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PubMed2002

Opsonization of apoptotic cells by autologous iC3b facilitates clearance by immature dendritic cells, down-regulates DR and CD86, and up-regulates CC chemokine receptor 7.

Immature dendritic cells (iDCs) do not mature after uptake of apoptotic cells and may play a role in the induction of peripheral tolerance to self antigens derived from apoptotic material. The integrins, alphavbeta3, alphavbeta5, and the scavenger receptor, CD36, have been shown to mediate uptake of apoptotic cells by iDCs. However, it is not known whether the complement system, also takes part in this process. In this study we investigated the ability of iDCs to bind to apoptotic cells opsonized by iC3b. Monocyte-derived dendritic cells were offered apoptotic Jurkat cells opsonized by autologous iC3b and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate. A significant increase (P < 0.001) in the amount of cleared apoptotic cells was seen at low ratios. Despite increased efficiency of uptake, interaction between iC3b-opsonized apoptotic cells and iDCs down-regulated the expression of major histocompatibility complex class II, CD86, CC chemokine receptor (CCR)2, CCR5, and beta2-integrins (P < 0.001), and up-regulated expression of CCR7 (P < 0.001). In addition, iDC maturation responses to CD40L and lipopolysaccharide were significantly inhibited. We conclude that opsonization of apoptotic cells by iC3b induces tolerant iDCs that are able to migrate to lymph nodes.

Antigens, CD↗

PubMed2004

Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells.

BACKGROUND: We have previously demonstrated that human bronchial epithelial cells engulf apoptotic eosinophils. OBJECTIVES: To compare and contrast the phagocytic capabilities of monocyte-derived macrophage and primary airway epithelial cells for apoptotic granulocytes. RESULTS: Here we compared phagocytosis of human apoptotic eosinophils and neutrophils by small and large airway epithelial cells (SAEC and LAEC) and monocyte-derived macrophages. Confocal microscopy of F-actin staining and scanning and transmission electron microscopy revealed phagocytic cup formation around apoptotic eosinophils by airway epithelial cells (AEC) membranes with evidence of their digestion. Resting and cytokine-stimulated AEC did not recognize and ingest apoptotic neutrophils. The latter were phagocytosed by macrophages that exhibited greater ingestion of and higher capacity for, apoptotic eosinophils over apoptotic neutrophils. Cytochalasin D completely abolished uptake of apoptotic eosinophils by SAEC, LAEC or macrophage monolayers. Ligation of epithelial cell CD44 receptors for 24 h increased phagocytosis of apoptotic eosinophils by SAEC and LAEC with a potency comparable with that of IL-1. Phagocytosis was a specific receptor-mediated process involving integrin- (alphavbeta3, alphavbeta5, CD36), phosphatidylserine receptor- and lectin-dependent mechanisms. No significant differences were observed in avarice for apoptotic eosinophils by SAEC or LAEC either resting, CD44 monoclonal antibodies- or cytokine- stimulated, or in their usage and expression of recognition receptors. CONCLUSION: These findings further suggest and define an important role for the bronchial epithelium in the selective removal of apoptotic eosinophils from the airways in asthma.

Apoptosis↗

PubMed1998

Integrins inhibit angiotensin II-induced contraction in rat aortic rings.

Many extracellular matrix proteins contain the tripeptide sequence arginine-glycine-aspartate (RGD). This RGD motif is recognized by integrins, a family of adhesion receptors present on vascular smooth muscle cells. In the present study, we examined the ability of different RGD-containing peptides to affect the contraction of rat aortic rings in response to different agonists. We found that the peptide RGDS inhibited angiotensin-induced contraction in a dose dependent manner. In contrast, the peptides RGDW and RGES had no effect on angiotensin-induced contractility. We show that function-blocking antibodies to the integrins alphavbeta3 and alpha5beta1 also inhibit angiotensin-induced contraction. These effects were observed in the absence of an intact endothelium. In contrast, neither an antibody directed against the beta1 subunit nor the peptide RGDS had an effect on phenylephrine or 5-hydroxytryptamine-induced contraction. These data suggest that interactions of vascular smooth muscle with components of the surrounding extracellular matrix may influence the response of smooth muscle to agonists.

