Localization of the cellular‐fibronectin‐specific epitope recognized by the monoclonal antibody IST‐9 using fusion proteins expressed in <i>E. coli</i>

Bárbara, Cristina; Borsi, Laura; Zardi, Luciano; Owens, Raymond J.; Baralle, Francisco E.

doi:10.1016/0014-5793(87)80160-6

Cited by 90 publications

(41 citation statements)

References 30 publications

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“…12, September 2001Cells were stained for ␣-SMA (anti-␣SM-1, IgG2a mAb; Skalli et al, 1986), F-actin (Phalloidin-Alexa 488, Molecular Probe, Eugene, OR), ␤-cytoplasmic (␤74, rbAb; Yao et al, 1995), ␣-sarcomeric actin (SR-1, IgM mAb; Dako), ␥-actins and ␣-SMA (AAL-20, rbAb), ED-A FN (IST-9, IgG1 mAb, gift from Dr. L. Zardi, National Institute for Cancer Research, Laboratory of Cell Biology, Genoa, Italy; Carnemolla et al, 1987), VSV-G-tag (anti-VSV-G-tag, rbAB, a gift of Dr. J.-C. Perriard, ETH, Zü rich, CH), the heavy chains (HC) of SMM and NMM (anti-SMMHC and anti-NMMHC, rbAbs; Benzonana et al, 1988) and DNA (DAPI). As secondary antibodies TRITC-and FITC-conjugated goat anti-mouse subclasses IgG1 and IgG2a (Southern Biotechnology Associates Inc., Birmingham, AL), IgG, IgM, and goat anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used.…”

Section: Fibroblast Contractionmentioning

confidence: 99%

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

Hinz

Celetta

Tomasek³

et al. 2001

MBoC

1,135

947

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To evaluate whether ␣-smooth muscle actin (␣-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of ␣-SMA, with that of lung fibroblasts (LFs), expressing high levels of ␣-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of ␣-SMA-positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for ␣-SMA. In a second approach, we measured the isotonic contraction of SCF-and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGF␤1 increased ␣-SMA expression and lattice contraction by SCFs to the levels of LFs; TGF␤-antagonizing agents reduced ␣-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with ␣-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with ␣-cardiac and ␤-or ␥-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased ␣-SMA expression is sufficient to enhance fibroblast contractile activity. INTRODUCTIONEarly during healing of an open wound, resident dermal fibroblasts proliferate from the wound margin and migrate into the provisional matrix composed of a fibrin clot. About 1 week after wounding, the provisional matrix is replaced by neo-formed connective tissue, known as granulation tissue, essentially composed of small vessels, extracellular matrix, and fibroblastic cells that become activated and modulate into myofibroblasts. The main feature of myofibroblasts is represented by an important contractile apparatus similar to that of smooth muscle (Gabbiani et al., 1971), and in particular by the neo-expression of ␣-smooth muscle actin (␣-SMA), the actin isoform typical of vascular smooth muscle cells (Skalli et al., 1986). Myofibroblasts are recognized to play a central role in closing the wound tissue, through their capacity to produce a strong contractile force (for review see Grinnell, 1994;Rønnov-Jessen et al., 1996;Powell et al., 1999;Serini and Gabbiani, 1999), possibly generated within stress fibers, similar to those present in cultured fibroblasts (Skalli et al., 1986;Serini and Gabbiani, 1999). Because wound contraction takes place when de novo expressed ␣-SMA is incorporated in stress fibers (Darby et al., 1990), it has been suggested that this actin isoform plays an important role in granulation tissue contraction (for review see Serini and Gabbiani, 1999). Although stress fibers have been proposed to function as contractile organelles (Burridge, 1981;Katoh et al., 1998) and their presence has been correlated with the production of isometric tension (Harris et al., 1981), at p...

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Section: Fibroblast Contractionmentioning

confidence: 99%

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

Hinz

Celetta

Tomasek³

et al. 2001

MBoC

1,135

947

View full text Add to dashboard Cite

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“…Four polyclonal antibodies to human plasma fibronectin were used in this study: two directed towards the entire molecule (Curtis et al, 1995;Kowalczyk et al, 1990); one to the 27 kDa Fib-1/Hep-1 fragment (P. J. M.-L. and T. S. Panetti, unpublished); and one specific for the 40 kDa gelatin binding fragment (P. J. M.-L. and Panetti, unpublished). Seven fibronectin domain-specific monoclonal antibodies were also used in the study: FDB3 antibody recognizes the VTHPGY sequence within the CS domain of fibronectin (Zheng et al, 1994); FDH2 is directed toward the Hep-2 domain of fibronectin; and IST-9 and 5C11F3 recognize the EDA fibronectin type III repeat (Carnemolla et al, 1987) et al, unpublished); Clone 568 to the eighth type III repeat of fibronectin was from Maine Biotechnology Services (Portland, ME); IST-5 to the fifth fibronectin type III repeat was purchased from Chemicon (Temecula, CA); and Clone 10, purchased from Transduction Laboratories (Lexington, KY), recognizes the second type III repeat in fibronectin (P.J.M.-L. and R.M.K., unpublished). A tetra-His monoclonal antibody (Qiagen, Valencia, CA) was used to monitor recombinant proteins.…”

