0000000000038371
AUTHOR
Jyrki Heino
"RKKH" peptides from the snake venom metalloproteinase of Bothrops jararaca bind near the metal ion-dependent adhesion site of the human integrin alpha(2) I-domain.
Integrin alpha(1)beta(1) and alpha(2)beta(1) are the major cellular receptors for collagen, and collagens bind to these integrins at the inserted I-domain in their alpha subunit. We have previously shown that a cyclic peptide derived from the metalloproteinase domain of the snake venom protein jararhagin blocks the collagen-binding function of the alpha(2) I-domain. Here, we have optimized the structure of the peptide and identified the site where the peptide binds to the alpha(2) I-domain. The peptide sequence Arg-Lys-Lys-His is critical for recognition by the I-domain, and five negatively charged residues surrounding the "metal ion-dependent adhesion site" (MIDAS) of the I-domain, when mu…
RGD motifs on the surface of baculovirus enhance transduction of human lung carcinoma cells.
Baculovirus vectors have been shown to enter a variety of mammalian cell lines and gene transfer with wild-type baculovirus (WT) has been demonstrated both in vitro and in vivo. Different protein motifs have been displayed on the viral surface to serve as ligands for cell-specific receptor molecules. We have generated recombinant baculovirus vectors displaying an RGD-motif, recognized by alphaV integrin, on the viral surface. The RGD motifs within the C-terminus of coxsackie virus A9 and human parechovirus 1 VP1 proteins were fused to the N-terminus of the major envelope glycoprotein, gp64, of Autographa californica multiple nucleopolyhedrovirus. The recombinant RGD-presenting viruses bound…
Production, crystallization and preliminary X-ray analysis of the human integrin alpha1 I domain.
Integrin α1β1 is one of the main collagen receptors in many cell types. A fast large-scale production, purification and crystallization method for the integrin α1 I domain is reported here. The α1 I domain was crystallized using the vapour-diffusion method with a reservoir solution containing a mixture of PEG 4000, sodium acetate, glycerol and Tris–HCl buffer. The crystals beong to the C2 space group, with unit-cell parameters a = 74.5, b = 81.9, c = 37.3 Å, α = γ = 90.0, β = 90.8°. The crystals diffract to 2.0 Å and a 94.2% complete data set to 2.2 Å has been collected from a single crystal with an R merge of 5.8%.
alpha 11beta 1 integrin recognizes the GFOGER sequence in interstitial collagens.
The integrins alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1) are referred to as a collagen receptor subgroup of the integrin family. Recently, both alpha(1)beta(1) and alpha(2)beta(1) integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O = hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, alpha(11)beta(1), recognizes collagens using C2C12 cells transfected with alpha(11) cDNA and the bacterially expressed recombinant a…
Integrins α5β1, αvβ1, and αvβ6 Collaborate in Squamous Carcinoma Cell Spreading and Migration on Fibronectin
Abstract The expression of αvβ6 fibronectin/tenascin receptor integrin is induced in malignant transformation of oral epithelium. In this study, we demonstrate the contribution of αvβ6 as well as other fibronectin receptor integrins in squamous cell carcinoma (SCC) cell adhesion and migration. Of 11 SCC cell lines isolated from the head and neck area, 8 (73%) expressed αvβ6 integrin on the cell surface. Three cell lines were chosen for further functional experiments: 1 with relatively high, 1 with moderate, and 1 with minimal surface expression of αvβ6 integrin. In addition to αvβ6, all 3 cell lines expressed α5β1 and αvβ1 fibronectin receptor integrins. Function-blocking experiments with i…
An arthritogenic alphavirus uses the α1β1 integrin collagen receptor
Ross River (RR) virus is an alphavirus endemic to Australia and New Guinea and is the aetiological agent of epidemic polyarthritis or RR virus disease. Here we provide evidence that RR virus uses the collagen-binding alpha1beta1 integrin as a cellular receptor. Infection could be inhibited by collagen IV and antibodies specific for the beta1 and alpha1 integrin proteins, and fibroblasts from alpha1-integrin-/- mice were less efficiently infected than wild-type fibroblasts. Soluble alpha1beta1 integrin bound immobilized RR virus, and peptides representing the alpha1beta1 integrin binding-site on collagen IV inhibited virus binding to cells. We speculate that two highly conserved regions with…
Integrin-mediated Cell Adhesion to Type I Collagen Fibrils
In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, α1β1 and α2β1 integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin α2I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin α1I and α2I domain avidity to collagen and to lower …
The Fibril-associated Collagen IX Provides a Novel Mechanism for Cell Adhesion to Cartilaginous Matrix
Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collage…
Clustering induces a lateral redistribution of α2β1 integrin from membrane rafts to caveolae and subsequent protein kinase C-dependent internalization
Integrin alpha 2 beta 1 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1- and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface alpha 2 beta 1 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of alpha 2 beta 1 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally alpha 2 beta 1 integrin was found inside caveolae …
Integrin alpha(2)I domain recognizes type I and type IV collagens by different mechanisms.
