Biology of the Cell

EXPRESSION OF AQPclc IN YEAST AND BACTERIA : SUBCELLULAR LOCALIZATION OF THE RECOMBINANT PROTEIN. LAGREE Valerie, CAVALIER Annie, THOMAS Dan&. PELLbRlN

Isabelle, DESCHAMPS Stephane. Biolagie (‘ellrdaire et Reprodnclrori, ( ‘NRS tJR.4256, lit~r~~er-.~ti de Rermes 1. Campus de Beaulieu, 35012 IENNLY C Ldex, l.rarxt~.

Water crosses the plasma membrane of most cells by diffusion through the lipid bilayer, however particular cells exhibit high water permeability due to

water selective membrane proteins Such proteins have been recently identified and gathered in the aquaporin family We have isolated and

sequenced a full-length cDNA from a filter chamber library of (,kadc//a viridi.r, an homopteran insect. This cDNA encode for a 26 kDa protein

whose sequence is 43% identical to the human water channel AQPI Expression of this protein in Xenopus oocytes membranes led to a lS-fold

increase of membrane water permeability We concluded that this protein is the first insect aquaporin and called it AQPcic. In order to produce sufftcient

quantities of AQPcic for further functional and structural studies, we have cloned its cDNA in different expression vectors. Two systems have been

used, bacteria (Lcherichia co/i) and yeast (Saccharomyccr cerewsiue), and the expression have been analyzed by western blotting and

immunocytochemistry In bacteria, the data show that AQPcic is mainly

produced in inclusions bodies. Indeed the protein was hard to solubilize and the product yield obtained was very low. In contrast, the expression of

AQPcic in yeast gives a significant amount of protein (0.4 m&l) and the protein is localized in all cell membranes. The recombinant AQPcic obtained

using this procedure is currently functionally tested

‘1 tlE LAMCl PROMOTER CONTAINS TWO SEQUENCES, INTE:RACFIN(; WITH ‘THE LARGE SUBUNIT OF REPLICATION FACTOR c AND sl’l, WHlCff ARE INVOLVED IN LAMININ yl EXPRESSION IN FAZA 567 RAT HEPATOMA CELI, LINf!,. Jocelvne LIETARD, Yoshihiko YAMADA*. and Bruno (‘Ll-MEN’1 Detoxication and Tissue Repair Group, Rennes I University School 01 Medicine, INSERM U-49, F-35033 RENNES, France and *Laboratory of Developmental Biology, N.I. D. K., BETHESDA MD 20892, IJSA.

Larninins, the major non-collagenous glycoproteins of all basement membranes are scarce in the normal adult liver. However, several Iarninin chains, particularly laminin yl, can be expressed at high levels in various liver diseases, such as fibrosis and carcinogenesis, as well as in 0th hepstocytes and hepatic stellate cells after a few days in culture and in henatoma cell lines. We have investigated the molecular mechanisms involved in the expression of laminin yt -in rat hcpatoma ceils. cis and rrurrs actine elements were identified in the promoter region of ILAMCI gene

L.

coding for laminin yt. Transfection of deletion mut&ts of the 5’ flanking r-egion of murine LAMCI gene in the Faza 567 hepatoma ccl1 line intlicatccl that two enhancers and a silencer segment which contains GC-and CTC- containing sequences, were located between -594 bp iirtd -04 bp. Two complexes wcrc formed when nuclear extracts from hepatoma cells were used in gel-shift analyses with double-stranded synthetic oligonucleotities corresponding to the CTC- and GCcontaining regions. Transactivation of IAMCI promoter was achieved when cotransfected in i?~o.vr~~1l7iiu cells with a Spt expression vector. Inmmnocytochemistry, western-blotting and northern-blot analyses indicated that Faza 567 hepatoma cells cxprcssed both Spl and Alp145, the lar-ge subunit of replication factor C (RFC). which interacted resnectivelv with the GC- and CTC-motives in the silcnccr regulatory eleme’nt of .LAMCI promoter. A combinatiort 01 immunoorecioitation and western-blotting with Faza 567 cell extracts revealed’that ‘A I~145 was in complex with PCNA. ‘These data show that L,AMCI gene expression in Fazu 567 hepatoma cells is governed by nuclear factors interacting with CTC- and GC-rich regions within the promoter. including RFC and Spl, thus suggesting that there is a rekttionship bctwcen laminin yi expression and the cell cycle

AQUAPOIUN-RELATED PROTEINS IN THE FILTER CHAMBER OF HOMOPTERAN INSECTS.

Francoise le Cahtrcc, Marie-The&e Guiliam, Anme Cavalier, Fabtennc

Beuron, Daniel Thomas, Jean-Francois Hubert and Jean Gouranton. Equipe canaux et rtcepteurs membranaires. URA CNRS 256, Campus dc

Beaulieu, Univetsite de Rennes I, 35042 Rennes cedex

I‘he filter chamber of homopteran insects is a water shuntmg coinpicx rn which water crosses plasma membranes through a transepithelial osmotic gradient. We previously purified and characterized from the plasma

membranes of Cicudellu viridis filter chamber a 22; kDn protein. Previous analysis showed that this protein belongs to the MIP family (major intrinsic

family, a group of membrane channels for small solutes). We demonstrated that it functions as a water channel (or aquaporin) when present in the

plasma membrane of xenopus oocytes This protein was called AQPdc. In the present study, we used polyclonal antibodies raised against AQPcic fil:

western blotting and immunocytochemical analysis of some homopteran insects intestinal tract. The species analyzed were Cercupis .satigumriitwta, l’hilaerms .~pmnurivs, Aphrophora nhr, 1~7tscelidi7i.r wukgulu.~ anil

Scu,r~/roi&r~.~ fitumn. The tatter insects are well known vectors for piant

pathology through mycoplasma transmission during sap sucking process Western blotting revealed that proteins immunologically related to the

aquaporin from (‘rcadella viridis are found in all species ‘The molecuhn weights of these proteins are 24-28 kDa Tltis is consistent ~vith the

molecular weight values reported for known aquaporins from t?thet biological systems (except lbr E77.sc:celicirv.s varicp/tu where a 15 kDa signal

was obtained) lmmunocytochemical studies on ultrathin sections rcveilied

that the antibody systematically labelled the membrane microvilli of ;“tltr! chamber epithclial cells. A good correlation thus exists between the presence

of the aquaporin related proteins and the water transfer function of these

cells

OVEREXPRES§I<~N OF MATRIX ME?‘ALLOPROTEfNAYE,-r AND THE JNNiBITOR TIMI’ IN LIVERS FRO-M PATIENTS WlTII GASTROINTESTINAL ADENOCARCINOMAS AND NO DETECTABLE METASTASIS.

Orlando MUSSY”, Nnthalic TtiERET*. Jean. t’rcrrr (‘,\MI’I(jN~, I;;:in- TURLINI. Olivier L.OREAL*, Annie L’I-I~I.,COIJAI,C~lI* arid B:iin:, CLEMENT*. “Groupc Dttonication el Reparation ‘i‘issulaire. Universtte dc Re~rnr,\ I. SClinrque Chirur-gicalc. and IlAnai. I’nthol. B, CHRIJ Pontchnillou. 35033 Rennes. France.

ii’c have invesligatcd whclher txi.iemcnt meirtbianc r-cinodciin~ :ir;t)’ pI,ty Ii role in the invasion of metastatic cells 111 the target liver. The expression ot both MMP2 and TIMP2 was analyzed in livers from patients with gastrointestinal adenocarcinornas and no detectable hepatic metastasis (jl=lZ). in matching pairs of tumoral and non tumoral livers frompatients with hepatic metastasis (n=9) and in control explanted livers (n=4). By seitli-yii3iititatls~~ RT-PCR, the steady-state MMl’2 arid ‘I‘lMP2 ntRNA lcvcis in iivc~s f’rcm patients with hepattc menstasis wcrc higher in tumoral areas comp;rrcd !(I non-tunlorai al-(335 with a ltrnl0i;ll/non ttrlmnal r-atio of 6.224 iifltf I 620.8, respectively. By zymograph!;. MMP2 activity was t-esoived 3s liilcnt :tnd active forms in the tumor-at are;rs whereas in non tumor:11 are;ls, flit: hIc11: form was predominant. In ;itu hybridization tlemonstratrd lMMI’2 2nd TIMP2 tr-anscripts in the stromrl cells of liver metastascs. III livcri from patients with gasuointestrnal ;IdenocarcinoIilas and no detectable mrtasmsis. MMP2 and TIMP2 mRNA levels were increased compared~with those of etther control livers (S-fold and ?.2-fold. respectively) or non tutnotal areas of Iivcrs from patients with metastascs (7.8.fold and 3-fold. respec tivstyl MrMP2 activity was detected by zymography mainly in latent form In situ hybridization rcvealcd no clta~lgc in the localisation of MMPZ and TIMI? transcripts. compared with non tnrnoral or control livers. Thcsc tiat:r sqgpes! that primary gastrointestinal adenocilrcinomiis induct the 0vercxprcsz:on 0: both MMP2 and TIMPZ n11iNA in the target liver. bclorc ritct:tst;tsis tx’come dCb3X1lk