A rhesus monkey model to characterize the role
of gastrin-releasing peptide (GRP) in lung
development. Evidence for stimulation of
airway growth.
K Li, … , S R Nagalla, E R Spindel
J Clin Invest.
1994;
94(4)
:1605-1615.
https://doi.org/10.1172/JCI117502
.
Gastrin-releasing peptide (GRP) is developmentally expressed in human fetal lung and is a
growth factor for normal and neoplastic lung but its role in normal lung development has yet
to be clearly defined. In this study we have characterized the expression of GRP and its
receptor in fetal rhesus monkey lung and determined the effects of bombesin on fetal lung
development in vitro. By RNA blot analysis, GRP mRNA was first detectable in fetal monkey
lung at 63 days gestation, reached highest levels at 80 days gestation, and then declined to
near adult levels by 120 days gestation; a pattern closely paralleling GRP expression in
human fetal lung. As in human lung, in situ hybridization localized GRP mRNA to
neuroendocrine cells though during the canalicular phase of development (between 63-80
days gestation) GRP mRNA was present not only in classic pulmonary neuroendocrine
cells, but also in cells of budding airways. Immunohistochemistry showed that
bombesin-like immunoreactivity was present in neuroendocrine cells, but not in budding airways,
suggesting that in budding airways either the GRP mRNA is not translated, is rapidly
secreted, or a related, but different RNA is present. RNase protection analysis using a probe
to the monkey GRP receptor demonstrated that the time course of receptor RNA expression
closely paralleled the time course of GRP RNA expression. In situ hybridization […]
Research Article
A Rhesus Monkey Model
to
Characterize the Role of
Gastrin-releasing Peptide
(GRP)
in
Lung Development
Evidence for Stimulation of Airway Growth
Kang Li,Srinivasa R. Nagalla, and Eliot R. Spindel
Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon97006
Abstract
Gastrin-releasing peptide (GRP) is developmentally ex-pressed in humanfetallung and isagrowthfactor for nor-mal and neoplastic lung but its role in nornor-mal lung
develop-ment has yet to be clearly defined. In this studywe have
characterizedtheexpression of GRPand its receptor infetal
rhesusmonkey lung anddetermined theeffectsof bombesin on fetal lung development in vitro. By RNA blot analysis,
GRP mRNAwasfirst detectable in fetalmonkey lungat63 days gestation, reachedhighest levels at80 days gestation,
and thendeclinedto nearadult levelsby120daysgestation;
apatterncloselyparalleling GRPexpressionin human fetal lung. As in human lung, in situhybridizationlocalized GRP mRNA toneuroendocrinecellsthoughduring the canalicu-lar phaseofdevelopment (between 63-80days gestation) GRPmRNAwaspresentnotonlyinclassicpulmonary neu-roendocrine cells, but also in cellsofbuddingairways.
Im-munohistochemistryshowed thatbombesin-like
immunore-activity
was present inneuroendocrinecells, butnotinbud-ding airways, suggestingthat inbudding airways eitherthe GRP mRNA is nottranslated,is rapidlysecreted, or a re-lated, butdifferentRNAis present. RNaseprotection
analy-sisusingaprobetothemonkey GRPreceptor demonstrated that the time course of receptor RNA expression closely paralleled the timecourseof GRP RNAexpression.In situ
hybridization showed that GRP receptors were primarily
expressed inepithelialcellsofthedeveloping airways.Thus
GRPwould appeartobe secretedfrom neuroendocrinecells to act ontargetcells indevelopingairways.This hypothesis wasconfirmedby organ cultureof fetalmonkey lung in the presenceofbombesin and bombesin antagonists. Bombesin treatment at1and 10 nMsignificantlyincreased DNA syn-thesis in airway epithelial cells and significantly increased the number and size ofairways in cultured fetal lung. In
fact, culturing60 d fetallungfor 5 d with 10 nM bombesin increased airway size and number nearly to that observed in cultured 80d fetal lung. The effects of bombesin could be blocked byspecific GRPreceptor antagonists. Thus this study demonstrates that GRP receptors are expressed on
Address correspondence to Eliot R. Spindel, Division of Neuroscience,
Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006.
Received for publication 22September 1993 and in revised form 27 June 1994.
airwayepithelialcells indevelopingfetal lungand that the interaction of GRP with the GRP receptor stimulates airway development. (J. Clin. Invest. 1994.
94:1605-1615.)
Keywords: bombesin * fetal lung *growth factors*airway
Introduction
Bombesin isatetradecapeptide initially purifiedfrom the skin of thefrog Bombina bombina in 1971 (1). One mammalian homolog of bombesin is the 27-amino acid peptide,
gastrin-releasing peptide
(GRP),'
initially isolated fromporcine stom-achby McDonaldetal. in 1979(2). 9 of the 10 COOH-terminal amino acids of bombesin and GRP are identical, and this COOH-terminal decapeptide(designated GRP-10) (3) appears to contain full receptor-binding and biological activities. Bombesin and GRP havenearlyidenticalpotenciesandphysio-logic effects(4-6), and thus amphibian bombesin isfrequently
usedtostudy the effects of GRP.
