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Am J Physiol Lung Cell Mol Physiol 292: L500-L509, 2007. First published October 6, 2006; doi:10.1152/ajplung.00052.2006
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Notch-1 regulates pulmonary neuroendocrine cell differentiation in cell lines and in transgenic mice

Lin Shan,1,2 Jon C. Aster,3 Jeffrey Sklar,3 and Mary E. Sunday1,2,3

1Department of Pathology, Children's Hospital and Harvard Medical School, Boston, Massachusetts; 2Department of Pathology, Duke University Medical Center, Durham, North Carolina; and 3Department of Pathology, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 11 February 2006 ; accepted in final form 28 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The notch gene family encodes transmembrane receptors that regulate cell differentiation by interacting with surface ligands on adjacent cells. Previously, we demonstrated that tumor necrosis factor-{alpha} (TNF) induces neuroendocrine (NE) cell differentiation in H82, but not H526, undifferentiated small cell lung carcinoma lines. We now test the hypothesis that TNF mediates NE cell differentiation in part by altering Notch gene expression. First, using RT-PCR, we determined that TNF treatment of H82, but not H526, transiently decreases notch-1 mRNA in parallel with induction of gene expression for the NE-specific marker DOPA decarboxylase (DDC). Second, we treated H82 and H526 with notch-1 antisense vs. sense oligodeoxynucleotides. Using quantitative RT-PCR and Western analyses we demonstrate that DDC mRNA and protein are increased in H82 by notch-1 antisense, whereas notch-1 mRNA and activated Notch-1 protein are decreased. mRNA for Hes1, a transcription factor downstream from activated Notch, is also decreased by Notch-1 antisense in H82 but not H526. After 7 days of Notch-1 antisense treatment, neural cell adhesion molecule (NCAM) immunoreactivity is induced in H82 but not H526. Third, we generated transgenic mice bearing notch-1 driven by the neural/NE-specific calcitonin promoter, which express activated Notch-1 in developing lung epithelium. Newborn NotchCal mouse lungs have high levels of hes1 mRNA, reflecting increased activated Notch, compared with wild-type. NotchCal lungs have decreased CGRP-positive NE cells, decreased protein gene product 9.5 (PGP9.5)-positive NE cells, and decreased gastrin-releasing peptide (GRP), CGRP, and DDC mRNA levels compared with normal littermates. Cumulatively, these observations provide further support for a role for Notch-1 signaling in regulating pulmonary NE cell differentiation.

small cell lung carcinoma; tumor necrosis factor-{alpha}; antisense oligodeoxynucleotides; DOPA decarboxylase; neural cell adhesion molecule

MAMMALIAN AIRWAY EPITHELIUM is composed of multiple cell populations, including pulmonary neuroendocrine cells (PNECs), which are distributed throughout airway epithelium from the lobar bronchus to the alveolar duct (60). PNECs occur both as solitary cells and clusters, the latter known as neuroepithelial bodies when they are innervated (39). During development, the first epithelial cells to differentiate in both human and rodent fetal airways are the PNECs (18, 60). Mammalian bombesin-like peptide (BLP) was identified as the first neuropeptide localized to PNECs (64). PNECs containing BLP immunoreactivity are present at high numbers in human newborn and fetal lung (64). BLPs include amphibian bombesin and mammalian/amphibian gastrin-releasing peptide (GRP) (45, 56), in which the bioactive amidated carboxy-terminal amino acid sequence is -His-Leu-Met-NH2. BLPs have been demonstrated to be potent mitogens for many small cell lung carcinoma cell lines (SCLCs) (17, 63), normal adult bronchial epithelial cells (52), and several fibroblast cell lines, including adult human pulmonary fibroblasts (1, 47). Previously, our laboratory (35) showed that BLPs stimulate cell proliferation and branching morphogenesis of murine embryonic lung buds, in which GRP receptors are localized mainly to the mesenchyme around developing blood vessels and airways. However, developmental mechanisms underlying PNEC differentiation have only recently been explored. Ito et al. (26) suggested that PNECs develop from immature lung epithelia because mesenchyme and nervous tissue are not required for this neuroendocrine (NE) differentiation to take place. Borges et al. (12) demonstrated that mice deficient for Mash-1, a proneural transcription factor, do not have any evidence of PNECs. Finally, Ito et al. (28) unambiguously showed that knockout of the Notch effector hes1 led to premature differentiation and increased numbers of PNECs in Hes1-null mice, and increased PNECs mice, in which notch gene expression and signaling were suppressed in parallel with decreased hes1. Cumulatively, all of these studies support a role for Notch signaling in PNEC differentiation. However, a direct role for Notch has not been previously addressed.