The collagens are recognized by the alphaI domains of the collagen receptor integrins. A common structural feature in the collagen-binding alphaI domains is the presence of an extra helix, named helix alphaC. However, its participation in collagen binding has not been shown. Here, we have deleted the helix alphaC in the alpha(2)I domain and tested the function of the resultant recombinant protein (DeltaalphaCalpha(2)I) by using a real-time biosensor. The DeltaalphaCalpha(2)I domain had reduced affinity for type I collagen (430 +/- 90 nM) when compared with wild-type alpha(2)I domain (90 +/- 30 nM), indicating both the importance of helix alphaC in type I collagen binding and that the collag…
The cell adhesion domain of type XVII collagen promotes integrin-mediated cell spreading by a novel mechanism.
Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)…
BioImageXD: an open, general-purpose and high-throughput image-processing platform
BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors.
Depletion of alphaV integrins from osteosarcoma cells by intracellular antibody expression induces bone differentiation marker genes and suppresses gelatinase (MMP-2) synthesis.
Integrin heterodimers sharing the common alphaV subunit are receptors for adhesion glycoproteins such as vitronectin and fibronectin. They are suggested to play an essential role in cell anchoring, differentiation, and survival. Here, we describe the construction of an expression plasmid coding for an intracellular single-chain antibody against alphaV integrin subunit. Saos-2 osteosarcoma cells transfected with this DNA construct showed an approximately 70-100% decrease in the cell surface expression of alphaVbeta3 and alphaVbeta5 integrins as shown by flow cytometry. Intracellular antibody expression had no effect on the mRNA levels of alphaV integrin. Pulse chase experiments of metabolica…
Fluorescent Small Molecule Probe to Modulate and Explore α2β1 Integrin Function
Collagen binding integrins are an important family of cell surface receptors that mediate bidirectionally signals between the interior of the cell and the extracellular matrix. The protein-protein interactions between cells and collagen are necessary for many physiological functions, but also promote diseases. For example, the interaction of α2β1 integrin and collagen has been shown to have an important role in thrombus formation and cancer spread. The fact that the discovery of small molecules that can block such protein-protein interactions is highly challenging has significantly hindered the discovery of pharmaceutical agents to treat these diseases. Here, we present a rationally designe…
Distinct Recognition of Collagen Subtypes by α1β1 and α2β1Integrins
Two integrin-type collagen receptors, α1β1 and α2β1, are structurally very similar. However, cells can concomitantly express the both receptors and they might have independent functions. Here, Chinese hamster ovary (CHO) cells, which lack endogenous collagen receptors, were transfected with either α1 or α2 integrin cDNA. Cells were allowed to adhere to various collagen types and their integrin function was tested by observing the progression of cell spreading. The cells expressing α1β1 integrin could spread on collagen types I, III, IV, and V but not on type II, while α2β1 integrin could mediate cell spreading on collagen types I-V. Type XIII is a transmembrane collagen and its interaction …
Functional display of an alpha2 integrin-specific motif (RKK) on the surface of baculovirus particles.
The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an α2 integrin, the α2I-domain. However, the interaction was not st…
Internalization of Echovirus 1 in Caveolae
ABSTRACT Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor α2β1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, α2β1 integrin, and …
Increased Expression of β6-Integrin in Skin Leads to Spontaneous Development of Chronic Wounds
Integrin alphavbeta6 is an epithelial cell-specific receptor that is not normally expressed by resting epithelium but its expression is induced during wound healing. The function of alphavbeta6-integrin in wound repair is not clear. In the present study, we showed that beta6-integrin expression was strongly up-regulated in the epidermis in human chronic wounds but not in different forms of skin fibrosis. To test whether increased beta6-integrin expression plays a role in abnormal wound healing we developed four homozygous transgenic mouse lines that constitutively expressed human beta6-integrin in the epithelium. The mice developed normally and did not show any histological abnormalities in…
Viral entry, lipid rafts and caveosomes.
