Increase in the expression of a family of small guanosine triphosphate binding proteins, rab proteins, during induced phagocyte differentiation

Increase in the expression of a family of small

guanosine triphosphate-binding proteins, rab

proteins, during induced phagocyte

differentiation.

I Maridonneau-Parini, … , M Bornens, B Goud

J Clin Invest.

1991;

87(3)

:901-907.

https://doi.org/10.1172/JCI115096

.

Rab is a newly identified family of small G-proteins that share 35-70% homology with the

yeast Sec4p and Ypt1p involved in the regulation of the secretory pathway. Mature

phagocytes display functions requiring organized intracellular traffic and, for this reason, we

questioned whether phagocyte differentiation could correlate with the increased expression

of rab proteins. Rabbit antisera raised against the recombinant proteins rab1Ap, 2p, 4p, and

6p were able to detect the corresponding proteins in the human monoblast leukemic cell

line U937. When these cells were induced to differentiate into monocyte/macrophage-like

cells displaying functional characteristics of a normal phagocyte, rab1Ap, 2p, 4p, and 6p

were increased and this correlated with an increase in the rab transcripts. Using a rab5

probe, we also observed an increased expression of the rab5 gene in differentiated cells.

Similarly, differentiation of the human leukemic myeloblast HL60 cell line along either

monocyte or granulocyte pathways induced an increased expression of the rab proteins.

Rab proteins were also detected in human neutrophils and in guinea pig alveolar

macrophages. As degranulation is one of the phagocyte functions acquired in the late stage

of differentiation, we investigated whether rab proteins would be involved in this process.

Although rab proteins were tightly membrane bound, none of them was detected in the

specific or azurophil granules purified from human neutrophils. The increased expression

[…]

Research Article

Increase in

the Expression of

a

Family of Small

Guanosine

Triphosphate-Binding Proteins,

Rab

Proteins, during

Induced

Phagocyte

Differentiation

Isabelle Maridonneau-Parini,* Chen-ZhiYang,MichelBornens,*and Bruno Goud

*Centre National dela Recherche Scientifique, Centre deGe'netiqueMoleculaire, 91190 Gifsur Yvette, France,

and Unite de Genetique Somatique (URA CNRS 361), Institut Pasteur, 75015 Paris, France

Abstract

Rabis a newlyidentified family of small G-proteins that share 35-70% homology with the yeast Sec4p and Yptlp involved in the regulation of the secretory pathway. Mature phagocytes displayfunctions requiring organized intracellular traffic and, forthis reason, we questioned whether phagocyte differentia-tion could correlate with the increased expression of rab pro-teins. Rabbit antisera raised against the recombinant proteins rablAp, 2p, 4p, and6p were able to detect the corresponding proteins in the human monoblast leukemic cell line U937. When these cells were induced todifferentiate into monocyte/ macrophage-like cellsdisplaying functional characteristics of a normalphagocyte, rablAp, 2p, 4p, and 6p were increased and this correlated withanincrease in the rab transcripts. Using a rab5 probe, we also observed an increased expression of the rab5 gene indifferentiated cells. Similarly, differentiation of the human leukemic myeloblast HL60 cell line along either monocyte or granulocyte pathways induced an increased ex-pression of the rab proteins. Rab proteins were also detected in humanneutrophils and in guinea pig alveolar macrophages. As degranulation isoneof the phagocytefunctionsacquired in the late stage ofdifferentiation, weinvestigated whether rab pro-teins would be involved in this process. Although rab propro-teins weretightly membrane bound, none of them was detected in the specificorazurophil granulespurifiedfrom human neutrophils. Theincreased expression of rabproteinsinmaturephagocytes suggests thatthey may promote functionshighlydevelopedin these cells. (J. Clin. Invest. 1991. 87:901-907.) Key words: granules * HL60 cells * neutrophils * small G-proteins U937 cells

Introduction

Many of the so-called "small guanosine triphosphate (GTP)-binding proteins" have been recentlyidentified(1,2). Theirmolecular masses are in the 20-30-kD region and they all possess consensus sequencesforGTPbinding and hydrolysis. Little is known about the specificfunctionsof these proteins. However, someof them seem to be involved in the regulation oftheintracellular traffic. Inyeast, Ypt 1 p and Sec4p control twostagesalongthe secretory pathway, between endoplasmic

AddressreprintrequeststoDr.Maridonneau-Parini,INSERMU332, InstitutCochin deGenetique Moleculaire, 22 rueMechain, 75014 Paris,France.

Receivedforpublication 2 May 1990 andinrevisedform16August 1990.

reticulum and Golgi apparatus and between Golgi and the plasma membrane, respectively (3-5). Sec4p is locatedonthe cytoplasmic face of both the plasma membrane and secretory vesicles intransit to the cellsurface (4). YptIpis present in the Golgi region (3). Among the mammalian smallG-proteins al-ready characterized, proteins from the rabfamily share 38-75% amino acid identity with Yptlp and Sec4p (6). In addi-tion, the domain corresponding to residues 35-40, described in p21 ras astheeffector-binding site, is highly conserved between rabproteins, Sec4p and Ypt lp (6). The rabfamilyis the only example of mammalian smallG-proteins possessingthe effec-tordomain homologoustoYpt 1 p and Sec4p. Therefore,rab proteins could also be involved in intracellular traffic. With the exceptionofrab3p,whichseems tobeonlypresent innervous andendocrine cells(7-12),rab lAp,2p, 4p, 5p,and6pappear tobeubiquitously distributed (6, 7).

Maturephagocytesareinvolved indifferent functions such asdegranulation, phagolysosome formation, and directed lo-comotion thatrequire organizedintracellulartrafficand sort-ing ofgranules andvesicles(13, 14). Theinvolvement ofsmall G-proteins inphagocytetrafficwassuggested bythe demonstra-tion that, in permeabilized neutrophils, guanine nucleotides stimulate degranulation through a mechanism distinct from thestimulationof the"largeG-proteins"involved in the trans-duction of activationsignalsacrosstheplasmamembrane(15). Inaddition,recentreports havedescribed thepresenceofsmall G-proteinsinphagocytes.Oneofthem,possiblyrap1,has been found associated with cytochromeb559(16),acomponent of the superoxide-generating enzyme NADPH oxidase. More-over,anovel class of ras-relatedproteins,rac1 andrac2,which are substrates for ADP ribosylation by botulinum toxin C3 ADP-ribosyltransferase, have beenidentifiedinleukemic my-eloblasts (17).Mostof thephagocyteactivities

require

mobili-zation ofintracellular

proteins

(13, 14),and thesefunctionsare acquiredinthe late stage ofphagocyticdifferentiation. There-fore, we questionedwhetherthis could correlate with the in-creasedexpressionofproteinsfromthe rabfamily.Inthe pres-ent study, the expression ofrab proteins was compared be-tween undifferentiated and differentiated phagocytes. The humanmonoblast-like cell lineU937,obtainedfroma histio-cytic lymphoma patient andthe human leukemiccell line HL60, derived from a promyelocytic leukemia, havebeen widely studiedasmodelsfortheunderstanding ofthe molecu-lar events associated with phagocyte differentiation (18). In contrast toU937whichcanonly maturealong themonocyte/ macrophage pathway, HL60 cells are characterized by a bi-modal differentiation potency that can be induced to either granulocytic or monocytic maturation. The synthesis ofrab proteins and the expression oftheir corresponding mRNAs wereincreasedin HL60 or U937 cells induced todifferentiate inphagocytes. Wealso report that, although rabproteinswere foundto bemostlymembranebound, theydonotbelongto phagocyte granules.

J.Clin. Invest.

©TheAmericanSocietyfor Clinical Investigation,Inc. 0021-9738/91/03/0901/07 $2.00

Methods

Cellisolation andcell culture

Humanneutrophils.Bloodwascollected fromhealthydonorsand neu-trophilswereisolated afterDextranT-500sedimentation and centrifu-gation through Ficoll-Hypaqueaspreviouslydescribed(19).Residual erythrocyteswereremoved by hypotonic lysis, andneutrophils were resuspendedinphosphate-buffered solution(PBS).

Alveolarmacrophages.The tracheaof anesthetizedHartley guinea-pigswascannulated andmacrophageswerecollectedby repeatedlung lavageswith sterile PBSat37°Caspreviouslydescribed(20).

HL60and U937 cells. HL60cellswerekindlyprovided by Dr. Petit

(Orsay, France)and U937 cellsby Dr. Polla(Geneva, Switzerland). ThecellsweregrowninsuspensioninRPMI1640supplemented with

10% heat-inactivated fetal calfserum (FCS) (Gibco Laboratories,

GrandIsland, NY),100U/mlpenicillin,and 100ug/mlstreptomycin at37°Cin5%CO2humidifiedatmosphere.

Maturationalongthemonocyticpathwaywasinduced byexposing HL60andU937(2-4 x 105 cells/ml)to20ng/ml phorbol myristate

acetate(PMA) (SigmaChemicalCo.,StLouis, MO)for 4d in

large-cul-ture Petri dishes. Stock solution of PMAwas I mg/ml in dimethyl

sulfoxide (DMSO). Atday2, culturesweresupplementedwithfresh

medium. At the endof thedifferentiation procedure,disheswere washedwith warmCa2'and

Mg2"-free

PBS(supplementedwith0.5

mMPMSF,3 Mg/ml leupeptin,0.6U/ml aprotinin,1 qg/mlpepstatin,

and 0.5 mM EDTA), and adherentcellsweregently scraped with a

rubberpoliceman.Thecellswerethencollectedby low-speed centrifu-gationandresuspended inwarm PBS, pH 7.4,andcellviabilitywas

assessedby trypan blue exclusion(> 85%).

HL60cellswerealsoinducedtodifferentiate insuspensionalong

thegranulocytic pathway by6d ofexposureto 1.3%DMSO (vol/vol),

freshmediumbeingaddedatdays2and 4. The cellswerethen washed

andresuspendedinPBS; the cellviabilitywas>95%.

Superoxide generation

U937 andHL60cellsexposedtoPMAwerecultured in24-wellplates.

Floatingcellswereremovedbywashing the wells twiceandImlofPBS containing50 MMnitroblue tetrazolium(NBT)'wasadded. The cells werestimulated withI uM A23 187(stocksolution: 10-2 Min DMSO) orwith 25 Mg/mlAA at37°C.NBT-positivecellswerecounted 30min

afteraddition of thestimulatingagent.A similarprocedurewasused withDMSO-differentiated HL60exceptthattheywerestimulated in suspension, and NBT-positive cells were counted in a Malassez

chamber.

Insomeexperimentsthe NBTtestwasreplaced by the superoxide

dismutase(SOD)-inhibitingreductionofferricytochromec(19),a

morespecificreactiontocontrol the cellabilityto generateO-.The sameexperimentalprocedurewasusedexceptthatNBTwasreplaced by80 MMcytochromecwithorwithoutSOD. At theendofthe

stimula-tionperiod,the incubationmediumwasremoved, centrifugedinorder

toeliminateanydetached cells and the supernatantsweremeasuredat

550nminadouble-beamspectrophotometer (Uvikon, Kontron, Swit-zerland) againstthe supernatantsobtained fromcellsincubated inthe

presenceof SOD (19). The sameexperimentswereperformed with

undifferentiated cells.

Cytometric analysis

U937 andHL60cellsexposedtoPMAweredetached from flasksby scrapingandprepared for DNAcontentanalysis accordingtothe

method described by Vindelov (21). 0.25 ml ofcell suspension

(- 107/ml)inTris buffer(100mMNaCl, 100 mMTris,pH7.6)was

addedto2 mlofice-cold solution A(10mMglycine,300 mMNaCI,

0.1% Triton X-100, pH 10),0.1mlboiled RNAsesolution(1%),and50 1.Abbreviationsused inthispaper:GAPDH,glyceraldehyde 3-phos-phate deshydrogenase; NBT, nitrobluetetrazolium;PMA, phorbol myristateacetate;SOD,superoxidedismutase.

,ulethidium bromide solution

(0.2

mg/ml).

Tubeswereinverted three times and keptat4°C. Relative DNAcontent wasassayedon aflow cytometer (Epics V, Coulter Electronics, Inc., Hialeah, FL) usingan argon laser (Spectra Physics Inc., Mountainview, CA) at 488 nm, 400 mW, and measuring the fluorescence above 590 nm.Some experi-ments were performed by replacing ethidium bromide with Hoechst 33342 (4,g/ml,final concentration) and using an argon laser at 351 +364 nm; the blue fluorescence of individual nucleiwasassessed at a flow rate of 1,000 objects s-' inaflowcytometer. Cell-cycle compart-ments were calculated from histograms using PARA 1 software (Coulter Electronics, Inc.) based upon an improved peak-reflect algo-rithm.

Cellfractionation

The method used has been described elsewhere (22). Briefly, neutro-phils suspended in relaxation buffer (100 mM KCI, 3 mM NaCl, 10 mMPipes, 3.5 mM MgCI2, andImM ATP, pH 7.3) were cavitated at 375 psi for 20 min in a nitrogen bomb at 4°C and collected in a tube containingEGTA (6 mM, finalconcentration), and the nondisrupted cells, the nuclei, and debris were removed by centrifugation at 400 g for 10 min at4°C. The supernatant was layered onadiscontinuous Percoll gradient and centrifuged at 45,000 g for 20 min at 4°C. Four fractions were obtained corresponding to the cytosol, cell membranes, and spe-cific and azurophil granules. Each fractionwascollected and centri-fugedfor 90 minat245,000gtoremovethe Percolland, except for the cytosol, fractionswereresuspended in 0.34M sucrosebuffered with 10 mMPipesatpH 7.0. Membranous and cytosolic fractions from U937 cellswereobtainedasdescribed above except that nitrogen cavitation wasperformed at 250 psi for 15 min at 4°C.

