 mM EDTA] for 75 min at 200 V. The gels were dried and exposed to x-ray film at -80 C. Specific bands were scanned using a Molecular Dynamics, Inc. laser densitometer and quantitated using ImageQuant software.

Zymography
Gelatinase activity in conditioned medium from MEC cultured for 7–10 days was analyzed by zymography on SDS-10% (wt/vol) polyacrylamide gels containing 1 mg/ml gelatin under nonreducing Laemmli buffer conditions. Samples were loaded on an equal cell number basis (conditioned medium from 20,000 cells for each sample). After electrophoresis, the gel was washed in 2% (vol/vol) Triton X-100 and incubated at 37 C overnight in substrate buffer [50 mM Tris-HCl, 5 mM CaCl₂, and 0.02% (wt/vol) sodium azide, pH 7.8, at 25 C]. After staining with Coomassie blue, the gelatin-degrading enzymes appeared as clear zones of lysis against a blue background.

Statistics
Data are presented as the mean ± SEM. When more than two groups were compared, statistical significance was evaluated using one-way ANOVA with the Tukey test for pairwise multiple comparisons. When two groups were compared, statistical significance was evaluated using Student’s t test. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TNF inhibits accumulation of WAP and ß-casein mRNAs in a time- and concentration-dependent manner, but does not alter transferrin mRNA levels
Our previous studies had demonstrated that TNF inhibits the accumulation of casein proteins (3, 4, 5) in isolated MEC; however, it was not known whether this effect was exerted at the mRNA level, or if other milk proteins were similarly inhibited. To assess this, MEC were cultured in optimal medium or, as a negative control, in medium lacking hydrocortisone for up to 10 days, and the effects of TNF on the expression of WAP and ß-casein mRNAs were examined. In the first experiment, 40 ng/ml TNF was added on day 7 of culture, and WAP mRNA levels were examined 1, 2, and 3 days thereafter; alternatively, TNF was present continuously from days 0–10. As shown in Fig. 1, WAP mRNA was decreased within 1 day of addition of TNF to the culture medium and remained below 20% of the control levels for up to 10 days of treatment. Additionally, as expected from its required role in WAP gene transcription (23), omission of hydrocortisone from the culture medium virtually suppressed WAP mRNA levels. Using a 10-day treatment period, we next examined the concentration dependence of this inhibitory effect of TNF. These experiments showed a 50% inhibition of WAP mRNA levels at 2 ng/ml TNF and close to maximal inhibition at 40 ng/ml (Fig. 2).

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of TNF on the time-dependent accumulation of WAP, ß-casein, and transferrin mRNAs. MEC in primary culture were grown in complete medium for 8–10 days and digested out of the RBM, and total RNA was isolated. TNF (40 ng/ml) or PBS vehicle was added to complete medium on day 0 (with harvest on day 10; 10 days of treatment) or on day 7 (with harvest on day 8, 9, or 10 of culture; 1, 2, or 3 days of treatment, respectively). Total RNA was isolated at each of these harvest times. A, Northern blots of WAP, ß-casein, transferrin, and their respective GAPDH mRNAs. B, Quantitation by scanning densitometry of autoradiograms from the experiment shown in A, normalized to GAPDH and calculated as a percentage of the appropriate vehicle control (i.e. controlled for time in culture). These experiments represent two separate experiments for each probe from MEC pooled from 12–15 rats in each experiment, each performed with a duplicate set of dishes and RNA isolation. An additional group of MEC was grown for 10 days in medium lacking hydrocortisone (HC) as a negative control. Quantitation by scanning densitometry of MEC grown in medium lacking HC revealed values of 3%, 11.7%, and 44.3% of the vehicle control values for WAP, ß-casein, and transferrin, respectively.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effects of various concentrations of TNF on the accumulation of WAP and ß-casein mRNAs. MEC in primary culture were grown in complete medium containing 0–40 ng/ml TNF for 10 days, digested out of the RBM, and total RNA was isolated. A, Northern blots of WAP, ß-casein, and their respective GAPDH mRNAs. RNA isolated from duplicate samples from the same experiment was loaded in adjacent wells. B, Quantitation by scanning densitometry of the duplicate lanes from the experiment shown in A, normalized to GAPDH and calculated as a percentage of the appropriate vehicle control. These experiments represent two separate experiments for each probe from MEC pooled from 12–15 rats in each experiment, each performed with a duplicate set of dishes and RNA isolation. An additional group of MEC was grown for 10 days in medium lacking hydrocortisone (HC) as a negative control. Quantitation by scanning densitometry of MEC grown in medium lacking HC revealed values of 1.8% and 13.9% of the vehicle control values for WAP and ß-casein, respectively.

