Primer extension

An antisense primer, P1 was designed 5' of the ATG translation site of the Jab1 gene and end-labeled with T4 polynucleotide kinase and ³²P-γ-ATP, followed by purification using Nu-Clean D25 columns (Shelton Scientific-IBI, Peosta, IA, USA). Total RNA was isolated from MDA-MD-231 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 20 μg of RNA was hybridized with 1 × 10⁶ c.p.m. of ³²P-labeled P1 oligonucleotide. The protocol for the Primer Extension System-AMV Reverse Transcriptase (Promega, Madison, WI, USA) was followed. The size of the extended product was determined by electrophoresis on a 6% polyacrylamide gel containing 6 M urea. To determine the size of the primer extended fragment, the labeled Promega marker and a sequencing reaction with Jab1 cDNA as template and primer P1 using the SequiTherm Excel II DNA Sequencing Kit (Epicentre, Madison, WI, USA) were run simultaneously on the gel.

Computer analysis

Transcription factor binding sites were predicted using Genomatix software (Munich, Germany); the MatInspector program, which uses TRANSFAC matrices; and the WebGene HCtata program's Hamming clustering method for TATA signal prediction in eukaryotic genes.

Cloning and analysis of the human Jab1 promoter

To clone the 5'-flanking region of the human Jab1 gene, a bacterial artificial chromosome clone containing the region corresponding to Jab1 was used as a template for PCR. The amplified DNA fragments were subcloned into the luciferase reporter vector pGL3 (Promega, Madison, W, USA). Progressive deletion mutants of the pGL3-Jab1 promoter were created by PCR. The integrity of constructs was confirmed by DNA sequencing. The following primers were used: +83R, -2006F, -2958F, -946F, -658F, -472F, -344F, -127F, and -59F (Table ​(Table11).

PCR and RT-PCR

The PCR reaction contained 100 ng of DNA template, 1.5 mM MgCl₂, 0.2 mM dNTP (Roche Applied Science, Indianapolis, IN, USA), 1 uM of primers, and Taq High Fidelity DNA polymerase (Invitrogen, Carlsbad, CA, USA).

The reverse transcriptase (RT) assay was performed from 2 μg of total RNA using Superscript II RT (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's procedure. A reaction without RT was performed in parallel to ensure the absence of genomic DNA contamination. PCR amplification was carried out as described previously [22]. Conditions for the PCR reaction consisted of an initial denaturation step at 94°C for 5 minutes, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 68°C. After a final extension at 72°C for 10 minutes, PCR products were resolved on 1.2% agarose gels and visualized by ethidium bromide transillumination under UV light. Primers used were: Jab1 F296-314, Jab1 R1094-1076, Jab1 R883-864, GAPDH F, and GAPDH R. For these and all following primer sequences please refer to Table ​Table11.

Transient transfection with reporter constructs and luciferase assay

MCF7, MDA-MB-231, and MDA-MB-468 cells were plated into 24-well tissue culture dishes at 4 × 10⁴ cells/well 24 hours before transfection. Transfections were performed in triplicate according to the manufacturer's protocol using Lipofectamine PLUS reagent (Invitrogen, Carlsbad, CA, USA). Briefly, 0.4 μg reporter plasmid Jab1-Luc (Firefly luciferase) together with 10 ng of pRL (Renilla luciferase) were cotransfected. Luciferase assays were performed 36 hours after transfection using a Dual-Luciferase Reporter Assay System (Promega, Madison, W, USA). Firefly and Renilla luciferase activities were read on a Monolight 3010 luminometer (BD Bioscience, Rockville, MD, USA). Firefly luciferase activity was normalized to Renilla luciferase readings in each well. Each experiment was conducted at least twice in triplicate.

Mutagenesis of the Jab1 promoter

Site-directed mutagenesis of CEBP and GATA1 was performed according to the QuickChange II method (Stratagene, La Jolla, CA, USA). The following mutagenic primers were used: CEBP-Mut, GATA1-Mut, and GATA1-Del (see Table ​Table11 for sequences). All mutants were verified by sequencing.

Nuclear extract preparation and electrophoretic mobility shift assay

Nuclear extracts were prepared as previously described [1]. Briefly, MCF7 and MDA-MB-468 cells were lysed in 10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). After incubation for 10 minute on ice, nuclei were recovered by centrifugation at 3,000 × g at 4°C for one minute and resuspended in 20 mM HEPES-KOH (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and protease inhibitor cocktail. Protein concentrations were determined using the DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). The following double-stranded DNA oligonucleotides were used in the electrophoretic mobility shift assays (EMSAs): -462/-436 CEBP-WT, -462/-436 CEBP-M1, -435/-417 GATA1-WT, and -435/-417 GATA1-M1 (see Table ​Table11 for sequences).