Notch receptors, members of a transmembrane protein family, play essential roles in cell fate decision in multiple developmental processes from Drosophila to humans (3, 4, 25, 48). Four Notch receptors (Notch 1–4) and five ligands (Jagged-1, Jagged-2, Delta-1, Delta-3, and Delta-4) have been identified in mammals (10, 37, 41, 65). The biochemical mechanisms of Notch signaling are conserved among the Notch receptors (43). Upon activation by Notch ligands, the Notch intracellular domain is cleaved and translocated into the nucleus. There it forms a complex with DNA-binding protein RBP-J{kappa} [CSL/CBF1/Su(H)/Lag-1] and directly activates expression of the negative basic helix-loop-helix (bHLH) hes neurogenic genes, which, in turn, repress expression of downstream neural/NE genes (10, 40). Notch signaling regulates how cells respond to intrinsic or extrinsic developmental cues that are necessary to unfold specific developmental programs. Notch activity affects differentiation, proliferation, and apoptotic programs, providing a general developmental tool to influence organ formation and morphogenesis (4, 34). For example, Notch signaling is important for development of pancreatic endocrine cells, differentiation of which is accelerated in mice deficient for Delta-like-1. Those studies showed that Notch signaling is critical for the decision between the endocrine and progenitor/exocrine fates in the developing pancreas (2). Similarly, Hes1 was shown to function as a general negative regulator of endodermal endocrine differentiation (31). However, the direct significance of Notch expression for normal PNEC differentiation has not been tested.

Notch has been identified on both epithelial and mesenchymal cells of embryonic lung (62). Using immunohistochemistry, Ito et al. (28) determined that non-NE epithelial cells in fetal mouse lung are immunopositive for Notch-1, Notch-3, and also Hes1. We hypothesized that Notch receptors might be important regulators of the non-NE cell phenotype in developing and neoplastic lung. Previous studies in our laboratory (22) demonstrated that tumor necrosis factor-{alpha} (TNF) induces NE cell differentiation in SCLCs. Through a mechanism involving NF-{kappa}B, TNF treatment of H82 cells induces NE gene expression and phenotypic features including immunostaining for proGRP and neural cell adhesion molecule (NCAM) and dense core neurosecretory granules by electron microscopy. We now test the hypothesis that TNF sustains NE differentiation of H82 cells at least in part by diminishing notch gene expression, which may be sufficient to reduce Notch signaling and to ultimately induce NE cell differentiation. To extend this investigation to normal PNEC differentiation, we also analyzed lungs of newborn mice overexpressing activated Notch in PNECs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. The NotchCal lineages were generated using a construct including the rat calcitonin/CGRP promoter (57) and the activated human notch-1 coding sequence called ICN1 (5, 6) followed by the simian virus 40 (SV40) polyadenylation signal sequences. Tail DNA was genotyped using PCR oligodeoxynucleotide (ODN) primers specific for notch-1 and SV40 sequences (Table 1). Three founder lines (707, 709, and 712) were generated, two of which were fertile and were bred onto an FVB background for more than six generations. Transgenic mice were compared with FVB wild-type littermates as negative controls. RT-PCR was performed on RNA prepared from the right lungs of newborn mice to confirm transgene expression. The left lung was fixed with 4% paraformaldehyde and paraffin-embedded for immunohistochemistry of epithelial cell markers.


