Biomedical subjects

D F Mosher

Publications and source records attributed to D F Mosher.

At least 37 records · Page 2Linked to original sources

PubMed2000

The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation.

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit beta1 (Y783 and Y795) to phenylalanines markedly reduces the capability of beta1A integrins to mediate directed cell migration. In this study, beta1-dependent cell spreading was found to be delayed in GD25 cells expressing beta1A(Y783/795F) compared to that in wild-type GD25-beta1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to beta1-dependent adhesion in GD25-beta1A(Y783/795F) cells compared to that in wild-type GD25-beta1A or mutants in which only a single tyrosine was altered (beta1A(Y783F) or beta1A(Y795F)). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via beta1A(Y783/795F) lies at the level of the initial autophosphorylation step. Indeed, beta1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the beta1A(Y783/795F) cells, consistent with the impairment in FAK activation. In contrast, p130(CAS) overall tyrosine phosphorylation was unaffected by the beta1 mutations. Despite the defect in beta1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-beta1A(Y783/795F) cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit beta1A are critical mediators of FAK activation and cell spreading in GD25 cells.

Amino Acid Substitution↗

PubMed2000

Sphingosine-1-phosphate and lysophosphatidic acid stimulate endothelial cell migration.

Endothelial cell migration is necessary for the formation of new blood vessels. We investigated the effects of 2 lysophospholipid mediators, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), on endothelial cell migration. S1P and LPA stimulated migration of fetal bovine heart endothelial cells (FBHEs) in a 3D-modified Boyden chamber assay with concentrations as low as 15 nmol/L stimulating a 2-fold change and concentrations in the 1- to 2-micromol/L range stimulating 14- to 20-fold changes. S1P specifically stimulated the migration of several endothelial cell strains but did not stimulate the migration of tumor cells or smooth muscle cells. LPA stimulated some endothelial and nonendothelial cell types to migrate. For FBHEs, S1P and LPA were mostly chemokinetic in checkerboard assays. S1P and LPA stimulated extracellular signal-regulated kinase 1/2 phosphorylation and enhanced paxillin localization to focal contacts, with no discernible change in the actin cytoskeleton in FBHEs. To characterize responsible receptor-dependent signaling pathways, we investigated the involvement of G(i), Rho, and phosphoinositide 3-OH kinase in S1P- and LPA-stimulated migration. Although perturbation of all 3 signaling molecules resulted in decreased migration, the mechanisms underlying the decreased migration were different. Pertussis toxin treatment, to target G(i), caused endothelial cells to develop dense bundles of F-actin and distribute paxillin staining to the cell periphery in response to S1P or LPA. Modification of Rho with C3 toxin disrupted the actin cytoskeleton. Inhibition of phosphoinositide 3-OH kinase decreased S1P- or LPA-induced endothelial cell migration with only minor disruption of the actin cytoskeleton. Inhibition of extracellular signal-regulated kinase kinase with PD98059 caused a loss of phosphorylation of extracellular signal-regulated kinase 1/2, similar to pertussis toxin, but only a minimal decrease in migration. These results indicate that S1P and, for some cells, LPA stimulate migration of endothelial cells through a mechanism that likely requires a balance between G(i) and Rho signaling to achieve the cytoskeletal remodeling necessary for cell migration.

Actins↗

PubMed2000

CD14(+) cells are necessary for increased survival of eosinophils in response to lipopolysaccharide.

There has been considerable interest in the effect that gram-negative bacterial endotoxin (lipopolysaccharide [LPS]) can have in asthma, given that inhalation of LPS has been shown to cause bronchial hyperresponsiveness. Further, there is evidence that the endotoxin-binding protein CD14 may be a marker for asthma. Inhaled LPS has been shown to cause an influx of eosinophils into the nasal airway and to increase the survival of CD16-negatively selected eosinophils in vitro. In this study, we compared survival of eosinophils isolated via CD16-negative selection with eosinophils that were isolated using both CD16- and CD14-negative selection criteria. Survival of CD16-negatively selected eosinophils was enhanced by LPS in a dose-dependent manner and was inhibited by the endotoxin antagonists polymyxin B or lipid X. In contrast, depletion of CD14(+) cells within the eosinophil preparations (CD14/CD16-negatively selected eosinophils) decreased the effect of LPS on survival. Preincubation of CD16-negatively selected eosinophils with antibody 60bd, which blocks LPS binding to CD14, prevented the survival-enhancing effect of LPS. However, CD14 was not detected on eosinophils by flow cytometry, even after incubation with LPS for up to 24 h. These results suggest that the survival-enhancing effect of LPS on eosinophils requires the presence of CD14(+) cells in the population. It is our hypothesis that enhanced eosinophil survival with LPS involves the contribution of another cell type.

