QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00778

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Klein, R. M. Articles by McKeown-Longo, P. J. Search for Related Content PubMed PubMed Citation Articles by Klein, R. M. Articles by McKeown-Longo, P. J. Social Bookmarking                         What's this?

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

Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA

View larger version (29K): [in a new window]   Fig. 1. Kinetics and localization of III1c binding to fibroblast monolayers. Human foreskin fibroblasts were grown to 90% confluence over three days in complete medium. (A) The growth medium was removed and replaced with fresh medium containing 5% fibronectin-depleted serum and 20 µM various His-tagged fibronectin modules for the indicated times: bound modules were detected by an ELISA using an antibody against the His tag. (B) Cell layers were incubated with 20 µM of III1c for the indicated times and bound III1c was detected by indirect immunofluorescence using an anti-His-tag (HIS) antibody. III1c was then visualized using a secondary antibody containing Alexa Fluor-594. Cells were costained for fibronectin (FN) using a polyclonal antibody against the entire molecule and a secondary antibody tagged with Alexa Fluor 488. Overlays of both images are shown in yellow. (C) Increasing concentrations of FN modules were incubated with the cell layers for 2 hours. Cell layers were washed and fixed, and bound protein was measured as described in A. In A and C, each point represents the average of triplicate samples. Error bars indicate standard error of the means. The data represents one of four experiments performed.

View larger version (20K): [in a new window]   Fig. 2. Effects of heparitinase on binding of III13 and III1c to cell layers. Human fibroblasts were grown as described in the legend to Fig. 1. Cell layers were pretreated for 2 hours with increasing amounts of heparitinase I. The monolayers were then incubated with 20 µM of either the III1c module or III13 module for 2 hours in the continued presence of heparitinase I. Wells were then processed to determine the amount of recombinant protein associated with the cell layers using a mAb to the His-tag. The data points represent the average of triplicate determinations, and error bars indicate the standard error of means. The graph represents one of three experiments performed. Loss of heparan sulfate in response to hepartinase was assessed by ELISA using an antibody against heparan sulfate (insert).

View larger version (54K): [in a new window]   Fig. 3. Effect of heparitinase on the localization of III1c in the cell layer. Human fibroblasts were seeded onto glass coverslips and grown as previously described. Wells were then incubated with or without 0.01 U/ml heparitinase I for 2 hours followed by the addition of 20 µM III1c or III13 for 2 additional hours. Cells were then washed, fixed, permeabilized and dual stained for fibronectin (FN) using a polyclonal antibody and the recombinant fragments using a monoclonal antibody against the His-tag (HIS) as indicated. Fields shown represent one experiment repeated three times.

View larger version (35K): [in a new window]   Fig. 4. Effect of III1c on levels of matrix fibronectin. Fibroblasts were seeded into wells and grown 3 days in the presence of complete medium containing 900 ng/ml 125I-labeled fibronectin to obtain a radiolabeled fibronectin matrix. Wells were then washed and monolayers incubated for 16 hours in the presence or absence of 20 µM of various recombinant fibronectin modules as indicated. Cell layers were extracted with 0.5% DOC buffer to obtain DOC-insoluble matrix. An equal proportion of the matrix from each treatment was analyzed by SDS gel electrophoresis under both reducing and nonreducing conditions and visualized by autoradiography. The figure represents one experiment repeated three times.

View larger version (53K): [in a new window]   Fig. 5. Effect of III1c on fibronectin matrix morphology. Fibroblasts were cultured on glass coverslips in the presence of fibronectin conjugated with Alexa Fluor-488 (10 µg/ml), indicated as AF-FN, for three days. Wells were washed and incubated with 20 µM III1c or carrier buffer as control for 20 hours. Cells were washed, fixed, permeabilized and stained for fibronectin using the designated mAbs. Alexa Fluor-488 labeled fibronectin was visualized and total matrix fibronectin was detected by indirect immunofluorescence using either an antibody against the CS domain (FDB3) or an antibody against the EDA domain (IST-9). The figure represents one experiment repeated three times.

View larger version (20K): [in a new window]   Fig. 6. Map of antibody binding sites on fibronectin. Incubation of cell layers with 20 µM of III1c resulted in the loss of IST-9 staining for fibronectin, but had no effect on the ability of any of the remaining ten antibodies to stain matrix fibronectin.

View larger version (33K): [in a new window]   Fig. 7. Effect of III1c on fibronectin epitopes. Fibroblasts, grown as described in the legend to Fig. 1, were incubated for 2 hours with increasing concentrations of Type III fibronectin modules in the presence of fibronectin monoclonal antibodies IST-9 (2 µg/ml) (A), 5C11F3 (8 µg/ml) (B), Clone 568 (2 µg/ml) (C) or IST-5 (2 µg/ml) (D). Following treatments, cell layers were washed and fixed, and bound antibody was determined by ELISA. Data shown represents the mean and standard error of an experiment performed in triplicate and represents one of three experiments.

View larger version (13K): [in a new window]   Fig. 8. Kinetics of III1c-induced loss of IST-9 binding. Fibroblast monolayers, grown as described previously, were incubated with 20 µM III1c or III11c for increasing times. The wells treated with carrier buffer were set as the 100% control. After incubation with fibronectin fragments, cell layers were washed, fixed and processed for ELISA using the monoclonal antibody IST-9 (2 µg/ml). Data points are the mean of triplicate wells. The graph depicts data from a representative experiment performed in triplicate.

View larger version (13K): [in a new window]   Fig. 9. Binding of III1c to EDA+ and EDA– fibronectins. Triplicate wells were coated with 20 µg/ml of either (EDA+) cellular fibronectin or (EDA–) plasma fibronectin, blocked with 1.0% BSA and incubated with increasing amounts of III1c for 1 hour. Bound III1c was measured by ELISA using a monoclonal antibody to the 6xHis tag.

View larger version (14K): [in a new window]   Fig. 10. Effect of III1c on IST-9 binding to EDA+ fibronectin. Wells were coated with 20 µg/ml of cellular fibronectin (EDA+) and then blocked with 1.0% BSA. Triplicate wells were incubated with increasing amounts of III1c for 1 hour. Following treatments, the binding of EDA specific mAbs, IST-9 (2 µg/ml), or 5C11F3 (8 µg/ml) was determined by ELISA. The graph depicts data from a representative experiment performed in triplicate.

View larger version (64K): [in a new window]   Fig. 11. Effect of III1c on p38 MAPK activation. Fibroblast monolayers, grown 3 days in complete medium, were washed and rendered quiescent by incubation for 36 hours in DMEM containing 0.1% BSA. Monolayers were then incubated with III1c (20 µM), III13 (20 µM) or EGF (25 ng/ml) in 0.1% BSA/DMEM for the indicated times. Cell lysates were electrophoresed and immunoblotted using an antibody against phosphorylated p38 (p-p38). The membranes were then stripped and reprobed with an antibody against p38 to ensure equal loading (p38). Blots shown are from one experiment performed three times.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter