Information processing in extracellular matrix/ Transferts d’informations dans le milieu extra cellulaire

Symposium organized by Daniel Rifkin from 5 to 10 October 2002

Participants

Ethan Bier (University of California, San Diego, La Jolla, USA), Ali Brivanlou (The Rockefeller University, New York, USA), Guido David (University of Leuven, Belgique), Kristina Downing (University of Oxford, Royaume-Uni), Susanne Eaton (Max Planck Institut, Dresden, Allemagne), Reinhard Faessler (Max Planck Institut, Martinsried, Allemagne), John T. Gallagher (Paterson Institute, Manchester, Royaume-Uni), Daniel S.Greenspan (University of Wisconsin, Madison, USA), Juan M. Hurle (Universidad de Cantabria, Santander, Espagne), Katri Koli (University of Helsinki, Finlande), Ulf Lindahl (Uppsala University, Suède), Joanne Murphy-Ullrich (University of Alabama at Birmingham, USA), Enid Neptune (John Hopkins School of Medecine, Baltimore, USA), Francesco Ramirez (Hospital for Special Surgery, New York, USA), Daniel Rifkin (New York University School of Medecine, USA), Lynn Y. Sakaï (Oregon Health and Science University, Shriners Hospital, Portland, USA), Jussi Taipale (Johns Hopkins University,  Baltimore, USA), Rupert Timpl (Max Planck Institut, Martinsried, Allemagne), Zena Werb (University of California, San Francisco, USA), Malcolm Whitman (Harvard Medical School, Boston, USA).

Report

From October 5th to October 10th, a meeting was convened on the topic of “Information Transfer in the Extracellular Environment.” Recently, several papers and reports have appeared highlighting the role of extracellular macromolecules in orchestrating the distribution and presentation of growth factors to their receptors. This concept of signal­ing molecules interacting with matrix had not been considered by any previous meeting, so such a conference appeared timely. Twenty individ­uals from various disciplines that impact upon growth factor presentation attended this conference. The expertise of those present covered a broad range of activities : including NMR structural studies, chemical sequenc­ing of carbohydrates, cell and developmental biology, and normal and pathophysiology, all of which potentially impact on understanding con­trol in the extracellular milieu. Below is a short summary of the presen­tations.

The meeting began with two presentations that indicated how earlier concepts of the passive role of insoluble elements of the matrix had to be reconsidered. E Ramirez, Mt. Sinai School of Medicine, New York, NY, gave a talk entitled “Extracellular Microfibrils ; Structural and Instructive Role in the Musculoskeletal System.” For some time, it has been appre­ciated that extracellular microfibrils, alone or associated with elastin in the elastic fibers, play a critical role during development and in adult­hood. Fibrillins 1 and 2 (Fbn1 and 2) are the major structural components of microfibrils and mutations in these proteins cause Marfan syn­drome and congenital contractura) arachnodactyly (CCA), respесtively. The Ramirez lab has generated a pull mutation in the Fbn2 gene. Inactivation of the Fbn2 gene in the mouse confirmed the structural role of the protein in establishing the mechanical properties of tendons and ligaments as the mutant mouse has a phenotype that mimics CCA. The mutation also uncovered an instructive role of Fbn2 in skeletal pattening and bone growth as Fbn2-/- mice display bilateral syndaсtyly in fore and hindlimbs. The patterning defect is the result of altered microfibril assem­bly in the developing autopod and impaired determination of naive mes­enchyme. Generation of doublу heterozygous Fbn2 +/-/Bmp7 +/- mice revealed an epistatic interaction between these two gene products. Thus, in a manner lot previously suspected, Fbn2 interacts with ВМР7. Preliminary in vitro data suggest that the interaction may be indirectly mediated by chordin binding to Fbn2. E. Neptune, Johns Hopkins Medical School, Baltimore, MD, next spoke on the “Role of TGF-ß in Lung Phenotypе of Marfan Mice.” Dr. Neptune emphasized how the Fbns play important structural roles in tissue homeostasis as demon­strated by the pleiotropic manifestations of Marfan Syndrome caused by Fbnl deficiency. Neptune and her colleagues showed that Fbnl deficient mice also display a primary failure of distal alveolar septation in addition to the well-known cardiovascular defects observed in Marfan Syndrome. The structural similarity between Fbnl and a component of the trans­forming growth factor beta (S) latent complex, e.g. the latent TGF-ß binding protein (LTBP), suggested that the pulmonary defects maу be manifestations of perturbations in TGF-ß signaling. Because LTBP, which binds TGF-ß, associates with Fbnl-containing microfibrils, Neptune suggested that the loss of Fbnl and subsequent microfibril dis­sociation might inappropriately release TGF-ß. Using both immunohis­tochеmistry and a novel transgenic reporter allele, Neptune showed that Fbnl deficient mice have a marked elevation in latent TGF-ß  activation and signaling in the developing lung. Administration of a TGF-ß neu­tralizing antibody to mutant pups rescued septation in vivo.

The first two presentations established the paradigm that ECM struc­tural components may influence growth factor/сytоkinе sequestration and thereby perturbation of the ECl can affect signaling and contribute to the pathogenesis of disease. The following presentations explored this idea first by considering the makeup of the matrix from a chemical per­spective of matrix components and their interactions and second by focusing on the modulatory interactions of individual signaling proteins with the matrix. R. Timpl, Max Planck Institute, Munich, Germany, dis­cussed “Molecular interaction in basement membrane assembly” The talk concentrated on recent X ray structures of diverse modules of basement membrane proteins involved in cell-matrix as well as matrix-matrix inter­actions. This included several LG modules of laminin alpha chains, which harbor overlapping binding epitopes for heparan sulfate (FIS) chains, sul­fatides and the dystroglycan receptor. Another example was a single LE module of laminin gamma 1 chain that is responsible for the high affin­ity binding of the laminin chain to the G3 domain of nidogens. This interaction is thought to stabilize laminin/collagen IV networks within basement membranes by providing essential connections to other com­ponents. One of these connectors is the nidogen G2 domain, which binds to a single IG module of the proteoglycan perlecan. The most complex structure elucidated so far is the hexameric NC1 domain of collagen IV (about 1300 residues), which is crucial for collagen IV self-assembly into networks. All these binding structures were elucidated by site-directed mutagenesis. The accumulated data have set the stage for genetic manip­ulations of these proteins in order to understand the biological signifi­cance of individual molecular interactions in the complex process that governs basement membrane assembly.

Integrins are transmembrane receptors that are critical transmitters of information from the extracellular environment across the cell mem­brane. Integrins are involved with the regulation of numerous cell-matrix phenomena such as differentiation, migration, and division. R. Faessler, Max Planck Institute, Munich, Germany discussed “ß 1 Integrin During Development and Disease”. Both the migration of hematopoietic stem cells during embryogenesis as well as the retention of adult hematopoi­etic stem cells (HSC) in the bone marrow environment are thought to be controlled by integrins. The Faessler group has demonstrated that ß 1 integrin expression is crucial for the homing of both yolk sac- and para­aortic splanchnopleura-derived HSC into the fetal liver. The homing defect is caused by an inability of HSC to interact with endothelial cells. Intеrestingly, the loss of ß 1 integrin expression does not interfere with the proliferation, survival and differentiation of HSC. This was shown in vitro and in vivo. Мorеover, Faessler showed that HSC are retained in the bone marrow. These findings are in contrast to previous reports, in which experimental results using blocking antibodies and/or peptides suggested an essential role for the integrin a4 ß 1 (VLA-4), VCAM-1 and fibronectin (FN) in the retention of HSCs in the BM and in the survival and expansion of hematopoietic progenitor cells. This presentation demonstrated the subtle but critical importance of integrins in the inter­actions of cells with the ЕСМ and how this sensing system integrates multiple cues for the ultimate outcome of various cell decisions.

The next two discussants focused on chemistry of the glycosaminо­glycans (GAG) with the goal of understanding the chemical moieties involved in oligosaccharides interaction with proteins in the ЕСМ. U. Lindahl, University of Uppsala, Uppsala, Swedеn, discussed some of the critical issues concerning the biology of the glycosaminoglycans (GAGs) in his presentation “Heparan Sulfate — A Versatile Signal Promoter.” Heparan sulfate (НS) binds to a variety of proteins. However, the pro­tein recognition motifs in HS are largely unknown. Lindahl presented data on the identification and analysis of oligosaccharide sequences of protein-binding HS. A series of binding motifs with partially overlapping specificities for FGFs and one of the FGF receptors was observed. The identification of protein-binding HS sequences, however, is hampered by the limited availability of authentic cognate saccharide species. Lindahl described a method for the generation of oligosaccharide libraries to explore interactions between HS and proteins. Such libraries were obtained by incubating end-group 3 1-labeled, chemically partially or fully 0-desulfated heparin oligosaccharides with 0-sulfotransferases in the presence of PAPE (sulfate donor). The products were probed by affinity chromatography on resins containing immobilized growth factors, followed by sequence analysis of bound and subsequently recovered oligosaccharides. The results demonstrated that the libraries expand the available HS sequence space. Additional studies showed that the library strategy could also be successfully applied to define substrate specificities of biosynthetic (0-srilfotransferases) and degradative (heparanase) enzymes. J. T. Gallagher, University of Manchester, UK, continued the discussion on the GAGs with his talk entitled “The Molecular and Cell Biology of Heparan Sulphate Proteoglycans.” Gallagher described efforts to understand the mechanisms used by HSPGs to bind extrinsic protein effectors and modulate their signaling activities. His group devised another novel method for structure and sequence analysis of the HS- polysaccharide chain and has identified some of the sequence require- ments for activation of HS-dependent growth factors such as FGF1/FGF2, HGF and VEGF165. Gallagher also described how mice harbouring a mutation in the HS-20ST gene, which transfers sulphate groups to C-2 of iduronic acid, display developmental abnormalities, the most striking being bilateral renal agenesis. The HS produced by these mice has an atypical structure in which the deficiency in 2-0-sulphation is compensated for by an increase in C-6 sulpfhation of glucosamine residues. Evidence was presented showing that this abnormal HS retains some of the protein binding functions of the normal polysaccharide. These findings call into question claims concerning strict sequence speci­ficity in HS-mediated growth factor/rnorphogen signaling.

The discussion now considered the ability of GAGs and PGs to act as signaling molecules. G. David, University of Leuven, Belgium, discussed the “Lytic Controls on Cell Surface HSPG signaling.” David focused on hydrolytic mechanisms, specifically heparanolysis and proteolysis, that may regulate or be required for the signaling functions of two proteo- glycans, the syndecans and the glypicans activators of FGF-induced FGFR signaling. However, soluble forms of these proteolycans released from cell surfaces with protease are inactive. Yet bacterial heparitinase converts these `shed” proteоglycans into potent activators of FGF-signal­ing. These findings can be reconciled with FGF-FGFR complex crystal structures that suggest that receptor dimerization is supported by end-structures in the GAGS. This implies that “proteolytic” shedding down­regulates FGF-signaling and that “heparanolytic” shedding up regulates FGF-signaling by retrieving codes hidden in the native HS structures. Two proteolytic pathwаys potentially regulate the signaling functions of the protein moieties of these proteoglycans. One is a pro-protein conver­tase (PC)-mediated processing of the glypican-3 core protein required for the cell-specific apoptotic effects of GPC3. The PC-mediated processing may uncover a protein determinant in the core-protein that sequesters anti-apoptotic signaling molecules. A second pathway is the turnover of the syndecans transmembrane-domain by presenilin-dependent regulated intramembrane protеolysis. presenilin-aсtivity is required for the rapid clearance of the syndеcan cytoplasmic domain from the plasmamem­braie, and in the absence of presenilin-activity these cytoplasmic domains accumulate in peri-nuclear vesicles.

J. Hurle from the Departmento de Anatomia y Biologia Celular, Facultad de Medicina, Spain, spoke on the “Regulation of the Chondrogenic and Apoptotic Fates of the Limb Mesoderm by Seсreted Signals.” In the developing vertebrate limb the formation of the digits involves the commitment of the undifferentiated mesodermal cells to either chondrogenic differentiation or to apoptosis. This process results in the establishment of the digital rays and the interdigital spaces. Hurle surveyed the interactions between FGFs, TGF-ß2, activins, BMPs and retinoic acid in the regulation of this process.

The discussion next moved to the consideration of the interactions of specific growth factors with effectors in the extracellular environment and how these interactions contribute to signaling molecule function. J. Taipale, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, described the “Extracellular Regulation of Нedgehog (Нh) Signaling.” Locаlly secreted Нh proteins diffuse from their source to form gradients, acting over long range to direct patterning of adjacent tissues. The establishment of these gradients is critical in regulating Нh action. Нh proforms undergo an autoprocess­ing reaction catalyzеd by their C-terminal domains (1h-C) to generate a 19 kDa mature Нh N-terminal fragment (IbN), which is esterified at its C-terminus with a cholesterol-moiеty and palmitated at its N-terminus, a reaction catalyzеd by the Skinаy Нedgehog (Ski) aсyltransferase. The doublуmodified Нh proteins are tightly associated with the membranes of secreting cells. Therefore, an important question is how Нh is mobi­lized. A specialized protein, Dispatched (Disp) releases Нh from the membrane. Transfer of Нh from the producing to the receiving cell requires НSPG. In the receiving cells, Нh binds to Patched (Ptc). Ptc helps shape the Нh gradient by sequestration of 1h, and signals down­stream by inhibiting the activity of the seven transmembrane receptor Smoothеnеd. Thus, at least five distinct macromolecules (Нh-C, Ski, Disp, НSPG and Ptc) affect or shape the Нh morphogen gradient. The multiple levels of control required to fine-tune the Нh signaling systеm reflects the precise control of Нh distribution and concentration essential for vertebrate development.

S. Eaton, Max Planck Institute of Molecular Cеll Biology and Genetics, Dresden, Germany, described a novel mechanism for growth factor movement in her talk “Argosomes and the Dispersal of Wingless Protein Through the Wing Epithelium.” Eaton described a process that disperses membrane fragments over large distances through the Drosophila imaginal disc epithelium. These membranes exovesicles, called argosomes, are derived from basolateral membranes and are pro­duced by many different regions of the disc. Argosomes travel through adjacent tissue where they localize predominantly in endosomes. The Wingless protein colocalizes with argosomes derived from Wingless-pro­ducing cells. The properties of argosomes are consistent with their being a vehicle for the spread of Wingless protein. Whether argosomes are unique to insects or are more widespread is not known. Manу vertebrate cells in culture produce particles or “blebs” that contain an assortment of proteins.

A major focus of the meeting was the interactions of members of the TGF-ß superfamily of signaling molecules with extracellular modifiers. The best understood system in this regard is the control of BMP signal­ing in the early Drosophila еmbryo where BMP plays key roles in eco­dermal patterning. E. Bier, University of California, San Diego, addressed this question. One role of BMP signaling is to act in a dose­dependent fashion to subdivide the dorsal ectoderm into distinct domains. A molecule that participates in shaping the dorsal BMP activity gradient is Sog, which has four cysteine rich domains (CR1-CR4). Sog diffuses dorsally from its ventral site of production and is distributed dor­sally in a graded fashion. The Sog gradient creates a reciprocal gradient of the key BMP family member decapentaplegic (Dpp). Formation of the Sog gradient also depends on degradation of Sog by the metalloprotease Tolloid (Tld). Another extracellular factor that influences BMP signaling is twisted gastrulation (Tsg) gene. Tsg can complex with Dpp and Sog and alter the effect of Sog and BMP signaling. In the absence of Tsg, Sog interferes selectively with the activities of the auхiliary BMPs Screw and Glass Bottom Boat, but not with Dpp itself. In the presence of Tsg, how­ever, a truncated form of Sоg (Supersog), containing only the first of the four CR domains, is generated. Supersog inhibits the function of broader range of BMPs than does Sog. Structure-function studies indicate that different CR domains can partially substitute for each other with respect to full length Sog activity, but only CR1 can generate Supersog fonction. These studies reveal the сomplexity and sophistication involved in gener­ating BMP activity gradients as well as funсtionally distinct BMP modu­latory factors.

Although it was originally considered a member of the BMP family, BMP-1 along with more recently discovered isoenzymes are the mammalian equivalents of the Drosophila protease Tolloid (Tld). Members of the family are important regulators of extracellular protein processing. D. S. Greenspan, University of Wisconsin, Madison, WI, described the biological consequences of BMP-1/Tolloid proteinases in a talk entitled, “BMP-1/Tolloid Proteinases as Morphogenetic Regulators.” The Greenspan lab examined the phenotypes of mice with pull alleles for Bmpl (the gene which encodes alternativеly spliced RNAs for BMP-1 and for the related protease mTLD) and for X111 (the gene which encodes the related protease m Tlll). Homozygous null Bmpl-/- mice are peri­natal lethal with a failure to close the ventral body wall and persistent herniation of the gut. However, despite the fact that BMP-1 has been shown to be a procollagen C-proteinase (PCP) nеcеssary for biosynthеtic processing of type I collagen — the major protein constituent of bone, axial and appendicular skeletal elements are grossly normal, thus indicat­ing genetic redundancy. Therefore, mTLL-1 null mice were generated. Homozygous null Tlll-/- mice are embryonic lethal at 13.5-dpc because of heart defects. The levels of PCP activity in Tlll-/- fibroblast cultures are indistinguishable from levels detected in wild type cells. These results cal into question whether mTLL-1, which has PCP activity in vitro, is a PCP in vivo. These results also suggest functional redundancy, perhaps provided by products of the Bmpl gene. Greenspan and his colleagues intercrossed Bmpl +/- and Tlll +/- heterozygotes and obtained embryos homozygous null for the two genes. Greenspan described studies to determine whether functional redundancy has indeed been removed in the doubly homozygous embryos and whether mTLL-1 is in fact an in vivo PCP. In support of the latter two possibilities, detectable PCP activ­ity was totally lacking in fibroblasts cultured from doubly homozygous null embryos. Thus, products of the Bmpl and Tlll genes are responsi­ble for all detectable PCP activity in mouse embryo fibroblasts. BMP-1 and m Tlll had previously been shown by Greenspan and his colleagues to be capable of cleaving Chordin, the vertebrate homolog of Sog, in vitro. Intact Chordin, while undetectable in cultures of cells derived from wild type, Bmpl-/- and Tll1-/- embryos, was readily detectable in cells derived from the doubly homozygous null embryos. Thus, products of the Bmpl and Tlll genes are responsible for processing Chordin aid modulation of BMP signaling in mammals. BMP-1, mTLD and mTLL‑1, which have now been demonstrated to be involved in biosynthetic processing of a number of structural components of the extracellular matrix, appear to orchestrate formation of the extracellular matrix with BMP signaling in morphogenetic events.

М. Whitman, Harvard Medical School, Boston, spoke about “The Control of Nodal Signaling in Early Embryonic Patterning”. Nodal lig­ands are essential for the establishment of both mesoderm and endoderm during еarly chordate development. Genetic evidence indicates that EGF­CFC factors are required for Nodal signaling, but the molecular basis for this requirement has been unknown. Whitman has shown that Cripto, an EGF-CFC factor neсessary for mouse development, acts as a co-receptor for Nodal, and that homologous EGF-CFC family members perform similar functions in fish and frogs. Cripto also requires a novel post-trans­lational modification, 0-fucosylation, to act as a nodal co-receptor. Whitman also reported that Nodal can inhibit BMP signaling by a novel, Cripto-independent mechanism. This inhibitory effect is mediated by intracellular hetero-dimerization between Nodal and BIPs. The affinity of Nodal for в1Р7 in dimerization is equal to the affinity of Nodal for itself, indicating that heterodimerization is likеly in tissues where thеy are expressed at similar levels. Heterodimerization inhibits both Nodal and BMP signaling, raising the possibility that direct BMP antagonism of Nodal signaling by heterodimerization is also an important regulatory mechanism.

Proteinases of both the serine proteinase and matrix metalloproteinase (MMP) families are important contributors to the regulation of extracel­lular events. Z. Werb, University of California, San Francisco, discussed “Matrix letaloproteinases as Regulators of Extracellular Signal Transduction.” The broadened understanding of ECl fonction during remodeling processes, and identification of many non-matrix substrates for MMPs has shown that these enzymes regulate multiple cellular func­tions during tumor development, e.g., cell proliferation, differentiation, survival, genomic (in) stability, angiogenesis, and malignant potential. Patterning of ramified epithelial tissues, like mammary gland, lung and kidnеy, requires branching morphogenesis, by which epithelial ducts invade into stromal tissue. This requires stromal remodeling and stromal­epithelial communication. During puberty, mouse mammаry epithelial ducts invade the stromal mammаry fat pad in a wave of branching mor­phogenesis to form a complex ductal tree. Werb described experiments using inhibitor-treated and mutant mice that demonstrate that mammary gland branching morphogenesis requires MMP activity for invasion and branch point selection. МMР-2 (gelatinase A), but not ММР-9 (gelati­nase B), facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of рuberty. Unexpectedly, МMР-2 also represses excessive lateral branching during mid-puberty. MMP-14 (MT1-IMP) is implicated in branching morphogenesis of embryonic lung and endothe­lial cell culture. They demonstrated a requirement for ММР-14 in the branching morphogenesis of the mouse mammary gland. IMP-14 funс­tions in three ways : by activating MМР-2 (which is also required for mammary branching morphogenesis) ; cleaving the MMP-dependent, mammary morphogen, epimorphin and by degrading collagen I to allow for invasion of ducts. In contrast, MМР-3 (stromelysin-1) induces sec­ondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during distinct phases of mammary gland branching morphogenesis. Thus, MMPs are centrally placed to modulate the balance of positive and negative growth and differentiation factors and to maintain homeostasis and mediate many aspects of tissue devel­opment, regeneration and pathogenesis. TGF-ß is unusual for a growth factor as it is secreted in a latent state. Latency is conferred by the con­tinued non-covalent interaction of the TGF-ß propeptide with the cytokine even after the two molecules have been separated proteolytically. The situation with the TGF-ß is further complicated by the fact that the latent complex is associated with a second gene product, LTBP. J. E. Murphy-Ullrich, University of Alabama at Birmingham, described the role of thrombospondin-1 (TSP 1) in the conversion of latent to active TGF-ß in her presentation “Latent TGF-R Thrombospondin-1 (TSP1) :

