484 J.M. MULLINTHE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:484–489 (1997)

© 1997 WILEY-LISS, INC.

JEZ 862

Potential Interplay Between Luminal GrowthFactors and Increased Tight Junction Permeabilityin Epithelial Carcinogenesis

JAMES M. MULLIN*The Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096

It has been generally accepted for many yearsthat carcinogenesis proceeds in at least two dis-tinct stages, the first being initiation, the secondbeing tumor promotion (Boutwell, ’74). Initiationhas been shown to be a heritable change imposedon DNA itself, whereas promotion is regarded asan extranuclear event, non heritable, and requir-ing chronic exposure to a tumor promoting agent(Diamond et al., ’80). One may think then of car-cinogenesis as a series of dominoes which mustfall in sequence in order to finally produce thecondition which we term cancer. One can likewiseconceive of it as a series of necessary and finallysufficient events, leading in the end to a tumor.

The majority of lethal cancers are epithelial inorigin (Fraumeni et al., ’89). The fact that epithe-lia line all of the major body fluid compartmentsplaces epithelial cells specifically in more pro-nounced contact with environmental carcinogenicagents than other somatic cells. In other words,if the process of carcinogenesis requires a tumorpromotion phase entailing chronic uninterruptedexposure to an exogenous tumor promoting sub-stance, epithelial cells in tissues such as lung andcolon would be repetitive targets.

There are two key characteristics of epithelia,two traits which are common among all of thevarious epithelial tissues. First, all epithelial cellsare intrinsically polar, having an apical cell sur-face facing the luminal fluid compartment, and abasal-lateral membrane facing the interstitialfluid compartment. These two membrane domainscontain not only different classes of membraneproteins, but distinct lipid compositions as well(Schneeberger and Lynch, ’92). The second char-acteristic common through all epithelial tissuesis the most basic function of any epithelium,namely its ability to form a barrier between thetwo fluid compartments which the epitheliumseparates. The common thread between these twomost fundamental characteristics is the tight junc-tion or zonula occludens. This protein (or protein

and lipid) circumferential band around the apicalpole of each epithelial cell is first and foremostthe selectively permeable diffusion barrier for theparacellular pathway. It prevents free flow of sol-utes from one fluid compartment to the otheralong the paracellular route between epithelialcells (Farquhar and Palade, ’65). The tight junc-tion also has a role in maintaining the apical/basal-lateral polarity of individual epithelial cells(Cereijido et al., ’89). When considering the scopeof epithelial cancers across such a variety of tis-sues, and the fact that according to the two-stagecarcinogenesis model an extranuclear change isnecessary for epithelial cancer, the primacy of epi-thelial polarity and barrier function makes themkey areas on which to focus attention.

Evidence for altered permeability of tight junc-tions in transformed epithelia has been known foralmost 30 years (Martinez-Palomo, ’70). A decreasein the number of tight junctional strands has beendescribed in transitional carcinoma of the urinarybladder (Saito, ’84). Decreased transepithelial im-pedance has been recorded across the colons ofmice treated with chemical carcinogens (Davieset al., ’89). Inflammatory bowel disease linkedwith increased cancer risk has itself been linkedwith increased tight junction permeability, notonly in affected patients but in unaffected rela-tives as well (Hollander, ’88). On a molecular level,the tight junctional protein, ZO-1 (Stevenson etal., ’89), has been shown to possess significant se-quence homology to a septate tumor suppressorprotein of Drosophila, whose mutation leads toneoplastic growth of epithelia within larvae(Willott et al., ’93; Woods and Bryant, ’91). Finally,the interaction of the normal APC (adenomatouspolyposis coli) colon cancer susceptibility geneproduct with the cell adhesion protein, β-catenin

*Correspondence to: James M. Mullin, The Lankenau Medical Re-search Center, 100 Lancaster Avenue, Wynnewood, PA 19096.

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(Su et al., ’93), the interaction of the APC-β catenincomplex with the DLG protein (the human ho-molog of the drosophila discs large tumor suppres-sor protein), partial homology of DLG with the tightjunctional proteins, ZO-1 and ZO-2 (Matsumine etal., ’96), and the immunoelectron microscopic lo-calization of APC to the intercellular junctionalregion of epithelia (Senda et al., ’96), all provideadditional support for a connection between al-tered tight junction dynamics and the develop-ment of epithelial tumors.

Not only do all epithelia serve as boundariesbetween two fluid compartments, but they engagein continuous thermodynamic work (reabsorptiveand secretory) on those compartments. The trans-epithelial transport processes entail not only wa-ter, salts, sugars, amino acids and other nutrients,but also proteins as well. Actually, the easily rec-ognized characteristics of the luminal fluid ofmany epithelial tissues results from the synthe-sis followed by vectorial secretion into the lumenof certain proteins such as the characteristic hor-mones of specific glands. Epithelial tissues suchas the thyroid, mammary or prostate are readilyrecognized as glandular, but other epithelial tis-sues such as the colon have less easily recogniz-able glandular properties. Given this perspective,it is not surprising that the luminal fluid of anyepithelial tissue contains significant levels ofgrowth regulatory proteins. The entire epidermalgrowth factor protein family (EGF, TGF,amphiregulin, betacellulin) is present in the lu-men of the gastrointestinal tract, some growth fac-tors coming into the lumen of the upper GI tractfrom saliva, but others being synthesized by theGI tract itself (Barnard et al., ’95). EGF itself isfound not only in the small intestinal lumen, butin the colonic lumen as well (Schaudies et al., ’89).Turning to the urinary tract, one finds the secondhighest concentration of EGF in the body to existin the urine (second only to saliva), with EGF ac-tually first isolated and identified (as urogastrone)from the urine (Gregory and Wilshire, ’75). The EGFpresent in the urine has been shown to not simplyresult from glomerular filtrate (i.e., plasma) but tobe synthesized and (luminally) secreted in the kid-ney (Jorgensen et al., ’90). Urine contains not onlythe 6kDa molecular weight form of EGF, but a highmolecular weight form as well (Nexo et al., ’92). Akey finding was to then demonstrate that even thehigh molecular weight form of EGF in urine (aswell as breast milk) is biologically active, capableof activating the tyrosine kinase activity of the EGFreceptor (Mroczkowski and Reich, ’93).

The purpose of growth factor proteins in the lu-men of epithelial tissues is thought to reside intissue repair processes. A key finding in this re-gard was the observation that ulceration any-where in the GI tract induces the development ofunique secretory cells (from the GI stem cells),which appear as buds from GI crypts, and secreteabundant amounts of immunoreactive EGF (Wrightet al., ’90). Indeed these authors pointedly mentionthat EGF is not absorbed from the gut, nor doesit seem to have any effect when introduced intothe normal GI lumen. Accordingly, instillation ofEGF by rectal enema into the colon of rats didnot produce any change in the cell kinetics of thecolon epithelium 5 cm from the rectum (Reeveset al., ’91). In vitro studies with rabbit duodenumin Ussing chambers showed, however, that EGFcan significantly shorten the recovery of transepi-thelial electrical resistance (barrier function) inacid-induced mucosal injury, presumably by firstincreasing cell motility (Riegler et al., ’96). Thesmall, highly stable trefoil peptides of the GI lu-men are likewise attracting a great deal of atten-tion for their ability to promote repair of injured GImucosa and thereby prevent disruption of the GIbarrier (Mashimo et al., ’96). This action has beenshown to be due at least in part to their ability toincrease cell migration (Dignass et al., ’94).

In all of the above studies, a common themeemerges: luminal growth factors seem to be with-out effect on normal epithelial tissue, except ifsome event such as tissue injury allows for growthfactor penetration below the epithelium. A veryclosely allied point is that the published litera-ture generally seems to support a case for (nor-mally) a near absence of growth factor receptorson the luminal (apical) surface of epithelial celllayers, a result of the intrinsic polarity of epithe-lial cells. In CACO-2 gastrointestinal epithelia,125I-EGF binding was observed to be 15-fold higherfrom the basal-lateral side, and as was also shownby Mullin and McGinn (’88) for renal epithelialLLC-PK1 cells, only basal-laterally administeredEGF was enhancing uptake of tritiated thymidineinto DNA (Bishop and Wen,’94). In the corticalcollecting duct, it was observed that whereasbasal-lateral EGF decreased transepithelial volt-age by 80%, apical EGF was without effect (Mutoet al., ’91). Although studies with isolated brushborder membranes have evidenced binding of 125I-EGF to a specific receptor in small intestine epi-thelia (Thompson, ’88), immunocytochemicalstudies show antibodies to EGF receptors label-ing only the basal-lateral membrane of GI epithe-

486 J.M. MULLIN

lia (Scheving et al., ’89). In a review concerning thepotential role of urinary EGF in bladder cancer,Messing and Reznikoff (’92) actually hypothesizethat the role of this biologically active protein willdepend upon the barrier properties of the uro-epithelium and changes in the normal (basal-lat-eral) distribution of EGF receptors on the surfaceof any given cell. It is to this specific question ofaccessibility of luminal growth factors to epithe-lial receptors (which both of the key functions ofthe tight junction help to determine) that our labo-ratory has focused its research.

In MDCK renal epithelial cell sheets, the phor-bol ester tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was observed to causea sharp decrease in transepithelial electrical re-sistance (Ojakian, ’81). Similarly in LLC-PK1 cellsheets, phorbol ester tumor promoters were ob-served to cause dome or hemicyst collapse (O’Brienet al., ’82). Both sets of observations pointed tothe ability of phorbol esters to increase tight junc-tion permeability, thereby decreasing the barrierfunction of the epithelium. This effect on tightjunctions was later confirmed by showing that theability of the phorbol esters, TPA and PDBU (phor-bol dibutyrate) to collapse transepithelial electri-cal gradients as well as domes was correlated withability to increase over 20-fold the transepithelialflux of the paracellular marker D-mannitol (Mul-lin and O’Brien, ’86). That the action of phorbolesters on tight junction permeability was beingmediated by its effect on the signal transductionenzyme, protein kinase C, and not a nonspecificeffect on the cell membrane was then shown byfive different approaches. First, the effect ofphorbol esters on tight junction permeability couldbe mimicked, although more slowly, by dia-cylglycerols (Mullin and McGinn, ’88). Second, thiseffect could also be produced by nonphorbol estertumor promoters also known to be protein kinaseC activators, such as teleocidin and aplysiatoxin,but not by non protein kinase C activating tumorpromoters such as palytoxin (Mullin et al., ’90,’91). Third, this effect of phorbol ester tumorpromoters on transepithelial paracellular perme-ability could be blocked completely and dose de-pendently by bisindolylmaleimide, a specificinhibitor of protein kinase C (Marano et al., ’95).Fourth, the time course of these effects on tightjunction permeability were observed to correlatevery closely with the translocation of the alpha(α) isoform of protein kinase C into the membrane-associated cell fraction, and the recovery of bar-rier properties of the epithelium correlated with

protein kinase C-α’s subsequent down regulation(Mullin et al., ’96). Fifth, the effect of phorbolesters on transepithelial permeability can be sig-nificantly slowed by the transfected overex-pression of a dominant/negative (nonfunctional)protein kinase Cα, and can be mimicked by thetransfected overexpression of (functional) proteinkinase C-delta (unpublished observations).

Recognizing that phorbol esters are highly po-tent mitogens in these epithelial cell sheets(Mullin and McGinn, ’88), we then undertook aseries of studies to show that it was not the mito-genic action of phorbol esters which precipitatedthe increased tight junction permeability. Utiliz-ing the fact that EGF was also highly mitogenicin LLC-PK1 cell sheets, we demonstrated thatwhereas phorbol esters cause sharply decreasedtransepithelial electrical resistance, EGF does notcause any immediate change in barrier proper-ties, but actually increases the resistance by over100% at the time that it is inducing cell divisionin the epithelium (Saladik et al., ’95). Similarly,we observed by the use of the electron dense dye,ruthenium red, that not only do tight junctionspersist in a mitogen-treated epithelium, but theypersist even through mitosis itself, and are ca-pable of even then blocking dye penetration intothe paracellular space (Peralta Soler et al., ’93).We conclude, therefore, that it is not the mitoge-nic action of phorbol esters which is responsiblefor their effect upon tight junction permeability.

We then observed a correlation between TPA’sability to produce abnormal cell architecture andits ability to cause tight junction permeabilityincrease. This came through a series of studiesfocusing on the action of chronic, long-term, un-interrupted exposure of epithelial cell sheets toTPA. The long-term exposure of LLC-PK1 cellsheets to TPA was observed to result in the ap-pearance of multilayered, polyp-like foci across anotherwise one cell layer thick epithelium (Mullinet al., ’92). The partial recovery of barrier func-tion which we observed in switching from acuteto chronic TPA exposure regimens was then ex-plained through the use of the electron dense dye,ruthenium red. We noted that when rutheniumred was applied to the apical surface of epithelialcell sheets chronically treated with TPA, the dyepenetrated tight junctions and stained the lateralintercellular membranes only in the multilayered,polyp-like foci, and never in the one cell layer thickareas between these foci. This contrasts with acell sheet treated acutely with TPA, where alljunctions (of the uniformly one cell layer thick

TIGHT JUNCTIONS AND EPITHELIAL CARCINOGENESIS 487

sheet) are permeable to the dye. Western im-munoblots of homogenates of cell sheets treatedchronically with TPA showed that although pro-tein kinase C-α was down regulated, a smallamount of this isoform always persisted in themembrane-associated fraction, whether the cellswere treated with TPA for 6 days or as long as 22weeks. Immunofluorescence shed additional lighton this issue by showing that whereas protein ki-nase C-α had apparently been completely downregulated in the one cell layer thick areas, it re-mained at high levels in the polyp-like foci. Cellsshed from the polyp-like foci have been subcul-tured in TPA medium and found to produce uni-formly multilayered cell sheets, with all tightjunctional pairs leaky to ruthenium red (Mullinet al., ’96).

In making the point that tight junctional sealsbecome leaky in response to the action of a givenagent, an often overlooked question is, leaky towhat? The nature of the types of solutes whichare then able to penetrate through the tight junc-tion, determine the biomedical significance of thepermeability change. Although a decrease intransepithelial electrical resistance is generallyreflected in an increased paracellular permeabil-ity to D-mannitol, this connection frequently doesnot exist, as we observed in LLC-PK1 cell sheetstreated for 24 hours with either EGF or the cy-tokine, tumor necrosis factor (TNF; Saladik et al.,’95; Marano et al., ’93), or has also been observedfor occludin-transfected MDCK cell sheets as well(Balda et al., ’96). Furthermore, in studies involv-ing acute TNF exposure of LLC-PK1 cell sheets,we have noted that even when a decline intransepithelial electrical resistance is paralleledby a rise in transepithelial flux of D-mannitol,increased flux of larger molecular weight solutesmay not occur. However, for both acute andchronic treatment of cell sheets with the phorbolester, TPA, the paracellular permeability increasehas been observed to extend even to dextrans of2,000,000 mw. To date, cationic ferritin (particlesize 9.4 nm) has been the only solute which wehave observed cannot pass across tight junctionsof cell sheets exposed to TPA (unpublished obser-vations). This very high molecular weight “cut-off” would indicate that the tight junctions ofphorbol ester-treated cell sheets will exhibit verysignificantly increased permeability to all proteingrowth factors. We have observed that when TPA-treated cell sheets exhibited over 40-fold increasesin transepithelial D-mannitol flux, they also ex-hibited over 35-fold increases in transepithelial

