MUC1 in Macrophage: Contributions to Cigarette Smoke–Induced Lung Cancer

Although an inflammatory microenvironment plays an important role in the lung cancer development (1, 2), how inflammation promotes lung carcinogenesis has not been clearly elucidated. Cigarette smoke, which elicits chronic pulmonary inflammation, is a major risk factor for lung cancer development (3, 4). Carcinogens derived from cigarette smoke such as benzo(a)pyrene induce lung cancer through DNA damage that results in mutations and epigenetic alterations (4, 5). In the microenvironment, inflammatory cells such as macrophages secrete cytokines that affect epithelial cells to favor carcinogenesis, which may involve suppression of DNA repair and promotion of apoptosis resistance, proliferation, metastasis, and secondary secretion of cytokines and growth factors (4, 6). TNF-α is an important proinflammatory cytokine that plays a key role in both inflammation and cancer development (7, 8). Therefore, in the tumor microenvironment, TNF-α produced by inflammatory cells may be a key mediator for inflammation-associated carcinogenesis (8, 9). However, the role and mechanism of TNF-α in lung cancer development are not well defined.

MUC1, a mucin-like glycosylated protein induced by airway inflammation and expressed on the bronchial epithelial cell membrane, plays an important role in the resolution of inflammation during respiratory tract infection (10, 11). MUC1 consists of two subunits derived from a single polypeptide: the N-terminal subunit containing highly conserved repeats of 20 amino acids that are modified by O-glycosylation and the transmembrane C-terminal subunit containing 72 amino acid residues that bind to various proteins involved in signal transduction (12, 13). MUC1 is regarded as a tumor antigen because it is aberrantly overexpressed in various cancers including lung cancer (14–16). In non–small cell lung cancer, MUC1 is overexpressed and correlated with poor patient survival (17). A variety of cellular partners for MUC1 have been identified, which may contribute to the malignancy of cancer cells and their resistance to chemotherapy (13). However, direct evidence for MUC1 in lung carcinogenesis is still lacking. Our previous study showed that chronic cigarette smoke carcinogen exposure induces persistent MUC1 overexpression in human lung bronchial epithelial cells, which facilitates cigarette smoke–induced cell transformation through EGF receptor (EGFR)–mediated cell survival signaling (18). Because MUC1 expression is sustained during chronic inflammation, MUC1 is likely involved in inflammation-associated cancer development (12).

Although cigarette smoke induces chronic pulmonary inflammation and MUC1 expression in airway epithelial cells, it is unclear if inflammation is involved in the upregulation of MUC1 in airway epithelial cells in smokers. Macrophages are the main inflammatory cell types that infiltrate into the lung, where they secrete cytokines such as TNF-α that promote recruitment of other inflammatory cells and production of other cytokines. Although its tumor-promoting effects have been documented, the role and mechanism of TNF-α in lung cancer development has not been well studied. Specifically, while TNF-α plays a role in MUC1 induction in airway epithelial cells during acute bacterial infection (19), whether it is involved in cigarette smoke–induced and MUC1-mediated lung cancer development is unknown. Furthermore, it is known that MUC1 is expressed in some immune cells (20, 21), but it is unclear if MUC1 is expressed and functions in macrophages.

In this study, we investigated the role and mechanism of cigarette smoke–induced inflammatory response in MUC1-mediated lung cancer development with a conditioned medium transfer approach and a validated human bronchial epithelial cell (HBEC) transformation model. The results identify a novel function for MUC1 in mediating cigarette smoke–induced macrophage activation that facilitates HBEC transformation. Together with its function in epithelial cells (18), our findings suggest that MUC1 plays a dual role in cigarette smoke–induced and inflammation-associated lung cancer development: to facilitate TNF-α secretion from macrophages and to potentiate transformation of HBECs.

Bisphenol A diglycidyl ether (BADGE) was obtained from Enzo Life Science. The inhibitors U0126 for extracellular signal—regulated kinase (ERK), SB203580 for p38, SC-514 for inhibitor of IκB kinase (IKK), and Necrostatin-1 for RIP1 kinase were purchased from Calbiochem. GW9662 and U0124, the negative control for U0126, were obtained from Millipore. siRNA (SiGenome SMARTpool) for MUC1, PPAR-γ, and TNF receptor 1 (TNFR1) and negative control siRNA were purchased from Dharmacon. The primary antibodies against Mucin 1 (GP1.4) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Santa Cruz Biotechnology. MUC1Ab-5, a hamster monoclonal antibody that recognizes the MUC1 CT domain, was purchased from Lab Vision. 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. Filters collected from the AMESA Type 1300 smoking machine, which generates mainstream cigarette smoke, were used to prepare cigarette smoke extract (CSE) by sequential extraction of material from cigarette smoke filter with dimethyl sulfoxide (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).

