Biomedical subjects

D T Woodley

Publications and source records attributed to D T Woodley.

At least 19 recordsLinked to original sources

PubMed2001

Nck/Dock: an adapter between cell surface receptors and the actin cytoskeleton.

In response to extracellular signals, cell surface receptors engage in connections with multiple intracellular signaling pathways, leading to the cellular responses such as survival, migration, proliferation and differentiation. The 'pY-->SH2/SH3-->effector' connection is a frequently used scheme by many cell surface receptors, in which SH2/SH3-containing adapters connect protein tyrosine phosphorylation to a variety of downstream effector pathways. Following the initial landmark finding that Grb2 adapter links the receptors to the Ras pathway leading to DNA synthesis, recent studies have revealed that the biological function of the SH2/SH3 adapter Nck/Dock is to link cell surface receptors to the actin cytoskeleton. For example, in the evolutionarily-conserved signaling network, GEF-Rac-Nck-Pak, Nck 'fixes up' the interaction of Pak with its upstream activator, Rac. The activated Pak then regulates the cytoskeletal dynamics. The fact that the majority of the more than 20 Nck-SH3-associated effectors are regulators of the actin cytoskeleton suggests that Nck/Dock regulates, via binding to distinct effectors, various cell type-specific motogenic responses. This review focuses on our current understanding of Nck/Dock function. Due to the number and complexity of the terminologies used in this review, a 'Glossary of Terms' is provided to help reduce confusions.

Actins↗

PubMed2001

The carboxyl terminus of type VII collagen mediates antiparallel dimer formation and constitutes a new antigenic epitope for epidermolysis Bullosa acquisita autoantibodies.

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC2 domain has been implicated in catalyzing the antiparallel dimer formation of type VII procollagen. In this study, we produced the entire 161 amino acids of the NC2 domain plus 186 amino acids of adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of collagen VII. Removal of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesis, we found that mutation of either cysteine 2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel dimer formation. In contrast, a single cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is associated with recessive dystrophic epidermolysis bullosa, was unable to form antiparallel dimers. Furthermore, autoantibodies from epidermolysis bullosa acquisita patients also reacted with NC2/COL. We conclude that NC2 and its adjacent collagenous segment mediate antiparallel dimer formation of collagen VII. Epidermolysis bullosa acquisita autoantibodies bound to this domain may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence.

Amino Acid Substitution↗

PubMed2001

Phosphorylation of focal adhesion kinase (pp125(FAK)) is increased in human keratinocytes induced to migrate by extracellular matrices.

During the healing process of skin wounds, human keratinocytes migrate across a provisional matrix of the wound bed. The mechanisms by which keratinocytes migrate on connective tissue are not known. In this study, we examined the role of focal adhesion kinase (FAK), an 125 kDa protein that co-localizes with focal adhesions in cells plated on extracellular matrix. We induced human keratinocytes into various states of migration by plating them on extracellular matrices that minimally, moderately, or strongly induce cellular migration, and then examined the expression of FAK at the protein level and its degree of tyrosine phosphorylation using Western immunoblotting and immunoprecipitation. In highly migratory human keratinocytes, we found that three proteins were predominantly tyrosine phosphorylated, one of them being FAK. Tyrosine phosphorylation of FAK tightly correlated with the level of cellular motility but not cell attachment to the matrix. Time course experiments demonstrated that in highly motile keratinocytes, tyrosine phosphorylation of FAK peaked at 12 h, the time when maximal migration on the matrix ensues. In contrast to FAK, the beta1 integrin subunit of human keratinocytes that configures with the alpha2, alpha3, and alpha5 integrin subunits to form integrin receptors for matrix, did not display tyrosine phosphorylation linked to motility. Using anti-sense oligonucleotides to FAK, we demonstrate that FAK is required for human keratinocyte migration, but not for focal adhesion formation.

Cell Movement↗

PubMed2000

Prokaryotic expression, purification, and reconstitution of biological activities (Antiprotease, antitumor, and heparin-binding) for tissue factor pathway inhibitor-2.

We report the expression of tissue factor pathway inhibitor-2 (TFPI-2) (also known as PP-5, placental protein-5; MSPI, matrix-associated serine protease inhibitor) in E. coli as a 25-kDa nonglycosylated protein with a glycine substituted for aspartic acid at the amino terminus. High-level expression of TFPI-2 was obtained with pRE1 expression vector under the transcriptional and translational controls of the lambdaP(L) promoter and lambdacII ribosome-binding site, respectively, with ATG initiation codon. TFPI-2 was produced as inclusion bodies and accounted for 25-30% of the total E. coli proteins. The inclusion bodies containing TFPI-2 were solubilized with urea, sulfitolyzed, purified, and refolded through a disulfide interchange reaction. The refolded E. coli TFPI-2 inhibited plasmin with an inhibition constant (K(i)) of 5 nM that is similar with the TFPI-2 expressed in a mammalian system. The refolded E. coli TFPI-2 bound heparin and also inhibited plasmin, regardless of whether the enzyme was in the fluid phase or was bound to the membranes of HT-1080 fibrosarcoma cells. In addition, refolded E. coli TFPI-2 inhibited radiolabeled matrix degradation and Matrigel matrix invasion by HT-1080 fibrosarcoma cells and B16-F10 melanoma cells. Together, our results suggest that glycosylation is not essential for antiprotease, antitumor, and matrix-binding activities of TFPI-2. Based on these collective data, we conclude that a biologically active nonglycosylated TFPI-2 can be produced in E. coli and that the protein can be produced in high-enough quantities to conduct in vivo studies for determination of the role of this inhibitor in tumor invasion and metastasis.

Amino Acid Substitution↗

PubMed2000

Development and characterization of a recombinant truncated type VII collagen "minigene". Implication for gene therapy of dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder of skin caused by mutations in the type VII collagen gene. The lack of therapy for DEB provides an impetus to develop gene therapy strategies. However, the full-length 9-kilobase type VII collagen cDNA exceeds the cloning capacity of current viral delivery vectors. In this study, we produced a recombinant type VII minicollagen containing the intact noncollagenous domains, NC1 and NC2, and part of the central collagenous domain using stably transfected human 293 cell clones and purified large quantities of the recombinant minicollagen VII from culture media. Minicollagen VII was secreted as correctly-folded, disulfide-bonded, helical trimers resistant to protease degradation. Purified minicollagen VII bound to fibronectin, laminin-5, type I collagen, and type IV collagen. Furthermore, retroviral-mediated transduction of the minigene construct into DEB keratinocytes (in which type VII collagen was absent) resulted in persistent synthesis and secretion of a 230-kDa recombinant minicollagen VII. In comparison with parent DEB keratinocytes, the gene-corrected DEB keratinocytes demonstrated enhanced cell-substratum adhesion, increased proliferative potential, and reduced cell motility, features that reversed the DEB phenotype toward normal. We conclude that the use of the minicollagen VII may provide a strategy to correct the cellular manifestations of gene defects in DEB.

Cell Line↗

PubMed2000

Autoantibodies to type VII collagen have heterogeneous subclass and light chain compositions and their complement-activating capacities do not correlate with the inflammatory clinical phenotype.