Oligonucleotides were end-labeled with [γ-³²P] ATP (MP Biomedicals, Solon, OH, USA) by T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) and purified with Quick Spin G-50 Columns (Roche Applied Science, Indianapolis, IN, USA). Pre-binding of 5 μg of nuclear extract to 0.05 mg/mL poly(dI:dC) (Roche, Applied Science, Indianapolis, IN, USA) was performed in a buffer containing 20 mM HEPES (pH 7.4), 0.1 mM EDTA (pH 8.0), 75 mM KCl, 2.5 mM MgCl₂, 1 mM DTT, and 5% glycerol for 20 minutes at room temperature (22 to 24°C) before addition of 60,000 c.p.m. of labeled probe. For competition assays, 25- and 100-fold excess cold competitor oligonucleotide duplex was added to the reaction buffer 10 minutes before addition of the labeled probes. For supershift assays, antibodies were added for 20 minutes at 4°C prior to addition of the labeled probe. Reactions were resolved by electrophoresis on a 4.5% nondenaturing polyacrylamide gel run in 0.5 × TBE, vacuum-dried with heat, and exposed to film at -80°C.

Chromatin immunoprecipitation (ChIP) assay

The manufacturer's protocol for the chromatin immunoprecipitation (ChIP) Assay Kit (Upstate Biotechnology, Temecula, CA, USA) was followed. Briefly, MCF7 cells or MDA-MB-231 cells transfected with control pcDNA vector or C/EBP-β2 and were incubated with 1% formaldehyde for 20 minutes at 37°C. Cells were collected, lysed, sonicated, and incubated with 4 μg of antibodies to C/EBP-α, C/EBP-β, GATA-1, Stat3, or β-actin overnight. PCR was used to amplify DNA bound to the immunoprecipitated histones after reversing the histone-DNA cross-links. The following primers were used for PCR: -472F and -344R (see Table ​Table11 for sequences).

Identification of the transcription elements located in the Jab1 promoter

Based on the determined location of the transcription start site, we analyzed the Jab1 5' flanking region for a functional promoter. Using the MatInspector program, which uses TRANSFAC transcription factor binding site matrices, we identified a number of putative transcription factor binding sites upstream of the Jab1 transcription start site (Figure ​(Figure1b).1b). A typical TATA box [23] was found 40 bp upstream of the transcription start site, along with a CAAT box at -99 bp. A number of putative transcription factor-binding elements were present in the promoter sequence, including STAT, ELK, p53, E2F, C/EBP, GATA, c-Myb, and AP-1.

Identification of Cis-Acting elements within the Jab1 gene promoter

To analyze the mechanisms responsible for transcriptional regulation of Jab1, luciferase constructs were generated to evaluate Jab1 promoter activity. A fragment of the human Jab1 promoter region from -2958 to +68 was amplified, sequenced, and subcloned into the luciferase reporter vector pGL3. To identify regulatory sequences that are important for transcriptional control of the Jab1 gene, we created a series of 5'-deletion constructs of the full length Jab1 promoter and analyzed for their ability to drive the expression of luciferase in MCF7 breast cancer cells (Figure ​(Figure2a).2a). Serial 5'-deletion mutations of the full-length promoter revealed a pattern of functional activity in transfected cells. The results suggest that the core promoter elements that drive maximal promoter activity are within the sequence spanning -472 and -345 upstream of the transcription initiation site, as the -344 Jab1-Luc construct displayed a marked loss of promoter activity (Figure ​(Figure2a).2a). Similar patterns of activity were seen in other breast cancer cell lines including BT-549, MDA-MB-231, SKBR3, and BT-474 (data not shown). Additionally, we evaluated the promoter strength in mammary tumor cells versus non-tumor cells. Jab1 promoter activity was three times higher in MCF7 breast cancer cells when observing the -472 construct than in MCF-10A normal mammary epithelial cells (Figure ​(Figure2b).2b). Further analysis of a panel of breast cancer cell lines including BT-474, ZR-75-1, MCF7, BT-549, and MDA-MB-453 revealed higher Jab1 promoter activity compared to normal mammary epithelial cells MCF-10A, HMEC and 184A (Figure ​(Figure2c).2c). Jab1 protein levels were also found to be higher in the panel of breast cancer cells compared with the normal mammary epithelial cells HMEC and MCF-10A (Figure ​(Figure2d).2d). Therefore, the transcription factors responsible for promoting Jab1 transcription are either not present in normal cells or not as active as in tumor cells. Another possibility is that normal cells may express transcription suppressors acting on this region. In Figure ​Figure2b,2b, the activity of the deletion constructs demonstrated differing ratios of luciferase activity between MCF7 and MCF-10A suggesting that different dominant regulatory factors may exist in each of these cell lines. However, interestingly, the same differential in promoter activity was seen between the -472 and -344 constructs in both cell lines.

