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B Accounts for the Repressor Effects of High Stromal Cell–Derived Factor-1
Levels on Tac1 Expression in Nontumorigenic Breast Cells1 Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey and 2 Department of Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey
Requests for reprints: Pranela Rameshwar, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, MSB, Room E-579, 185 South Orange Avenue, Newark, NJ 07103. Phone: 973-972-0625; Fax: 973-972-8854. E-mail: rameshwa{at}umdnj.edu
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Abstract |
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(SDF-1) is a CXC chemokine that interacts with CXCR4 receptor. Tac1 encodes peptides belonging to the tachykinins, including substance P. SDF-1 production is decreased in Tac1 knockdown breast cancer cells and is also reduced in these cancer cells following contact with bone marrow stroma when Tac1 expression is increased. Here, we report on the effects of relatively high and low SDF-1 levels on Tac1 expression in nontumorigenic breast cells MCF12A. Reporter gene assays, Northern analyses, and ELISA for substance P showed increased Tac1 expression at 20 and 50 ng/mL SDF-1 and reduced expression at 100 ng/mL. Omission of the untranslated region showed a dose-dependent effect of SDF-1 on reporter gene activity, suggesting that receptor desensitization cannot account for the suppressive effects at 100 ng/mL SDF-1. Tac1 expression at high SDF-1 involves an intracellular signaling pathway that incorporates the activation of phosphatidylinositol 3-kinase-phosphoinositide-dependent kinase-1-AKT-nuclear factor-
B (NF-B). The major repressive effect occurs via NF-B located within exon 1. In summary, NF-B is involved in the repression of Tac1 at higher levels of SDF-1 in MCF12A. These results are relevant to dysfunction of Tac1 in breast cancer cells and also provide insights on the behavior of breast cancer cells as they traverse across gradient changes of SDF-1. (Mol Cancer Res 2007;5(4):373–81) |
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and SDF-1ß (3). Both transcripts are identical, except for four additional amino acids in the COOH terminus of the ß variant (2). SDF-1 acts as a chemoattractant to different types of stem cells and immune cells (4). The movement of cells by SDF-1 is partly facilitated by the mobilization of intracellular calcium and activation of the focal adhesion complex (4). In the bone marrow, stromal cells constitutively produce SDF-1, thereby facilitating the sequestration of CXCR4-expressing hematopoietic stem cells for their retention within the bone marrow compartment (5, 6). We have recently reported that SDF-1 regulates the expression of the Tac1 gene in bone marrow stroma (7). This regulation depends on SDF-1 levels, with the lower concentrations mediating stimulation and inhibition at higher concentrations. Consequently, this regulation is important for normal hematopoietic functions. Tac1 (also referred to as preprotachykinin-A) is a single copy gene that is conserved by evolution. It is ubiquitously expressed in the nervous, hematopoietic, and immune systems (8). The Tac1 gene produces several peptides belonging to the tachykinin family, of which substance P and neurokinin-A are its major products (9, 10). In most peripheral cells, including breast epithelial cells, substance P and neurokinin-A interact with the seven-transmembrane, G protein–coupled receptors neurokinin-1 and neurokinin-2 (11, 12). In nontransformed cells, neurokinin-1 and neurokinin-2 exhibit intracellular cross-talk so that each regulates the biological effects of the other (13).
Several endocrine-related cancers, including breast cancers, show constitutive expression of the Tac1 gene (14). For breast cancer and neuroblastoma, a cause-effect relationship between Tac1 expression and their proliferation has been shown (14-17). Both in vitro and in vivo models have shown a requirement for the Tac1 gene to facilitate the entry of breast cancer cells into the bone marrow (14-16). Tac1 peptides also mediate functions amenable to breast cancer development, such as protection of cancer cells from radiation damage, apoptosis, proliferative properties, induction of cytokines with angiogenic functions, and sensitivity to hypoxia (18-21). Tac1 expression also induces the expression of a truncated form of neurokinin-1, which is linked to malignancy (22).