Lipid rafts and caveolae are detergent-insoluble plasma membrane microdomains, involved in cellular endocytic processes and signalling. Several viruses, including a human pathogen, echovirus 1, and an extensively studied simian virus 40 utilize these domains for internalization into the host cells. Interaction of viruses with receptors on the cell surface triggers specific conformational changes of the virus particle and can give rise to signalling events, which determine the mechanisms of virus entry. After internalization via cell surface lipid rafts or caveolae, virus-containing vesicles can fuse with caveosomes, pre-existing cytoplasmic organelles, or dock on other intracellular organel…
BioImageXD - Free Microscopy Image Processing Software
Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008
The collagen receptor subfamily of the integrins
The four collagen receptor integrins, alpha1beta1, alpha2beta1, alpha10beta1 and alpha11beta1, form a structurally and functionally distinct subgroup when compared to other members of the integrin family. In this review, we discuss the structures of these receptors and their differences in collagen binding and signalling function.
Endothelial cell-matrix interactions.
Dynamic interactions between endothelial cells and components of their surrounding extracellular matrix are necessary for the invasion, migration, and survival of endothelial cells during angiogenesis. These interactions are mediated by matrix receptors that initiate intracellular signaling cascades in response to binding to specific extracellular matrix molecules. The interactions between endothelial cells and their environment are also modulated by enzymes that degrade different matrix components and thus enable endothelial invasion. Recent reports on gene targeting in mice have confirmed the role of two classes of matrix receptors, integrins and cell surface heparan sulfate proteoglycans…
Novel α2β1 integrin inhibitors reveal that integrin binding to collagen under shear stress conditions does not require receptor preactivation.
The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm(2)). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent ad…
Induction of collagenase-3 (MMP-13) expression in human skin fibroblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase.
Collagenase-3 (matrix metalloproteinase-13, MMP-13) is a recently identified human MMP with an exceptionally wide substrate specificity and restricted tissue-specific expression. Here we show that MMP-13 expression is induced in normal human skin fibroblasts cultured within three-dimensional collagen gel resulting in production and proteolytic activation of MMP-13. Induction of MMP-13 mRNAs by collagen gel was potently inhibited by blocking antibodies against alpha1 and alpha2 integrin subunits and augmented by activating antibody against beta1 integrin subunit, indicating that both alpha1 beta1 and alpha2 beta1 integrins mediate the MMP-13-inducing cellular signal generated by three-dimens…
Integrin α2β1 Mediates Isoform-Specific Activation of p38 and Upregulation of Collagen Gene Transcription by a Mechanism Involving the α2 Cytoplasmic Tail
Two collagen receptors, integrins alpha1beta1 and alpha2beta1, can regulate distinct functions in cells. Ligation of alpha1beta1, unlike alpha2beta1, has been shown to result in recruitment of Shc and activation of the Ras/ERK pathway. To identify the downstream signaling molecules activated by alpha2beta1 integrin, we have overexpressed wild-type alpha2, or chimeric alpha2 subunit with alpha1 integrin cytoplasmic domain in human osteosarcoma cells (Saos-2) lacking endogenous alpha2beta1. The chimeric alpha2/alpha1 chain formed a functional heterodimer with beta1. In contrast to alpha2/alpha1 chimera, forced expression of alpha2 integrin resulted in upregulation of alpha1 (I) collagen gene …
Transcription of α2 Integrin Gene in Osteosarcoma Cells Is Enhanced by Tumor Promoters
Integrin alpha2beta1 is a heterodimeric transmembrane receptor for collagens. In osteogenic cells the expression of alpha2beta1 integrin is induced by both Kirsten sarcoma virus and chemical transformation. The association of alpha2 integrin with transformed cell phenotype was studied further by testing the effects of two tumor promoters, 12-O-tetradecanoylphorbol 13-acetate (TPA) and okadaic acid (OA), on human MG-63 osteosarcoma cells. TPA, an activator of protein kinase C, increased the cell surface expression of alpha2 integrin and the corresponding mRNA levels. Nuclear run-on assays indicated that TPA activated the transcription of alpha2 integrin gene. TPA also slightly increased the …
Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1▿
ABSTRACT Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. …
Jararhagin-derived RKKH Peptides Induce Structural Changes in α1I Domain of Human Integrin α1β1
Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides,…
Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1.
