Biomedical subjects

S A Corbett

Publications and source records attributed to S A Corbett.

15 recordsLinked to original sources

PubMed2006

Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly.

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D.

Animals↗

PubMed2000

Regulation of fibronectin matrix assembly by activated Ras in transformed cells.

Fibronectin extracellular matrix plays a critical role in the microenvironment of cells. Loss of this matrix frequently accompanies oncogenic transformation, allowing changes in cell growth, morphology, and tissue organization. The HT1080 human fibrosarcoma cell line is deficient in formation of fibronectin matrix fibrils but assembly can be induced by the glucocorticoid dexamethasone. Here we show that fibronectin assembly can also be restored by stimulation of alpha5beta1 integrin with activating antibody or with Mn2+ suggesting that integrin activity is reduced in these cells. While dexamethasone promoted actin stress fiber formation, actin filaments remained cortical following Mn2+ treatment showing that the dexamethasone effect is not due solely to cytoskeletal changes. HT1080 cells have one activated allele of N-ras and PD98059 inhibition of signaling from Ras through ERK increased fibronectin matrix accumulation. Conversely, the p38 MAP kinase inhibitor SB203580 blocked induction of matrix and increased ERK phosphorylation. Thus, two MAP kinase pathways contribute to the control of integrin-mediated fibronectin assembly. ERK activity and fibronectin assembly were linked in three different ras-transformed cell lines but not in SV40- or RSV-transformed cells indicating that oncogenic Ras uses a distinct mechanism to down-regulate cell-fibronectin interactions.

3T3 Cells↗

PubMed1999

Requirements for alpha(5)beta(1) integrin-mediated retraction of fibronectin-fibrin matrices.

Retraction of the blood clot by nucleated cells contributes both to hemostasis and to tissue remodeling. Although plasma fibronectin (FN) is a key component of the clot, its role in clot retraction is unclear. In this report, we demonstrate that the incorporation of FN into fibrin matrices significantly improves clot retraction by nucleated cells expressing the integrin alpha(5)beta(1). Further, we show that FN-fibrin clots support increased cell spreading when compared with fibrin matrices. To determine the structural requirements for FN in this process, recombinant FN monomers deficient in ligand binding or fibrin cross-linking were incorporated into fibrin clots. We show that recombinant FN monomers support clot retraction by Chinese hamster ovary cells expressing the integrin alpha(5)beta(1). This process depends on both the Arg-Gly-Asp (RGD) and the synergy cell-binding sites and on covalent FN-fibrin binding, demonstrating that cross-linking within the clot is important for cell-FN interactions. These data show that alpha(5)beta(1) can bind to FN within a clot to promote clot retraction and support cell shape change. This provides strong evidence that alpha(5)beta(1)-FN interactions may contribute to the cellular events required for wound contraction.

Animals↗

PubMed1999

Beta3 integrin activation improves alphavbeta3-mediated retraction of fibrin matrices.

BACKGROUND: Integrins are heterodimeric transmembrane glycoproteins that mediate cell interactions with the extracellular matrix. In vivo, integrin affinity can be modulated by intracellular signaling events. This can be simulated by a point mutation (D723R) in the cytoplasmic tail of the beta3 integrin subunit which results in constitutive activation. The effects of beta3 integrin activation on the function of alphavbeta3, an integrin which is important to the adhesive events of multiple cell types, were addressed using Chinese hamster ovary cells expressing either the wild-type alphavbeta3 integrin or the mutant alphavbeta3(D723R). The interactions of these cell lines with fibrin matrices were compared. METHODS: Receptor expression levels were confirmed by FACS analyses using a monoclonal anti-alphavbeta3 antibody. Cell attachment to fibrin-coated dishes was determined after 1 h by fixation and crystal violet staining followed by elution of the dye and OD measurement. Fibrin clot retraction was measured by culturing cells in fibrin clots for 24 h. The clots were detached from the dish and the surface area was calculated at individual time points. RESULTS: CHO alphavbeta3(D723R) cells displayed a greater than twofold increase in attachment to fibrinogen or to fibrin matrices when compared to wild-type transfectants. Further, CHO alphavbeta3(D723R) cell retraction of fibrin matrices was significantly greater at nearly all time points. CONCLUSION: Activation of the beta3 integrin subunit significantly improves the interaction of alphavbeta3 with fibrin and may play a role in the integrin-mediated signaling events which occur following vascular injury.

Animals↗

PubMed1999

Nitric oxide mediated vasoreactivity during fracture repair.

