Induction of CEACAM6 by expression of recombinant (r)TTF-1. Human fetal lung epithelial cells were transduced by exposure to Ad12A2 overnight and then cultured in control medium (Waymouth). A: representative Western blot for dose response. Exposure to Ad12A2 resulted in dose-dependent increases in rTTF-1 and CEACAM6 (lanes 3–8) compared with both AdGFP-treated cells (lanes 1 and 2) and control (C) cells (lane 9), with both proteins reaching levels manyfold greater than achieved with DCI (lane 10). PFU, plaque-forming units. B: densitometric quantification of Western blot data. Fold induction for DCI (•) is shown for comparison. C: representative Western for time course. Expression of rTTF-1 protein (Ad12A2 at 6 PFU/cell) was maximal by ∼16 h after transduction, whereas CEACAM6 protein increased markedly only after 36 h. D: densitometric quantification of Western blot data. E: immunofluorescent staining for TTF-1 and CEACAM6 in cultured cells. The intensity of nuclear TTF-1 staining (red) was variable for cells transduced with Ad12A2 (4 PFU/cell, center) or treated with DCI (left). CEACAM6 staining (green) was primarily punctate with both treatments, and more cells were CEACAM6 positive after Ad12A2 transduction compared with DCI treatment. Staining in AdGFP-treated control cells (right) was at low background level.

DCI induction of CEACAM6 mRNA requires de novo protein synthesis. Epithelial cells were cultured in control medium for 3 days (to deplete endogenous TTF-1), DCI was added, and at various times thereafter (16–38 h) cycloheximide (CX, 1 μg/ml) was added to some dishes for the remainder of the 48-h culture. In response to DCI, TTF-1 protein (filled bars) increased maximally within 16 h (4-fold vs. control) and was not affected by cycloheximide. Induction of CEACAM6 mRNA (13-fold at 48 h, open bars) was sensitive to inhibition of protein synthesis for up to 24 h after addition of hormones. Data are means + SE of 3–4 replicates in 2 experiments expressed as % of 48-h value (100%). *P < 0.01 vs. 48-h value.

CEACAM6 secretion from cultured epithelial cells and in vivo. A: representative secretion experiment. Release of CEACAM6 was approximately linear for 7 h for both DCI-treated and Ad12A2-transduced (6 PFU/cell) cells. Western blot (inset) shows reduced secretion after 1 h at 4°C (1a lane) compared with 1 h at 37°C (1 lane) for both DCI-treated and Ad12A2-transduced cells. B: secretion summary. At 7 h, secretion of CEACAM6 was ∼5-fold higher for DCI-treated cells than for Ad12A2-transduced cells (n = 3, P < 0.05). C: effect of mannosamine on secretion. Cells were cultured 72 h in DCI with or without mannosamine (10 mM) added for the last 48 h to inhibit mannose-related glycosylation. Less CEACAM6 was released into the medium for mannosamine-treated cells (2nd and 3rd lanes) compared with control (1st lane) and conversely treated cells had higher CEACAM6 content. TTF-1 expression (bottom) was similar for cells with or without mannosamine. In 3 experiments the recovery ratio (+mannosamine/−mannosamine) in the medium was 0.48 ± 0.09 (P < 0.01 for n = 5). D: CEACAM6 in tracheal aspirate (TA) fluid from a newborn infant. By Western blot analysis with antibody recognizing both CEACAM5 and CEACAM6, both proteins were detected in both the large-aggregate surfactant pellet and supernatant from a ventilated premature infant, with samples obtained on different postnatal days as shown in lanes 1–6. CEACAM6 and CEACAM5 bands migrated identically to those in cultured cells (lanes 7–9). Results are representative of 3 infants with serial sampling of tracheal aspirates.

Developmental and hormonal regulation of carcinoembryonic cell adhesion molecule (CEACAM)6. A: developmental increase in CEACAM6. Western blots were performed on specimens of fetal and postnatal lung. Relative densitometric units (du), normalized to GAPDH, are shown as means ± SE with n = 3–7/group except 2 yr (n = 2). B–F: fetal lung epithelial cells were cultured in the absence (control) or presence of dexamethasone (Dex), cAMP+IBMX (cAMP), or both agents (DCI). B: Western blot from representative experiment. C: summary protein data from 3 experiments (means ± SE). *P < 0.05 vs. control, **P < 0.05 vs. cAMP and Dex alone. D: transcription rate for CEACAM6 mRNA. Nuclei were isolated after exposure of cells to DCI or diluent (control) for 2–48 h. Data are normalized to GAPDH mRNA and expressed as means ± SE for fold difference relative to control cells. *P < 0.05 vs. control. E: representative Western blot for time course of CEACAM6 and thyroid transcription factor (TTF)-1 induction. F: densitometric quantification of Western blots. An increase in CEACAM6 was delayed >24 h, consistent with TTF-1 dependence for CEACAM induction (n = 3).

Effect of TTF-1 and CEACAM6 knockdown. Freshly isolated epithelial cells were electroporated with small inhibitory RNA (siRNA) oligonucleotide (si) or scrambled control oligonucleotide (si-ct) and then cultured for 72 h. A: Western blot in representative experiment of TTF-1 knockdown. DCI alone (lane 3) increased TTF-1, CEACAM6, and SP-B compared with control (lane 4, Waymouth medium). Addition of TTF-1 siRNA oligos in the presence of DCI (lane 1) reduced levels of TTF-1, CEACAM6, and SP-B compared with the scrambled control oligo (lane 2). B: densitometric data comparing knockdown of TTF-1 vs. knockdown of CEACAM6. Knockdown of TTF-1 (filled bars) significantly reduced content of TTF-1 (46 ± 6% of control), CEACAM6 (20 ± 2% of control), and SP-B (26 ± 5% of control) in 4 experiments (all *P < 0.01). In contrast, knockdown of CEACAM6 (n = 3, gray bars) reduced content of CEACAM6 (12 ± 3% of control, *P < 0.01) but did not affect either TTF-1 or SP-B.

Induction of CEACAM6 is cell type specific. A: representative Western blot of enriched epithelial cells and fibroblasts from the same lung cultured with or without DCI for 4 days. Both TTF-1 and CEACAM6 expression were markedly increased in type II epithelial cells (TII) but not in fibroblasts (Fb). The weak TTF-1 band observed in lane 7 is likely due to contaminating epithelial cells. Left portion of image is adapted from Kolla et al. (31). B: densitometric analysis of Western blots. Data are means and ranges from 2 experiments and demonstrate DCI induction of CEACAM6 in epithelial cells but not in fibroblasts. C: fluorescence immunostaining of CEACAM6 induction in explant cultures (5 days) of human fetal lung (19 wk of gestation). CEACAM6 staining was markedly greater in DCI-treated (right) vs. control (left) explants and was detected only in the epithelial cell layer (arrow) lining the air spaces (a), not in the mesenchymal (m) region. D: localization of CEACAM6 in DCI-treated cells by confocal microscopy. Left: cells were immunostained with monoclonal anti-CEACAM6 and anti-mouse Alexa 488-tagged secondary antibody (green). CEACAM6 was localized to large perinuclear vesicles (lamellar bodies) and also to plasma membrane (arrow). Center and right: cells were stained sequentially with anti-CEACAM6 antibody (as above) and then rabbit polyclonal anti-SP-B (Chemicon), with anti-rabbit-Cy3 tagged secondary antibody (red). CEACAM6 colocalized with SP-B (yellow color) to large perinuclear vesicles characteristic of lamellar body staining by SP-B in these cells. At higher power (right), colocalization of CEACAM6 (green) and SP-B (red) was apparent in most vesicles. N, nucleus.

CEACAM6 effects on apoptosis and surfactant function. A and B: annexin V and TOTO-3 staining at 24 h (A) and 72 h (B) by confocal microscopy. Freshly isolated epithelial cells were electroporated with anti-CEACAM6 oligonucleotides (siRNA, top) or scrambled control oligonucleotides (scram control, bottom). Cells treated with siRNA (top left, A) showed more annexin V (conjugated to Alexa 488) binding to the plasma membrane compared with cells treated with scrambled control oligos (bottom left). TOTO-3 stained few cells at 24 h (center), indicating good membrane integrity. At 72 h increased annexin V binding (conjugated to Alexa 594; B, left) occurred with knockdown of CEACAM6 similar to the response at 24 h (A). In addition, there was increased nuclear TOTO-3 staining, consistent with more cell death at this later time interval. The cellular density was similar for both groups as shown in the differential interference contrast images (right). Images are representative of 5 fields in 3 experiments. C: CEACAM6 knockdown increases caspase-3 activity. Freshly isolated cells were electroporated with anti-CEACAM6 si oligonucleotide, scrambled control oligonucleotide, or no oligonucleotide [control (cont) and DCI] and cultured 18–24 h in the absence (cont) or presence (all other groups) of DCI. Caspase-3 activity was elevated ∼3-fold in cells with knockdown of CEACAM6 compared with si control oligonucleotide value (n = 4; *P < 0.05 vs. cont). D: CEACAM6 protects surfactant from protein inhibition. Freshly isolated cells were electroporated with either CEACAM6-specific siRNA (80% knockdown by Western blot) or scrambled control oligonucleotide and cultured 4 days in the presence of DCI. Conditioned media (CM) were concentrated to a similar total protein level (range 0.2–0.6 mg/ml) in each experiment. Infasurf was diluted to 1.5 mg phospholipid/ml with fresh Waymouth medium (Infasurf alone control) or with conditioned medium, and surface tension was assessed in a pulsating bubble surfactometer. Infasurf alone rapidly achieved a low minimum surface tension (∼0 mN/m). In conditioned medium with low content of CEACAM6 (si oligo), the presence of exogenous protein increased equilibrium surface tension (time 0), delayed the time to minimum surface tension (∼550 s), and increased minimum surface tension (∼14 mN/m). In the presence of CEACAM6-replete conditioned medium (control oligo), a low minimum surface tension (∼2 mN/m) was achieved by ∼250 s.