SDF-1 is involved in cancer metastasis (23). Its involvement partly occurs through its chemotactic properties for CXCR4-expressing cells, including breast cancer cells (23). The density of CXCR4 on breast cancer cells is proportional to the invasiveness of the cancer (24). CXCR4-SDF-1 system is currently being targeted for cancer treatment with the development of CXCR4 antagonists (25). We have recently reported a close relationship between SDF-1 and Tac1 expression in the integration of breast cancer cells within bone marrow stroma, located close to the endosteum (26). This reported model mimics an early event in breast cancer invasion into bone marrow. Because this early period could generate breast cancer resurgence from the bone marrow, a detailed understanding of how the cancer cells adapt a quiescence phenotype by SDF-1 and Tac1 peptides needs to be studied. We have reported reduced levels of SDF-1 in Tac1-silenced breast cancer cells (15). In addition, contact of breast cancer cells with bone marrow stromal cells formed gap junctions and concomitantly reduced SDF-1 production and increased Tac1 expression in the breast cancer cells (26). This observation, combined with recent studies showing a decrease in Tac1 expression at relatively high levels of SDF-1 in stromal cells (7), suggests that an understanding of how SDF-1 regulates Tac1 in the behavior of breast cancer needs in-depth studies. To avoid confounds by multiple mutations, we investigated the effects of SDF-1 on Tac1 expression in nontumorigenic breast cells.
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affects Tac1 gene regulation using the nontumorigenic cell line MCF12A. We therefore investigated the validity of the cell model by ensuring that MCF12A expresses SDF-1 receptor CXCR4. To address this question, we used membrane extracts from MCF12A and did immunoprecipitation/Western blots for CXCR4. Positive control used membrane extracts from the CXCR4-expressing MDA-MDB-231 breast cancer cells (27). Negative control used CXCR4 knockdown T47D breast cancer cells. Representative blots shown in Fig. 1
indicate a band at the predicted size for MCF12A (Fig. 1, left lane). This weak band for MCF12A is expected because these cells are nontumorigenic compared with the strong band for the highly metastatic MDA-MDB-231 (Fig. 1, middle lane). There was no detectable band for CXCR4 knockdown MDA-MDB-231 (Fig. 1, right lane). Thus, the studies have established MCF12A as an authentic cell model to address the central hypothesis of this report based on the expression of CXCR4.
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Induces the Production of Substance P in MCF12A have been shown to induce the expression of Tac1 in stroma cells (7), we next tested if MCF12A can exhibit similar function with regard to the response of the cell to SDF-1. To this end, MCF12A cells were stimulated with 50 ng/mL SDF-1 for 24 h in serum-free medium or unstimulated. Cell-free supernatants were analyzed for substance P production by ELISA. The results, shown in Table 1
, indicate significant (P < 0.01) increase in substance P levels following stimulation. To verify that the increase level of substance P was not due to an artifact, the experiment was repeated with another nontumorigenic cell line, MCF10, and the results also show significant (P < 0.05) increase in substance P production (Table 1). Because T47D breast cancer cells have been shown to produce high levels of substance P (8), they served as positive control.
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by repeating the stimulation in the presence or absence of 10 ng/mL CXCR4 antagonist AMD3100. The concentration of AMD3100 was based on dose-response studies ranging between 0.1 and 100 ng/mL. We observed significant (P < 0.01) reduction in substance P production in the presence of AMD3100, indicating specific effects through CXCR4 (Table 1).
Effects of SDF-1 on the Activity of the 5' Flanking Region of Tac1
As nontumorigenic cells responded to SDF-1 by producing substance P, and the latter is the major peptide of the Tac1 gene, we studied the regulation of Tac1 at the level of transcription using reporter gene activities. MCF12A cells were transfected with pGL3 containing two different fragments of the 5' flanking region of the Tac1 gene: PPT-I/1.2 and PPT-I/N0 (Fig. 2A
). Transfectants were stimulated for 16 h with SDF-1 at 20, 50, and 100 ng/mL. These concentrations were selected because the range spanned the opposing effects of this chemokine with regard to activation of Tac1 (7). At 20 ng/mL, SDF-1 is stimulated with Tac1 activity in bone marrow stroma following a gradual decrease up to 100 ng/mL (7). Cell extracts were quantitated for luciferase activities. Because SDF-1 could affect the promoter in pß-gal, luciferase activities were calculated per microgram of total protein. Baseline luciferase activities were determined in transfectants with pGL3 alone and were determined to be <5 relative luciferase units/µg of total protein.
