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

First published online 17 January 2006
doi: 10.1242/jcs.02771

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 Naik, M. U. Articles by Naik, U. P. Search for Related Content PubMed PubMed Citation Articles by Naik, M. U. Articles by Naik, U. P. Social Bookmarking                         What's this?

Junctional adhesion molecule-A-induced endothelial cell migration on vitronectin is integrin _vß₃ specific

¹ Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA
² Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
³ Delaware Biotechnology Institute, University of Delaware, Newark, DE 19716, USA

View larger version (97K): [in a new window]   Fig. 1. Overexpression of JAM-A induces HUVEC migration. (A) Western blot of protein lysate (50 µg) from mock- and JAM-A-transfected HUVECs. The lower blot shows the upper blot after reprobing using anti--tubulin to ensure equal loading. (B) Densitometric analysis of (A) indicates JAM-A overexpression is about three times that of endogenous JAM-A expression. (C) A wound-induced migration assay on vitronectin matrix was performed using mock- and JAM-A-transfected cells in the absence of serum or growth factors. The extent of cell migration into the wounded area was photographed under phase-contrast microscopy at 0 hour and after 24 hours. Magnification, x100. Data shown are representative of three separate experiments.

View larger version (87K): [in a new window]   Fig. 2. JAM-A-induced HUVEC migration is vitronectin specific. (A) A haptotactic transwell motility assay using mock- and JAM-A-transfected cells was performed on inserts pre-coated with vitronectin. Images are representative of the migration of JAM-A-overexpressing cells in three separate experiments performed in triplicate. Magnification, x200. (B) A haptotactic transwell motility assay using mock- and JAM-A-transfected cells was performed on inserts pre-coated with indicated ECM proteins. (C) Data collected in (B) represented as fold increase over mock. A two- to fivefold increase in migration of JAM-A-overexpressing cells on vitronectin was routinely observed, and was dependent upon the passage number of transfected cells.

View larger version (19K): [in a new window]   Fig. 3. JAM-A does not bind to vitronectin. (A) HUVECs stably transfected with JAM-A or mock transfected were allowed to adhere to various concentrations of vitronectin as indicated for 1 hour. Bound cells were quantified as described in the Materials and Methods. (B) ELISA standard curve using soluble JAM-A as indicated. (C) In vitro binding assay of soluble JAM-A to immobilized vitronectin. Data shown are expressed as mean ± s.e.m. of three separate experiments.

View larger version (23K): [in a new window]   Fig. 4. JAM-A-induced HUVEC migration on vitronectin is integrin vß3 specific. HUVECs stably transfected with JAM-A or mock transfected were pre-treated with various concentrations of antibodies or inhibitors as indicated. Cell migration assays on vitronectin or fibronectin were performed as described in the Materials and Methods. (A) Pre-treatment with anti-vß3 antibody (*P<0.05), (B) pre-treatment with anti-vß5 antibody, (C) pre-treatment with XT199 RGD-based antagonist specific to integrin, (D) same as in (C) on fibronectin.

View larger version (93K): [in a new window]   Fig. 5. Integrin vß3 associates with JAM-A. (A) Western blot showing the precence of JAM-A in the immunoprecipitate (IP) of anti-vß3 from total cell lysate (Input) of serum-starved untransfected HUVECs treated with or without RGDS as indicated. Isotype-specific IgG (cIgG) was used as a negative control, and the blot was reprobed with anti-ß3 antibody, to ensure equal loading. (B) Immunofluorescence images of live, adherent HUVECs incubated with FITC-XT199. (i-vi) XT199 accumulation at the cell-cell junction was monitored by confocal microscopy from time 0-20 minutes. Arrowheads indicate the position of the cell-cell junctions. (v) FITC-XT199 accumulation at the cell-cell junction of live HUVECs was ablated as a result of competitive inhibition by the addition of excess 1 mM RGDS. (vi) Live HUVECs were pre-treated with 100 µM RGDS compound for 20 minutes to allow integrin vß3 accumulation at the cell-cell junction. The live cells were then fixed and treated with anti-vß3 antibody to ensure the accumulation was vß3 specific (arrowhead). Data shown are representative of three separate experiments. Bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 6. Signaling through JAM-A, PI 3-kinase and PKC is required for JAM-A-induced migration. (A) Western blot of protein lysate (50 µg) from mock-, JAM-A- and JAM-A -257-transfected HUVECs, indicating level of expression of JAM-A. [Note: deletion of the cytoplasmic domain (-257) results in a lower-sized band.] (B) Cell migration on vitronectin using cells as in A. (C) Western blot showing the absence of HA-tagged JAM-A -257 in the immunoprecipitate (IP) of anti-vß3 from total cell lysate (Input) of serum-starved HUVECs transfected with the JAM-A -257 construct and treated with or without RGDS as indicated. Isotype-specific IgG was used as a negative control (cIgG), and the blot was reprobed with anti-ß3 antibody, to ensure equal loading. Mock-transfected or JAM-A-overexpressing cells were pre-treated with various concentrations of anti-JAM-A antibody (D), or with DMSO or wortmannin or Bis (E) for 20 minutes and then assayed for migration on vitronectin. *P<0.05.

View larger version (26K): [in a new window]   Fig. 7. JAM-A enhances sustained MAPK activation. (A) Immunoblot of phospho-ERK1/2 activity from serum-starved mock-transfected and JAM-A-overexpressing HUVEC samples plated on vitronectin for up to 60 minutes. Corresponding total ERK1/2 amounts are shown to indicate equal loading. (B) Densitometric analysis of normalized data from A, presented as mean ± s.e.m. (C) Immunoblot of phospho-ERK1/2 activity from serum-starved mock-transfected HUVECs, and JAM-A- and -257-overexpressing HUVEC samples plated on vitronectin for various times as indicated. Corresponding total ERK1/2 amounts are shown to indicate equal loading. (D) Densitometric analysis of normalized data from C, presented as mean ± s.e.m. Data shown are representative of at least three independent experiments.

View larger version (41K): [in a new window]   Fig. 8. JAM-A upregulates FAK activation. (A) Immunoblot of phosphorylated Tyr397 (pY 397) from serum-starved mock-transfected HUVECs, and JAM-A- and -257-overexpressing HUVEC samples in suspension or plated on vitronectin for 10 minutes. Corresponding total FAK is shown to indicate equal loading. (B) Densitometric analysis of normalized data from A, presented as mean ± s.e.m.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter