MUC1 in macrophage contributions to cigarette smoke-induced lung cancer

Reagents

Bisphenol A diglycidyl ether (BADGE) was obtained from Enzo Life Science. The inhibitors U0126 for ERK, SB203580 for p38, SC-514 for IKK, and Necrostatin-1 for RIP1 kinase were purchased from Calbiochem. GW9662 and U0124, the negative control for U0126, were obtained from Millipore. Small interfering RNA (siRNA; SiGenome SMARTpool) for MUC1, PPAR-γ, and TNFR1 and negative control siRNA were purchased from Dharmacon. The primary antibodies against Mucin 1 (GP1.4) and GAPDH were from Santa Cruz Biotechnology. MUC1Ab-5, a hamster monoclonal antibody that recognizes the MUC1 CT domain was purchased from Lab Vision (Fremont, CA). Antibodies against β-Tubulin and β-Actin were from Sigma-Aldrich. Antibodies against PPAR-γ and phospho-PPAR-γ (S112) were purchased from Abcam. Antibodies against ERK, and phospho-ERK (Y185/187) were from Invitrogen. The human TNF-α detection ELISA kit was purchased from eBioscience (San Diego, CA). Filters collected from the AMESA Type 1300 smoking machine, which generates mainstream CS, were used to prepare cigarette smoke extract (CSE) by sequential extraction of material from CS filter with DMSO (for dissolving water-insoluble components) and culture medium (for dissolving water-soluble components). Before use, the water-soluble and -insoluble fractions were proportionally mixed to make total CSE. Total particulate material (TPM) was determined by weighing the filter before and after extraction (22).

Cell Culture

THP-1 and U937 macrophages were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM of L-glutamine, 100 U/ml of penicillin, and 100μg/ml of streptomycin. These cell lines, obtained from ATCC (Manassas, VA), were used within 6 months of receipt. The immortalized human bronchial epithelial cells, HBEC-13, were generously provided by Drs. Shay and Minna, Southwestern Medical Center, Dallas, TX (23). HBEC cells were maintained in Keratinocyte serum free medium (KSFM) (Invitrogen), supplemented with 5 μg/L of human recombinant EGF and 50 mg/L of bovine pituitary extract in plates coated with fibronectin (Athena ES).

Transfections

THP-1 and U937 macrophages were cultured in the presence of 50 nM PMA for 24 h. The differentiated cells were transfected with MUC1 and control small interfering RNA (siRNA) with INTERFERin^™ siRNA Transfection Reagent (Polyplus-transfection) according to manufacturer’s instructions. MUC1 protein level was determined by Western blot at 48 h after transfection.

Western Blot

Cells were washed twice with cold PBS, collected, and lysed with M2 buffer (20 mM Tris-HCl, pH 7.6, 0.5 % Nonidet P-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM β-glycerophosphate, 1 mM sodium vanadate, and 1 μg/ml leupeptin). Concentration of protein was measured by Bradford assay (Bio-Rad). Equal amounts of proteins were separated by 12 % SDS-polyacrylamide gels and then transferred to PVDF membranes. The proteins were detected by enhanced chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate, Millipore). The intensity of the individual bands was quantified by densitometry (ImageJ) and normalized to the corresponding input control (β-Actin) bands. Fold changes were calculated with the control taken as 1.

Detecting TNF-α by ELISA

Cells were plated at 70–80% confluence in 12-well plates. After differentiation, cells were treated as described in the figure legends. After CSE treatment for 1 h, treated culture media was removed and cells were maintained in fresh RPMI 1640 without serum. Twenty-four hours later, the culture media was collected and following the manufacturer’s instructions the concentration of TNF-α was detected by ELISA analysis with the Human TNF alpha ELISA Ready-SET-Go!® (eBioscience).

Cell Viability Assay

Cell Viability was assessed using a 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay as previously described (24). Briefly, cells were seeded in 12-well plates at ~70% confluence, differentiated for 24h in the presence of PMA (50 nM), and then treated as indicated in the figure legends. After treatment, cells were washed twice with PBS, and were incubated in MTT solution for 2–3 h. The percentage of viable cells was calculated using the following formula: Cell viability (%) = (Absorbance of treated sample/Absorbance of control) ×100. The experiments were performed in triplicate.

