Cytokine control of cell motility: Modulation and mediation by the extracellular matrix

Progress in Growh

Farm Research. Vol. 5. p. 223-248, I994 Copyright 6 1994 Elsevier Science Ltd Prmted in Great Britain. All tights reserved 0955-2235194 $26.00

CYTOKINE CONTROL OF CELL MOTILITY: MODULATION AND MEDIATION BY THE EXTRACELLULAR MATRIX Seth L. Schor School of Biological Sciences The Medical School, 3rd Floor Oxford Road University of Manchester Manchester, Ml3 9PT. U.K.

Cytokines are multifunctional regulators of cell behaviour aflecting such diverse activities as cell proliferation, gene expression and motility. Matrix macromolecules ir@ence a similarly wide range of cell functions. A review of the available literature suggests that cytokines may affect cell motility by (a) directly injuencing the motility apparatus, and (b) indirectly as a consequence of the altered expression of genes coding for matrix macromolecules, their respective cell surface receptors and matrix degrading enzymes and their inhibitors. Conversely, the composition and supramolecular organisation of the matrix plays a central role in defining cellular response to potentially multifunctional cytokines. Such complex and reciprocal interactions between cytokines and the matrix elicit both positive and negative reiterative feedback loops which must be taken into account when interpreting the results of migration assays in vitro and e.utrapolating them to in vivo processes. Keywords: Cytokines, matrix macromolecules, cell movement.

INTRODUCTION Cell migration is a prominent feature of embryonic development, involving both the coordinate movement of cell sheets and the translocation of single cells through complex macromolecular matrices. Tissue cells in the adult tend to lead considerably more sedentary lives, although they may be coaxed into re-expressing the more mobile attributes of their fetal progenitors during wound healing. Inappropriate cell migration is also a prominent feature of various disease processes, such as tumour invasion, where it is often accompanied by the lysis and remodelling of the surrounding extracellular matrix. Animal model studies and recent clinical trials have implicated numerous cytokines and matrix macromolecules in the control of cell migration in these processes [l-6]. Complementary in vitro investigations have revealed that these same molecules regulate the initiation, directionality and cessation of cell migration in a number of different assay systems. These investigations have further indicated that the effects of individual cytokines and matrix macromolecules 223

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cannot be viewed in isolation, but must be considered within the framework of multicomponent networks. The dynamic and reciprocal nature of these cytokine-matrix interactions has been revealed by observations indicating that (a) the precise effects of a potentially multi-functional cytokine on cell behaviour is modulated by the “context” of the extracellular matrix, and (b) cytokines affect the synthesis and degradation of numerous matrix macromolecules, as well as the quantity, diversity and activity of their respective cell surface receptors [7-91. Seen in the light of these findings, it has become increasingly apparent that a deeper understanding of the complex interrelationship between cytokines and matrix macromolecules is paramount to the interpretation of results obtained in vitro and their rational extrapolation to the in vivo situation. With these various points in mind, the objectives of this review are (a) to describe the principal assay systems used to assess cell migration in vitro, (b) indicate the diversity of cytokines and matrix macromolecules reported to be biologically active in these assays, and (c) highlight the available evidence indicating that matrix macromolecules may both modulate and mediate cellular migratory response to cytokines. MIGRATION

ASSAYS

Numerous assays have been devised to assessthe influence of cytokines and matrix macromolecules on cell migration in vitro. In spite of manifold differences in detail, these assays may be subdivided into three main groups on the basis of the substrata employed, these being 2D artificial surfaces, 3D macromolecular matrices and explanted biological tissues. The transmembrane assay is the most commonly used experimental system [IO]; this is a 2D substratum assay based on the assessment of cell migration through a porous polycarbonate membrane separating an upper and lower medium compartment containing the putative effector molecule. Assessing cell migration in response to different concentrations of cytokine in the two chambers makes it possible to distinguish between chemokinesis (defined as the random stimulation of cell migration by a homogenous concentration of soluble effector molecule) and chemotaxis (defined as the directional stimulation of cell migration in response to an imposed concentration gradient of the effector). These two modes of cytokine action may be independently assessed by the “checkerboard” method of analysis [1 11. Using this assay system, numerous cytokines have been reported to affect cell migration by both chemokinetic and chemotactic mechanisms. Related assays are based on measuring the effects of cytokines on the motility of cells cultured on 2D tissue culture substrata by a variety of techniques, including the measurement of phagokinetic tracks [12], time-lapse video microscopy of individual cells [13, 141, determination of colony diameter and/or inter-cell distance [15-l 71, and cell dispersal from multicellular spheroids onto the tissue culture substratum [18]. Soluble matrix macromolecules have similarly been reported to exert both chemokinetic and chemotactic effects upon cell migration in transmembrane assays [19-231. These molecules may additionally affect cell migration by huptotactic mechanisms involving the directional stimulation of cell motility in response to a concentration gradient of adsorbed matrix molecule across the polycarbonate membrane [21, 241. The effects of adsorbed (insoluble) matrix constituents on cell migration have also been studied in related assays in which the relevant molecules are applied to a suitable 2D tissue culture substratum, either uniformly or in discrete tracks [25, 261.

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In view of the complex interdependency between cytokines and matrix macromolecules, as well as in an attempt to develop assay systems more closely approximating the in vivo milieu, various workers have developed migration assays involving cell movement within 3D macromolecular matrices. In one of the earlier systems employed, cells were plated onto the surface of 3D gels of type I collagen fibres and their subsequent migration down into the fibrillar collagen matrix measured by microscopic observation [ 151. Progressively more complex substrata may be produced by the stepwise addition of other matrix macromolecules (such as fibronectin and glycosaminoglycans) [27-301. 3D matrices consisting of other potentially relevant macromolecules, such as fibrin [21, 321 and “reconstituted basement membrane” (Matrigel) have also been used. In this regard, Twamoto and Sugioka [33] have examined the ability of various tumour cell lines to penetrate porous membrane filters coated with a thin 3D film of Matrigel in response to chemotactic factors produced by NIH 3T3 cells. They report that there was a good correlation between the chemotactic response of these cells in vitro and their respective metastatic potential in vivo. Interestingly, Simon et al. [34] have assessed the migratory behaviour of various normal, transformed and tumour-derived cell lines in the same assay system. In contrast, they report that normal cells (human skin fibroblasts) and non-tumourigenie cell lines (3T3) were able to penetrate the Matrigel coating, whilst various metastatic tumour cell lines (MCF7, MCF7 ras, BeWo and JAR) were not; the authors conclude that cell migratory behaviour in this particular assay system is not a reliable indicator of in vivo metastatic potential. We have similarly found little correlation between the ability of cells to migrate into 3D gels to type I collagen and metastatic potential [ 151; indeed, as was the case with Simon et al., we found that cells of mesenchymal or neural crest origin (whether normal or transformed) migrated into collagen gels, whilst, in the absence of exogenous chemotactic factors, the majority of epithelial cells (both normal and transformed) remained on the gel surface. The results obtained with such 3D macromolecular matrix assays have both confirmed and conflicted with those accrued in the more commonly used 2D substrata assays, For example, members of the TGFP have been reported to stimulate the migration of human skin fibroblasts in transmembrane assays [35, 361, whilst other workers [37, 381 failed to observe any migration stimulating activity in these same assays using a variety of target cell lines, including human fetal fibroblasts, human foreskin fibroblasts, rat lung fibroblasts and Swiss 3T3 cells. Our own unpublished observations have also failed to detect an effect of TGF-/?I on the migration of human skin fibroblasts in the transmembrane assay; interestingly, the same batches of TGF/? were actually found to inhibir the migration of human skin fibroblasts in the 3D collagen matrix assay. Although the reasons for these discordant inter- and intraassay results are not clearly understood, potential underlying mechanisms may involve the modulation of cellular response to TGFP by the precise nature of the macromolecular substratum (as discussed below) and temporal differences in the two assay protocols; i.e. in the transmembrane assay cell migration is assessed within a 2-3 h timescale, whilst the 3D collagen gel assay requires 334 days and is therefore more sensitive to amplification mechanisms mediated via cytokine-induced effects on other aspects of cell behaviour (e.g. the synthesis and deposition of matrix macromolecules). More complex substrata are afforded by explanted pieces of biological tissues, such as cornea [39] and retina [40]. Although it may be argued that such substrata provide

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the best approximation of the in viva environment, their complex and poorly defined nature complicates the interpretation of studies specifically designed to elucidate the contribution of individual matrix constituents to the control of cell motility. CYTOKINES

AFFECTING

CELL

MOTILITY

The extensive literature dealing with the effects of cytokines on cell migration has been the subject of several previous reviews [4143]. Such studies have revealed that numerous cytokines affect cell migration by both chemokinetic and chemotactic mechanisms; these include such well characterised “growth factors” as epidermal growth factor (EGF), platelet derived growth factor (PDGF), members of the transforming growth factor-p family (TGFP), members of the heparin binding growth factor family, such as acidic and basic fibroblast growth factors (a- and bFGF), interleukins, and haematopoietic growth factors, such as granulocyte/ macrophage colony stimulating factor (GM-CSF). Several more recently identified cytokines, such as scatter factor (SF), autocrine motility factor (AMF), autotaxin (ATX) and migration stimulating factor (MSF) have been referred to as motility factors as a consequence of their effects on cell migration in the various assays employed in their initial identification [42,43]. Scatter factor was originally identified in the conditioned medium of fetal and transformed fibroblast cell lines using the dispersal of MDCK cells from tight colonies as a sensitive bioassay [44]. Subsequent studies indicated that SF is identical to hepatocyte growth factor (HGF) and that SF/HGF is produced by a wide variety of different cell types and affects the migration of a similarly broad range of target cells [4S, 461. SF/HGF binds to the c-met receptor [4648]. The nature of the signal transduction cascade initiated by c-met ligation has been the subject of several studies. These have indicated that SF/HGF activity is inhibited by agents which activate adenylate cyclase (e.g. forskolin, theophylline) and markedly enhanced by PMA and agents which inhibit PKC activity (e.g. Staurosporine) [49]; in contrast, modulators of CaZ+/ calmodulin mediated pathways had little effect on SF/HGF activity. AMF has a molecular mass of 55 kDa and was originally shown to be produced by a melanoma cell line [50]. It was initially referred to as an “autocrine” factor by virtue of the fact that the producing melanoma cell line was also a responsive target. AMF modulates cell motility as a consequence of its binding to a 78 kDa cell surface receptor (gp78AMFR), which subsequently becomes phosphorylated; the gene coding for gp78AMFR has been cloned and reported to display significant amino acid sequence homology with p53[51]. ATX is an apparently unrelated 125 kDa migration stimulating cytokine also produced by melanoma cells [52]. The bioactivity of ATX is blocked by prior exposure of target melanoma cells to pertussis toxin, thereby suggesting the involvement of a pertussis toxin-sensitive G protein in signal transduction. In our early studies with 3D collagen matrix assay, we observed that fetal fibroblasts produced a migration stimulating factor (MSF) not made by their normal adult counterparts [53,54]. Although not producing MSF themselves, confluent adult fibroblasts retain responsiveness to it, as evidenced by their elevated migration when exposed to fetal fibroblast conditioned medium. Using this migratory response of adult fibroblasts as a convenient bioassay for monitoring MSF activity, we developed a protocol for purifying MSF from fetal fibroblast conditioned medium [55].

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Subsequent biochemical characterisation of MSF indicated that it is a protein with apparent molecular weight of 119 kDa (although this appears to degrade easily during the purification procedure to a number of lower molecular weight derivatives) [56]. Both the 119 kDa species and certain of its lower molecular weight derivatives stimulate fibroblast migration (with half maximal activity in the region of 0. l-l .O pg/ ml). N-Terminal amino acid sequence analysis of the 119 kDa molecule and its biologically active degradation products have revealed the presence of a peptide domain exhibiting significant homology with the gelatin-binding fragment of fibronectin, as well as two other apparently unique domains. Both SF/HGF [57, 581 and AMF [59] have recently been reported to stimulate the proliferation of certain target cell types; it would therefore appear that many (if not all) of these “motility factors” resemble the “growth factors” in that they too affect several fundamental aspects of cell behaviour and should consequently be considered multifunctional with respect to their spectrum of biological activities. Stoker and Gherardi [43] coined the term motogenic to refer to the effects of cytokines on cell motility and clearly distinguish these from the better characterised nritogenic activities of these molecules. A number of general statements may be made regarding the motogenic activities of cytokines. These include: 1. The eft%ects of cytokines on cell migration may be mediated via mechanisms directly aflecting the cytoskeleton and associated ‘inotility engine“. As is the case with

their better understood effects on cell proliferation, the initial event required for the mediation of cytokine motogenic activity clearly involves ligand binding to an appropriate cell surface receptor. These receptors have been classified into several families on the basis of the molecular nature of their extracellular domains and the presence or absence of intrinsic tyrosine kinase activity within the cytoplasmic segment of the molecule [60]. Various well characterised signal transduction cascades are known to be elicited as a result of receptor ligation and (where relevant) the consequent activation of tryosine kinase activity: these include activation of phospholipase C-r, the generation of diacylglycerol and phosphatidylinositol metabolites, changes in C&+ ion flux, and elevation of cytoplasmic pH [60-62]. The key issue with respect to the mediation of motogenic signals is that receptor ligation may directly affect such relevant parameters as plasma membrane ruffling, the formation of focal adhesion plaques and actin assembly. Although many of the details remain to be established, such direct effects of cytokines on the cellular motile machinery appear to involve changes in the phosphorylation of both actin [63, 641 and various actin regulatory molecules (such as profilin) [65, 661 by ras-related GTPases (such as rho and rat) [67-69]. 3-. Individual members of cytokine “superfamilies” commonly display striking isoform speciJicity with regard to their motogenic activity. In this regard, Nister et al. [70] reported that PDGF-BB and PDGF-AB were chemotactic for human foreskin fibroblasts in the transmembrane assay, but that the PDGF-AA isoform was not. Koyama et al. [71] similarly found that both PDGF-BB and PDGF-AB stimulated the migration of smooth muscle cells, whilst PDGF-AA was devoid of demonstrable activity when used on its own. In contrast, PDGF-AA has been reported to stimulate the migration of 3T3 cells [72] and type 2 astrocyte precursor cells [73]. Finally, isoforms of TGFP have been reported to exhibit cell-dependent differences in motogenic activity. Merwin et al. [74] noted that TGF-PI was a potent inhibitor of

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bovine aortic endothelial cell migration, whilst TGF-/?2 displayed only modest migration inhibiting activity; in contrast, the migration of bovine aortic smooth muscle cells was significantly stimulated by TGF-/?l and unaffected by TGF-@. 3. Cytokines are multifunctional modulators of cell motility, capable of either not affecting, stimulating or inhibiting migration. Koyama et al. [71] have reported that

PDGF-AA inhibited the migration stimulating activity of both PDGF-AB and PDGF-BB for smooth muscle cells, but had no appreciable effect on cell migration when examined on its own. Grotendorset [75] reported that EGF inhibited the migration stimulating activity of PDGF for 3T3 cells, whilst Adelmann-Grill et al. [37] found that either concurrent exposure or preincubation of fibroblasts to TGF/? completely abolished the migration stimulating activity of EGF. In contrast to previous reports, these latter authors did not observe any direct stimulatory effect of TGFB on fibroblast migration when examined on its own. Finally, Koyama et al. [76] found that although TGFPl and PDGF both stimulated smooth muscle cell migration when assessed independently, TGF/%l was a potent inhibitor of PDGFinduced migration in co-incubation studies, displaying a dose-dependent inhibitory activity in the’ range of 1.O pg/ml-1 .O rig/ml. 4. The proltferative state of the target cells used in transmembrane assays may afict their motogenic response to cytokines. In this regard, Grotendorst [75] reported

that the motogenic response of (density arrested) quiescent NIH/3T3 cells to PDGF was 25-fold higher than subconfluent, exponentially growing cells and that the response of cells at intermediate densities was proportional to cell density. This difference was not due to alterations in PDGF binding, as the low density cells actually bound more cytokine than the confluent ones. Interestingly, both subconfluent and confluent cells showed similar motogenic responsiveness to fibronectin. More recently, Hughes and McCulloch [77] have used computer-assisted image analysis and double labelling techniques to confirm that cycling human gingival fibroblasts displayed a greatly reduced motogenic response to PDGF compared to non-cycling cells within the same assay; indeed, cells in S phase were actually totally unresponsive to PDGF. 5. The motogenic activity of cytokines commonly follows a bell shaped doseresponse curve. A number of cytokines, including TGF-@I, PDGF, EGF and MSF

have been reported to display a bell-shaped dose-response when assessed in transmembrane assays [30, 35, 36, 781. Similar bell-shaped dose-response curves have also been obtained with respect to the effects of cytokines on other aspects of cell behaviour (e.g. proliferation and gene expression) and presumably result from common underlying mechanisms, such as cytokine binding to different classes of cell surface receptors and/or the effect of differential receptor occupancy [71, 799811. MATRIX

MACROMOLECULES

AFFECTING

CELL

MOTILITY

As is the case with cytokines, there is an extensive literature documenting the effects of matrix macromolecules on cell motility. Using various assay systems, these studies have indicated that cell migration is modulated by ubiquitous macromolecular constituents of the extracellular matrix, such as collagen, fibronectin, laminin and proteoglycans. A detailed survey of this literature is beyond the scope of the present discussion and may be found in several recent reviews [82--841.

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Cell adhesion to the matrix is a requisite for cellular movement involving the generation of tractional forces. These cell-matrix interactions must be made and broken in a temporally coordinated fashion during the process of cellular translocation. Apart from this physical requirement for the cyclical establishment of attachment foci between cell and matrix, a number of points relating to cell-matrix interactions are particularly germane to the role of matrix macromolecules in the modulations and mediation of cytokine motogenic activity; these may be briefly summarised as follows: 1. The ligation of matrix receptors results in the inhibition of diverse second messenger cascades which may directly a#ect motility apparatus. The integrin family of cell surface receptors have been particularly well characterised in this regard [85]. The cytoplasmic domains of these receptors are capable of establishing functional connections with the cytoskeleton (via interactions with molecules such as crcactinin and talin), thereby providing the structural basis for altering cytoskeletal structure and function. Integrin-mediated cell signalling has been reported to employ various second messenger transduction strategies, including the phosphorylation of receptorassociated kinases, activation of phospholipase C, the induction of Ca’+ fluxes and cytoplasmic “alkylisation” [86]. The tyrosine phosphorylation of proteins present in focal adhesions (such as p~l25~~~, paxillin and tensin) have been recently reported and appear to play an important role in signal transduction [87-891. The initiation of calcium transient.s is a common feature of cytokine signal transduction. Recent data have indicated that the ligation of integrin receptors elicits similar rapid changes in calcium flux [90, 911 and that exogenous calcium and magnesium may modulate integrin-mediated cell motility [92-941. Apart from these types of “outside-in” signalling, integrin activity may also be modulated by changes in their affinity for specific ligands 195, 961. These latter types of “inside-out” signalling may be elicited by a number of means, including cell interaction with cytokines. Taken together, these observations suggest various mechanisms by which cytokine motogenic activity may be mediated by primary effects on matrix and/or integrin expression. 2. The diverse efects of matrix macromolecules on cell motility are mediated bJ distinct intramolecular domains. The amino acid sequences of these various domains have recently been reviewed by Zetter and Brightman [82]. Fibronectin was the first matrix macromolecule to be functionally dissected in this fashion. A number of initial studies clearly indicated that the effects of fibronectin on cell adhesion and migration were mediated by cell interaction with the RGDS motif found in the cell-binding domain [ 19, 97, 981; subsequent studies revealed that cells of neutral crest origin also recognised additional motifs found in other functional domains [22, 99, 1001. In studies assessing cell migration in transmembrane assays, concentrations of native fibronectin and the central cell binding domain in the region of l-50 pg/ml were reported to be required to elicit a motogenic response; in these same assays, other fragments of fibronectin (including the 43 kDa gelatin-binding domain) were completely devoid of migration stimulating activity. In collaboration with Drs Martin Humphries (University of Manchester) and Dean Mosher (University of Wisconsin), we have recently examined the effects of fibronectin fragments on fibroblast motility in the 3D collagen matrix assay. As the data in Fig. 1 indicate, we found that concentrations of the 43 kDa gelatin-binding fragment

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q

5

FN

n

Gel domain

n

Cell

domain

l

HEP-2

r

----II li1 1

FIGURE 1. The effects adult fibroblasts.

of native

fihronectin

Concentration (pg/ml) and its proteolytic fragments

on the migration

of confluent

(GFB) in the region of 0.1-1.0 pg/ml exerted a significant stimulation of cell migration. This biological activity of GBF displayed a bell-shaped dose-response curve, with migration being maximal at concentrations of 10 pg/ml-1 rig/ml. In contrast, native fibronectin, the 110 kDa cell binding fragment, 29 kDa Hep-l/Fib-l fragment and 20 kDa Fib-2 fragment were completely devoid of migration stimulating activity over the extended concentration range examined. Identical results were obtained with recombinantly engineered fragments of fibronectin containing the gelatin-binding domain, as well as a 10 kDa proteolytic degradation product of GBF. The motogenic activities of both the proteolytic and recombinant GBF peptides were completely neutralised by monoclonal antibodies directed against epitopes present in the gelatin-binding domain, but unaffected by antibodies recognising epitopes in other domains. Our results indicate that GBF produced by the proteolytic degradation of fibronectin expresses a potent (cytokine-like) motogenic activity at femtomolar concentrations when assayed on a 30 collagenfibre matrix. The discrepancy between the motogenic activities of native fibronectin and its various proteolytic fragments in the transmembrane and 3D collagen matrix assays support the view that the nature of the substratum employed in the assay may profoundly modulate cellular migratory response to exogenous cytokines and matrix macromolecules. The apparently distinct biological activities of GBF and native

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fibronectin in the 3D collagen matrix assay are similarly intriguing. These observations are consistent with previous reports describing similar cryptic activities of other fibronectin fragments with respect to the stimulation of monocyte migration in transmembrane assays [loll and the induction of protease gene expression by adherent rabbit synovial fibroblasts [102]. Although previous studies have not demonstrated an effect of GBF on cell migration, this proteolytic fragment of fibronectin has been shown to possess a number of other biological properties, including the enhancement of morphological transformation in virally transformed cells [IO31 and the expression of collagenase activity [104]. Nabeshima et al. [105] have reported that degradation products of collagen stimulate the migration of various tumor cell lines in the transmembrane assay; interestingly, native collagen did not exhibit such chemotactic activity. Taken together with our results, these findings suggest that the insoluble matrix may provide a reservoir of potentially motogenic degradation products. As cytokines have been reported to induce the expression of various matrix degrading enzymes [ 106, 1071, it is possible that the generation of motogenically active fragments of matrix macromolecules by these enzymes may provide one means of mediating cytokine effects on cell motility. A considerable amount is also known about the particular domains mediating the motogenic activities of other matrix constituents. Vaughan [108] reported that a particular isoform of tanascin resulting from alternative splicing of the fibronectin type III domain (ten220) is differentially expressed during the development of the chick cornea and, in distinction to other tenascin isoforms, is both temporally and spatially correlated with epithelial cell migration; Husmann et al. [109] went on to demonstrate that different regions of the fibronectin type III repeat in tenascin were involved in mediating the migration and outgrowth of cultured neural cells. Taraboletti et al. [23] reported that the chemotactic and haptotactic motogenic activities of thrombospondin were mediated by distinct domains. Aznavoorian et al. [21] have presented similar evidence that the chemotactic and haptotactic activities of laminin and type IV collagen (as assessed in the transmembrane assay) are mediated via distinct signal transduction cascades. Several cytokines have been observed to stimulate the expression of specific isoforms of matrix macromolecules. For example, TGF-@I has been variously reported to enhance the synthesis of fibronectin isoforms containing the ED-A, ED-B and alternatively spliced IIICS domains [110-l 131. Taken together, these observations suggest that cytokine-dependent alterations in matrix isoform expression may provide a possible mechanism for the matrix mediation of cytokine motogenic activity. Developmental and transformation-related changes in the relative expression of fibronectin isoforms have similarly been reported to correlate with changes in cell migratory behaviour. Fibronectin obtained from aged fibroblasts is relatively deficient in its content of both the ED-A and ED-B domains [114] and displays a diminished ability to support cell attachment and migration [115]; related studies have revealed that aged fibroblasts display a decreased migratory activity compared to cells derived from younger donors /116]. In contrast, other studies have demonstrated that fetal and transformed fibroblasts secrete relatively elevated levels of the ED-B fibronectin isoform [I 171 and display relatively elevated levels of migratory activity [53] compared to their normal adult counterparts.

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Gordon et al. [118] have recently reported that the migration of a human bladder carcinoma cell line is differentially affected by the extracellular matrix deposited by control and ras transfected fibroblasts. The identity of the molecular constituent or constituents responsible for this effect are not as yet known, although it would appear that fibronectin is a reasonable initial candidate, as normal and transformed cells produce different fibronectin isoforms. 3. Specific classes and/or isoforms of cell surface receptors are involved in mediating the motogenic activities of matrix macromolecules. A considerable amount is known

regarding the involvement of different integrin subunits in mediating the motogenic activities of matrix macromolecules, such as fibronectin, lamin and tenascin [96, 119, 1201; these observations have highlighted the role of the /I, subunit, as it appears to be involved in mediating the effects of several matrix constituents. The motogenic activity of hyaluronic acid (HA) is mediated via its interaction with at least two distinct types of cell surface receptor, RHAMM and CD44. RHAMM (receptor for hyaluronan mediated motility) is a 58 kDa glycoprotein which has recently been cloned [121]; it is relatively abundant at the tips of advancing lamellipodia in migrating cells. Two isoforms of CD44 have been identified; the H isoform (CD44H) binds HA, whilst the E isoform (CD44E) does not; Thomas et al. [ 1221 demonstrated that induction of CD44H (but not CD44E) in melanoma cells resulted in a stimulation of cell migration on HA coated substrata. The motogenic activity of cytokines may therefore be indirectly mediated via cytokine-induced changes in the repertoire of matrix receptors expressed by target cells (as discussed in more detail below). 4. Cytokines bind to matrix macromolecules and may be presented to cells in this form. The interaction of members of the heparin-binding family of growth factors

(e.g. aFGF, bFGF) with heparin and heparan sulphate proteoglycan alters their affinity for their respective cell surface receptors [123]. Other cytokines, including GM-CSF, IL-3, IGF-1, and LIF bind to macromolecular constituents of extracellular matrix and remain biologically active in this form [124127]. TGF-/?l stimulates the expression of the core protein of decorin; it avidly binds to this protein, in which state it is rendered biologically inactive [128]. TGF-pl has also been reported to bind to fibronectin [129]. The motogenic activity of cytokines may therefore also be modulated indirectly via the binding of cytokines to cytokine-induced matrix proteins, Matrigel has recently been reported to contain a complex mixture of tightly bound cytokines which appear to be responsible for mediating some of the reported biological activities of this matrix preparation [ 1301. This is an important finding and clearly indicates that great care must be taken to exclude the possibility that adsorbed cytokines are actually responsible for the reported motogenic activities of matrix macromolecules. 5. The molecular conformation of matrix constituents and their 30 spatial organisation exert profound eflects upon cell migration, Early studies in our laboratory

indicated that cell adhesion and migration were differentially affected by denatured collagen (gelatin), a thin coating of native type I collagen and a 3D meshwork of native type I collagen fibres [15, 13 11. Subsequent studies have confirmed these observations and indicated that the presence of a minimal triple helical tropocollagen organisation [132], as well as the physical properties of the macromolecular substratum [133], have profound effects upon ccl1 migration. In these early studies we also reported that HeLa cells plated on the surface of collagen gels did not migrate down

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into the 3D macromolecular matrix [15]; in contrast, these same cells displayed significant migratory activity (as assessed by colony morphology) when they were initially plated within the 3D collagen matrix as a single cell suspension. We subsequently demonstrated that the phenotype of endothelial cells was similarly influenced by their precise mode of interaction with the collagen substratum; cells plated on the gel surface were induced to display a”cobblestone” phenotype, whilst cells plated within the gel matrix rapidly adopted a “sprouting cell” phenotype [ 134, 1351. Marx et al. [136] have reported that the expression of PDGF receptors by kidney mesangial cells was profoundly affected by culture on and within such 3D collagen matrices. These various findings indicate that the supramolecular organisation of the extracellular matrix and the spatial distribution of cellular interactions with the matrix (i.e. only on the basal cell surface or isotropically over the entire cell surface) play a key role in defining cell phenotype.

MATRIX

MACROMOLECULES MOTOGENIC

MAY MODULATE ACTIVITY

CYTOKINE

The polycarbonate membranes used in transmembrane assays must be coated with a matrix macromolecule (such as native collagen, gelatin or fibronectin) in order to promote cell adhesion. The potential effects of these matrix molecules in defining cellular migratory response to cytokines has not been adequately addressed, although various lines of evidence coming from both transmembrane and other 2D substratum assays suggest that they may be significant. For example, Bade and Nitzgen [137] studied the effects of various matrix macromolecules in defining the migratory response of rat liver epithelial cells to EGF; they found that fibronectin inhibited the motogenic activity of EGF in a dose-dependent fashion, whilst both laminin and type IV collagen were without effect. McIntosh et al. [138] reported that capillary endothelial cells migrated significantly better on filters coated with fibronectin compared to either native collagen or gelatin; cell attachment and proliferation occurred to the same extent (with minor variation) on substrata coated with all three matrix molecules. Soluble fibronectin was not effective in promoting cell migration. Kondo er al. [ 1391 found that neither PDGF, aFGF, bFGF nor TGFB stimulated the migration of human skin fibroblasts on uncoated tissue culture substrata in the presence of serum-free medium; cells plated on substrata coated with type I collagen. fibronectin or heparin displayed a similarly low level of migratory activity in the presence of serum free medium. In contrast, cell migration was significantly stimulated by combinations of PDGF and type I collagen. No other combination of cytokine and matrix macromolecule was found to be effective. Taraboletti et al. [ 1401 reported that thrombospondin inhibited the chemotactic response of endothelial cells to bFGF. In a similar study, Tucker et al. [141] investigated the role of the extracellular matrix in defining the motogenic activity of aFGF and TGFa. They observed that the rat bladder carcinoma line NBT-II formed tight colonies when grown on plastic dishes or dishes coated with either fibronectin or laminin; in contrast, cells grown on a collagen substratum displayed an inherent degree of dispersion which was significantly increased by exposure to both cytokines. Our data suggest that the matrix may also play an important role in modulating the

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cell density (xlO%m-*I FIGURE 2. The effects of cell density on the migration of adult and fetal Ahroblasts.

