JOURNAL OF PATHOLOGY, VOL. 164: 95-100 (1991)

C. L. OAKLEY LECTURE (1991)

CELL ADHESION AND EPITHELIAL DIFFERENTIATION*

STEWART FLEMING

Department of Pathology, University of Edinburgh, Teviot Place, Edinburgh EH9 8AG, U.K.

SUMMARY The differentiated function of epithelial tissues is dependent on the molecules which mediate the cohesion and

polarization of the epithelium. Separate molecular families are responsible for the adhesion between adjacent cells and the adhesion beween cells and the substratum. Using the kidney as a model of epithelial differentiation it has been shown that the assembly of a basal lamina containing the glycoprotein laminin and cadherin mediated intercellular adhesion are key events. The Wilms’ tumour anti-oncogene plays a regulatory role in renal epithelial differentiation. Tissue modelling and tumour formation are both accompanied by reduced cell adhesion molecule function.

KEY woRDs-Cell adhesion, kidney, epithelium, differentiation, Wilms’ tumour.

INTRODUCTION

In this lecture I intend to present evidence that molecules which promote adhesion of cells to each other and to the extracellular matrix are crucial in the differentiation of epithelium and that circum- stances in which the function of these molecules is impaired results in loss of the differentiated epithelial phenotype associated with tumour forma- tion and progression. An epithelium is a cohesive sheet of one or more layers of cells which lines a body surface and is attached to a connective tissue base. Epithelial integrity is critical in the differen- tiated function of many tissues such as skin, intes- tine, kidney, and lung, where epithelia perform barrier, secretory or absorptive functions. The for- mation of epithelial tissues is, therefore, of great biological importance but the reverse process, loss of the differentiation of epithelium, may be equally important to us as pathologists. Most common cancers are epithelial and one of the challenges facing us is an understanding of the factors which allow malignant epithelial cells to break contact

* 1991 C. L. Oakley Lecture delivered at the winter meeting of the Pathological Society of Great Britain and Ireland, held at the Clinical School, Addenbrooke’s Hospital, University of Cam bridge.

with their neighbours, invade adjacent connective tissues and eventually metastasize.

The kidney has been a valuable model system for the investigation of the factors which control epithelial differentiation for two main reasons. Although predominantly an epithelial organ the kidney is formed from the embryonic mesoderm, therefore during organogenesis there is a mes- enchymakpithelial transition.’ Study of the events involved in this phenotypic alteration has provided data on the regulation of epithelial differentiation in the kidney which may be applicable to other tissues. Secondly the renal e ithelium is a stable tissue as

injury occurs by cell division and developmental plasticity of the mature epithelium.

Morphological changes can be recognized during the sequential formation of nephrons from the metanephric blastema when induced by ingrowing ureteric bud branches. Induced cells first condense then, with the formation of a central lumen, form the renal vesicle. This structure elongates and twists to give the S-shaped tubule (Fig. I). The different segments of the nephron are determined at this stage, although further differentiation occurs including fusion of the S-shaped tubule with the ureteric bud.3

defined by Leblond; Y thus regeneration following

0022-341 7/91/05009546 $05.00 0 1991 by John Wiley & Sons, Ltd.

96 S. FLEMING

Fig. 1-The developing renal epithelium has condensed from the undifferentiated blastema, acquired a central lumen and elongated to form a n S-shaped tubule (Foetal kidney)

In immunocytochemical and biochemical studies of foetal kidney it is clear that not only does structural maturity precede function but that func- tionally important molecules such as brush border enzymes are not expressed until structural differen- tiation has occurred. For example, the peptidases of the renal brush border detected by antibodies of the CDlO and CD13 clusters are not seen until after fusion of the S-shaped tubule and the ureteric bud.4

Thus, by the time the molecules which are respon- sible for the specific function of the renal tubule, glomerulus, etc. are expressed the embryonic tubule is already recognizable as epithelial. .This cohesive layer of cells forming the immature nephron is struc- turally epithelial but has formed from a mesenchy- ma1 tissue. This suggests that the factors which control the structural integrity of the epithelial phenotype are those responsible for the initial mesenchymal-epithelial switch.