An experimental model of fracture healing has been used to investigate whether nitric oxide mediated vascular reactivity, determined using laser Doppler flowmetry, is present in bone after a fracture. Times corresponding to Days 0, 1, 3, 7, 14, and 28 after fracture were used to study the injured and contralateral limbs in response to bolus intravenous administration of nitric oxide inhibitor, N-nitro-L-arginine methyl ester, and nitric oxide stimulator, acetylcholine. N-nitro-L-arginine methyl ester administration (1 mumol/kg, 10 mumol/kg, and 100 mumol/kg) caused a dose dependent increase in systemic blood pressure in each of the assessment groups; however, there was no statistical difference between the groups. Doppler flow readings at the fracture site showed measurable changes in local vascular reactivity after drug administration. At Day 1 after fracture, the magnitude of unit change in vascular reactivity in response to N-nitro-L-arginine methyl ester (1 mumol/kg, 10 mumol/kg, and 100 mumol/kg) was significantly higher in the fractured limb compared with the contralateral limb and also when compared with other points of assessment. These results show that nitric oxide mediated vasoreactivity is present about a fracture site and is maximal in the early healing phase, before returning to basal levels as healing progresses. This is compatible with an initial restoration of blood flow at a fracture site by nitric oxide dependent vasodilation of preexisting blood vessels, followed by ingrowth of less nitric oxide dependent angiogenic vessels during the later phase of repair.

Acetylcholine↗

PubMed1999

Nitric oxide in fracture repair. Differential localisation, expression and activity of nitric oxide synthases.

Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair.

Animals↗

PubMed1998

Modulation of cell-extracellular matrix interactions.

Changes in extracellular matrix (ECM) structure and composition, such as occur during morphogenesis, can have important regulatory effects on cell behavior. Two fibronectin (FN)-based systems have been developed to dissect how cells respond to different types of ECM. One system mimics the provisional matrix of the wound and is composed of FN cross-linked into a fibrin clot matrix. Unlike cells on FN alone, cells on an FN-fibrin matrix are smaller with cortical distribution of actin filaments and membrane ruffles. Addition of the ECM protein tenascin to the FN-fibrin matrix induces a different cell morphology. Thus, matrix composition can have profound effects on cell phenotype. Cells also interact with FN while assembling it into a fibrillar matrix. Using recombinant FNs, a domain that is required for normal progression of FN fibril formation has been identified. During assembly of this recombinant matrix, formation of actin stress fibers and focal adhesions is delayed, demonstrating that changes in FN matrix structure can affect intracellular organization and activation of signaling pathways.

Actins↗

PubMed1998

Dexamethasone up-regulates cadherin expression and cohesion of HT-1080 human fibrosarcoma cells.

The synthetic glucocorticoid dexamethasone markedly decreases the invasiveness of HT-1080 human fibrosarcoma cells. We show here that dexamethasone treatment of HT-1080 cell aggregates more than doubles their cohesivity from 3.9 to 9.7 dyne/cm. Western blot analysis shows a corresponding increase in cadherin expression. This was accompanied by an increase in the rate of calcium-dependent aggregation. Dexamethasone-treated aggregates spread to form a monolayer in Matrigel spreading assays, but the cells remained much more contiguous than their untreated counterparts. Invasion-suppression by dexamethasone may therefore be due, at least in part, to a previously unsuspected increase in cadherin-mediated cohesion.

Antineoplastic Agents, Hormonal↗

PubMed1998

Fibronectin-fibrin cross-linking: a regulator of cell behavior.

At sites of tissue injury or inflammation, extravasation of plasma proteins leads to the formation of a complex fibrillar matrix composed primarily of fibrin and plasma fibronectin (pFN). This protein meshwork serves not only to reestablish the integrity of the vascular system but also to provide a scaffold for cell migration and subsequent wound repair. The interactions between cell surface receptors and this provisional extracellular matrix (ECM) provide important cues that can modulate the cellular response at the injury site, leading to alterations in cell growth and gene expression. Key determinants of this response may lie in the structure and composition of this "injury-associated" ECM.

Journal Article↗

PubMed1998

Nitric oxide in the local host reaction to total hip replacement.

