The solid state environment orchestrates embryonic development and tissue remodeling

Kidney International, Vol. 51(1997), pp. 1427—1433

The solid state environment orchestrates embryonic development and tissue remodeling CAROLINE H. DAMSKY, AMR MouRsI, YAN ZHOU, SUSAN J. FISHER, and RUTH K. GLOBUS Departments of Stomatology, Anatomy, Pharmaceutical Chemistry, and Obstetrics, Gynecology and Reproductive Endocrinology, University of California San Francisco, San Francisco, and NASA-Ames Research Center, Moffitt Field, California, USA

The solid state environment orchestrates embryonic development and tissue remodeling. Cell interactions with extracdllular matrix and with other cells play critical roles in morphogersesis during development and in tissue homeostasis and remodeling throughout life. Extracellular matrix is

information-rich, not only because it is comprised of multifunctional structural ligands for cell surface adhesion receptors, but also because it contains peptide signaling factors, and proteinases and their inhibitors. The functions of these groups of molecules are extensively interrelated. In this review, three primary cell culture models are described that focus on adhesion receptors and their roles in complex aspects of morphogenesis and remodeling: the regulation of proteinase expression by fibronectin and integrins in synovial fibroblasts; the regulation of osteoblast differentiation and survival by fibronectin, and the regulation of trophoblast differentia-

tion and invasion by integrins, cadherins and immunoglobulin family adhesion receptors.

During development, the embryo establishes a series of preliminary body plans, each of which is a remodeled and refined version of the previous plan. Although the genetic blueprint is crucial, the position of cells within the developing embryo, and the solid-state

factors and their ligands, and matrix remodeling proteinases and their inhibitors provide a solid state environment that orchestrates the morphogenetic program during development, as well as tissue

homeostasis. The activities of these groups of molecules are interrelated. Thus, the addition of growth factors such as vascular

endothelial growth factor stimulates expression of a distinctive repertoire of integrins [4] and increases proteinase production [5] by endothelial cells undergoing an angiogenic response. Similarly, an addition of TGF-13 enhances ECM and ECM receptor production and reduces net proteolytic activity in many cell types [61. The

equation can also work in the other direction. In our own work, using synovial fibroblasts, we have shown that subtle changes in ECM composition can trigger enhanced expression of MMP

[7—to]. The kinds of changes in ECM composition that are capable of triggering enhanced MMP are remarkably specific (Table 1). However, one generalization appears to be valid at least

for fibroblasts. Intact matrix components that are not subject to intricate regulation in a given tissue, such as FN, collagen I and vitronectin, promote basal levels of proteinase expression. Howenvironment of other cells and extracellular matrix (ECM), ever, certain fragments of these molecules [such as the central cell provide a continually changing context within which to interpret binding domain of FN (FN 1117b0, or 12OFN)J, that would, for and execute the genetic program. ECM is extremely complex, example, be generated in wounds or sites of inflammation [11, 12], containing a large number of modular structural components, induce elevated MMP expression [7, 9]. In addition, specific such as collagens, proteoglycans and glycoproteins that interact combinations of an intact ECM component and any one of the with one another and with cells. Although the ECM plays a critical matricellular matrix components [131 that are transiently exrole as a scaffold on which and within which to organize tissues, pressed at sites of morphogenesis or tissue remodeling and repair the ECM also contains an immense amount of information. Cells [such as tenascin (TN), decorin, or secreted protein acidic and must be able to decode this information, respond to it and modify rich in cysteine (SPARC)1 are also inductive [8, 10, 141. Three the information within the ECM continually, not only during their points are illustrated by these data. Firstly, cell surface receptors differentiation, but throughout their lifespan in the mature organ- for ECM are important sensors and regulators of ECM homeostaism. Thus, cells require a broad and highly regulated repertoire of Secondly, many specific combinations of ECM components receptors for ECM (and for other cells) to be able to take sis. can trigger enhanced MMP expression in fibroblasts, and thirdly, advantage of the information in this solid state environment. The the effects of these ECM components on MMP do not necessarily integrin and cell surface-associated proteoglycan (such as synde- correlate with the degree of cell spreading or the ability to organize cans, CD44) families of receptors provide such a repertoire for focal contacts (FC) [9], two other kinds of signals that cells receive structural ECM components. Structural ligands are not the only critical molecules in the ECM, however. Growth/differentiation from ECM. Thus, cells independently process multiple signals from even a simple, highly defined ECM environment. factors (peptide signaling factors [11) and proteinases and their A further feature of the regulation of MMP expression by ECM inhibitors [2, 3] are also sequestered in the ECM, or are tethered is that multiple signaling pathways are possible. One pathway is to the cell surface. The ECM and receptors for ECM and other cells, growth largely cell shape/FC-independent, highly substrate-specific and direct. This is illustrated by the example of the I 2OFN cell binding

domain, 12OFN + the C-terminal heparin binding domain of FN, or the combinations of TN with intact FN [7—9]. All these matrices

© 1997 by the International Society of Nephrology

promote extensive spreading and, with the exception of I2OFN, 1427

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Damsky et al: Adhesion receptors in development and remodeling

Table 1. Regulation of metalloproteinase expression by extracellular matrix composition in synovial fibroblasts

ECM composition Fibronectin (FN) Collagen I (Col I) Vitronectin (VN) 12OFN

I2OFN + CS-F' L2OFN + HBDC

anti-integrin aS anti-a5 + anti-cr4 FN + tenascin (TN)

ColI+TN TN+VN

VN + decorin Col I + decorin FN + decorin SPARCd and fibrillar collagens

MMP-1 expression Low Low Low High Low High High Low High Low Low High Low

Low High

Immobilization of ECM ligands described in [7—10] CS-i, amino acids 1-25 of the IIICS (or V) alternatively spliced domain of FN, binds cs4fll. h

HBD: the C-terminal heparin binding region of FN. Type III repeats 12—14 d SPARC (secreted protein, acidic and rich in cysteine) in the appropriate ECM contexts regulates MMP indirectly by inducing a soluble factor that triggers MMP [14]

MMP, the level of specificity in the matrix combinations that are

inductive for MMP is remarkable. Thus, TN/collagen I and TN/VN are not inductive, whereas TN/FN is inductive [8]. In another example, decorin/VN is inductive but decorin/collagen I and decorin/intact FN are not, even though decorin binds to all three components [10]. The ability of decorin to regulate MMP

expression in certain ECM contexts may be relevant to the observation that decorin limits matrix accumulation, and is there-

fore important for maintaining matrix homeostasis in normal tissues. This property has intrigued investigators interested in therapeutic targets to block excessive matrix accumulation in fibrotic diseases. Since decorin sequesters TGF-p, a known inducer of ECM, it is logical to propose that decorin suppresses ECM accumulation by limiting the availability of TGF-/3 [16]. There are now extensive data to support this idea [17, 18]. In addition, however, it is possible that in certain matrix environments where VN and decorin are found together, decorin could also limit ECM accumulation and help to promote resolution of an acute fibrotic response by enhancing MMP expression and therefore ECM turnover. Additional pathways for ECM regulation of MMP expression differ in important ways from the direct ligand specific pathway described above. For example, triggering cell rounding in fibroblasts by undermining integrin-mediated cell spreading will induce MMP regardless of which ligand was interacting with the

focal contact formation, in synovial fibroblasts. They all also cells. A second example involves an indirect pathway. In this case induce enhanced levels of MMP (in the case of the mixed FN/TN SPARC in combination with many different ECM components matrix, the region of the TN that initiates the proteinase inductive signal is distinct from that which disrupts focal contact formation by fibroblasts [8]). In this direct pathway, the signal is transduced by integrins (such as a5f31 in the case of FN fragment inducers) and the induction of mRNA for MMP-1 (interstitial collagenase) is rapid. Within 10 minutes of plating synovial fibroblasts on an inductive ECM (such as 12OFN or FN/TN), c-fos, a component of

the AP-1 transcription factor complex, is translocated to the

(FN or fibrillar collagens, but not laminin) first induces a soluble factor that then stimulates MMP expression. Since the domain in SPARC responsible for this induction pathway is distinct from that which undermines FC formation by cells on FN or collagens, this pathway, like the direct pathway, is cell-shape independent [14]. At this time, both the receptor(s) responsible for transducing the inductive signal in the SPARC pathway and the identity of the

intermediate soluble factor produced are unknown. Taken tofor the subsequent up-regulation of MMP-1, as inclusion of gether, our results in the synovial fibrohiast system illustrate antisense oligonucleotides for c-fos blocks elevation of MMP-1 clearly, at the cellular level at least, the principle stated at the levels. AP-1 and PEA-3 binding sites in the MMP-1 promoter are outset of the review that the principal groups of molecules required for expression in response to the 12OFN signal [15]. Since residing in the ECM act coordinately to modulate the cellular immobilized anti-integrin a5 monoclonal antibody can replace environment, and that alterations in the environment then act nucleus. Transcription of additional AP-i components is required

12OFN as an inductive substrate, it is clear that the a5/31 integrin

back on the cells. The relatively simple system just described, that

is, primary synovial fibroblasts on highly defined ECM, is well suited for continuing to work out how signals from specific ECM inductive signals from FN fragments is unclear, as are the environments are transduced to the transcription machinery of identities of the signal transducing receptors for the FNITN and the nucleus to regulate ECM homeostasis. The discussion above relates to the regulation of matrix degradVN/decorin signals. Since anti-integrin a5, I 2OFN and smaller FN fragments that ing proteinase expression by signals transduced by integrins (and contain the RGD sequence all induce elevated MMP when perhaps other classes of ECM receptor). An intriguing new twist immobilized on the substrate, whereas intact FN signals cells to to the relationship of adhesion and proteolysis relates to the produce a low basal level of MMP, other region(s) of FN must plasminogen activator system [19, 20]. Both urokinase plasminocontain information to control the inductive signal from the gen activator receptor (uPAR) and plasminogen activator inhibcentral cell binding domain. Interestingly, the 25 amino acid CS-i itor (PAl-i) can bind vitronectin, an ECM component. The ratio sequence from the alternatively spliced V region of FN is neces- of the inhibitor (PAl-i) and the proteinase (uPA) appears to sary and sufficient to suppress the MMP-inducing 12OFN signal. regulate uPAR adhesion to vitronectin [19]. Furthermore, uPAR This sequence binds the a4/31 integrin, which is highly expressed (which is glucosyl-phosphatidylinositol-linked to the cell surface) on synovial fibroblasts. Thus, two different /31 integrins coopera- can form stable complexes with integrins and inhibit their normal tively regulate MMP expression in vitro in response to FN in these ligand interactions. The integrin-uPAR complexes are suggested cells [9]. to transduce signals from vitronectin [201. This intimate structural In the direct pathway described above for signaling elevated and functional interaction between uPAR and integrins blurs the is able to transduce the 12OFN signal [7, 9, 15]. Whether other integrins or cell surface proteoglycans participate in transducing

a

Damsky et al: Adhesion receptors in development and remodeling

distinctions between the roles of proteinases and adhesion molecules and provides a novel mechanism for focalized proteolysis of ECM such as occurs in processes such as angiogenesis or tumor cell invasion ([3] has a thorough discussion of focalized matrix remodeling). Tissues and organ systems operate in very complex environ-

A

must be maintained with great care throughout the lifespan of the

B

ments that are constructed over time during development and

gain insight into this higher order of complexity, it is important to establish more comprehensive model systems both in culture and

in transgenic/knockout animals in which the cells of interest recapitulate complex activities over an extended time period. We are taking this approach to study the roles of adhesion receptors in the stepwise differentiation programs of osteoblasts and trophoblast cells, the workhorses of two fascinating tissues, bone and placenta, which exhibit intensive remodeling activity, not only during their initial differentiation, but throughout their lifespan. The studies to be described below illustrate two general principles that are important to keep in mind when considering the initial differentiation program, normal physiology and pathology of any tissue. The first, which we illustrate with osteoblasts, is that cells

can respond to the same matrix signal differently at different stages of differentiation and maturation. The second, which we illustrate with the placenta, relates to the extraordinary invest-

Osteoblasts

,0 Matrix

organism despite assaults that threaten tissue homeostasis. To

D3

+

Matrix Matrix

C

1429

D8

+

Matrix

Dl 0

Matrix Osteocyte

D

Osteoblasts

Mineralized Mineralized ma matrix

ment some cell types make in intricately regulating their adhesion

molecule phenotype during their differentiation and while they

are executing their physiologic function. In the case of the placenta's principal cell type, the cytotrophoblast, this turns out to be absolutely essential for normal placentation, and is defective in the important hypertensive pregnancy disorder, preeclampsia.

Fibronectin is a differentiation-promoting factor for preosteoblasts, and a survival factor for mature osteoblasts In the flat bones of the skull (calvaria), bone forms directly from

condensed mesenchyme (intramembranous bone formation) without requiring the prior laying down of a cartilage model that matrix-secreting osteoblasts and matrix-resorbing osteoclasts then remodel and replace (endochondral bone formation). We have

Dl 4

Matrix Fig. 1. Fibronectin is required for morphogenesis of immature osteoblasts and for suivival of mature osteoblasts. Rat fetal calvarial osteoblasts form

mineralized nodules over a 2 to 3 week period in vitro. A. Immature proliferating osteoblasts form a confluent monolayer by D3. B and C. In

the presence of ascorbate, the culture undergoes multilayering and nodules form and mature from days 7 to 10. Nodule formation, but not multilayering, is inhibited if anti-FN or central cell binding fragments of FN are present continuously starting from day 3 [23]. D. In the presence of f3-glycerolphosphate, nodules mineralize by day 14. If anti-FN is added

at day 13 or later, about 95% of the cells in the culture undergo programmed cell death [30].

used a well-established culture model [2 1—231 to study the roles of

specific ECM constituents and their integrin receptors in different stages of osteoblast differentiation. In this model (Fig. 1), preosteoblasts are isolated from E21 rat calvaria and over a two to three week period in culture these cells proliferate, multilayer, and, in the presence of ascorbate and f3-glycerolphosphate, form nodules that consist of mature plump osteoblasts that secrete and organize an extensive fibrillar type I collagen-rich matrix, which becomes mineralized. The mature nodules also contain osteocyte-like cells

genic differentiation of MG-63 osteosarcoma cells [27], while amplification of the integrin a51 FN receptor promotes differentiation of these cells [28]. We therefore treated osteoblast cultures at different stages with antibody against FN, or with soluble fragments representing

production is closely regulated and that at different stages differentiating osteoblasts have a distinctive matrix phenotype [22—24]. It is also clear that although type I collagen is known to comprise more than 90% of bone ECM, several other matrix molecules are present, some of which are highly characteristic of bone (such as bone sialoprotein and osteocalcin). FN is also present [25, 26] and we were curious about its role in osteogenesis, given previous data showing that perturbing cell-FN interactions suppresses osteo-

differentiation, respectively. Nonimmune IgG-treated control cul-

several regions of the FN molecule: both kinds of treatments were attempts to disrupt normal osteoblast-FN interactions [23]. Addi-

tion of anti-FN to confluent cultures of immature osteoblasts embedded within the mineralized matrix core of the nodule. (culture day 3) inhibited nodule formation by more than 75% and When such cultures are analyzed for the presence of ECM and suppressed expression of mRNA for alkaline phosphatase and other proteins that are characteristic of bone, it is clear that ECM osteocalcin, which are early and late markers for osteoblast tures exhibited normal nodule formation starting at day 7 to 8. The anti-FN treatment was non-toxic and reversible: when the antibody was removed, differentiation and nodule formation resumed. Similar results occurred as long as the anti-FN was added prior to nodule initiation (day 6). Addition to the cultures of soluble fragments of FN containing the central cell binding domain, but lacking other regions of FN, also blocked nodule

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Damsky et al: Adhesion receptors in development and remodeling

At the start of this remarkable journey, the CTB stem cells of formation [23], as did a function-perturbing antibody against the the anchoring chorionic villus form a polarized monolayer on the cs5 subunit of the specific integrin a5j31 FN receptor [29]. Surprisingly, when anti-FN was added to mature osteoblast trophoblast basement membrane, continuous with the mononu-

cultures (day 13), in which nodules had formed and become clear CTB layer of floating villi (Fig. 2). This monolayer covers the mineralized, over 90% of the cells in the culture died within 24 stromal core of the villi, which contain fetal blood vessels and hours [301. Although there was some cell shape change following connective tissue cells. These CTB express several cell surface addition of the anti-FN, the cells remained attached, and no adhesion receptors characteristic of their epithelial origin, includfloating cells were observed during the 24 hour period. Cell nuclei ing the cs64 integrin laminin receptor and E-cadherin, a calciumwere condensed and apoptotic bodies were evident throughout dependent cell-cell adhesion molecule that is ubiquitously exthe culture in both nodular and internodular cells. The nuclear pressed by epithelia [35, 36]. These receptors promote firm changes were evident prior to extracellular lactate dehydrogenase, attachment of CTB to their ECM and to one another, respecan indicator of plasma membrane integrity, suggesting that the tively. As CTB differentiate along the invasive pathway, they cells in these mature osteoblast cultures were undergoing pro- intricately modulate their adhesion phenotype such that there are grammed cell death in response to anti-FN. Addition of antibod- at least five distinguishable stages of differentiation in which the ies against TN or type I collagen to mature osteoblast cultures had no effect on survival, indicating that the response was related to perturbing osteoblast—FN interactions.

CTB adhesion phenotypes are distinctive. These stages are clearly

epithelial-endothelial transformation as they differentiate, a process that is defective in the systemic hypertensive pregnancy disorder,

CTB, which recapitulate their differentiation in vitro [38], indicate

preeclampsia. During establishment of the functional placenta in humans (placentation), a subset of fetal cytotrophoblast (CTB) stem cells leaves the fetal compartment and invades the uterine stroma (interstitial invasion) and vasculature (endovascular invasion) thereby anchoring the fetus to the mother and establishing the flow of oxygenated blood that bathes the fetal portion of the placenta (Fig. 2). Once CTB have invaded the superficial vascular network in the decidua, they work their way upstream in the spiral arteries, but not veins, and replace the maternal endothelium on the vessel wall as far as the first third of the myometrium. Once on the vessel wall, they extensively degrade and remodel the muscular tunica media of these spiral arterial segments, converting them from narrow-bore, high-resistance to wide-bore low-resistance vascular channels capable of efficient transport of arterial blood to the intervillous space, which surrounds the floating chorionic villi

cadherin restrains CTB invasion [36, 39]. Thus, the switching of the integrin and cadherin family adhesion receptors by differentiating CTB is related to their acquisition of an invasive phenotype. We hypothesize that the Ig family vascular adhesion molecules that are up-regulated by CTB contribute to the successful takeover of the spiral arterial walls by CTB once they have entered these vessels. Thus, if intravascular CTB must contact maternal

laid out spatially at the fetal-maternal interface (Fig. 2) and are therefore amenable to analysis by immunocytochemistry of plaAlthough the mechanisms are still unclear, the extensive cell cental bed biopsies obtained at the time of elective termination of death in these cultures may be an exaggerated manifestation of a pregnancies (gestational ages 8 to 18 weeks) or at term following more targeted normal process of regulating the number of Cesarean deliveries (34 to 40 weeks). After an initial survey of integrin subunit distribution, we osteoblasts in bone. It is estimated that of the number of matrix-secreting osteoblasts present at a growing bone surface, determined that those CTB within the uterine interstitium and less than 30% can be accounted for by the number of osteocytes spiral arterioles lost staining for a6/34 and instead stained strongly and mature bone lining cells present at the same location in a for alf3l laminin/collagen receptor [37]. Integrin al/31 is widely subsequent resting phase. These data suggest that programmed expressed on endothelial cells during development, and this gave cell death may be an important natural mechanism for local us the first hint that CTB may undergo a comprehensive transcontrol of osteoblast number [31]. Regardless of the mechanisms, formation of their adhesion phenotype so as to resemble the however, the conclusion is inescapable that in this culture model endothelial cells they replace. Subsequent staining of 10 to 18 at least, immature osteoblasts go from being FN-dependent for week placental bed biopsies for cell adhesion receptors from all differentiation to being FN-dependent for survival. The next step the major families (integrins, cadherins, immunoglobulins, selecis to determine whether this is also the case in vivo. If so, tins) confirmed this hypothesis. Thus, as they leave the trophodissecting the mechanisms underlying this biphasic response to blast basement membrane, CTB show reduced staining for EFN on the part of osteoblasts will be extremely interesting. This cadherin and start staining for VE-cadherin (cadherin 5), which to study illustrates clearly that changing either the context of the date has only been found on vascular endothelial cells. Interstitial, extracellular environment or that of intracellular signaling net- and especially endovascular CTB, stain strongly for this cadherin, works can lead to distinctive responses to ECM signals. Thus, it is and also express other adhesion receptors characteristic of endocritical when designing therapeutic agents directed at ECM thelial cells, including the crV/33 integrin which is known to be components, that one is familiar with the role of that component up-regulated on endothelium during angiogenesis [4], vascular endothelial adhesion molecule (VCAM)-1 and the a4 subunit of at a variety of developmental and physiologic stages. its integrin receptors (a4pl and a47), and platelet endothelial Placentation and preeclampsia adhesion molecule (PECAM)-1 [36]. Invasion assays using antiDuring placentation in humans, cytotrophoblasts undergo an integrin subunit-specific antibodies and isolated primary villus

of the fetal placenta [32—34].

that both al/31 and aVf33 promote CTB invasion, while E-

endothelium directly as part of the replacement process, they

could use a4 integrins and aV/33 to contact VCAM-1 and PECAM-1, respectively, on the endothelium. Once CTB reach the vessel wall, homophilic PECAM-1 and VE-cadherin interactions

could promote the organization of the CTB into a functional endothelial-like monolayer. Finally, since once they have colonized the vessel wall, CTB continue to invade the muscular tunica media of the spiral arterioles, they might use their alf3l and aVI33

Damsky et al: Adhesion receptors in development and remodeling

Mother (uterus)

Fetus (placenta)

Cytotrophoblast

Uterine blood vessels

stem cells AV

Basement membrane

Zone I Adhesion receptors on CTB

1431

Zone II

Zone Ill

Zone IV

ZoneV

cV5

-

aV6 ctVI33

a64

cx64

(a64)a

—

—

—

a5131

cz5J31

—

—

—

alfll

E-cad

E-cad

(Ecad)a

(Ecad)a

—

V-cad

V-cad

V-cad

V-cad

-

PECAM-1

PECAM-1

PECAM-1

VCAM-1

VCAM-1

VCAM-1

VCAM-1

-

integrins along with their high output of proteinases, especially MMP-9 [35, 38], to migrate through and remodel these vessels. Testing this hypothesis will require a more sophisticated in vitro

—

—

alIll

Fig. 2. Diagram of a 12 to 16 weeks gestation anchoring villus (A V), which spans the fetal and maternal compartments of the human placenta. Cytotrophoblast (CTB) stem cells (Zone I) differentiating along the invasive pathway leave the trophoblast basement membrane, form an aggregate (cell column: Zones II & III) and invade the uterine wall (Zone IV) and maternal vasculature (Zone V). Zones I-V demarcate regions in which CTB express distinct adhesion molecule repertoires. As they differentiate, CTB adopt an adhesion phenotype similar to that of endothelial cells. In the pregnancy disorder preeclampsia, E-cadherin and a6134 staining remain strong and endothelial adhesion receptors are not detected. aparentheses indicate reduced staining. Data are pooled from references [35, 36, 41].

endothelial adhesion phenotype switch executed by CTB in normal placental tissues. Preeclamptic CTB that are present in

model in which endothelium and CTB are co-cultured in a

the superficial decidua interstitium and vasculature, the limit of their penetration, retain strong staining for epithelial markers

geometry and in the presence of physical forces (for example,

(iritegrin a6J34 and E-cadherin), and stain very weakly or not at all

flow) that more closely mimic in vivo conditions. The importance of this extensive adhesion molecule switching by CTB for normal placentation is brought home forcefully when considering the hypertensive pregnancy disorder, preeclampsia. In the form of the disease considered here, pregnant women with no previous history of hypertension present with hypertension, proteinurea and fetal intrauterine growth retardation in the second or third trimester, depending on the severity of the disease. Analysis

for integrins a11 and aV3, both of which have been shown in functional studies to be required for invasion by normal CTB in

of placental bed biopsies taken at Cesarean delivery document

vitro [36, 39]. They also do not stain for VE-cadherin, PECAM-1 and VCAM-1 [33, 421. These findings lead us to hypothesize that

CTB in preeclampsia fail to adopt an adhesion phenotype required for optimal interstitial and endovascular invasion to the appropriate depth in the uterine wall. The resulting failure to modify the maternal vasculature adequately likely contributes significantly to the relatively poor perfusion of the fetal placenta by maternal blood and to the relatively hypoxic environment of

that both interstitial and endovascular invasion by CTB are restricted to the superficial decidua [33, 40, 41]. As a result, the fetal-maternal interface in preeclampsia (discussed in [43]). maternal spiral arteriole segments in the deep decidua and Preeclampsia is a complex set of diseases with a multifactorial superficial myometrium are not modified, and remain small-bore and highly muscular. Thorough immunocytochemical analysis of

etiology. Although it is a systemic hypertensive disease by the time

the patients experience symptoms, it is likely that the disease the adhesion receptor repertoire of preeclamptic CTB in situ originates early in pregnancy in the developing placenta. In the indicates that they start, but fail to complete, the epithelial-to- subset of patients defined above, defective adhesion molecule

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Damsky et al: Adhesion receptors in development and remodeling

switching by CTB is but one factor (although we think an

factor that stimulates basic fibroblast growth factor and urokinase

important one) contributing to the disease process. Preeclamptic CTB show other abnormalities as well, including low expression

6. ADZICK NS, LORENZ HP: Cells, matrix, growth factors, and the surgeon. The biology of scarless fetal wound repair. Ann Surg 220:

levels of protcinases (such as MMP-9) likely to be critical for invasion [38], and reduced expression of the trophoblast-specific

HLA class-i molecule HLA-G, that is hypothesized to play an important role in the ability of the fetus to avoid rejection by the maternal immune system [44]. Since none of the genes for these molecules is abnormal (the babies are normal if their prematurity does not compromise their ability to thrive), preeclampsia is a disorder in which abnormal regulation of hormones, peptide signaling factors and/or transcription factors is likely to be associated with the primary cause. Although the primary cause(s) are still unclear, we believe that altered expression of adhesion receptors, proteinases and HLA-G in preeclamptic CTB, especially as they affect the maternal vasculature of the uterine wall, are proximal sequellae of the primary cause, and contribute not only to the local abnormalities in placentation, but also to the processes that result in systemic disease. In summary, the systems discussed in this review as well as other

informative models [45] underscore the critical and varied roles

played by adhesion receptors in normal morphogenesis and differentiation and in ECM homeostasis. Important as they are, however, they function in a solid state environment with which peptide signaling factors and proteinases and their inhibitors are also associated. The functions of these classes of molecules are interdependent, and together orchestrate morphogenesis and tissue and ECM homeostasis. Acknowledgments This manuscript summarizes a presentation made to the International Society of Nephrology Frontiers in Science Conference (September 12—15, 1996). Work in the authors' laboratories was supported by P50-DE10306,

HD22210, and the Arthrifis Foundation to C.H.D., HD22210 and HD30367 to S.J.F., and NAGWA-4625 and NCC 2-655 from N.A.S.A. to R.K. G.A.M. was supported by an NRSA fellowship T32 DE07204 and Y.Z. was supported by a Rockefeller Foundation postdoctoral fellowship. The laboratories of C.H.D., S.J.F. and R.K.G. contributed equally to the work discussed.

Reprint requests to Dr. Caroline H. Damsky, HSW 604, UCSF, 513 Parnassus Avenue, San Francisco, California 94143-0512, USA. E-mail: damsky(itcgl.ucsfedu

Appendix Abbreviations are: CTB, cytotrophoblast; ECM, extracellular matrix; FC, focal contacts; FN, fibronectin; MMP, matrix metalloproteinase; PEAM-1, platelet endothelial adhesion molecule-i; SPARC, secreted protein, acidic and rich in cysteine; TN, tenasein; VCAM-1, vascular endothelial adhesion molecule-i; VN, vitroneetin.

References

1. Si'oN MB: The importance of context in cytokine action.(this issue) Kidney lot 51:000—000, 1997 2. ASUKENAS J, DAMSKY CH, BISSELL MJ, WERB Z: Integrins, signaling

and the remodeling of extracellular matrix, in The Integrins, edited by CHERESH D, MEd-lAM R, New York, Academic Press, 1994 3. BASBAUM C, WERB Z: Focalized proteolysis: spatial temporal regulation of extracellular matrix degradation at cell surface. Curr Opin Cell Blot 8:731—739, 1996

4. Btooi.s PC, CLARK RA, CLIERESH DA: Requirement of vascular Integrin alpha v and beta 3 angiogenesis. Science 264:569—571, 1994 5. PEVERALI FA, MANDRIOTA SJ, CIANA P, MARELLI R, QUAX P, RIFKIN

DB, DELLA VALLE G, MIGNAT-ri P: Tumor cells secrete an angiogenic

expression in vascular endothelial cells. J Cell Physiol 161:1—14, 1994 10—8, 1994 7. WERB Z, TREMBLE PM, BEHRENDTSEN 0, CROWLEY E, DAMSKY CII:

Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J Cell Blot 109:877—889, 1989 8. TREMBLE P, CHIQUET-EHRISMANN R, WERB Z: The extracellular

matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts. Mol Blot Cell 5:439—453, 1994 9. HUHTALA P, HUMPHRIES MJ, MCCARTHY JB, TREMBLE PM, WERB Z,

DAMSKY CH: Cooperative signaling by alpha 5 beta 1 and alpha 4 beta

I integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibroneetin. J Cell Biol 129:867—879, 1995 10. HUTTENLOCHER A, WERB Z, TREMBLE P, HUIITAI..& P, ROSENBERG L,

DAMSKY CH: Decorin regulates collagenasc gene expression in fibroblasts adhering to vitronectin. Matrix Biol 15:239—250, 1996 11. Xw DL, MEYERS R, HOMANDBERG GA: Fibroneetin fragments in ostcoarthritie synovial fluid. J Rheumatol 19:1448—1452, 1992 12. WYsOcKI AB, STAIAN0-Colco L, GRINNELL F: Wound fluid from

chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9. J Invest Dermatol 101:64—68, 1993 13. BORNSTEIN P: Diversity of function is inherent in matricellular proteins: An appraisal of thrombospondin if Cell Biol 130:503—506, 1995 14. TREMBLE PM, LANE TF, SAGE EH, WERB Z: SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway. J Cell Blot 121:1433—1444, 1993 15. TREMBLE P, DAMSKY CH, WERB Z: Components of the nuclear

signaling cascade that regulate collagenase gene expression in response to integrin-derived signals. J Cell Biol 129:1707—1720, 1995

16. BORDER WA, N0BI.E NA, YAMAMOTO T, HARPER JR, YAMAGUCHI Y, PIERSCHHACHER MD, RUOSLAHTI E: Natural inhibitor of transforming

growth factor-13 protects against scarring in experimental kidney disease. Nature 360:361—364, 1992

17. BORDER WA, NOBLE NA, KETFELER M: TGF-beta: A cytokine mediator of glomeruloselerosis and a target for therapeutic intervention. Kidney mt 47(Suppl 49):S59—S6l, 1995 IS. ISAKA Y, BREES DK, IKEGAYA K, KANEDA Y, IMAI E, NOBLE NA,

BORDER WA: Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney. Nat Med 2:418—423, 1996 19. DENG G, CURRIDEN CS, WANG S, ROSENBERG S, LOSKUTOFF Di: Is

plasminogen activator inhibitor-I the molecular switch that governs urokinase receptor-mediated cell adhesion and release? J Cell Blot 134:1563—1572, 1996 20. WE! Y, LUKASHEV M, SIMON DI, BODARY SC, ROSENBERG 5, DOYLE

MV, ChAPMAN HA: Regulation of integrin function by the urokinase receptor. Science 273:1551—1555, 1996 21. BELLOWS CG, AUBIN JE, HEERSCHE JN, ANTOSZ ME: Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue mt 38:143—154, 1986 22. STEIN GS, LIAN JB, OWEN TA: Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J 4:3111—3123, 1990 23. MOURSI AM: Integrin receptors for flbronectin regulate osteogenesis. JBone Miner Res 11:137, 1996 24. GRZESIK WJ, ROBEY PG: Bone matrix RGD glycoproteins: Immuno-

localization and interaction with human primary osteoblastie bone cells in vitro. J Bone Miner Res 9:487—496, 1994 25. GRONOWICZ GA, DEROME ME, MCCARTHY MB: Glucocorticoids inhibit fibronectin synthesis and messenger ribonucleic acid levels in cultured fetal rat parietal bones. Endocrinologt 128:1107—1114, 1991 26. WINNARD RG, GERSTENFELD LC, TOMA CD, FRANCESCHI RT: Fibroneetin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts. J Bone Miner Res 10:1969—1977, 1995 27. DEDHAR S: Signal transduction via the beta 1 integrins is a required

intermediate in interleukin-1f3 induction of alkaline phosphatase activity in human osteosarcoma cells. Exp Cell Res 183:207—214, 1989 28. DEDHAR 5, ARGRAVES WS, SUZUKI S, RUOSLAHTI E, PIERSCHBACHER