A Growth Factor-Matricellular Protein Couple with Implications for Fibrotic Tissue Remodeling.” TGF-ß regulates cell proliferation, inflam­matory responses and ECM remodeling and is a key agent in the patho­genesis of fibroproliferative diseases. Мurphy-Ullrich has identified a mechanism for the activation of latent TGF-ß through interactions with TSP1. A sequence (KRFK) in the TSP1 type 1 repeat interacts with a conserved sequence (LSKL) at the amino-terminus of the propeptide (LAP) of the latent TGF-ß complex : binding of TSP1 or KRFK induces activation of latent TGF-ß. Circular dichroism studies indicate that bind­ing of TSP-1 to LAP induces conformational changes. TSP1-mediated activation of latent TGF-ß at the cell surface may involve multi-molecu­lar complexes. TSP-dependent control of TGF-ß bioactivity plays a role in the post-natal development of epithelial structures, in dermal wound healing and mау also be a key mediator of the increased TGF-ß bioactiv­ity in diabetic and hypertensive conditions, two diseases with fibrotic complications. In an animal model of anti Thy 1 induced glomeruloscle­rosis, peptide antagonists of TSP1-dependent TGF-ß activation amelio­rate the development of fibrotic complications. Thus, interactions of TSP1 with the latent form of TGF-ß regulate the bioaсtivity of TGF-ß, which, in turf, modulates matrix deposition and tissue remodeling. This pathway suggests a model by which autocrine factors determine the state of the ЕСМ.

D. B. Rifkin, New York University School of Medicine, New York, reported on his group’s analysis of the functions of LTBP. Three of the known four LTBPs associate covalently with the latent forms of TGF-ß, mediate their efficient secretion, and subsequently associate with the ЕСМ. The phenotype of the LTBP-3-/- mouse is consistent with a lack of TGF-(3 signaling in the synchondroses as well as in long bones, which develop osteopetrosis and osteoarthritis. Both of these phenotypes have been described previously in animals with impaired TGF-ß signaling. Therefore, the reported results with the LTBP-3 Hull miсe are consistent with a role for LTBP-3 in TGF-ß secretion, activation, or botte. The Rifkin group has also analyzеd regions of LTBP necessary for activation of latent TGF-ß by integrins. They reported that sequences within the LTBP hinge region are necessary. These results imply that LTBP in the latent TGF-ß complex must interact both with TGF-ß and a matrix or membrane molecule. This interaction, when coupled with integrin bind­ing to LAP, may provide a force necessary for latent TGF-ß  activation.

K. Koli, University of Helsinki, Finland, described several studies on the biology of LTBPs. LTBP-3 is the smallest of the known LTBPs, and the gene consists of 28 exils including one alternatively spliced exil. The splice variant contains an additional epidermal growth factor-like repeat in the C-terminus of the protein. The gene is transcribed to —4.6 kb mRNA and is expressed at high levels in human heart, skeletal mus­cle, prostate, ovaries and in certain osteosarcoma and fibroblastic cell fines. Efficient secretion of hLТВP-3 requires ci-expression of TGF-ß, which results in the release of high molecular weight complexes. Thus, LTBP-3 has an essential role in the secretion and targeting of TGF-ß1 in specific tissues. Ву using a gene trap strategy, Koli and collaborators have produced an Ltbp-4 hypomorphic mouse. Mice homozygous for the dis­rupted allele develop severe pulmonary emphysema and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic liber structure aid with a reduced deposition of TGF-ß in the extracellular space. Epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myс, and undergo uncon­trolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural compilent of the extracellular matrix and as a local regulator of TGF-ß tissue deposition aid signaling.