Nitric oxide is emerging as one of the most studied molecules in many aspects of human physiology and pathophysiology. Because of the inflammatory nature of aseptic loosening of total hip replacement, it is likely that nitric oxide plays a major role in this condition as well. Nitric oxide is known to interact with cyclooxygenase enzymes that produce prostanoids. Nitric oxide can stimulate synthesis and activity of the inducible, proinflammatory isoform of the enzyme, namely, cyclooxygenase 2. Interactions between the cytokine inducible nitric oxide synthase and cyclooxygenase 2 pathways serve to regulate bone cell viability such that cyclooxygenase 2 activity can protect against nitric oxide mediated programmed cell death. In the pseudomembrane these two pathways are coactivated in CD68 positive macrophages, fibroblasts, lining cells, and in vascular smooth muscles. Particle generation from wear of the prosthesis has a significant role as an inducer of nitric oxide synthase and cyclooxygenase 2; macrophages laden with small size particles and positive for inducible nitric oxide synthase and cyclooxygenase 2 are a frequent finding. Nitric oxide readily reacts with superoxide to form peroxynitrite, which is a strong oxidant species. In pseudosynovial interface membrane, detection of nitrotyrosine provides evidence for the formation and activity of peroxynitrite. These findings show evidence that nitric oxide, superoxide, and peroxynitrite mediated cellular damage is part of the pathophysiology of aseptic loosening of joint implants. These new findings suggest that antiinflammatory compounds can be useful to treat early aseptic loosening of joint implants.

Animals↗

PubMed1997

Covalent cross-linking of fibronectin to fibrin is required for maximal cell adhesion to a fibronectin-fibrin matrix.

In a blood clot, fibrin and plasma fibronectin (pFN) are covalently cross-linked by activated factor XIII (factor XIIIa) to form pFN-fibrin multimers. To determine the functional significance of covalent pFN-fibrin interactions, we have developed an in vitro model which allows the incorporation of recombinant FN (recFN) molecules into a covalently cross-linked recFN-fibrin matrix. Using the baculovirus expression system, we have expressed recFN monomers composed of the amino-terminal 70-kDa region and the first 11 type III repeats (WT) with mutations in the glutamines at positions 3 and 4 (Q2) or at 3, 4, and 16 (Q3). Examination of the covalent incorporation of these recFNs into fibrin clots confirms that glutamines 3 and 4 are major participants in FN-fibrin cross-linking as the mutation of these sites reduces cross-linking efficiency by 65%. Additional mutation of the glutamine at position 16, however, eliminates >99% of cross-linking suggesting that it also may be factor XIIIa reactive. When the Q3 recFN-fibrin clots were used as substrates for cell adhesion, there was a decrease in both cell attachment and spreading when compared with the WT recFN-fibrin clots. These data demonstrate that for maximal cell attachment to a FN-fibrin clot, FN must be cross-linked to fibrin by factor XIIIa.

3T3 Cells↗

PubMed1997

Modulation of protein tyrosine phosphorylation by the extracellular matrix.

UNLABELLED: Fibronectin (FN) cross-linked to fibrin following injury provides the provisional matrix required for cells to begin tissue repair. Our previous work has demonstrated that fibroblasts adherent to multimeric FN within the context of a fibrin matrix (FN-fibrin) exhibit clear phenotypic differences from those adherent to a dimeric FN-coated surface. We hypothesize that this response to multimeric FN may be mediated by altered protein tyrosine phosphatase activity following integrin activation. METHODS: NIH 3T3 cells were plated in the presence or absence of pervanadate (PV), a phosphotyrosine phosphatase inhibitor, on wells coated with FN or FN-fibrin matrix. Spread cell areas were measured after increasing incubation times and are recorded as mean cell area (mm2) +/- SEM. Alternatively, cells were lysed and equal amounts of protein were analyzed by immunoblot using a monoclonal antibody specific for phosphotyrosine. RESULTS: PV significantly inhibited cell spreading on FN-fibrin matrices. In contrast, PV treatment had little effect on cell area on FN alone. Analysis of cell lysates revealed that protein tyrosine phosphorylation events differ in a substrate-dependent manner. CONCLUSION: Cell attachment to a FN-fibrin matrix induces distinct cell shape and cytoskeletal organization. Inactivation of tyrosine-specific phosphatases enhances this distinction and inhibits the spreading of cells attached to this substrate. The phosphotyrosyl protein content of treated cells on FN-fibrin matrix is also diminished. These results suggest that cell-extracellular matrix interactions affect the tyrosine phosphorylation balance of the cell, thus modifying cytoskeletal organization and related signaling events.

3T3 Cells↗

PubMed1997

Modulatory roles for integrin activation and the synergy site of fibronectin during matrix assembly.

