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A Plasma Membrane ProteinIsInvolvedinCellContact-mediated RegulationofTissue-specific Genes inAdult Hepatocytes

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We have identified theliver-regulating pro- tein(LRP), a cellsurfaceproteininvolvedinthe maintenanceofhepatocytedifferentiation when cocul- turedwithratliverepithelial cells(RLEC) .LRP was definedby immunoreactivityto a monoclonal antibody (mAb L8)
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  A Plasma Membrane Protein Is Involved in Cell Contact-mediatedRegulation of Tissue-specific Genes in AdultHepatocytes Anne Corlu, * Bernard Kneip, * Corinne Lhadi, * Geneviève Leray, * Denise Gaise, * Georges Baffet, Domnique Bourel,t and Christiane Guguen-Guillouzo* *INSERM U49, Unité de Recherches Hépatologiques ; and t CentreRégional de Transfusion Sanguine, Hôpital Pontchaillou, 35033 Rennes Cedex, France Abstract   We have identified the liver-regulating pro- tein (LRP), a cell surface proteininvolved inthe maintenance ofhepatocyte differentiation when cocul- turedwith rat liver epithelial cells (RLEC)   LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC   mAb L8 specifically detected two polypeptidesof85 and 73 kD in immu- noprecipitation ofboth hepatocyte- and RLEC- iodinated plasma membranes   The involvement of thesepolypeptides, which areintegral membrane pro- teins, in cell interaction-mediated regulation ofhepato- cytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC   Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping C ELL-cell interactions are offundamentalimportance in the development and organizationof multicellular organisms and in their physiology and pathology   The general assumption is that these interactions are medi- ated by cell adhesionmolecules, substrate adhesion mole- cules, and cell junctional molecules   All are morpho-regula- tory molecules during the early stagesof development and permssively regulate primary processes such as cell move- ment and division   They give rise tothe signals which leadto differential gene expression and thusto embryonic induc- tion   Various histogenetic examples support this proposal (Gallin etal   1986 ; Edelman, 1987 ; Jessel, 1988)   How- ever, the molecular mechanisms involved in these coordi- nated regulationsare still poorly understood   Cell-cell communications also playa veryimportant role in maintaining the differential gene expression of the mature cells ofadult tissues   Pertinent examples are provided by the central and peripheral nervoussystems (Rathjen et al   1987 ; Seilheimer et al ., 1989)   An interesting example is also provided by the adult liver   Disrupting livertissue to isolate hepatocytes leadstoadecline in the transcription of most liver-specific genes (Clayton etal ., 1985)   Furthermore, es- tablishment in culture of homotypic interactions between he- patocytes failsto preserve their adult phenotype (Jefferson etal ., 1984),whileproteoglycans induce gap junction ex- © The Rockefeller University Press, 0021-9525/91/10/505/11  2 .00 The Journal of CellBiology, Volume 115, Number 2, October 1991505-515 gene expression, cytoskeletal organization and deposi- tion of extracellular matrix (ECM)   An early cytoskeletal disturbance was evidencedand a marked alteration ofhepatocyte functional capacity was ob- served in the presenceof the antibody, together with aloss of ECM deposition   By contrast, cell-cell aggre- gation or cell adhesion to various extracellular matrix components werenot affected   These findings suggest that LRP is distinct from an extracellular matrix receptor   The fact thatearly addition of mAb L8 dur-ing cell contactestablishment was necessaryto be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotalrole in the coordinated metabolicchanges which lead to the differentiated phenotype of mature hepatocytes   pression and restoretranscription of tissue-specific mRNAs in primary cultures (Fujita etal ., 1987)   This suggested a po- tent rolefor nonparenchymal cells on the differential tran-scription of liver-specific genes in adult hepatocytes   Dfferent nonparenchymal epithelial cell lines have been established from bothneonataland adult ratlivers that show phenotypic simlarities to oval cells (Grisham1980 ; Fausto etal ., 1987)   Moreover, they canexpress some ofthe func- tionscharacteristic of hepatocytes (Germain etal ., 1988)   The exact srcin of these cells in theliver is still incompletely solved   However, according to Fausto (1990) they can be de- finedasa population of stemlike cells, most likely originat- ing from bileductules,that have retained their broad devel- opmental capacities   Several cell lines (rat liverepithelial cell [RLEC]') with simlarcharacteristics were obtained in our laboratory (Morel-Chany etal ., 1978)   We have shown that the establishment of cell-cell contacts between adult hepatocytes and these nonparenchymal epithelial cells,al- lows hepatocytes torestore cell polarity and to maintain their functional activitiesfor several weeks (Guguen-Guil-louzo etal ., 1983)   An increasedexpression of liver-specific 1   Abbreviations used inthis paper   CAM, cell adhesion molecule ; ECM, Extracellular matrix ; EHS, Engelbreth-HolmSwarm mouse sarcoma tu- mor ; LRP, liver regulatingprotein ; RLEC, ratliver epithelial cell   505   on S  e p t   em b  er 1 4  ,2  0 1  5  j   c  b .r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published October 15, 1991  genes was observed, resulting, at least in part, from activa- tion of transcription (Fraslin etal ., 1985)   In addition,secre-tion and deposition ofvarious matrix components gave rise toa complex extracellular matrix (ECM) network which sur- rounded and coveredhepatocyte colonies   Interestingly, both liver-specific gene activation and matrix deposition occurred in a coordinated manner (Baffet et al 1982)   Furthermore, it has been established that cell-cell contacts with RLEC, but not via gap-junction communications, are requiredwhile soluble factors did not seem to be involved to any major ex- tent (Mesnil etal ., 1987)   Therefore, this hepatocyte cocul- lure system represents auseful model to study thecausal re- lationship between the different biological events leading to stable differentiation in adult liver   Inthe present study, we haveexplored the possibility that a plasma membrane protein expressed on RLEC mght playa critical role in mediating cell-cell recognition and leading to increased tissue-specific gene transcription   We found that a mAb directedagainst an RLEC cell surfaceprotein in- hibited liver-specific gene activation when added early to hepatocyte-RLEC cocultures, and concomtantly altered matrixdeposition and cytoskeletonorganizationin hepato- cytes   This report describes in some detail this biological phenomenon and analyzes its specificity   It presents evidence for the involvement ofa novel plasma membrane liver- regulating protein (LRP)   Materials and Methods Reagents Lamnin-entactin complex, lamnin,heparan-sulfate proteoglycan, and col- lagen IV were extracted from the Engelbreth-Holm-Swarm mouse sarcoma tumor(EHS) (Kleinman et al ., 1982)according to Timpl et al   (1979) wth slight modifications (Hassel et al ., 1985)   Fibronectin was from Boehringer Mannheim (Meylan, France)   Hybridization probes for mRNA analysis were as follows   pRSA8 for al- bumn gene (Sargent et al   1979), A4C9 for aldolase B gene (Simon et al ., 1983)and procollagen a,  I (Cavel etal ., 1989)   Anticytokeratin 8 and 18 antibodies (anti-CK55 andanti-CK49) of known specificity (Leroux-Ncollet et al ., 1983) were a gift of Dr   N   Mar- ceau (HotelDeu, Quebec)   Cell Isolation and Culture Adult normal hepatocytes were obtained by perfusing rat livers (Sprague- Dawley ; 150-200 g) wth 0 .025   collagenase solution (Boehringer Mann- heim) buffered wth 0 .1 M Hepes (pH 7 .4) accordingto the method ofSeglen (1973) wth slight modifications (Guguen et al ., 1975) . Adulthepatocytes were maintained eitherin pure culture or in coculture   For pure culture, hepatocytes were plated ina mxture of 75 MEM and 25 medium 199, supplemented wth 10 FCS and containingper m   penicillin (100 IU), streptomycin sulfate (100wg), bovine insulin(5 pg), and BSA (1 mg)   Themedium added wth 7 x 10 -5 M hydrocortisonehemsuccinate, was renewed 4 h later and everyday thereafter   In some as- says, pure cultures of hepatocytes weremaintained in a medium containing 25 mM nicotinamde(Paine et al ., 1979)   Cocultures wereprepared according to the conditions previouslyde-scribed (Guguen-Guillouzo et al ., 1983)   Briefly, 4h after hepatocyte seed- ing, the medium was discarded and nontransformed RLEC or other cell types, suspended inafresh medium were added   24 h later, the medium was supplemented wth 7x 10-5 M hydrocortisone hemsuccinate andrenewed every day thereafter   Mouse 3T3 fibroblasts (Rheinwald et al   1975), bovine cornealen- dothelial cells (Gospodarowicz etal   1979), human skin fibroblasts, and rat liver myofibroblasts were maintained in the serum-supplemented medium described above,wthout insulin,corticosteroids, andalbumn   Two RLEC cell lines (SDIII, SDVI)were established from the liver of 10-d-old Sprague-Dawley rats in our laboratory, according to the procedureofWlliams et al   (1971) and used between passages 10 and 30   In addition, The Journal of CellBiology, Volume 115, 1991 a woodchuck liver epithelial cellline (WLEC) was obtained from an im- ported eastern American woodchuck   RLEC, WLEC, and bovine lens epithelialcells (Arruti and Courtois, 1978) were grown in theWlliams E medium supplemented wth 10 FCS For RNA analysis, hepatocytes from cocultures were selectively sepa- rated from RLEC by incubation for 10 mn wth a calciumfree collagenase solution (0 .05% pH 7 .6 buffered wth 0 .1 M Hepes (Fraslin etal ., 1985)   Hepatocytes that were more sensitiveto low concentration of Ca++ became rounded and detached in clumps, whereas RLEC remained well spread   Production and Selection of mAbs Balb/c mcewere immunized wth a cell suspensionof 10' live RLEC, and spleen cells were fused wth the SP2/O-Ag 14 myeloma cells (Bourel et al ., 1984)   Culture supernatants were screened for theproduction of an Ig by using an indirect immunofluorescent assay on both RLEC and hepatocytes   Subsequently,the positive clones were selected according to both theirabil- ity to recognize a plasma membrane protein and their inability toreact wth nonhepatic cells, e .g   human skin fibroblasts and bovine cornealen- dothelial cells   Cell suspensions were fixedina 4   paraformaldehyde solu- tion buffered wth 0 .1 M sodium cacodylate (pH 7 .5) for 15 mn at 4°C   The ability of mAbs to modify the functional capacities of hepatocytes in coculture waschecked by adding 100,200, and 500 pl of supernatants from thepreviously selected hybridoma clones to the cocultures upon RLEC seeding, and then every day at each medium renewal   Viability of hepatocytes was tested andalbumn secretion was measured after 5or6 d   In some assays, euglobulin-precipitated IgM L8 preparedby dialyzing 2 nil of asciticfluid against distilled water at pH 5 was used as described elsewhere(Goding, 1986 ; Garcia-Gonzalez et al ., 1988)   Albumn secretionrate was quantified in the culture medium by laser im munonephelometry (Lescoat et al   1985)   Indirect Immunolocalization Hepatocyte pure cultures and cocultures were fixedin a 4 paraformalde-hyde solution bufferedwth sodium cacodylate 0 .1 M (pH 7 .4) for 30 mn at 4°C   mAbs were localized using either the indirect immunoperoxidase or inununofluorescencetechniques(Guillouzo et al ., 1982)   Incubations were carried out wthor wthout 0.2 saponin   Second antibody