The interaction of the TGF-ß propeptide with LTBP occurs only with the third 8-cys or ТВ domain in LTBP. As additional non-reactive 8-cys domains exist in LTBP, and in the fibrillins, it has been of interest to understand what controls the reactivity of these specific motifs in the TGF-ß-binding LTBPs. Thus, covalent interaction is mediated bу exchange of an internal 8-cys 3 disulfide to form two disulfide bridges with the LAP1 dimer. A. K. Downing, University of Oxford, UK, spoke on the “Specificity of the Latent Transforming Growth Factor-(3 Binding Protein-1-LAP1 Interation.” The Downing lab has determined the solu­tion structure оf ТВ3 based on nuclear magnetic resonance data. The spe­cific rеactivity of 8-cys 3 appears to result from an insertion of two amino acids in loop between cys 6 and 7. This alters the structure so that the disulfide bond between cys 2-6 is more exposed to the solvent. This structure provides a hypothesis concerning the molecular basis of the ini­tial 8-cys-LAP1 interaction as the original disulfide 2-6 bond in LTBP 8­-cys 3 is surrounded by a ring of negatively charged amino acids. Using a combination of molecular biological and biophysical methods, Dr. Downing has identified acidic residues, which participate in macromole­cular recognition. These data, which are relevant to the control of TGF-ß 1 activity, mаy assist in the design of small molecules that regulate TGF-ß 1 activity.

Ali Brivanlou, Rockefeller University, NY, gave a presentation entitled “The latеnt TGF-ß-binding protein-1 (LTBP-1) is expressed in the organ­izer and regulates nodal and activin signaling.” Dr. Brivanlou and collab­orators isolated the Xenopus LTBP-1 (xLTBP-1) cDNA. The cDNA encodes a protein similar to the mammalian LTBP-1 in both size and domain structure. In addition, theу found a novel longer splice isoform of xLTBP. The RNAs for both forms of xLTBP display temporal regula­tion and the shorter transcript is expressed maternally. Both transcripts also display spatial regulation aid are found in the dorsal mesoderm of the organizer. In animal cap experiments, LTBP-1 potentiates the activity of activin aid nodal. The activity of LTBP-1 did not appear to require covalent association with activin as the addition of medium containing activin and LTBP-1 to animal caps enhanced the activin effect. These results indicate that LTBP-1 mау be part of the regulatory system that establishes the threshold of morphogen activity for activins and nodals in the dorsal side of the embryo during gastrulation. An exciting aspect of this presentation was that the reported data suggest that LTBPs may affect signaling molecules other than mау and the interactions with LTBP may be quite different than those of the TGF-ß.

In the final presentation, Dr. Lynn Y. Sakai, Shriner’s Hospital for Children, Department of Вiochemistry and Molecular Вiology, Oregon Health and Science University, Portland, discussed “Targeting Growth Factors to Extracellular Microfibrils.” Dr. Lynn Sakai hypothesized that an important function of fibrillins is to target growth factors to the ECM. A corollary to this hypothesis is that each of the three fibrillins mаy tar­get different growth factors or ray target the same growth factors dif­ferentially. Therefore, variations in the composition of fibrillins may influ­ence processes critical to development, growth, and aging. Each fibrillin contains 7 copies the 8-cys domin. Whereas it has been shown that an 8­cys module in certain LTBPs can bind covalently to propeptides of TGF-ßs, fibrillins do not do so. Dr. Lynn Sakai described experiments demonstrating specific binding of fibrillin 8-cys2 to the propeptide of BMP-7 and 8-cysl to ВМР-4. Immunolocalization experiments using human, mouse and chicken tissues demonstrated colocalization of BIP­7 and fibrillin, supporting the hypothesis that fibrillins target growth fac­tors to the extracellular matrix. Together with the documented LTВP/ TGF-ß interactions, these data suggest that 8-cys modules mediate noncovalent interactions with propeptides of the TGF-ß -related growth factor family.