GRP is widely distributed in brain, gastrointestinal tract, and lung, and functions as a neurotransmitter, paracrine hor-mone, andgrowth factor (7-9). In the lung, GRP is located in the pulmonary neuroendocrine cells (PNEC) (10), and high levels are found in human fetal and neonatal lung (11-14), and in small celllungcarcinoma(SCLC) (15, 16). Peak GRP mRNAexpression occurs from 14to30 wkgestation and while GRPpeptide and mRNA consistently colocalize in neuroendo-crine cells inearlygestation, by lategestation, many cells that contain GRPpeptidenolonger contain GRPmRNA (17).
GRP and bombesin havebeen showntostimulate the clonal
growth of normal bronchial epithelial cells and the growth of SCLC cells both in vitro and in vivo(6, 18-20). Cuttittaetal.
(18)have demonstrated that antibodiestobombesincaninhibit thegrowth of SCLC lines in vitro and in vivo and have sug-gested that GRP is an autocrine growth factor for SCLC. The ability of GRPtostimulate the growth of SCLC cell lines corre-lates with theexpression of the GRP-preferring bombesin recep-torsubtype.The GRP receptor has been cloned and shown to
belong to the seven-membrane spanning domain superfamily
(21-23). Specific antagonists of the GRP receptor (GRP-R) have beendeveloped (24) andcaninhibit growth of SCLC cell lines that express GRP receptors (25, 26). Although the pres-enceof GRP-R in SCLC is well documented, the location, time course andexpression of GRP-Rin normal fetal lung has yet tobeclearly demonstrated.
The observation of peak expression of GRP mRNA in hu-man fetal lung at midgestation (17), combined with
growth-1. Abbreviations usedin thispaper: GRP, gastrin-releasing peptide;
GRP-R,GRP receptor;PNEC, pulmonaryneuroendocrinecells; SCLC,
small celllungcarcinoma.
J. Clin. Invest.
©The AmericanSociety forClinicalInvestigation, Inc.
0021-9738/94/10/1605/11 $2.00
promoting effects on some SCLC cell lines (18, 19, 27) and pulmonary epithelial cells (6, 20), are highly suggestive that GRPgene activation plays a role in normal fetal lunggrowth. In the mouse, Sunday et al. and King et al. (28, 29) have demonstrated that bombesincan stimulate DNA andsaturated phosphatidylcholine (SPC) synthesis in fetal lung. Similarly, Aguayoet al. (30)have alsodemonstrated that bombesin can stimulate airway branching in mouse lung explants. However, it is noteworthy that the GRP is expressed relatively later in gestationin mice thanin humans (17, 28), and that in contrast tohumans, GRP immunoreactivity cannotbelocalizedtomouse PNEC (31). Thusananimal model thatmoreclosely parallels human patterns offetal pulmonary GRP expression isneeded tocharacterize the role of GRPinhuman lung development.In this paperwe demonstrate thatthe pattern ofGRPexpression in fetalrhesus(Macaca mulatta) monkeylung closelyparallels that of human fetal lung and demonstrate that bombesin and GRP significantly increase both the number and size of small airways inorgancultures of fetal monkey lung.
Methods
Lung tissues were collected from fetal (50-154 d gestation), neonatal and adult rhesus monkeys. For mRNA measurement, tissues were frozen on liquid nitrogen and stored at -80'C. For immunohistochemistry,
tissue were fixed in Zamboni's fixative (1.5% paraformaldehyde and 1%picric acid)and for in situhybridization, tissueswerefixed in 4% paraformaldehyde in borate buffer, pH 9.5, (32).
Generationofspecificmonkey GRP and GRP receptor cRNA probes. PCR was used to generate specific monkey GRP and GRP-R cDNA probes. Primers correspondingtothebeginning and end of the human GRP exon-3(33)andGRP-R gene exon-3(23, 34)wereusedtoamplify
250-bp fragments of DNA from monkeygenomicDNA. Primers chosen foramplification were highly conserved between ratand human (34, 35)and thuswere likelyconserved between human and monkey. The
amplifiedDNA wassubclonedinto thevectorPCR 1000 (Invitrogen,
SanDiego, CA)andtheiridentityconfirmedbyDNA sequenceanalysis. 32P-labeled antisense GRP and GRP-R cRNA probes were prepared
by transcribing with T7 RNA polymerase as described by Melton
etal.(36).
mRNA determination. Total RNA wasprepared bythe method of
Chirgwinetal.(37) by homogenizationoflungtissue inguanidinium
thiocyanate followedby centrifugation throughCsCl. Total RNAwas
subjected to electrophoresis through 1.5%formaldehyde-agarose gels
and transferredtoDuralonnylonmembranes(Stratagene,LaJolla, CA) by capillary blotting,andUV-crosslinked(38).Gelswerestained with ethidium bromidetovisualize the 28S and 18Sribosomal RNA bands
to check fordegradationand recovery of RNA. Hybridization wasin
5x SSC; 5X Denhardt's solution (lx Denhardt's solution = 0.02% Ficoll-400, 0.02% bovineserumalbumin,0.02% polyvinylpyrrolidone-40); 50% formamide; 50 mM sodiumphosphate, pH 7.0;0.2% SDS andsonicated, denaturedsalmon sperm DNA(200
Itg/ml)
at65°Cfor 18 h. Membraneswere washed for 2 x 15 min in 2xSSC/0.1%SDSatroom temperature followedby 2 X 15 min washes in O.1x SSC/ 0.1% SDS at 65°C, andexposedfor 24-72 hat -85°Cwith Kodak
XARfilms withintensifying screens.