Integrins are cell surface receptors for several microbial pathogens including echovirus 1 (EV1), a picornavirus. Cryo-electron microscopy revealed that the functional domain (alpha(2)I) of human alpha(2)beta(1) integrin binds to a surface depression on the EV1 capsid. This three-dimensional structure of EV1 bound to alpha(2)I domain provides the first structural details of an integrin interacting with a picornavirus. The model indicates that alpha(2)beta(1) integrin cannot simultaneously bind both EV1 and the physiological ligand collagen. Compared with collagen binding to the alpha(2)I domain, the virus binds with a 10-fold higher affinity but in vitro uncoating of EV1 was not observed as…
A Raft-derived, Pak1-regulated Entry Participates in α2β1 Integrin-dependent Sorting to Caveosomes
We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered alpha2beta1 integrin does not enter…
A peptide inhibiting the collagen binding function of integrin alpha2I domain.
Integrin alpha2 subunit forms in the complex with the beta1 subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special "inserted" domain (I domain) in the integrin alpha2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet alpha2beta1 integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant ralpha2I domain to type I collagen led us to study jararhagin further. Synthetic peptides represe…
Molecular mechanism of α2β1 integrin interaction with human echovirus 1
Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did …
The collagen receptor integrins have distinct ligand recognition and signaling functions
Distinct collagen subtypes are recognized by specific cell surface receptors. Two of the best known collagen receptors are members of the integrin family and are named alpha1beta1 and alpha2beta1. Integrin alpha1beta1 is abundant on smooth muscle cells, whereas the alpha2beta1 integrin is the major collagen receptor on epithelial cells and platelets. Many cell types, such as fibroblasts, osteoblasts, chondrocytes, endothelial cells, and lymphocytes may concomitantly express both of the receptors. We have studied the cell biology of these integrins at two levels. First, we have analyzed their ligand binding mechanism and specificity. Second, we have studied their signaling function inside th…
Entry of Human Parechovirus 1
ABSTRACT Human parechovirus 1 (HPEV-1) is a prototype member of parechoviruses, a recently established picornavirus genus. Although there is preliminary evidence that HPEV-1 recognizes α V integrins as cellular receptors, our understanding of early events during HPEV-1 infection is still very limited. The aim of this study was to clarify the entry mechanisms of HPEV-1, including the attachment of the virus onto the host cell surface and subsequent internalization. In blocking experiments with monoclonal antibodies against different receptor candidates, antibodies against α V and β 3 integrin subunits, in particular in combination, appeared to be the most efficient ones in preventing the HPE…
The Selective Regulation of αVβ1 Integrin Expression Is Based on the Hierarchical Formation of αV-containing Heterodimers
The integrin beta1 subunit can form a heterodimer with 12 different alpha subunits. According to the present model, the expression level of any alphabeta complex is regulated by the availability of the specific alpha subunit, whereas beta1 subunit is constantly present in a large excess. The expression of several heterodimers containing the alphaV subunit seems to be regulated by an identical mechanism. The fact that many cells express alphaVbeta1 heterodimer, and that this fibronectin/vitronectin receptor may be selectively regulated, compromises the present model of the regulation of beta1 and alphaV integrins. We have tried to solve this problem by assuming that distinct alphabeta hetero…
Integrin alpha 2 beta 1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3 beta.
The integrins are a large family of heterodimeric transmembrane receptors composed of α and β subunits (22). In addition to mediating cell-matrix interactions, integrins have been shown to activate intracellular signaling pathways which, in collaboration with growth factor-induced signals, regulate cellular functions (46). Some integrin signaling cascades are activated via the β subunit cytoplasmic domain, and they are therefore triggered by several integrin heterodimers. These signals include the activation of protein tyrosine kinases of the Src and focal adhesion kinase (FAK) families (9, 47). More-recent studies have revealed signaling events that are activated specifically by an α subun…
Selective Binding of Collagen Subtypes by Integrin α1I, α2I, and α10I Domains
Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collag…
Collagenase-3 (MMP-13) Enhances Remodeling of Three-Dimensional Collagen and Promotes Survival of Human Skin Fibroblasts
Collagenase-3 (MMP-13) is a matrix metalloproteinase capable of cleaving a multitude of extracellular matrix proteins in addition to fibrillar collagens. Human MMP-13 is expressed by fibroblasts in chronic cutaneous ulcers, but not in normally healing adult skin wounds. However, MMP-13 is produced by fibroblasts in adult gingival and in fetal skin wounds characterized by rapid collagen remodeling and scarless healing. Here, we have examined the role of human MMP-13 in remodeling of three-dimensional (3D) collagenous matrix by primary adult human skin fibroblasts. The high level of human MMP-13 expression by fibroblasts achieved by adenoviral gene delivery resulted in potent enhancement of r…
Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin
Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCa…