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J F Talts

Publications and source records attributed to J F Talts.

At least 19 recordsLinked to original sources
PubMed2001

Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane.

Basement membranes are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Our previous analysis of a fibroblast growth factor (FGF) receptor mutation raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. Here we test this hypothesis and demonstrate that constitutively active Akt/PKB, an important downstream element of phosphoinositide 3'-kinase signaling, induces the synthesis of laminin-1 and collagen IV isotypes and causes their translocation to the basement membrane. By using promoter-reporter constructs, we show that constitutively active phosphoinositide 3'-kinase-p110 or Akt/PKB activates, whereas dominant negative Akt/PKB inhibits, transcription of laminin beta1 and collagen IV alpha1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary-T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of basement membrane formation. The possible role of Akt/PKB-induced laminin and collagen IV synthesis in cell survival and differentiation will be discussed.

Animals↗
PubMed2001

Glucocorticoids down-regulate the extracellular matrix proteins fibronectin, fibulin-1 and fibulin-2 in bone marrow stroma.

Glucocorticoids regulate hematopoietic cell interactions with the bone marrow microenvironment, but the molecules involved in the regulation are still largely unknown. We have studied the effect of glucocorticoids on mRNA expression and protein synthesis of the major extracellular matrix adhesion protein fibronectin and three other extracellular proteins, fibulin-1, fibulin-2 and nidogen-1, in mouse bone marrow cultures and in a hematopoiesis supporting the stromal MC3T3-G2/PA6 cell line. Glucocorticoids suppressed mRNA expression and protein synthesis of fibronectin, fibulin-1 and fibulin-2, but not nidogen-1, in adherent cells of bone marrow cultures, as shown by Northern blot analysis and immunoprecipitation. mRNA levels of all four proteins were down-regulated by dexamethasone in MC3T3-G2/PA6 cells, indicating a direct glucocorticoid effect on cells synthesizing extracellular matrix proteins. Dexamethasone down-regulated fibronectin mRNA rapidly, within 2 h of treatment, in the stromal cells. This effect did not require mRNA or protein synthesis, as shown by Northern blot analysis after treatment by actinomycin D and cycloheximide. Interferon-alpha, which also has been reported to modulate haematopoietic cell-matrix interactions, did not affect mRNA expression of the proteins in MC3T3-G2/PA6 cells. Our results indicate that glucocorticoids down-regulate expression of several mesenchymal-type extracellular matrix molecules in bone marrow, but with a variable effect on different proteins. Thus one mechanism by which glucocorticoids regulate haematopoiesis may be by altering the relative proportions of extracellular matrix proteins.

Animals↗
PubMed2001

Dystroglycan binding to laminin alpha1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro.

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.

Animals↗
PubMed2000

Structural and functional analysis of the recombinant G domain of the laminin alpha4 chain and its proteolytic processing in tissues.

The C-terminal G domains of laminin alpha chains have been implicated in various cellular and other interactions. The G domain of the alpha4 chain was now produced in transfected mammalian cells as two tandem arrays of LG modules, alpha4LG1-3 and alpha4LG4-5. The recombinant fragments were shown to fold into globular structures and could be distinguished by specific antibodies. Both fragments were able to bind to heparin, sulfatides, and the microfibrillar fibulin-1 and fibulin-2. They were, however, poor substrates for cell adhesion and had only a low affinity for the alpha-dystroglycan receptor when compared with the G domains of the laminin alpha1 and alpha2 chains. Yet antibodies to alpha4LG1-3 but not to alpha4LG4-5 clearly inhibited alpha(6)beta(1) integrin-mediated cell adhesion to laminin-8, indicating the participation of alpha4LG1-3 in a cell-adhesive structure of higher complexity. Proteolytic processing within a link region between the alpha4LG3 and alpha4LG4 modules was shown to occur during recombinant production and in endothelial and Schwann cell culture. Cleavage could be attributed to three different peptide bonds and is accompanied by the release of the alpha4LG4-5 segment. Immunohistology demonstrated abundant staining of alpha4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for alpha4LG4-5, indicating their loss from tissues. Immunogold staining demonstrated an association of the alpha4 chain primarily with microfibrillar regions rather than with basement membranes, while laminin alpha2 chains appear primarily associated with various basement membranes.

Adipose Tissue↗
PubMed2000

Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae.

The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.

Animals↗
PubMed2000

Structure of the C-terminal laminin G-like domain pair of the laminin alpha2 chain harbouring binding sites for alpha-dystroglycan and heparin.

The laminins are large heterotrimeric glycoproteins with fundamental roles in basement membrane architecture and function. The C-terminus of the laminin alpha chain contains a tandem of five laminin G-like (LG) domains. We report the 2.0 A crystal structure of the laminin alpha2 LG4-LG5 domain pair, which harbours binding sites for heparin and the cell surface receptor alpha-dystroglycan, and is 41% identical to the laminin alpha1 E3 fragment. LG4 and LG5 are arranged in a V-shaped fashion related by a 110 degrees rotation about an axis passing near the domain termini. An extended N-terminal segment is disulfide bonded to LG5 and stabilizes the domain pair. Two calcium ions, one each in LG4 and LG5, are located 65 A apart at the tips of the domains opposite the polypeptide termini. An extensive basic surface region between the calcium sites is proposed to bind alpha-dystroglycan and heparin. The LG4-LG5 structure was used to construct a model of the laminin LG1-LG5 tandem and interpret missense mutations underlying protein S deficiency.

Amino Acid Sequence↗
PubMed2000

Structure and function of laminin LG modules.

Laminin G domain-like (LG) modules of approximately 180-200 residues are found in a number of extracellular and receptor proteins and often are present in tandem arrays. LG modules are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. The recently determined crystal structures of LG modules of the laminin alpha2 chain reveal a compact beta sandwich fold and identify a novel calcium binding site. Binding epitopes for heparin, sulfatides and alpha-dystroglycan have been mapped by site-directed mutagenesis and show considerable overlap. The epitopes are located in surface loops around the calcium site, which in other proteins (agrin, neurexins) are modified by alternative splicing. Efficient ligand binding often requires LG modules to be present in tandem. The close proximity of the N- and C-termini in the LG module, as well as a unique link region between laminin LG3 and LG4, impose certain constraints on the arrangement of LG tandems. Further modifications may be introduced by proteolytic processing of laminin G domains, which is known to occur in the alpha2, alpha3 and alpha4 chains.

Amino Acid Sequence↗
PubMed1999

Mutation of a basic sequence in the laminin alpha2LG3 module leads to a lack of proteolytic processing and has different effects on beta1 integrin-mediated cell adhesion and alpha-dystroglycan binding.

A RRKRRQ sequence unique to the LG3 module of the laminin alpha2 chain was previously shown to be sensitive to endogenous proteolysis during the recombinant production of the tandem array alpha2LG1-3. Mutation of RQ surrounding the cleaved peptide bond did not prevent this processing and intracellular degradation. Alanine mutagenesis of three alternate basic residues, however, was shown to prevent the cleavage in alpha2LG1-3, allowing for the alpha2LG3 module to be obtained as a folded, globular fragment. The mutation did not change heparin and sulfatide binding or cell adhesion of alpha2LG1-3 which can be mediated by alpha3beta1 and alpha6beta1 integrins. It did, however, cause a 10-fold reduction in alpha-dystroglycan binding. The data favor the interpretation that binding epitopes for heparin/sulfatides, beta1 integrins and alpha-dystroglycan occupy different parts of the alpha2LG1-3 structure.

Animals↗
PubMed1999

Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.

The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly.

Animals↗
PubMed1999

The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin.

Laminin G-like (LG) modules in the extracellular matrix glycoproteins laminin, perlecan, and agrin mediate the binding to heparin and the cell surface receptor alpha-dystroglycan (alpha-DG). These interactions are crucial to basement membrane assembly, as well as muscle and nerve cell function. The crystal structure of the laminin alpha 2 chain LG5 module reveals a 14-stranded beta sandwich. A calcium ion is bound to one edge of the sandwich by conserved acidic residues and is surrounded by residues implicated in heparin and alpha-DG binding. A calcium-coordinated sulfate ion is suggested to mimic the binding of anionic oligosaccharides. The structure demonstrates a conserved function of the LG module in calcium-dependent lectin-like alpha-DG binding.

Agrin↗
PubMed1999

Tenascin-C modulates tumor stroma and monocyte/macrophage recruitment but not tumor growth or metastasis in a mouse strain with spontaneous mammary cancer.

The local growth of tumors and their ability to metastasize are crucially dependent on their interactions with the surrounding extracellular matrix. Tenascin-C (TNC) is an extracellular matrix protein which is highly expressed during development, tissue repair and cancer. Despite the high levels of TNC in the stroma of primary and metastatic tumors, the function of TNC is not known. In the present study we have crossed TNC-null mice with a mouse strain where both female and male mice spontaneously develop mammary tumors followed by metastatic disease in the lungs. We report that the absence of TNC had no effect on the temporal occurrence of mammary tumors and their metastatic dissemination in lungs. Furthermore, the number and size of tumors, the number and size of metastatic foci in the lungs, the proliferation rate and apoptosis of tumor cells and tumor angiogenesis were not altered in the absence of TNC. Histological examination revealed that the tumor organisation, however, was modulated by TNC. In the presence of TNC both primary as well as metastatic tumors were organised in large tumor cell nests surrounded by thick layers of extracellular matrix proteins. In the absence of TNC these tumor cell nests were smaller but still separated from each other by extracellular matrix proteins. In addition, the TNC-null stromal compartment contained significantly more monocytes/macrophages than tumor stroma from TNC wild-type mice. Using in vitro coculture experiments we show that TNC-null tumor cells were still able to activate the TNC gene in fibroblasts which express low basal levels of TNC. Altogether these data indicate that TNC has a very limited role during the spontaneous development and growth of mamary tumors and their metastasis to the lungs.