Angiotensin II↗

PubMedDate not supplied

Therapeutic effect of arginine-glycine-aspartic acid peptides in acute renal injury.

1. Previous studies from our laboratory have suggested that arginine-glycine-aspartic acid (RGD) peptides, serving as a decoy, may prevent tubular obstruction in the ischaemic model of acute renal failure. Specifically, we have demonstrated that: (i) stressed tubular epithelial cells reverse the polarity of integrin receptors from the predominantly basolateral location to the apical cell membrane as a part of a more generalized process of the loss of epithelial cell polarity; (ii) depletion of integrins expressed on the basal cell surface leads to the loss of anchorage to the basement membrane and cell desquamation; (iii) expression of integrin receptors on the apical cell membrane leads to indiscriminate interactions (e.g. the adhesion of desquamated cells to the cells remaining in situ), thus initiating the process of tubular obstruction; and (iv) conglomeration of the desquamated cells via integrin receptors further aggravates tubular obstruction. 2. Importantly, these integrin-based interactions can be blocked by synthetic RGD peptides. The linear RGD peptide injected into the renal artery upon release of the renal artery clamp prevented the elevation of proximal tubular hydrostatic pressure characteristically seen in animals with renal ischaemia that received injection of the vehicle of an inactive peptide. 3. In vivo study of RGD peptides in ischaemic acute renal failure in rats demonstrated attenuation of renal injury and accelerated recovery of renal function. 4. Using linear RGD peptide labelled with 99mTc; we have shown that this probe was retained in ischaemic kidneys. 5. To visualize RGD binding sites at the cellular level, we performed a mapping using fluorescent derivatives of two RGD peptides, a cyclic biotinylated (Bt)-RGD peptide and a linear Rhodamine green-labelled (RhoG)-RGD peptide. 6. The findings suggest that the binding sites for RGD peptide are represented by the alphaVbeta3 integrin in the vasculature and some desquamated cells, whereas the majority of the desquamated cells bind Bt-RGD via beta1 integrins. 7. These findings were further tested using cultured endothelial cells co-incubated with leucocytes. When co-incubation experiments were performed in the presence of cyclic RGD pentapeptide, the adhesion of HL-60 cells to both control and hypoxic endothelial monolayers was significantly reduced.

Acute Kidney Injury↗

PubMed2003

Major mutations in calf-1 and calf-2 domains of glycoprotein IIb in patients with Glanzmann thrombasthenia enable GPIIb/IIIa complex formation, but impair its transport from the endoplasmic reticulum to the Golgi apparatus.

The crystal structure of integrin alphavbeta3 comprises 3 regions of contact between alphav and beta3. The main contact on alphav is located in the beta-propeller while calf-1 and calf-2 domains contribute minor interfaces. Whether or not contacts between calf-1 and calf-2 domains of glycoprotein (GP) IIb (alphaIIb) and GPIIIa (beta3) play a role in GPIIb/IIIa complex formation has not been established. In this study we analyzed the effects of 2 naturally occurring mutations in calf-1 and calf-2 domains on GPIIb/IIIa complex formation, its processing, and transport to the cell membrane. The mutations investigated were a deletion-insertion in exon 25 located in calf-2 and an in-frame skipping of exon 20 located in calf-1. Mutated GPIIb cDNAs were cotransfected in baby hamster kidney cells with normal GPIIIa (beta3) cDNA. Analysis by flow cytometry failed to demonstrate detectable amounts of GPIIb or GPIIb/IIIa complex on the surface of cells transfected with each mutation, but immunohistochemical staining revealed their intracellular presence. GPIIb was mainly demonstrable as pro-GPIIb by immunoprecipitation of cell lysates expressing each mutation. Differential immunofluorescence staining of GPIIb and cellular organelles suggested that most altered complexes were located in the endoplasmic reticulum. Homology modeling of normal GPIIb based on the alphavbeta3 crystal structure revealed similar contacts between alphav and beta3 and between alphaIIb and beta3. Introduction of the mutations into the model yielded partial disruption of the normal contacts in the corresponding domains. These data suggest that despite partial disruption of calf-1 or calf-2 domain, GPIIb/IIIa complex is formed but its transport from the endoplasmic reticulum is impaired.