TACE activity assay

Following the manufacturer’s instructions, TACE activity was assessed using SensoLyte^® 520 TACE (α - Secretase) Activity Assay Kit (ANASPEC). After treatment, cells were washed with PBS, collected, and lysed with assay buffer. TACE enzymatic activity was quantified by continuous measurement of fluorescence intensity in a microplate fluorometer (λex 485 nm and λem 535 nm). TACE activity was normalized with the total protein level of each sample as determined by Bradford protein assay (Bio-Rad, Hercules, CA, USA).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from each sample using TRIzol^® Reagent (Life Technologies). Two micrograms of RNA were used as a template for cDNA synthesis with a reverse transcription kit (Promega). An equal volume of cDNA product was subjected to PCR analysis with some modifications according to ref. 18. The following primers were used in the PCR reactions: MUC1, forward primer 5′-ACAATTGACTCTGGCCTTCCG-3′ and reverse primer 5′-TGGGTTTGTGTAAGAGAGGCT-3′; β-actin, forward primer: 5′-CCAGCCTTCCTTCCTGGGCAT-3′ and reverse primer 5′-AGGAGCAATGATCT TGATCTTCATT-3′; TNF-α, forward primer 5′-AATCGGCCCGACTATCTCGACTTT-3′ and reverse primer 5′-TTTGAGCCAGAAGAGGGT-3′. For MUC1 and TNF-α, the PCR cycles were 32 and 31, respective, while for β-actin the cycles were 23. The amplified PCR products were resolved on 2% agarose gels with 0.5 μg/ml ethidium bromide, visualized, and photographed.

Detection of MUC1 in mouse lung by immunohistostaining

Lungs from A/J mice exposed to CS or filtered air (FA) for 20 weeks were paraformaldehyde-fixed and stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was carried out using the VECTASTAIN® ABC Kit with peroxidase labeling and DAB (3, 3′-diaminobenzidine) Peroxidase Substrate Kit (Vector laboratories, Burlingame, CA). Briefly, the slides were deparaffinized in xylene and rehydrated in gradient ethanol. Antigen retrieval was done by boiling the slide in 0.1 % citrate buffer for 15 min. Then, the slide was treated with 3% H₂O₂ for 20 min followed by blocking with 5% normal rabbit serum in PBS for 2 h at room temperature. After blocking the slide was incubated with primary antibody against MUC1 (rabbit antibody, Santa Cruz) overnight at 4°C. The ABC kit was then applied and the slide was developed with DAB according to instructions from the manufacturer.

Soft agar assay

HBEC-13 cells were exposed to CSE (20 μg/ml TPM) with or without TNF-α every three days for a total of 4 treatments. For each treatment, the cells were pre-treated with TNF-α (100 pg/ml) for 30 min followed by CSE treatment for 1 h. After CSE treatment, cells were cultured in fresh KSFM. A total of 1×10⁴ cells in each well (12-well plate) were seeded in soft agar for colony formation. To study the influence of conditioned medium from CSE-treated macrophages on cell transformation, HBEC-13 cells were exposed to conditioned medium with or without anti-TNF-α neutralizing antibody for 24 h prior to CSE (20 μg/ml TPM) treatment. Cells were then treated similarly as mentioned above. After 3-week incubation, colonies in the agar were photographed and counted. The average number of colonies ± S.D. was determined using 6 randomly selected fields (18). All experiments were run in triplicate.

Chromatin immunoprecipitation assay (CHIP)

Antibodies specific to PPAR (Abcam) were used to capture protein–DNA complexes. Mouse immunoglobulins were used for isotype controls. qPCR was carried out using Power SYBR Green PCR Master Mix (Applied Biosystems) with the ABI PRISM 7900HT. Results were generated in triplicate independent experiments. Primer sequences are: forward primer: 5′-GACCGGTATAAAGCGGTAGG-3′ and reverse primer: 5′-GTCATGGTGGTGGTGAAATG-3′. Results were quantified as fold enrichment using a 2^−DDCT method.

Statistics

All data are expressed as mean ± S.D. Statistical significance was examined by one-way analysis of variance. In all analyses, p<0.05 was considered statistically significant.

Increased MUC1 expression in CS exposed mouse lung macrophages and human macrophage cell lines

To investigate the effect of CS on MUC1 expression in lung cells, we first compared Muc1 expression in lung tissues from A/J mice chronically exposed to CS or filtered air (FA) for 20 weeks by immunohistostaining (25). Compared to Muc1 expression in FA exposed mice, Muc1 expression in airway and alveolar cells of CS exposed mice was significantly increased (Fig. S1). Interestingly, while CS exposure increased infiltration of inflammatory cells into the lung, a clear signal for Muc1 was also detected in lung macrophages. Both the number of Muc1-positive macrophages and the extent of Muc1 expression were promoted by CS exposure (Figs 1A and S1).

In human macrophage cells (THP-1 and U937), MUC1 expression was confirmed by Western blot with antibodies against both the extracellular domain (MUC1-N) and the C-terminal domain (MUC1-CT) (Fig. 1B, 1C). To validate the specificity of the Western blot, MUC1 siRNA was used to specifically block MUC1 expression. The MUC1 signal was effectively eliminated with MUC1 siRNA transfection (Fig. 1D). These results strongly suggest that MUC1 is expressed in macrophages. Furthermore, treating the human macrophage cells with CS extract (CSE) strikingly induced the expression of MUC1 (Fig. 1B, 1C). Collectively, these results suggest expression of MUC1 in lung macrophages is strongly induced by CS. Thus, we proceeded to examine if MUC1 plays a role in CS-induced inflammatory response in macrophages.

MUC1 is required for CSE-induced TNF-α production from macrophages

CS induces pulmonary macrophage infiltration and MUC1 was reported to regulate an inflammatory response in epithelial cells (26, 27). Therefore, we examined the role of MUC1 in CSE-induced inflammatory response in macrophages in an in vitro cell culture system. CSE strongly induced TNF-α secretion from both THP-1 and U937 cells, starting at 4h post-treatment and lasting for over 24h (Fig. 2A). Strikingly, knockdown of MUC1 expression by RNA interference effectively reduced CSE-induced TNF-α secretion from both THP-1 and U937 cells (Figs. 2B and 2C). MUC1 knockdown was confirmed by Western blot (Figs. 2B and 2C, insert). Remarkably, knockdown of MUC1 had no effect on cell viability with or without CSE treatment in both THP-1 and U937 cells (Figs. 2B and 2C). These results strongly suggest that MUC1 is required for TNF-α secretion in CSE-treated human macrophages.

CSE-induced MUC1 expression and TNF-α secretion involve PPAR-γ

To investigate how CSE induces MUC1 expression in macrophages, MUC1 mRNA level was detected by RT-PCR. In THP-1 cells, CSE treatment robustly increased MUC1 transcript beginning at 30 min and persisting for 2 h (Fig. 3A). Consistently, CSE also induced MUC1 mRNA expression in U937 cells (Fig. 3B). These results indicate that CSE activates MUC1 transcription.

The MUC1 promoter is under control of several transcription factors such as NF-κB, SP1 and PPAR-γ (27–29). To explore which transcription factor is involved in CSE-induced MUC1 expression, THP-1 cells were pre-exposed to a number of inhibitors before CSE treatment. The PPAR-γ inhibitors BADGE and GW9662, but not inhibitors for NF-κB, MAPKs (JNK, ERK, and p38) or ROS, remarkably attenuated CSE-induced MUC1 mRNA expression (Figs. 3C, 3D and S2A). Further, CSE caused an early activation of PPAR-γ in THP-1 cells (S2B). These results are in agreement with reports that PPAR-γ activates MUC1 gene transcription in human lung and gastric epithelial cells, and mouse placenta (27–29). Consistently, suppressing PPAR-γ with either BADGE or PPAR-γ siRNA dramatically suppressed CSE-induced MUC1 protein expression (Fig. 3D, 3E). Further, in a CHIP assay, binding of PPAR-γ to the MUC1 promoter was robustly induced by CSE (Fig. 3F). Thus, these results suggest that CSE induces MUC1 expression in human macrophages through PPAR-γ mediated transcription.

To corroborate the role of PPAR-γ in MUC1 expression and function, BADGE was used to treat macrophages before CSE treatment for TNF-α secretion. BADGE strongly inhibited CSE-induced TNF-α secretion, while it had no detectable cell cytotoxicity in macrophages (Fig. 3G). These results demonstrate that PPAR-γ-mediated MUC1 expression plays a critical role for CSE-induced TNF-α secretion from macrophages.

ERK is involved in CSE-induced MUC1 expression and TNF-α secretion in macrophages

It has been reported that the ERK pathway is involved in MUC1 expression (28). Thus, we also examined if this MAPK is involved in CSE-induced MUC1 expression. The ERK inhibitor U0126, but not the negative control U0124, effectively suppressed CSE-induced MUC1 protein expression (Fig. 4A). Consistent with this observation, CSE effectively activated ERK, beginning at 1 h and persisting for more than 4h after treatment (Fig. 4B). Interestingly, ERK inhibition had no detectable effect on CSE-induced MUC1 mRNA expression (Fig. 3C). These results suggest that the ERK pathway is likely involved in CSE-induced MUC1 expression in macrophages through a posttranscriptional mechanism. Indeed, the stability of MUC1 protein was reduced in ERK-inhibited and CSE-treated cells. The half-life of MUC1 was 2h in ERK-inhibited cells compared to 4.5h in control cells (Fig. 4C). In addition, U0126 also significantly blocked CSE-induced TNF-α secretion from macrophages with no cytotoxicity to the cells (Fig. 4D). Altogether, these results suggested that ERK is involved in CSE-induced MUC1 expression and TNF-α secretion in macrophages.

