Fibronectin regulates latent transforming growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding protein-1

Latent transforming growth factor-beta-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGF beta in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-beta (TGF beta) into the ECM of osteoblasts and fibroblasts. Immunolocalization studies suggested that fibronectin provides an initial scaffold that precedes and patterns LTBP1 deposition but that LTBP1 and fibronectin are later localized in separate fibrillar networks, suggesting that the initial template is lost. Treatment of fetal rat calvarial osteoblasts with a 70-kDa N-terminal fibronectin fragment that inhibits fibronectin assembly impaired incorporation of LTBP1 and TGFbeta into the ECM. Consistent with this, LTBP1 failed to assemble in embryonic fibroblasts that lack the gene for fibronectin. LTBP1 assembly was rescued by full-length fibronectin and superfibronectin, which are capable of assembly into fibronectin fibrils, but not by other fibronectin fragments, including a 160-kDa RGD-containing fragment that activates alpha5beta1 integrins. This suggests that the critical event for LTBP1 assembly is the formation of a fibronectin fibrillar network and that integrin ligation by fibronectin molecules alone is not sufficient. Not only was fibronectin essential for the initial incorporation of LTBP1 into the ECM, but the continued presence of fibronectin was required for the continued assembly of LTBP1. These studies highlight a nonredundant role for fibronectin in LTBP1 assembly into the ECM and suggest a novel role for fibronectin in regulation of TGF beta via LTBP1 interactions.     

Potential role for heparan sulfate proteoglycans in regulation of transforming growth factor-beta (TGF-beta) by modulating assembly of latent TGF-beta-binding protein-1

Latent transforming growth factor-beta-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-beta in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-beta into the ECM of osteoblasts and fibroblasts. Here we provide evidence that heparan sulfate proteoglycans may mediate binding between LTBP1 and fibronectin. We have localized critical domains in the N terminus of LTBP1 that are required for co-localization with fibronectin in osteoblast cultures and have identified heparin binding sites in the N terminus of LTBP1 between residues 345 and 487. Solid-phase binding assays suggest that LTBP1 does not bind directly to fibronectin but that the binding is indirect. Heparin coupled to bovine serum albumin (heparin-BSA) was able to mediate binding between fibronectin and LTBP1. Treatment of primary osteoblast cultures with heparin or heparin-BSA but not with chondroitin sulfate impaired LTBP1 deposition onto fibronectin without inhibiting expression of LTBP1. Inhibition of LTBP1 incorporation was accompanied by reduced incorporation of latent TGF-beta into the ECM, with increased amounts of soluble latent TGF-beta. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by beta-d-xylosides also reduced incorporation of LTBP1 into the ECM. These studies suggest that heparan sulfate proteoglycans may play a critical role in regulating TGF-beta availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin.     

Matrix association of latent TGF‐beta binding protein‐2 (LTBP‐2) is dependent on fibrillin‐1

The components of the extracellular matrix (ECM) and their differential expression patterns play important roles in tissue formation. The deposition of latent TGF‐β binding proteins (LTBPs) to the ECM exhibit distinct distribution profiles. We have analyzed here the temporal and spatial ECM association of latent TGF‐β binding protein LTBP‐2 in cultured human embryonic lung fibroblasts. We found that LTBP‐2 was not assembled to the ECM until by confluency of cultures following the deposition of fibronectin (FN) and fibrillin‐1. In 5‐day‐old cultures LTBP‐2 was rapidly secreted from cells and it subsequently associated with the ECM as shown by metabolic labeling and immunoprecipitation. LTBP‐2 colocalized transiently with fibronectin and failed to assemble to the ECM of FN deficient mouse fibroblasts. Analysis of different cultured human cell lines revealed partial colocalization of LTBP‐2 and fibrillin‐1 in the ECM of fibroblasts, MG‐63 osteosarcoma cells and human vascular endothelial cells. Silencing of fibrillin‐1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP‐2. Current results suggest that LTBP‐2 is not an element of the provisional ECM of fibroblasts but is more likely a component of more mature ECM and indicate that matrix association of LTBP‐2 depends on a pre‐formed fibrillin‐1 network. J. Cell. Physiol. 221: 586–593, 2009. © 2009 Wiley‐Liss, Inc.     

Extracellular matrix proteins regulate epithelial–mesenchymal transition in mammary epithelial cells

Mouse mammary epithelial cells undergo transdifferentiation via epithelial–mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland.     

BMP1 controls TGFbeta1 activation via cleavage of latent TGFbeta-binding protein

Transforming growth factor beta1 (TGFbeta1), an important regulator of cell behavior, is secreted as a large latent complex (LLC) in which it is bound to its cleaved prodomain (latency-associated peptide [LAP]) and, via LAP, to latent TGFbeta-binding proteins (LTBPs). The latter target LLCs to the extracellular matrix (ECM). Bone morphogenetic protein 1 (BMP1)-like metalloproteinases play key roles in ECM formation, by converting precursors into mature functional proteins, and in morphogenetic patterning, by cleaving the antagonist Chordin to activate BMP2/4. We provide in vitro and in vivo evidence that BMP1 cleaves LTBP1 at two specific sites, thus liberating LLC from ECM and resulting in consequent activation of TGFbeta1 via cleavage of LAP by non-BMP1-like proteinases. In mouse embryo fibroblasts, LAP cleavage is shown to be predominantly matrix metalloproteinase 2 dependent. TGFbeta1 is a potent inducer of ECM formation and of BMP1 expression. Thus, a role for BMP1-like proteinases in TGFbeta1 activation completes a novel fast-forward loop in vertebrate tissue remodeling.     

In vivo effects of TGFbeta1 on the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGFbeta) wanes in the milk of lactating rat, an increase in TGFbeta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGFbeta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGFbeta1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGFbeta1 while TbetaRII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p<0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p<0.05). Importantly, TGFbeta1 inhibited cell proliferation (p<0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p<0.05). Also, TGFbeta1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p<0.001), and by morphological features at 12 h (p<0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGFbeta1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TGFbeta1 in gastric growth during postnatal development.     

In-vitro analysis of the expression of TGFbeta -superfamily-members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture

Traditional surgical methods for the reconstruction of cartilage defects rely on the transplantation of autologous and allogenous tissues. The disadvantages of these techniques are the limited availability of suitable tissues and the donor site morbidity of transplants. In addition, in cultured chondrocytes, the dedifferentiation of cells seems unavoidable during multiplication. In this study, we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) and human mesenchymal stem cells (MSC) in cell culture using microarray technique, immunohistochemistry and RT-PCR. Transforming growth factor beta (TGFbeta) is a multifunctional peptide that plays play a crucial role in inducing and maintaining chondrogenic differentiation. In dedifferentiating chondrocytes, the gene for TGFbeta1 was constantly expressed, while the gene for TGFbeta2 was never expressed. The genes for TGFalpha, TGFbeta4 and TGFbetai were activated with ongoing dedifferentiation. TGFbeta-receptor 3 was constantly expressed, while the genes for the TGFbeta-receptors 1 and 2 were never expressed. Immunohistochemical staining for TGFbeta beta 3 revealed upregulation in the course of dedifferentiation. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation, whereas the gene for LTBP3 was constantly expressed, and negative results were obtained for the gene for LTBP4. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation. During chondrogenic differentiation, the MSCs constantly expressed TGFbeta1, beta2, beta3 and beta4. LTBP1, LTBP2 and TGFbeta-R3 were downregulated. In conclusion, TGFbeta3, TGFbeta4, TGFbetai, LTBP1 and LTBP2 may assist the process of dedifferentiation, while TGFbeta1 and beta2 might not be involved in this process. Of the TGFbeta-receptors, only type 3 might be involved in dedifferentiation.     

Glucocorticoid enhances transforming growth factor-beta effects on extracellular matrix protein expression in human placental mesenchymal cells

Extracellular matrix (ECM) proteins synthesized by human placental mesenchymal cells (PMCs) provide structural support for the villus. Aberrant expression of ECM proteins by PMCs has been associated with intrauterine growth restriction (IUGR). To provide insight into the mechanisms of ECM protein regulation in the stroma of the placental villus, in the current study, we examined the interaction of glucocorticoid (GC) and transforming growth factor-beta (TGFbeta) in the modulation of ECM proteins in cultures of PMCs isolated from human term placentas. Initial results obtained by ELISA showed that combined treatment with dexamethasone (DEX) and TGFbeta enhanced oncofetal fibronectin (FFN) protein levels in serum-free culture medium severalfold in a dose-dependent manner. Northern blotting and real-time polymerase chain reaction (PCR) analyses revealed a similar enhancement in levels of FN mRNA in cells treated with TGFbeta and DEX. Real-time PCR results also revealed that DEX and TGFbeta enhanced collagen (Col) I and Col IV expression, but did not affect levels of Col III or laminin, indicative of selective stimulation of ECM proteins. Hypoxic treatment moderately enhanced FFN levels in control cells but not in those treated with DEX and TGFbeta. In contrast with the results obtained with PMCs, we noted that DEX treatment suppressed FFN levels in untreated and TGFbeta-treated cytotrophoblasts, suggesting that GC and TGFbeta modulate FFN expression in placenta in a cell-type-specific manner. We conclude that GC and TGFbeta are key regulators of ECM protein synthesis in PMCs, suggesting a role in modulating placental architecture in uncomplicated pregnancies and those associated with aberrant ECM protein expression.     

Disruption of transforming growth factor beta-regulated laminin receptor function by expression of antisense laminin, a chain RNA in human colon cancer cells

Transforming growth factor beta1 (TGFbeta) simultaneously induces the expression of fibronectin, fibronectin receptor, laminin, and laminin receptor (alpha6beta1 integrin) in the human colon cancer cell line Moser (Int J Cancer, 57:742, 1994). Induction of fibronectin and induction of fibronectin receptor by TGFB are tightly coupled, and disrupting fibronectin induction disrupts the induction of fibronectin receptor and cellular adhesion to fibronectin (J Cellular Physiol, 170:138, 1997). We recently demonstrated the efficacy of using antisense chain-specific laminin RNA expression vectors to disrupt the induction by TGFP of the multichain laminin molecule (J Cellular Physiol, 178:296, 1999). We now show in this report that Moser cells used alpha6 and beta1 integrins to adhere to laminin, and, as is the fibronectin and fibronectin receptor system, disrupting the induction by TGFbeta of the ligand laminin by the expression of antisense laminin A chain RNA disrupted the induction of 125I-laminin binding and cellular adhesion to laminin. Disrupting laminin induction also blocked the induction of alpha6 and beta1 integrin laminin receptor by TGFbeta. We conclude that disrupting the induction of the ligand laminin by TGFbeta disrupts TGFbeta-regulated laminin receptor function by suppressing the induction of alpha6 and beta1 integrins. Therefore, targeted disruption of the ligand laminin may be an effective means in disrupting the function of both the ligand and its receptor in cells that utilize the laminin and laminin receptor system in malignant cell behavior.     

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE₂ stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells. © 1995 Wiley-Liss, Inc.     

Molecular and functional aspects of latent transforming growth factor-β binding protein: just a masking protein?

Latent transforming growth factor-beta (TGFbeta) binding protein (LTBP), a component of the high-molecular-weight latent TGFbeta complex, is found in various cell and tissue types. Originally described as a TGFbeta-masking protein, recent detections of four isoforms and numerous splice variants provide new aspects of its putative functional role. Regulation and sequestration of TGFbeta activity and structural remodeling of the extracellular matrix (ECM) seem to be the main tasks, but other possible functions might exist. The mechanism by which LTBP interacts with cell surface molecules or cellular receptors and ECM components remains unclear. Cellular, molecular and functional aspects will be discussed.     

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

The predicted secretomes of Monosiga brevicollis and Capsaspora owczarzaki,close unicellular relatives of metazoans,reveal new insights into the evolution of the metazoan extracellular matrix

The extracellular matrix (ECM) is a major mediator of multi-cellularity in the metazoa. Multiple ECM proteins are conserved from sponges to human, raising questions about the evolutionary origin of ECM. Choanoflagellates are the closest unicellular relatives of the metazoa and proteins with domains characteristic of metazoan ECM proteins have been identified from the genome-predicted proteome of the choanoflagellate Monosiga brevicollis. However, a systematic analysis of M. brevicollis secretory signal peptide-containing proteins with ECM domains has been lacking. We analysed all predicted secretory signal-peptide-containing proteins of M. brevicollis for ECM domains. Nine domains that are widespread in metazoan ECM proteins are represented, with EGF, fibronectin III, laminin G, and von Willebrand Factor_A domains being the most numerous. Three proteins contain more than one category of ECM domain, however, no proteins correspond to the domain architecture of metazoan ECM proteins. The fibronectin III domains are all present within glycoside hydrolases and none contain an integrin-binding motif. Glycosaminoglycan-binding motifs identified in animal thrombospondin type 1 domains are conserved in some M. brevicollis representatives of this domain, whereas there is little evidence of conservation of glycosaminoglycan-binding motifs in the laminin G domains. The identified proteins were compared with the predicted secretory ECM domain-containing proteins of the integrin-expressing filasterean, Capsaspora owczarzaki. C. owczarzaki encodes a smaller number of secretory, ECM domain-containing proteins and only EGF, fibronectin type III and laminin G domains are represented. The M. brevicollis and C. owczarzaki proteins have distinct domain architectures and all proteins differ in their domain architecture to metazoan ECM proteins. These identifications provide a basis for future experiments to validate the extracellular location of these proteins and uncover their functions in choanoflagellates and C. owczarzaki. The data strengthen the model that ECM proteins are metazoan-specific and evolved as innovations in the last common metazoan ancestor.     

Three key residues underlie the differential affinity of the TGFbeta isoforms for the TGFbeta type II receptor

TGFbeta1, beta2, and beta3 are 25kDa homodimeric polypeptides that play crucial non-overlapping roles in development, tumor suppression, and wound healing. They exhibit 70-82% sequence identity and transduce their signals by binding and bringing together the TGFbeta type I and type II receptors, TbetaRI and TbetaRII. TGFbeta2 differs from the other isoforms in that it binds TbetaRII weakly and is dependent upon the co-receptor betaglycan for function. To explore the physicochemical basis underlying these differences, we generated a series of single amino acid TbetaRII variants based on the crystal structure of the TbetaRII:TGFbeta3 complex and examined these in terms of their TGFbeta isoform binding affinity and their equilibrium stability. The results showed that TbetaRII Ile53 and Glu119, which contact TGFbeta3 Val92 and Arg25, respectively, together with TbetaRII Asp32, Glu55, and Glu75, which contact TGFbeta3 Arg94, each contribute significantly, between 1 kcal mol(-1) to 1.5 kcal mol(-1), to ligand binding affinities. These contacts likely underlie the estimated 4.1 kcal mol(-1) lower affinity with which TbetaRII binds TGFbeta2 as these three ligand residues are unchanged in TGFbeta1 but are conservatively substituted in TGFbeta2 (Lys25, Ile92, and Lys94). To test this hypothesis, a TGFbeta2 variant was generated in which these three residues were changed to those in TGFbetas 1 and 3. This variant exhibited receptor binding affinities comparable to those of TGFbetas 1 and 3. Together, these results show that these three residues underlie the lowered affinity of TGFbeta2 for TbetaRII and that all isoforms likely induce assembly of the TGFbeta signaling receptors in the same overall manner.     