Animals↗

PubMed2004

Effects on rotavirus cell binding and infection of monomeric and polymeric peptides containing alpha2beta1 and alphaxbeta2 integrin ligand sequences.

Integrin-using rotaviruses bind MA104 cell surface alpha2beta1 integrin via the Asp-Gly-Glu (DGE) sequence in virus spike protein VP4 and interact with alphaxbeta2 integrin during cell entry through outer capsid protein VP7. Infection is inhibited by the alpha2beta1 ligand Asp-Gly-Glu-Ala (DGEA) and the alphaxbeta2 ligand Gly-Pro-Arg-Pro (GPRP), and virus-alpha2beta1 binding is increased by alpha2beta1 activation. In this study, we analyzed the effects of monomers and polymers containing DGEA-, GPRP-, and DGEA-related peptides on rotavirus binding and infection in intestinal (Caco-2) and kidney (MA104) cells and virus binding to recombinant alpha2beta1. Blockade of rotavirus-cell binding and infection by peptides and anti-alpha2 antibody showed that Caco-2 cell entry is dependent on virus binding to alpha2beta1 and interaction with alphaxbeta2. At up to 0.5 mM, monomeric DGEA and DGAA inhibited binding to alpha2beta1 and infection. At higher concentrations, DGEA and DGAA showed a reduced ability to inhibit virus-cell binding and infection that depended on virus binding to alpha2beta1 but occurred without alteration in cell surface expression of alpha2, beta2, or alphavbeta3 integrin. This loss of DGEA activity was abolished by genistein treatment and so was dependent on tyrosine kinase signaling. It is proposed that this signaling activated existing cell surface alpha2beta1 to increase virus-cell attachment and entry. Polymeric peptides containing DGEA and GPRP or GPRP only were inhibitory to SA11 infection at approximately 10-fold lower concentrations than peptide monomers. As polymerization can improve peptide inhibition of virus-receptor interactions, this approach could be useful in the development of inhibitors of receptor recognition by other viruses.

Cell Line↗

PubMed2005

Convenient solid-phase synthesis of diethylenetriaminepenta-acetic acid (DTPA)- conjugated cyclic RGD peptide analogues.

Solid-phase synthesis of radiometal chelator-conjugated peptides can facilitate the creation of radioactive peptide libraries to be utilized in high throughput in vivo screening of targeted nuclear-imaging agents. In this study, a new diethylenetriaminepentaacetic acid (DTPA) derivative, 1-(p-succinamidobenzyl)- DTPA penta-t-butyl ester [DTPA(But)(5)-Bz-NH-SA], and its precursor molecule, 1-(p-aminobenzyl)- DTPA penta-t-butyl ester (DTPA(But)(5)-Bz-NH(2)), were applied to the solid-phase synthesis of DTPA-conjugated cyclic peptides containing the Arg-Gly-Asp (RGD) motif with high efficiency. The resulting conjugates, DTPA-Bz-NH-SA-c(Lys-Arg-Gly-Asp-phe) [DTPA-Bz-NH-SA-c(KRGDf)] and DTPA-Bz-NHc( Glu-Arg-Gly-Asp-phe) [DTPA-Bz-NH-c(KRGDf)], demonstrated similar in vitro biologic activities as their corresponding parent peptides. (111)In-labeled, DTPA-conjugated RGD peptides showed selective binding to integrin alphavbeta3 in human melanoma M21 tumors grown in nude mice. Furthermore, (111)In-DTPABz- NH-c(ERGDf) showed lower retention in the liver and the kidney than (111)In-DTPA-Bz-NH-SAc( KRGDf) did, which contributed to higher target to nontarget ratio for (111)In-DTPA-Bz-NH-c(ERGDf). The method reported here can be extended to the construction of peptide libraries containing DTPA for high throughput in vitro and in vivo screening of molecularly targeted imaging agents.

Amino Acid Motifs↗

PubMed2000

Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells.

OBJECTIVE: Based on their differentiation properties and facilely of ex vivo expansion, human bone marrow mesenchymal progenitor cells (MPC), are considered as attractive targets to deliver foreign genes to the bone marrow or other mesenchymal tissues. In this study we investigated the feasibility of transduce MPC with adenoviral vectors (Adv). METHODS: MPC were expanded ex vivo and transduced with replication-defective Adv-containing reporter genes (lacZ or GFP) under the control of CMV promoter. Transfection efficiency was assessed by microscopical scoring or by flow cytometry. Expression and involvement of Adv-attachment (CAR) and Adv-internalization (integrins alphav) receptors were evaluated by flow cytometric studies. RESULTS: Transgene expression analysis showed that only 19%+/-3% of cells expressed the transgenes at high levels. MPC express the attachment and internalization receptors required for Adv infection. While integrins alphavbeta3 and alphavbeta5 are expressed by all MPC, CAR is solely expressed by a fraction of low size cells. Antibodies against CAR and alphavbeta5, but not against alphavbeta3, blocked Adv-mediated gene transfer into MPC, showing that CAR and alphavbeta5 are required for infection. Because alphavbeta5, as compared with CAR, is overexpressed in MPC, the results suggest that the efficiency of Adv-mediated gene transfer into MPC depends on the level of CAR expression. CONCLUSION: These findings demonstrate that Adv may be useful to engineer a subpopulation of ex vivo expanded human mesenchymal progenitors, with a high level of transgene expression.

Adenoviridae↗

PubMed1996

Human microvascular endothelial cells adhere to thrombospondin-1 via an RGD/CSVTCG domain independent mechanism.

Thrombospondin-1 (TSP-1), 450-kDa glycoprotein secreted by platelets and endothelial cells at sites of tissue injury or inflammation, plays an important role in angiogenesis, inflammation, and vascular occlusive skin diseases. Many of the physiologic and pathologic activities of TSP-1 are dependent upon its interactions with endothelial cells. To better understand the basis of these activities, we examined the mechanisms mediating the binding of human dermal microvascular endothelial cells (HDMEC) to immobilized TSP-1. HDMEC bound to but did not spread on TSP-1 in a concentration-dependent manner. Monoclonal antibodies (MoAbs) which recognize two purported TSP-1 binding proteins, CS36 and the alphav integrin chain, or TSP-1-derived peptides CGRGDS and CSVTCG, alone or in combination with heparin, did not inhibit HDMEC adhesion to immobilized TSP-1. Furthermore, CSVTCG-ovalbumin conjugates failed to support HDMEC adhesion. Although RGD-containing peptides immobilized on plastic wells supported HDMEC binding, they also induced cell spreading not characteristic of cell binding to TSP-1 and binding was inhibited by free RGD peptide. Two MoAbs against different domains of TSP-1 (A 4.1 and C 6.1) failed to block HDMEC binding to TSP-1, but both MoAbs inhibited G361 human melanoma cell binding to TSP-1 by 60%. Acid treatment of TSP-1 almost completely abrogated its ability to support HDMEC binding, while acid treatment inhibited G361 binding by 50%. However, either antibody completely abrogated G-361 cell binding to acid-treated TSP-1. These data demonstrate that HDMEC bind to immobilized TSP-1 in an RGD- and CSVTCG-independent manner via an acid labile epitope(s) which recognized via a receptor or receptors distinct from CD36 or alphavbeta3 integrin receptor.

Amino Acid Sequence↗

PubMed2000

Analysis of the alpha4beta1 integrin-osteopontin interaction.

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.

Amino Acid Sequence↗

PubMed2002

The human melanoma associated protein melanotransferrin promotes endothelial cell migration and angiogenesis in vivo.

Melanotransferrin is a member of the transferrin family, which is comprised of serum transferrin, lactoferrin and ovotransferrin, and is highly expressed on melanoma cells compared to normal melanocytes. Since melanoma is an highly vascularized tumour that expresses melanotransferrin at high levels, we tested purified recombinant melanotransferrin for its capability to induce angiogenesis in the chick chorioallantoic membrane. Macroscopic and microscopic evaluation of the vascular density demonstrated that melanotransferrin exerts an angiogenic response quantitatively similar to that elicited by fibroblast growth factor-2. Overexpression of vascular endothelial growth factor-receptor-2 was observed in newly formed vessels, suggesting that the angiogenic activity of melanotransferrin may depend on activation of endogenous vascular endothelial growth factor. In addition, when antibodies against vascular endothelial growth factor were included in the assay, the angiogenic response was inhibited by 50%. In a Boyden chamber assay purified recombinant melanotransferrin induced chemotactic migration of vascular cells, which was decreased in the the presence of anti-vascular endothelial growth factor antibodies suggesting an involvement of vascular endothelial growth factor present in endothelial cells also in this assay. However, melanotransferrin was found not to directly bind to integrin alphavbeta3 or the vascular endothelial growth factor-receptor-2 as assessed in a BlAcore assay. A possible correlation between vascularization occurring during melanoma progression and the expression of melanotransferrin and vascular endothelial growth factor was established by immunolocalization of the two factors in sections of melanoma at different clinical steps of melanoma progression. These latter data strongly imply that melanotransferrin may participate in the vascularization of solid tumours and that inhibition of melanotransferrin could form the basis for intervention in tumours which use this pathway.

Adult↗

PubMed1998

Role of beta1 and beta3 integrins in human smooth muscle cell adhesion to and contraction of fibrin clots in vitro.

The degree of lumen narrowing in advanced lesions correlates poorly with the amount of intimal mass accumulated in the atherosclerotic plaque. As an alternate mechanism of stenosis, we propose that human smooth muscle cells bind to fibrin deposited in the matrix and exert contractile forces to cause a narrowing of the lumen. In the present study we demonstrated in vitro that human newborn aortic smooth muscle cell lines can contract and adhere to fibrin clots composed of either fibronectin-depleted plasma ("plasma") or recombinant fibrin. By using neutralizing antibodies and RGD peptides, we showed that members of the integrin family mediated the interaction between human newborn smooth muscle cells and fibrin. Neutralizing antibodies against the integrin alphavbeta3 (c7E3 Fab and LM609) did not inhibit either plasma clot contraction or recombinant fibrin clot contraction by human newborn smooth muscle cells. In contrast, antibodies against alpha5, beta1, and alpha5/beta1 inhibited contraction of clots composed of either plasma or recombinant fibrin. Anti-alphavbeta3, anti-alphav, anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies inhibited human newborn smooth muscle cell adhesion to plasma clots; however, only anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies significantly inhibited adhesion to recombinant fibrin. While the linear RGD peptides had no effect, the cyclic peptide penRGD inhibited adhesion to plasma clots and recombinant fibrin. However, it did not block contraction of recombinant fibrin clots. These results suggest that during the interaction of human newborn smooth muscle cell lines with fibrin, alpha5beta1 plays a significant role. This interaction is of potential interest as a target for efforts to block vascular contraction.

Antibodies↗

PubMed2004

Interaction of human fibrinogen receptor (GPIIb-IIIa) with decorsin.

AIM: To build up the structure of human fibrinogen receptor GPIIb-IIIa, subsequently combined with its antagonist decorsin, and to investigate the interaction between decorsin and its receptor GPIIb-IIIa at the molecular level. METHODS: A three-dimensional (3D) molecular model of human fibrinogen receptor GPIIb-IIIa was generated by InsightII, a distance geometry-based homologous modeling package. The structure of human fibrinogen receptor GPIIb-IIIa was built by the InsightII/Homology module using the corresponding of integrin alphaVbeta3 (PDB filecode 1JV2) as the template. Then the primary structures were optimized by energy minimization. Subsequently the structural model was docked with its antagonist decorsin (PDB filecode 1dec). RESULTS: A good substrate-receptor interaction model was achieved. The interaction sites with decorsin converge at domain 8 (betaA domain of beta3 subunit) of GPIIb-IIIa. The direct interatomic contacts were made between 16 GPIIb/IIIa residues and 10 decorsin amino-acid residues. These included van der Waals contacts, electrostatic interaction, hydrogen bond, and salt bridge. Residues in contact were concentrated in four dispersed regions of human GPIIb-IIIa: the RGD reaction motif (118-132 of GPIIIa), the span from 210 to 213 of GPIIIa, Thr182 residue and Asp251 residue of GPIIIa; and they were distributed over five segments of decorsin: Asp10 residue, Asn18 and Lys19 residues, Arg28 residue, RGD motif, and Asp35-Pro36-Tyr37 segment. CONCLUSION: This complex model plays an important role in development and research of some new drugs, especially a new guided fusion-type fibrinogen receptor antagonist.

Amino Acid Sequence↗

PubMed2000

Identification of a stromal cell type characterized by the secretion of a soluble integrin-binding protein, MFG-E8, in mouse early gonadogenesis.

Mfge8 (milk fat globule-EGF-factor 8) encodes a soluble integrin-binding protein containing two Notch-like EGF domains and two discoidin domains. It mediates cell-to-cell interaction by binding to integrin alphavbeta3 via the RGD motif of its second EGF domain. Mfge8 was first expressed at 10.0 dpc in cells of the coelomic epithelium covering the mesonephros, and at 10.5 dpc Mfge8-expressing cells were found in the mesenchyme underneath the coelomic epithelium of the genital ridges. At 11.5-12.5 dpc, Mfge8 expressing cells were found in the stromal tissues subjacent to the coelomic epithelium that envelop the fetal gonad of both sexes. MFG-E8 protein was accumulated extracellularly in the interstitial tissues at the boundary of the mesonephros and the genital ridges. A comparison of the expression domains of Mfge8 and several gene markers showed that Mfge8 expression did not significantly overlap with the expression domain of Wt1 or Emx2, but partially with that of Lhx9 in 11.5-day XY gonads. Comparison of the expression pattern of Mfge8 with that of Hsd3beta1 in the 12.5-day testes revealed that the Mfge8-positive cells constitute a previously uncharacterized somatic cell type which is distinct from Sertoli cells, Leydig cells, peritubular myoid cells and the endothelial cells.

Animals↗

PubMed2003

Coordinate expression of fibulin-5/DANCE and elastin during lung injury repair.

Fibulin-5, previously known as DANCE and EVEC, is a secreted extracellular matrix protein that functions as a scaffold for elastin fiber assembly and as a ligand for integrins alphavbeta3, alphavbeta5, and alpha9beta1. Fibulin-5 is developmentally regulated in the lung, and lung air space enlargement develops in mice deficient in fibulin-5. Fibulin-5 is also induced in adult lung following lung injury by hyperoxia. To further examine the role of fibulin-5 during repair of lung injury, we assessed fibulin-5 expression during elastase-induced emphysema in C57/b mice. Mice were treated with either saline or elastase via the trachea, and the lung was examined 20 days after treatment. Fibulin-5 mRNA was induced almost fourfold, whereas elastin mRNA was minimally elevated. Immunohistochemistry studies showed that fibulin-5 was induced in cells within the alveolar wall following elastase treatment. Western analysis demonstrates that fibulin-5 was strongly expressed in isolated primary lung interstitial fibroblasts. Fibulin-5 protein was localized to the fibroblast cell layer in culture, and brief elastase treatment degraded the protein. Intact fibulin-5 did not accumulate in the culture media. Treatment of fibroblasts with the proinflammatory cytokine interleukin-1beta abolished fibulin-5 mRNA expression. Our results indicate that fibulin-5 is coordinately expressed and regulated with elastin in lung fibroblasts and may serve a key role during lung injury and repair.