Epidermolysis bullosa acquisita and bullous systemic lupus erythematosus are blistering skin diseases characterized by IgG autoantibodies that predominantly target the noncollagenous domain 1 of type VII collagen, a skin basement membrane component. The basic immunologic events leading to the blistering processes in these diseases remains unclear. We defined the subclass and light chain compositions of the IgG autoantibodies in 15 patients, in order to gain insight into the blistering mechanism. Immunofluorescence correlated the patients' in vivo-bound and circulating antibasement membrane autoantibodies. Four eukaryotic recombinant proteins, including one full-length and three truncated noncollagenous domain 1 proteins generated by sequential deletion of C-terminal amino acids, were used to perform enzyme-linked immunosorbent assay to detect the patients' anti-type VII collagen autoantibodies. The majority of patients' autoantibodies contained both complement-activating and non-complement-activating IgG subclasses. The presence or absence of complement-activating IgG autoantibody subclasses did not correlate with the inflammatory or noninflammatory clinical phenotype. The majority of tested sera contained both kappa and lambda light chain autoantibodies. All sera that reacted to the full-length noncollagenous domain 1 also reacted to the smallest truncated protein containing the cartilage matrix protein and the first three fibronectinlike repeats. The patients' anti-type VII collagen autoantibodies, likely to be polyclonal in nature, may contribute to the pathogenesis of the blistering process by both complement-dependent inflammatory injury and complement-independent mechanical disruption of the anchoring function of type VII collagen. The N-terminal region of the noncollagenous domain 1 may contain an important antigenic epitope targeted by the IgG autoantibodies.

Autoantibodies↗

PubMed2000

Induction of keratinocyte IL-8 expression and secretion by IgG autoantibodies as a novel mechanism of epidermal neutrophil recruitment in a pemphigus variant.

A subset of pemphigus herpetiformis, a rare pemphigus variant, is characterized histopathologically by subcorneal acantholysis and neutrophilic infiltration. The mechanism of neutrophil infiltration is unknown, but chemokines such as IL-8 may play a role. We investigated the possible role of IL-8 in two such cases. Direct and indirect immunofluorescence studies demonstrated in vivo-bound and circulating IgG epithelial cell surface-binding autoantibodies, both predominated by IgG4 subclass. ELISA and immunoblotting studies revealed that the patients' IgG autoantibodies recognized recombinant desmoglein 1 but not desmoglein 3. Preadsorption of the patients' sera with recombinant desmoglein 1 completely removed the epidermal cell surface immunostaining. Significantly, immunohistochemistry demonstrated intense expression of IL-8, co-localized with in vivo-bound IgG, in the upper epidermis, where the acantholysis took place. Affinity-purified sera IgG from these two patients, a normal individual, and a pemphigus vulgaris patient containing desmoglein 1 autoantibodies, were incubated with normal human keratinocytes in vitro. Cells treated with these patients' IgG secreted a seven-to-nine-fold increase of IL-8 (30-37 pg/ml) compared with the controls (2-4 pg/ml) and expressed a higher intensity of cytoplasmic IL-8 staining. These data demonstrate a novel functional role for IL-8 in the pathogenesis of the neutrophil-dominant subset of pemphigus herpetiformis. The autoantibody-induced epidermal cell IL-8 expression may represent a novel mechanism of epidermal neutrophil recruitment.

Adult↗

PubMed2000

Colony-stimulating factor-1 receptor utilizes multiple signaling pathways to induce cyclin D2 expression.

Colony-stimulating factor-1 (CSF-1) induces expression of immediate early gene, such as c-myc and c-fos and delayed early genes such as D-type cyclins (D1 and D2), whose products play essential roles in the G1 to S phase transition of the cell cycle. Little is known, however, about the cytoplasmic signal transduction pathways that connect the surface CSF-1 receptor to these genes in the nucleus. We have investigated the signaling mechanism of CSF-1-induced D2 expression. Analyses of CSF-1 receptor autophosphorylation mutants show that, although certain individual mutation has a partial inhibitory effect, only multiple combined mutations completely block induction of D2 in response to CSF-1. We report that at least three parallel pathways, the Src pathway, the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and the c-myc pathway, are involved. Induction of D2 is partially inhibited in Src(-/-) bone marrow-derived macrophages and by Src inhibitor PP1 and is enhanced in v-Src-overexpressing cells. Activation of myc's transactivating activity selectively induces D2 but not D1. Blockade of c-myc expression partially blocks CSF-1-induced D2 expression. Complete inhibition of the MEK/ERK pathway causes 50% decrease of D2 expression. Finally, simultaneous inhibition of Src, MEK activation, and c-myc expression additively blocks CSF-1-induced D2 expression. This study indicates that multiple signaling pathways are involved in full induction of a single gene, and this finding may also apply broadly to other growth factor-inducible genes.