Section: Reagentsmentioning

confidence: 99%

Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin

Klein

Zheng

Ambesi

et al. 2003

Journal of Cell Science

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The fibronectin matrix contains cryptic sites which are thought to modulate cellular biological responses. One of these sites, located in fibronectin's first type III repeat (III1c), influences signaling pathways that are relevant to cytoskeletal organization and cell cycle progression. The purpose of this study was to identify possible mechanisms responsible for the effects of III1c on cell behavior. Recombinant peptides representing various type III repeats of fibronectin were compared for their effects on fibronectin matrix organization and activation of intracellular signaling pathways. III1c and III13 but not III11c or III10 bound to monolayers of human skin fibroblasts in a dose- and time-dependent manner and were localized to the extracellular matrix. Binding of III13, but not III1c, to matrix was sensitive to heparitinase, suggesting that the association of III1c with the matrix was not dependent on heparan sulfate proteoglycans. Quantitative and morphological assessment indicated that, in contrast to previously published reports, the binding of III1c to cell layers did not result in the loss or disruption of matrix fibronectin. Binding of III1c but not III13 to the extracellular matrix did result in the loss of a conformationally sensitive epitope present within the EDA type III module of cellular fibronectin. III1c-induced loss of the EDA epitope did not require the presence of cells, occurred within 1 hour and was associated with the activation of p38 mitogen-activated protein kinase (MAPK) followed by the formation of filopodia. Maximal phosphorylation of p38 MAPK occurred within 1 hour, whereas cytoskeletal changes did not appear until 12 hours later. These findings are consistent with a model in which the binding of III1c to the extracellular matrix results in a conformational remodeling of the fibronectin matrix, which has both short- and long-term effects on cell physiology.

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“…For the identification of fibronectin, we used an undiluted mouse monoclonal antibody (Ist-9) that recognizes ED-A sequence of fibronectin, the specificity of which has been reported [12,13]. Rabbit affinity-purified antibodies against synthetic peptides corresponding to the intracellular juxtamembrane parts of the receptors were used for the identification of TGF-b type I and II receptors at a concentration of 3 m g/ml [14,15].…”

Section: Immunohistochemical Study Of Fibronectin and Tgf-b Receptorsmentioning

confidence: 99%

Transforming growth factor-β receptor and fibronectin expressions in aortic smooth muscle cells in diabetic rats

et al. 1997

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We have previously reported that cultured aortic smooth muscle cells (SMC) and medial layers of arteries of diabetic rats and rabbits express more platelet-derived growth factor (PDGF) b -receptor than those of non-diabetic animals [1,2]. In addition, diabetic SMC grow much faster than control SMC specifically in reponse to A-B heterodimer and B-B homodimer of PDGF, suggesting that the overexpression of PDGF b -receptor in SMC is a causative element in the accelerated growth of diabetic SMC. In an in vivo study, SMC-dominant intimal thickening was enhanced in diabetic rabbits compared with non-diabetic rabbits at 2 weeks after balloon catheter injury of the carotid arterial wall [2]. Taken together, the above results suggest that the change in growth properties of SMC by the overexpression of PDGF b -receptor plays a Diabetologia (1997) 40: 383-391 Transforming growth factor-b receptor and fibronectin expressions in aortic smooth muscle cells in diabetic rats b -receptor compared with controls. Fibronectin, one of the increased components of extracellular matrices in diabetic arteries, plays an important role in the phenotypic change of smooth muscle cells from the contractile to the synthetic type with the expression of the PDGF b -receptor. Moreover, fibronectin synthesis is regulated by transforming growth factor-b (TGF-b). In this study, we report on the expression of TGF-b receptors in diabetic smooth muscle cells, by immunohistochemistry, cross-linking of 125 I-TGF-b1 to cells and quantitative reverse transcription-polymerase chain reaction. We also report on the effects of TGF-b1 on fibronectin synthesis of diabetic smooth muscle cells by use of ELISA and immunoprecipitation, in order to clarify the role of TGF-b-fibronectin pathway in forming characteristic changes of diabetic smooth muscle cells. Cultured aortic smooth muscle cells of diabetic rats expressed TGF-b type II receptor about 8.7 times that of controls at the protein level and 5.7 times at the mRNA level, whereas the expression of the type I receptor did not differ between the two types of smooth muscle cells. These changes were accompanied by increased fibronectin synthesis in diabetic smooth muscle cells in response to TGF-b1. Furthermore, protein expression of fibronectin, and mRNA and protein of TGF-b type II receptor were increased in the diabetic aorta compared with the control aorta in vivo, implying the importance of the TGF-b-fibronectin pathway for the unique biology of smooth muscle cells in the diabetic artery. [Diabetologia (1997) 40: 383-391]

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Localization of the cellular‐fibronectin‐specific epitope recognized by the monoclonal antibody IST‐9 using fusion proteins expressed in E. coli

Cited by 90 publications

References 30 publications

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin

Transforming growth factor-β receptor and fibronectin expressions in aortic smooth muscle cells in diabetic rats

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