MUC1 mediates CSE-induced activation of TNF-α-converting enzyme (TACE)

TNF-α secretion is mainly mediated by shedding of the cell membrane-bound pro-TNF-α by TNF-α-converting enzyme (TACE) (30). To explore the underlying mechanism by which CSE induces TNF-α secretion, we examined if MUC1 is involved in regulation of TACE activity. Throughout the time period assessed, CSE induced TACE activity in a time-dependent manner (Fig. 5A). CSE-induced TNF-α secretion was effectively blocked by TACE inhibitor TAPI-1, confirming that CSE activates TACE activity to induce TNF-α secretion (Fig. S3). Interestingly, knockdown of MUC1 significantly attenuated CSE-induced TACE activity (Fig. 5B), suggesting that the induction of TACE activity by CSE was MUC1 dependent. CSE treatment or MUC1 knockdown had no effect on TACE expression (Fig. 5C), implying that CSE modulates TACE protease activity but not expression through MUC1. Similarly, suppression of ERK by U0126, which suppresses MUC1 expression, had no effect on TACE expression (Fig. 5C). CSE-induced MUC1 expression inhibited by treatment with BADGE or U0126 (Fig. 3D, 4A) effectively blocked CSE-induced but not basal TACE activity (Fig. 5D). These results are consistent with the important role of PPAR-γ and ERK in CSE-induced MUC1 expression. Altogether, these results demonstrate that CSE induces TNF-α secretion from macrophages through MUC1-dependent activation of TACE activity.

TNF-α secreted from macrophages significantly induces MUC1 expression in human bronchial epithelial cells (HBECs)

Because our previous study showed that MUC1 in HBECs plays an important role in CS carcinogen-induced HBEC transformation and TNF-α is a potential cytokine involved in MUC1 induction (18, 19), we investigated if TNF-α secreted from macrophages induces MUC1 expression in HBECs. Conditioned media from CSE-treated macrophages significantly increased MUC1 expression in HBEC-13 cells, while the control medium from untreated macrophages showed little effect on MUC1 expression (Fig. 6A). It is interesting that CSE by itself had a minor induction of MUC1 expression whereas conditioned medium from CSE-treated macrophages had a much stronger effect. On the other hand, the combination of CSE and conditioned medium from CSE-treated macrophages had no additive effect in MUC1 expression (Fig. 6B). These results suggest that the inflammatory response plays a major role in MUC1 expression in HBECs, although the direct induction of MUC1 by CSE in HBECs may have some minor contribution to MUC1 expression. Importantly, a TNF-α neutralizing antibody, but not a negative control antibody effectively blocked MUC1 induction by conditioned medium from CSE-exposed macrophages, suggesting that the MUC1 induction is TNF-α-dependent (Fig. 6C). Further, recombinant TNF-α was used to substantiate the role of TNF-α in MUC1 induction in HBECs. Indeed, MUC1 expression was strongly induced by the recombinant TNF-α (Fig. 6D). Taken together, these results establish the role of TNF-α secreted from macrophages in stimulating MUC1 expression in HBECs.

TNF-α secreted from macrophages facilitates CSE-induced transformation of HBECs

We next investigated the influence of conditioned media from CSE-treated macrophages on CSE-induced HBEC transformation. For induction of MUC1 expression, HBEC-13 cells were exposed to conditioned medium for 24 h, followed by exposure to CSE (Fig. 6A). Cell transformation was assayed by colony formation in soft agar assay. While CSE modestly induced colony formation, the conditioned medium from CSE-treated macrophages robustly enhanced CSE-induced cell transformation (Fig. 7A, 7B). Importantly, the effect of the conditioned medium was completely abolished by incubation with a TNF-α neutralizing antibody, but not the negative control antibody (Figs. 7A and 7B). In HBEC-13, recombinant TNF-α effectively and consistently augmented CSE-induced colony formation (Fig. S4). Together with our previous finding that MUC1 plays an important role in CS carcinogen-induced HBEC transformation (18), these results suggest that TNF-α secreted from macrophages potentiates CSE-induced transformation through the induction of MUC1 expression in HBECs.