Animals↗

PubMed2000

Nckbeta adapter regulates actin polymerization in NIH 3T3 fibroblasts in response to platelet-derived growth factor bb.

The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckalpha (Nck) and Nckbeta (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckbeta in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckbeta but not Nckalpha blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckbeta abolishes its interfering effect. Nckbeta binds at Tyr-1009 in human PDGF receptor beta (PDGFR-beta) which is different from Nckalpha's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckbeta but not an anti-Nckalpha antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckbeta but not Nckalpha blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckbeta acts in parallel to or downstream of Rac1. This is the first report of Nckbeta's role in receptor tyrosine kinase signaling to the actin cytoskeleton.

3T3 Cells↗

PubMed1999

Matrix localization of tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor (TFPI-2/MSPI) involves arginine-mediated ionic interactions with heparin and dermatan sulfate: heparin accelerates the activity of TFPI-2/MSPI toward plasmin.

Human tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a Kunitz-type serine protease inhibitor, inhibits plasmin, trypsin, chymotrypsin, plasma kallikrein, cathepsin G, and factor VIIa-tissue factor complex. The mature protein has a molecular mass of 32-33 kDa, but exists in vivo as two smaller, underglycosylated species of 31 and 27 kDa. TFPI-2/MSPI triplet is synthesized and secreted by a variety of cell types that include epithelial, endothelial, and mesenchymal cells. Because the majority (75-90%) of TFPI-2/MSPI is associated with the extracellular matrix (ECM), we examined which components of the ECM bind TFPI-2/MSPI. We found that TFPI-2/MSPI bound specifically to heparin and dermatan sulfate. Interaction of these two glycosaminoglycans (GAGs) with TFPI-2/MSPI involved one or more common protein domains, as evidenced by cross-competition experiments. However, binding affinity for TFPI-2/MSPI with heparin was 250-300 times greater than that for TFPI-2/MSPI with dermatan sulfate. Binding of TFPI-2/MSPI to GAGs was inhibited by NaCl or arginine but not by glucose, mannose, galactose, 6-aminohexanoic acid, or urea, suggesting that arginine-mediated ionic interactions participate in the GAG binding of TFPI-2/MSPI. This supposition was supported by the observation that only NaCl or arginine could elute the TFPI-2/MSPI protein triplet from an ECM derived from human dermal fibroblasts. Reduced TFPI-2/MSPI did not bind to heparin, suggesting that proper disulfide pairings and conformation are essential for matrix binding. To determine whether heparin modulates the activity of TFPI-2/MSPI, we determined the rate of inhibition of plasmin by the inhibitor with and without heparin and found that TFPI-2/MSPI is more active in the presence of heparin. Collectively, our results demonstrate that conformation-dependent arginine-mediated ionic interactions are responsible for the TFPI-2/MSPI triplet binding to fibroblast ECM, heparin, and dermatan sulfate and that heparin augmented the rate of inhibition of plasmin by TFPI-2/MSPI.

Arginine↗

PubMed1999

Alpha 2 beta 1 integrin mediates dermal fibroblast attachment to type VII collagen via a 158-amino-acid segment of the NC1 domain.