was a peroxidase-labeled anti-mouse IgM (Nordic Immunological Laboratories, Tilburg, The Netherlands) or anFITC anti-mouse Ig (Dagnostics Pasteur, Marnes-la-Coquette,France)   For EM cells were postfixed wth 2 .5 glutaraldehyde and subse-quently incubated for 30 mn in 1 osmum solution in 0 .1 M cacodylate buffer   The cells were then dehydrated in graded ethanol andembedded in Epon   ECM Deposition The reticulin staining by silver impregnationof the extracellular matrix, was carried out according tothe method of Gordon and Sweets (1936) in cocultures fixed wth a mxture of 4 paraformaldehydeand 2 .5 glutaraldehyde in cacodylate buffer (pH 7 .4) for 15 mn at 4°C   Time-lapse Cinephotomcroscopy A 1-d-oldcoculture, grownon a glass coverslip coatedwth fibronectin was mounted in a Rose chamber   Thechamber was put onto the mcroscope stage 90mn after epithelial cell seeding andunder a controlled constant temperatureof 37°C   Cell behavior wasmonitored wth a Zeiss ICM 405 mcroscope equippedwth Normarsky differential interferencecontrast,a Zeiss objective 100 x NA 1 .25 plan and a 16-mm Arriflex camera   The time-lapse device gavean impulse every 2 s, and a photomcrograph was taken wth Agfa copexpan rapid film   Cell-Cell and Cell-Substratum Adhesion Assays The effect of mAb L8on hepatocyte attachment to RLEC was examned   Dshesof 3 .5 cm in diameter covered by a confluent RLEC monolayer were preincubated wth 2 /A of either SP2/O-Ag or L8 asciticfluid per m of serumfree medium for 1 h   Freshly isolated hepatocytes (10 6 cells) were then seeded and maintained at 37°C inthe same medium containing SP2/O-Ag or mAb L8   Media of vialsin duplicate were harvested at 2, 3, 4, 7, and 22 h, and the number of unattachedhepatocytes was estimated by measuring thelactate dehydrogenase activityafter cell lysis wth PBS containing 0.2 Triton X-100 (Rubin et al   1986)   The effect of mAb L8on hepatocyteattachment to various substrata in- 506   on S  e p t   em b  er 1 4  ,2  0 1  5  j   c  b .r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published October 15, 1991  cluding EHS gel, fibronectin, collagen IV, lamnin,lamnin-entactin com- plex, and heparan-sulfate proteoglycan, was also examned   2,ugof protein were coated on 0 .32-cm well tissue cultureplates containing 100 al of serum-free medium After2 h, 3   BSA was added to a final concentration of 1 .5   for a further 30 mn   Medium wasremovedand hepatocytes previ- ouslyincubated for 30 mn in aserumfree medium containing increasing amounts of mAb L8 or SP2/OAg ascitic fluid and 0 .02 BSA were seeded   After 1 h, the plates were gently washed twcewth PBS   The num- bei l of attached and unattachedhepatocytes was measured as described above   Extraction of RNA and Northern Blot Hybridization Total RNA was prepared from hepatocytes, using5   guanidium thiocya- nate/CsCtechnique (Chirgwn et al ., 1979)   Total RNA (20,ug) was resolved by electrophoresis and transferred ontoa nitrocellulose filter (Thomas, 1980)   The filter was prehybridized accord-ingto Andrews etal   (1982) and hybridized wth3 x 106 cpmm of 32p nick-translated cDNA probe for 18 h at 65°C   In Vitro Ranslationof mRNA Total RNA (5 pg) was added to25plof rabbit nuclease-treated reticulocyte lysate (Pelham and Jackson, 1976) containing 50ACi of [ 35 S]-methionine (Amer sham Les Uis, France)   Translation was carried out2 h at 30°C . TCA-precipitable material (4 x 105 cpm) was run on SDS-PAGE   Labeling of Plasma Membrane Proteins Radioiodination of cell surfaceproteins was performed according to a pro- ceduredescribed elsewhere(Cément etal ., 1989) wth some modifica- tions   Monolayer cultures (150-cm 2 Petridishes containing 18 x 106hepa- itocytes and 50 x 10 6 epithelial or endothelial cells) werewashed three timeswthcold PBS and labeled at room temperature with 3 m of PBS containing 2 mCi carrier-free Na[ 115I] (Amersham) by lactoperoxidase- catalyzed iodination   Plasma Membrane ProteinExtraction Cells were washed three times wth PBS, scraped offthePetridish,pelleted in the mcrofuge at 6,500 rpm for 10 s and solubilized ineither 1 Triton X-100, 0 .01 SDS, 2 mM EDTA, 130 mM NaCl, 10 mM Tris-HCI (pH 7 .4), or in 1   Triton X-114, 130 mM NaCl, 10 mM Tris-HC (pH 7 .4), by pass- ing five times through a G26 needlewth a syringe and maintained 30 mn at 4°C   Two protease inhibitors, aprotinin (100 IU/m) and phenylmethyl- sulfoxide (2 mM), were included in all buffers   Cell lysates were thencen- trifugedfor 10 mn at 13,000 rpm When extracted in Triton X-114,the soluble material was submtted to phase separation according to Bordier (1981), and thenthe two phases were adjusted byadding either Triton X-114 orthe buffer in order to obtain the same salt and surfactant contents in the different samples for immunopre- cipitation   Immunoprecipitation Lysates were incubated 90 mn wth3 ul of ascitic fluid Tb elimnate aspecific boundaries, ascitic fluid was treated by protein A-Sepharose be- fore use Subsequently, the samples were incubated with goat anti-mouse IgM (Nordic Immunological Laboratories) followed by protein A-Sepha- rose (Pharmacia, St Quentin-Les-Yvelines, France)   The affinity beadswerewashed wth lysis buffer and bound material was eluted wth 100 Al of sam ple buffer according to Laemntli (1970) for molecular weight determnation orwth 100 pl of lysis buffer according to OFarell (1975) for pI determna- tion   SDS-PAGE electrophoresis was performed in 4-15   or 7 .5-15   poly- acrylamde linear gradient slabs in a Laemmi buffer system IEF was per- formed wth Ampholines pH 3-10 (Pharmacia) in OFarellbuffer systemGels were stained by Coomassie blue, dried, and autoradiographed using Kodak X-Omat AR films   Results Selection of a mAb (mAb L8) Able to Alter the Hepatocyte-differentiated Phenotype in CocultureTo identify a cell surfaceproteininvolved in the interactions Corlu etal   A Liver Regulating Plasma Membrane Protein Figure 1   Specific effect of m b L8 on the survival of rat hepato- cytes cultured with RLEC . Phase-contrast mcrographsof(A) 2-d-old hepatocytepure culturetreated wth L8 hybridoma super-natant ; (B) 5-d-old hepatocyte coculture ; (C) 5-d-oldhepatocytecoculture treated wth L8 hybridoma supernatant . m b L8, 500 pl per m of medium,was added upon starting the culture and then every day at each medium renewal   Note that hepatocytes died in coculture with m b L8, whereas RLEC remained alive and wellspread   Bar, 50 jam of hepatocyteswith RLEC incoculture, mAbs were gener- ated afterfivedifferent fusions, using live RLEC as immu- nogen   Hybridoma culturesupernatants were first screened for positive immunofluorescence with freshlyisolated hepa- tocytes and RLEC   From 400 positive hybridoma cultures, 95   were positive with RLEC and hepatocytes and 5 were positive onlywith RLEC   Antibodies from both groups were selected according totheir ability to bind antigens lo- cated on plasma membranes   m bs that reacted with human skin fibroblasts and bovinecorneal endothelial cells were discarded . Finally, 24 hybridomas activelysecreting liver plasma membrane antibodies were selectively cloned and grown for further analysis   The ability of m bs to inhibit thecoculture effect was tested by adding every day for one week increasing concen- trations of hybridoma culturesupernatants into the media of hepatocyte pure cultures and cocultures . Only m b L8 was able to markedly reduce the survival of hepatocytes in cocul- tures . By day 3, the typical cuboidal shape of hepatocytesmaintained in cocultures was not observed in the presence of this antibody   Most hepatocytesdied after 5 or 6 d as in pure cultures, whereas RLECs were unaffected (Fig   1)   No effect on hepatocyte viability was detectedwith time in pure cultures   These results indicated that m b L8 did not induce 50 7   on S  e p t   em b  er 1 4  ,2  0 1  5  j   c  b .r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published October 15, 1991  A 30E 0 L 20 c á BE 3 20 c Q C 30 E =20 c Q 0 30 02468 10 Days 02468 10 Days 0   0 2 4 6 8 10 Days Figure 2   Effect of mAb L8on serum albumn secretion by hepato- cytes in coculture under the followingconditions   (A) The antibody was added at different concentrations   Serum albumn was mea- sured in the medium collected daily frompure cultures (--Fi--) and cocultures  t) maintained without mAb L8, ascontrols, and from cocultures added wth2 .5 (-0-),510 and 20 pg/m(-~-) of partiallypurified mAb L8 . Antibody was added upon RLEC seeding and every day at each medium renewal   Serum albumn was measured by immunonephelometry   (B) The antibody was removed early or late from thecoculture   Serum albumn was measured in the medium from cocultures maintained without mAb The Journal of Cell Biology, Volume 115, 1991 toxic effects, but mght altercell-cell interactions between hepatocytes and RLEC   The capacity of the cells to secrete albumnwas inves- tigated in culturestreated daily with mAb L8   While high- ly maintained in untreated cocultures, albumn secretion dropped in those to which 10 or 20hg of partially purified antibody per m of medium had been added (Fig . 2 A) . The remaining production observed in mAb L8-treatedcocul- ture couldcorrespond toa release of theprotein from cells that did not detach asfast as in pure cultures or resulted from anotherhepatocyte regulating process   mAb L8was characterized as an IgM by the Ouchterlony technique   The effects of mAb L8 on hepatocyte cocultures were verified to be unrelated tothe globulin isotype by treat- ingthecocultures with the supernatant of another hybridoma selected for its ability to secrete mAb ofthe same isotype (data not shown)   The mAb L8-related E fect TakesPlaceduring Cell ContactsEstablishment To determne the optimal timeof mAb L8 addition that affects hepatocyte cocultures, two experiments were per- formed   Firstly, mAb L8 wasadded at the onset of cell seed-ing and the treatment was stopped 1, 2, 3, or 4d thereafter(Fig . 2 B)   Secondly, the coculture was initiated without an- tibody and the treatment was begun only after 1, 2, or 3 d (Fig . 2 C)   The mAb L8 effect was evaluated by measuring the capacityof hepatocytes to survive and tosecrete albumn in the medium We found that suppression of mAb L8 treat- ment byday 1, aswell aslate addition of antibody at day 3, hadno significant influence on cellviability andalbumn production   An inhibitory effect was only evidenced when the antibody was present between days 1 and 2 after RLEC seeding, corresponding tothe establishment of cell-cell con- tacts . The first day lag-phase represented the time neededby RLECs to attach, grow, and reachconfluencywithhepato- cyte colonies   The effect of mAb L8 appeared fully reversible when thecoculture was treated for 1 d (Fig   2B) . However, thealbu- mn secretion levels didnot completely reach those of un- treated cocultures after 2d or more of mAb L8 treatment, even when the cells were allowed to recover for up to 12 d . This correlated with previousdata showing theexistenceofa critical state between days 3 and 4, beyond which initiation of coculture was no longer possible(Fraslin etal ., 1985)   In addition, mcrocinematographywasperformed to ex- plore the hepatocytebehavior during establishment of cell contacts with RLEC   1-h filmof a liverepithelial cell mak- ing contactwithhepatocytes revealed that a flat and clear L8 as control (--a-) and from cocultures added wth 20 ug/m of partially purified mAb L8 at RLEC seeding and removed at days 1 2 3(-*-), or 4 (-0-), or maintained during all the culture time (-A-)   (C) The antibody was added early or late to thecoculture   Serum albumn was measured in the medium from cocultures wthout mAb L8 as control   - f-), and from cocultures to which 20 j,g/m