RNaseprotection assay. RNaseprotection analysisof theexpression
of GRP-R mRNA in rhesusmonkey lungwasperformedasdescribed by Ausubeletal.(38).Total RNAs from fetal and adultmonkey lungs
werehybridizedwithgel purified, 32P-labeled GRP-R cRNAprobein
hybridizationbuffer(40mMPipes, pH 6.4;0.4MNaCl;1 mMEDTA;
80%formamide)at45°Cfor18h, digestedwith 40rg/mlribonuclease
Aand 2
/sg/ml
ribonucleaseT1 at37°Cfor 1 h, phenol extractedand ethanolprecipitated.Sampleweresubjectedtoelectrophoresis on1.5-mm-thick 5% urea-acrylamide gels, the gels dried at 80'C for 45 min andexposed for 16 h at-850Cwith Kodak XAR films with intensifying screens.
Immunohistochemistryand antibody preparation. The monkey GRP prohormone exists in two forms which both identically encode GRP, as we have previously described for human GRP (39, 40). The sequence
of the monkey GRP prohormoneswere derived from cDNA cloning
and will be fully described elsewhere (note, monkey GRP is identical insequence to human GRP). Two synthetic peptides corresponding to the COOH-terminal regions of the monkey GRP prohormones were
synthesized and used as immunogens for antiserum production. The synthetic peptides corresponding to monkey prohormones type 1 and 2 wereMCI: Tyr-Lys-Asp-Leu-Val-Asp-Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Gln-Leu-Asn-Gln-Gln and MC2:
Tyr-Asn- Pro-Pro-Ala-Glu-Pro-Ala-Met-Thr-Met-Met-Ala-Ser-Leu-Lys-Gly-Glu-Lys-Gln-Asn-Pro.Thepeptides wereconjugatedtoKeyhole Limpet Hemocyanin using N-ethyl-N'(3-dimethylaminopropyl) carbodiimide
(Calbiochem-Behring,LaJolla,CA).NewZealand White rabbits were immunized subcutaneously with 100
pg
peptides using complete Freund's adjuvant in the initial injections and incomplete Freund'sadju-vantin the subsequent injections.
Immunohistochemistry wasperformed using monoclonal antibody
(2A1
1)
against amphibian bombesin that cross reacts equally with GRP ( 18) and with the antisera against the two synthetic monkey GRPpro-hormoneCOOH-terminal peptides (MCI and MC2). Bound antibody
was visualized with theavidin-biotincomplex (ABC) (Vector Labora-tories, Inc.,Burlingame, CA) immunoperoxidase techniqueusing diami-nobenzadineasthesubstrate as described by Watts and Swanson (41). Controlsconsisted of nonimmune rabbit serum, or anti-bombesin
antise-rum(2A1
1)
preadsorbedwithbombesin (10qg/ml),
orGRPprohor-moneantisera adsorbed withMCIorMC2. Nopositivereactions were detected in anyofthecontrols.Combinedin situ hybridization-immu-nohistochemistry was performed as described by Watts and Swanson
(41)asmodifiedbyLorangetal.(42). After immunohistochemically-processed sections weredeveloped withdiaminobenzadine, they were rinsed four times in .02 M potassium-phosphate-buffered saline
(KPBS),pH 7.4, driedovernightinavacuum desiccator thensubjected
toin situhybridizationasdescribed below.
Serial sections of some monkey fetal lung (60, 80, and 125 day gestation) were also stained forchromograninA and neuronspecific
enolasebythe ABC immunoperoxidase technique.
In situ hybridization. In situ hybridization was performed as de-scribedbyAngereretal.(43)and Lorangetal. (42).Inbrief,tissues
werefixedin 4%paraformaldehydefor 6-8h, and transferredto 15% sucrose in0.1MPBS for 16 hat4°C.Thetissueswerethen embedded in Tissue-TEK O.C.T. compound (Miles, Elkhart, IN) and frozen in
powdered dry ice,and storedat-80°C. Tissue sectionswere cut on a
cryostat at 5-10
tim,
placed onto poly-L-lysine and gelatine coatedslides,anddriedovernightundervacuum. Slideswererefixed with 4%
paraformaldehydefor 15 min, washed2xwith0.1 MPBS,treated with 2
qg/ml
proteinase K, 0.1 M triethanolamine, and 2.5ttl/ml
aceticanhydride. Hybridization was carriedout with monkey GRP receptor cRNAprobe preparedasdescribed above.35S-labeledprobe (10 x 106 cpm)wasappliedtoeach slide ina volume of 70
1.d
ofhybridizationbuffer(60% formamide, 12% dextransulfate,0.25 MNaCl, 2x
Den-hardt' solution, 1 mMEDTA,20mMDTT,0.5 mg/ml tRNA,and 10 mMTris, pH 8.0). Coverslipswereappliedand slides wereincubated inamoist closedchamberat55°Cfor 20-24 h. Slideswerewashed in
4x SSC for 5 min x 4,treated with 20
jtg/ml
ribonucleaseAtodigestany remaining unhybridized probes, and then washed sequentially at
roomtemperature in2x, lx and 0.5x SSC 10 min for eachwash,and in 0.1x SSC at 65°C for 30 min. Sections were then dehydrated in
ethanol, air-dried,anddippedinKodak NTB-2autoradiographic emul-sion. Slides were exposed for 7-14d, developed and slides not
pre-viously processedforimmunohistochemistry werecounterstained with
hematoxylin.