CELL SUBSTRATUM ADHESION

The earliest changes seen during the induction of renal tubulogenesis are in the extracellular matrix. The synthesis of the mesenchymal collagens I and 111 ceases and collagen IV, laminin and fibronectin

are expressed, initially in a diffuse pattern but rap- idly becoming restricted to the basal pole of the cells of the renal ~ e s i c l e . ~ Ekblom has demonstrated that the key event in this stage is the expression of the 400 kD A chain of laminin. In his murine in vitro experiments the 200 kD B1 and B2 chains of laminin are expressed early but are insufficient to induce tubular epithelial cell attachment and apical-basal polarization. Once laminin A chain mRNA and protein were detectable in his experimental system epithelial cell polarization occurred. These results demonstrate that laminin A chain is the important factor in the initial epithelial phenotypic change which is the establishment of a specific extracellular matrix at the basal pole.'

The differentiating epithelium must adhere to the newly synthesized basal lamina in order to establish polarity. There are two main types of cell receptors for the laminin molecule, a 68 kD rece tor and

evidence suggests that the laminin receptors in- volved in the mesenchymal epithelial switch are of the integrin group. Integrins, which are a p hetero- dimers recognizing Arg-Gly-Asp (RGD) sequences in the ligand, not only provide a mechanism of cell attachment but also link the substratum to the cytoskeleton.' The integrin chains are transmem- brane proteins the cytoplasmic domain of which interacts with proteins of the cytoplasmic plaque of focal cell substratum contacts such as vinculin and talin. These components in turn bind to the actin micro filament^.^

In foetal kidney members of the pl family of integrins are expressed during the mesenchymal epithelial transition and continue to be expressed by the renal epithelium in the adult kidney. Anti- bodies against integrin receptors impair murine renal tubulogenesis in vitro."

In the differentiated epithelium of the renal tubule the basal pole of the cells is adapted for their trans- port function. The cell membrane is arranged into alternating areas of attachment to the basement membrane and membrane infoldings. The ability of the cell substratum to organize the cytoskeleton and hence this complex cell structure in human renal epithelium in v i m has been investigated (M. Rahilly, manuscript in preparation). Human renal epithelial cells in primary culture attached to and grew on glass or on substrata composed of the indi- vidual extracellular matrix components laminin and fibronectin. The cells were then fixed and stained using monoclonal antibodies for integrins, talin, vinculin and actin filaments. Cells grown on glass

members of the integrin receptor family. P Current

CELL ADHESION AND EPITHELIAL DIFFERENTIATION 97

attached at the cell periphery where integrins and vinculin were expressed. These cells synthesized and secreted laminin at this peripheral site. The cyto- skeleton of these cells was poorly organized. By con- trast cells grown on fibronectin or laminin substrata exhibited a highly organized array of integrin, vin- culin and talin containing cell substratum contacts with organization of actin filaments into orientated bundles (Fig. 2 ) . The extracellular matrix and its receptors expressed during renal tubulogenesis appear to organize the cytoskeleton and structural differentiation of the basal pole characteristic of the renal epithelial phenotype.

CELL-CELL ADHESION

There are several distinct mechanisms of cell-cell adhesion.” These can be divided into junctional or non-junctional mechanisms depending on the rec- ognition of a morphologically defined junctional structure usually by electron microscopy. There are two main adhesive junctions to be considered. Des- mosomes, composed of a series of transmembrane glycoproteins, desmogleins, and proteins residing in the cytoplasmic plaque, desmoplakins, are intercel- Mar junctions which are linked to the intermediate filament cytoskeleton. Adherens junctions (zonula adherentes) contain a different family of transmem- brane adhesion molecules and are linked to the actin cytoskeleton. Neither gap junctions nor tight junc- tions are adhesive junctions but are rather struc- tures involved in the passage of small molecules by intercellular or paracellular routes.

The main adhesive molecules of the adherens junctions are members of the cadherin family.” These molecules are responsible for calcium depen- dent intercellular adhesion. The cadherins exhibit homophilic binding, that is a cadherin on one of a pair of cells binds to an identical molecule on the other cell. Transfection experiments have shown that cadherin expression and the resulting cell-cell adhesion causes phenotypic changes in the trans- fected cells. Fibroblasts transfected with cDNA en- coding uvomorulin, an epithelial cadherin, adhere to each other and adopt an epithelioid m~rphology. ’~ These cells also show polarity in the distribution of the Na/K ATPase. The non-transfected cells express this enzyme over the whole cell surface, but in the adherent transfected cells the enzyme is confined to the baso-lateral membrane.I4 Thus, adhesion mol- ecules of this type not only promote intercellular adhesion but induce the polarity and morphology characteristic of an epithelial phenotype.

To test whether cadherins are involved in the regulated conversion of mesenchyme to epithelium which occurs during renal differentiation, mono- clonal antibodies were used in immunocytochemi- cal experiments. Cadherin expression was seen in the developing renal tubular epithelium as it con- densed from the metanephric blastema. The epi- thelium continued to express these molecules in the adult kidney although the podocytes of the glom- erulus cease their expression at the S-shaped tubular stage. By using monoclonal antibodies for the dif- ferent cadherin families, in immunocytochemical and Western blotting experiments, it was found that human renal epithelial cadherin was A-CAM (Fig. 2) , a member of the N-cadherin family. This is dif- ferent from most animal species in which the renal cadherin is uvomorulin (L-CAM), a member of the E-cadherin family.I5

Identification of desmosomal components has been similarly performed. Desmoglein I (DG 1) and desmoplakins (DPIJI) were first seen in the condensing renal tubular epithelium approximately simultaneously with the cadherins. At this develop- mental stage the desmosomes identified by electron microscopy were poorly formed and immature. DGI and DP1,II became localized into discrete structures and morphologically identifiable desmo- somes appeared with further differentiation. How- ever, in the podocytes desmosomes and cadherins were lost as the foot processes of these cells formed.I6

These results suggest that expression of cadherins and desmosomal components are important in the formation and cohesion of the renal epithelium.

REMODELLING AND ADHESION

Adhesion between cells would appear to be im- portant in regulating the epithelial phenotype, but structural changes of the nephron including dif- ferentiation into different segments occurs and necessitates a degree of flexibility in intercellular binding.

Intercellular adhesion is maintained by both adherens junctions and desmosomes in epithelium but their formation and stability differ. The for- mation of intercellular junctions by podocytes in glomerular disease was studied by immunocyto- chemistry and electron microscopy. Adherens junc- tion formation was found to be a frequent response to injury in podocytes. This change occurred in a variety of diseases including lesions such as minimal

Fig. 2-Immunofluorescence preparations viewed on a Zeiss laser scanning microscope. (a) Renal tubular epithelial cells grown on glass coverslips and stained with an antibody to vinculin (Sigma) show weak cytoplasmic staining and small discrete areas of intense staining at thecell periphery. (b) In contrast to (a) when the same cells are grown on laminin coated coverslips, highly organized arrays of vinculin containing focal contacts are assembled over the entire ventral surface of the cell. (c) Cells from a renal carcinoma grown on laminin fail to organize their focal contacts, vinculin reactivity being confined to small peripheral patches. (d) Renal tubular cells switched from normal calcium to low calcium medium show loss of cell-cell adhesion. A-CAM (GC4, Sigma) reactivity in this immunogold preparation is seen on the separated cell surfaces. This reactivity is lost after I5 min in low calcium medium. (e) Restoration of normal calcium concentration is followed several hours later by A-CAM expression and re-adhesion. A-CAM is initially found on the entire cell surface becoming concentrated at sites of cellkell contact. (f) Eventually cohesive renal epithelial cell monolayers form and A-CAM reactivity is found on cellkell interfaces

CELL ADHESION AND EPITHELIAL DIFFERENTIATION 99

change disease which is known to be reversible. By contrast, desmosomal components were only seen in podocytes overlying areas of permanent glom- erular scarring. These data suggested that in renal epithelium desmosomes stabilize adhesion initially established in a labile, potentially reversible form by adherens junctions.”

The adhesive functions of cadherins and desmo- somes are calcium dependent. Primary cultures of renal tubular and glomerular epithelial cells in vitro were exposed to low calcium conditions, Within 1-2 min cell separation occurred although thin intercel- lular bridges persisted. Cadherin immunoreactivity was seen on the separated cellular surfaces although this was eventually lost within 10 min in low calcium medium. Restoration of the calcium concentration to physiological levels allowed the cells to re-adhere although this required several hours in normal cal- cium concentrations and was accompanied by reap- pearance of A-CAM on the cell surface (Fig. 2).’* Desmosome disruption occurred at a slower rate. These data support the hypothesis that cadherin dependent adhesion of the adherens junction type is labile while stability of cohesion in the differen- tiated tissue is achieved by desmosomes and the intermediate filament cytoskeleton.

REGULATION OF THE MESENCHYMAL- EPITHELIAL TRANSITION

Obviously during the mesenchymal-epithelial transition several genes are switched off and others switched on in a closely regulated manner. Infor- mation on the basis of the regulation of the tran- sition has come from studying the pathology of renal differentiation in Wilms’ tumour. Knudson showed from study of familial and sporadic cases of Wilms’ tumour that an autosomal recessive ‘anti- oncogene’ was involved. Familial cases inherit one abnormal copy and acquire an abnormality, mutation or loss, in the remaining good copy of the gene, resulting in tumour formation. In sporadic cases two acquired events occur at the genetic locus. Cytogenetic data have placed the Wilms’ tumour locus on the short arm of chromosome 1 I .

One candidate gene from 1 lp l3 has recently been isolated and sequenced. The gene encodes a poly- peptide composed of four zinc finger domains and thus is similar to many gene transcription regulatory proteins. in situ hybridization and Northern blot techniques have been used to study the pattern of

expression of this gene in foetal kidney. The candi- date Wilms’ tumour gene mRNA was detected in high levels in the cells undergoing the mesenchy- mal-epithelial transition in renal embryogenesis.” The signal fell to low levels in the maturing tubular epithelium although some persistance in podo- cytes was seen. In adult kidney only low levels of expression were seen. A series of Wilms’ tumours studied showed a close correlation between levels of expression of this gene and epithelial differentiation in the tumour. In other fetal tissues expression of the candidate Wilms’ tumour gene was seen in developing mesothelium, sex cord epithelium and uterus. The common feature of these several tissues is that they are amongst the few which undergo mesenchymakpithelial transition as part of their development.

These results raise the prospect that the Wilms’ tumour gene encodes a DNA transcription regulat- ory protein involved in coordination of epithelial differentiation in the kidney and in other epithelia derived from the embryonic mesoderm.

LOSS O F THE ADHESIVE PHENOTYPE IN RENAL NEOPLASMS

Several tumour types show morphological evi- dence of loss of cohesion, indeed in some techniques this is a valuable diagnostic criterion.21 Changes in the expression of integrins, basement membrane components and members of the N-CAM family have been shown to occur in a variety of epithelial

Most adult renal tumours are carcinomas. These frequently ‘invade’ the adjacent kidney as a cohesive mass.’’ A small proportion of renal carcinomas show a more infiltrative behaviour especially those of a sarcomatoid type. Immunocytochemistry has shown that sarcomatoid renal carcinomas with an infiltrative pattern of invasion lack expression of DGI. Preliminary cell culture data have shown that some renal carcinomas have reduced levels of A-CAM expression and a reduced ability to form organized adhesive substratum contacts when grown on laminin (Fig. 2). Well differentiated papil- lary renal carcinomas, which are known to have a better prognosis, continue to express A-CAM and cell-substratum contacts. These data are prelimi- nary but suggest that loss of adhesion molecules may be an important progression factor in renal carcinoma.

100 S. FLEMING

CONCLUSIONS

The structural features of epithelial differen- tiation are dependent on the regulated function of various cell-cell and cell-substratum adhesion mol- ecules. The expression of these molecules may be under the control of regulatory genes, abnormalities of which are involved in the pathogenesis of tu- mours including Wilms’ tumour. Loss of function of cell adhesion molecules is important in phenotypic changes and progression of epithelial neoplasms.

ACKNOWLEDGEMENTS

I wish to thank Drs Leigh Biddlestone, Maeve Rahilly and Barbara Langdale-Brown for helpful discussion of the manuscript. I would also like to thank them and many other colleagues for their invaluable contributions to my research. Different parts of this work were supported by the National Kidney Research Fund, Scottish Hospitals Endow- ment Trust and the Scottish Home and Health Department .

REFERENCES

1. Ekblom P. Developmentally regulated conversion of mesenchyme to epithelium. Fuseh J 1989;3: 2141-2150.

2. Leblond CP. Classification of cell populations on the basis of their proliferative behaviour. Nail Cuncer Ins1 Monogruphs 1964; 1 4 119-1 50.

3. Saxen L, Koskimies 0, Lehti A. Miettinen H, Rapola J, Waartiovaara J. Differentiation of kidney mesenchyme in an experimental system. Ails Morphogenesis 1968; 7: 25 1-284.

4. Fleming S . Thesis MD, University of Glasgow, 1988.

5.

6.

7.

8.

9.

10.

I I.

12.

13.

14.

15.

16.

17.

18. 19.

20.

21.

22.

Ekblom P, Sariola H, Thesleff 1. Basement membrane and organo- genesis. In: Developmental Mechanisms: normal and abnormal. ed. Lash J, Saxen L. New York: Alan Liss, 1984; pp 109-122. Ekblom M, Klein G. Mugrauer G, el a/. Transient and locally restric- ted expression of laminin A chain mRNA by developing epithelial cells during kidney organogenesis. CeN 1990; 60: 337-346. Buck CA, Horwitz AT. Cell surface receptors for extracellular matrix molecules. Ann Rev Ce// Biol 1989; 3 179-206. Burridge K , path K , Kelly T, Nicholls A, Turner C. Focal adhesions: Transmembrane junctions between the extracellular matrix and the cytoskeleton. Ann Rev Cell B i d 1988; 4: 487-526. Otey CA, Pavalko FM. Burridge K. An interaction between a-actinin and thep, integrin subunit in vilro. JCeNBiol 1990; 111: 721-729. Sorokin L, Sonnenbourg A, Aumailley M, Timpl R, Ekblom P. Rec- ognition of the laminin E8 cell kidney site by an integrin possessing the ah subunit is essential for epithelial polarisation in developing kidney tubules. JCeNBiol 1990; 111: 1265-1273. Garrod DR. Desmosomes, cell adhesion molecules and the adhesive property of cells in tissues. J CeU Sci 1986; Suppl. 4: 221-237. Takeichi M. The cadherins. Cell-cell adhesion molecules controlling animal morphogenesis. Development 1988; 102: 639-655. Mage R-M, Matsuzaki F, Gallin WS, Goldberg J I , Cunningham BA, Edelman GM. Construction of epithelioid sheets by transfection of mouse sarcoma cells with cDNAs for chick cell adhesion molecules. Proc Nut/ AcudSci USA 1988; 85: 7272-1278. McNeill H, Ozawa M, Kember R, Nelson WJ. Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity. Cell 1990; 6 2 309-316. Biddlestone LR, Fleming S . Morphological evidence that A-CAM is the major intercellular adhesion molecule in human kidney. J Parhol 1990 (in press). Garrod DR, Fleming S. Early expression ofdesmosomal components during kidney tubule morphogenesis in human and animal embryos. Developmenr 1990; 108: 3 13-32 I. Fleming S, Langdale-Brown B, Biddlestone LR. Cell adhesion mol- ecules on glomerular podocytes. Ncphrol Diaf Trnnsplunf 1990; 5: 677. Biddlestone LR, Fleming S. In preparation. Pritchard-Jones K, Fleming S , Davidson D ern/. The Wilms’ tumour gene is involved in normal human genitourinary development. Narure 1990; 346: 19&197. Olsen TS. Tumours of the kidney and urinary tract. Copenhagen: Munksgaard. 1984. Orell SR, Sterrett GF, Walters MN-I, Whittaker D. Fine Needle Aspiration Cytology. Edinburgh: Churchill Livingstone. 1986; 90. Fearon ER, Cho KR, Nigro J M er ul. Identification o f a chromosome 1 8q gene that is altered in colorectal cancers. Science 1990; 247: 49-56.