Animals↗

PubMed2000

Contraction of collagen matrices mediated by alpha2beta1A and alpha(v)beta3 integrins.

The (beta)1-null fibroblastic cell line GD25 and its derivatives were studied to gain an understanding of the roles of (beta)1 and (beta)3 integrins in the initial (1-hour) contraction of collagen gels. Stable transfectants of GD25 cells expressing the (beta)1A splice variant of (beta)1 ((beta)1A-GD25) did not express (alpha)2(beta)1A and did not adhere to collagen. After transfection of (alpha)2 into (beta)1A-GD25 cells, the (alpha)2(beta)1A-GD25 transfectants contracted collagen gels in the presence of serum, whereas (beta)1A-GD25 cells did not. The GD25 parental cells, however, also contracted collagen gels. Collagen gel contraction by GD25 cells was blocked by antibodies to (alpha)v(beta)3 or a RGD-containing peptide, indicating that (alpha)v(beta)3 is the integrin responsible for mediation of contraction by GD25 cells. Collagen gel contraction by (alpha)2(beta)1A-GD25 cells was not inhibited by antibodies to (alpha)v(beta)3 or RGD-containing peptide, but was inhibited by anti-(alpha)2 antibody. Flow cytometry demonstrated negligible expression of (alpha)v(beta)3 by (beta)1A-GD25 and (alpha)2(beta)1A-GD25 cells when compared to GD25 cells. Platelet derived growth factor (PDGF) and sphingosine-1-phosphate (S1P) enabled gel contraction by (alpha)2(beta)1A-GD25 and GD25 cells, respectively, in the absence of serum. PDGF-stimulated contraction by (alpha)2(beta)1A-GD25 cells was attenuated in the presence of inhibitors of phosphatidylinositol-3-kinase whereas such inhibitors had no effect on S1P-stimulated contraction by GD25 cells. These experiments using the (beta)1-null GD25 cells and (beta)1A and (alpha)2(beta)1A transfectants demonstrate that (alpha)2(beta)1A and (alpha)v(beta)3 independently mediate collagen gel contraction and are regulated by different serum factors and signaling pathways.

Cell Line↗

PubMed1999

Engagement of alpha4beta7 integrins by monoclonal antibodies or ligands enhances survival of human eosinophils in vitro.

Asthma is characterized by an airway inflammatory infiltrate that is rich in eosinophilic leukocytes. Cellular fibronectin and VCAM-1, ligands for alpha4 integrins, are enriched in the fluid of airways of allergic patients subjected to Ag challenge. We therefore hypothesized that ligands of alpha4 integrins can promote eosinophil survival independent of cell adhesion. Cellular fibronectin and VCAM-1 increased viability of human peripheral blood eosinophil in a dose- and time-dependant manner whether the ligand was coated on the culture well or added to the medium at the beginning of the assay. Eosinophils cultured with cellular fibronectin were not adherent to the bottom of culture wells after 3 days. Treatment with mAb Fib 30 to beta7, but not mAb P4C10 or TS2/16 to beta1, increased eosinophil survival. The increased survival of eosinophils incubated with Fib 30 was blocked by Fab fragments of another anti-beta7 mAb, Fib 504. Eosinophils incubated with soluble cellular fibronectin or mAb Fib 30 for 6 h demonstrated a higher level of GM-CSF mRNA than eosinophils incubated with medium alone. Addition of neutralizing mAb to GM-CSF during incubation, but not mAbs to IL-3 or IL-5, reduced the enhancement of eosinophil survival by soluble cellular fibronectin or mAb Fib 30 to control levels. Thus, viability of eosinophils incubated with cellular fibronectin or VCAM-1 is due to engagement, probably followed by cross-linking, of alpha4beta7 by soluble ligand (or mAb) that stimulates autocrine production of GM-CSF and promotes eosinophil survival.

Antibodies, Monoclonal↗

PubMed1999

Synergism among lysophosphatidic acid, beta1A integrins, and epidermal growth factor or platelet-derived growth factor in mediation of cell migration.

GD25 cells lacking the beta1 integrin subunit or expressing beta1A with certain cytoplasmic mutations have poor directed cell migration to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), ligands of receptor tyrosine kinases, or to lysophosphatidic acid (LPA), a ligand of G-protein-coupled receptors (Sakai, T., Zhang, Q., Fässler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538 and Sakai, T., Peyruchaud, O., Fässler, R., and Mosher, D. F. (1998) J. Biol. Chem. 273, 19378-19382). We demonstrate here that LPA synergizes with signals induced by beta1A integrins and ligated EGF or PDGF receptors to modulate migration. When LPA was mixed with EGF or PDGF, migration was greater than with EGF or PDGF alone. The enhancement was greater for beta1A-expressing cells than for beta1-null cells. Cells expressing beta1A with mutations of prolines or tyrosines in conserved cytoplasmic NPXY motifs had blunted migratory responses to mixtures of LPA and EGF or PDGF. The major effects on beta1A-expressing cells of LPA when combined with EGF or PDGF were to sensitize cells so that maximal responses were obtained with >10-fold lower concentrations of growth factor and increase the chemokinetic component of migration. Sensitization by LPA was lost when cells were preincubated with pertussis toxin or C3 exotransferase. There was no evidence for transactivation or sensitization of receptors for EGF or PDGF by LPA. EGF or PDGF and LPA caused activation of mitogen-activated protein kinase by pertussis toxin-insensitive and -sensitive pathways respectively, but activation was not additive. These findings indicate that signaling pathways initiated by the cytoplasmic domains of ligated beta1A integrins and tyrosine kinase receptors interact with signaling pathways initiated by LPA to facilitate directed cell migration.

ADP Ribose Transferases↗

PubMed1999

Sphingosine 1-phosphate stimulates fibronectin matrix assembly through a Rho-dependent signal pathway.

Fibronectin matrix assembly is a cell-dependent process mediated by cell surface binding sites for the 70-kD N-terminal portion of fibronectin. We have shown that Rho-dependent cytoskeleton reorganization induced by lysophosphatidic acid (LPA) or the microtubule-disrupting agent nocodazole increases fibronectin binding (Zhang et al, Mol Biol Cell 8:1415, 1997). Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in mitogenesis and cytoskeletal remodelling. Both LPA and S1P are present in increased amounts in serum as compared with plasma as a result of platelet activation. Addition of S1P to human osteosarcoma MG63 cells or human foreskin fibroblasts increased cell-mediated binding and assembly of fibronectin. MG63 cells expressed the Edg-2 and Edg-4 G-protein-coupled receptors for bioactive lipids, whereas foreskin fibroblasts expressed Edg-2, Edg-3, and Edg-4. The stimulatory effect of S1P on the binding of fibronectin or the N-terminal 70-kD fragment of fibronectin was dynamic and due to increases in both the number and affinity of binding sites. The stimulation of 70-kD fragment binding by nanomolar S1P, like stimulation of binding by LPA or nocodazole, was blocked by inactivation of Rho with C3 exotoxin but not by pertussis toxin-mediated inactivation of Gi. These results indicate a common signal pathway leading to control of cellular fibronectin matrix assembly by bioactive lipids generated during blood coagulation.

Binding Sites↗

PubMed1999

Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers.

The assembly of focal adhesions and actin stress fibers by cells plated on fibronectin depends on adhesion-mediated signals involving both integrins and cell-surface heparan sulfate proteoglycans. These two cell-surface receptors interact with different domains of fibronectin. To attempt to identify the heparan sulfate proteoglycans involved, we used fibronectin-null (FN-/-) mouse fibroblasts to eliminate the contribution of endogenous fibronectin during the analysis. FN-/- fibroblasts plated on the cell-binding domain of fibronectin or on antibodies directed against mouse beta1 integrin chains attach but fail to spread and do not form focal adhesions or actin stress fibers. When such cells are treated with antibodies directed against the ectodomain of mouse syndecan-4, they spread fully and assemble focal adhesions and actin stress fibers indistinguishable from those seen in cells plated on intact fibronectin. These results identify syndecan-4 as a heparan sulfate proteoglycan involved in the assembly process. The antibody-stimulated assembly of focal adhesions and actin stress fibers in cells plated on the cell-binding domain of fibronectin can be blocked with C3 exotransferase, an inhibitor of the small GTP-binding protein Rho. Treatment of cells with lysophosphatidic acid, which activates Rho, results in full spreading and assembly of focal adhesions and actin stress fibers in fibroblasts plated on the cell-binding domain of fibronectin. We conclude that syndecan-4 and integrins can act cooperatively in generating signals for cell spreading and for the assembly of focal adhesions and actin stress fibers. We conclude further that these joint signals are regulated in a Rho-dependent manner.

Actin Cytoskeleton↗

PubMed1999

Dominant effects of the bcr-abl oncogene on Drosophila morphogenesis.

We targeted expression of human/fly chimeric Bcr-Abl proteins to the developing central nervous system (CNS) and eye imaginal disc of Drosophila melanogaster. Neural expression of human/fly chimeric P210 Bcr-Abl or P185 Bcr-Abl rescued abl mutant flies from pupal lethality, indicating that P210 and P185 Bcr-Abl can substitute functionally for Drosophila Abl during axonogenesis. However, increased levels of neurally expressed P210 or P185 Bcr-Abl but not Drosophila Abl produced CNS defects and lethality. Expression of P210 or P185 in the eye imaginal disc produced a dominant rough eye phenotype that was dependent on dosage of the transgene. Drosophila Enabled, previously identified as a suppressor of the abl mutant phenotype and substrate for Drosophila Abl kinase, had markedly increased phosphotyrosine levels in Bcr-Abl expressing Drosophila, indicating that it is a substrate for Bcr-Abl as well. Drosophila, therefore, is a suitable model system to identify Bcr-Abl interactions important for signal transduction and oncogenesis.

Animals↗

PubMed1999

Functional beta1-integrins release the suppression of fibronectin matrix assembly by vitronectin.

beta1-null GD25 fibroblasts adherent to vitronectin fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin or to assemble fibronectin (Sakai, T., Zhang, Q., Fässler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538). We have made four observations that extend this finding. First, the presence of vitronectin on a substrate that otherwise can support fibronectin assembly has a dominant-negative effect on assembly. Second, the dominant-negative effect is lost when active beta1A is expressed. Third, beta1A containing the extracellular D130A inactivating mutation has a dominant-negative effect on fibronectin assembly. Fourth, beta1-null cells adherent to vitronectin are flat and lack filopodia, whereas beta1-null cells adherent to fibronectin or beta1A-expressing cells adherent to either vitronectin or fibronectin are contracted and exhibit numerous filopodia. These results reveal, therefore, that GD25 cells adherent to vitronectin can only assume a shape suitable for assembly of fibronectin when there is a countervailing signal from functional beta1-integrins.

Amino Acid Sequence↗

PubMed1999

Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin.

Many properties have been assigned to the procollagen and properdin (Type I) modules of thrombospondin-1 (TSP1) based on activities of large proteolytic fragments of TSP1 or peptides containing TSP1-derived sequences. To examine the activities of the modules more exactly, we expressed the first properdin module (P1); the third properdin module (P3); the first and second properdin modules (P12); the first, second, and third properdin modules (P123); and the procollagen module with the first, second, and third properdin modules (CP123) in the GELEX expression vector (GE1) using the baculovirus system. GE1 encodes the pre-pro sequence, the transglutaminase cross-linking site(s), the protease-sensitive site, and the gelatin binding domain from the amino terminus of rat fibronectin. All five recombinant proteins were expressed by insect cells, secreted into the culture medium, and purified by gelatin-agarose affinity chromatography. P123 shared with TSP1 a resistance to trypsin unless reduced and alkylated. P12/GE1, P123/GE1, and CP123/GE1 bound poorly to heparin-agarose except in the absence of sodium chloride, whereas peptides based on P2 are known to bind to heparin in up to 150 mM sodium chloride. In cross-linking experiments employing activated recombinant factor XIII and the transglutaminase cross-linking site in the fibronectin-derived sequence, P12/GE1, P123/GE1, CP123/GE1, and P3/GE1 but not P1/GE1 became incorporated into a fibrin clot more than GE1 alone. Analysis of the complex indicated that cross-linking was to the portion of the fibrin alpha-chain remaining in the D-dimer of plasmin digests. P123 also cross-linked to the Aalpha-chain of unclotted fibrinogen. P123 competed for 125I-TSP1 incorporation into the fibrin clot. P123 did not cross-link to plasminogen, histidine-rich glycoprotein, fibronectin, or plasma globulins other than fibrinogen/fibrin. These results indicate that the properdin modules of TSP1 specifically interact with fibrinogen/fibrin but not with heparin under physiologic conditions.

Animals↗

PubMed1999

Segmental antigen challenge increases fibronectin in bronchoalveolar lavage fluid.

Fibronectin may contribute to asthma pathogenesis by recruitment and activation of inflammatory cells, and by promotion of subepithelial fibrosis. Fibronectin is produced by several types of airway cells, including epithelial cells, fibroblasts, and alveolar macrophages. To test the hypothesis that antigen-induced airway inflammation is associated with increased local generation of fibronectin, segmental bronchoprovocation (SBP) with antigen and saline was performed in 17 atopic patients. Bronchoalveolar lavage (BAL) was performed at 5 min and 48 h after segmental challenge with saline or antigen. Fibronectin concentrations in BAL fluid, measured by enzyme-linked immunosorbent assay (ELISA), increased more than 5-fold 48 h after antigen challenge (65 [47 to 110] versus 407 [240 to 697] ng/ml, median and 25 to 75% interquartiles, p < 0.05). Fibronectin concentrations 48 h after antigen challenge correlated with histamine concentrations 5 min after antigen challenge and numbers of eosinophils, neutrophils, macrophages, and total cells in BAL fluid 48 h after antigen challenge. BAL was more enriched in fibronectin 48 h after challenge than would be predicted solely from increased permeability of plasma proteins. Western blot analysis showed that fibronectin in BAL fluid was largely intact and contained the extra domain-A (ED-A) splice variant of cellular fibronectin, indicative of local production. We conclude that antigen challenge in atopic subjects causes increased production of fibronectin by airway cells and speculate that this response may contribute to airway remodeling in allergic inflammation.

Adult↗

PubMed1999

Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex.

Adrenal Cortex↗

PubMed1998

Restoration of beta1A integrins is required for lysophosphatidic acid-induced migration of beta1-null mouse fibroblastic cells.

Cells lacking the beta1 integrin subunit or expressing beta1A with certain cytoplasmic mutations have poor directed cell migration to platelet-derived growth factor or epidermal growth factor, ligands of receptor tyrosine kinases (Sakai, T., Zhang, Q., Fässler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538). We investigated the effect of expression of beta1A integrins on lysophosphatidic acid (LPA)-induced migration of fibroblastic cells derived from beta1-null mouse embryonic stem cells. These cells expressed edg-2, a G-protein-linked receptor for LPA, as well as the related edg-1 receptor. Cells expressing wild type beta1A demonstrated enhanced cell migration across filters coated with gelatin or adhesive proteins in response to LPA, whereas beta1-deficient cells lacked LPA-induced cell migratory ability. Checkerboard analyses indicated that LPA causes both chemotaxis and chemokinesis of beta1-replete cells. Cells expressing beta1A with mutations of prolines or tyrosines in conserved cytoplasmic NPXY motifs, threonine in the inter-motif sequence, or a critical aspartic acid in the extracellular domain had low migratory responses to LPA. These findings indicate that active beta1A integrin is required for cell migration induced by LPA and that the cytoplasmic domain of ligated beta1A interacts with pathways that are common to both receptor tyrosine kinase and G-protein-linked receptor signaling.

Animals↗

PubMed1998

Modulation of beta1A integrin functions by tyrosine residues in the beta1 cytoplasmic domain.

beta1A integrin subunits with point mutations of the cytoplasmic domain were expressed in fibroblasts derived from beta1-null stem cells. beta1A in which one or both of the tyrosines of the two NPXY motifs (Y783, Y795) were changed to phenylalanines formed active alpha5 beta1 and alpha6 beta1 integrins that mediated cell adhesion and supported assembly of fibronectin. Mutation of the proline in either motif (P781, P793) to an alanine or of a threonine in the inter-motif sequence (T788) to a proline resulted in poorly expressed, inactive beta1A. Y783,795F cells developed numerous fine focal contacts and exhibited motility on a surface. When compared with cells expressing wild-type beta1A or beta1A with the D759A activating mutation of a conserved membrane-proximal aspartate, Y783, 795F cells had impaired ability to transverse filters in chemotaxis assays. Analysis of cells expressing beta1A with single Tyr to Phe substitutions indicated that both Y783 and Y795 are important for directed migration. Actin-containing microfilaments of Y783,795F cells were shorter and more peripheral than microfilaments of cells expressing wild-type beta1A. These results indicate that change of the phenol side chains in the NPXY motifs to phenyl groups (which cannot be phosphorylated) has major effects on the organization of focal contacts and cytoskeleton and on directed cell motility.