Dermal fibroblasts are in apposition to type VII (anchoring fibril) collagen in both unwounded and wounded skin. The NC1 domain of type VII collagen contains multiple submodules with homology to known adhesive molecules, including fibronectin type III-like repeats and a potential RGD cell attachment site. We previously reported the structure and matrix binding properties of authentic and recombinant NC1. In this study, we examined the interaction between dermal fibroblasts and the NC1 domain of type VII collagen. We found that both recombinant and authentic NC1 vigorously promoted human fibroblast attachment. Adhesion of fibroblasts to NC1 was dose dependent, saturable, and abolished by both polyclonal and monoclonal antibodies to NC1. Cell adhesion to NC1 was divalent cation dependent and specifically inhibited by a monoclonal antibody directed against the alpha2 or beta1 integrin subunits, but not by the presence of RGD peptides. Furthermore, the cell-binding activity of NC1 was not conformation dependent, since heat-denatured NC1 still promoted cell adhesion. Using a series of recombinant NC1 deletion mutant proteins, the cell binding site of NC1 was mapped to a 158-aa (residues 202-360) subdomain. We conclude that human dermal fibroblasts interact with the NC1 domain of type VII collagen and this cell-matrix interaction is mediated by the alpha2beta1 integrin and is RGD independent.

Binding Sites↗

PubMed1999

Bullous systemic lupus erythematosus with autoantibodies recognizing multiple skin basement membrane components, bullous pemphigoid antigen 1, laminin-5, laminin-6, and type VII collagen.

BACKGROUND: Bullous systemic lupus erythematosus is a generalized subepidermal blistering skin eruption in patients suffering from systemic lupus erythematosus. Type VII collagen was initially identified as the target antigen. OBSERVATION: We studied an unusual patient who had bullous systemic lupus crythematosus. The patient fulfilled the criteria of systemic lupus with an antinuclear antibody titer of 1:5120. Immunopathological testing revealed in vivo deposition of all IgG subclasses, secretory IgA1, and both light chains at the patient's skin basement membrane. The in vivo-bound IgG and IgA were localized at the hemidesmosomes and lamina densa. The patient's IgG and IgA circulating autoantibodies labeled both the epidermal roof and the dermal floor of salt-split skin and recognized the hemidesmosomal protein BP230 as well as the full-length native form and the recombinant noncollagenous domain 1 of type VII collagen (anchoring fibril). In addition, the patient's IgG autoantibodies recognized the anchoring filament proteins laminin-5 and laminin-6 (alpha3 chain and gamma2 chain). CONCLUSIONS: We conclude that patients with bullous systemic lupus erythematosus may have autoantibodies to multiple basement membrane components critical for epidermal-dermal junctional adhesion. Possible pathogenic mechanisms in this patient's clinical diseases include provocation of organ-specific disease (bullous disease) by systemic autoimmunity (lupus) and the "epitope spreading" immune phenomenon.

Adolescent↗

PubMed1999

NC1 domain of type VII collagen binds to the beta3 chain of laminin 5 via a unique subdomain within the fibronectin-like repeats.

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous, globular domains, NC1 and NC2. Approximately 50% of the molecular mass of the molecule is consumed by the NC1 domain. We previously demonstrated that NC1 binds to various extracellular matrix components including a complex of laminin 5 and laminin 6 (Chen et al, 1997a). In this study, we examined the interaction of NC1 with laminin 5 (a component of anchoring filaments). Both authentic and purified recombinant NC1 bound to human and rat laminin 5 as measured by enzyme-linked immunosorbant assay and by binding of 125I-radiolabeled NC1 to laminin 5-coated wells, but not to laminin 1 or albumin. NC1 bound predominantly to the beta3 chain of laminin 5, but also to the gamma2 chain when examined by a protein overlay assay. The binding of 125I-NC1 to laminin 5 was inhibited by a 50-fold excess of unlabeled NC1 or de-glycosylated NC1, as well as a polyclonal antibody to laminin 5 or a monoclonal antibody to the beta3 chain. In contrast, the NC1-laminin 5 interaction was not affected by a monoclonal antibody to the alpha3 chain. Using NC1 deletion mutant recombinant proteins, a 285 AA (residues 760-1045) subdomain of NC1 was identified as the binding site for laminin 5. IgG from an epidermolysis bullosa acquisita serum containing autoantibodies to epitopes within NC1 that colocalized with the laminin 5 binding site inhibited the binding of NC1 to laminin 5. Thus, perturbation of the NC1-laminin 5 interaction may contribute to the pathogenesis of epidermolysis bullosa acquisita.

