Connexin43 mediates direct intercellular
communication in human osteoblastic cell
networks.
R Civitelli, … , S T Geist, T H Steinberg
J Clin Invest. 1993;91(5):1888-1896. https://doi.org/10.1172/JCI116406.
We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye
diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for
connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by […]
Research Article
Find the latest version:
Connexin43
Mediates Direct Intercellular Communication
in Human
Osteoblastic Cell
Networks
RobertoCivitelli, Eric C. Beyer,* Pamela M. Warlow, AudraJ. Robertson,t StevenT. Geist,t andThomasH.Steinbergt
DivisionofEndocrinology andBoneand MineralDiseases,the Jewish HospitalofSt. Louis;*Divisions ofHematology/Oncology,and
*InfectiousDiseases; DepartmentsofMedicine, Pediatrics,andCellBiology and Physiology,
WashingtonUniversitySchool ofMedicine, St. Louis, Missouri 63110
Abstract
We haveexamined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromalcells(BMC)and trabecular bone osteo-blasts(HOB),and in the humanosteogenicsarcomacellline, SaOS-2. Both HOB and BMC cells were functionally coupled, sincemicroinjectionofLuciferyellow resulted in dye transfer to neighboring cells, with averages of 3.4±2.8 (n = 131) and 8.1±9.3(n =51) coupled cells perinjection, respectively.In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4±1.8 coupled cells per injection, n =100). Dyediffusionwasinhibited by octanol (3.8 mM), an inhibitorof gap junctional communication.Allof the osteoblas-tic cellsexpressedmRNAfor connexin43 andconnexin45,but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similarquantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced signifi-cantlylessCx43proteinthan either HOBorBMC,asassessed by immunofluorescence andimmunoprecipitation.Conversely,
more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and
transformedhumanosteoblastic cells correlates with the level ofexpressionof
Cx434
which appearstomediateintercellularcommunication in these cells. Gap junctionalcommunication may serve as a means by which osteoblasts can work in synchrony and propagatelocally generated
signals
throughoutthe skeletaltissue.(J. Clin.Invest. 1993.91:1888-1896.) Key
words:gapjunctions* connexins * osteoblasts * dye-coupling-cell-cellcommunication
Introduction
Bone remodeling is the result ofa
complex
and coordinatedseries ofevents
occurring
in the skeletal tissue. Theseprocesses includesequential degradation
of bone matrix and dissolutionofthe mineral
phase
(bone resorption),
followedby deposition ofnewbonematrixand mineralization(bone
formation) (1,2). This
organized
pattern ofeventsrequires
a coordinatedPortionsof this workwerepresentedatthe 13th annualmeeting of the AmericanSocietyfor Bone and Mineral Research, 24-28 August 1991, SanDiego, CA,and haveappeared in abstract form (No. 672).
Addresscorrespondence to RobertoCivitelli, M. D., Division of BoneandMineralDiseases,TheJewishHospitalof St. Louis, 216 S. Kingshighway Boulevard, St. Louis, MO 63110.
Receivedfor publication 19October 1992 andin revisedform28
December 1992.
actionby the cellsofbone: osteoblasts, osteoclasts, and osteo-cytes(3). Cells of the osteoblastic lineage are pivotal to bone
remodeling, sincemost, ifnot all, chemical, mechanical, and probably electrical stimuli are processed by these cells. Not only are the osteoblasts directlyinvolved in bone formation, butthey also function astransmitters ofbone resorptive signals totheosteoclast (4).Infact osteoblasts, rather than osteoclasts, express receptors forhormoneswithbone resorptive activity, such asparathyroid hormone, parathyroid hormone-related
peptide,and 1,25-dihydroxy-cholecalciferol(5-7).
This coordinated activity among bone cells implies the exis-tenceof intercellular communication,butits mechanisms are not wellcharacterized. Intercellular communication may oc-cur via secretion ofsoluble factors, direct cell-cell contact
throughcelladhesion/recognitionmolecules, or by metabolic andelectriccoupling throughgapjunctions.There is evidence thatosteoblasticcells secrete solublefactors when they are stim-ulatedby hormonesorcytokines, and that these factors may transmit theresorptive signal from osteoblasts to osteoclasts
(8-12).However,nospecific factorhas been isolated or char-acterizedto date. In contrast, the presenceof gap junctions between osteoblasts has been documented morphologically in
sectionsofnormal bone(13-15), and in rat calvaria cells in culture(16). Thesegapjunctionshave been observed between
adjacent osteoblasts,osteoblasts andperiostealfibroblasts, and osteoblasts andosteocyticprocesses insections of normal bone
(13-15). Recently, the gap junction protein connexin43
(Cx43)'
has been identified in normal and transformedratosteoblasticcells(17, 18).
Inthe present study, we have characterized gap junction
communicationin human bone-derivedosteoblasticcells and in a humanosteogenicsarcomacellline. We found that Cx43,
originally identified fromratheart, appearstobe theprotein
that mediates gapjunction communicationin human
osteo-blasticcells. Another gapjunction protein,Cx45, was
identi-fiedin thesecells,but itsexpressiondoesnotcorrelatewith dye coupling.
Methods
Reagents and chemicals
Lucifer yellowwaspurchased from Molecular Probes (Eugene, OR). The stockwasdissolved indistilled,deionized water, toa10-mM
con-centration,andstoredat-200Cin the dark.
[35SItrans-S
wasobtained from ICN Biomedicals (Boston, MA). Polyclonal rabbit antiserum directedagainstconnexin43wasproduced byimmunizingrabbits withasynthetic peptide (252-271 fragmentofratconnexin43) (19).
Rab-bitpolyclonalantiserumagainstCx45wassimilarly generatedusingan
oligopeptidecorrespondingtoamino acids 285-298 ofdogCx45(20).
1.Abbreviations used inthis paper:BMC, bone marrowosteogenic
stromalcells; Cx,connexin; HOB,human trabecularosteoblasts.
J.Clin. Invest.
© The AmericanSocietyfor ClinicalInvestigation,Inc.
0021-9738/93/05/1888/09 $2.00
All other chemicals and thetissue culture media were from Sigma
Chemical Co.(St.Louis, MO),unlessotherwise indicated.Octanol, an inhibitor of gap junctioncommunication,wasused toconfirm that dye transferwasmediated by gapjunctions. Octanol dilutions were pre-paredfresh,before use,followingaproceduredescribed by Musil et al. (21 ).Briefly,20 ul ofa1:4solution of octanol in ethanol were added to 10 mlof culture medium (see below) at370C,and vortexedvigorously
for 2 min to ensure properdissolution. This procedure yields final
concentrations of 3.8 mM octanol, and 0. 15% ethanol.
Cell cultures
Humantrabecular osteoblasts (HOB).Cellsderived fromhuman tra-becular bonearefelttorepresent thedifferentiatedosteoblasts thatline
bonesurfaces(3).HOBwereprepared accordingtothe method of
Gehron-RobeyandTermine (22),withmodifications (23).The
tra-becular boneobtained fromhumanspecimensasdescribed abovewas
minced and washed several timesinDME/Ham's F-12 (1:1). The bone chips thus obtainedwere incubatedwith type IV collagenase (SigmaChemicalCo.) for90 minat370Ctorelease cellsfrom the bone
surfaces.Thereleased cellswerethen seededonplasticflasks,and cul-tured in thesamemedium, supplemented with 10%FCS, 100
Ag/ml
streptomycin,and100 U / mlpenicillin.The bonechipswerewashed in
serum-containing mediumtoinhibit collagenaseactivity,and cultured
separatelyusingthesamemedium used for the collagenase-released
cells.Afterafew days,cellsmigrated from withinthe boneparticlesand
begantomultiply;andwithin3-4 wktheyreachedconfluence. Thereaf-ter, bothcollagenase-releasedcells and those outgrown from the bone
chips were released with trypsin-EDTA (0.05-0.02%, respectively, Sigma Chemical Co.), counted,andsubculturedinmedium contain-ing10% FBS.
HOB are large cells that possess several cytoplasmic processes which typically extend up to 50-60 um (Fig. 1 A). These processes allowcell-cellcontact at adistancefrom the cellbody.As demon-strated inFig.1 A, HOB stainforalkalinephosphatase. Althoughmost
cells showspecific staining,notall cells show thesamedegree of stain-ing.Thisvariabilitymayreflecttheheterogeneity withinthe
popula-tionofosteoblasticcells.HOBproduce osteocalcinbothconstitutively
and understimulation with 1,25(OH)2D3 (23). These features are
characteristics ofadifferentiated osteoblastic phenotype (3).
Bonemarrowosteogenicstromal cells (BMC). BMCarederived fromhuman bone marrow, andarebelievedtorepresent less differen-tiatedosteoblast precursors(24).BMCwereisolated and cultured
us-ingamodification ofthe methodsdescribed byAshtonetal.(25),and Long et al.(26). Surgical specimensofribs wereobtained from volun-tary donorsundergoingchest surgery forvarious medical problems.
The boneexplants weresplit longitudinallyand the bone marrow was collectedbygentlyvortexinginserum-freeDME/Ham's F-12 ( 1:1 ). Theextract wasmixedwith an equal partofaMEM,and themixture
layered onto adiscontinuousFicoll-hypaque gradient. The mononu-clear cellpopulationwasrecoveredfromthe upper level interface
be-tween the medium and the higher density Ficoll-hypaque, washed
twiceinaMEM, resuspendedin thesamemedium,and seeded intissue
cultureflasksorontoglasscoverslips.The cultureswereincubatedin
aMEM, supplementedwith 5%heat-inactivated FCS,and100 gg/ml
streptomycinand100 U/mlpenicillin.
Asevident inFig. 1 B, only a small numberofBMC cells exhibit alkaline phosphatasestaining, suggestiveofalessdifferentiated pheno-type. Furtherdifferentiation of stromal cells toward an osteoblastic phenotypecanbeinduced by incubation with dexamethasone (24).
Therefore,someBMC cultureswereincubated inl0-7M
dexametha-sone forat least7 d. Atthis time the cells appear larger andmore
flattened,and possessnumerous short cytoplasmicprocesses. These
morphologicfeatures resemble those ofthemoredifferentiated trabecu-larbone-derived cells (see above). Inaddition, cells incubatedwith dexamethasone also express higher levels of alkaline phosphatase (Fig.
I C),andproducegreateramountsofosteocalcin,bothconstitutively
B
Figure1.Phasecontrastmicrograph of alkaline phosphatase-stained
human osteoblasts(HOB, A),bonemarrowstromal cells(BMC)
aftera7-dincubation without (B),orwith10-7Mdexamethasone(C).
and understimulation,than stromal cells growninthe absenceof dexa-methasone(24).
Osteogenicsarcomacells.Theclonal cell line SaOS-2 wasobtained
from theAmerican Type Culture Collection (Rockville, MD).These
cellsarederived fromahumanosteogenicsarcoma, and characterized
ashavinganosteoblasticphenotype (27,28). Theyweremaintained in DME/Ham's F-12 (1:1), supplemented with 10% FBS, as de-scribed(29).
Dye
coupling
Gapjunctioncommunicationwasstudiedbyintercellular diffusion of Luciferyellow(30).Cellsweresubculturedonglasscoverslips (31-mm
mounted on athermostatted tissue chamber (Biophysica Technology,
Baltimore, MD), covered with growth medium, and placed on the stageofaninverted epifluorescence microscope (Diaphot;Nikon, Gar-den City, NY),immediatelybefore themicroinjections. Borosilicate
glasscapillaries (outer diameter1.2mm),withaninternalglass fiber (Omega Dot;StoeltingCo.,Wood Dale, IL)wereused topull micropi-pettes. Luciferyellow (10 mM)wasintroduced intothecapillariesby
backfilling. Individual cells(sixtoeightpercoverslip)were selected from within confluent areas, andmicroinjected (1,100-1,200hPa for 0.3 s)usingaZeiss-Eppendorfmicroinjector-micromanipulator(type 5242;Eppendorf, Hamburg, Germany), attached to the microscope
stage.The volumeofinjected material didnot cause any
morphologi-calchangesofmicroinjected cells,asdetectedbylight microscopywith phase contrast.
Luciferyellow was excited using a microspectrofluorometric sys-tem(CMl1II;SpexIndustries, Edison, NJ),whose monochromator was set at 435 nm. Theexcitationlightwasdirectedtothe cell cultures
byadichroic mirror with455-nmcut-offwavelength.Thelightemitted by Luciferyellow wasfiltered through a460-nm barrierfilter, with 10-nm bandpass (BV-lA assembly cube; Nikon). Lucifer
yellow-emittedfluorescencewasmonitored usingalowlight silicon intensified
target camera(SIT-66;DageMTI,Michigan City, IN)connected to the lateral portofthemicroscope. Fluorescence imagesweredisplayedon a
monitor,and recorded ontovideotapes. Beforethemicroinjection,a
snapshot ofthe selectedfieldunderphasecontrastilluminationwas also recorded. Toavoidphotobleachingof Lucifer yellow, the excita-tion lightwasdirectedtothesampleonly forafewseconds atdifferent times duringtheexperiment. The numberofcellsadjacenttoeach
injection site exhibiting fluorescence 3-5 minaftertheinjectionwas recorded. In someexperiments, photographic imagesweretakenby replacingthevideo camerawithaphotographiccamera. Results
ob-tainedinanumberofseparatecoverslipsforeach cell type and condi-tion,werepooled.Quantitative analysis ofcellcouplingwasperformed bycalculatingthefrequency distribution ofthe numberofcellscoupled
toeachsinglecellmicroinjectedwith Luciferyellow,andnormalizing
the resultsto100%.
RNA
blots
RNAblots wereperformed aspreviously described (19).Cells were grown toconfluencein100-mmplasticdishes,and totalcellular RNA wasisolatedusing guanidinium isothiocyanate (31). Samples(10Ig/
lane)wereseparatedonformaldehydeagarosegelsandtransferredto nylon membranes. The membranes werehybridized (0.75Msodium
phosphate, 1.0%SDS, 100 ug/ml salmon sperm DNA, 65°C)and washed underhighstringency conditions (30mMsodium phosphate,
1.0%SDS, 65°C).Thefollowing32P-labeledcDNAprobeswereused: ratconnexin26(32),connexin32 (33), connexin37 (34), connexin40 (35), connexin43 (36), connexin45 (20), andconnexin46 (37). A cDNAforhumanfibroblast y-actin wasagiftofDr. Stanley
Kors-meyer,WashingtonUniversity,St. Louis.
Immunoprecipitation
of
connexin43Cellswereculturedon 100-mmtissuecultureplatesto75-85%
con-fluence, incubatedinmethionine-free medium for 30 min, and
meta-bolicallylabeled with300-,uCi/plate [35S]trans-S(ICNBiomedicals)
for 6 h. Cells were thenwashedandsolubilized in 150 mMNaCl, 1% TritonX-100,0.5%deoxycholate,0.1%SDS,0.5%BSA,50 mM Tris pH 8.Radioactivitywas measured in a 5-,ulaliquotof each solubilized cellsample,andappropriatedilutionsweremadetoachieveauniform concentration of radiolabeled proteins before the samples were
im-munoprecipitated. Sampleswerevortexed, incubated5min at room
temperature,vortexed again, andcentrifugedat 12,000 rpm for 5 min at4°C. Supernatants containingsolubilizedproteinsweredivided into
aliquotsandincubatedovernight with either preimmune or anticon-nexin43 immune serum at4°C.Theimmune complexes were
precipi-tatedusing protein A-Sepharose (Sigma Chemical Co.), and boiledin
Laemmlisamplebufferfor 10 min. Proteinswereseparatedby
electro-phoresison 10%polyacrylamide gelswithPromegaCorp.(Madison, WI)mid-molecularweight proteinstandards. Gelswerestainedwith Coomassieblue R-250, dried, andexposed to XAR-5 radiographic
film.
Immunofluorescence
Cells grownonglasscoverslipswerefixed inmethanol/acetone(1:1) for2minatroomtemperature,washed,andincubated in PBS
contain-ing2%heat-inactivatedgoatserumand1%TritonX-100for10 min.
Thesampleswereincubated witha1:200 dilution ofeitherpreimmune
serum oranticonnexinserumfor 45minatroomtemperature.After three washes inPBS,a 1:500dilutionofrhodamine-conjugatedgoat anti-rabbit IgG (BoehringerMannheimBiochemicals,Indianapolis, IN)wasadded for 45minatroomtemperature.The coverslipswere
washed twice inPBS,mountedonglassslides with glycerol/PBS(90/ 10),and sealed with nail polish. Theepifluorescence photomicro-graphsweretaken withaZeissAxioskopmicroscope.
Results
Dye
coupling
inhuman osteoblasts. Diffusion ofLuciferyellow
between osteoblastic cells was used to assess gapjunctional
communication. As illustrated inFig. 2,both HOB and BMC demonstratedahigh degreeofcell coupling, whereas the
osteo-genicsarcomaSaOS-2 cellswerepoorlycoupled. Dyediffusion primarilyoccurredbetweentwocellbodies, although
occasion-ally theareaofcontactbetweentwocellswassmall, suggestive ofcontactviaacytoplasmicprocess.
A quantitative analysis of cell coupling in HOB,SaOS-2, BMC, and in BMC cultures treated with dexamethasone is shown in Fig. 3. About 80% of the HOB were found to be
functionallycoupledtoother cells, withanaverageof 3.4±2.8
(n= 131),andarather widescatterofthe frequency distribu-tion. Despite the high degree of coupling observed in some
HOB(dyediffusionto > 10cellsfromasingle cell), - 20% of
theHOBwerenotcoupled. This variabilitywas notdependent
onthe cell isolateordonor: variable resultswereobtained in monolayers derived from thesamedonor.
The number of coupled cellswashigher in the stromal cells than in trabecular bone osteoblasts (Fig. 3). Theaverage
cou-pling for BMC in resting conditionswas8.1±9.3 (n=51), with
onecoupled cellperinjected cellasthe mode. BMCgrownin thepresenceofdexamethasone exhibitedanarrower rangeand loweraverage(4.9±3.7;n =74)ofcoupledcells percell,than those observed in culturesgrowninthe absence of the steroid. However, the modewasthesame,and thetwofrequency distri-butions were not significantly different
(Kolmogorov-Smir-novtest).
In contrast, the transformed osteogenic sarcoma SaOS-2 cellswerepoorly coupled. No diffusion of Luciferyellow
oc-curredfrom 40% of microinjectedcells, and theaverage
cou-plingwas only 1.4±1.8 (n = 100) coupled cellspercell. In addition, dyediffusion in the SaOS-2 linewasslower thanin
anyother cell type. While for HOB and BMC cells the spread-ingof Luciferyellowwascompletewithin 3-5min fromthe
microinjection, in the transformed cell line it requiredatleast 10-12mintoreachmaximal propagation, inthe caseswhere diffusion occurredatall.
Effect
of octanol on intercellular communication. We next1.8±3.0, n = 28 for BMC grown in the presence of dexametha-sone.
Expression ofconnexin43 in human osteoblasts. To iden-tifytheproteinsthat mediate gapjunctional communication between osteoblasts, we asked whether any of the known membersoftheconnexin family ofgapjunction proteins was expressedby these cells. We isolated total cellular RNA from the osteoblasts, and performed RNA blots using 32P-labeled cDNA probes forCx26, Cx32, Cx37, Cx4O, Cx43, Cx45, and Cx46. BMC, HOB, andSaOS-2, all expressed mRNA for Cx43 and Cx45 (see below), butnotfor Cx26, Cx32, Cx37, Cx4Oor Cx46 (not shown).
RNAisolatedfrom allofthehuman osteoblastic cells con-tainedabandthathybridized with theratCx43cDNAprobe, and this bandwasthe samesizeas that of the control RNA isolated fromratheart(Fig.5).AlthoughBMC and HOB were morehighlycoupled than theSaOS-2cells, all ofthe osteoblas-tic cells contained similar quantitiesofCx43 RNA. To confirm theintegrityof themRNA present ontheseblots, membranes werereportedwithanactioncDNA probe;actinbandsof simi-lardensitywere detectedineachsample (datanotshown).
Theexpression of Cx43 proteinin the normaland
trans-50
40
30
20
cj
0
'4-. '
0
4 0
a 50
j
>1 40
a
- 30 0)
Figure 2. Diffusion of Lucifer yellow between human trabecular os-teoblasts(HOB;A), stromal bonemarrowcells(BMC;B), and SaOS-2 cells(C).Cells grownonglasscoverslipsweremicroinjected
with Lucifer yellow, and observed under fluorescence illumination (435-nmexcitation, 455-465-nm emission).Micrographsweretaken 5-10 minafter microinjectionof the dye. The cell microinjected in eachmicrograph is indicated by the letteri.
illustrated in Fig. 4, the inhibitoryactionofthe alcohol was complete for - 25-30minof incubation. Thereafter,the cells
progressively regained their ability to transfer dye. On this
basis,dyecouplingwasexamined in bothtrabecular and bone marrow-derived osteoblasts within 20minofexposure to oc-tanol.Inthepresenceofoctanol, the number of coupled cells perinjection wassignificantlyreduced in allcases,with aver-agesof
1.4+2.0,
n=23for HOB; 3.7±5.0,n=11 forBMC; and20
10
0
0 1 2 3 4 5 6 78 9 10>10 0 1 2 3 4 5 6 7 8
50
I
-20 CD
0
(D
10 (D
00
CD60 <
50 N 0
40
''f-0 30
20
00
Number of coupled cells per cell
Figure 3. Cell coupling in human osteoblasic cells. Cells grown on glasscoverslipswerepreparedasdescribed in Methods, and
microin-jectedwithLucifer yellow. The number of cells coupled to each cell
microinjectedwasdetermined either 3-5 min (HOB and BMC) or 10-12 min(SaOS-2)after -microinjection. Data for each cell type werepooledfrom differentexperiments performed in different
cover-slips (3-10 microinjectionspercoverslip), andwerenormalizedto
100%toallowcomparisons among groups.
C)
u
0.
Ca
UM
a) 0
(L)
0
4-a,
0) .0
z 12
1 1
10
6
5
4
3
2
0
0 10 20 30 40 50 60
Time after octanol addition (min)
Figure 4.Time-courseofoctanol inhibitionofcellcoupling.Human
osteoblasticcells grown onglasscoverslipsweremicroinjectedwith
Lucifer yellow.The numberofcellscoupledto asinglecell microin-jected withthefluorescent probewasrecordedatthedifferent time points. Onlyonemicroinjectionpercoverslipwasperformedinthese experiments,to ensure exacttiming.Betweentwoandfivecellswere
studied foreachtimepoint.Data represent the average and standard
deviations,whenapplicable.
formed osteoblastic cellswasassessedbyimmunoprecipitation after metaboliclabelingwith
[35S]
methionine. Cx43waspre-cipitated from all the cell types
(Fig. 6).
In HOB andBMCcultures,labeledprotein bands of- 40 and 42 kDwere
precipi-tated with immune serum but not with
preimmune
serum.These bandscorrespond, respectively, to
nonphosphorylated
and phosphorylated forms ofCx43, as demonstrated bythe presence of only the lower molecularmassform of theprotein
inphosphatase-treated samples (datanotshown,andreference
21 ). A43-kD cross-reactivebandwaspresent in
precipitates
using both preimmune andimmune serum. Its presence in
immunoprecipitates performedin the presenceof100
,gg/ml
oftheimmunizingpeptidedemonstrates thatthisbandisnot a
Cx43-specificband(19). Treatmentwith dexamethasone did notsignificantly affecttheexpressionof Cx43protein byBMC cells(Fig. 6, right panel).
I)
m
0
0
X0
I 0n
Figure 5. Expressionofconnexin43mRNAby human osteoblasts. Total RNA was isolated from nearly confluent cultures of BMC, HOB, and SaOS-2 cells, andformaldehyde-agarosegels were run us-ing 10jigof sample for each cell type. Rat heartRNAwasusedas acontrol.
kD Figure 6. Expression of
55 connexin43protein by
humanosteoblasts.
40 - _ Cellsweregrownto
confluenceintissue cul-turedishes,solubilized, and immunoprecipi-I)(j) tatedwith anti-Cx43
~~~+
8 c o serum orpreimmune
0
(,
co co serum.InHOBandBMCcells,Cx43 ap-pearedastwobandsthatmigratedat 40 and 42 kD, representing
nonphosphorylatedand phosphorylated forms of the protein,
respec-tively.The blot ontheleftpanelshowingHOB and SaOS-2 cells was
exposed twiceaslong as the blotinthe right panel, showing BMC cells incubatedinthe presence or in the absence of dexamethasone.P, preimmuneserum;I,immune serum.
Incontrast with the equivalent expression of Cx43 mRNA in HOB, BMC, and SaOS-2 cells, much less Cx43 protein was
detected in SaOS-2 cells than in the nontransformed osteo-blasts(Fig.6). In addition, there was a consistent difference in the stateof phosphorylationof Cx43: while in HOB and BMC themajor Cx43band wasofthe slowermobility phosphory-latedform oftheprotein,in SaOS-2 cells most of the precipi-tatedCx43migratedatthe levelofthe nonphosphorylated
pro-tein.
Wealsostudiedthe cellulardistribution ofconnexin43
pro-tein by immunofluorescence. Both the HOB and BMC cells
displayedalinearpunctatedistribution of fluorescenceat the border between adjacent cells that was not detected in cells
incubated with preimmune serum (Fig. 7). In SaOS-2 cells,
specificCx43stainingwasalso seenat the plasma membrane (Fig. 8, a andb),although it was much less abundant than in
nontransformedcells.
ExpressionofCx45inhuman osteoblastic cells. Under high stringency conditions, a dog Cx45 cDNA probe hybridized withRNAisolatedfromthe human osteoblastic cells (Fig. 9). Incontrast totheresultsobtainedwith Cx43, the SaOS-2 cell
line expressed more Cx45 mRNA than did HOB and BMC
cultures. The Cx45 band migratedmore slowly thandidthe
hybridizingband detected in the control rat uterus RNA(Fig.
9), butatthesamelevelasthehybridizingband detected in human heart RNA (Kanter, H.L., and E.C. Beyer,
unpub-lisheddata).
Immunofluorescence studies using an antibody directed
againstacytoplasmicsequenceofCx45 demonstrated that in SaOS-2cells, Cx45waslocatedontheplasmamembrane be-tweenadjacent cells(Fig. 8, cand
d).
Specific Cx45 stainingwas notobserved in HOB, or in BMC cultures (not shown), consistent withthelowlevelof Cx45 mRNA detected in these cells.
Discussion
I
Figure7.Immunofluorescence stainingfor connexin43 in human osteoblastic cells. Human trabecular bone osteoblasts(HOB;AandB), and bonemarrowstromal cells(BMC;C and D) were grown on glasscoverslips, fixed, and incubated with eitherpreimmuneserum(A andC),or
anti-Cx43 serum (B and D), followed by rhodamine-conjugated goat anti-rabbit IgG antibody. Bar=20,m. Gapjunctions form conduitsbetween cells, thereby
estab-lishing intercellularelectricalandchemicalcoupling. The func-tionofgapjunctions incardiacmuscle seemsrelativelyclear, since electrical impulses must be transmitted rapidly forthe heart tofunction as aneffectivepump. In othertissues,where the needfor electrical coupling isnotevident,thephysiologic roleofgapjunctions islessclear.
The skeletal tissue presents several interesting features in termsofthe potentialrolesforgapjunction proteins.Themain physiologicprocesses, boneresorptionandformation,are en-acted byspecificbutstrictlycooperatingcell types, osteoclasts and osteoblasts, respectively. Closeinteraction between these cell types occursduringthecomplex boneremodelingprocess, aphenomenon called "coupling"(40). Although remarkable efforts havebeen expended in theidentification of potential soluble factors that may mediate osteoblast-osteoclast cou-pling,thepossibility of direct cell-cell communication inbone has beengivenlittle attention. Afewmorphologic studies are available demonstratingtheexistence ofgapjunctionsbetween adjacent osteoblasts,osteoblasts and osteocytes andosteocytic processes (13-15), and in culturesofrat calvaria cells ( 16).
Communication between osteoblasts and osteocytes through cytosolicprocessesmay beinvolved inskeletalgrowth regulation. Sinceosteocytesarecompletely embeddedwithin thecalcified bone,they areinanoptimal positionto senseand transduce mechanical forcesthat are applied to the skeleton.