Immunoblot

analysis

Proteinsfrom intact cells or subcellular fractions were dissolved in sodium dodecyl sulfate(SDS)-samplebuffer, heated at 100°C for 10 min, separated on SDS-PAGE (12% polyacrylamide slab gel) using the Mini-Protean II cell(Bio-Rad Laboratories, France) and transferred to nitrocellulose using the MultiphorIINovablot system asdescribed by thesupplier (LKB Produkter, Bromma, Sweden). After saturation with 1% gelatin, 0.5% bovine serum albumin (BSA) in 150 mM NaCl, 0.1% Tween 20, and 10 mM Tris, pH 7.4, the nitrocellulose sheetswere subsequentlyincubated with rabbit polyclonalantibodies(1:500 dilu-tion)followed by incubation with1251-proteinA(0.1MuCi/ml)and were analyzed byautoradiography. Eachgel lane was loaded with 25 ,g protein (the protein concentration was measured according to Brad-ford[23])orwith 50 ng of recombinant rabproteins.Thelinear range of these assays has been monitoredusingrecombinant rabproteinsand wasfoundto coverthe rangefrom 5 to 250 ng.

Antibodies

Polyclonal rabbit antibodiesweregeneratedagainst humanrab pro-teins produced inEscherichia coliandpurifiedtohomogeneity by col-umnchromatographyasdescribedbyZahraouietal.(6).RabIAp has beencoupledtokeyholelimpet hemocyaninwithglutaraldehyde(24). Theimmunization schemewasessentiallyasdescribedby Louvardet al.(25).

Northern blot

analysis

Total RNAs from undifferentiated and PMA-differentiated U937 cells were extractedwith4 Mguanidinium thiocyanateandpurifiedby cen-trifugationthrough5.7 Mcesium chloride(26).Theconcentration of RNAwasmeasuredby absorbanceat260 nm; 10MgRNAper well wereelectrophoresed in 1%agarose gels containing 2.2 M formalde-hydeandtransferredto anylon membrane (Hybond, Amersham, Bucks., UK)by thecapillaryblotmethod in 20XSSC(IxSSCis 0.88% NaCI, 0.44% sodiumcitrate,pH 7). The membranewasprehybridized

rl)

- C*4 v

(9

.0 .0 .0 .0

L. L. E..

.0 co

C

Cl)

.0 .0 .0

C-o

.0

U/)

(9 qt

<

C*4 (

'_,

.0::

(a

Sw6 M 0 L

Figure 1.Characterization of the specificity of the rabbit immunesera against rabproteins by

immunoblotting.Theimmunesera (IS) wereobtainedasdescribedin the Methods section. 0.5 ytg of recombinant rabproteinswas

loaded onthegelexceptfortherab protein against which the tested IS wasdirected (50ng wereloaded). As anexample,whenIS anti-rab A was tested, 50 ngof recombinant rablApwereloaded and 0.5 Agof rab2p, rab4p, andrab6p.The proteins were runon a 12% SDS-PAGEgelandtransferred electrophoreticallytonitrocellulose. Thefilterswerethenincubated with IS anti-rab1,anti-rab2,anti-rab4 (1:500). Bandswerevisualizedusing

'251-protein

A.

Co.) andpolyvinylpyrrolidone, 0.5% SDS, and 100

Ag/ml

salmon sperm DNA.Themembranewasthenhybridized for24 hwithcDNA probes labeledwith[a-32P]dCTP(sp act, 2x 106cpm/ml). The probe obtained fromthe ratrab 1 cDNAis thesame asin Zahraoui (6).The following probes obtained fromthe ratrabcDNAs were used:rab2,a 900-pb EcoRI fragment containing 20 bp ofthe3'noncoding region and50bp ofthe 5'noncoding region; rab5,a 1.3 kbpEcoRI-PUVII fragmentcontaining 800 bpfromthe 5'noncoding domain. Rab6 probe, aEcoRI fragment containing 1.4kbp with 220 bpfromthe 3' and 550 bp from the 5' noncoding regions, was obtained from the human rab cDNA. Afterhybridization,themembraneswerewashed twiceat room temperaturefor15 min in 2xSSC supplementedwith 0.1% SDSandoncefor60 min at65°C in 0.5x SSC supplemented with 0.1% SDS. The membraneswerethenexposedto ahyperfilm-MP (Amersham) at -80°C. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)mRNAlevelswere used as aninternalstandardusing the probedescribedelsewhere(27). Thesignalsobtainedbydensitometric scanning of autoradiogramswereintegrated using thevisILoGsoftware (Noesis, France).

Results

Presence

of

rab proteins in alveolar macrophages and neutro-phils. Usingpolyclonal antibodies, we have analyzed the ex-pression ofrab proteins in guinea pig alveolar macrophages and humanneutrophils.Inthat the membersofthe rabfamily share 30-45%amino acididentity(theregion ofbest homology beingthedomain involvedin GTPbinding/hydrolysis)(6), we first tested the specificity of the immune sera generated against eachof thepurifiedrabproteinsexpressed inE.coli. As shown inFig. 1, antiseraagainstrabiAp andrab2p recognized only rablApandrab2prespectively. The monospecificity of the an-tiserum anti-rab6p has been reported elsewhere (28). Anti-serumagainst rab5pwas notspecificenoughtobeused. Anti-serum against rab4pfaintly recognized purified rab2p. How-ever, in cell lysates, no signal was detected atthe migration positionof rab2p (Fig. 2) withanti-rab4pantiserum probably because the level of endogenous rab2pwas lowcompared to

theamountof recombinantrab2p(0.5 ,ug)appliedtothegelin Fig. 1. In addition, none of the antisera recognized purified Sec4porp21ras(data notshown), furtherindicatingthat the antibodieswere notdirectedagainstthehighlyconserved GTP binding/hydrolysisdomainscommontothe rab and the other ras-related proteins.

.0

Q

LM

.

>

CM

Q. Q

Lo

(0

.0

Co

0

>

97o

*SF

Figvure2.Immunoblotanalysis ofrabproteinsinguineapigalveolar macrophages.RecombinantrablAp,rab2p,rab4p, andrab6p (50 ng) (p)andproteins (25,g/well)fromtotalcelllysates(/y)wereseparated

on a12%polyacrylamide-SDSgel, transferredtonitrocellulose,and probedwith immune seraagainst rablAp,rab2p, rab4p,andrab6p respectivelyas in thelegend ofFig.1.Molecularweightstandardsare

shownontheleft (X10-3).

a

?'-

a

>.

..

*0

TableI.Generation ofO° byDifferentiatedU937 andHL60Cells

°i Differentiating

Cell line agent AA A23187

nmol/l06cells

U937 None UD UD

HL60 None UD UD

U937 PMA 5.81±1.28 7.23±1.07

HL60 PMA 2.78±0.72 0.37±0.09

HL60 DMSO 2.11±0.93 ND

U937 andHL60 cellswereexposed for4 dto20ng/ml PMAorfor 6 dto 1.3%DMSO. The cellswerestimulated for 20 min with25 ,ug/mlAA orwith 1 ,uM A23 187 and the reduction ofcytochromec

wasmeasured inthepresence(reference cuve)and in the absence (sample cuve) of SOD inadouble-beamspectrophotometer. Abbre-viations: ND,notdetermined; UD, undetectable.

Westernblot analysis showed that the antisera directed against rablAp,rab2p, rab4p,and rab6p reactedwithproteins fromtotalmacrophage lysates (Fig. 2). It should be pointed out thattheendogenous rab proteins, especially rab6p, migrated

slightly

fasteron SDS-PAGEthan thecorresponding proteins producedinE.coli,suggesting thatposttranslational modifica-tionsmayoccurin mammalian cells. Inaddition, the minor bandsdetected bytheimmunesera were alsodetected by the preimmune sera (data not shown). Immunoblots performed with lysates from human neutrophils gave results similar to alveolar macrophages(data not shown).

Expressionofrab proteins during induced myeloid cell

dif-ferentiation.

We then attempted to determine whether the ex-pression ofrabproteinsvaries as afunctionof phagocyte differ-entiation.Toaddressthis question, U937 andHL60celllines wereinducedtodifferentiateusing PMA (20 ng/ml) orDMSO (1.3%). The terminal maturation ofthe two celllines was estab-lished by (a) their superoxide (O°)generating capacity as mea-suredbyNBT or cytochrome creduction,(b)their adhesion to the substratumwhentheydifferentiate intomonocyte, macro-phage-likecells, and (c) theirarrestin theG0-G1 phase of the division cycle. Undifferentiated HL60orU937 cells were un-abletogenerate

O2

or to adhere. U937 orHL60cells exposed toPMAdifferentiated into monocyte-likecells. They became

0

0

<~~~

hm

OC

ca.

HL60

adherentafter 24 h and the maximal number ofNBT-positive cells was reached after 2 d for U937 cells(91±5%ofadherent cells, n = 5) and 3 d for HL60 cells (87±6%, n = 3). HL60 cells exposedto1.3% DMSO were inducedtodifferentiate in suspen-sion along the granulocytic pathway and required 5 d to reach the maximal number of NBT-positive cells (78±6%, n = 3). To study the expression of rab proteins in cells that have termi-nated thedifferentiation process, we worked with U937 and HL60 cellsexposed to PMAfor4d or toDMSOfor 6 d,sinceat this stage, theyfully express a typical phagocyte function

(02

generation) andthey are arrested in the GO-G 1 phase ofthe cell divisioncycle. The generation ofO2 was measured upon stimu-lation with the calcium ionophore A23187

(10-'

M) or with AA (25 ,g/ml) (Table I). Undifferentiated and differentiated cells were analyzed by flow cytometry. The following results areexpressed asmean±SD, n = 3: 53±7% of undifferentiated U937 cells were in GO-GI phase, 35±6% in S phase, and 12±3% in G2-M phase. Undifferentiated HL60 cells were 48±6% inGO-G1 phase, 32±5% in S phase and 20±5% in G2-Mphase. Exposure to PMAfor4dincreased theproportionof U937 cells in theGO-G 1 phase (92±5%). Similarly, inhibition of cell growth was observed with PMA-differentiated HL60 cells, 91±4% of the cellsbeingin the G0-G1 phase. The re-maining 8-9% corresponded to bi- or polynucleated cellsas observedby optical microscopy.