>1
'Al >4 >% Co
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30 40 50 75 78 88 Percent ofgestation
organculture methodwasusedasdescribedby Jaskolletal.(44).Fetal
lungs from 60-, 70-,and80-d-gestationalrhesusmonkeyswereexcised
onice andcutinto1mm3pieces using aseptic technique. Sectionswere
takenonly from peripheral partsoflungtoavoidlarge central airways and blood vessels. This also served to minimize chances of biasing resultsby having unequal distribution of centrallyandperipherally de-rived sections inexperimental groups.Thelung explants wereplaced onTranswellmicroporousmembrane inserts in 6 well culture clusters
(Costar, Cambridge, MA) and cultured in a serum free, chemically defined medium (Fitton-Jackson modification BGJb, GIBCO BRL,
GrandIsland, NY) containing100U/,ig penicillin/streptomycinin5%
C02/airat37TC. Tostudy the effects of bombesin and itsantagonists
onthegrowth of monkey fetal lunginvitro, lungs wereincubatedin
thepresenceorabsenceofbombesin(1and 10nM);theGRPreceptor
antagonists, D-Phe6 bombesin (6-13) propylamide (BIM-26147) (45)
or D-fluorylamide-Phe 6-bombesin(6- 13)-methylester (BIM-26226) (46).Peptideswerepresentthoughtheentire cultureperiodand media + peptides werechanged dailytolimitpeptide degradation. After 5 d inculture, fetal lung explantswere fixedin Zamboni'sfixative or4%
paraformaldehyde in borate buffer, pH 9.5, and then sectioned and
subjected tohematoxylin/eosin staining andor immunohistochemical staining and in situ hybridization. Image analysis was performed on
sectionsasdescribed below.
S-Bromo-2'-deoxy-uridine (BrdU) labeling. BrdU-labeling was
used to measure the effects of bombesin and its antagonist on DNA
synthesis and cell proliferation. BrdU-labeling was performed using
reagents andprotocols supplied by Amersham (Cell Proliferation Kit, AmershamCorp., Arlington Heights, IL). Fetal lung froma
60-d-gesta-tionrhesusmonkeywascultured in BGJb medium either withorwithout
addedbombesin (10 nM) in 5% C02/airat37TCfor 48 h, then pulse-labeledwithBrdU(final dilution= 1:1,000) for 60 min. After labeling,
tissues werewashed in PBS for 15 min,fixed in Zamboni's fixative,
andprocessed for frozen sectioning. BrdU labelingwasvisualized by immunohistochemistry usingaspecific BrdU monoclonal antibody and
sectionscounterstainedwith0.25% thionin.
Morphologicandstatistical analysis. The Optimas Bioscan image analysisprogram(v.4.0)wasusedtoquantifyairway size and number and tocountBrdU-labeled cells in cultured lung. For eachtreatment,
atleast ten fields (field area = 2 mm2) were analyzed. Within each treatment,fields countedwerechosenatrandom from multiple sections,
Figure 1.Analysis of GRPmRNA
ex-pressioninfetal and adult monkey lung. (A) Northern blot analysisof GRP
mRNAs. 10 jg of totalRNA from
tis-suesshownweresubjectedto
electro-phoresis and hybridized toamonkey GRPcRNAprobeasdescribed in
Meth-ods. (Lanes 1-6)Fetal monkey lung from thegestationalagesshown;(lane 7) adult monkey lung; (lane 8) 19-wk
humanfetallung.The mobilities of the
28 and 18 S ribosomal RNAsare as
shown.Thearrow shows theposition
of thehybridizingGRP mRNA.(B) Comparisonof the timecourseof GRP
mRNAexpression in monkey and
hu-manfetal lung.Data forGRPmRNA
expression inhuman fetallungis from
Spindeletal.(17).
tominimize bias ofchoosing adjacentsections with similar sized
air-ways. Within each field, all airways were counted and the internal
diameterof eachairwayrecorded inaspreadsheet (Excel 4.0,Microsoft
Inc,Redding,WA)toallowconstruction ofhistogramsofairwaysize
andnumber.Comparisons ofairwaysize andnumberweremadeby 1-,
2-,or3-way ANOVA usingeither StatView 2.01 orSuperAnova 1.11
(Abacus Concepts, Berkeley, CA)onaMacintosh IIFX. Post hoctests
wereperformed with Fisher's LSD.
The numberof unlabeled and BrdU-labeledcells indeveloping
air-ways werecounted in 10fieldsper group.Within eachfield, inevery
airwaythatcontainedlabeledcells,the numberof labeled and unlabeled cells was determined. This allowed calculation of the percentage of labeledepithelialcellsperfield and theaveragenumberofBrdU labeled
cellsperairway.