Animals↗

PubMed1999

Thalidomide increases human keratinocyte migration and proliferation.

Thalidomide is reported to have therapeutic utility in the treatment of pyoderma gangrenosum, Behçet's disease, aphthous ulcers, and skin wounds. We investigated the effect of thalidomide on human keratinocyte proliferation and migration, two early and critical events in the re-epithelialization of skin wounds. Thalidomide at concentrations less than 1 microM did not affect keratinocyte viability. Using a thymidine incorporation assay, we found that thalidomide, at therapeutic concentrations, induced more than a 2. 5-fold increase in the proliferative potential of the cells. Keratinocyte migration was assessed by two independent motility assays: a colloidal gold assay and an in vitro scratch assay. At optimal concentrations, thalidomide increased keratinocyte migration on a collagen matrix more than 2-fold in the colloidal gold assay and more than 3-fold in the scratch assay over control. Although pro-migratory, thalidomide did not alter the level of metalloproteinase-9 secreted into culture medium. Thalidomide did, however, induce a 2-4-fold increase in keratinocyte-derived interleukin-8, a pro-migratory cellular autocrine factor. Human keratinocyte migration and proliferation are essential for re-epithelialization of skin wounds. Interleukin-8 increases human keratinocyte migration and proliferation and is chemotactic for keratinocytes. Therefore, thalidomide may modulate keratinocyte proliferation and motility by a chemokine-dependent pathway.

Cell Division↗

PubMed1998

Molecular cloning and characterization of a cDNA encoding canine type VII collagen non-collagenous (NC1) domain, the target antigen of autoimmune disease epidermolysis bullosa acquisita (EBA).

Type VII collagen, the major component of anchoring fibrils, serves as tight adhesion of skin basement membrane zone (BMZ) through its amino-terminal non-collagenous (NC1) domain. The NC1 domain is targeted by autoantibodies of an acquired blistering skin disease termed epidermolysis bullosa acquisita (EBA) naturally occurring in humans and dogs. We cloned the full-length canine type VII collagen NC1 domain cDNA and delineated its molecular and immunological characteristics. The canine NC1 domain cDNA consists of 3759 nucleotides encoding for 1253 amino acids, with a molecular mass of approx. 134 kDa and a 17 amino acid signal peptide. The expression of canine type VII collagen was confirmed by Northern blot analysis and by a rabbit antibody raised against a 17 amino acid peptide deduced from canine NC1 sequence. Comparison of canine NC1 with the corresponding human sequence indicated 86.7% and 87.6% identity at the nucleotide and deduced amino acid levels respectively. The protein homology reached greater than 95% within two immunodominant epitope areas. Furthermore, human EBA autoantibodies and a rabbit anti-human NC1 cross-reacted with canine skin BMZ and the newly synthesized canine type VII collagen. The molecular and immunological identities between human and canine NC1 domains suggest that NC1 may be critical for the EBA development.

Amino Acid Sequence↗

PubMed1998

HT-1080 fibrosarcoma cell matrix degradation and invasion are inhibited by the matrix-associated serine protease inhibitor TFPI-2/33 kDa MSPI.

The urokinase-urokinase receptor system plays a dominant role in the degradation and invasion of extracellular matrix (ECM) by tumor cells. In this system, urokinase bound to its cell receptor converts plasminogen to plasmin, a broad-spectrum serine protease that participates in the degradation and invasion of connective tissues by tumor cells. In this study, we evaluated whether these activities of plasmin are inhibited by a newly characterized human 32 kDa recombinant serine protease inhibitor called trypsin/tissue factor pathway inhibitor-2 (rTFPI-2). We found that rTFPI-2 dose-dependently inhibited fluid-phase plasmin as well as plasmin generated on the ECM and/or the cell surface of HT-1080 fibrosarcoma cells. The degradation of radiolabeled matrix as well as Matrigel invasion by these tumor cells is also inhibited by rTFPI-2 in a dose-dependent fashion. We have reported that rTFPI-2 is identical to 33 kDa extracellular matrix-associated serine protease inhibitor (33 kDa MSPI), whereas the 31 and 27 kDa MSPIs are under-glycosylated forms of the 33 kDa MSPI. We therefore evaluated the ability of MSPIs from the ECM of dermal fibroblasts to inhibit plasmin and found that the plasmin activity was effectively blocked by the MSPIs. We have also evaluated whether the HT-1080 cells synthesize and secrete the MSPIs and found that the synthesis and secretion of the MSPIs was undetectable in these cells. Collectively, our results suggest that rTFPI-2/33 kDa MSPI inhibits plasmin on the tumor cell and ECM surfaces as well as the degradation and invasion of matrix by HT-1080 fibrosarcoma cells.

Antineoplastic Agents↗

PubMed1998

Integrin alpha 5 beta 1 expression is required for inhibition of keratinocyte migration by ganglioside GT1b.

Polysialoganglioside GT1b, a keratinocyte membrane glycosphingolipid, inhibits normal keratinocyte adhesion and migration on a fibronectin matrix. The specificity of the inhibition for cells plated on a fibronectin matrix and competition of GT1b inhibition with peptide RGDS suggest that GT1b abrogates the alpha 5 beta 1/fibronectin interaction. We examined the effects of GT1b on the adhesion and migration of keratinocyte-derived cell lines and correlated GT1b responsiveness and alpha 5 beta 1 integrin expression. GT1b (5 nM) significantly inhibited migration of normal human keratinocytes, immortalized keratinocytes, and squamous cell carcinoma SCC12F2 cells on fibronectin, but not on collagen I. Concentrations as high as 5 microM had no effect on SCC13 or HaCaT cells. Likewise, GT1b inhibited fibronectin-dependent cell adhesion of normal human keratinocytes, immortalized keratinocytes, and SCC12F2 cells, but had no effect on SCC13 or HaCaT cells. Flow cytometric and Western immunoblot analysis of integrin expression showed significantly decreased alpha 5 and beta 1 integrin expression in SCC13 and HaCaT cells compared to normal keratinocytes, immortalized keratinocytes, and SCC12F2 cells. Incubation with TGF-beta 1 increased alpha 5 beta 1 integrin expression and induced responsiveness to GT1b in HaCaT cells. These data imply that GT1b "response" requires sufficient expression of alpha 5 beta 1 and further suggest that the mechanism of the inhibitory effect of GT1b involves GT1b/alpha 5 beta 1 interaction.

Carcinoma, Squamous Cell↗

PubMed1998

Epitope spreading: lessons from autoimmune skin diseases.

Autoimmune diseases are initiated when patients develop aberrant T and/or B cell responses against self proteins. These responses presumably are directed to single immunogenic epitopes on these proteins. Recent data in animal models of autoimmune diseases suggest that the targets of immune responses in autoimmunity do not remain fixed, but can be extended to include other epitopes on the same protein or other proteins in the same tissue, a phenomenon termed "epitope spreading." The "epitope spreading" phenomenon also applies to situations in which tissue damage from a primary inflammatory process causes the release and exposure of a previously "sequestered" antigen, leading to a secondary autoimmune response against the newly released antigen. In experimental autoimmune animal diseases, "epitope spreading" seems to have significant physiologic importance in determining the course and duration of disease. In this paper, we review the current concepts in animal models of autoimmune diseases in order to define the "epitope spreading" phenomenon, and we then propose how this phenomenon might play a significant role in the development and the course of autoimmune skin diseases. Hopefully, an understanding of "epitope spreading" will help the dermatology community to better understand the pathogenesis of autoimmune skin diseases and to rationally fashion disease-specific immune therapy in the future.

Animals↗