THP-1 and U937 macrophages were cultured in RPMI-1640 supplemented with 10% FBS, 2 mmol/L of l-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. These cell lines, obtained from American Type Culture Collection, were used within 6 months of receipt. The immortalized HBECs, HBEC-13, were generously provided by Drs. Shay and Minna (Southwestern Medical Center, Dallas, TX; ref. 23). HBECs 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 (AthenaES).

THP-1 and U937 macrophages were cultured in the presence of 50 nmol/L phorbol 12-myristate 13-acetate (PMA) for 24 hours. The differentiated cells were transfected with MUC1 and control siRNA with the INTERFERin siRNA Transfection Reagent (Polyplus-transfection) according to the manufacturer's instructions. MUC1 protein level was determined by Western blot analysis at 48 hours after transfection.

Cells were washed twice with cold PBS, collected, and lysed with M2 buffer (20 mmol/L Tris–HCl, pH 7.6, 0.5% Nonidet P-40, 250 mmol/L NaCl, 3 mmol/L EDTA, 3 mmol/L EGTA, 2 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L β-glycerophosphate, 1 mmol/L 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-PAGE and then transferred to polyvinylidene difluoride (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.

Cells were plated at 70% to 80% confluence in 12-well plates. After differentiation, cells were treated as described in the figure legends. After CSE treatment for 1 hour, 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-α ELISA Ready-SET-Go! (eBioscience).

Cell viability was assessed using a MTT cell proliferation assay as previously described (24). Briefly, cells were seeded in 12-well plates at approximately 70% confluence, differentiated for 24 hours in the presence of PMA (50 nmol/L), 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 to 3 hours. 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.

Following the manufacturer's instructions, TNF-α–converting enzyme (TACE) activity was assessed using the 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 (λ_ex 485 nm and λ_em 535 nm). TACE activity was normalized with the total protein level of each sample as determined by Bradford protein assay (Bio-Rad).

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′-AGGAGCAATGATCTTGATCTTCATT-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, respectively, whereas 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.

Lungs from A/J mice exposed to cigarette smoke or filtered air 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). 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 minutes. Then, the slide was treated with 3% H₂O₂ for 20 minutes followed by blocking with 5% normal rabbit serum in PBS for 2 hours at room temperature. After blocking, the slide was incubated with primary antibody against MUC1 (rabbit antibody; Santa Cruz Biotechnology) overnight at 4°C. The ABC Kit was then applied and the slide was developed with DAB according to the manufacturer's instructions.

HBEC-13 cells were exposed to CSE (20 μg/mL TPM) with or without TNF-α every 3 days for a total of four treatments. For each treatment, the cells were pretreated with TNF-α (100 pg/mL) for 30 minutes followed by CSE treatment for 1 hour. 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 hours before 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 ± SD was determined using six randomly selected fields (18). All experiments were run in triplicate.

Antibodies specific to PPAR (Abcam) were used to capture protein–DNA complexes. Mouse immunoglobulins were used for isotype controls. Quantitative PCR (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 the |$2^{-{\rm \Delta \Delta} C_{\rm T}}$| method.

All data are expressed as mean ± SD. Statistical significance was examined by one-way ANOVA. In all analyses, P < 0.05 was considered statistically significant.

To investigate the effect of cigarette smoke on MUC1 expression in lung cells, we first compared Muc1 expression in lung tissues from A/J mice chronically exposed to cigarette smoke or filtered air for 20 weeks by immunohistostaining (25). Compared with Muc1 expression in filtered air–exposed mice, Muc1 expression in airway and alveolar cells of cigarette smoke–exposed mice was significantly increased (Supplementary Fig. S1). Interestingly, while cigarette smoke 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 cigarette smoke exposure (Fig. 1A and Supplementary Fig. S1).

In human macrophage cells (THP-1 and U937), MUC1 expression was confirmed by Western blot analysis with antibodies against both the extracellular domain (MUC1-N) and the C-terminal domain (MUC1-CT; Fig. 1B and C). To validate the specificity of the Western blot analysis, 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 CSE strikingly induced the expression of MUC1 (Fig. 1B and C). Collectively, these results suggest that expression of MUC1 in lung macrophages is strongly induced by cigarette smoke. Thus, we proceeded to examine whether MUC1 plays a role in a cigarette smoke–induced inflammatory response in macrophages.

Cigarette smoke 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 4 hours posttreatment and lasting for over 24 hours (Fig. 2A). Strikingly, knockdown of MUC1 expression by RNA interference (RNAi) effectively reduced CSE-induced TNF-α secretion from both THP-1 and U937 cells (Fig. 2B and C). MUC1 knockdown was confirmed by Western blot analysis (Fig. 2B and C, inset). Remarkably, knockdown of MUC1 had no effect on cell viability with or without CSE treatment in both THP-1 and U937 cells (Fig. 2B and C). These results strongly suggest that MUC1 is required for TNF-α secretion in CSE-treated human macrophages.

To investigate how CSE induces MUC1 expression in macrophages, MUC1 mRNA level was detected by reverse transcription PCR (RT-PCR). In THP-1 cells, CSE treatment robustly increased MUC1 transcript beginning at 30 minutes and persisting for 2 hours (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 preexposed to a number of inhibitors before CSE treatment. The PPAR-γ inhibitors BADGE and GW9662, but not inhibitors for NF-κB, mitogen—activated protein kinases (MAPK; JNK, ERK, and p38), or reactive oxygen species (ROS), remarkably attenuated CSE-induced MUC1 mRNA expression (Fig. 3C and D and Supplementary Fig. S2A). Furthermore, CSE caused an early activation of PPAR-γ in THP-1 cells (Supplementary Fig. 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 and E). Furthermore, in a chromatin immunoprecipitation (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.

It has been reported that the ERK pathway is involved in MUC1 expression (28). Thus, we also examined whether 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 hour and persisting for more than 4 hours 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 2 hours in ERK-inhibited cells as compared with 4.5 hours 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.

TNF-α secretion is mainly mediated by shedding of the cell membrane–bound pro-TNF-α by TACE (30). To explore the underlying mechanism by which CSE induces TNF-α secretion, we examined whether 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 (Supplementary 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 (Figs. 3D and 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.

Because our previous study showed that MUC1 in HBECs plays an important role in cigarette smoke carcinogen-induced HBEC transformation and TNF-α is a potential cytokine involved in MUC1 induction (18, 19), we investigated whether TNF-α secreted from macrophages induces MUC1 expression in HBECs. Conditioned media from CSE-treated macrophages significantly increased MUC1 expression in HBEC-13 cells, whereas 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). Furthermore, 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.

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 hours, followed by exposure to CSE (Fig. 6A). Cell transformation was assayed by colony formation in soft agar assay. Although CSE modestly induced colony formation, the conditioned medium from CSE-treated macrophages robustly enhanced CSE-induced cell transformation (Fig. 7A and B). Importantly, the effect of the conditioned medium was completely abolished by incubation with a TNF-α–neutralizing antibody, but not the negative control antibody (Fig. 7A and B). In HBEC-13, recombinant TNF-α effectively and consistently augmented CSE-induced colony formation (Supplementary Fig. S4). Together with our previous finding that MUC1 plays an important role in cigarette smoke 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.

In this study, we provide evidence showing a novel function for MUC1 in a cigarette smoke–induced inflammatory response in macrophages. MUC1 was modestly expressed in mouse lung macrophages and was robustly induced by chronic cigarette smoke exposure. The expression of MUC1 in human macrophages was also upregulated by CSE in vitro. Suppressing MUC1 expression significantly attenuated CSE-induced TACE activation and TNF-α secretion from macrophages. Furthermore, we found that conditioned medium from CSE-treated macrophages significantly induced MUC1 expression in HBECs and potentiated CSE-induced HBEC transformation in a TNF-α–dependent manner. Therefore, together with our previous finding that MUC1 in HBECs plays an important role in cigarette smoke carcinogen-induced transformation (18), our observations establish a dual role for MUC1 in cigarette smoke–induced and inflammation-associated lung cancer development: to facilitate TNF-α secretion in macrophages and to potentiate transformation in HBECs (Fig. 7C).

We found that the induction of MUC1 in macrophages by CSE is dependent on PPAR-γ–mediated transcription and ERK-mediated protein stabilization. The expression and function of MUC1 in respiratory mucosal epithelial cells are well documented (12). MUC1 expression was also found in immune cells such as T lymphocytes and dendritic cells (20, 21). However, whether MUC1 is functionally expressed in macrophages has not been determined. In this report, we carefully determined the expression of MUC1 in vivo in mouse macrophages by IHC and in vitro in human macrophages by Western blot analysis and RT-PCR. The detection of MUC1 was validated by RNAi. Furthermore, we determined that MUC1 is inducible by CSE and is functional in macrophages. Knockdown of MUC1 effectively attenuated CSE-induced TNF-α secretion. Thus, our results for the first time establish the expression and function of MUC1 in macrophages. The pro-inflammatory function of MUC1 in macrophages is contradictory to reports showing an anti-inflammatory function for MUC1 in epithelial cells (27). The reason for this discrepancy is currently unknown but it is likely that MUC1 may function distinctively in different cell types.

We further identified the main pathways for cigarette smoke–induced MUC1 expression in macrophages. The direct binding of PPAR-γ to the MUC1 promoter induced by CSE was detected by CHIP assay. The blockage of PPAR-γ effectively inhibited CSE-induced MUC1 mRNA and protein expression in macrophages. This is consistent with reports that PPAR-γ directly binds and activates MUC1 transcription (27–29). Also, suppressing PPAR-γ blocked CSE-induced TNF-α secretion from macrophages, further substantiating the role of PPAR-γ–mediated MUC1 expression as a proinflammatory response in macrophages. Supporting our observation, it was reported that the PPAR-γ inhibitor, BADGE, suppressed TNF-α production in the murine macrophage cell line RAW 264.7 (31). In addition, expression of PPAR-γ was increased in monocytes/macrophages in smokers (32). We further found that ERK is also required for CSE-induced MUC1 expression in macrophages at a posttranscriptional level. Thus, MUC1 expression in macrophages is cooperatively controlled by PPAR-γ and ERK at multiple levels.

The anti-inflammatory role of MUC1 in epithelial cells is likely through suppression of Toll-like receptor signaling (11, 33). We show here that MUC1 plays a proinflammatory role in macrophages involving TACE activation. It would be interesting to determine whether the contradictory roles of MUC1 in different cell types are due to different molecular partners or different isoforms of MUC1. Similarly, the role of PPAR-γ in the cigarette smoke–induced inflammatory response seems to also be complex, being either anti- or proinflammatory (27, 31, 32, 34). The complexity of the response may be due to different cell types (i.e., epithelial cells vs. macrophages), different stimulations (i.e., cigarette smoke vs. PMA), or different PPAR-γ–modulating agents used (27, 31, 32, 34, 35). Nevertheless, our results clearly show a proinflammatory role for PPAR-γ in macrophages in response to CSE through activation of MUC1 expression. Elucidation of the mechanisms for the functional interaction of PPAR-γ and MUC1 in a cell's response to inflammatory stimuli would help us to understand the complex roles of these molecules in inflammation.

Because TNF-α is able to induce MUC1 expression in HBECs, we examined whether TNF-α functions in an autocrine manner to induce MUC1 expression in macrophages. Knockdown of TNFR1, the major TNF-α receptor, had no effect on CSE-induced MUC1 expression and TNF-α secretion (Supplementary Fig. S5A and S5B). A similar result was obtained when TNFR2 was knocked down (data not shown). Consistently, knockdown of TNFR1 had no effect on CSE-induced ERK activation in macrophages (Supplementary Fig. S5C), suggesting that the CSE-induced ERK activation is independent of TNF-α–induced signaling. Therefore, it is unlikely that MUC1 expression in macrophages is triggered by autocrine TNF-α. Finally, we determined that MUC1-mediated TNF-α secretion from macrophages potentiates cigarette smoke–induced transformation of HBECs. This process occurs at least in part through the induction of MUC1 expression in HBECs. Previously, we have shown that increased MUC1 in HBECs potentiates EGFR-mediated cell signaling for cigarette smoke carcinogen-induced HBEC transformation (18). Therefore, our results suggest that by functioning in different cell types in the tumor-prone microenvironment, MUC1 substantially contributes to lung carcinogenesis. Specifically, MUC1 plays a dual role: to facilitate TNF-α secretion in macrophages and to potentiate transformation in HBECs. Further studies are warranted to determine whether these MUC1-mediated mechanisms can be targeted for prevention against lung cancer development.

No potential conflicts of interest were disclosed.

Conception and design: X. Xu, K. Kato, K.C. Kim, Y. Lin

Development of methodology: X. Xu, B. Li, K. Kato

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Xu, C. Tellez, S.A. Belinsky, K.C. Kim

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Xu, K. Kato, C. Tellez

Writing, review, and/or revision of the manuscript: X. Xu, M.T. Padilla, C. Tellez, S.A. Belinsky, K.C. Kim, Y. Lin

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Xu, M.T. Padilla, B. Li, A. Wells

Study supervision: Y. Lin

The authors thank Kieu Do for her help with the CHIP assay.

This study was supported by grants from NIEHS/NIH (R01ES017328) and Office of Science (BER), U.S. Department of Energy (DE-FG02-09ER64783).

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.