Actins↗

PubMed1998

Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers.

We developed a method using a single set of degenerate oligonucleotide primers for amplification of the conserved active site of transglutaminases by reverse transcription-polymerase chain reaction (RT-PCR) and identification of the PCR products by cleavage with diagnostic restriction enzymes. We demonstrate amplification of tissue transglutaminase (TGC), keratinocyte transglutaminase (TGK), prostate transglutaminase (TGP), the a-subunit of factor XIII, and band 4.2 protein from different human cells or tissues. Analysis of normal human keratinocytes revealed expression of a transglutaminase different from the expected and characterized transglutaminase gene products. A full-length cDNA for the novel transglutaminase (TGX) was obtained by anchored PCR. The deduced amino acid sequence encoded a protein with 720 amino acids and a molecular mass of approximately 81 kDa. A comparison of TGX to the other members of the gene family revealed that the domain structure and the residues required for enzymatic activity and Ca2+ binding are conserved and showed an overall sequence identity of about 35%. Two transcripts with an apparent size of 2.2 and 2.8 kilobases were detected with a specific probe for TGX on Northern blots of human foreskin keratinocyte mRNA, indicating the presence of alternatively spliced mRNAs. cDNA sequencing revealed a shorter TGX transcript lacking the sequence homologous to that encoded by exon III of other transglutaminase genes. TGX expression increased severalfold when keratinocyte cultures were induced to differentiate by suspension or growth to postconfluency, suggesting that TGX contributes to the formation of the cornified envelope.

Amino Acid Sequence↗

PubMed1998

Enhanced expression of thrombospondin-1 and hypovascularity in human cholangiocarcinoma.

Cholangiocarcinoma (CCC) is relatively hypovascular, in contrast to hepatocellular carcinoma (HCC), which is often highly vascular. We investigated if the diminished vascularity of CCC is related to altered expression of thrombospondin-1 (TSP-1), an antiangiogenic factor, and/or vascular endothelial growth factor (VEGF), a potent angiogenic factor, comparing the relationships with those of high- and low-vascular HCC. We also investigated the relationship between the mutation of the p53 gene and TSP-1 expression or VEGF expression. Northern blot analysis and immunohistochemical staining were performed on surgically resected human CCC and HCC. The ratios of TSP-1 mRNA level in cancer cells versus adjacent noncancerous cells (T/N ratios) were significantly higher in CCC (n = 11) than in HCC with high vascularity (n = 15). In contrast, T/N ratios of VEGF mRNA level in CCC (n = 11) were comparable with those in HCC with low vascularity (n = 5). In CCC, the cancer cells and fibroblasts were positively stained with anti-TSP-1 antibody. We observed that T/N ratios of VEGF mRNA level, but not those of the TSP-1 mRNA level, were significantly correlated with vascularity in HCC. The relative increase in TSP-1 and the relative decrease in VEGF in tumors compared with normal tissue may underlie the limited angiogenesis of CCC. The p53 gene did not affect the expression of TSP-1 in CCC or VEGF in HCC.

Adult↗

PubMed1998

Restricted localization of thrombospondin-2 protein during mouse embryogenesis: a comparison to thrombospondin-1.

Thrombospondin-1 and -2 (TSP1 and TSP2) are multifunctional, multimodular extracellular matrix proteins encoded by separate genes. We compared the distributions of TSP1 and TSP2 in mouse embryos (day 10 and later) by immunohistochemistry. TSP1 was detected on day 10 in the heart and intestinal epithelium, on day 11 in megakaryocytes, and on day 14 in the lung. TSP2 was not detected until day 14, with strongest staining in mesenchymal condensation that gives rise to cartilage and bone. The distribution of TSP2 was different from but overlapped with the distribution of TSP1. TSP1 was found in cartilage proper with diminished staining around chondrocytes undergoing differentiation and hypertrophy, whereas TSP2 was restricted to the matrix surrounding chondrocytes of the growth zone cartilage. TSP2 and TSP1 were both expressed in centers of intramembranous ossification that form the skull bones, in reticular dermis, on the apical surface of nasal epithelium, in skeletal muscle, and in the sheath surrounding vibrissae. Areas of exclusive staining for TSP2 included the perichondrium surrounding the cartilage of the nasal cavities, developing bone of the lower mandible, and adrenal gland. The distinct localizations of TSP1 and TSP2 indicate that the two proteins have specific functions during mouse embryogenesis.

Animals↗