migratory response of cells to cytokines in the 3D collagen matrix assay. In this regard, data presented in Fig. 2a indicate that the ‘base-line’ migratory behaviour of control cells in this assay is significantly influenced by cell density and must be taken into account before the superimposed effects of cytokines can be investigated. In this study, both adult and fetal fibroblasts were plated at various densities and the percentage of cells within the 3D collagen matrix determined after a 4 day incubation period. Fibroblasts plated at the lower densities (l-5 x lo3 cells cm-*) formed a subconfluent cell layer within an hour after plating and did not significantly alter their relative degree of confluency during the subsequent 4 day incubation period; in contrast, fibroblasts plated at densities of lo4 cells cm-2 and higher established a confluent monolayer immediately after initial attachment. As previously reported [53], these data indicate that the migratory activity of adult skin fibroblasts is dependent upon plating cell density, exhibiting a significant decline as the degree of confluency on the surface of the collagen matrix increased. Fetal fibroblasts displayed a distinct migratory phenotype, this characterised by an apparent cell densityindependence of migratory activity. Data presented in Fig. 2b summa&e results obtained with 100 fetal and adult fibroblast lines at a standard subconfluent and confluent plating density. These results indicate that the migratory activity of fetal and adult fibroblasts at subconfluent cell densities are indistinguishable (with mean values of 22.8 f 4.6 and 24.3 f 2.4, respectively); in contrast, fetal and adult fibroblasts displayed significantly different migratory activities at confluent cell densities, with corresponding mean values of 20.3 f 1.7 and 3.2 f 1.9. Several mechanisms may be responsible for the differential effect of cell density on the migration of fetal and adult fibroblasts [142]; these may involve a number of parameters, including density-dependent differences in (a) the reorganisation of the collagen fibre matrix by the plated cells, (b) the deposition of matrix macromolecules, (c) the secretion of soluble factors, (d) the production of other potentially relevant molecules, such as proteases, and (e) cell-cell social interactions.

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THE MOTOGENIC ACTIVITY OF CYTOKINES IS DEPENDENT UPON AND/OR MEDIATED BY INDUCED ALTERATIONS IN CELGMATRIX INTERACTIONS Several motogenic cytokines have been reported to modulate the composition of the extracellular matrix and/or cell adhesion to individual matrix constituents. These observations have prompted a number of workers to suggest that the observed motogenic activity of these cytokines may be a secondary consequence of these various primary effects on cell-matrix interactions. Unfortunately, demonstrating such a mechanistic relationship unambiguously is fraught with a number of experimental difficulties. Several criteria must be met, including the demonstration that (a) the effect of the cytokine on cell-matrix interactions and migration exhibit the same dose-response, (b) the effect of the cytokine on cell-matrix interaction precedes its effect on migration, (c) interference with the effect of the cytokine on cell-matrix interaction (as with an antibody directed against the matrix molecule) also blocks its motogenic activity, and (d) the motogenic activity of the cytokine can be mimicked by an independent induction of the same change in cell-matrix interactions (as by adding a particular matrix macromolecule whose synthesis may be stimulated by the cytokine). A considerable amount of work done in this area has focused on members of the TGF/? family. These cytokines have been reported to modulate the composition of the extracellular matrix by several means, including up-regulating the expression of various matrix macromolecules (e.g. fibronectin, collagen and proteoglycans), downregulating the expression of matrix degrading enzymes, and up-regulating the synthesis of inhibitors of these enzymes [144-1461. Related studies have indicated that members of the TGFP family also modulate the expression of various integrin subunits [147,148]. A number of reports have suggested the possibility of a mechanistic link between these effects of TGFP on cell-matrix interactions and its motogenic activity. For example, Nickoloff et al. [149] reported that TGF/I stimulated the migration of keratinocytes (as assessed in the transmembrane and agarose drop assays). This motogenic activity of TGFjl was paralleled by a stimulation of fibronectin synthesis and blocked by anti-fibronectin antibodies; exogenous fibronectin was found to elicit a similar stimulation of cell migration. Mooradian et al. [150] reported that TGFP increased the adhesion of A549 human lung carcinoma cells to type I collagen coated substrata; this effect of TGFj? on cell adhesion was paralleled by a stimulation of cell invasion into 3D collagen gels and haptotactic response to type I collagen in transmembrane assays. Similar suggestive evidence supporting a mechanistic link between TGFPinduced alterations in cell adhesion to matrix macromolecules and cell motility have been provided by Delannet and Duband [ 15 I]. These authors reported that TGFP reduced the time required for neural crest cell outgrowth from premigratory regions of explanted neural tube; this effect of TGFP on cell motility was temporally correlated with an increased adhesion to various matrix macromolecules. Basson et al. [I 521 have recently provided direct evidence that TGFP up-regulates the expression of both the p, and & integrin subunits in endothelial cells in a manner which parallels its observed motogenic activity. TGF/? has generally been regarded as an anabolic cytokine in that it induces an increase in matrix deposition. Various recent studies have. however, indicated that it

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may also lead to a degradation of matrix as a consequence of up-regulating the expression of matrix degrading enzymes in certain target cells. In this regard, Salo et al. [106] demonstrated that TGFj? up-regulated the expression of both the 72 and 92 kDa species of gelatinase (at both the protein and mRNA level) in human oral mucosal and dermal keratinocytes, but not in gingival fibroblasts. Wahl et al. [ 1531 have similarly reported that TGF-P up-regulated the expression of these two species of gelatinase, as well as the cl, and p, integrin subunits in monocytes. The motogenic activities of several other cytokines have also been linked with their respective effects on cell-matrix interactions. Nishida et al. [ 1541 studied the effects of IL-6 on cornea1 epithelial cell migration on exposed cornea1 stroma. They found that this cytokine stimulated cell migration in a dose-dependent fashion which was inhibited by exposure to either anti-fibronectin or anti-GRGDSP antibodies. The adhesion of cornea1 epithelial cells pre-incubated with IL-6 to fibronectin-coated substrata was increased, thereby suggesting that the motogenic activity of IL-6 was probably a result of a primary effect on the expression of fibronectin receptors. Basson et al. [155] similarly examined the cooperative interactions between cytokines and matrix in regulating the migration of a human colonic cell line (Caco-2). These cells attached and spread equally well on substrata coated with collagen (types I, III, IV and V) and laminin, although cell migration over laminin coated substrata was significantly lower compared to that on collagen. Interestingly, EGF stimulated cell migration on laminin, but not collagen; this motogenic effect of EGF was independent of its stimulation of cell proliferation, which was the same on all substrata. They further reported that the pool of cell surface crl integrin subunit was increased by EGF in cells cultured on laminin coated substrata, but decreased in cells cultured on collagen. No detectable differences were observed in the quantity of the ~12integrin subunit, although its spatial distribution was different in migrating and nonmigrating cells. We have reported that hyaluronic acid (HA) plays a central role in mediating the motogenic activities of several cytokines in the 3D collagen matrix assay. We initially reported that MSF exerted a biphasic effect on HA synthesis by confluent adult skin fibroblasts which paralleled its effect on cell migration [156]. Further evidence suggesting a mechanistic link between MSF-induced changes in HA synthesis and cell migration came from observations indicating that (a) co-exposure of cells to MSF and testicular hyaluronidase completely abolished the stimulation of cell migration, (b) exposure of cells to exogenous HA in the absence of MSF induced a similar bellshaped dose-response to that elicited by MSF, and (c) brief exposure of confluent adult fibroblasts growing on plastic dishes to MSF resulted in a persistent up regulation in HA synthesis; these pre-incubated cells displayed an elevated level of migration in the collagen matrix assay in the absence of further MSF which was inhibited by co-exposure to hyaluronidase [30, 1561. More recent data have extended these observations by indicating that the motogenit activities of several, but not all, cytokines are similarly dependent upon their primary effect on HA synthesis. This apparent mechanistic diversity with respect to HA dependence is well illustrated by EGF and TGFa. Both cytokines have been reported to stimulate the migration of fibroblasts [37,157], as well as a variety of epithelial [17,158,159] and tumour cells [ 181 in transmembrane and related assays. Data presented in Fig. 3 indicate that (a) both cytokines stimulate the migration of confluent adult fibroblasts, (b) the motogenic activity of EGF, but not that of TGFcr,

Cvtokine-Mutrix

Interactions

in Cell Motility

control

EGF

TGF-alpha

piGcxzq FIGURE 3. The e&&s of EGF and TGFaon adult tibroblaskx migration and HA syntbeais. Adult tibroblasts were plated onto collage0 gels at conflaent ceil density in the presence of 10 rig/ml EGF aml TGFa( f 1 IU/ml testicular hyal~~~~nidase). Migration assessed after a 4 day incubation period. loset: Parallel cultares were labelled with ~H-ghcosamir~ to assess the effects of these cytokines on HA synthesis; control HA synthesis indicated by dotted line.

is paralleled by a corresponding stimulation of HA synthesis (inset), and (c) the stimulation of cell migration by EGF is partially abrogated by co-exposure to hyaluronidase, whilst that of TGFa is not affected. Neither cytokine had an effect upon the migration of subconfluent adult fibroblasts (data not shown). EGF and TGFaexert a distinct effect upon the migration of fetal fibroblasts which also appears to be related to their respective effects on HA synthesis. We have previously reported that fetal and adult skin fibroblasts differ with respect to the effect of cell density on HA synthesis [160]; fetal (and transformed) fibroblasts do not down regulate HA synthesis upon reaching confluence, whilst adult fibroblasts do so. Data presented in Fig. 4 indicate that the constitutively elevated level of HA synthesis by confluent fetal fibroblasts (dotted line) is not further stimulated by exogenous EGF or TGFG~ (inset). These observations further indicate that exposure of control fetal fibroblasts to hyaluronidase resulted in a significant inhibition of cell migration; hyaluronidase partially inhibited the migration of fetal cells co-exposed to EGF and did not significantly affect the migration of cells co-exposed to TGFa. Taken together, these observations are consistent with the interpretation that (a) the stimulation of confluent adult fibroblast migration by EGF is mediated via both HA-dependent and HA-independent mechanisms, (b) the relatively elevated level of migration displayed by confluent fetal fibroblasts is dependent upon their constitutively elevated level of HA synthesis, and (c) the stimulation of adult and fetal fibroblast migration by TGFn involves an HA-independent mechanism. It is possible that the distinct effects of EGF and TGFa may involve different signal transduction pathways elicited by engagement of these cytokines to the same cell surface receptor. Subsequent studies indicated that differences in the matrix deposited by subconfluent and confluent adult fibroblasts contribute to the observed cell density effects on cell migration. In these studies, adult fibroblasts were plated onto collagen gels at a

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238

control

EGF

TG

In- HA’ase

-alpha

Y +HA’ase

1

FIGURE 4. The e&b of EGF aod TGFa on fetal libroblasts migration end HA synthis. Fetal Bbroblasts were plated onto collagen gels at coolhot cell dermity in the pmeence of 10 &ml EGF and TGFd( f 1 IIJ/ml testicolar byaiuronidase). Migration ssessed after a 4 day incubation period. Ioseti ParnJM cuitures were labelled with ~H-glacoa~ to assessthe effects of these cytokines on HA syntbeeiq control HA synthesis indicated by dotted line.

1

control

I

EGF

TGF-alpha

FIGURE 5. The effects of cehnodUled substrate on the migration of subconthot ad& fibroblasts reeporrse to EGF and TGFa Adolt Rbroblasts were plated at a sobconfhn t eelI de&y onto control collagen gels and co&sent cell CMS in the presence and ahsence of EGF and TGFcr.

confluent cell density and incubated for a period of 3 days; the cells were then removed from the gels in a manner which left the c&-modified substratum (CMS) intact [143]. Freshly harvested fibroblasts were plated onto control collagen gels and CMS at a s&co&rent cell density in the presence and absence of EGF and TGFcr. As the data in Fig. 5 indicate, (a) subconfluent cells plated on confluent cell CMS

Cytokine-A4atri.x Interactions in Cell Motility

.?3Y

(O-HABsa\

pm-incubate EXPOSURE

In assay TO TGF-beta

FIGURE 6. Tbe effects of pre-incubation of adult fibroblasts with TGF-/3 on their subsequent migration and syntbesii of HA when cultured ou 3D collagen cells. Adult fibroblasts growing on plastic tissue culture dii were incubated for 24 b with TGF-/J. These pre-incubated cells were then plated onto 3D collagen gels in tbe presence and absence of hyaluronidase; non-pre-incubated ceUs were also plated onto collagen gels in tbe presence and absence of TGF-/? and byaluronidase. Cell migration down into tbe collagen matrix was measured after a 4 day incubation period. Tbe dotted line indicates tbe level of migration achieved by (control) non-pre-incubated ceUs in the absence of TGF-B. Inset: Parallel cultures were used to compare the effects of TGF-B pre-incubation on HA synthesis by cells cultured on collagen gels during tbe same 4 day period, HA synthesis by non-pre-incubated ceils indicated by dotted line.