Results
GRPandGRP-Rgeneexpressionwasanalyzed bythree
differ-entmethods. Northern blot analyses wereperformed to
deter-mine GRP mRNAexpression in fetal and adult monkey lung; RNaseprotection assays wereperformed todemonstrate GRP-R expression in lung tissue and in situ hybridization analyses
wereperformed todetermine cell typeand relative number of cellscontaining GRP and GRP-R mRNAs.
By Northern blot analysis, GRP mRNAwasnotdetectable in 50-d fetal lung; first detectableat63 dgestation, reached highest levelsat80days gestation and then declinedtonearadult levels
by 120 dgestation (Fig. 1 A). Adjusting for length of gestation
(160 d for monkey, 40 wk for human), this timecourseis quite
similarto thepattern ofGRP expression we previously reported in humanfetallung (Fig. 1B) ( 17). In both monkeys and humans,
lung GRP mRNA expression beginsat30% of gestation, reaches
peak levelsby 50% gestation, and then rapidly declines toadult levelsby 75% of gestation (Fig. 1 B).
In situ hybridization localized GRP mRNA to PNEC and neuroepithelial bodies in both bronchi and bronchioles in fetal and neonatalmonkey lung (Fig. 2 A). That these cells contained
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Figure2. In situhybridizationandimmunohistochemistryof GRP expression in fetal monkey lung. (A) In situ hybridization of 125-d gestation
monkey lungwith35S-labeled antisenseGRPcRNAdemonstrateshybridizingfoci (arrowheads) corresponding to GRP-positiveneuroendocrine
cells(x180). (B) Immunohistochemical stainingof125-dgestation monkey lung with bombesin antibody
2A1
1 (18)demonstrates GRP-positive neuroendocrine cells(arrowheads)atthebifurcationsof a bronchiole (X225).(C and D) Dark field (C) and lightfield(D)photographs of in situhybridizationof 80-dgestation monkey lungwith35S-labeledantisenseGRP cRNAshowingthe presence of GRP mRNA in cells ofbudding airways(arrowheads) (Cand Dx290).GRPwasdemonstratedbystrongstainingwith bombesin
mono-clonal antibody 2AII (18) (Fig. 2 B) and by staining with antibodies to the monkey GRP prohormones (not shown). GRP-positive neuroendocrine cells werelocalized both
proxi-mally anddistally,predominantlyinbronchioles, frequentlyat points of bifurcation (Fig. 2 B). Absorption of the anti-bombesin antiserum with anti-bombesin and theprohormone antise-rum with peptides MCI and MC2 blocked staining in serial sections. Cells wereidentified asPNECby morphology, loca-tion andbypositive stainingwith ahumananti-chromogranin antibody (17, 47, 48).Thussimilar cell types express GRP in
monkeyand human fetallung.
Between 63 and 80 d gestation GRP mRNA was present notonly intheneuroendocrinecells,butalsoin cellsofbudding airways (Fig. 2, CandD). These cells however didnotshow anystainingwithbombesinantibody2A11 norwith the
antibod-ies against the monkey GRP prohormones. Thus, GRP mRNA istransiently expressed inairwayprogenitor cells but maynot be translated.
The presence of GRP-RmRNA was determined by RNase protection analysis of total RNA from monkey fetal and adult lungs using a probe to exon 3 of the monkey GRP receptor
(Fig. 3). GRP-Rexpressionwasdetectable inmonkey lung by
63 dgestation, plateauedfrom 80to140 dgestationand mark-edly decreased in adult lung (Fig. 3). Thus this time course
parallelsthe timecourseobserved for GRPmRNAexpression. UsingthesameGRP receptorexon3probeand bombesin
anti-body2A11,combinedGRP-R in situhybridization/GRP
immu-nohistochemistry was performedtodetermine which cells ex-pressedtheGRP-RmRNA(Fig. 4).AsshowninFig.4, GRP-R was expressedprimarilybycellslining developingairways.
As evident inFig.4 C, hybridization is locatedluminally and
t.
;. 'k,-.. 4C4
.."o i. .
f:..
.v li
.t': -
no
en_cu vD <
ODu 71- Ca C0 CL
Figure3. RNaseprotection analysis of GRP-RmRNA ex-pression infetal and adult
mon-key lung. Adult monmon-key
stom-ach (lane 6, 10
1g)
served as a positive control (61).Overex-posureof undigested probe shows>98% purity. 10
Jsg
of total RNAprepared frommon-keyfetal and adult lungswere
subjectedtosolution hybridiza-tion with the monkey GRP re-ceptorcRNAprobeasdescribed
inMethods. Sizeof input probe (including flanking vector) se-quences=320bases; size of protectedfragment=250 bases. Protectedfragmentswereof ap-propriatesizesasdeterminedby
DNAsequencing ladders(not
shown).
An
I
in areas of cellular thickening. The expression of GRP-R in
airwaysappears bimodal in that either many cells inanairway
section express the GRP-R or no cells in an airway section express the GRP-R. The proximity of the GRP-Rexpressing epithelial cells to the GRP expressing PNEC suggests that GRP
released from PNECmayact ontheairway epithelialcells. As shown in Fig. 4, D and E silver grains also overlayed some PNEC suggesting that some GRP-expressing cells may also express theGRP-R.
