Nuclear Factor 1-C2 Contributes to the Tissue-specific Activation of a Milk Protein Gene in the Differentiating Mammary Gland
2002; Elsevier BV; Volume: 277; Issue: 20 Linguagem: Inglês
10.1074/jbc.m105979200
ISSN1083-351X
AutoresMarie Kannius‐Janson, Eva Johansson, Gunnar Bjursell, Jeanette Nilsson,
Tópico(s)RNA Interference and Gene Delivery
ResumoMembers of the nuclear factor 1 (NF1) transcription factor family have been postulated to be involved in the regulation of milk genes. In this work we have been able to identify the splice variant NF1-C2 as an important member of a tissue-specific activating complex that regulates the milk gene encoding carboxyl ester lipase (CEL). Mutation of the NF1-binding site in the CEL gene promoter results in a drastic reduction of the gene expression to about 15% in mammary epithelial cells. Furthermore, we demonstrate that the NF1-C2 protein interacts with a higher affinity to the NF1-binding site in the CEL gene promoter than other NF1 family members do and that NF1-C2 in the mouse mammary gland is a phosphorylated protein. During development of the mouse mammary gland, binding of NF1-C2 to the CEL gene promoter is induced at midpregnancy, in correlation with the induction of CEL gene expression. The fact that the NF1-C2 involving complex remains throughout the lactation period and decreases during the weaning period, when the CEL gene is down-regulated, supports its importance in the regulation of CEL gene expression. To our knowledge, this is the first report identifying a specific, endogenously expressed NF1 isoform to be involved in the tissue-specific activation of a gene. Members of the nuclear factor 1 (NF1) transcription factor family have been postulated to be involved in the regulation of milk genes. In this work we have been able to identify the splice variant NF1-C2 as an important member of a tissue-specific activating complex that regulates the milk gene encoding carboxyl ester lipase (CEL). Mutation of the NF1-binding site in the CEL gene promoter results in a drastic reduction of the gene expression to about 15% in mammary epithelial cells. Furthermore, we demonstrate that the NF1-C2 protein interacts with a higher affinity to the NF1-binding site in the CEL gene promoter than other NF1 family members do and that NF1-C2 in the mouse mammary gland is a phosphorylated protein. During development of the mouse mammary gland, binding of NF1-C2 to the CEL gene promoter is induced at midpregnancy, in correlation with the induction of CEL gene expression. The fact that the NF1-C2 involving complex remains throughout the lactation period and decreases during the weaning period, when the CEL gene is down-regulated, supports its importance in the regulation of CEL gene expression. To our knowledge, this is the first report identifying a specific, endogenously expressed NF1 isoform to be involved in the tissue-specific activation of a gene. Mammary epithelial cell differentiation is a complex process in which quiescent ductular cells proliferate and form alveolar structures that express their specialized products, the milk proteins. The differentiation process is driven by the cooperative action of multiple steroid and peptide hormones (1Topper Y.J. Freeman C.S. Physiol. Rev. 1980; 60: 1049-1056Crossref PubMed Scopus (719) Google Scholar). As milk protein genes are expressed only in differentiating epithelial cells, they act as markers for the differentiation of these cells. Studies of the regulation of milk protein genes are therefore of great interest for the understanding of mammary gland development and function. Extensive studies have defined multiple cis-acting elements and transcription factors involved in the regulation of milk protein production. These include binding sites for the glucocorticoid receptor (2Welte T. Philipp S. Cairns C. Gustafsson J.A. Doppler W. J. Steroid Biochem. Mol. Biol. 1993; 47: 75-81Crossref PubMed Scopus (48) Google Scholar), signal transducers and activators of transcription (3Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (714) Google Scholar, 4Li S. Rosen J.M. Mol. Cell. Biol. 1995; 15: 2063-2070Crossref PubMed Scopus (158) Google Scholar), CAAT/enhancer-binding protein (5Doppler W. Welte T. Philipp S. J. Biol. Chem. 1995; 270: 17962-17969Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and nuclear factor 1 (NF1) 1The abbreviations used are: NF1nuclear factor 1CELcarboxyl ester lipasePAPpotato acid phosphataseEMSAelectrophoretic mobility shift assayWAPwhey acidic proteinHAhemagglutininRTreverse transcriptaseGAPDHglyceraldehyde-3-phosphate dehydrogenaseUSFupstream stimulating factorBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol 1The abbreviations used are: NF1nuclear factor 1CELcarboxyl ester lipasePAPpotato acid phosphataseEMSAelectrophoretic mobility shift assayWAPwhey acidic proteinHAhemagglutininRTreverse transcriptaseGAPDHglyceraldehyde-3-phosphate dehydrogenaseUSFupstream stimulating factorBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (4Li S. Rosen J.M. Mol. Cell. Biol. 1995; 15: 2063-2070Crossref PubMed Scopus (158) Google Scholar). nuclear factor 1 carboxyl ester lipase potato acid phosphatase electrophoretic mobility shift assay whey acidic protein hemagglutinin reverse transcriptase glyceraldehyde-3-phosphate dehydrogenase upstream stimulating factor 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol nuclear factor 1 carboxyl ester lipase potato acid phosphatase electrophoretic mobility shift assay whey acidic protein hemagglutinin reverse transcriptase glyceraldehyde-3-phosphate dehydrogenase upstream stimulating factor 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol In a previous paper (6Kannius-Janson M. Lidberg U. Hulten K. Gritli-Linde A. Bjursell G. Nilsson J. Biochem. J. 1998; 336: 577-585Crossref PubMed Scopus (23) Google Scholar), we demonstrated that a member(s) of the NF1 family plays an important role in the expression of the milk protein gene carboxyl ester lipase (CEL). The CEL gene is highly expressed in the mouse mammary gland during pregnancy and lactation and in the mouse exocrine pancreas (7Kannius-Janson M. Lidberg U. Bjursell G. Nilsson J. Biochem. J. 2000; 351: 367-376Crossref PubMed Scopus (16) Google Scholar). However, we showed that the involvement of NF1 was mammary gland-specific. Initially, NF1 was identified as a factor required for the replication of adenovirus DNA (reviewed in Ref. 8de Jong R.N. van der Vliet P.C. Gene (Amst.). 1999; 236: 1-12Crossref PubMed Scopus (66) Google Scholar) but has since been recognized as a potent transcriptional regulator of many viral and cellular genes (9Miksicek R. Borgmeyer U. Nowock J. EMBO J. 1987; 6: 1355-1360Crossref PubMed Scopus (99) Google Scholar,10Cato A.C. Skroch P. Weinmann J. Butkeraitis P. Ponta H. EMBO J. 1988; 7: 1403-1410Crossref PubMed Scopus (122) Google Scholar). The NF1 family in vertebrates is composed of four members, NF1-A, NF1-B, NF1-C, and NF1-X, that are all differentially spliced and expressed in unique but overlapping patterns (11Apt D. Liu Y. Bernard H.U. Nucleic Acids Res. 1994; 22: 3825-3833Crossref PubMed Scopus (76) Google Scholar, 12Santoro C. Mermod N. Andrews P.C. Tjian R. Nature. 1988; 334: 218-224Crossref PubMed Scopus (492) Google Scholar, 13Inoue T. Tamura T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1990; 265: 19065-19070Abstract Full Text PDF PubMed Google Scholar). NF1 proteins bind to DNA as homo- or heterodimers to the consensus binding site, TTGG(C/A)(N5)(G/T)CCAA. Functional NF1-binding sites have been characterized in genes expressed in almost every tissue. They have been shown to regulate both ubiquitous and tissue-specific genes (reviewed in Ref. 14Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (429) Google Scholar). With such a diverse set of tissue-specific and developmentally regulated genes under the control of NF1 proteins, it appears likely that NF1 proteins play a major role in development. Streuli et al. (15Streuli C.H. Edwards G.M. Delcommenne M. Whitelaw C.B. Burdon T.G. Schindler C. Watson C.J. J. Biol. Chem. 1995; 270: 21639-21644Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) have shown that there is a connection between NF1 binding and the differentiated stage of the mammary gland epithelium. This is further supported by Furlong et al. (16Furlong E.E. Keon N.K. Thornton F.D. Rein T. Martin F. J. Biol. Chem. 1996; 271: 29688-29697Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) who described a switch in expression and binding of different NF1 proteins as mammary epithelial cells move from the fully differentiated stage to the involution stage. These findings suggest that different NF1 family members might be important for both the development and the regression of the mammary gland epithelial cells. However, the particular forms of NF1 that are active in these processes have not been characterized. Here we report that the particular isoform NF1-C2 binds to the NF1-binding site in the mouse CEL gene promoter. The DNA binding activity of NF1-C2, an ∼50-kDa phosphoprotein, is increased at day 13 of pregnancy and decreased at involution in mice, which is in concordance with the expression of the CEL gene. We also show that binding of NF1-C2 increases the expression of the CEL gene in the mouse mammary epithelial cell line HC11 and that this activation is mammary gland-specific. Mutation of the NF1-binding site in the CEL gene promoter reduced the CEL gene expression to about 15%. Furthermore, by showing that NF1-C2 binds to the NF1-binding site with higher affinity than NF1-A1, we provide evidence that different NF1 family members can bind with different affinity to the same site. The inguinal mammary glands from different stages of development were dissected from F1:C57Bl6 × CBA mice. Preparations of nuclear extracts for EMSA and Western experiments were carried out as described previously (17Ausubel, F., Brent, R., Moore, D., Smith, J., Seidman, J., and Struhl, K. (eds) (1987) Current Protocols in Molecular Biology, pp. 12.1.1–12.1.4, Wiley Interscience, New YorkGoogle Scholar). Protein concentrations of the extracts were determined by the method of Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215575) Google Scholar), and the extracts were stored in aliquots at −70 °C before use. The mouse mammary epithelial cell line HC11, kindly provided by Dr. R. Ball, Friedrich Miescher-Institute, Basel, Switzerland, was grown at 37 °C in a 5% CO2, 95% air atmosphere in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 5 μg/ml insulin, and 10 ng/ml epidermal growth factor. The rat pancreatoma cell line AR4–2J (ATCC) was cultured at 37 °C in a 5% CO2, 95% air atmosphere in Dulbecco's modified Eagle's medium containing 2 mm glutamine and 4.5 g/liter glucose and supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. HC11 cells were transiently transfected using Lipofectin in Opti-MEM (Invitrogen) with 5 μg of either of the pCHNF1A1.1, pCHNF1B2, pCHNF1C2, or pCHNF1X2 expression plasmids (expressing HA-tagged mouse NF1-A1, NF1-B2, NF1-C2, and NF1-X2) (kindly provided by Dr. R. M. Gronostajski, Lerner Institute (19Chaudhry A.Z. Lyons G.E. Gronostajski R.M. Dev. Dyn. 1997; 208: 313-325Crossref PubMed Scopus (177) Google Scholar)) per 6-cm culture dish. After 24 h the medium was switched to the RPMI 1640 medium supplemented as described above. After about 40 h the cells were harvested and nuclear proteins were prepared. The complementary oligonucleotides (5′-GTTCTGCTTGGCGTGTTATCAAG-3′ and 5′-AGCAACCTTGATAACACGCCAAG-3′) were annealed and radiolabeled by filling in with Klenow polymerase in the presence of [α-32P]dCTP, creating a probe referred as the NF1 oligonucleotide, representing −1792 to −1764 of the mouse CEL promoter (6Kannius-Janson M. Lidberg U. Hulten K. Gritli-Linde A. Bjursell G. Nilsson J. Biochem. J. 1998; 336: 577-585Crossref PubMed Scopus (23) Google Scholar). A GC box oligonucleotide was created by annealing and end labeling the complementary oligonucleotides 5′-CTGAGGGGGTAGAGGGGAGGGAGTGC-3′ and 5′-TCAGGCACTCCCTCCCCTCTACCCCC-3′ and a USF oligonucleotide by annealing and end labeling the complementary oligonucleotides 5′-TCTGTCCCAGAAGTCACGTG-3′ and 5′-CCGAGCACGTGACTTCTGGGA-3′. The WAP oligonucleotide was the same as described previously (20Mukhopadhyay S.S. Wyszomierski S.L. Gronostajski R.M. Rosen J.M. Mol. Cell. Biol. 2001; 21: 6859-6869Crossref PubMed Scopus (56) Google Scholar). Nuclear extracts (4–8 μg) were incubated with 1.5 μg of poly(dI-dC) and 25,000 cpm of labeled probe in EMSA binding buffer (20 mm Hepes, pH 7.9, 50 mm KCl, 10% glycerol, 2 mm MgCl2, 0.5 mmEDTA, 0.1 mg/ml bovine serum albumin, 0.5 mmdithiothreitol) in a 20-μl reaction volume for 15 min at room temperature. For supershift experiments, 2 μl of anti-NF1 antibody (rabbit polyclonal antiserum, 8199, reacting with the C-terminal half of NF1-C, kindly provided by Dr. N. Tanese, New York University Medical Center, New York) or 2 μl of anti-Stat 5a antibody (Santa Cruz Biotechnology) were included in the binding reaction and preincubated for 15 min before addition of probe. DNA-protein complexes were resolved on a 6% polyacrylamide gel (Tris glycine, 5% glycerol). Nuclear extracts (20 μg) were electrophoresed through a 10% SDS-polyacrylamide gel followed by electroblotting onto Hybond-P filter (Amersham Biosciences). For the dephosphorylation experiments, nuclear extracts (20 μg) were treated with 1.5 units of potato acid phosphatase (PAP) (Sigma) in 0.1m BisTris buffer (pH 6.0) in a 60-μl reaction volume at 30 °C for 1 h. To detect endogenous NF1-C, filters were incubated with a 1/1000 dilution of anti-NF1 antibody (8199), and the primary antibody was detected with peroxidase-conjugated anti-rabbit IgG using the BM Chemiluminescence Blotting Substrate peroxidase (Roche Molecular Biochemicals) and ECL films (Amersham Biosciences). In the overexpression experiments the overexpressed NF1 proteins were detected by incubating the filters with anti-HA antibody (Roche Molecular Biochemicals) or anti-NF1-C antibody (8199). The primary antibodies were detected with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Roche Molecular Biochemicals), respectively. Nuclear extracts (20 μg) were incubated with 4.5 μg of poly(dI-dC) and 50,000 cpm of labeled probe in EMSA binding buffer in a 50-μl reaction volume for 15 min at room temperature. The reactions were UV cross-linked in a UV Stratalinker 2400 (Stratagene) at 254 nm for 15 min and separated on a 10% SDS-polyacrylamide gel. The gel was dried and exposed to an x-ray film at −70 °C. The inguinal mammary glands from different stages of development were dissected from F1:C57Bl6 × CBA mice. Total RNA was extracted from these glands, HC11 cells, and AR4–2J cells by Trizol (Invitrogen) according to the manufacturer's instructions. Poly(A)+ RNA was purified using Oligotex mRNA kit (Qiagen). RT-PCR experiments were carried out using the TitanTM One Tube RT-PCR System (Roche Molecular Biochemicals). The primers used for NF1-C amplification were “FwdC,” 5′-GCCGGCATGAGAAGGACTCTACCCA-3′ (bp 1164–1188, GenBankTM accession number Y07693), and “RevC,” 5′-AGGAGGGATGGGAAGGCAACCTCGG-3′ (bp 1736–1760, GenBankTMaccession number Y07693), yielding a 597-bp fragment of the mouse NF1-C2 and a 520-bp fragment of the mouse NF1-C5. For mouse GAPDH amplification, the primers used were 5′-CACCACCATGGAGAAGGCCGGGGCC-3′ and 5′-TTGAAGTCGCAGGAGACAACCTGGT-3′, yielding a 554-bp fragment. For each reaction, 10 ng of poly(A)+ RNA was used, and the reactions were incubated at 50 °C for 30 min, 97 °C for 2 min, followed by 10 cycles of 1 min at 97 °C, 1 min at 55 °C, and 4 min at 68 °C, and 30 cycles (or 20 cycles for GAPDH) of 30 s at 97 °C, 30 s at 55 °C, and 1 min at 68 °C. From cycle 11 the 68 °C step was extended by 5 s every cycle. Finally the reactions were incubated at 68 °C for 7 min. For Northern blotting, poly(A)+ RNA was separated on a 1% agarose/formaldehyde gel and transferred to a GeneScreen PlusTM (PerkinElmer Life Sciences) nylon filter. The filter was hybridized with probes detecting NF1-C (a 550 bp-fragment excised by NaeI/BglII digestion from pCHNF1-C2 kindly provided by Dr. R. M. Gronostajski, Lerner Institute (19Chaudhry A.Z. Lyons G.E. Gronostajski R.M. Dev. Dyn. 1997; 208: 313-325Crossref PubMed Scopus (177) Google Scholar)) or human β-actin (CLONTECH). HC11 cells, stably transfected with CEL promoter/luciferase constructs with an intact (mCEL-1831Luc) or mutated (mCEL-1831NF1:1mutLuc) NF1-binding site (previously described (6Kannius-Janson M. Lidberg U. Hulten K. Gritli-Linde A. Bjursell G. Nilsson J. Biochem. J. 1998; 336: 577-585Crossref PubMed Scopus (23) Google Scholar)) were grown to different degrees of confluence and then harvested as described previously (21Lidberg U. Kannius-Janson M. Nilsson J. Bjursell G. J. Biol. Chem. 1998; 273: 31417-31426Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Luciferase assays were performed using the Promega kit with 50 μl of cell lysate and assayed in a luminometer (Berthold). The luciferase activity was normalized to the protein concentration of each extract, determined by the method of Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215575) Google Scholar). Our earlier promoter studies in mouse mammary gland-derived cells revealed that a major positive element in the CEL gene promoter interacts with a member(s) of the NF1 family (6Kannius-Janson M. Lidberg U. Hulten K. Gritli-Linde A. Bjursell G. Nilsson J. Biochem. J. 1998; 336: 577-585Crossref PubMed Scopus (23) Google Scholar). The CEL gene is activated between day 11 and 14 of pregnancy in mice, and we wanted to analyze if this activation correlates with binding of NF1 at this specific stage of differentiation. EMSA analysis with an oligonucleotide including the NF1-binding site and nuclear extract prepared from mouse mammary gland tissue at different stages of development revealed that there is an increased intensity of the NF1 complex at day 13 of pregnancy (P13) (Fig. 1A). The intensity is maintained at day 16 of pregnancy (P16) and at day 1 of lactation (L1) but is reduced 2 days after weaning (W2). An EMSA with the same extracts but an unrelated probe containing a GC box confirmed that the difference in NF1 binding to the NF1-binding site was not due to unequal quantification of the extracts (Fig. 1A). Epithelial cells have been shown previously (12Santoro C. Mermod N. Andrews P.C. Tjian R. Nature. 1988; 334: 218-224Crossref PubMed Scopus (492) Google Scholar) to express factors of the NF1-C family. We preincubated the EMSA binding reaction with an anti-NF1 antibody that specifically recognizes the C-terminal domain of NF1-C proteins (Fig. 1B). Because the NF1 complex was supershifted, we could conclude that it contains NF1-C. No supershifted band was observed with a control anti-Stat5a antibody. The specificity of the antibody is shown in Fig. 1C. Together these results indicate that NF1-C binding could be coupled to the developmental regulation of the CEL gene. Furthermore, by EMSA and supershift analysis we have demonstrated that NF1-C also interacts with the promoter of the rat whey acidic protein (WAP) gene (data not shown), another milk protein gene induced simultaneously with the CEL gene. This suggests that NF1-C is important for the expression of different milk genes induced at midpregnancy. Earlier reports (22Roy R.J. Guerin S.L. Eur. J. Biochem. 1994; 219: 799-806Crossref PubMed Scopus (30) Google Scholar, 23Reifel-Miller A.E. Calnek D.S. Grinnell B.W. J. Biol. Chem. 1994; 269: 23861-23864Abstract Full Text PDF PubMed Google Scholar) have shown that NF1 proteins can range in sizes from 30 to 100 kDa, which is due to the many isoforms and different kinds of post-translational modifications such as phosphorylation and glycosylation (14Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (429) Google Scholar). To examine the sizes of the NF1-C proteins in the extracts used, a Western blot was performed. This analysis revealed that the expression patterns of the NF1-C proteins vary during mammary gland development (Fig. 2). The NF1-C protein of ∼50 kDa increases and decreases with the degree of differentiation in a pattern similar to the binding pattern observed in the EMSA experiment. Hence, we conclude that this protein is most likely responsible for the interaction with the NF1-binding site in the CEL gene promoter. The ∼74-kDa NF1 protein appearing in W2 is presumably the same as that described earlier (16Furlong E.E. Keon N.K. Thornton F.D. Rein T. Martin F. J. Biol. Chem. 1996; 271: 29688-29697Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) as an NF1 protein triggered in early involution of the mouse mammary gland. Our data demonstrate that even this factor is an NF1-C protein since we used an NF1-C-specific antibody. This shows that different NF1-C proteins are present in the mammary gland during development and regression. One way to determine the size of DNA-binding proteins is by covalently cross-linking the proteins to its regulatory sequence using UV light. Because the efficiency of UV cross-linking is usually low, it is exceedingly rare for more than one cross-linking event to occur in a complex. Consequently, the observed molecular masses, when using the NF1-binding site, are likely to be those of monomers rather than dimers. Hence, this gives us the opportunity to investigate if the ∼50-kDa protein is responsible for the interaction with the NF1-binding site. As can be seen in Fig. 3, the indicated species with high intensity generated in the extract from L1 were reduced in the extract from W2. The band corresponding to free, unbound probe was estimated to ∼30 kDa, which was subtracted from the size of the bound species. The resulting size of about 50 kDa is in agreement with that of the NF1-C protein observed in the Western blot analysis. To analyze the activation potential of NF1-C we used the mammary gland epithelial cell line HC11. The cells were grown to different degrees of confluence in media containing epidermal growth factor and insulin, because increased confluence has been shown previously to affect the differentiation ability. EMSA analysis of nuclear extracts from the different stages of confluence showed a change in NF1-C binding activity (Fig. 4A). Surprisingly, there was a higher intensity of the NF1-C complex, in the extract from cells grown to the lowest degree of confluence (stage 1), although in extract from cells grown to the highest degree of confluence (stage 3) there was no binding of this complex at all. We observed no difference in DNA binding activity for these extracts to the control GC box oligonucleotide (data not shown). Western blot analysis also confirmed that the ∼50-kDa proteins were most abundant at stage 1, and minimal levels were found in the extract from stage 3 (Fig. 4B). Hence, HC11 cells provided us with a suitable system to investigate the activation potential of the ∼50-kDa protein in a mammary epithelial context, because we now had a stage where the factor was present and one in which it was not. HC11 cells stably transfected with the mCEL-1831Luc and the mCEL-1831NF1:1mutLuc constructs (6Kannius-Janson M. Lidberg U. Hulten K. Gritli-Linde A. Bjursell G. Nilsson J. Biochem. J. 1998; 336: 577-585Crossref PubMed Scopus (23) Google Scholar) were grown to different degrees of confluence and then analyzed with a luciferase reporter gene assay and EMSA. The overall activity of the two constructs increased with increasing degrees of confluence (Fig. 4C). However, the proportional difference in luciferase activity between the two constructs decreased as the cells became more confluent (Fig. 4D). At stage 1 the activation potential was 7-fold higher for the wild type construct compared with that of the mutant construct. At the highest stage of confluence there was almost no difference at all. By comparing the results from the reporter gene analysis and the EMSA we suggest that the presence and binding of the ∼50-kDa NF1-C protein to the NF1-binding site in the CEL gene promoter activates the CEL gene. Mutation of the NF1-binding site precludes binding of NF1-C, and accordingly the expression was reduced to ∼15%. Because the ∼50-kDa protein is the dominant NF1-C protein in HC11 cells as well as in the mammary gland at P13 to L1, we wanted to investigate which NF1-C isoform it represents. Based on the sequence of the mouse NF1-C gene, we devised an RT-PCR strategy that allowed us to detect and distinguish the different NF1-C isoforms that are known to exist in mouse (Fig. 5A). Poly(A+) RNA from HC11 cells at stages 1 and 2 of confluence was isolated and subjected to RT-PCR (Fig. 5B). Two NF1-C products were detectable corresponding to the isoforms NF1-C2 (exon 9 spliced) and NF1-C5 (exons 9 and 10 spliced). However, the expression level of NF1-C5 was barely detectable which implies that the ∼50-kDa protein is NF1-C2. Subcloning and sequencing verified the identity of the NF1-C2 transcript. As shown in Fig. 1A and Fig. 2, the binding of NF1-C2 to the NF1-binding site in the CEL gene promoter increased and decreased congruently with the expression of the NF1-C2 protein. To investigate if the amount of NF1-C was regulated at the transcriptional level, a Northern blot was performed with mRNA prepared from different stages of the mammary gland (Fig. 6). Two major transcripts of ∼4 and 6.5 kb were detected throughout mammary gland development, from day P10 to L1, whereas the mRNA level was drastically reduced in the involution stage (W2). The relationship between these two transcripts is not clear, although it is believed that the larger transcript is a precursor of the smaller mRNA (24Nebl G. Mermod N. Cato A.C. J. Biol. Chem. 1994; 269: 7371-7378Abstract Full Text PDF PubMed Google Scholar). NF1-C transcripts were also detected in the virgin stage (V) at levels comparable with those at day P10 to L1 (data not shown). Our data are in contrast with Mukhopadhyay et al. (20Mukhopadhyay S.S. Wyszomierski S.L. Gronostajski R.M. Rosen J.M. Mol. Cell. Biol. 2001; 21: 6859-6869Crossref PubMed Scopus (56) Google Scholar) who could not detect any NF1-C transcripts in the lactating mouse mammary gland. The reason for this discrepancy is unclear, but the fact that we could detect not only NF1-C transcripts but also NF1-C proteins in the lactating mammary gland, as well as similar findings by Kane et al. (25Kane R. Finlay D. Lamb T. Martin F. Adv. Exp. Med. Biol. 2000; 480: 117-122Crossref PubMed Google Scholar), demonstrates that NF1-C proteins are indeed present. The presence of NF1-C transcripts in early stages of the mammary gland development (V and P10) indicates that the expression of NF1-C proteins at day P13 is not regulated at the transcriptional level. It is known that NF1 proteins can be phosphorylated in vivo (26Cooke D.W. Lane M.D. Biochem. Biophys. Res. Commun. 1999; 260: 600-604Crossref PubMed Scopus (33) Google Scholar, 27Yang B.S. Gilbert J.D. Freytag S.O. Mol. Cell. Biol. 1993; 13: 3093-3102Crossref PubMed Scopus (56) Google Scholar, 28Bisgrove D.A. Monckton E.A. Packer M. Godbout R. J. Biol. Chem. 2000; 275: 30668-30676Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We therefore wanted to investigate if the NF1-C2 protein in the mammary gland is a phosphorylated protein. Nuclear extracts from HC11 cells at stage 1 of confluence were treated with potato acid phosphatase (PAP) and analyzed by Western blot using the anti-NF1-C antibody (Fig. 7). This analysis revealed that PAP converted the ∼50-kDa NF1-C2 protein to a faster migrating species with the size of about ∼35 kDa. The data indicate that the NF1-C2 protein involved in binding to the NF1-binding site in the CEL gene promoter is a phosphoprotein. Analysis of the same PAP-treated extract by EMSA and supershift experiment revealed that PAP converted the NF1-C2 complex to the faster migrating complex that can be seen in Fig. 1A in the extracts P13-L1 (data not shown). The existence of these less phosphorylated NF1-C2 proteins in extracts not treated with PAP is probably an artifact that results from endogenous phosphatase activity in the nuclear extracts because the intensity of this complex is increased with longer incubation times at room temperature. Taken together, these data suggest that the NF1-C2 protein responsible for the interaction with the NF1-binding site in the CEL gene promoter is a phosphorylated protein. However, a phosphorylation/dephosphorylation event cannot be responsible for regulating the binding of this factor because NF1-C2 proteins of only one degree of phosphorylation, the ∼50-kDa proteins, is detected during mammary gland development. Northern blot analysis revealed that not only the NF1-C gene, but also the NF1-A, -B, and -X genes are expressed in the mammary gland during pregnancy and lactation as well as in HC11 cells (data not shown). However, the facts that the NF1 complex binding to the NF1-binding site in the CEL gene promoter can be supershifted with the NF1-C-specific antibody and that only a few strong bands appear in the UV cross-linking experiment suggest that this site might be specific to NF1-C2 or that NF1-C2 binds to this site with higher affinity than other NF1 family members. To investigate this we overexpressed the NF1-C2 or NF1-A1 proteins in HC11 cells, prepared nuclear extracts, and investigated DNA binding with EMSA (Fig. 8A). When overexpressing NF1-A1, a new, weak, and slower migrating band appeared, but the endogenous NF1-C2 complex remained at the same intensity. In contrast, when overexpressing NF1-C2 the new band that appeared had strong intensity, whereas the endogenous band was almost outcompeted. EMSA with an oligonucleotide containing a binding site for upstream stimulating factor (USF oligonucleotide) confirmed that the different extracts were equally quantified. A Western blot analysis
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