Animals↗
PubMed1998

Endochondral ossification is dependent on the mechanical properties of cartilage tissue and on intracellular signals in chondrocytes.

Skeletal elements are formed either by replacing a performed cartilagenous matrix template in a process called endochondral ossification or directly from mesenchyme by a process known as membranous ossification. Longitudinal growth of bones is achieved by growth plates where calcified cartilage is converted into bone. To investigate the role of extracellular matrix as well as intracellular signaling pathways in the formation and growth of bone, the genes coding for type II collagen and cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (cGK) II, were disrupted. It is demonstrated that loss of Col2a1 or cGKII led to abnormal endochondral ossification and skeletal development. In cGKII -/- mice, bones derived by membranous ossification developed normally while bones derived by endochondral ossification were shortened. This growth defect was not associated with a general metabolic disturbance. In Col2a1 knockout mice, endochondral ossification was completely absent, whereas membraneous ossification was not affected. Despite the defects in bone formation, invasion of blood vessels into bone cavities and formation of bone marrow occurred in Col2a1-null mice. Taken together, the phenotypes of these two knockout mice show that chondrocytes need a well-functioning extracellular matrix scaffold and a normal cGMP-signaling system for endochondral ossification to form a normal skeleton.

Animals↗
PubMed1998

Structural analysis and proteolytic processing of recombinant G domain of mouse laminin alpha2 chain.

Four individual LG modules from the C-terminus of the laminin alpha2 chain (LG1, LG2, LG4 and LG5) and combinations of these modules were prepared as recombinant products from transfected mammalian cells. This demonstrated that LG modules represent autonomously folding protein domains. Successful production depended on proper alignment of module borders and required a sequence correction at the C-terminus which added an extra cysteine. The LG modules were glycosylated and shown by electron microscopy to have a globular shape, indicating proper folding. Evidence is provided for the splicing of a 12 bp exon in LG2, although this did not impair folding. Proteolytic cleavage at the C-terminus of a basic sequence was observed close to the N-terminus of LG3. A similar processing also occurs in tissue-derived laminin-2 and -4 which contain the alpha2 chain.

Amino Acid Sequence↗
PubMed1998

Expansion of the nonadherent myeloid cell population by monoclonal antibodies against tenascin-C in murine long-term bone marrow cultures.

Tenascin-C, a predominantly mesenchymal extracellular matrix protein, has a restricted distribution in adult tissues. It has previously been shown that this protein is expressed in the bone marrow. In this paper we show that murine myeloid and lymphoid long-term bone marrow cultures differ in their expression of tenascin-C splice variants. In the adherent stromal layer of myeloid cultures, the 260-kDa polypeptide encoded by the 8-kb mRNA was the major splice variant, whereas in the stromal layer of lymphoid cultures both the shorter 210-kDa polypeptide encoded by the 6-kb mRNA and the 260-kDa polypeptide were abundantly expressed. However, in both culture systems the larger 260-kDa tenascin-C polypeptide was the major isoform secreted in the culture supernatant. This finding is in agreement with previous reports indicating that the smaller 210-kDa isoform is preferentially deposited in the stroma, whereas the alternatively spliced segment in the 260-kDa tenascin-C may contain anti-adhesive domains. Glucocorticoids in myeloid long-term bone marrow cultures and in the MC3T3-G2/PA6 cell line downregulated the expression of tenascin-C. In the present study we observed that this was due primarily to downregulation of the 8-kb major splice variant of the tenascin-C mRNA. We also studied the possible role of tenascin-C in the bone marrow by using antibodies against tenascin-C in long-term bone marrow cultures. We found that three monoclonal antibodies against the carboxyterminal type III fibronectin repeats of tenascin-C (TNCfn 7-8) increased the number of the non-adherent myeloid cells in myeloid long-term bone marrow cultures. It has recently been suggested that the TNCfn 6-8 domain of tenascin-C binds to the alpha8beta1 integrin. Using Northern blotting, we found that the integrin alpha8 subunit was expressed in adherent cells in bone marrow cultures, raising the possibility that tenascin-C acts in bone marrow cultures by binding to the alpha8beta1 integrin.

Animals↗
PubMed1997

Importance of nidogen binding to laminin gamma1 for branching epithelial morphogenesis of the submandibular gland.

Epithelial-mesenchymal interactions are major driving forces for the development of most solid organs. The importance of these interactions was first shown for the embryonic submandibular gland more than 40 years ago. We here present evidence that interactions between two basement membrane components, nidogen (entactin) and laminin gamma1 chain, could be important for epithelial-mesenchymal interactions in this gland. Nidogen mRNA was detected by in situ hybridization in the mesenchyme, and yet the protein was detected in epithelial and endothelial basement membranes. The role of nidogen-laminin interactions for epithelial morphogenesis was studied by applying antibodies to submandibular gland organ cultures. Antibodies reacting strongly with the nidogen-binding site of laminin gamma1 chain drastically perturbed branching epithelial morphogenesis. Electron microscopy of the epithelial-mesenchymal interface showed that blocking antibodies disrupted the formation of the basement membrane. Epidermal growth factor was shown to increase the expression of nidogen in mesenchyme, and could counteract the effect of the blocking antibodies. We suggest that nidogen could be an important mesenchymal factor for submandibular gland development.

Animals↗
PubMed1997

Characterization of monoclonal antibodies against tenascin-C: no apparent effect on kidney development in vitro.

Tenascin-C is an extracellular matrix glycoprotein found in embryonic mesenchyme. The precise biological function of tenascin-C is unknown, but different parts of the molecule have effects on cell adhesion and other cellular activities. We studied the expression and role of tenascin-C in the embryonic mouse kidney. By Northern blots, no tenascin-C was detectable in uninduced mesenchyme from day 11 embryonic kidneys, but after 24 hours of in vitro culture both major splice variants of tenascin-C were detected. The larger variant was the predominant form. By in situ hybridization tenascin-C mRNA in 13-day old embryonic kidneys was detected in the mesenchyme surrounding newly formed epithelial structures. In 17-day old embryonic kidneys, tenascin-C mRNA was detected in mesenchyme around the forming epithelial structures in the cortex, and expression was also seen in mesenchyme surrounding the capsular epithelium of glomeruli. In newborn kidneys, expression had shifted to the medulla but was still confined to mesenchymal areas. We have characterized 6 new monoclonal antibodies against mouse tenascin-C, which all stain embryonic kidneys from different stages in a pattern consistent with earlier reports and with the mRNA data. The binding sites of the monoclonal antibodies on the tenascin-C molecule were mapped to discrete regions of tenascin-C. These six and five previously described antibodies against tenascin-C were tested in antibody perturbation experiments. Three of these have been shown by in vitro assays to perturb function of other cell types. Despite this, none of them inhibited development of mouse kidneys in organ culture, although they were tested at 1 mg/ml. It raises the possibility that tenascin-C is not crucial for kidney development. Alternatively, tenascin-C has more subtle functions which could not be identified with the assays used here.

Animals↗
PubMed1995

Regulation of mesenchymal extracellular matrix protein synthesis by transforming growth factor-beta and glucocorticoids in tumor stroma.

We have here studied the composition and regulation of stromal extracellular matrix components in an experimental tumor model. Nude mice were inoculated with WCCS-1 cells, a human Wilms' tumor cell line. In the formed tumors the stroma was found to contain mesenchymal extracellular matrix proteins such as tenascin-C, fibulins-1 and 2 and fibronectin, but no nidogen. Nidogen was confined to basement membranes of tumor blood vessels. Since glucocorticoids have been shown to downregulate tenascin-C expression in vitro, we tested whether dexamethasone can influence biosynthesis of extracellular matrix components during tumor formation in vivo. A downregulation of tenascin-C mRNA and an upregulation of fibronectin mRNA expression by dexamethasone was noted. Transforming growth factor-beta 1 mRNA levels were unaffected by the dexamethasone treatment. Glucocorticoids can thus downregulate tenascin-C synthesis although local stimulatory growth factors are present. The competition between a negative and a positive extrinsic factor on synthesis of stromal extracellular matrix components was studied in a fibroblast/preadipocyte cell line. Transforming growth factor-beta 1 stimulated tenascin-C synthesis but did not affect fibronectin or fibulin-2 synthesis. Dexamethasone at high concentrations could completely suppress the effect of transforming growth factor-beta 1 on tenascin-C mRNA expression. Transforming growth factor-beta 1 could in turn overcome the downregulation of tenascin-C mRNA expression caused by a lower concentration of dexamethasone. We therefore suggest that the limited expression of tenascin-C in part is due to a continuous suppression by physiological levels of glucocorticoids, which can be overcome by local stimulatory growth factors when present in sufficient amounts.

Animals↗