behaved like confluent cells in that they exhibited a relatively reduced level of migration compared to cells on control gels, (b) the constitutively high level of migration displayed by subconfluent cells on collagen gels was not affected by either EGF or TGFcl, and (c) the relatively low migratory activity of subconfluent cells on the confluent cell CMS was significantly stimulated by both cytokines. Studies currently underway in our laboratory are concerned with identifying the molecules present within (and/or the physical properties of) the confluent cell CMS which are responsible for these observed effects on the migration of subconfluent cells. HA also appears to be involved in mediating the rather complex (matrixdependent) effects of TGFP on cell migration. In this regard, we have recently reported that TGFj?does not affect either the migration of confluent adult fibroblasts or their synthesis of HA when cultured on collagen gels [30]. In contrast, we have recently found that TGFP induces an up-regulation of HA synthesis by these same cells when cultured on plastic dishes and that this stimulation of HA synthesis persists when the cells are plated onto collagen gels in the absence of further TGF-P (Fig. 6, inset). Interestingly, only a brief exposure of adult skin fibroblasts growing on plastic dishes to TGFP was sufficient to result in a persistent stimulation of both HA synthesis and cell migration when the cells were subsequently plated at confluent density on 3D collagen matrices in the absence of further TGFP(Fig. 6) (Ellis et al., in preparation); the relatively elevated level of migration displayed by these preincubated cells was completely neutralised by co-exposure to hyaluronidase. As previously reported, the migration of non-pre-incubated cells exposed to TGF-P

240

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FIGURE 7. Cell-matrix interactions in the control of cell motility. Ligation of the ceil surface receptors for cytokines and matrix macromolecules elicit a number of related signal transduction cascades which direttly affect cell migration. In addition, reciprocal interactions between cytokines and matrix macromolecules result in reiterative feedback loops which affect the expression of genes coding for these effector molecules and their respective cell surface receptors.

during the 4 day duration of the assay was not significantly different from control cells incubated in the absence of cytokine (Fig. 6, dotted line). These data indicate that (a) TGFP is a bifunctional modulator of HA synthesis in adult skin fibroblasts, with its precise effect being determined by the nature of the extracellular matrix, and (b) preincubation of adult fibroblasts growing on plastic dishes to TGFB results in a persistent up-regulation of HA synthesis which is not apparently diminished by subsequent culture on 3D collagen matrices and is responsible for the elevated level of migration these cells displayed on this substratum. Related unpublished observations indicate that the motogenic activities of several other cytokines, including PDGF, aFGF and bFGF, are also dependent upon a primary effect on HA synthesis. Heldin et al. [161] have previously reported that PDGF-BB, EGF, bFGF and TGF/%l stimulated HA synthesis by both subconfluent and confluent fibroblasts growing on plastic tissue culture dishes. Samuel et al, [162] have recently reported that both the addition of exogenous TGFP and the induction of TGFP secretion by appropriately transfected fibroblasts resulted in a stimulation of cell migration on 2D tissue culture substrata. This motogenic activity of TGFP was shown to result from a coordinate cytokine-induced up regulation in the synthesis of both HA and one of its cell surface receptors (RHAMM). Seen in the context of our observations regarding the substratumdependence of TGFP control of HA synthesis, it is interesting to note that the cells used in this study were cultured on plastic tissue culture dishes. Information is not yet available regarding the effect of TGFP on RHAMM expression by cells cultured on 3D collagen matrices. CONCLUSIONS

There is a substantial literature indicating that the motogenic activity of cytokines may be both modulated and mediated by cell interaction with the extracellular matrix. The complex interplay between cytokines and matrix macromolecules with respect to cell motility is depicted in Fig. 7. According to this scheme, interaction of

Cytokine-Matrix

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241

cytokines and matrix macromolecules with their respective cell surface receptors elicits a number of signal transduction cascades. Molecules generated by these cascades affect cell migration by a variety of both ‘direct’ and ‘indirect’ mechanisms. In mechanisms falling into the former category, the evoked signal transduction cascade directly affects the motility machinery itself, e.g. the organisation and functional activity of the cytoskeleton. In contrast, mechanisms falling into the ‘indirect’ category commonly involve a primary effect on the expression of genes coding for (a) cytokines and their respective receptors, and/or (b) matrix macromolecules, their cell surface receptors and matrix degrading enzymes; these newly synthesised molecules may in turn affect cell migration by various direct and indirect means. Seen in this perspective, the various effects of cytokines and matrix macromolecules on gene expression may result in complex and reiterative loops involving both negative and positive feedback. Taken together, these findings suggest that the relative stability of the matrix may provide a long-term “memory” for modulating and defining the specific response of a cell to a potentially multifunctional cytokine. The scheme presented in Fig. 7 highlights several further features of cytokine-~ matrix interactions which have been raised in this review. For example: 1. Although much is now known about signal transduction, the “black box” in Fig. 7 indicates that a significant number of gaps in our knowledge remain to be filled. For example, as distinct transduction pathways have been shown to mediate chemotactic and haptotactic response to the same matrix macromolecule [21], we must ask what factors determine which particular (of the several potential) signal transduction cascades are elicited by receptor ligation. Are the same receptors involved in transducing these signals and/or is the elicited signal affected by receptor association with extracellular, membrane-bound or intracellular “transduction modifiers”? Data should soon be available regarding the signal transduction cascades involved in mediating the apparently differential response of fibroblasts to ligation of the same receptor with either EGF or TGFa and the manner in which this is modified by the extracellular matrix. 7A. Signal transduction by both cytokines and matrix macromolecules appears to be mediated by similar signal transduction cascades. As more information is acquired, it may prove that a relatively small number of convergent and common signal transduction strategies are used to mediate the rather diverse effects of these effector molecules. 3. The observed motogenic activity of a particular cytokine may reflect the combined effects of multiple direct and indirect mechanisms, these perhaps involving distinct signal transduction cascades and different lag periods after receptor ligation. In this regard, Humes et al. [163] have reported that the migratory response of renal proximal tubule cells to TGF-/?I is dependent upon cytokine induced alterations to both cytoskeleton organisation (early response) and proteoglycan synthesis (late response). This is an important consideration in selecting a suitable in vitro migration assay; clearly assays which take only several hours to complete will not be suitable for investigating cytokine motogenic activity mediated by indirect mechanisms which may take several days to become apparent. 4. Finally, as so many cytokines and matrix macromolecules are pleiotropic regulators of cell behaviour, we must also ask what possible advantage might be conferred by a single molecule affecting several rather diverse aspects of cell behaviour.

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C’.vtokitle-Matrix 76.

77.

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