When equal amountsoftotalproteinsfrom undifferen-tiated and PMA- or DMSO-differentiated HL60 cells were loaded(Fig.3), we observed that rablAp,2p, 4p, and6p were expressed in nondifferentiated cells but their amounts were higherin mature cells. A similar increase in the expression of rabproteinswasdetectedin lysatesfromU937 cellsinducedto differentiate with PMA(Fig. 3).Inundifferentiated U937cells, rab6pwasdetectableafteralongertimeexposureof the X-ray film.

Asthe smallGTP-binding proteinsappear toexistin both membrane-bound and solubleforms(29), weinvestigatedthe immunoreactive signals in themembrane and cytosolfractions prepared from mature and immatureU937 cells. The signal washigherin the membranefractionfrom PMA-treated cells than inundifferentiatedprogenitorcells(Fig. 4).Rabl Ap and rab6p wereweakly detected in the cytosol fraction (Fig. 4). These resultsindicatethat theincreasedamounts ofrab pro-teins inmaturephagocytesaccumulate in the membrane frac-tionand not inthecytosol.RablAp, 2p,4p, and 6p aretightly bound to the membrane since treatment ofthe membranes

0

< c/

.0

_o

.a~~~~~~~O .. .

X, _41j

",...vow

U937I_p

Figure3. Effect of induced phagocyte differentiationonthe expressionof rabproteins. Equal amountsofproteinsfrom nondifferentiated(nd)andPMAor

DMSO-differentiated HL60 cell lysatesweresubjectedto

immunoblottingasdescribed inthe legend ofFig. I in paralleltothe recombinant rablAp,rab2p, rab4p, andrab6pproteins (50 ng).Similar experimentswereperformedwith U937cellsexceptthattheywere not

a

z

a

E

0..E

rablA

rab2

I

rab4 m

rab6

Figure4.Distribution ofrabproteins between thecytosolandmembrane fractions in nondifferentiatedU937 cells(ND) andPMA-induced U937 celldifferentiation (D). Cell cavitation * wasperformedin a nitrogen bomb.

Cell membranes (m)andcytosol(cyt) wereseparated bydensity

El centrifugationon aPercollgradientas

described in the Methods section. The proteins(25,ug/well)were

immunoblotted using immune sera againstrablAp, 2p,4p,and6p.

with the chaotropic agent urea (6 M), orwith 0.2 Msodium carbonate(pH 11), orhigh saltconcentration(1 MNaCl) did not induce thedissociation ofrab proteins. However, mem-brane exposure to 1% Triton X-100 for 2 min at 4°Cfully solubilizedthe rabproteins(data not shown).

Theaccumulationofrabproteinsin thedifferentiatedcells could be the resultof changesatthetranscriptionalor transla-tional levels. In PMA-differentiated U937 cells, the level of rabl,2,5, and 6 mRNAsincreasedgreatly(Fig.5). Rab4 probe gavenonspecificbandsmaking it uselessinthisstudy (data not shown). Asobserved in human fibroblasts (6), rab1 cDNA probe revealed two transcripts, rab2 probe detected three mRNAs, and rab5 and rab6 were each expressed as a single

mRNA (Fig. 5). We verified that theamount oftransferred mRNAdid not vary significantly betweenthe nondifferen-tiated and the differennondifferen-tiatedcellsusingthe GAPDHprobe(Fig. 5). Toevaluate the relative increaseinrabtranscriptsin differ-entiated cells, densitometricscansofNorthern blotswere per-formed. The relative level ofthe low molecularweight tran-script of rab1 wasincreased3.8-foldin differentiatedcells,the secondtranscriptwasincreased2.2-fold. Rab2 mRNAswere increased 1.9-, 3.7-,and 3.0-fold for thelow, intermediate,and high molecularweight transcripts, respectively. Rab5 mRNA was increased 10.1-foldand rab6 mRNA 2.6-fold. These re-sults suggest that the increase in rabproteinsinmature phago-cytes isregulatedatthetranscriptional level.