MD: Human osteosarcoma cells resistant to detachment by an

Damsky et al: Adhesion receptors in development and remodeling

Arg-Gly-Asp-containing peptide overproduce the fibronectin receptor. J Cell Biol 105:1175—1182, 1987

29. MouRsi A, DAMSKY CH, LULL J, ZIMMERMAN D, DOTY SB, AOTA S-I,

GLOBUS R: Fibronectin regulates calvarial osteoblast differentiation. J Cell Sci 109:1369—1380, 1996 30. GLOBUS R, HOLMUHAMEDOV E, LULL J, LOPEZ V, DOTY SB, MOURSI

A, DAMSKY CH: Fibronectin is a survival factor for differentiated osteoblasts. (abstract) Mol Biol Cell 7:584a 31. PARFITr AM: Bone-forming cells in clinical conditions, in Bone, edited by HALL BK, New Jersey, Telford Press, 1990, p 351 32. ENDERS AC, KING BF: Early stages of trophoblastic invasion of the

maternal vascular system during implantation in the macaque and baboon. Am J Anat 192:329—346, 1991 33. ZHOU Y, DAMSKY CH, CHIU K, ROBERTS JM, FISHER SJ: Preeclamp-

sia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts. J Clin Invest 91:950—960, 1993 34. CROSS JC, WERB Z, FISHER SJ: Implantation and the placenta: Key pieces of the development puzzle. Science 266:1508—1518, 1994 35. FISHER SJ, CUI TY, ZHANG L, HARTMAN L, GRAHL K, ZHANG GY, TARPEY J, DAMSKY CH: Adhesive and degradative properties of human placental cytotrophoblast cells in vitro. J Cell Biol 109:891—902, 1989

36. ZHOU Y, FISHER SJ, JANATPOUR M, GENBACEV 0, DEJANA E, WHEELOCK MJ, DAMSKY CH: Human cytotrophoblasts adopt a vascular phenotype as they differentiate: A strategy for successful endovascular invasion? J Clin Invest (in press) 37. DAMSKY CH, FITZGERALD ML, FISHER SJ: Distribution patterns of

extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo. J Clin Invest 89:210—222, 1992

1433

38. LIBRACH CL, WERB Z, FITZGERALD ML, CHIU K, CORWIN NM, ESTEVES RA, GROBELNY D, GALARDY R, DAMSKY CH, FISHER SJ:

92-kD type IV collagenase mediates invasion of human cytotrophoblasts. J Cell Biol 113:437—449, 1991 39. DAMSKY CH, LIBRACH C, LIM KR, FITZGERALD ML, MCMASTER MT,

JANATPOUR M, ZHOU Y, LOGAN SK, FISHER SJ: Integrin switching

regulates normal trophoblast invasion. Development 120:3657—3666, 1994 40. BROSENS IA, ROBERTSON WB, DIxoN HG: The role of the spiral

arteries in the pathogenesis of preeclampsia. Obstet Gynecol Annu 1:177—191, 1972

41. REDMAN CW: Current topic: Pre-eclampsia and the placenta. Placenta 12:301—308, 1991

42. Zs-iou Y, DAMSKY CH, FISHER SJ: Preeclampsia is associated with

failure of human cytotrophoblasts to mimic a vascular adhesion phenotype: One cause of defective endovascular invasion in this syndrome? J Clin Invest (in press) 43. GENBACEv 0, Josj R, DAMSKY CH, POLLIOTFI BM, FISHER SJ: Hypoxia alters early gestation human cytotrophoblast differentiation!

invasion in vitro and models the placental defects that occur in

preeclampsia. J Clin Invest 97:540—550, 1996 44. MCMASTER MT, LIBRACH CL, ZHOU Y, LIM KH, JANATPOUR MJ, DEMARS R, KOVATS S, DAMSKY C, FISHER SJ: Human placental

HLA-G expression is restricted to differentiated cytotrophoblasts. J Immunol 154:3771—3778, 1995 45. ROSKELLEY CD, SREBROW A, BISSELL MJ: A hierarchy of ECM-

mediated signalling regulates tissue-specific gene expression. Curr Opin Cell Biol 7:736—747, 1995