Transferrin is a milk protein that is synthesized at high levels in mammary gland from pregnant and lactating mice, but is relatively insensitive in vitro to the lactogenic hormones that regulate expression of WAP and casein (24, 25). Given this difference in regulation, it was of interest to compare the effects of TNF on transferrin expression with its effects on the other two milk proteins. Figure 1 demonstrates that in contrast to the inhibitory effect of TNF on WAP and ß-casein, TNF at 40 ng/ml had no effect on transferrin mRNA levels and, if anything, slightly stimulated transferrin mRNA levels at 48 h; lower concentrations had no effect (not shown). This observation suggests that TNF may be acting selectively to modulate WAP and casein. As shown in this figure, transferrin levels were modestly reduced in MEC cultured in the absence of glucocorticoid.

TNF decreases WAP and ß-casein mRNA levels by decreasing the stability of the transcripts and by decreasing transcription
Actinomycin D studies. TNF could reduce the steady state levels of WAP and ß-casein mRNAs by decreasing the stability of the mRNAs, decreasing the transcription of the genes, and/or a combination of both mechanisms. This might occur, for example, if TNF induced a factor mediating either of these activities. Our first approach was to use the transcriptional inhibitor actinomycin D to determine whether it would interfere with the ability of TNF to inhibit the accumulation of WAP or ß-casein mRNAs. In preliminary studies we found that normal MEC were extremely sensitive to actinomycin D-induced toxicity, so to address this question, cells were treated with vehicle or TNF for 24, 48, or 72 h, with 1 µg/ml actinomycin D added only during the last 6 h of culture. This drug concentration did not inhibit cell growth within this time period, but inhibited [³H]uridine incorporation into RNA by about 70% (data not shown). With this protocol, actinomycin D did not alter steady state levels of WAP, ß-casein, or transferrin mRNAs in either the presence or absence of TNF (data not shown).

The lack of effect of actinomycin D in this initial study could suggest that the inhibitory effect of TNF is not mediated at the transcriptional level and/or that a TNF-induced decrease in transcript stability does not depend on transcription. However, it is also possible that the effect of TNF is initiated early, and that when actinomycin D is added only for the final 6 h of culture, it is too late for it to exert an effect, as the gene responsible for initiating the effect of TNF may already have been transcribed.

To address this question, a second set of experiments was performed in which actinomycin D was added simultaneously with TNF, and its effects on WAP, ß-casein, and transferrin mRNAs were determined after 4, 8, 14, and 24 h of culture. Longer time periods were not evaluated because cell growth is rapidly inhibited by actinomycin D (Fig. 3); for this reason also all quantitations of the milk protein mRNAs were normalized to cell number as well as to GAPDH to allow appropriate comparisons. Similar results were observed if the mRNAs were normalized to 18S or 28S RNA instead of to GAPDH (data not shown). Two significant observations can be made from this experiment. First, the inhibition of WAP mRNA accumulation by TNF was rapid and was seen as early as 14 h after the addition of TNF and to an even greater extent at 24 h (Fig. 4). Second, although actinomycin D alone decreased WAP mRNA levels, when added simultaneously with TNF, actinomycin D partially interfered with the inhibitory action of TNF. This can be seen at the 24 h point as well as by comparing the slower rate of decline of WAP mRNA levels between 4 and 24 h in the TNF plus actinomycin D group compared with that in the group given TNF alone. As discussed in more detail below, these data are consistent with the hypothesis that TNF induces transcription of a factor that reduces the stability of WAP mRNA.

View larger version (50K): [in this window] [in a new window]   Figure 3. Effects of TNF and actinomycin D on viable MEC number. MEC in primary culture were grown in optimal medium for 7 days. On day 7, the medium was changed to optimal medium containing TNF (0 or 40 ng/ml) and/or actinomycin D (0 or 1 µg/ml), and the cells were cultured for an additional 4, 8, 14, or 24 h. Viable cell number was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Each bar represents the mean ± SEM of triplicate wells. *, Statistically significant difference from the appropriate vehicle control; **, statistically significant difference from the TNF group.

View larger version (71K): [in this window] [in a new window]   Figure 4. Effect of concurrent exposure to the transcriptional inhibitor actinomycin D on TNF modulation of accumulation of WAP mRNA. MEC in primary culture were grown in optimal medium for 7 days. On day 7, the medium was changed to optimal medium containing TNF (0 or 40 ng/ml) and/or actinomycin D (0 or 1 µg/ml), and the cells were cultured for an additional 4, 8, 14, or 24 h. MEC were then digested out of the RBM, and total RNA was isolated. A, Northern blot of WAP and GAPDH mRNAs and ethidium bromide-stained 28S RNA (negative image). Three independent Northern blots were performed from triplicate cell cultures, and the blots were reprobed for each milk protein as well as for GAPDH. A representative blot and 28S band are shown. Although the absolute levels of GAPDH and WAP mRNAs appear to be equally decreased by actinomycin D at 24 h in this experiment, when replicate experiments were evaluated, actinomycin D decreased GAPDH to 48 ± 5% and TNF to 5 ± 1% of the 24 h control value, respectively (n = 3). B, Quantitation by scanning densitometry of the triplicate experiments, normalized to GAPDH, with the WAP/GAPDH ratio then normalized for cell number. Each bar represents the mean ± SEM of three experiments. *, Statistically significant difference from the appropriate vehicle control.

View larger version (61K): [in this window] [in a new window]   Figure 5. Effect of concurrent exposure to the transcriptional inhibitor actinomycin D on TNF modulation of accumulation of ß-casein mRNA. MEC in primary culture were grown in optimal medium for 7 days. On day 7, the medium was changed to optimal medium containing TNF (0 or 40 ng/ml) and/or actinomycin D (0 or 1 µg/ml), and the cells were cultured for an additional 4, 8, 14, or 24 h. MEC were then digested out of the RBM, and total RNA was isolated. A, Northern blot of ß-casein and GAPDH mRNAs. Three independent Northern blots were performed from triplicate cell cultures, and the blots were reprobed for each milk protein as well as for GAPDH. A representative blot is shown. Note that this is the same RNA sample for which the representative 28S band is shown in Fig. 4. B, Quantitation by scanning densitometry of the triplicate experiments, normalized to GAPDH, with the ß-casein/GAPDH ratio then normalized for cell number, and expressed as a percentage of the time zero vehicle control value. Each bar represents the mean ± SEM of three experiments.

View larger version (70K): [in this window] [in a new window]   Figure 6. Effect of concurrent exposure to the transcriptional inhibitor actinomycin D on TNF modulation of accumulation of transferrin mRNA. MEC in primary culture were grown in optimal medium for 7 days. On day 7, the medium was changed to optimal medium containing TNF (0 or 40 ng/ml) and/or actinomycin D (0 or 1 µg/ml), and the cells were cultured for an additional 4, 8, 14, or 24 h. MEC were then digested out of the RBM, and total RNA was isolated. A, Northern blot of transferrin and GAPDH mRNAs. Three independent Northern blots were performed from triplicate cell cultures, and the blots were reprobed for each milk protein as well as for GAPDH. A representative blot is shown. Note that this is the same RNA sample for which the representative 28S band is shown in Fig. 4. B, Quantitation by scanning densitometry of the triplicate experiments, normalized to GAPDH, with the transferrin/GAPDH ratio then normalized for cell number and expressed as a percentage of the time zero vehicle control value. Each bar represents the mean ± SEM of three experiments. *, Statistically significant difference from the appropriate vehicle control.