Extracellular matrix assembly and 3D organization during paraxial mesoderm development in the chick embryo

The extracellular matrix (ECM) is a major player in the microenvironment governing morphogenesis. However, much is yet to be known about how matrix composition and architecture changes as it influences major morphogenetic events. Here we performed a detailed, 3D analysis of the distribution of two ECM components, fibronectin and laminin, during the development of the chick paraxial mesoderm. By resorting to whole mount double immunofluorescence and confocal microscopy, we generated a detailed 3D map of the two ECM components, revealing their supra-cellular architecture in vivo, while simultaneously retaining high resolution cellular detail. We show that fibronectin assembly occurs at the surface of the presomitic mesoderm (PSM), where a gradual increase in the complexity of the fibronectin matrix accompanies PSM maturation. In the rostral PSM, where somites form, fibronectin fibrils are thick and densely packed and some occupy the cleft which comes to separate the newly formed somite from the PSM. Our 3D approach revealed that laminin matrix assembly starts at the PSM surface as small dispersed patches, which are always localized closer to cells than the fibronectin matrix. These patches gradually grow and coalesce with neighboring patches, but do not generate a continuous laminin sheet, not even on epithelial somites and dermomyotome, suggesting that these epithelia develop in contact with a fenestrated laminin matrix. Unexpectedly, as the somite differentiates, its fibronectin and laminin matrices are maintained, thus initially containing both the epithelial dermomyotome and the mesenchymal sclerotome within the somite segment. Our analysis provides unprecedented details of the progressive in vivo assembly and 3D architecture of fibronectin and laminin matrices during paraxial mesoderm development. These data are consistent with the hypothesis that progressive ECM assembly and subsequent 3D organization are active driving and containing forces during tissue development.     

Pancreatic tumor cells influence the composition of the extracellular matrix

The malignant behavior of cancers depends on the microenvironmental context. We investigated compositional alterations of the extracellular matrix (ECM) in pancreatic cancer, with special emphasis on the proteoglycans decorin, lumican, and versican. Compared with normal controls (n=18), marked overexpression of these proteoglycans was observed in pancreatic cancer tissues (n=30) by quantitative RT-PCR (p<0.0001). Immunohistochemistry revealed abundance of proteoglycans in the ECM of pancreatic cancer specimens, whereas tumor cells themselves were devoid of either decorin, lumican or versican. RT-PCR confirmed pancreatic stellate cells (PSCs) as the major source of these proteins. Interestingly, TGFbeta1 and conditioned medium derived from pancreatic cancer cell lines synergistically suppressed the expression of known anti-tumor factors decorin and lumican, but stimulated the expression of pro-metastatic factor versican in cultured PSCs. These findings indicate that malignant cells can actively influence the composition of the ECM through TGFbeta1 and other soluble factors, altering their microenvironment in a tumor-favorable way.     

Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.     

Site-restricted expression of cytotactin during development of the chicken embryo

The sequential appearance of the extracellular matrix (ECM) protein, cytotactin, was examined during development of the chicken embryo by immunohistochemical techniques. Although cytotactin was identified as a molecule that mediates glia-neuron interactions, preliminary immunohistochemical localization of the molecule suggested that it was an ECM protein with a widespread but nonetheless more restricted distribution than either fibronectin or laminin. In the present study, it was found that cytotactin is first present in the gastrulating chicken embryo. It appears later in the basement membrane of the developing neural tube and notochord in a temporal sequence beginning in the cephalic regions and proceeding caudally. Between 2 and 3 d of development, the molecule is present at high levels in the early neural crest pathways (surrounding the neural tube and somites) but, in contrast to fibronectin and laminin, is not found in the lateral plate mesoderm or ectoderm. At later times, cytotactin is expressed extensively in the central nervous system, in lesser amounts in the peripheral nervous system, and in a number of nonneural sites, most prominently in all smooth muscles and in basement membranes of lung and kidney. Cytotactin appears in adult tissues with distributions that are similar to those seen in embryonic tissues. The findings raise the possibility that certain ECM proteins contribute to pattern formation in embryogenesis as a result of their restricted expression in a spatiotemporally regulated fashion at some sites but not at others.