Regulation of upa in mammary tumor cells: Synergism between retinoic acid and 8-bromo-cyclic AMP

10

FIBRINOLYSIS

29 IDENTlFlCATlON OF A SPECK

RECEPTOR FOR PLASMIN ON

GROUP A STREPTOCOCCI RMwdLottenbeg,

MkhaelD.f?Boyle, chrlstoplwrC.hder

TlmA

Lheseker,LucyE~n8miQregoryO.vcmMcring Univers$yof Horkfa Collegeof Medicine, GainesMe, FL and Me&al Cdlege

ofOhio,Toledo, OH,

USA

We have identified a unique receptor for human plasmin which is expressed by certain strains of streptococci. The bacterium-bound plasmin retains its enzymatic ectivky and is not inhibited by aipha 2-atiplasmin. We have characterized the interaction of grorq A strep&cccus strain 84/i 4 with plasmii and demcnslrate that binding occurs opIlmally at physlologlc pH and ionic stmngth. The Kd was 5 x

III-” M end there were qp~ximetely

&Xl recepdors per bftctefium.

This binding was inhibited by lysine and lysine analogs in a ccncentmtiiondependent manner suggesting a role for plesmins hi affinity lysine binding site intheinteraction of plasmin with the bacteria. Lys-plasminogen also binds to these bacteria while gl~asminogen, other kringle containing proteins and other seriie proteeses, do not demonstrate significant bin&g aclfvlty. Mutanolysin treaknent of the bacteria yielded soluble receptor activity. Purfficatkm of Ihe receptor by affinitychromatography reveals that it is physico&emically distinct from skeptokinase and does not demonstrate plasminogen activator activity. The bincfrngof active plasmin to the surface of pathogenic streptococci may play a slgniIicant de in the infectious &ease P-

30

REGULATION OF UPA IN MAMMARY TUMOR CELLS: SYNERGISM BETWEEN RETINOIC ACID AND 8-BROMO-CYCUC AMP R. Mira-y-Lopez

Fcotprintlng and gel retardation experimsnts with nuclear protein extracts from HeLa cells have shown protein bindlng to at least eight sites in the 5’flanking region of the UPA gene. Four of these sites are highly homologous to previously characterbed bincfmgsites for proteins AP-1, AP-2, CRB/ATF and E2aE+Beta. The remaining four sites show Ilttle or no homdogy to known transcription factor binding sites, and are de+rated human u-PA bindiig sites (HUBS) I-IV. The enhancer reglon, upstream of -1870, spans at least three SiteS:

1. A CREB/ATF sequence around -1970. 2. An E2aEcBeta consensus around -1870. 3. HUBS IV, around -1890. The putative silencer (-1825 through -1570) shows binding at four sites: a. HUBS Ill around -1790. b. A complex site around -1675, blndng two proteins, in a non-exdusive way. One protein binds to an AP-1 core seqrence, and the binding can be abolished by mutaIlon wiihin this core seqence. This AP-i/HUBS11 site is a candii for being respcnslble for the deregulation of u-PA gene expression observed in human transformed cells, since AP-l-like activky has been shown lb bs aclfvated by transforming oncogenes (Wasylyk et al., Embo J., 7, 24752483, lQ88). c.AnAP-2slteat-1585,whichcoinckleswlthageneficallymapped phorbol ester responsive element (sea abstract by Verde et al., this volume). Also in this case, mutation of the Ap-2 consensus sqence abolishes binding ackvity in the gel retardation assay, indicating that the protein involved is indeed AP-2. Deletions and point mutatbns are presentry being studed to elucidate the function of these sites, the proteins they bind and their rde in regulation of human U-PA gene exptession

32 flBRlN CLOT OPALESCENCE INTRODUCED BY Retinoic acid and cholera toxin interact synergistically to increase primary rodent and human mammary tumor UPA secretion in organ culture. We have begun a study of Ihe mechanism of this synergism in Shionogi SC1 15 mcuse mammary carclncma cells. In these cells, the following mean increases in UPA secretion were seen after 24 hrs oftreatmantin&senceofsetum: lmM8-bmmo-cAM P, 15fold 100 nMretinoicadd,2fokl;endlmM&bom~PtlWnMretinoic a&f, 30 fdd over control. Similar results were obtained when 1 nM cholera toxin was used instead of 8-btunocAM P. Retinoidswlthout Ihe acid funcIion dd not synegize with CAMP. Synergism with reIlndc acid (RA) was half-maximal at 3 nM and maximal at 100 r&I. Time course experiments showed that the effect of 8-bromo-cAMP reached a plateau after 6 hours while the mild effect of RA and the synergism with cAMP ran a slower, parallel course, being first detected at 12 hts and increasing progressively Lp to at least 48 hrs. This suggested that RA induced a state of heightened sensitivity to CAMP. Thii was confirmed by pretreatment of SC1 15 cells with RA, whii caused a synergistic UPA increase upcn treaIment with -P in Ihe absence of RA. This effect of RA was irreversible, as demonstrated by its persistence after extensive washing following pretreatment Western blot analysis with anti-mouse UPA antibodies indiied that increased extracellular UPA activity reflected changes in uPA concentraIlon. RNA and protein synthesis inhibitors blocked UPA induction. Retinoic acid exerted a mikf but persistent effect on SC1 15 cell growth, increasing population dc&ling Iime frcm 17 to 20 hcurs. Further experiments will abn to clarify whether the effect of RA is mediated by inductionof dlffe~on

31

DNA BINMNG PROTUNS AND REGULATlON OF THE EXPRESSION OF THE HUMAN UROKlNASE-TYPE PLASMINOGEN ACTlVATOR GENE C&us Neriov, MortemJohnsen, Pasquale Vet& and Rancesco B/as/ Insfiiute of Mcr&iokqy,

UniwMy

of Cqenhagen,

Denmark

The human urokinase (u-PA) gene contains at least 2.1 kbp of 5’ regulatorysequence,whi~indudesanenhancer(rTwbeamofpobition -1870), and a possble silencer (between -1825 d-1570). %quenca homologies indicate the presence of response elements for phorbol esters, glucowrticoids, estrogens ar&yclicAMP(Verds et al., Nudekr Acids Res., 18, November 1988).

INCORPORATION OF PLASMINOGEN OR tPA lslscJar.mn Now Reswch Irstkte, DK.2880 Bagsvaercl Denmark lbuntbln-Induced fibrin dot formation was monitored by the change in optical density (O&JO&. This was strongly krcreased when tPA or Lysrr-plasmlnogen was present In contrast, the flbrln dot dansity was only slightly affeded by the presence of Glul- or Val+&asminogen. The increase in Oh,,,, as a fundicn of tPA - or Lysrrplasminogen concentrationfolbwed saturation curves suggestlngtfratthe change in opecily reflected a change in clot shucture induced by binding of tPA or Lysnplasminogen tofibrinin I :I complexes. Complex formation as monitored by 00~ could be prevented by the presence of trans-4-(aminomethyl)cyclohexane-1 carboxylic acid (tAMCA). Displacement from fibrin by tAMCA was described by simple dissociationcurves, & = 30 @I IAMCA for Lysnplasminogen and & = 1 mM for IPA dsplacement. Measurements of fibrin enhanced Lysrrplasminogen acilvatlon catalyzed by tPA revealedthat the curve for tAMCA inhibition of this adivity was slperirrposable on the Lysf7plasminogen displacement curve measured by clot opacity. This indicates that the inhibition was caused by displacement of plasminqen and not tPA from fibrin. Ad&ion of Lysnplasmincgen immediately after fibrin dot lysis resulted in precipitation of a complex between Lysrrplasminogen and soluble fibrin degradation products (fibrin fragments X, Y, and D). No precipitation was &served lpon further fibrin degradation. These results in&ate that complex form&x-r between fibrin and LysTlplasmincgen or tPA might induce an increased lateral association of fibrin protofibrils to form thicker bundles, and association of sokble high molecular fibrin degradation productstofoml preciplhltes.

33 THE SPEClFlC ACTlvlTY OF t-PA DELETION MUTANTS Is

STRONGLY DEPENDENT ON THE TYPE OF ASSAY USED; PROBLEMS WHEN INTERPREllNG m DATA Gunndb~CMstar~llrlstda~ksirom,CnUtefinasBrlry, hnaftRi9nsson, and worn Lowenadkf KabGenAB,

andKabiA6,

S-11281

Sfodchofm, Sweden.

SIX t-PA deletion mutants were constructed, all whll lacked the growth-factor (G) domain andthe first kringle domaii (Kl) and had the potential glyc.osylatlcnsite at Asn184 chanted to Gln. The mutants