Gap junctions on thecytosolic process may allow the osteo-cytes totransmitthesesignalstothe cells on the endosteal and periostealsurface, and therefore direct the architectural devel-opmentoftheskeleton. Indeed,stretch-activatedion channels have beenidentified on the plasma membrane of bone cells (41 ), and, more recently,propagationofa
Ca2"
signal through osteoblasticmonolayershasbeen demonstratedto occur after mechanicalstimulation(42).Gapjunctional communication mayalso allowthe osteo-blasts liningthe bone surface to work insynchrony duringthe production anddeposition of organic matrix and subsequent calcification. Thus,onecouldspeculate that osteoblasts operat-ing within a boneremodelingunit(3, 43) may befunctionally interdependent via direct cell-cellcontacts,which would pro-vide themechanism fora coordinated actionwithineachsingle unit. Osteoblastsarealso ableto establishchemical coupling withneighboring cells in their environment at anytime during their differentiation.Asdemonstratedbymorphologicstudies (13-15), heterotypic coupling is possible, thus allowing the osteoblaststoexchangesignals withcells on thebone surface.
Analysisof intercellular relationshipsbetweenbonecells is difficult because ofthe structural complexity of this tissue. However,thedifferent cell models employedinthis study al-low someinitial extrapolationstothepossibleroleofgap junc-tions in osteoblasts. These cells undergoarelatively long
differ-entiation process from osteogenic stromal cells ofthe bone
Figure8. Immunofluorescence localization of connexin43andconnexin45in SaOS-2 cells. Adherent SaOS-2 cellswerefixedandstained for eitherCx43 (A andB)orCx45(CandD), followedbyrhodamine-conjugatedgoatanti-rabbitIgGsecondaryantibody.(AandC)preimmune
serum; (B andD) immuneserum.Bar=20,um.
marrowto differentiatedosteoblasts liningthe bone surfaces.
Subsequently,someof the osteoblasts inactively formingbone remain embeddedwithin the calcified tissueandbecome osteo-cytes, theterminal differentiation step alongthe osteoblastic
lineage(3). In ourstudies, both the stromal BMC, probably representinganearlystageof
differentiation,
and themoredif-ferentiated HOB, derived fromcells
residing
onthe bonesur-faces, demonstratedahighdegreeofdye-couplingand expres-sion ofconnexin43. Further differentiationofBMCby incuba-tion with dexamethasone was notassociated with significant
28S
18S
-0 1
I I CO D
Figure9.Expressionof connexin45 mRNA by
human osteoblastic
cells. Total RNAfrom
HOB, BMC, and SaOS-2 cells washybridized
with a32P-labeled
cDNA probe for Cx45,
usingrat uterus RNA as a control.
inhibition ofdye-coupling and Cx43 expression. Therefore,
ourdataseem toindicate that thepresenceof functionally ac-tive gapjunctionsis notdependentonanyparticularphaseof
osteoblastdifferentiation. However, it is possiblethat gap
junc-tional conductance, and/or Cx43 production andassembly intoconnexonsmaybedifferentiallyregulated at each stageof differentiation, perhaps reflecting different functional
require-mentsforgapjunctional communication.
Thisworkdemonstrates thatCx43is the major gap junc-tionprotein expressed by humanosteoblasts, and that expres-sion ofCx43 is correlated with the degree of cell coupling. These resultsareconsistent witha recent studies in rat calvaria cells (17), and in rat osteogenic sarcoma cells (ref. 18, and
Steinberg, T. H., R. Civitelli, et al., manuscript in prepara-tion),allpointingtoCx43asthe gap junction protein responsi-ble for cell-cell communication between osteoblastic cells. Connexin43wasfirstisolatedfromhearttissue(36),but it has been lateridentifiedinanumber of other cell types,including
kidney, brain,ovary, smoothmuscle,and some epithelia(21, 44).The accumulated data on osteoblasts providefurther evi-dence that expression of this gapjunction protein is wide-spread.
Connexin43 mRNAand protein were also expressed by the poorly coupled osteogenic sarcoma cells SaOS-2. Although these cellsexpressedasmuchCx43mRNA as thewell-coupled
immunofluorescence. Although the reason for the dissociation between mRNA and protein expression in these cells is un-known, the relatively low Cx43 protein expression is commen-surate to the degreeofdye-coupling exhibited by theSaOS-2 cells comparedto thenormalosteoblastic cells. This is perhaps the consequence ofan abnormalpatternof oncogene
expres-sioncharacteristic of tumoral cells,andwhichmayinhibitthe
productionof gap junction proteins,asdemonstratedtooccur incellsexpressingv-rasor v-src(45-47). Alternatively, SaOS-2cells may lack an adhesion protein requiredforcellcoupling. The lack of Cx43phosphorylationin theSaOS cells is reminis-cent of the findings ofMusil etal.(48)intheSt180 sarcoma line. These cells appeared to be deficientintheexpression of
adhesion proteins;transfection of thecells withE-cadherin re-sultedin Cx43phosphorylationandintercellular
communica-tion.
Interestingly,the SaOS-2cellsexpressed higheramounts of Cx45 mRNAthandidtheHOB or BMC.Thus,theexpression
ofCx45 inversely correlatedwithdye couplinginthesecells,in
spite ofthe fact that in SaOS-2 cells Cx45wasshownby
immu-nofluorescenceto be localized to the plasma membrane, ina patterncharacteristicof functional gap junctions.These obser-vations suggest that in humanosteoblasticcells Cx45 may not be functional, atleastintheconditions used for these experi-ments. Thisconsideration,coupled to the very lowexpression ofCx45 by HOB and BMC, suggeststhatCx45does notplaya
majorrole in cell-cellcommunicationbetweenhuman osteo-blasts.
Insummary, the present work establishes that human osteo-blasts are functionally interconnected by gap junctions, and that Cx43, themajorgapjunction proteinexpressed by these cells,mediatesintercellular communicationinthesecells. The ability to exchange anddiffusenutrients and regulatory mole-culesthroughout the bone environment may represent an
effi-cient meansof coordinatingthe actionofa variety of cells in a highly complex tissue.
Acknowledgments
The authors areindebtedtoSu-Li Cheng,LeonardRifas,andLindaR. Halsteadforthe humantissuecultures;toElizabeth Hickfor perform-ing immunofluorescence studies; and to Michael Scott and Marilyn Roberts for technical assistance.