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  		Table 1. Oligonucleotide sequences

 
In situ hybridization. Nonisotopic in situ hybridization was carried out to localize mRNA in lung tissue sections, as described previously (51). Briefly, lungs were fixed for 3–4 h in 4% paraformaldehyde and then embedded in paraffin. Five-micrometer sections were placed on Fisher Plus slides. NotchCal transgene sequences were amplified by PCR and subcloned into the TA cloning vector. The PCR primers were designed to include SV40 polyadenylation signal sequences together with the T7 or T3 promoter sequences (underlined) as follows: T7-notch-1, 5'-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGA GTC CTT GGG GTC TTC TAC CTT TC-3'; and T3-sv40, 5'-AAG CGC GCA ATT AAC CCT CAC TAA AGG GAA CAA AAG CTG GGT CCT ATG GAA CTG ATG AAT GGG AGC-3'. The cRNA probes were labeled with biotin-14-dCTP according to the manufacturer's specifications (Invitrogen, Carlsbad, CA). Frozen sections were incubated with proteinase K (4 µg/ml) and then hybridized at 42°C overnight with biotin-14-labeled cRNA (0.25 µg/ml hybridization buffer). After washing, slides were developed with streptavidin-alkaline phosphatase conjugate in blocking buffer (100 mg/ml of blocking reagent) and then with nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate solution (Roche Molecular Biochemical, Mannheim, Germany). Sections were counterstained with 2% aqueous methyl green, and coverslips were applied using aqueous mounting medium.

Cell cultures. NCI-H82 and NCI-H526 cells were obtained from American Type Culture Collection. These are "variant" SCLC cell lines that lack NE-specific cell markers and demonstrate high levels of c-myc mRNA, consistent with an undifferentiated cell phenotype. All cell lines were maintained in RPMI 1640 medium supplemented with 2.5% fetal calf serum, hydrocortisone-insulin-transferrin-estradiol-selenium (HITES), 2% L-glutamine, and 10 ml of penicillin-streptomycin at 37°C in a humidified incubator in 5% CO2. Cells grown to high densities (~106 cells/ml) were stimulated with 400 U/ml TNF for varying times: 0, 5, 10, 30, 60, or 120 min, or 24 h. Total RNA was prepared from cultured cells using TRI reagent according to the manufacturer's instructions. cDNA was prepared using the SuperScript first-strand synthesis system. One microliter of this reaction was used for each PCR.

RT-PCR. Comparisons of relative levels of gene expression were made by using either semiquantitative or quantitative real-time RT-PCR (QRT-PCR). The sequences of oligonucleotide primers used for RT-PCR are given in Table 1.

Semiquantitative RT-PCR was carried out as described in detail previously (22). The number of cycles was determined for each primer pair such that RNA detection was in the midlinear segment of the curve when specific amounts of positive control RNA were used at 0.05–10 µg total RNA in the RT reaction.

QRT-PCR was carried out with an intercalating fluorescent dye (SYBR Green I) in ABI Prism 7700 Thermocycler. Differences in the number of PCR cycles required to detect gene expression were evaluated using a threshold of fluorescent activity set above background and occurring during the exponential phase of the reaction. Normalization for variation in cDNA loading was performed by the simultaneous amplification of GAPDH in each well. Primers used are given in Table 1. All quantitative PCR reactions were carried out at least in triplicate. Amplifications were performed in a final reaction volume of 50 µl containing cDNA templates, universal PCR Master Mix, and primers. Cycling conditions were 40 cycles of 95°C for 15 s and 60°C for 1 min.

Phosphorothioate ODN treatment. Phosphorothioate-modified antisense ODN (AS-ODN) were 22 nucleotides in length. Notch-1 AS-ODN has the sequence of 5'-GGTTGTCAATCTCCAGGTAGAC-3'. No significant homology of the sequence was found with any other genes by a BLAST search of the GenBank database. The corresponding sense phosphorothioate ODN was used in parallel cultures as a negative control for the Notch-1 AS-ODN. All ODN were synthesized and purified by MWG Biotech. ODN (1 µM) were incubated with H82 and H526 cell lines in RPMI 1640 medium. This medium was changed daily with fresh ODN added. Cytospins of the cultured cells were prepared using 5 x 105 cells per slide. Immunostaining included at least 10 cell clusters (over 200 cells) for each marker. Experiments were done in triplicate for both cell lines and evaluated in a blinded fashion.