of partially purified mAb L8 were added either upon hepatocytes seeding (-A-) or RLEC seeding   - fl - ), or at day 1 (-ß-), day2   orday 3   andeveryday there-after   50 8   on S  e p t   em b  er 1 4  ,2  0 1  5  j   c  b .r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published October 15, 1991  Figure 3 Normarsky mcrographs of hepatocytes when establishing contacts wth RLEC One epithelialcell justestablishes contact wth hepatocytes (A)   A flat and clear zone at the contact site started to form within 20 mn (B) and was wider and more evident 50 mn (C) and 60 mn later (D)   Note that cytoplasmc organelles graduallyrearranged more radially toward thecontacting site   Bar, 10 um zonewasformed at the contact site within 20 mn while in- tracytoplasmcorganelles were reoriented in a radial manner toward this site (Figs   3, A-D)   This indicatedthat establish- ment of cell-cell contacts with RLEC induced very early changes in hepatocyte organization   The Molecule Localized by mAb L8 Is Expressedby Both Hepatocytes and RLEC Immunopositive reaction with rnAbL8 was located all along the plasma membrane ofboth freshlyisolated hepatocytes and hepatocyte pure cultures for only the4 first days and thendisappeared   By contrast, mAb L8 inununoreacted with he- patocyte plasma membranes in cocultures for 2 wk (Fig   4B)   The reactioninthese cells gradually decreasedwithtime thereafter   Immunopositive reaction was also observedon RLEC, at a lower intensity   It was slightly increased incells closeto hepatocytes   Electronmcroscopicexamnation re- vealed electron-densedeposits distributed all along the cell surface ofboth hepatocytes and RLEC (Fig   4 C)   The antigen recognized by mAb L8 was present in thenor- mal liver   Antigenic sites were generally distributed in the hepatic lobule, but restricted to the sinusoids (Fig   4 A)   Cells Immunoreacting with mAb L8 Are Capable of Interactingwith Hepatocytes in Coculture Two different RLEC cell lines (SDIII and SDVI) and one woodchuck liverepithelial cell line (WLEC) interacted with rat hepatocytes infavoring survival and albumn secretion   Furthermore, addition of mAb L8 strongly reduced these Corlu etal   A LiverRegulating Plasma Membrane Protein effects   In parallel, these cells were immunoreactive to mAb L8 (Table I This feature was not characteristic of all cells with an epithelial originor all cells from liver tissue since bovine lens epithelialcells and ratliver myofibroblasts failed to interact withhepatocytes in coculture and that mouse 3T3 fibroblasts succeeded in it (Kuri-Harcuch and Mendoza- Figueroa, 1989)   Moreover, immunoreactivity of mAb L8on cells was limtedtothose from rat and woodchuck origin   Immunopositive reaction was not detected on mouse 3T3 cells and cocultures with 3T3 cells were not affected by mAb L8 in consequence ofthespecies specificity expected of this mAb   In addition, assays were performed whichcombined hepa- tocytes from different species with RLEC Hepatocytes from mouse,woodchuck, dog, baboon, and man were all ableto interact with RLEC and to maintain a higher functional sta-bility in coculture   mAb L8 Specifically Suppresses the Liver-specific Gene Expression in Coculture Total hepatocyte RNA was extracted from pure hepatocyte cultures andfrom untreated and mAb L8-treated cocultures at different times and was analyzed by Northern blot   Hy- bridization was performed with cDNA probes correspond- ing to rat albumn, aldolase B, and procollagen a,  I As expected from previousdata (Fraslin etal ., 1985),the steady state of albumn mRNA level gradually decreasedwithtime in pure cultures, while thatof procollagen a,  I mRNA strongly increased after 4d of culture (Fig   5 B)   The steady- 509   on S  e p t   em b  er 1 4  ,2  0 1  5  j   c  b .r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published October 15, 1991
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