flux of the growth factor, EGF (Mullin andMcGinn, ’87). We have likewise found a very simi-lar situation for transepithelial flux of insulin acrossTPA-treated cell sheets. Moreover we have very re-cently shown that this tumor promoting, phorbolester-enhanced flux of insulin can result in signifi-cant stimulation of cell division of a fibroblast cellpopulation situated in the basal-lateral fluid com-partment below an epithelial cell sheet, when theinsulin is presented to the apical (luminal) fluidcompartment above the epithelial cell sheet (unpub-lished observations). The increased growth factorflux is therefore bringing biologically active proteininto the interstitial fluid compartment.

If in any epithelial tissue there exist foci of twoor more cells whose DNA has been permanentlyaltered by “initiating” carcinogens or spontaneousmutation, the chronic exposure to tumor promot-ing carcinogens may then become a sufficient con-dition to not only engender massive tight junctionpermeability increases, but neoplasia as well. Ourstudies suggest that the paracellular permeabil-ity increases arising from tumor promoters willextend to one or more of the growth factor pro-teins existing at very high levels in the luminalfluid of any epithelial tissue. The downhill con-centration gradient for these growth factors acrossthe epithelium should direct these proteins intothe lateral intercellular space and interstitial fluidcompartments. Three outcomes with importantbiological implications can then ensue: 1) the per-meating ligands can bind to and activate recep-tors on the basal-lateral surface of the epithelia,leading to epithelial proliferation; 2) the ligandscan bind to and activate receptors on the fibro-blasts (and other cell types) within the lamina pro-pria, leading to their proliferation; and/or 3) theligands can trigger receptors on the cells in thelamina propria, and these cells are then them-selves induced to secrete growth factors which canact upon the epithelium in a paracrine loop. Inany outcome, proliferation of the epithelia cantheoretically ensue.

If these leaky foci in the epithelium involve cellswhose DNA has already been mutationally al-tered, the risks for uncontrollable cell growth maybe significant. The difference between luminalgrowth factors serving a beneficial purpose inwound healing, versus a potentially lethal func-tion in carcinogenesis, can then be one of dura-tion. In the first instance the exposure of thebasal-lateral surface and interstitium to luminalgrowth factors is transient, lasting as long as thewound requires to reseal the barrier. In the sec-

488 J.M. MULLIN

ond scenario, the chronic nature of the tumor pro-moter exposure, and the irreversible nature of theDNA mutation, can combine to place foci of epi-thelial cells under constant stimulation from lu-minal growth factors. In certain types of DNAmutation one can envision hyperplasia as a re-sult, but in others, the outcome could be signifi-cantly less controlled. If the leakage of proteinsfrom luminal compartments into the interstitiumcontinues on into the vasculature, the occurrenceof neoplastic and perhaps preneoplastic foci maybe signaled by the appearance in the bloodstreamof proteins normally sequestered in luminal com-partments. Since the proteins would be specificfor individual epithelial tissues, a useful new ap-proach for early diagnosis of epithelial cancersmay be available.

ACKNOWLEDGMENTSArnost Kleinzeller allowed me the freedom in

my thesis work to branch out of epithelial physi-ology. He not only allowed, but encouraged me togo off in my own direction. He therefore not onlyintroduced me to the potential of epithelial cellcultures and the significance of epithelial barri-ers, but made possible my crosstraining in car-cinogenesis in the laboratories of Leila Diamondand Tom O’Brien. For me Arnost will always em-body the virtue of believing passionately in one’sresearch, regardless of totalitarianism, sickness,or study sections. I hope to never be tested as hehas, but wish more simply that my work will be afitting tribute to his guidance and fire.

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