Western blot analyses. Protein lysates were prepared from cultured cells, and Western blots were carried out as described previously (13). In brief, 40 µg of each protein was loaded in each well of a precast polyacrylamide gel, which was then electroblotted onto nitrocellulose and baked before incubation with a rabbit antiserum to activated notch (cat. no. 2,421; Cell Signaling Technology, Beverly, MA). Positive bands were detected using chemiluminescence according to standard protocols. Blots were reprobed with an antibody to beta-actin (cat. no. ab1801; AbCam, Cambridge, MA) as a positive control to normalize for protein loading.

Immunoperoxidase analyses. Newborn mice were harvested after CO2 asphyxiation as described. All of the animal studies were approved by the Independent Animal Care and Use Committee at Children's Hospital in Boston and also at Duke University. Lungs were fixed for 18–24 h in 4% paraformaldehyde before being processed into paraffin, as described previously. Three-micrometer paraffin sections were prepared on Fisher Plus slides. Immunostaining for protein gene product 9.5 (PGP9.5) was carried out using the avidin-biotin complex immunoperoxidase technique, with diaminobenzidine as substrate and methyl green as counterstain. The PGP9.5-monoclonal antibody was used at 1:2,000 dilution (Novacastra Lab, Newcastle, UK). Horse anti-mouse IgG was used as the secondary antibody at 1:250 dilution. NCAM monoclonal antibody (Dako, Carpinteria, CA) was used at 1:10 dilution, followed by horse anti-mouse IgG secondary antibody at 1:200.

Statistical analyses. Numerical data were analyzed using the unpaired Student's t-test. Results are expressed as mean group values ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NE differentiation in SCLC cells. To verify that the current passages of H82 and H526 cell lines used for these experiments had the same functional responses to TNF treatment as previously (19, 22), we used semiquantitative RT-PCR. In the lung, central and peripheral nervous systems, and many other organs, DOPA decarboxylase (DDC, also known as L-amino acid decarboxylase) is neural/NE-specific (8, 9, 16, 22, 42, 44), although it has also been described in Drosophila epidermis (36) and gastric mucosa, the latter likely to originate in gut endocrine cells and/or neurons (21). As shown in Fig. 1, DDC mRNA is induced in H82 cells within 5 min after adding TNF, peaks at 30–120 min, and gradually declines to baseline levels by 2 wk (data not shown). TNF-treated H526 cells do not express the DDC gene under the same experimental conditions (Fig. 1).


Figure 1
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  		Fig. 1. Notch family gene expression in TNF-{alpha} (TNF)-treated H82 and H526 cells. Semiquantitative RT-PCR was used to compare gene expression in H82 (A) and H526 (B) cells after adding TNF (400 U/ml) at 0, 5, 10, 30, 60, and 120 min. A: compared with untreated H82 cells (time 0), Notch-1 mRNA is decreased in TNF-treated H82 in parallel with increased dopa decarboxylase (DDC) mRNA. B: H526 cells had no change in Notch-1 or DDC mRNA after treatment with TNF.

 
In parallel, TNF treatment of H82 cells decreases notch-1 gene expression within 5 min, with undetectable notch-1 mRNA levels after 10 min (Fig. 1). By QRT-PCR, H82 demonstrates a similar significant decline in hes1 mRNA levels by 40% after 5–10 min of TNF treatment that is sustained up to 60 min (the last test point, data not shown). However, there is no significant change in gene expression for Notch-2, Notch-3, or Jagged-2 (unpublished data). In addition, Notch-4 and Jagged-1 mRNAs are modestly (<2-fold) increased, peaking at 120 min (unpublished data). These observations suggest that TNF could potentially sustain NE cell differentiation of H82 at least in part by downregulating notch-1 or hes1 gene expression.

In contrast, TNF treatment of H526 cells did not alter relative mRNA levels for Notch-1 (Fig. 1B), -2, -3, or -4, or Jagged-1 or -2 (unpublished data). TNF-treated H526 cells have a significant but slight (20–30%) increase in hes1 mRNA by QRT-PCR after 10–60 min (data not shown).

Notch signaling regulates NE gene expression in H82 cell line. To evaluate whether Notch-1 might alter NE-specific gene expression, H82 cells were treated with AS-ODN completely specific for notch-1 or the corresponding sense control as the negative control. At different time points, the cells were harvested, and mRNA levels for GRP, another NE cell marker known to be upregulated by TNF, were measured using QRT-PCR. After 2 h of treatment with notch-1 AS-ODN, GRP gene expression became detectable and reached peak levels after 6 h (Fig. 2).


Figure 2
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  		Fig. 2. Gastrin-releasing peptide (GRP) mRNA in H82 cells treated with Notch-1 antisense oligodeoxynucleotides (AS-ODN). GRP mRNA was assessed by semiquantitative RT-PCR (A) and quantified by quantitative real-time RT-PCR (QRT-PCR) (B) in H82 cells treated with notch-1 AS or sense ODN (1 µM each). A: a representative RT-PCR experiment demonstrates increased GRP mRNA as early as 1 h after adding notch-1 AS-ODN. Peak GRP mRNA levels occur 6–12 h later. There is no GRP mRNA in parallel sense-treated H82 cultures. beta-actin mRNA for Notch-1 AS-ODN-treated cells is shown; beta-actin for sense-treated H82 was essentially the same (unpublished data). B: pooled results from 3 QRT-PCR experiments are shown. Two hours after adding notch-1 antisense, GRP mRNA (normalized for relative amounts of GAPDH mRNA) was increased, with peak levels occurring 6–12 h later and then returning to undetectable levels by 48 h. Control H82 cells were age-matched cells from the same passage but treated with notch-1 sense. These sense-treated H82 cells had no decrease in notch-1 mRNA and no induction of GRP gene expression. *P < 0.01 and **P < 0.05 compared with time 0.

 
To confirm and extend these results, expression of Notch-1 and DDC, another NE-specific gene, were evaluated using QRT-PCR (Fig. 3). DDC mRNA was detected 2 h after adding Notch-1 AS-ODN and peaked at 6 h. Neither GRP nor DDC gene expression were detected in Notch-1-sense-treated H82 cells (Fig. 2 and unpublished data). To verify that the classic Notch signaling pathway was implicated in the induction of H82 NE differentiation, we also evaluated relative mRNA levels for Hes1, another neurogenic signaling molecule that functions downstream from Notch. As shown in Fig. 3, hes1 mRNA levels began to decline at 2 h, in parallel with notch-1, with reduced hes1 gene expression present at 6 and 24 h (Fig. 2). The reduced hes1 mRNA level at 6 h was statistically significant (P < 0.05).


Figure 3
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  		Fig. 3. Changes in notch-1, hes1, and DDC mRNAs in H82 and H526 cells treated with Notch-1 AS-ODN. QRT-PCR was used to quantify mRNA levels for notch-1, hes1, and DDC in H82 cells (A) and H526 cells (B) treated with notch-1 AS or sense ODN (1 µM each). The values in AS-treated cells were normalized by subtracting values in the corresponding sense-treated cultures and then defining the sense-treated control value at time 0 as 0 (that is, no change from itself as baseline). Changes are expressed with respect to this baseline as percentages above (for increases) or below (negative values for decreases). A: in H82 cells, DDC mRNA levels increase as notch-1 and hes1 mRNA levels decline. {dagger}P = 0.066, *P < 0.006, **P < 0.04 (all compared with time 0). B: in H526 cells, there is a decrease in notch-1 mRNA at 24 h, but hes1 mRNA levels are unchanged. DDC levels are doubled at 6 and 24 h, but the baseline DDC levels in H526 are significantly lower than those in H82 by a factor of ~1/103. *P < 0. 006.

 
In contrast, in H526 notch-1 AS-ODN led to a small decline in notch-1 mRNA later, at 24 h, but without any significant change in hes1 mRNA (Fig. 3). The late increase in DDC mRNA at 6 and 24 h may be due to spontaneous differentiation of these SCLC cells in culture, but these DDC levels were still less than ~1/103 of the baseline levels for H82 (unpublished data).

To confirm that decreased notch-1 mRNA levels lead to a drop in protein levels for activated Notch-1, we carried out Western blots, normalized for beta-actin. Representative results from one of two experiments are given in Fig. 4. We detected activated Notch-1 2 h after adding notch-1 AS-ODN (Fig. 4, A and bottom), whereas no decrease was observed with the notch-1 sense control. Interestingly, levels of activated Notch-1 were also reduced for H526 treated with notch-1 AS-ODN, but not H526 treated with the sense control (Fig. 4, B and bottom).


Figure 4
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  		Fig. 4. Changes in activated Notch-1 protein levels in H82 and H526 cells treated with Notch-1 AS-ODN. Western blot analyses were carried out using H82 cells (A) and H526 cells (B) treated with notch-1 AS or sense ODN (1 µM each). Cultured cells were harvested at time 0 and after 30, 60, and 120 min, or 4–8 h of culture. Western blots were reprobed with an antibody to beta-actin to correct for protein-loading fluctuations. The band areas were quantified by densitometry, and then activated Notch-1 protein levels were normalized by dividing by the corresponding actin band measurement. Bottom: over the culture period, activated Notch-1 was markedly diminished as early as 2 h after adding notch-1 AS but not by notch-1 sense in both H82 and H526.

 
After 2 wk of treatment in vitro, cultured H82 and H526 cell lines were harvested for immunohistochemical detection of NCAM, a neural/NE-specific marker that we previously demonstrated to be induced in H82 by TNF (22). Compared with untreated controls (Fig. 5, A and C), treatment of H82 with TNF or notch-1 AS-ODN increased NCAM staining (Fig. 5, B and D, respectively). In contrast, in H526, there was no effect of either TNF or notch-1 AS-ODN on NCAM immunostaining compared with the matched negative controls (unpublished data).


Figure 5
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  		Fig. 5. Neural cell adhesion molecule (NCAM) immunostaining in H82 cells treated with TNF or Notch-1 antisense. H82 cells were treated for 2 wk with media alone (A), TNF (B), notch-1 sense ODN (C), or notch-1 antisense ODN (D). After 2 wk of culture, cells were used to prepare cytospins on glass slides, and NCAM immunostaining was performed. Bar, 50 µm.

 
Altered PNECs in Notch-1-overexpressing transgenic mice. Transgenic mice were generated on an FVB background with the calcitonin/CGRP-promoter driving a well-characterized, activated human Notch-1 construct that lacks the transmembrane domain, termed ICN1 (6). The resultant "NotchCal" mice had apparently normal growth and development by routine histopathological examination (data not shown). To verify transgene expression in the lung, RT-PCR showed that NotchCal was consistently expressed in the lungs of the newborn transgenic mice (unpublished data). To localize NotchCal expression in the lung, in situ hybridization was carried out using the SV40 polyadenylation sequences as a cRNA probe, which is entirely transgene-specific (57). Using the antisense cRNA probe, NotchCal mRNA was localized to the airway epithelium of transgenic mice (Fig. 6A), compared with no signal in sections probed with the sense control cRNA (Fig. 6B).


Figure 6
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  		Fig. 6. NotchCal transgene expression and Notch signaling in newborn mouse lung. A: NotchCal mRNAs are localized to airway epithelium (a few representative areas are indicated by long arrows) using the AS cRNA probe. B: a serial section probed with the sense cRNA shows no hybridization signal. Bar, 50 µm; L, airway lumen; V, blood vessel. C: QRT-PCR of newborn mouse lung shows hes1 mRNA levels are significantly elevated in NotchCal transgenics (n = 3) compared with wild-type (WT) littermates (n = 3). *P < 0.05.

 
To verify that the NotchCal mice are overexpressing other members of the Notch signaling pathway, we carried out QRT-PCR for hes1, normalized for beta-actin. As shown in Fig. 6C, hes1 mRNA levels in newborn lung are over 20-fold higher in NotchCal pups compared with wild-type controls.

Finally, we assessed relative numbers of PNECs by immunostaining for PGP9.5 (Fig. 7, A–C) and CGRP (Fig. 7D). As shown in Fig. 7A, newborn wild-type littermates (1 day after birth) have easily detectable PGP9.5-positive cell clusters in the airway epithelium (short arrow) as well as prominent peribronchiolar nerve fibers (long arrows). In contrast, there are very few PGP9.5-positive NE cells in airways of NotchCal mice. One weakly stained PNEC is indicated by a short arrow in Fig. 7B. However, there are still abundant strongly PGP9.5-positive nerve fibers in NotchCal lungs (Fig. 7B, long arrows).


Figure 7
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  		Fig. 7. Immunostaining for pulmonary neuroendocrine cells (PNECs) in NotchCal transgenic mice. A: WT littermate control has numerous protein gene product 9.5-positive (PGP9.5+) PNECs present in airway epithelium of the lung at 1 day after birth. Small arrow indicates a small cluster of PGP9.5+ cells. Long arrows indicate peribronchiolar PGP9.5+ nerve fibers. Bar, 25 µm. B: in contrast, lungs of newborn NotchCal littermates have only rare NE cells present. Small arrow indicates one weakly PGP9.5+ epithelial cell. Long arrows indicate peribronchiolar PGP9.5+ nerve fibers. Bar, 25 µm. C and D: morphometric analyses of numbers of PGP9.5+ (C) and CGRP+ (D) PNECs in WT and NotchCal transgenic mice. The data are expressed as numbers of PGP9.5+ or CGRP+ cells per focus, numbers of PGP9.5+ or CGRP+ foci per square inch of lung tissue, and numbers of PGP9.5+ or CGRP+ cells per square inch of lung tissue. *P < 0.004, **P < 0.01, and {dagger}P < 0.0001 comparing WT and transgenic mice.

 
Morphometric analyses were carried out to evaluate the overall prevalence of PNECs in NotchCal newborns compared with wild-type littermates. The results of these analyses are given in Fig. 7, C and D. As shown in Fig. 7C, NotchCal mice have significantly fewer PGP9.5-positive cells per focus/cluster (P < 0.004), fewer PGP9.5-positive foci per square inch (P < 0.0001), and fewer PGP9.5-positive cells per square inch (P < 0.0001) (chosen for normalization because NE cells can appear in alveolar ducts in newborn mice). Essentially, the same results were obtained for CGRP-positive cells (Fig. 7D).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that Notch-1 downregulation can promote NE differentiation in an undifferentiated SCLC cell line and in developing normal PNECs. TNF-treated H82 cells demonstrate decreased notch-1 gene expression in parallel with induction of DDC mRNAs, whereas TNF-treated H526 cells have no change in notch-1 or DDC mRNAs. We (22) previously showed that the immediate induction of DDC and GRP mRNAs by TNF appears to be linked to NF-{kappa}B activation. We became interested in whether TNF could also promote NE differentiation by reducing Notch gene expression. We (22) hypothesized that decreased Notch signaling could be one mechanism contributing to the sustained increase in DDC and GRP gene expression and the ultimate NE phenotypic induction after 7–14 days.

Our system was originally developed using two SCLC cell lines, H82 and H526, both of which are undifferentiated and are classified as "variant" SCLC cell lines (15, 19). At baseline, these cell lines have no detectable NCAM immunoreactivity or mRNA for the NE markers DDC and GRP (22). Similar to retinoic acid-induced NE differentiation of H82 (46), we demonstrated that TNF treatment of H82 can induce mRNAs for the NE markers GRP and DDC within 5–10 min and NE phenotypic features after 7–14 days in culture.

The notch gene encodes a receptor protein that is involved in many cellular processes during development. Best understood is its role in neurogenesis, whereby Notch inhibits neural/NE differentiation in neighboring cells. Using retrovirus-mediated gene expression in the embryonic chick retina, it was shown that progenitor cells exposed to the Notch ligand Delta-1 were blocked from neuronal differentiation. A converse effect was seen in cells expressing a dominant-negative form of Delta-1. Thus Delta-Notch signaling controls a cell's choice between remaining as a progenitor and differentiating as a neuron (23). Similar effects have been observed with altered Notch gene expression in the chick retina (7). Cumulative evidence indicates that repressive bHLH factors such as Hes1 are also regulated by Notch signaling (29, 30, 49).

In the present study, we demonstrate that notch-1 gene "knockdown" with AS-ODN is sufficient to induce NE cell differentiation in H82 cells, similar to TNF treatment of H82 (61). Neither H82 nor H526 expresses NE phenotypic markers at baseline. In contrast to H82, H526 cannot be induced to differentiate into a NE phenotype by either TNF or retinoic acid, suggesting that H526 could be lacking a functional downstream mediator of Notch signaling (27, 54). This is also suggested by the presence of decreased activated Notch-1 protein with Notch-1 AS-ODN treatment of both H82 and H526.

It should be noted that TNF-treated H82 cells have ~6-fold increased DDC mRNA 2 h after adding TNF by QRT-PCR, compared with untreated H82 (data not shown), whereas Notch antisense-treated H82 has ~2-fold increased DDC mRNA (Fig. 3). In comparison, well-differentiated SCLC or carcinoid cell lines, such as H345, H69, or H720, have DDC levels that are over 150-fold greater than untreated H82 (20). These data suggest that we are inducing just the beginning of NE cell differentiation as determined by several known molecular markers (20). It should also be noted that even within the same tumor there is marked variation in DDC levels from one cell to another, and DDC levels are much higher in tumor cell lines than in the original tumor (8).

Decreased PNECs in NotchCal transgenic mice overexpressing activated Notch-1 suggests that Notch can block NE cell differentiation in normal lung development and not just in neoplastic cells. Thus Notch-1 expression in epithelial precursor cells would inhibit NE differentiation and downregulate calcitonin/CGRP promoter activity, effectively leading to a negative feedback loop in this system. However, we cannot rule out the possibility that supraphysiological Notch activity in the target cells could be proapoptotic (11) or growth inhibitory (59) rather than acting purely on differentiation. Activation of Notch is known to repress the achaete-scute genes, which regulate transcription of Delta (50). In our Notch-1 transgenic mice, the transgene was detected in many airway epithelial lining cells, especially in smaller airways. The 1.5-kb fragment of the calcitonin/CGRP promoter that we used has been shown to be expressed at ~100 times higher levels in NE cells compared with non-NE cells (32, 55). The current data suggest that progenitor cells expressing the transgene at low levels might be blocked from NE cell differentiation, leading to their widespread distribution throughout the non-NE airway epithelium. We cannot rule out the possibility that the transgene might be "leaky." However, we (32) did previously demonstrate that the same promoter is expressed in undifferentiated epithelial cells in embryonic lung and differentiated PNECs after birth. The presence of a few PGP9.5+ CGRP+ cells suggests that some CGRP+ cells fail to express the transgene during development. Our data together with that of Ito et al. (27) strongly suggest that notch gene expression is an important regulator of PNEC differentiation. Finally, it should be noted that even in the absence of Hes1, there remain some fetal airway epithelial cells that do not differentiate into NE cells (27). These observations suggest that there is no single master regulator of NE cell differentiation, not even Hes1 (27). Although thyroidal C cells are derived from the neural crest, similar mechanisms involving Notch signaling pathways appear to regulate NE differentiation of these cells in both the developing thyroid and in medullary carcinomas of the thyroid (38, 53).

In conclusion, our observations confirm that Notch signaling plays a pivotal role in PNEC differentiation in lung development and in lung tumors. The ability of Notch downregulation to induce NE-specific gene expression and NE phenotypic features can occur both in neoplastic cells and in normal lung development. It is speculated that the ability of some SCLCs to differentiate into tumors with non-SCLC phenotypic features (33), or, conversely, the expression of NE markers in non-SCLC tumors (14), could depend in part on the function of activated Notch signaling, although the significance of NE differentiation of non-SCLCs remains controversial (14, 24, 58).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-44984.


   ACKNOWLEDGMENTS
 
We thank Dr. Rodica Emanuel for excellent technical assistance. Present address of J. Sklar: Department of Pathology, Yale University Medical Center, New Haven, CT.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. E. Sunday, Dept. of Pathology, Duke Univ. Medical Center, Research Drive, Carl 0043, Durham, NC 27710 (e-mail: mary.sunday{at}duke.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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