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and significant (P < 0.05) reduction at 100 ng/mL SDF-1 (Fig. 2B). In the absence of exon 1/intron 1 (PPT-I/N0), luciferase activities were directly proportional to SDF-1 concentration (Fig. 2C). In summary, although SDF-1 exerts a biphasic response for PPT-I/1.2, its effect on PPT-I/N0 was dose dependent.
Role of Nuclear Factor-B on the Activity of pGL3-PPT-I/1.2
The difference between PPT-I/1.2 and PPT-I/N0 is the omission of exon 1 and intron 1 (Fig. 2A). We therefore propose that this omitted region might consist of the repressor activity observed at 100 ng/mL SDF-1 for PPT-I/1.2 (Fig. 2B). Because this region has a nuclear factor-B (NF-B)-binding site (28), we next determined whether NF-B could be involved in the repressive effects of the highest SDF-1 studied. MCF12A cells were cotransfected with pGL3-PPT-I/1.2 and/or the following: pCMV-IB (wild-type IB) and pCMV-IBM (mutant IB). The mutant IB is resistant to phosphorylation and cannot be ubiquitinated, thereby retaining NF-B in the cytosol. We have selected 50 and 100 ng/mL of SDF-1 for this experiment because they exert opposing functions on PPT-I/1.2 (Fig. 2B).
Transfectants were stimulated with SDF-1 at 50 and 100 ng/mL, and after 16 h, luciferase activities were determined. As expected (Fig. 2B), optimum luciferase activity was observed at 50 ng/mL (Fig. 3A, left group, left diagonal column
). However, similar studies in which wild-type IB vector (pCMV-IB) was cotransfected showed significant (P < 0.05) reduction of luciferase (Fig. 3A, middle group). In parallel studies, cotransfection with dominant-negative IB (pCMV-IBM) showed significant (P < 0.05) increase in luciferase activities at both concentrations of SDF-1 (Fig. 3A, right group). In summary, this section describes studies showing repressor activities by NF-B on the activation of pGL3-PPT-I/1.2 in cells stimulated with 50 and 100 ng/mL of SDF-1.
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on Tac1 mRNA (ß-PPT-A)B at the level of reporter gene constructs correlate with endogenous expression of Tac1. To address this question, Northern analyses were done for ß-PPT-A with total RNA obtained from MCF12A cells, unstimulated or stimulated with SDF-1 at 20, 50, or 100 ng/mL. Representative studies (Fig. 3B) indicate optimum mRNA bands at 50 ng/mL SDF-1 (Fig. 3B, top row), which is consistent with the reporter gene activities (Fig. 2B). Because the mutant IB reversed the inhibitory effects of SDF-1 on luciferase activities (Fig. 3A, right group), we asked whether this observation correlates with Tac1 mRNA (ß-PPT-A) levels. To this end, we transfected MCF12A with pCMV-IBM (mutant), and after 24 h, the transfectants were unstimulated or stimulated with SDF-1 at 20, 50, or 100 ng/mL in serum-free medium. After 6 h, total RNA was analyzed by Northern blot for ß-PPT-A. The results show dense bands for all concentrations of SDF-1 (Fig. 3B, third row). This indicates that blocking of NF-B activation by the mutant IB prevented the inhibitory effects (Fig. 3B, first row) of SDF-1 at 100 ng/mL.
Western Blot for NF-B p65 Subunit in SDF-1–Stimulated MCF12A
Dominant-negative and wild-type IB indicate roles for activation of NF-B in the repression of Tac1 expression (Fig. 3). This section verified these observations by examining the cells for changes in nuclear p65 subunit of NF-B after stimulation with SDF-1. MCF12A was unstimulated or stimulated with SDF-1 at 20, 50, or 100 ng/mL. After 30 min, nuclear and cytoplasmic extracts were analyzed by Western blots for p65. Representative studies, shown in Fig. 4A
, indicate strong bands in the nuclear extracts from stimulated cells (Fig. 4A, top row). This contrasted with unchanged bands in cytoplasmic extracts from unstimulated and stimulated MCF12A. The bands obtained with nuclear extracts were normalized with ß-actin (Fig. 4A, bottom row) and then presented as fold change over unstimulated cells (Fig. 4B). The change at 100 ng/mL SDF-1 was 4-fold over unstimulated cells. The results show increased levels of nuclear p65 in MCF12A stimulated with 100 ng/mL SDF-1.