Rablp, 2p, 4p, and 6p are notassociated with

neutrophil

granules.Oneofthemaincharacteristics ofphagocyte differen-tiation is the accumulation of secretorygranulesandthe devel-opment offully active stimulus-secretion coupling mecha-nisms. We wondered whether rabproteins mightbelocatedon granules and, therefore, involved in the control ofregulated secretion. Sinceinduced maturation of HL60 cells failsto de-velopspecific granules ( 18), wedecidedtoinvestigatewhether rabproteins would be locatedat thegranulelevel in human neutrophils. Usingapreparationthatprovidesahigh level of purification of

specific

and

azurophil granules (22),

we ob-served that none oftheantisera

against

rab

proteins

reacted with both granule types(Fig. 6) obtained from differentblood donors. Rab I Ap,2p, 4p,and6p appearedtoaccumulateinthe membranefraction,aweaksignal beingdetected in the

cytosol

with thefourantisera. Itshould bepointedoutthattwo addi-tional bandsmigratingmoreslowlythanrab2pwerealso de-tectedwiththe antiserum in the membrane fraction from

hu-N

LO

(

CO QO QO

m co

1

z

a

z

a

z

a

z

*-~~~~~~N-S

Figure5. mRNAexpression of the rabgenesin nondifferentiated (ND) and PMA-differentiated U937 cells(D). Total RNAs from

nondifferentiated cells and from cells inducedto

differentiate by4-d exposure to PMA were electrophoresed (10mgperwell),blotted to a

nylonmembrane, andhybridized withrabl, rab2, rab5,rab6, andGAPDHprobesas described in the Methodssection. The membraneshybridized withtherabprobeswere

subjectedtoautoradiographyat-80°C for16 hand the membraneshybridized with GAPDH probe wereexposed for30 min.Positions of28S and 18S ribosomalRNAsareindicatedonthe left.

L.

I

0.

CD

0

a0z

0

28S

*

o E 3

N Figure6.Distributionof rab

proteins

between

cytosol,

membrane,and

rab1Ap~

. * "'

specific

and

azurophil granules

from

human

neutrophils.

Postnuclear supernatant from

disrupted

human rab2 3

neutrophils

wasfractionated

_by

density

centrifugation

ona rab4 l * discontinuous Percollgradientas

describedin Methods. Proteins from . eachfraction

(25

,ug perwell)were

rab6 * immunoblottedwith immunesera

against

rablAp,

rab2p,

rab4p,

and

rab6p.

man

neutrophils (Fig. 6).

These bands werenotobserved in U937 and

HL60

cells;

wedonot knowatpresent what

they

represent.

Discussion

Inthisarticlewe_{report that rab}mRNAsand rab

proteins

are

more

highly

_expressed

inmature

phagocytes

thanintheir

pro-genitor

cells. The

_proteins

arefoundinbotha membrane-asso-ciated and

_cytosolic

form in undifferentiated and

differentiated

cells,

the membrane-bound form

being predominant

before andafter

phagocyte

differentiation. This

induction

of the

ex-pression

of rab

proteins

in

_myeloid

cells

_acquiring

the

pheno-type

of

_phagocytes

wasobserved

using

eitherPMAor

DMSO,

suggesting

thatthis

induction

ismore

likely

torepresentastep in the

differentiation

programthana

_{pharmacological}

effect of the

_{differentiating}

_agent.

The increase in the

expression

of small

_G-proteins

isnota

_general

feature

associated with

phago-cyte differentiation

since

_transcripts

fromrac1a

_(a

small

G-pro-tein C3 botulinum toxin

_substrate)

increased in thecourseof

HL60

cell

differentiation

anddecreased in

maturing

U937 cells

(I17).

In

_addition,

theincrease in rab

expression

doesnot_appear to be a

_{general phenomenon}

associated with cell differentia-tion.

Indeed,

rab

_transcripts

are notincreased when mouse B

lymphocytes

differentiate in vitro into

IgG-secreting

cellsupon stimulation with

_{lipopolysaccharides}

_(C.

Z.

_Yang

andB.

Goud,

manuscript

in

_{preparation).}

These results suggest that the in-creased

expression

of rab

proteins

in

differentiated

phagocytes

may be associated with functions

highly developed

in these cells.

The_presenceofsomeother small

G-proteins

hasbeen

re-cently

describedin

_phagocytes.

Inhuman

_neutrophils,

a pro-teinfromtheras

superfamily,

_possibly

_rap-l,

hasbeenshown

tobeassociated with

_cytochrome

b559

_(16),

a_{component of}

the

_{02-generating}

enzyme NADPHoxidase. In

addition,

two

nonidentifiedsmall

_G-proteins

have beendescribedto

copurify

withthe membrane_receptortothechemotacticagent FMLP from

differentiated HL60

cells

_(30).

Itis

_interesting

to notethat

cytochrome

b559and_{FMLP receptor}are

poorly expressed

in

undifferentiated

phagocytes (18, 31).