This work has beensupported,in part, by National Institutes of
Health grants AR-41255 (R.Civitelli),GM-45815 (T. H.Steinberg),
and HL-45466 andEY-08368 (E. C. Beyer). E.C. Beyer and T. H.
SteinbergareEstablishedInvestigatorsof the American Heart Associa-tion.
References
1.Marcus, R. 1987. Normal and abnormal bone remodeling in man.Annu.
Rev.Med.38:129-141.
2. Parfitt,A.M. 1988. Bone remodeling: relationshipto theamountand structureofbone, and the pathogenesis and prevention of fractures.In Osteoporo-sis:Etiology, Diagnosis, and Management. B.L.Riggsand L. J.Melton, editors. Raven Press, Ltd.,NewYork. 45-93.
3.Rodan, G.A.,andL.G. Raisz. 1990. Cellular basis forboneturnover.In
MetabolicBoneDiseases and Clinically Related Disorders.L. V.Avioliand S. M.
Krane, editors.W. B.Saunders Co., Philadelphia. 1-41.
4.Rodan, G.A.,andT. J.Martin. 1981. Role of osteoblastsinhormonal controlof bone resorption-a hypothesis.CalcifTissueInt. 33:349-351.
5. Silve, C. M., G. T. Hradek, A.L.Jones,andC.T.Arnaud.1982.
Parathy-roidhormonereceptor in intact embryonic chicken bones: characterizationand
cellularlocalization.J.CellBiol.94:379-386.
6. Narbaitz, R., W. E. Stumpf, M. Sar, S. Huang, and H. F. DeLuca. 1983.
Autoradiographic localization oftarget cells for 1,25-dihydroxyvitamin D3 in bonesfrom fetalrats.CalcifTissueInt. 35:177-182.
7.Juppner, H., A. B. Abou-Samra, S. Uneno, W.-X. Gu, J. T. Potts, Jr., and G. V. Segre. 1988. The parathroid hormone-like peptide associated withhumoral
hypercalcemiaofmalignancy and parathyroid hormone bind to the same recep-tor on the plasma membrane of ROS 17/2.8 cells. J.Biol. Chem. 263:8557-8560.
8. McSheehy, P. M. J., and T. J. Chambers. 1986.Osteoblast-likecells in the presence of parathyroid hormone release soluble factor that stimulates osteoclas-tic bone resorption.Endocrinology. 119:1654-1659.
9. McSheehy, P. M. J., and T. J. Chambers. 1987. 1,25-dihydroxyvitaminD3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclas-tic bone resorption. J. Clin. Invest. 80:425-429.
10. Morris, C. A., M. E. Mitnick, E. C. Weir, M. Horowitz, B. L. Kreider, and K. L. Insogna. 1990. The parathyroidhormone-relatedprotein stimulates human
osteoblast-likecells to secrete a 9,000 daltonbone-resorbingprotein.
Endocrinol-ogy.126:1783-1785.
1 1. Thomson, B. M., G. R. Mundy, and T. J. Chanbers. 1987. Tumor necrosis factors a and j3 induce osteoblastic cells to stimulate osteoclastic bone resorption. J. Immunol. 138:775-779.
12. Thomson, B. M., J. Saklatvala, and T. J. Chambers. 1986. Osteoblasts mediateinterleukin-1 stimulation of bone resorption by rat osteoclasts. J. Exp. Med. 164:104-112.
13. Jeasonne, B. G., F. A. Faegin, R. W. McMinn, R. L.Shoemaker,and W. S. Rehm. 1979. Cell-to-cellcommunicationofosteoblasts. J. Dent. Res.
58:1415-1419.
14. Doty, S. B. 1981. Morphological evidence of gap junctions between bone cells.Calcif TissueInt. 33:509-51 1.
15. Doty, S. B. 1988. Cell-to-cellcommunicationin bone tissue. In The Biolog-ical Mechanisms of Tooth Eruption and Root Resorption. Z. Davidovitch, edi-tor. EBSCO Media,Birmingham,AL. 61-69.
16. Shen, V., L. Rifas, G. Kohler, and W. A. Peck. 1986. Prostaglandins change cell shape and increase intercellular gap junctions in osteoblasts cultured from rat fetal calvaria. J. Bone Miner. Res. 1:243-249.
17. Schirrmacher, K.,I.Schmitz, E. Winterhager,0.Traub, F. Brummer, D. Jones, and D. Bingmann. 1992.Characterizationof gap junctions between osteo-blast-like cells in culture.Calcif TissueInt. 51:285-290.
18. Schiller, P. C., P. P. Mehta, B. A. Roos, and G. A. Howard. 1992. Hor-monal regulation of intercellular communication: parathyroid hormone in-creases connexin 43 gene expression and gap-junctional communication in
osteo-blasticcells.Mol. Endocrinol.6:1433-1440.
19. Beyer, E. C., and T. H. Steinberg. 1991. Evidence that the gap junction proteinconnexin-43is the ATP-induced pore of mouse macrophages. J. Biol. Chem.266:7971-7974.
20. Kanter, H. L., J. E. Saffitz, and E. C. Beyer. 1992. Cardiac myocytes express multiple gapjunctionproteins. Circ. Res. 70:438-444.
21. Musil, L. S., E. C. Beyer, and D. A. Goodenough. 1990. Expression of the gapjunction protein connexin43in embryonic chick lens: molecular cloning, ultrastructural localization, biosynthesis, and post-translationalphosphorylation.
J.Membr.Biol. 116:163-175.
22.Gehron-Robey,P., and J. D. Termine. 1985. Human bone cells in vitro.
CalcifTissue Int. 37:453-460.
23. Rifas, L., L. R. Halstead, W. A. Peck, L. V. Avioli, and H. G.Welgus.
1989. Human osteoblasts in vitro secrete tissue inhibitor ofmetalloproteinases
and gelatinase but not interstitial collagenase as major cellular products. J. Clin. Invest. 84:686-694.
24. Cheng, S.-L., L. Rifas, J. Yang, and L. V. Avioli. 1991. Characterization of
dexamethasone-induced human bone marrow stromal osteoblast-like cells. J. Bone Miner. Res.6:S89.(Abstr.)
25. Ashton, B. A., F. Abdullah, J. Cave, M. Williamson, B. C. Sykes, M. Couch, and J. W. Poser. 1985.Characterizationofcells with high alkaline phos-phatase activity derived from human bone and marrow: preliminary assessment of theirosteogenicity.Bone(Elmsford).6:313-319.
26. Long, M. W., J. L. Williams, and K. G. Mann. 1990. Expression ofhuman
bone-related proteinsin thehematopoieticmicroenvironment. J. Clin. Invest.
86:1387-1395.
27.Boland, C. J., R. M. Fried, and A. H. Tashijan, Jr. 1986. Measurement of cytosolic freeCa2"concentrationsin human and ratosteosarcomacells: actions of boneresorption-stimulatinghormones.Endocrinology.118:980-989.
28. Shupnik, M. A., and A. H. Tashijan, Jr. 1982. Epidermal growth factor and phorbol ester action on human osteosarcoma cells: characterization of re-sponsive and nonrere-sponsive cell lines. J. Biol.Chem.257:12161-12164.
29. Civitelli, R., Y. S. Kim, S. L. Gunsten, A. Fujimori, M. Huskey, L. V. Avioli, and K. A. Hruska. 1990. Nongenomic activation of the calcium message system by vitamin D metabolites in osteoblast-like cells. Endocrinology. 127:2253-2262.
31. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isola-tion byacidguanidiniumthiocyanate-phenol-chloroformextraction.Anal. BiO-chem. 162:156-159.
32.Zhang, J. T., and B. J.Nicholson. 1989.Sequenceandtissuedistribution
ofasecondproteinofhepaticgapjunctions, Cx26,asdeducedfromitscDNA. J.
CellBiol. 109:3391-3401.
33. Paul, D. L. 1986. Molecularcloning ofcDNAforratlivergapjunction protein.J.Cell Biol. 103:123-134.
34.Willecke,K., R.Heynkes, E. Dahl, R. Stutenkemper, H. Hennemann,S.
Jungbluth, T. Suchyna, and B. J. Nicholson. 1991. Mouse connexin37:cloning
andfunctionalexpressionofa gapjunctiongenehighly expressedinlung.J.Cell Biol. 114:1049-1057.
35. Beyer, E. C.,K. E.Reed, E. M.Westphale,H. L. Kanter,and D. M. Larson. 1992. Molecularcloning and expression ofratconnexin40,agap junc-tionprotein expressed in vascular smooth muscle.J.Membr. Biol. 127:69-76.
36.Beyer,E.C.,D.L.,Paul,and D. A.Goodenough. 1987. Connexin43:a
protein fromrathearthomologousto agapjunctionprotein from liver. J.Cell
Biol. 105:2621-2629.
37. Paul, D. L., L.Ebihara,L. J.Takemoto,K. I.Swenson, and D.A. Good-enough. 1991.Connexin46,anovel lensgapjunctionprotein,induces
voltage-gated currents innonjunctionalplasmamembrane of Xenopus oocytes. J. Cell
Biol. 115:1077-1089.
38. Burt, J.M.,and D.C.Spray. 1988.Single-channeleventsandgating
behavior of the cardiac gapjunction channel. Proc. Nail. Acad. Sci. USA.
85:3431-3434.
39.Chanson, M., R.Bruzzone,D. Bosco,and P. Meda. 1989.Effects of
n-alcohols onjunctionalcouplingandamylase secretion ofpancreatic acinar cells.J.Cell.Physiol. 139:147-156.
40.Parfitt, A. M. 1982. Thecouplingofbone formation to bone resorption: a critical analysis ofthe concept and ofits relevance to the pathogenesis ofosteopo-rosis.Metab. Bone Dis. & Relat. Res. 4:1-6.
41.Duncan, R. L., and S. Misler. 1989. Voltage-activated and stretch acti-vatedBa2+-conducting channels in an osteoblast-like cell line (UMR 106-01). FEBS(Fed. Eur.Biochem. Soc.) Lett. 251:17-21.
42.Xia, S.-L., and J. Ferrier. 1992. Propagation of a calcium pulse between osteoblastic cells. Biochem. Biophys. Res. Commun. 186:1212-1219.
43. Frost, H. M. 1986. IntermediaryOrganization ofthe Skeleton. CRC Press, Inc., Boca Raton, FL.
44.Beyer, E.C.,J.Kistler, D. L. Paul, and D. A. Goodenough. 1989. Antisera directedagainst connexin43 peptides react with a 43-kD protein localized to gap junctions inmyocardiumandothertissues.J.CellBiol 108:595-605.
45.Atkinson, M. M., and J. D. Sheridan. 1988. Altered junctional permeabil-ity between cells transformed by v-ras, v-mos, or v-src. Am. J. Physiol. 255:C674-C683.
46.Bignami, M., S. Rosa, G. Falcone, F. Tato, F. Katoh, and H. Yamasaki. 1988.Specific viral oncogenes causedifferentialeffects on cell-to-cell
communica-tion,relevant to thesuppressionof the transformedphenotype by normalcells.
Mol.Carcinog. 1:67-75.
47.Brissette, J. L., N. M. Kumar, N. B. Gilula, and G. P. Dotto. 1991. The tumorpromoter 12-o-tetradecanoylphorbol-13-acetate and the ras oncogene modulateexpressionandphosphorylationofgapjunctionproteins.Mol. Cell. Bid. 11:5364-5371.
48.Musil, L. S., B. A.Cunningham, G. M. Edelman, and D. A. Goodenough. 1990.Differential phosphorylationof the gapjunction protein connexin43 in junctional communication-competent and-deficient cell lines. J. Cell Bid.
