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,
Daniele Guardavaccaro,1, Patricia G. Santamaria,1 Ryo Koyama-Nasu,1 Esther Latres,1,
Roderick Bronson,2 Lili Yamasaki,3 and Michele Pagano1*
Department of Pathology and NYU Cancer Institute, New York University School of Medicine,1 Biological Sciences, Columbia University, New York, New York,3 Tufts University School of Veterinary Medicine, North Grafton, Massachusetts2
Received 22 April 2004/ Returned for modification 27 May 2004/ Accepted 23 June 2004
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ABSTRACT |
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B family of transcription factors. Here we show that mammary glands of ßTrcp1–/– female mice display a hypoplastic phenotype, whereas no effects on cell proliferation are observed in other somatic cells. To investigate further the role of ßTrcp1 in mammary gland development, we generated transgenic mice expressing human ßTrcp1 targeted to epithelial cells under the control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. Compared to controls, MMTV ßTrcp1 mammary glands display an increase in lateral ductal branching and extensive arrays of alveolus-like protuberances. The mammary epithelia of MMTV ßTrcp1 mice proliferate more and show increased NF-B DNA binding activity and higher levels of nuclear NF-B p65/RelA. In addition, 38% of transgenic mice develop tumors, including mammary, ovarian, and uterine carcinomas. The targeting of ßTrcp1 to lymphoid organs produces no effects on these tissues. In summary, our results support the notion that ßTrcp1 positively controls the proliferation of breast epithelium and indicate that alteration of ßTrcp1 function and expression may contribute to malignant behavior of breast tumors, at least in part through NF-B transactivation. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, etc.) (reviewed in references 11, 29, and 31). Indeed, FBPs are subunits of ubiquitin ligases named SCFs because they comprise Skp1, Cul1, and one of many FBPs.
ßTrcp1 (ß-transducin repeat-containing protein), the mammalian ortholog of Xenopus ßTrCP (53), was identified by using either Skp1 or the pseudosubstrate Vpu as bait in two-hybrid screens (13, 41). Mammalian ßTrcp1 and the paralogous protein ßTrcp2 have been reported to be involved in the degradation of IB (inhibitor of NF-B [nuclear factor B]) family members in response to NF-B-activating stimuli (17, 21, 24, 25, 32, 45, 49, 52, 59-61). Several studies have reported that ßTrcp1 and ßTrcp2 also control ß-catenin stability in mammalian cultured cells (23, 24, 30, 37, 43, 59). Furthermore, additional ßTrcp substrates have been proposed: Atf4/Creb2 (35), Smad3 (18), Smad4 (57), the alpha interferon receptor (33), the prolactin receptor (38), and the disks large tumor suppressor (40). More recently, ßTrcp1 and ßTrcp2 have been implicated in cell cycle control. During the S and G2 phases, these two FBPs keep Cdk1 inactive by inducing the degradation of its activating phosphatase Cdc25a (9, 28). At the G2/M transition, ßTrcp1 and ßTrcp2 change their specificity by targeting the Cdk1-inactivating kinase Wee1 for degradation (58), resulting in Cdc25a accumulation and Cdk1 activation. Finally, in mitosis ßTrcp1 and ßTrcp2 promote the degradation of Emi1 (22, 42, 47), an inhibitor of the anaphase-promoting complex/cyclosome, thereby attenuating Cdk1 activity via the anaphase-promoting complex/cyclosome-mediated degradation of two activating cyclin subunits (cyclin A and cyclin B). Thus, ßTrcp1 and ßTrcp2 contribute to "turning off" Cdk1 in the S and G2 phases, turning it on at the G2/M transition, and turning it off again in late mitosis.
Previously, we have shown that ßTrcp1 loss of function in mice does not affect viability but induces an impairment of spermatogenesis and reduced male fertility (22). In the present study, we show that mammary glands of ßTrcp1–/– mice display a hypoplastic phenotype. To investigate further the role of ßTrcp1 in mammary gland development, we generated and characterized a transgenic mouse model in which we targeted ßTrcp1 expression to epithelial cells using the mouse mammary tumor virus (MMTV) promoter. In addition, the epithelial-tissue-specific phenotype observed in both the loss-of-function and gain-of-function mouse models was confirmed by generating a transgenic mouse mutant in which ßTrcp1 expression was targeted to the lymphoid organs using the CD4 promoter and in which no phenotype was observed. The results of these studies are herein presented.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA extraction and reverse transcription-PCR (RT-PCR) analysis. Total RNA was extracted using RNeasy (QIAGEN) according to the manufacturer's instructions. RNA was quantified, and its purity was determined by standard spectrophotometric methods. cDNA was synthesized from 1 µg of the total RNA by using the Omniscript RT kit (QIAGEN). The following primers specific to human ßTrcp1 were used: F, 5'-AATTCCTCAGAGAGAGAAGACT-3', and R, 5'-TCTGGCAAAACACTAAATATAT-3'. To amplify mouse GAPDH, the following primers were used: F, 5'-GGGTGGAGCCAAACGGGTC-3', and R, 5'-GGAGTTGCTGTTGAAGTCGCA-3'. cDNA was amplified using 1 U of Taq DNA polymerase (Fermentas Inc., Hanover, Md.), and amplification was performed in a PTC-100 thermal cycler (MJ Research Inc.) for 35 cycles (denaturation at 94°C for 1 min, annealing for 1 min, and extension at 72°C for 2 min). The annealing temperatures were as follows: for human ßTrcp1, 55°C; for mouse GAPDH, 58°C. The amplification reaction products were resolved on 2.0% agarose-Tris-acetate-EDTA gels, electrophoresed at 100 V, and visualized by ethidium bromide staining.
Histology and whole-mount preparation. The animals were monitored daily. Necropsies were performed on all animals that died spontaneously during the observation period. For histological analysis, mammary glands and other tissues were fixed overnight or longer in 10% phosphate-buffered formalin, embedded in paraffin, and sectioned. They were stained with hematoxylin and eosin for histological analysis. For whole-mount tissue preparations, mammary glands were processed as described previously (27).
Immunohistochemistry and bromodeoxyuridine (BrdU) incorporation. For immunohistochemical staining, deparaffinized sections were immersed in methanol containing 0.03% hydrogen peroxide for 30 min to block endogenous peroxidase activity. The slides were subjected to microwaving in 10 mM citrate buffer three times for 5 min and then incubated with Protein Block Serum-Free (Dako, Tucson, Ariz.) for 30 min to block the nonspecific antibody binding sites. The slides were treated with an anti-p65/RelA polyclonal antibody (1:100; Santa Cruz) at 4°C overnight and then with EnVision+ (Dako) for 30 min. Peroxidase activity was developed using 3,3'-diaminobenzidine tetrahydrochloride in 50 mM Tris-HCl (pH 7.5) containing 0.001% hydrogen peroxidase. The sections were slightly counterstained with Mayer's hematoxylin. For the negative control, the primary antibodies were replaced with normal mouse serum.
For BrdU incorporation, a sterile solution containing 10 mg of BrdU (Sigma)/ml and 0.6 mg of fluorodeoxyuridine (Sigma)/ml in phosphate-buffered saline was injected intraperitoneally (100 µl/10 g of body weight). After 2 h, the mice were sacrificed, mammary glands were collected and fixed, and 5-µm sections were prepared. BrdU incorporation was visualized by immunohistochemistry using a BrdU detection kit (Zymed Laboratories), and the nuclei were counterstained with hematoxylin. For quantification, 10 random fields per section at a magnification of 40x were documented by photomicroscopy, and the percentage of BrdU-positive epithelial-cell nuclei relative to the total number of epithelial-cell nuclei was calculated.
Electrophoretic mobility shift assay.
The electrophoretic mobility shift assay was performed as described previously (4, 46). Briefly, 3 µl (approximately 3 µg) of cell extract was incubated for 20 min at room temperature in 20 µl of buffer (20 mM Tris [ph 7.4], 5% glycerol, 0.1% Tween 20, 0.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 50 mM KCl) containing 2 µg of poly(dI-dC) and a B probe (100,000 cpm) labeled using Klenow fill-in. The probe was the palindromic B probe previously described (6). The mixture was then separated on a native polyacrylamide gel that was dried and exposed for autoradiography.
Immunoblotting and Northern blotting.
Immunoblotting and Northern blotting were performed as previously described (12, 22). Antibodies to cyclin A (3), Emi1, ß-catenin, and IB
(22) were previously described. Antibodies to IBß, NF-B1/p105, and keratin 18 were from Santa Cruz Biotechnology, antibody to c-Myc was from Sigma, and antibody to cyclin D1 was from Zymed.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thus, we conclude that ßTrcp1 is necessary for the proper development of the mouse mammary gland under conditions of low hormonal stimuli.
Targeted expression of ßTrcp1 induces hyperplasia and transformation in epithelial organs. To further analyze the role of ßTrcp1 in the development of the mammary gland epithelium, we generated ßTrcp1 transgenic mice by placing the human ßTrcp1 cDNA under the control of the MMTV long terminal repeat promoter (Fig. 2A to C). Using this construct, three independent ßTrcp1 transgenic lines of mice were obtained, but all the experiments described herein were performed with two MMTV ßTrcp1 transgenic lines (lines 2 and 19) that expressed similar ßTrcp1 levels in the breast epithelium (Fig. 2A).