Initiation of fibronectin (FN) matrix assembly is dependent on specific interactions between FN and cell surface integrin receptors. Here, we show that de novo FN matrix assembly exhibits a slow phase during initiation of fibrillogenesis followed by a more rapid growth phase. Mn2+, which acts by enhancing integrin function, increased the rate of FN fibril growth, but only after the initial lag phase. The RGD cell-binding sequence in type III repeat 10 is an absolute requirement for initiation by alpha5beta1 integrin. To investigate the role of the cell-binding synergy site in the adjacent repeat III9, a full-length recombinant FN containing a synergy mutation, FN(syn-), was tested for its ability to form fibrils. Mutation of this site drastically reduced FN assembly by CHOalpha5 cells. Only sparse short fibrils were formed even after prolonged incubation, indicating that FN(syn-) is defective in progression of the assembly process. These results show that the synergy site is essential for alpha5beta1-mediated accumulation of a FN matrix. However, the incorporation of FN(syn-) into fibrils and the deoxycholate-insoluble matrix could be stimulated by Mn2+. Therefore, exogenous activation of integrin receptors can overcome the requirement for FN's synergy site as well as modulate the rate of FN matrix formation.

Animals↗

PubMed1997

Aseptic loosening of total hip replacement. Macrophage expression of inducible nitric oxide synthase and cyclo-oxygenase-2, together with peroxynitrite formation, as a possible mechanism for early prosthesis failure.

Aseptic loosening is a major cause of failure of total hip arthroplasty. The adverse tissue response to prosthetic wear particles, with activation of cytokine and prostanoid production, contributes to bone loss around the implants. We have investigated the possibility that inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) are expressed in macrophages in the pseudomembrane at the bone-implant interface, thereby contributing to the periprosthetic bone resorption. We also assessed whether peroxynitrite, a nitric oxide (NO)-derived oxidant associated with cellular injury, is generated in the membrane. Enzymatic activity of iNOS was measured using the arginine-citrulline assay technique and prostaglandin E2 (PGE2), as an indicator of COX-2 activity, was measured using an enzyme immunoassay. Cellular immunoreactivity for iNOS, nitrotyrosine (a marker of peroxynitrite-induced cellular injury) and COX-2 was assessed by quantitative peroxidase immunocytochemistry while immunofluorescence methods were used for subsequent co-localisation studies with CD68+ macrophages. The presence of calcium-independent iNOS activity and PGE2 production was confirmed in the homogenised interface membrane. Immunocytochemistry showed that periprosthetic CD68+ wear-debris-laden macrophages were the most prominent cell type immunoreactive for iNOS, nitrotyrosine and COX-2. Other periprosthetic inflammatory and resident cell types were also found to immunolocalise nitrotyrosine thereby suggesting peroxynitrite-induced protein nitrosylation and cellular damage not only in NO-producing CD68+ macrophages, but also in their neighbouring cells. These data indicate that both iNOS and COX-2 are expressed by CD68+ macrophages in the interface membrane and peroxynitrite-induced cellular damage is evident in such tissue. If high-output NO and peroxynitrite generation were to cause macrophage cell death, this would result in the release of phagocytosed wear debris into the extracellular matrix. A detrimental cycle of events would then be established with further phagocytosis by newly-recruited inflammatory cells and subsequent NO, peroxynitrite and prostanoid synthesis. Since both NO and PGE2 have been implicated in the induction and maintenance of chronic inflammation with resulting loss of bone, and peroxynitrite in the pathogenesis of disease states, they may be central to the pathogenesis of aseptic loosening.

Adolescent↗

PubMed1996

Changes in cell spreading and cytoskeletal organization are induced by adhesion to a fibronectin-fibrin matrix.

Plasma fibronectin (pFN) cross-linked to fibrin during the injury response provides a provisional matrix required for cells to begin tissue repair. Using a synthetic matrix of pFN and fibrin as a substrate for cell adhesion and spreading, we have determined that pFN covalently cross-linked to fibrin into a complex multimer is functionally distinct from pFN immobilized onto a plastic surface. NIH-3T3 cells on a FN-fibrin matrix reach 50% of the maximal cell area of cells spread on FN-coated plastic. They neither attach nor spread on cross-linked fibrin alone. Cells on pFN-fibrin matrices form few prominent stress fibers and exhibit clear differences in membrane ruffling and filopodial extension when stained with rhodamine-labeled phalloidin. Interestingly, these differences are enhanced by upregulation of protein kinase C. These data suggest that cell-FN interactions can be modified by the molecular context of the protein within the extracellular matrix resulting in distinct cell morphology and cytoskeletal organization.

3T3 Cells↗