To determine theeffects of GRP on airway development,
organ culturewasperformedon60-, 70-,and 80-d fetalmonkey
lung,periods of maximalGRPand GRP-R expression.Tissues incubated for 5 d maintained morphology characteristic of
healthy lungtissue asshowninFig. 5.Therepresentative sec-tion shown inFig.5 shows the stimulationofairwaysize and
number causedby 10 nM bombesin. Theeffects of bombesin andantagonists werequantifiedby image analysis andresults
arepresentedinFigs. 6-8. Figure 6 shows ahistogram of the number ofairways of the given sizes (in 10
tim
increments) for each treatment. Bombesinatboth1 and 10 nMsignificantlyincreasedairway number and sizeat60,70, and 80 dgestation.
Theeffect of bombesin was completely blocked by the specific
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analysisof GRP-R and GRPexpressioninfetalmonkey lung. All sections wereexposedtobombesinantibody2A11Iandprobedwith the antisenseor sense35S-labeledGRP-R cRNAprobe.Allfieldsarefrom 80-dfetalmonkey lungat x150. ArrowspointtoGRP-immunostainingcells.(A)BrightfieldofhybridizationtoGRP-Rsenseprobe showingnospecific hybridization
aroundairway.(BandC)
Brighffield
ofhybridizationtoGRP-R antisenseprobe.Notehowhybridizationtendstobeluminal. and inareasof cellularthickening. (DandE)Darkfield withpolarized-epiluminescentilluminationshowinghow GRP-R hybridizationoverliessome,butnot all,
GRP-positiveneuroendocrine cells.
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GRPreceptoantagonistsiD-Phe bombesicntl (6-13)preopyamidem (45, 46) (Fig.w6)ys wefelldabcmariseond GRPhirecep cantago-ie
bhoerbtdsidnfcndecreasesthinumesolarger-sized airwayswihastg deiscriedtbelow(Fig. 7).
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large(>v71onm)n sies.leAs shon iparntFig.ras7, stogesoefwtsthe
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Bomb ( OnM) Bomb (1nM)
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Figure 6. Effects ofbombesinandGRPreceptor antagonists on size and number of airways in fetal lung organ culture. Fetal monkey lungs of thegestationalages shownwerecultured without anytreatment (con-trol),in the presence ofbombesin(1 nMand 10 nM), GRP receptor
antagonist(100 nM) and bombesin +antagonist (10nM + 100 nM).
Airwaysin102-mm2fields per groupwerecountedasdescribed in the Methods. Data shown is for the GRP receptorantagonist D-Phe6
bombesin(6-13) propylamide (45).Similar resultswereobtainedwith
asecond GRP receptor antagonist, BIM-26226 (46)(resultsnot
shown). *P<0.01 comparingthe overall number ofairways in 1 and 10 nM bombesin treatedlungstocontrollungculturesby 3-way
ANOVAusingFisher's LSD post hoctest.
d fetallung culturedin thepresenceof 10 nMbombesin(Fig.
8). Bombesin increased the number of airways in each size category, such thatthere were nolongersignificantdifferences between the treated 60-d lung and the untreated 80-d lung
(Fig.
8).
repli-90
60
Day
Lung
80
-- 70 *
6060
850
-40*
CU
30-20
10
0
Small
Medium
Large
Total
90
70
Day
Lung
80 *
70-6060
50->1 40*
30
*-20 -*
10
0
Small
MediumLarge
Total
._0
.
_z
Small
MediumLarge
TotalFigure 7. Histogram showing the effects of bombesin and its antagonistsonthe total airway number and number of small, medium and large sized airways(small = 10-40Mlm,medium=41-70 ,um,large= 70-150[Sm)in fetalmonkey lungs.Bombesinat 1 and 10 nMsignificantlyincreased totalairways in 60, 70, and 80-d lungs. Bombesinsignificantlyincreased the number of medium andlarge airways,butnotthe number of small airways.Antagonisttreatmentdidnotsignificantlyaffect overall number ofairways,butdidsignificantlydecrease the number oflarge airwaysand increase the number of small airwaysat mosttimepoints. *P <0.05comparedtonumberof controlairwaysof thesameage and sizeby 1 way ANOVA followedby Fisher's LSD post hoctests.
cation and to determine the target cells, BrdU labeling was performed. Asshown in Fig. 9,themajority of cells thattook up BrdU lined developing airways. Treatment with 10 nM bombesinsignificantlyincreasedboth the number and percent-ageofBrdU-labeled airwayepithelial cellsascomparedto un-treated lung (Fig. 9). The majority of cells labeled by BrdU were located in theepithelium of developing airways and rela-tivelyfewermesenchymal cells werelabeled (Fig. 9 A).
Discussion
Northern blotanalyses demonstrated that GRP mRNAs are ele-vated in fetal rhesus monkey lung from 63 to 80 d gestation. This transient increase of GRP mRNA in midgestational fetal monkey lungis similar to the increasedGRPmRNAexpression observedfrom 16 to 30 wkgestationin human fetal lung (17). Also like human lung, in situ hybridization localized the GRP-mRNAin fetalmonkey lungtoneuroendocrine cells. GRP im-munostaining appeared at about the same time when GRP mRNAexpressionwasfirstdemonstrated, andwasconsistently present in the neuroendocrine cells in both fetal and neonatal
lungs.This strongsimilaritybetweenmonkey andhumanin the pattern and time course of GRP gene expression makes the monkeyapotentially valuable modeltocharacterizethe roleof GRPinfetal lung development.