In preliminary studies we used Northern blot analysis to establish that 40 ng/ml TNF reduced ß-casein mRNA levels in HC11 cells to 44.3 ± 13.9% and 38.6 ± 8.5% (mean ± SEM) of the control value after 2 and 3 days of treatment, respectively, thus validating the use of this cell line for these studies. In follow-up nuclear run-on experiments, we found that TNF decreased transcription of both the ß-casein and WAP genes as early as 4 h after its addition to culture; as noted in the primary culture model, however, TNF did not affect transcription of transferrin, again demonstrating its selectivity (Fig. 7). The transcriptional effect was lost after 48 h of culture, suggesting that a decrease in mRNA stability may be a more important influence on transcript levels at this time.

View larger version (34K): [in this window] [in a new window]   Figure 7. Effect of TNF on transcription of WAP, ß-casein, and transferrin genes in HC11 mouse MEC (nuclear run-on assay). HC11 cells were cultured in lactogenic medium for 3 days, then fresh lactogenic medium containing 0 or 40 ng/ml TNF was added, and the cells were cultured for an additional 4 or 48 h. Nuclei were prepared, and in vitro labeled transcripts were hybridized to probes for WAP, ß-casein, transferrin, and GAPDH immobilized on nylon membrane. The figure shown is representative of four samples run in two independent experiments.

An induction of NFB and/or an induction in MMP-9 may contribute to the mechanism by which TNF inhibits ß-casein and WAP expression

View larger version (68K): [in this window] [in a new window]   Figure 8. Effect of TNF on binding of HC11 nuclear extracts to an NFB consensus sequence. A, HC11 cells were cultured in lactogenic medium for 4 days, then fresh lactogenic medium containing 40 ng/ml TNF was added, and the cells were cultured for an additional 12 h. Nuclear extracts were prepared, incubated with a 50-fold excess of unlabeled NFB probe (cold NFB) or with antibodies against p65 (Rel A), c-Rel, p52, or p50, as described in Materials and Methods, and then analyzed by EMSA. The arrows indicate the bands that are competed by the unlabeled NFB. From the supershift (arrowheads) and/or loss of intensity of these specific bands after antibody treatment, the upper and lower specific bands are identified as the p65/p50 heterodimer and the p50 homodimer of NFB, respectively. B, HC11 cells were cultured in lactogenic medium for 4 days, then fresh lactogenic medium containing 0 or 40 ng/ml TNF was added, and the cells were cultured for 2 or 4 h. Nuclear extracts were prepared and analyzed by EMSA. The positions of the two specific NFB DNA-binding complexes are indicated. This figure is representative of four independent samples. The intensities of the two specific bands varied with different batches of HC11 cells.

View larger version (53K): [in this window] [in a new window]   Figure 9. Effects of TNF and actinomycin D on MMP-9 mRNA levels. MEC in primary culture were grown in optimal medium for 7 days. On day 7, the medium was changed to optimal medium containing TNF (0 or 40 ng/ml) and/or actinomycin D (0 or 1 µg/ml), and the cells were cultured for an additional 4, 8, 14, or 24 h. MEC were then digested out of the RBM, and total RNA was isolated. A, Northern blot of MMP-9 mRNA. Three independent Northern blots were performed from triplicate cell cultures, and each blot was probed for MMP-9, the milk proteins ( Figs. 4–6), and GAPDH. A representative blot is shown. Note that this is the same RNA sample for which the representative 28S band is shown in Fig. 4. B, Quantitation by scanning densitometry of the triplicate experiments, normalized to GAPDH, with the MMP-9/GAPDH ratio then normalized for cell number. Each bar represents the mean ± SEM of three experiments. *, Statistically significant difference from the appropriate vehicle control.

View larger version (41K): [in this window] [in a new window]   Figure 10. Effects of TNF and actinomycin D on gelatinase activity of MMP-9 in conditioned medium from MEC treated with TNF and/or actinomycin D. MEC in primary culture were grown in optimal medium for 7 days. On day 7, the medium was changed to optimal medium containing TNF (0 or 40 ng/ml) and/or actinomycin D (0 or 1 µg/ml), and the cells were cultured for an additional 14 or 24 h. Conditioned media were collected and evaluated by zymography. A, Zymogram showing MMP-9 activity in conditioned medium from normal MEC in primary culture in response to treatment with vehicle, TNF, or actinomycin D, as indicated. Identity of the 97-kDa (nonactivated) and 95-kDa (activated) MMP-9 bands was confirmed by Western blot (6 ). The results shown are representative of eight separate samples for each group. B, Quantitation by scanning densitometry of the zymograms. Loading of sample was normalized for cell number; specifically, conditioned medium from 20,000 cells was loaded in each lane. The bar in the vehicle control group represents the mean of eight separate samples; the bars in the three treatment groups represent the mean ± SEM of eight separate samples for each group. *, Statistically significant difference from the actinomycin D alone group; **, statistically significant difference from the TNF alone group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The ability of actinomycin D to partially (WAP) or completely (ß-casein) block the inhibitory effect of TNF on the mRNA levels of these genes is consistent with the possibility that TNF inhibits their transcription. Indeed, a transcriptional mechanism was confirmed in the nuclear run-on studies as well as in other experiments in which TNF was found to inhibit ß-casein (30) (Zhang, H., and M. M. Ip, unpublished) and WAP (Zhang, H., and M. M. Ip, unpublished) promoter activity in transiently transfected HC11 cells. In addition, however, we cannot rule out an effect on mRNA stability, because the actinomycin D results are also consistent with the postulate that TNF induces transcription of factors that decrease the stability of WAP and/or ß-casein transcripts. Interestingly, the ability of actinomycin D alone to significantly decrease WAP mRNA levels suggests that transcription is normally required to stabilize WAP mRNA. TNF might then act to directly inhibit the transcription of the responsible gene(s) or to induce the expression of other genes that decrease the stability of the WAP transcript. Finally, the observation in the nuclear run-on experiments that TNF inhibited transcription of the WAP and ß-casein genes after 4 h of TNF treatment, but not after 48 h, suggests that both transcription and transcript stability are important components in the mechanism by which TNF exerts its effects.

Several possible mechanisms may explain the ability of TNF to inhibit the expression of the WAP and ß-casein genes. A likely candidate is the transcription factor NFB, which has been shown to be induced by TNF in many cell types (31), including the HC11 mammary epithelial cell line (herein and Ref. 30) and normal MEC in primary culture (39). It could be proposed that NFB acts to directly inhibit transcription by binding to one of the NFB-like sequences in either the WAP or ß-casein promoters or may indirectly modulate the activity of one of the known transcriptional regulators. For example, Geymayer and Doppler (30) demonstrated that the NFB p65/p50 heterodimer indirectly interferes with the ability of STAT5 (signal transducer and activator of transcription-5) to activate transcription of the ß-casein gene coincident with a reduced phosphorylation of STAT5. Negative cross-talk between STAT5 and NFB has also been reported in other models as well (35). This may suggest that TNF-induced NFB directly inhibits transcription of the ß-casein gene by interfering with STAT5 activity.

An important role for the extracellular matrix (ECM) in regulating transcription of the WAP and ß-casein genes was previously established. For example, the ECM-induced formation of alveolar MEC colonies appears to be required for the expression of WAP in vitro (36). Moreover, an ECM-responsive element has been identified in the ß-casein promoter (BCE1) (37). MMPs are a family of enzymes that play a critical role in remodeling of the ECM. Recently, we observed that MMP-9, an enzyme that is expressed in both rat and mouse mammary glands (Lee, P.-P. H., and M. M. Ip, unpublished), is secreted by the mammary epithelium in response to TNF (6). Moreover, MMP-9 activity is required (6) for the extensive TNF-induced three-dimensional branching morphogenesis that occurs when MEC in primary culture are cultured within a reconstituted basement membrane (3, 5, 6). The studies reported herein demonstrate that expression of MMP-9 mRNA as well as the activity of secreted MMP-9 protein are rapidly induced in MEC in response to TNF, and it is tempting to speculate that the subsequent disruption of the ECM may contribute to the inhibition of expression of both ß-casein and WAP. An alternative possibility is that MMP-9 stimulates the processing of cytokines/growth factors and/or their receptors or the release of matrix-bound growth factors, thus altering the hormonal milieu surrounding the MEC in such a way as to inhibit functional differentiation.

Of interest was the observation that TNF stimulation of MMP-9 mRNA was similar in the absence or presence of the transcriptional inhibitor actinomycin D; in contrast, gelatinase activity of secreted TNF-induced MMP-9 protein was completely blocked by actinomycin D. This demonstrates that MMP-9 can be regulated at multiple levels. First, the inability of actinomycin D to block TNF stimulation of MMP-9 mRNA suggests that in MEC, TNF does not regulate transcription of MMP-9, but, rather, increases the stability of the MMP-9 transcript. A similar observation was reported with transforming growth factor-ß, which was shown to exert its stimulatory effect on MMP-9 mRNA in human prostate cancer cell lines by increasing the stability of the message (38). This would suggest that TNF-induced NFB does not increase transcription of MMP-9 in MEC. Second, the fact that actinomycin D did block the gelatinase activity of MMP-9 in the conditioned medium suggests that transcriptional regulation is involved in the translation, processing, stability, and/or secretion of this enzyme. Taken together with the ß-casein and WAP data in Figs. 4 and 5, it is possible to make some tentative conclusions with respect to the potential role that MMP-9 may play in the regulation of expression of these two genes. Specifically, the ability of actinomycin D to block TNF-induced MMP-9 activity directly correlated with the activity of this transcriptional inhibitor to block the TNF-mediated decrease in WAP and ß-casein transcripts, thus raising the possibility that TNF could exert its effects on these milk protein genes in part by stimulation of MMP-9. Unfortunately, we did not have sufficient MMP-9-neutralizing antibody to address this question more directly. Finally, it should be noted that TNF also stimulates MMP-9 mRNA levels in HC11 cells (data not shown), and although these cells are grown on plastic, the cells are allowed to become superconfluent before the addition of lactogenic medium and, as a consequence, may make their own extracellular matrix. It is thus conceivable to invoke an MMP-9-mediated mechanism for the regulation of WAP and ß-casein transcription, although we believe that this is just one of the ways in which TNF regulates expression of these genes.

In summary, the work described herein demonstrates that TNF is a key regulator of functional differentiation in MEC and extends our previous studies, which demonstrated that TNF stimulates the proliferation and branching morphogenesis of the mammary epithelium (3, 4, 5, 6, 7). In this paper we report that TNF inhibits WAP and ß-casein expression by both transcriptional and posttranscriptional mechanisms. Current studies in the laboratory are focused on identifying the TNF-responsive regions in the promoters of both of these genes and determining whether NFB plays a functionally significant role in their regulation or whether other transcriptional regulators may mediate the effect of TNF. In any case, a further understanding of how TNF modulates the transcription of milk protein genes not only will have implications for mammary gland biology, but may also shed light on the mechanism of TNF action in other cell types as well.

	Acknowledgments

 
The authors are grateful to Mr. Larry Mead for preparation of the figures, to Laura Lee for the control NFB/EMSA studies, to Dr. Jeffrey Rosen for providing us with the rat ß-casein and WAP probes, to Dr. Lynn Matrisian for the rat MMP-9 probe, to Drs. M. Griswold and S. Sylvester for providing us with the rat transferrin probe, to Asahi Chemical Industry Co. for providing us with human recombinant TNF, and to Ms. Jane Ehrke and Drs. Haitao Zhang and Linda Varela for their critical review of this manuscript.

	Footnotes

 
¹ This work was supported by NIH Grant CA-77656 and the shared resources of NIH Core Grant CA-16056.

² Current address: Department of Obstetrics and Gynecology, University of South Florida, Tampa, Florida 33606.

Received October 30, 2000.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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