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B in SDF-1–Stimulated MCF12AB is involved in the repression of Tac1 activity at 100 ng/mL SDF-1, we next sought to identify the upstream candidates. Because SDF-1 has been shown to activate NF-B through the phosphatidylinositol 3-kinase (PI3K) pathway (29), then inactivation of phosphorylated PI3K at 100 ng/mL SDF-1 should lead to an increase in Tac1 expression. To this end, we transfected MCF12A with pGL3-PPT-I/1.2 and then stimulated the transfectants with 100 ng/mL SDF-1 in the presence or absence of the PI3K inhibitor LY294002 at 5, 10, 30, and 50 µmol/L. After 16 h, cell extracts were quantitated for luciferase activities and the results (Fig. 5A
) show a direct proportion between luciferase activities and the concentrations of LY294002. These observations indicate that PI3K is involved in the activation of PPT-I/1.2.
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B. This was addressed by stimulating MCF12A with 100 ng/mL SDF-1 in the presence or absence of 50 µmol/L LY294002. After 30 min and 1 h, we isolated nuclear and cytoplasmic extracts and then analyzed the extracts for p65. Inhibition of PI3K with LY294002 led to undetectable nuclear p65 (Fig. 5B). We therefore deduce that SDF-1 at high concentrations (100 ng/mL) leads to the activation of PI3K and then to the activation of NF-B.
Figure 5 shows SDF-1 activating PI3K and downstream NF-B. We now sought to identify molecules between PI3K and NF-B. MCF12A cells were stimulated with different concentrations of SDF-1 (20, 50, or 100 ng/mL) in serum-free medium, and after 1 h, nuclear and cytoplasmic extracts were analyzed for the following: phosphorylated phosphoinositide-dependent kinase-1 (PDK-1), AKT, and phosphorylated AKT. The results show increases in the phosphorylated forms of PDK and AKT (Fig. 6A, rows 1 and 3
). Their phosphorylation states have been reported to correlate with the downstream activation of NF-B (29). The results support the mechanism that SDF-1 activates NF-B through the PI3K-PKD-AKT pathway (Fig. 6B). Thus, these observations explain the mediators between stimulation by 100 ng/mL SDF-1 to the repression of PPT-I/1.2 (Fig. 2B).
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Discussion |
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, at a high concentration, inhibits the induction of the Tac1 gene in nontumorigenic mammary epithelial cells. We have found that the concentrations of SDF-1 affect the activity of the 5' flanking region of Tac1 (Fig. 2B). Whereas relatively low concentrations (20 and 50 ng/mL) of SDF-1 activated the 5' flanking region of Tac1 and induces its expression, a higher concentration (100 ng/mL) was inhibitory (Fig. 2B). Our unpublished studies indicate SDF-1 at <50 ng/mL SDF-1 produced by high and low invasive breast cancer cells. Our studies also showed that SDF-1 is involved in autocrine regulation of Tac1 in breast cancer cells (T47D and MCF7) because its knockdown led to reduced substance P levels in both cell lines. Low production would be an advantage to the cancer cells because the CXCR4 receptor will need to be free of ligand as the cancer cells move to distant sites. In addition, low SDF-1 levels would maintain high Tac1 expression, observed in breast cancer cells (26). The mechanisms by which SDF-1 regulates Tac1 will be insightful to understand dysregulation in breast cancer cells.
Although receptor desensitization at high concentrations of SDF-1 for the G protein–coupled receptors, such as CXCR4 receptor, could be a possibility (30), our studies have ruled out this mechanism. We show a dose-dependent effect in reporter gene activities in the absence of sequences at the 3' end of the Tac1 fragment that encompasses exon 1 and intron 1 (Fig. 2A and C). This indicates that mechanisms other than receptor desensitization are operative.
Further studies indicate that the inhibitory effect could be explained by NF-B activation at 100 ng/mL SDF-1. Functional studies combined with Western blots show upstream pathway involving the activation of PI3K (Figs. 5 and 6). This is consistent with the ability of CXCR4 to activate PI3K through Gi (31). This activation seems to be followed by the phosphorylation of AKT and PDK-1 (Fig. 6A). As activation of NF-B is downstream of PI3K-AKT-PDK-1 (32), we concluded that this pathway is operative for relatively high levels of SDF-1 (Fig. 6). At low SDF-1 levels, NF-B also exhibits suppressive effects on the expression of the Tac1 gene. This was shown by a significant reduction in reporter gene activity in the presence of wild-type IB and increase in the presence of dominant-negative IB (Fig. 3A). Studies with nuclear extracts (Fig. 4A) show similar density of p65 bands at 20 and 50 ng/mL of SDF-1. Because the reporter gene assays that omitted the NF-B sites (Fig. 2C) were decreased, although eliminating the repressive effects at 100 ng/mL SDF-1 (Fig. 2B and C), we deduce that there are sequences within the omitted region that are responsible for enhanced activity. These are ongoing studies because the changes in activated NF-B with small differences in SDF-1 could be relevant to breast cancer as the cells migrate through gradients of SDF-1, such as the bone marrow cavity (33, 34). In addition, the findings would be relevant to cancer cells in which the genes for SDF-1 and Tac1 are coexpressed in breast and other types of malignancies (14, 15). Because cytokines can activate NF-B, the role of this transcription factor is relevant for sites of metastasis where cytokine production is high, such as the bone marrow (34). It is interesting to note that Tac1 expression is increased in regions where SDF-1 levels are relatively lower (15), further supporting the biphasic effects observed in this study for MCF12A. These similarities suggest that, at least for breast cancer cells in the bone marrow, the use of MCF12A might be representative of the mechanisms found in the bone marrow.
Similar to SDF-1, the experimental level of Tac1 expression is also changed in breast cancer cells as they traverse the bone marrow cavity toward the region of high SDF-1 levels (14, 34). At cell cycle quiescence when breast cancer cells form contact with bone marrow stroma, Tac1 expression is increased and SDF-1 levels are significantly reduced in the breast cancer cells (15, 35, 36). Because this report shows a repressor function at high levels of SDF-1, and an enhancing property at low SDF-1 levels, perhaps this might explain why SDF-1 levels are required to be down-regulated in regions close to the stromal compartment.
NF-B activation has been shown to up-regulate many prometastasis and proangiogenic genes (37, 38). Similar reports have shown that NF-B activation by SDF-1 leads to an up-regulation of CXCR4 and consequently an increase in cell motility (37). The present report, combined with others (37) on the importance of Tac1 in breast cancer entry into the bone marrow (14), has begun to compose a mechanistic understanding of how breast cancer cells could take advantage of the bone marrow microenvironment for their survival and perhaps their quiescence nature (33).
Based on the above discussion on the relevance of the genes for Tac1 and SDF-1 in breast cancer metastasis into the bone marrow (14, 23), we propose that understanding the relationship between these two genes in a nontumorigenic system could provide insights into mechanisms that facilitate early metastasis and integration of breast cancer and perhaps other cancers into the bone marrow. The clinical significance is that cancer cells are entering the bone marrow at a time long before the tumor is clinically evident. At this time, the cancer cells, by unidentified mechanisms, can evade treatment, causing cancer resurgence from the bone marrow long after remission. Based on the functions of the discussed cells, it seems that these cancer cell subsets are molecularly and behaviorally different from the more aggressive or highly proliferative breast cancer cells that are detectable. This induced dormancy of the breast cancer cells in the bone marrow microenvironment makes these cells a far greater threat to clinical outcome than the actively metastatic breast cancer cells, which are easier to treat with conventional therapies.
Based on our study of the SDF-1/Tac1 pathway in the nontumorigenic model, we can conclude that the quiescent cancer cells in the bone marrow are likely to follow the pathways shown in this study with nontumorigenic cells, where high levels of SDF-1 are capable of down-regulating Tac1 expression. Furthermore, the information described in this study can be used to compare different grades of breast cancer cells at the molecular level and determine if there are mutations in this pathway in late-stage tumorigenic cells that would lead to active bone metastasis rather than dormancy.
The major findings of this study are shown as a cartoon (Fig. 6B). Although the study began with varying concentrations of SDF-1 on Tac1 expression, we focused on the level that caused repression, which is depicted as relatively high level in the cartoon (Fig. 6B). The studies used molecular and biochemical analyses to show that SDF-1 activates PI3K to phosphorylate AKT followed by phosphorylation of IB. Consequently NF-B is translocated to the nucleus where it could interact with Tac1/exon 1 (7). Because Tac1 is expressed in breast cancer cells where NF-B is highly activated, these studies are relevant to future studies that examine the tumorigenic effects of Tac1 and also to understand how the PI3K-AKT pathway might explain the different behavior of breast cancer cells in bone marrow (26, 39).
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was purchased from R&D Systems (Minneapolis, MN). Rabbit anti-p65 and horseradish peroxidase (HRP)-conjugated goat anti-murine IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphorylated PDK-1, AKT, and phosphorylated AKT antibodies; HRP-conjugated goat anti-rabbit IgG; and LY294002 PI3K inhibitor were components of the Phospho-Akt Pathway Sampler kit from Cell Signaling Technology (Beverly, MA). Rabbit anti-human CXCR4 was purchased from Affinity BioReagents (Golden, CO). HRP-goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology.
Cell Lines
MCF12A and MCF10 nontumorigenic mammary epithelial cells and T47D breast cancer cell line were purchased from the American Type Culture Collection (Manassas, VA) and cultured according to the American Type Culture Collection instructions. MDA-MDB-231 was kindly provided by Dr. Ian Whitehead (University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ). MDA-MDB-231 was stably knocked down for CXCR4 using the small interfering RNA vector (kindly provided by Dr. Si-Yi Chen, Department of Molecular and Human Genetics, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; ref. 27).
Immunoprecipitation
Membrane proteins were prepared from MCF12A and MDA-MDB-231, unmanipulated and knocked down for CXCR4 as described (22). Membrane proteins (3 mg) were immunoprecipitated with anti-CXCR4 at a final dilution of 1:1,000 by overnight incubation at 4°C while rotating the samples. After this, protein G-Sepharose beads (Roche Diagnostics, Indianapolis, IN) were added to mobilize all Fc fragments. This mixture was rotated for 90 min at 4°C. The mixture was pelleted and boiled in sample buffer followed by electrophoresis on 12% reducing SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes (Perkin-Elmer Life Sciences, Boston, MA) and then incubated overnight with primary antibody (anti-CXCR4). After this, membranes were incubated with HRP-goat anti-rabbit IgG. The primary and secondary antibodies were used at final dilutions of 1:1,000 and 1:2,000, respectively. HRP was developed with chemiluminescence detection reagent (Perkin-Elmer Life Sciences).
Western Blot
MCF12A cells were unstimulated or stimulated with SDF-1 (20, 50, or 100 ng/mL). In parallel studies, MCF12A cells were stimulated with SDF-1 (100 ng/mL) and/or the PI3K inhibitor LY294002 (50 µmol/L). After 1 h, nuclear proteins were extracted using the N-Extract kit (Sigma) and the total protein concentrations were determined using the Bio-Rad detergent-compatible protein assay (Bio-Rad, Hercules, CA). Extracts (15 µg) were analyzed by Western blots using 12% SDS-PAGE, and the proteins were transferred onto polyvinylidene difluoride membranes. The membranes were incubated overnight with primary antibody. Except for anti-ß-actin, rabbit was the source of all primary antibodies. Primary antibodies were detected by 2-h incubation with HRP-conjugated IgG. All primary and secondary antibodies were used at final dilutions of 1:1,000 and 1:2,000, respectively. HRP was developed with chemiluminescence detection reagent.
Substance P ELISA
Competitive ELISA quantitated substance P as described (8). MCF12A and T47D were stimulated for 16 h with SDF-1 at 50 ng/mL or unstimulated. Streptavidin-coated plates (Sigma) were incubated with biotinylated substance P. Cell-free media were collected and then added to 96-well plates, precoated with streptavidin-biotinylated substance P (100 µL) of unknown samples, or standard substance P and rabbit anti-substance P (Biogenesis, Brentwood, NH) at 1:3,000 were added to triplicate wells. After this, wells were incubated with alkaline phosphatase–conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) and Sigma 104 phosphatase substrate. Substance P levels were calculated from a standard curve produced from 12 serial dilutions of known substance P concentrations, and the absorbance was read at 405 nm.