In humanlung, as inrhesus monkey the periodofgestation during whichtheGRPgeneis activatedcorrespondstothe latter partof thepseudoglandular stageandto the canalicular phase ofpulmonary development(17,49,50). Thisphaseis charac-terizedbyconsiderableextension and branching ofthe respira-tory bronchioles which are lined by cuboidal epithelium and situated close to the capillary system. Atplaces where direct contactbetweenbronchiolesandcapillariesoccurs, the epithe-lial cells may be flattened to squamous epithelium (49, 50). Developing monkey lung generally follows a similar time
course asdoes human lung, and alveolarization does not
sig-nificantly startuntil day 95 of gestation (51).Thus in the pri-mate, GRP isexpressedin thepseudoglandularand canalicular phase during initial airway formation. By contrast, in rodents, GRP is expressedattheendofgestation (28, 29). This would suggestthat inprimates, GRPmay influenceairway branching, but in rodents, GRP has more ofan effect in terminal airway
E[
Antag
Antag
+bomb
* Control
0
Bomb
(1 nM)
A # #w* 4
* ;;f
150
120
-o
E
cc
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>1
90
60
30
80daycontrol Bomb(1OnM)
r..V.
...
.^
..*X
., ,
Figure8.Histogram showingthe apparentabilityofbombesinto accel-erateairwaygrowth. The size and number ofairwaysiscompared
between 60-d fetallungcultured in the absence ofbombesin,60-d fetal lungculturedin the presence of 10 nM bombesin and 80-d lung cultured in theabsenceof bombesin.2-way-ANOVAfollowed by Fisher's LSD shows nodifferenceinairwaynumber between 80-dculturedlung and
60-d lungculturedwith bombesin.
differentiation (28, 29). Interestingly, Aguayo et al. (30) has recently reported thatbombesin-likepeptides can stimulate lung branching infetalmouse lung explants,despite theinabilityto detect GRP immunoreactivity in fetal mouselung. This raises thepossibility that other, as yet undescribedbombesin-like pep-tides may be active in rodent lung.
Asurprisingfinding inthis study wasthatatmidgestation, GRP mRNAwaspresentnotonly in the neuroendocrine cells, but also in the epithelial cells ofbudding airways. Thisfinding
is similar to the recent report of calcitonin gene-related peptide inserouscellsofrattrachea (52).In contrast toGRP expression in neuroendocrine cells in which strong immunostainingcould bedemonstrated,noGRPorGRPprohormone immunostaining
could be detected in the budding airways. This suggests that GRP mRNA is present, butnottranslated inairwayprogenitor cells. This would be similartothe many humanlung neoplasms
which also express but do not translate the GRP mRNA and thus may helpexplain why so many differentlung neoplasms
express the GRP mRNA(53,54). Itis alsopossiblethat GRP is presentatlow concentrations orisrapidly secreted, butnot stored in thesecells,thusmaking immunohistochemical detec-tion difficult. Another possibility which cannot-be ruled out is the presence ofarelated, but different GRP-mRNA whose translationproduct isnotdetected bytheantibodies used.
Pre-liminary screening of a cDNA library from 80-d fetal lung
however didnotreveal any clonesencodingnewbombesin-like peptides. The expression of some but not all neuroendocrine markers (i.e., no chromogranin expression) raises the still poorly understood question of the relation ofairway progenitor
cellstoneuroendocrine cellsor totheprogenitorsof neuroendo-crine cells.
Bombesin-likepeptides havebeen implicated as autocrine growth factors influencing pathogenesis and progression of someSCLC (18, 19, 27) and the expression ofGRP receptor
C
Percent labeled Average labeled cells per field cells per airway
Figure9. Effectof bombesinonincorporation ofBrdUintoculturedfetal monkeylung.604d fetal monkey lungwascultured in the (A) presence of bombesin (10 nM)or(B)absence of bombesin andpulse-labeledwith
BrdU.Sections (X550)wereprocessedandstainedforBrdUasdescribed inMethods. Asexamples,arrowheadspointout someofthe BrdU-posi-tive cells. (C) Percentage of BrdU-posiBrdU-posi-tivecells per2-mm2 field and
averagenumberof BrdU cellsperairway inbombesin-treated and un-treatedlung.Airwaysin 102-mm2fieldswerecounted pergroup; the
total numberof BrdU-positivecells/total cells countedareshown over
in SCLC is well documented (23, 34, 55-57). The expression ofGRP ligand in fetal and neonatallung of human and other mammals is also well described (8, 10, 13, 14, 17), but the expression of GRP receptors in normal fetalprimatelung has notbeendescribed. Inthis study,wehave demonstrated GRP-Rgeneexpression in normalmonkey fetal lungby RNase pro-tection and in situ hybridization analyses. Insituhybridization
showsmarkedstaining around thedevelopingairways (Fig.4) which suggests thatdeveloping airway epithelial cells may be thecells thatrespond to themitogenic effects of GRPduring
lungdevelopment. Consistent with this, both GRP ligandand receptorareexpressed in monkey fetal lungatabout thesame stages. Thus onelikely hypothesis is that GRP synthesized by PNEC isaparacrine growth factor forepithelial cells of devel-opingairways which express GRP-R.
Theobservation of GRP-R inairway epithelial cells is con-sistent with the observationsbyWilleyetal. (6)andSiegfried etal. (20) that bombesin and GRP aremitogenic for human bronchial epithelialcells. Thedouble in situ
hybridization/im-munohistochemical analyses suggest that somePNEC express both GRP and GRP-R. This is consistent with thefindings by Speirs et al. (58) that GRP stimulates the growth of rabbit PNEC. This is also consistent with the expression of GRP-R bysomeSCLC and the existenceofanautocrinefeedbackloop
as first proposed by Cuttitta et al. (18) in which SCLC both secrete and respond to GRP. While our data shows GRP-R hybridizationoverlying PNEC andstrongly suggests that PNEC expressGRP-R, without confocalmicroscopywe cannot
defini-tively rule out that the hybridization observed derives from overlying cells.Inexamining theexpression of GRP-R in
devel-oping airways, it is strikingthatnotall airwaysexpress GRP-R, ratheronlyasubsetof airways express GRP-R,andtypically, in an"activated" airway many cells will express GRP-R. This suggests that there may bealocal, paracrine factor thatturns onGRP-Rexpression indeveloping airways.The identification of this factor is likely tohave significance both for normal as wellasneoplastic lung growth.
To determine if the peak expression of GRP mRNA in developing oflung coincided with effects of bombesin-like pep-tides on lung development, organ culture was performed. As shown inFig. 6,bombesinincreased inadose-dependent man-nerbothairway size and number. Whileourmethodologies did notallow a specific quantitation of branching morphogenesis, the increase in number suggests a concomitant increase in branching. Interestinglywedidnotobserveanincrease in small airways which might be expected with increases in branching. This may relatetotherelative rapidity of stimulation ofairway
growth by bombesin oralternately that rather than stimulating airwaybranching, bombesin simply stimulates increases in air-way lineargrowth and diameter. That this increase was achieved by stimulation of cell division wasconfirmed by labeling with BrdU, inwhich bombesin increased DNA synthesis primarily in the epithelial cells of budding airways and had relatively weakereffectsonmesenchymal cells.
Considering that animportant aspect of both the pseudo-glandular and the canalicular phase is extension and branching ofairways (49, 50), the stimulatory effects of bombesin we observed inorgan culture suggests that the peak expression of GRP in neuroendocrine cells in fetal lung is related to the rapid
airway developmentthat isongoingatthat time.Itis, however, necessarytokeepinmind that besides bombesin, other growth
factors, such as epidermal growth factor (EGF) and trans-forming growth factor-/31 (TGF-/61), may play a synergistic role in fetal lung development (59, 60). Both EGF and EGF receptor have been demonstrated in primitive airways. For ex-ample, addition of exogenous EGFtolungs in culture resulted in significant stimulation of branching morphogenesis and in-crease of DNA, RNA and protein content (59). The interaction between thesefactors and GRP clearly deserves further study. Stimulatory effects of bombesin wereobserved in60-, 70-,
and 80-d lung. Whether bombesinwould continuetobe stimula-tory in lungs from older monkeys will likely correlate with expression of GRP-R inairway cells. Thus criticaltothe regula-tion of airway growth will be what regulates GRP and GRP-R expression.
That the effects ofbombesin could becompletely blocked by two different GRP receptor antagonists is consistent with theexpression of theGRP-R as detected by RNase protection and in situ hybridization. This suggests that endogenous bombesin-like peptides acttostimulateairway development by interaction with the GRP-R. Based on the immunohistochemical stainingand Northern blotanalysis it is reasonable to conclude this peptide is GRP. Itispossible thattheeffects of bombesin on airway development in rodents may bemediated by other as yetuncharacterized bombesin-like peptides as GRP cannot bedetected in early gestation rodentlung. The failure of GRP-receptorantagonists by themselves to blockairway development inorgan culture may derive from insufficient concentrations of antagonists at the site of endogenous peptide secretion, high local concentrations of endogenous peptide, or may relate to a lack of secretion ofendogenous GRP because of loss of regula-tory mechanisms.
In conclusion, thepresent study demonstrates that GRP and the GRP-R genes are expressed in monkey fetal lung primarily during the pseudoglandular and canalicular phase of pulmonary development. The pattern andtime course of expression of GRP gene in developing fetal lung in monkey is similar to that in human; suggesting this will be a valuable model to determine theeffects of bombesin-like peptides on lung development. The clear stimulatory effects on airway development induced by bombesin suggests potential therapeutic uses of bombesin in very early threatened or actual premature deliveries. Future in vivo studies to administer bombesin-like peptides to fetal mon-keys in utero will help determine the extent of bombesin-like peptides role inlung development.
Acknowledgments
This research was supported by National Institutes of Health grants CA39237, CA53584, by the cell culture and morphology cores of Na-tional Institute of Child Health and Human Development Population Research Center grantP30-HD18185 and by grants from the Council onTobacco Research. GRP antibody2A11 was generously supplied by FrankCuttitta and GRP receptor antagonists were generously supplied by David Coy and by Biomeasure Incorporated.
The authors wish to thank David Warburton for advice on lung organ culture and Richard Simerly for assistance with double in situ immunohistochemical analysis.
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