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ßTrcp1 activates NF-B in breast epithelium.
ßTrcp1 mediates the ubiquitination of several cellular substrates including IB family members (i.e., IB, IBß, and IB
) resulting in their proteasome-dependent degradation and consequent activation of the NF-B transcription activity. To determine whether ßTrcp1 expression in mammary glands was associated with an increased activation of NF-B, we examined the DNA binding activity of NF-B in the mammary, lachrymal, and salivary glands, in the uterus, and in the ovary. Mammary gland, ovary, and uterus of 10-week-old virgin transgenic females showed higher NF-B activity in comparison with that of wild-type females (Fig. 5A and data not shown). This increase was also evident in mammary glands of older mice (6 and 22 months old) and in breast tumors in MMTV ßTrcp1 transgenic mice (Fig. 5A, panel b).
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B pathway (7), we examined by immunohistochemistry the nuclear localization of p65/RelA, which increases in response to NF-B activation. The nuclear localization of p65/RelA in mammary epithelial cells of 10-week-old MMTV ßTrcp1 transgenic mice was much higher than that in wild-type animals (Fig. 5B).
Lastly, we analyzed by immunoblotting the expression of cell cycle regulatory proteins and members of the NF-B and ß-catenin pathways in mammary glands from 10-week-old virgin wild-type and ßTrcp1 transgenic mice (Fig. 5C). In 75% of the cases (eight mice for each genotype), we observed a significant increase in the levels of cyclin D1 in mammary glands from ßTrcp1 transgenic mice compared with those in wild-type glands. In contrast, the levels of IB, IBß, NF-B1/p105, NF-B2/p100, ß-catenin, cyclin A, Emi1, Wee1, and c-Myc were identical in wild-type and ßTrcp1 transgenic mammary glands (Fig. 5C and data not shown).
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DISCUSSION |
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To confirm a role for ßTrcp1 in the development of the mammary gland, we generated an MMTV ßTrcp1 transgenic mouse mutant (Fig. 2A). We found that targeting expression of ßTrcp1 to the mammary gland enhances proliferation and induces hyperbranching and hyperplastic nodules (Fig. 3A), a phenotype exactly opposite to that observed in the loss-of-function model. The phenotype observed in MMTV ßTrcp1 mice is most evident in aged animals, in which mammary hyperplasia is observed in 80% of cases (Fig. 3B). Moreover, 38% of MMTV ßTrcp1 mice developed tumors (Fig. 4D) localized to the breast, ovary, uterus, and lung, all organs in which the MMTV promoter induces ßTrcp1 expression (Fig. 2B). In contrast, targeting ßTrcp1 expression to lymphoid organs using the T-cell-specific promoter CD4 did not produce any effect on either cell proliferation (data not shown) or tumor development (Fig. 4C).
NF-B is the generic term for a family of transcription factors that regulates key genes involved in cell proliferation, apoptosis, immune responses, and inflammation (19). NF-B is activated by a wide variety of different stimuli, including proinflammatory cytokines such as tumor necrosis factor alpha and interleukin-1, bacteria, and bacterial lipopolysaccharide, viruses, viral proteins, double-stranded RNA, and physical and chemical stresses. After stimulation, the IB kinase, IKK, mediates phosphorylation of IB on two critical serine residues, leading to the immediate recognition by ßTrcp and consequent polyubiquitinylation of IB via the SCFßTrcp complex (21, 24, 25, 32, 45, 49, 52, 59-61). The consequent rapid degradation of IB by the 26S proteasome exposes the nuclear localization sequence of NF-B family members (e.g., p50 and p65/RelA), resulting in their translocation to the nucleus, where they induce the transcription of target genes. NF-B1/p105 functions both as a precursor of NF-B/p50 and as a cytoplasmic inhibitor of NF-B, and its degradation is also mediated by ßTrcp (26, 34).
We found that the increase in cell proliferation observed in MMTV ßTrcp1 mice associates with an activation of the NF-B pathway determined by measuring both B DNA binding activity and the abundance of nuclear p65/RelA (Fig. 5A and B). The positive regulation of NF-B activity by ßTrcp1 is likely to represent one of the reasons for the changes in cell proliferation observed in the mammary glands of our mouse models. Consistent with this hypothesis, an increase or a decrease in BrdU incorporation was observed in breast epithelium lacking IB (7) or expressing an inactive IKK (10), respectively. These two former studies also agree with ours on another issue: in all three cases, no significant differences in apoptosis rates were observed in mammary glands upon NF-B activation, despite the well-established role of this transcription factor in inhibiting apoptosis. Since NF-B is particularly activated during pregnancy, peaking around days 15 to 16 post coitum and during involution, it is possible that a further increase in activity is not achievable because of a plateau. Finally, the work by Cao and colleagues shows that cyclin D1 is a critical effector of the network that controls mammary epithelial proliferation in response to NF-B activation (10). Similarly, we found elevated levels of cyclin D1 in mammary glands of ßTrcp1 transgenic mice (Fig. 5C), which display high NF-B activity.
NF-B activation not only plays an important role in the development of the mouse mammary gland (7, 8, 10), but it is also known as a key player in oncogenesis, promoting proliferation and inhibiting apoptosis (reviewed in reference 39). Elevated and/or constitutive NF-B activation is found in a variety of tumors (20), including breast cancers (14, 44, 50, 51). In addition, transgenic mice overexpressing the NF-B family member c-Rel in breast epithelium develop late-onset mammary carcinomas (48). Our data confirm and extend this notion by showing a role for ßTrcp1, an activator of NF-B, in controlling proliferation of breast epithelial cells and in contributing to their transformation. Given the well-established role of cyclin D1 in the growth control and transformation of breast epithelium, it is possible that the effect of ßTrcp1 on breast tumorigenesis is, at least in part, mediated by the increase in cyclin D1 levels (Fig. 5C). In agreement with our studies of breast tumors, ßTrcp1 has been found to be overexpressed in human colorectal carcinomas, and Wnt signaling induces the expression of ßTrcp1 in colon cancer cell lines with a consequent upregulation of NF-B transactivation (54). Furthermore, ßTrcp2 is upregulated in tumor cell lines (including breast cancer lines) (55) and in chemically induced mouse papillomas and squamous-cell carcinomas, and this overexpression is associated with constitutive activation of NF-B (5).
How does ßTrcp1 activate NFB in the MMTV ßTrcp1 mice? Are other substrates affected by the lack or overexpression of ßTrcp1 in breast and other epithelial cells? The SCFßTrcp complex targets several substrates for degradation, including IB family members, NF-B1/p105, ß-catenin, Emi1, Cdc25a, Wee1, Atf4/Creb2, Smad3/4, the alpha interferon receptor, the prolactin receptor, and the disks large tumor suppressor. In addition, ßTrcp1 activates NF-B by inducing the processing of NF-B2/p100 (1, 16). We could not observe in MMTV ßTrcp1 or ßTrcp1–/– mice any significant changes in the levels of ßTrcp1 substrates by either immunoblotting (for IB, IBß, NF-B1/p105, NF-B2/p100, ß-catenin, Emi1, and Wee1) or immunohistochemistry (ß-catenin) (Fig. 5C and data not shown). However, we could observe a clear effect on NF-B activation. A possible explanation for the lack of changes in ßTrcp1-deficient mice is the redundancy with ßTrcp2, which is well expressed in breast epithelial cells (Fig. 1h). In transgenic animals the explanation is more difficult; however, it needs to be reiterated that while on one hand ßTrcp induces IB degradation, on the other, by activating NF-B, it induces the synthesis of IB, obscuring IB proteolysis. In addition, it is possible that levels of endogenous ßTrcp1 are limiting and that the forced expression of ßTrcp1 induces small perturbations in the degradation of IB, IBß, IB, and NF-B1/p105 and in the processing of NF-B2/p100, the sum of which produces, as a final result, a prolonged and enhanced activation of NF-B.
So far, of more than 70 human FBPs, only 4 have been well characterized and matched to downstream substrates: ßTrcp1, ßTrcp2, Skp2, and Fbw7. Of these, Skp2 is the product of a protooncogene, while Fbw7 is a tumor suppressor protein. The results herein demonstrate a role for ßTrcp1 in the development of the mammary gland. Furthermore, our results indicate that ßTrcp1 might contribute to the malignant behavior of breast and other epithelial cells by providing a gain of function, at least in part via the overactivation of NF-B.
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ACKNOWLEDGMENTS |
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This work was supported by a fellowship from the Japanese Ministry of Culture (2001) and an IARC fellowship (2002) to Y.K., an American-Italian Cancer Foundation fellowship (1999 to 2000) and a Susan Komen Breast Cancer Foundation fellowship (2001 to 2004) to D.G., a Bernard B. Levine Foundation award to R.K.-N., an Irma T. Hirschl scholarship and grants from the NIH (R01-CA76584 and R01-GM57587) to M.P., and a Pew scholarship and an NIH grant (R01-CA79646) to L.Y.
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FOOTNOTES |
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Y.K. and D.G. contributed equally to this work.
Present address: Department of Oral Maxillofacial Pathobiology, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Present address: Regeneron Pharmaceuticals, Tarrytown, N.Y.
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