Amino Acid Sequence↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed1998

Activation of MMP-2 by human GCT23 giant cell tumour cells induced by osteopontin, bone sialoprotein and GRGDSP peptides is RGD and cell shape change dependent.

We show that osteopontin (OPN), bone sialoprotein (BSP) and GRGDSP peptides, in solution, induce activation of metalloproteinase-2 (MMP-2) secreted by human GCT23 giant cell tumour cells. Activation of MMP-2 is RGD sequence dependent, possibly involves anti-alphaVbeta3 integrins, is preceded by a change from spread to rounded cell morphology and is mimicked by the actin depolymerising agent cytochalasin B. Cells that had spread on OPN, BSP and GRGDSP substrata failed to activate MMP-2, but subsequent addition of soluble GRGDSP induced rounding and MMP-2 activation. Activation induced by GRGDSP and cytochalasin B was cell mediated, inhibited by EDTA, tissue inhibitor of metalloproteinase-2 (TIMP-2) and carboxyl terminal MMP-2 consistent with a role for membrane type (MT)-MMP but did not involve urokinase, plasmin or thrombin activity. Activation induced by GRGDSP and cytochalasin B, but not cell rounding, was inhibited by herbimycin A, cycloheximide and actinomycin D, suggesting a role for tyrosine kinases, protein and RNA synthesis, but was not associated with changes in mRNA for MT-MMP-1, MMP-1, MMP-2, TIMP-1 or TIMP-2. GRGDSP and cytochalasin B enhanced levels of membrane-associated pro- and active form MMP-1 and MMP-2 but not MT-MMP-1, stimulated cell surface MMP-1 staining and induced that of MT-MMP-1, MMP-2 and TIMP-2. This was consistent with the possible relocation of constitutive MT-MMP-1 to the cell surface as a prerequisite for subsequent cell surface MMP-2/TIMP-2/MT-MMP-1 complex formation and to the potential induction of conditions favourable for reciprocal cell surface MMP-1/MMP-2 activation. Our data provide a novel insight into interactions between RGD containing bone matrices, GCT cells and MMPs of potential relevance to GCT pathology.

Carcinoma, Giant Cell↗

PubMed1998

Quantitative assessment of leading edge adhesion: reattachment kinetics modulated by injury-derived intracellular calcium predict wound closure rates in endothelial monolayers.

Migrating cells continually develop new substrate attachments at the leading edge (LE) in order to maintain traction for movement. This study evaluates the relationship between LE adhesion and wound closure by modulating injury-derived intracellular free Ca2+ ([Ca2+]i) signaling in endothelial cell (EC) monolayers following scrape-wounding. These data show that brief treatment with increased extracellular Ca2+ ([Ca2+]e) during wounding accelerated wound area closure rates by 50-65%, while brief treatments with calcium influx inhibitors reduced rates by 30-50%. Fura-2 studies in wounded monolayers indicated supranormal [Ca2+]e during wounding increased (by 52%), while influx-inhibitors decreased (by 36%) the percentage of cells exhibiting elevated plateau [Ca2+]i levels. Quantitative time-lapse interference reflection microscopy (IRM) together with indirect alphavbeta3 integrin immunofluorescence was used to measure the effects of 100 microM Gd3+ and 5 mM [Ca2+]e treatment on fractional LE adhesion after wounding. Influx inhibition blocked development of increased injury-derived LE adhesion. Measurements indicated a linear relationship (r2 = 0.99, 0.98) between LE adhesion, development rates (quantified as an association rate constant) and steady state wound closure rates. Changes in filopodial activity, as indicated by phase contrast microscopy, did not correlate with changes in wound closure rates, but an association existed between the percentile peak [Ca2+]i response and the initiation of filopodial activity, suggesting a role for filopodia in mediating Ca2+-sensitive acceleration. Taken together, our data suggest that injury-derived [Ca2+]i signaling may regulate wound closure rates by an adhesion-mediated mechanism.

Animals↗