In mature

_neutrophils,

both

_cytochrome

b559 and FMLP receptorare, forapart, stored in the

_{specific granules}

and are translocated tothe

plasma

membrane

_during

the

degranulation

process

(22,

32,

33).

This

suggests

that,

in

_parallel

totheincreased

expression

of

cytochrome

b559and thechemotacticreceptor

along

phago-cyte maturation, their associated small G-proteins may also increase and control either theinsertionoftheirassociated pro-teins into theplasmamembrane(i.e., cytochrome b559hasto beatthe end of the electron transport chain NADPH oxidase [34])or toparticipateinto the fusion of thegranules with the plasma membrane upon phagocyteactivation. Phagocyte dif-ferentiation appearsto correlate with both theacquisition of degranulationcapacity (13,18)and with theincreased expres-sion of rabproteins (this work)whicharecandidatesfor partici-pating in the intracellular traffic.Therefore,it waspossible that one(or more) of the rabproteins may be located on the gran-ules.However,noneof the rabproteinsstudied herewas asso-ciated witheither specific or azurophilgranules purified from human neutrophils. This suggests that small G-proteins dis-tinctfrom rab proteins probably remain to be characterized on specific and azurophil granule membranes.

At present, noneof the physiological functions of the rab proteins has been precisely determined in mammaliancells. Since they share high homology with Sec4p and Yptlp, it is likely that they participate in the regulation ofintracellular traffic. This hypothesis is sustainedby the resultsrecently ob-tainedby several laboratories on the subcellular localizationof rabproteins.Rab lAp, which is the mammalian homologue of Yptlp(6, 35), a protein apparently required for protein trans-port from endoplasmic reticulum to Golgi complex (36), is probably associated with the Golgi apparatus (3, B. Goudand N.Touchot,unpublished observation).Rab6p has been found tobeassociated with the surface of Golgi cisternae, probably medial and trans, in a wide variety of animal cells (28). Rab l Ap andrab6p are probably associated to the Golgi appara-tus inhumanneutrophils since Golgi membraneswerepresent inthe membrane fraction obtained by centrifugation on Per-collgradient (37) as demonstrated by galactosyl transferase(a specific Golgi marker) immunoreactivity usingan antiserum kindly provided by E. Berger(Zurich, Switzerland; data not shown). Similarly, in U937 cells induced to differentiate with PMA, rab6pwasimmunofluorescently detected intheGolgi area(unpublished data). The very recent observation that syn-thetic peptides analogous to the rab effector domain inhibit vesiculartransportbetweenendoplasmic reticulum andGolgi apparatus and betweenthecompartments oftheGolgiin semi-intactcells(38) furthersupportthepotentialroleof rab pro-teins in vesicular transport in mammalian cells. Rab2p has been localized to an intermediate compartment between the endoplasmicreticulum and theGolgiapparatus andrab5phas beenfound associated withthe cytoplasmicface of both the plasma membrane and early endosomes (39).The intracellular localization of rab4p is still unknown. The distinctsubcellular localizations oftheproteins fromthe rabfamily is inagreement withthe modelofBourne(29) proposingthat different trans-portsmallG-proteinsmay ensurethe unidirectionaltransfer of carrier vesicles between twosubcellular compartments. The high expressionin maturephagocytes of theubiquitousrabl p, 2p,4p,6p,andprobablyrab5psuggests thattheymay promote functionshighly developedinthese cells. Apossiblerole for rab proteinswould betheirinvolvement inthe processof mem-brane

recycling. Ordinary

membranerecyclingoccurs

membrane mass, via vesicles from theGolgi apparatus,atthe regionofthe cell surface thatbecomes theleading edge ( 14, 41). Cells that cannotpolarizetheir membranerecyclingare

gener-allyunable to locomote(14).Wesuggestthat rabprotein over-expressionin phagocytes could reflect theirinvolvement in eachstepof the very active process ofmembranerecycling.

Inconclusion,ourdatasuggestthatthe increased transcrip-tion of rab genes could be one aspect of the differentiation program ofphagocytes. In addition, theacquisition of well-characterized cell functions by maturing phagocytes may render this cell modelausefultool in theunderstandingof the physiologicalrole of thesmallG-proteinsfromthe rabfamily. Acknowledgments

We are_greatly indebted to NicolasTouchot, AhmedZahraoui,and

Armand Tavitian for thegiftof rabcDNAs and therecombinantrab

proteins.Wegratefully acknowledge BrigitteOlofsson forprovidingthe

GAPDH_{probe; Spencer}Brown andDominiqueMariefor theirexpert assistanceinthecytometry;Eric G.Bergerforthegiftof theantiserum

againstgalactosyl transferase;and Babette B.Weksler,Armand

Tavi-tian,and_{Mary McCaffrey}for criticalreadingof themanuscript.

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