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Figure 2

5' deletion of the Jab1 promoter analysis reveals increased activity in breast cancer cells compared with normal cells. (a) Identification of the minimal promoter region of Jab1 using progressive deletion of the 5' region in luciferase reporter constructs. The plasmids were cotransfected with Renilla luciferase plasmid pRL-null (transfection control) into MCF7 cells, and luciferase activity was assayed. Values represent mean ± standard error of triplicate experiments. Luciferase assays shown in subsequent figures were performed similarly. (b) Jab1 promoter 5' deletion constructs were transfected into MCF7 and MCF-10A cells and subjected to luciferase reporter assays. Promoter activity was higher in MCF7 epithelial breast cancer cells than in non-tumorigenic epithelial MCF-10A cells. Deletion of the region -472 to -344 resulted in a loss of promoter activity in both cell lines. (c) A panel of breast cancer cells and normal mammary cells were transfected with the -472 Jab1-Luc construct. Significance is noted as * P < 0.05 and ** P < 0.01 and was determined by t-test. (d) Jab1 protein levels for the cell lines in (c) were detected by western blotting, β-actin is shown as a control. Jab1, c-Jun activation domain-binding protein-1.

Identification of C/EBP and GATA transcription elements located in the -472/-345 region of the Jab1 promoter

Our data suggest that the -472 to -345 (-472/-345) region is key to driving the transcription of Jab1. We therefore sought to identify the transcription factors that regulated Jab1 through this region. Using the TRANSFAC database, we identified a number of putative transcription factor binding sites in the -472/-345 region, including sites for C/EBP (-444/CAAC/-441) and GATA-1 (-435/TATCT/-431) (Figure ​(Figure1b).1b). Because these transcription factors are activated in some cases of tumorigenesis [24,25], we speculated that they contributed to the increased transcription of Jab1.

C/EBPs are a family of basic leucine-zipper transcription factors, including C/EBP-α, C/EBP-β, -∂, -ε, -γ, and -ζ [26]. Of these, C/EBP-α, -β, and -∂ are expressed in the mammary gland, with C/EBP-β proposed to play a role in breast cancer [27,28]. The GATA family of transcription factors is required for erythroid and megakaryocytic differentiation [29]. Unlike other GATA members, GATA-1 has not been associated with any solid tumors, but mutations in GATA-1 are associated with essentially all cases of acute megakaryoblastic leukemia in children with Down syndrome [30].

To pinpoint the functional significance of the C/EBP and GATA-1 binding sites detailed within the Jab1 promoter we performed promoter and EMSA analysis of these regions. Cloning of this region in front of the -105/+83 sequence containing the TATA and CAAT boxes was sufficient to drive Jab1 promoter activity (Figure ​(Figure3a).3a). To determine whether these putative elements are capable of binding transcription factors, we performed a series of EMSA experiments with nuclear extracts from MCF7 breast cancer cells. The oligonucleotides and mutants for the C/EBP and GATA-1 binding sites are shown in Figure ​Figure3b.3b. The -462/436 and -435/-417 probes showed transcription factor binding activity to the DNA containing the C/EBP and GATA-1 binding sites, respectively (Figure ​(Figure3c).3c). The cold specific oligonucleotides competed efficiently for binding, whereas a cold mutant competitor containing a mutation in the C/EBP or GATA-1 binding site did not. Further, a supershift was observed when the oligonucleotides were incubated with antibodies to C/EBP-β and GATA-1 (Figure ​(Figure3c3c).

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Figure 3

Functional analysis of the Jab1 promoter. (a) The -472/-312 region contains a regulatory sequence that is sufficient to activate transcription of the Jab1 promoter when cloned in front of the TATA and CAAT boxes. MCF7 cells were transfected with either the -105/+83-Luc or -472/-312, and -105/+83 Jab1-Luc constructs and subjected to luciferase reporter assays. Values were compared with the -105 Jab1-Luc reporter by Student's t test, * P < 0.01. (b) Sequence of the probes used in EMSA assays. The probes span the sequence from -440 to -414 or from -412 to -389 of the Jab1 promoter and encompass the C/EBP or GATA sites. (c) Binding of C/EBP and GATA-1 to the Jab1 promoter. EMSAs showing binding of nuclear proteins C/EBP and GATA-1 to the C/EBP and GATA-1 binding sites of the Jab1 promoter. End-labeled oligonucleotide probes corresponding to the -440/-414 (C/EBP) or -413/-389 (GATA-1) regions of the WT Jab1 promoter sequence were incubated with nuclear extract proteins from MCF7 cells and separated on a 4.5% polyacrylamide gel. Lane 1 shows binding between probe and nuclear extract. Specificity was determined by addition of 50 and 100 molar excess of unlabeled specific cold probe, and mutant probe as specific and nonspecific (NS) competitors as indicated. Lane 4 shows complexes observed in the presence of probes containing mutations in C/EBP or GATA-1. Supershift of the complex was observed following incubation with C/EBPβ or GATA-1 antibodies as shown in the right panels. (d) MCF7 cells were transfected with the -472 Jab1-Luc, -472 C/EBP mutant (Mut1 ^C/EBP), -472 GATA-1 mutant (Mut 2 ^GATA1), Mut1+2, or -344 Jab1-Luc plasmids. Mutation of the C/EBP(Mut1^C/EBP) and GATA-1 (Mut2^GATA1) binding sites reduced Jab1 promoter activity by about 60% and 20%, respectively, and by 75% when mutated together. The firefly luciferase activity of each sample was normalized to that of Renilla luciferase (pRL). Data present the mean ± standard deviation of three independent experiments. All values were compared with the -472 Jab1-Luc reporter by Student's t test. C/EBP, CCAAT/enhancing binding protein; EMSA, electrophoretic mobility shift assay; Jab1, c-Jun activation domain-binding protein-1; NS, nonspecific binding; SS, supershift.

To assess whether any of these sites is important for expression of Jab1, each was mutated individually in luciferase reporter plasmids. We introduced mutations in the -472/-345 region of the Jab1 promoter to disrupt C/EBP and GATA-1 binding and compared their activity with the -472 Jab1-Luc promoter construct in transient transfection assays. Mutation of either C/EBP or GATA-1 binding sequence reduced Jab1 promoter activity by approximately 40% and 20%, respectively, and mutation of both sites resulted in a reduction of approximately 75% (Figure ​(Figure3d).3d). Interestingly, C/EBP and GATA binding sites homology from the human and mice promoter regions were found very well conserved [see Figure S1 in Additional file 1].

C/EBP-α, C/EBP-β, and GATA-1 transactivate the Jab1 promoter

Next, we examined which of the C/EBP and GATA family members are important for Jab1 promoter activity. We cotransfected different members of the C/EBP and GATA family, C/EBP-α, C/EBP-β, or C/EBP-δ, or GATA1-6, into MCF7 cells, along with the -472 Jab1-Luc plasmid (Figure ​(Figure4a).4a). Compared with control cells transfected with vector alone, cells transfected with C/EBP-α, C/EBP-β, or GATA-1 showed the greatest increase in -472 Jab1-Luc reporter activity (2.2-, 3.0-, and 2.6-fold, respectively). GATA-2 and GATA-3 also showed a significant increase in activity but for the purpose of this study were not studied in detail.

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Figure 4

C/EBP-α, C/EBP-β and GATA-1 transactivate the Jab1 promoter. (a) -472/+83 Jab1-Luc reporter plasmid was cotransfected with expression plasmids for C/EBP-α, C/EBP-β, C/EBP-δ, and GATA-1-6. The results are presented as fold activation compared with cells transfected with pcDNA3.1 vector control. All values were compared with the vector control by Student's t test. (b) Left, LAP1 (C/EBP-β1), LAP2 (C/EBP-β2), or LIP (C/EBP-β3) plasmids, along with the -474 Jab1-Luc promoter construct, were cotransfected with the Renilla luciferase plasmid pRL-null (transfection control) into MCF7 cells, and luciferase activity was assayed. Right, western blot analyses to demonstrate the protein expression of LAP1, LAP2 and LIP. (c) Endogenous expression of the C/EBPβ isoforms (LAP1, LAP2, and LIP) and GATA-1 were detected at higher levels in breast cancer cell lines compared with normal mammary epithelial cells by western blot analysis. (d) Identification of proteins in complexes by ChIP assays. Antibodies to β-actin, C/EBP-α, C/EBP-β, or GATA-1 were used to immunoprecipitate protein/DNA complexes from the sonicated lysates of MCF7 cells. After reversing the cross-linking, DNA was precipitated and PCR was performed using primers to amplify the -472/-344 promoter DNA. As a negative control, primers for the upstream promoter region -642/-475 were used. Bottom panel represents quantification of the data with ImageJ software. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun activation domain-binding protein-1; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitory protein; PCR, polymerase chain reaction; pRL, Renilla luciferase reporter vector; RT-PCR, reverse transcription polymerase chain reaction.

Interestingly, the transcription factor C/EBP-β has been associated with breast cancer [27,31]. It is translated into three different isoforms: C/EBP-β1, a 55-kDa liver-enriched activating protein (LAP1); C/EBP-β2, a 42- to 46-kDa protein also called LAP2 and; C/EBP-β3 a 20-kDa liver-enriched inhibitory protein (LIP) [25,32]. Of the three C/EBP-β isoforms, LAP2 has been specifically observed in breast cancer, and overexpression of this isoform can induce epithelial-mesenchymal transition [28]. LIP, however, is unable to activate gene transcription, but is still able to bind to DNA and dimerize, and therefore acts as a dominant negative [27]. It has been suggested that the LAP/LIP ratio may be an important indicator of C/EBP-β transcription [25]. We found that LAP2 activates the Jab1 promoter (over two-fold), whereas LAP1 had little effect and LIP decreased activity by about 16% (Figure ​(Figure4b).4b). Further, analysis of the C/EBPβ isoforms in breast cancer cells compared with normal, revealed higher levels of the LAP-1 and -2 isoforms in MCF7, MDA-MB-468 and MDA-MB-231 breast cancer cells compared with normal mammary epithelial cells HMEC and MCF-10A (Figure ​(Figure4c).4c). Interestingly, the LIP expression was also expressed at higher levels in the breast cancer cells compared with normal (Figure ​(Figure4c).4c). This in line with the increased endogenous Jab1 expression detected in these breast cancer cells compared with the normal mammary epithelial cells as shown in Figure ​Figure2d.2d. We also detected higher GATA-1 expression in the breast cancer cells compared with normal mammary epithelial cells and taken together could be a driving force in leading Jab1 expression in breast cancer.

C/EBP-α, C/EBP-β, and GATA-1 bind to the Jab1 promoter

To further determine whether C/EBP isoforms and GATA-1 bind to the Jab1 promoter, we performed ChIP assays on chromatin obtained from MCF7 cells (Figure ​(Figure4d).4d). When chromatin was incubated with β-actin, no product was observed. When chromatin was incubated with antibodies to C/EBP-α, C/EBP-β, and GATA-1 and the -472/-344 region was amplified by PCR, a product was observed. These data suggest that C/EBP-α, C/EBP-β, and GATA-1 bind specifically to the -472/-344 region of the Jab1 promoter.

Stat3 enhances the activity of C/EBP-α and C/EBP-β on the Jab1 promoter

The C/EBP transcription factors form heterodimers with other C/EBP family members and other transcription factors. We observed that Jab1-driven luciferase reporter activity increased upon addition of conditioned medium to MCF7 cells (data not shown). One of the pathways activated by conditioned medium is the Stat3 pathway, which has also been linked with breast tumorigenesis [33]. Moreover, Stat3 has been shown to interact with the C/EBP transcription factors and increase their activity [24]. We therefore investigated whether Stat3 can transactivate the Jab1 promoter, alone or in combination with C/EBP-α or C/EBP-β. Stat3 alone increased Jab1 promoter activity, but Stat3 along with C/EBP-α and C/EBP-β synergistically increased Jab1 promoter activity (Figure ​(Figure5a),5a), suggesting that Stat3 interacts with C/EBP on the Jab1 promoter. To that end, we observed a STAT general consensus site (TTN5AA) [34] that overlaps the C/EBP site within the Jab1 promoter sequence (-472 to -345) and was not identified by transcription factor database searches (Figure ​(Figure5b).5b). Stat1, Stat3, Stat4, and Stat92E all bind to this generalized consensus site [34,35].

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Figure 5

Stat3 can synergize with C/EBP-α and C/EBP-β to transactivate the Jab1 promoter. (a) The -472/+83 Jab1-Luc reporter plasmid was cotransfected with expression plasmids for C/EBP-α, or C/EBP-β. Plasmid amounts were normalized by the addition of empty expression plasmid. Following transfection, MCF7 cells were incubated for another 36 hours. The results are presented as fold activation compared with cells transfected with pcDNA vector control. (b) A potential STAT-binding element (TTN5AA consensus sequence) overlaps the C/EBP binding site (CAAC). (c) Binding of Stat3 to the Jab1 promoter is enhanced by the addition of C/EBP. MDA-MB-231 cells were transfected with either control of C/EBP-β2 and collected for ChIP assays using an antibodies against Stat3 and primers for the -472/-344 region of the Jab1 promoter. Right panel represents quantification of the band intensities and normalized to the input with ImageJ software. (d) MDA-MB-468 cells were cotransfected with either pcDNA control, a Flag-tagged Stat3 (Flag-Stat3), or Flag-tagged dominant negative (DN)-Stat3^EEE/VV, along with the -472 Jab1-Luc construct, and subjected to luciferase reporter assays. (e) Overexpression of Flag-tagged Stat3 increased Jab1 protein levels while a reduction was seen with Flag-DN- Stat3^EEE/VV. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun activation domain-binding protein-1.

We next performed a ChIP assay to determine whether Stat3 could bind with C/EBP-β2 co-operatively to the Jab1 promoter using MDA-MB-231 cells, which have constitutively activated Stat3. Since C/EBP-β2 (or LAP2) seems to be a major activator of the Jab1 promoter (Figure ​(Figure4),4), we next focused only on C/EBP-β2. Binding of Stat3 to the Jab1 promoter was increased greater than seven-fold when C/EBP-β2 was transfected into the cells (Figure ​(Figure5c).5c). Taken together, these data suggest that C/EBP-β2 and Stat3 bind to the Jab1 promoter to increase Jab1 promoter activities.

We further investigated whether inhibition of Stat3 affects Jab1 promoter activity in MDA-MB-468 cells, which have constitutively activated Stat3. Expression of a dominant-negative mutant of Stat3 reduced over 80% Jab1 promoter activity (Figure ​(Figure5d).5d). The EEE/VV mutation renders the protein incapable of DNA binding [36]. Expression of exogenous wild type Stat3 increased Jab1 expression, whereas the dominant-negative EEE/VV mutation reduced Jab1 protein levels (Figure ​(Figure5e5e).

Inhibition of Stat3 and Src decrease Jab1 promoter activity and protein expression

To further demonstrate the regulation of Jab1 by Stat3, we examined the effect of inhibition of Stat3 and also its upstream activator Src using siRNA [37,38]. Inhibition of Stat3 and Src by siRNA resulted in a dramatic reduction of Jab1 promoter activity (Figure ​(Figure6a),6a), mRNA levels (Figures ​(Figures6b6b and ​and6c),6c), and Jab1 protein expression (Figure ​(Figure6d).6d). Similar results were obtained with MDA-MB-231 cells (data not shown). Collectively, these results demonstrate that the Src/Stat3 signaling pathway plays an important role in the regulation of Jab1 transcription.

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Figure 6

Inhibition of Stat3 and Src by siRNA reduces Jab1 promoter activity in breast carcinoma cells. (a) MDA-MB-468 cells were cotransfected with siRNA targeting Stat3 (siStat3) or Src (siSrc), along with either the -472 Jab1-Luc construct or -472-Mut1^C/EBP Jab1-Luc (as a control) and subjected to luciferase reporter assays, western blot analyses to demonstrate the efficient knockdown of Stat3 and Src protein levels are shown in the right panel. (b) MDA-MD-468 cells were transfected with increasing doses of siStat3 and siSrc and Jab1 was detected by RT-PCR, quantification of Jab1 RNA levels is shown in (c). (d) Western blot analysis demonstrates that Jab1 protein levels decreased following inhibition of Stat3 and Src by siRNA. Jab1, c-Jun activation domain-binding protein-1; Mut, mutation; siRNA, small interfering RNA; RT-PCR, reverse transcription polymerase chain reaction; STAT, signal transducer and activator of transcription.