Reporter Gene Assay
pGL3-basic containing inserts of the 5' flanking regions of the Tac1 gene (PPT-I/1.2 and PPT-I/N0) was described previously (28). Briefly, the insert of PPT-I/1.2 is 1.2 kb and includes intron 1, exon 1, and upstream sequences. Exon 1 and intron 1 are omitted in PPT-I/N0. Mercury IB dominant-negative vector set was purchased from BD Bioscience Clontech (Palo Alto, CA). This study used both pCMV-IBM and pCMV-IB. pCMV-IB allows for activation of NF-B, whereas pCMV-IBM sequesters NF-B in the cytosol by preventing the phosphorylation of IB.
MCF12A cells were transfected with Effectene reagent (Qiagen, Valencia, CA), and the transfectants were stimulated with SDF-1 at 20, 50, and 100 ng/mL in serum-free medium. After 16 h, cells were scraped in 30 µL of lysis buffer (Promega, Madison, WI) and then lysed by repeated freeze-thaw cycles in a dry ice/ethanol bath. Cell-free lysates were obtained by centrifugation at 15,000 x g for 5 min at 4°C. Luciferase activities were quantitated with 10 µL of lysates using the Luciferase Assay System (Promega). Luciferase activity was presented per microgram of total protein in levels normalized with cells transfected with vector alone. Total protein was determined with a kit purchased from Bio-Rad.
Northern Analyses
Northern blots were done for Tac1 mRNA (ß-PPT-A) as described (40). Total RNA was extracted from MCF12A cells that were unstimulated or stimulated with SDF-1 (20, 50, or 100 ng/mL). In parallel studies, MCF12A cells were transfected with pCMV-IBM (mutant) and then stimulated with SDF-1 (20, 50, or 100 ng/mL). The total RNA (15 µg) was analyzed with specific cDNA probe described previously (15). The probe was randomly labeled with 3,000 Ci/mmol/L [-32P]dATP (DuPont/NEN, Boston, MA) as described (28). Membranes were stripped and reprobed with cDNA for 18S rRNA. Hybrids were detected by exposures in phosphoimager cassettes (Molecular Dynamics, Sunnyvale, CA). Cassettes were scanned on Typhoon 9410 Molecular Imager phosphoimager (Molecular Dynamics).
Data Analyses
Statistical evaluations of the data were done with ANOVA and Tukey-Kramer multiple comparisons test. A P value of <0.05 was considered significant.
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: In partial fulfillment for a Ph.D. thesis of K.E. Corcoran.
Received 11/30/06; revised 1/21/07; accepted 1/30/07.
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-chemokine, stromal cell-derived factor-1, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J Biol Chem 1998;273:23169–75. in the circulation. Blood 2005;105:4561–8. and stromal-derived factor-1ß explains functional diversity. Blood 2004;103:2452–9. stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C. Blood 2000;95:2505–13.: another mediator in neural-emerging immune system through Tac1 expression in bone marrow stromal cells. J Immunol 2007;178:2075–82. and activation of caspase-3 in hypoxia-reoxygenated bone marrow stroma is negatively regulated by the delayed production of substance P. J Immunol 2001;167:4600–8. regulation in breast cancer cells contacting bone marrow stroma is critical for normal hematopoiesis. Blood 2006;108:3245–52.B kinase, and nuclear factor-B and induces cell-cell adhesion and aortic smooth muscle cell proliferation. J Biol Chem 2004;279:3188–96.B promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J Biol Chem 2003;278:21631–8.B in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2002;4:301–10.This article has been cited by other articles:
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  K. E Corcoran, A. Malhotra, C. A Molina, and P. Rameshwar Stromal-derived factor-1{alpha} induces a non-canonical pathway to activate the endocrine-linked Tac1 gene in non-tumorigenic breast cells J. Mol. Endocrinol., March 1, 2008; 40(3): 113 - 123. [Abstract] [Full Text] [PDF]
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