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

doi: 10.1242/10.1242/jcs.00145

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 Lu, M. Articles by Schnapp, L. M. Search for Related Content PubMed PubMed Citation Articles by Lu, M. Articles by Schnapp, L. M.

Integrin 8ß1 mediates adhesion to LAP-TGFß1

¹ Pulmonary and Critical Care Medicine, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA
² Departments of Medicine and Cell Biology, NYU School of Medicine, New York, NY 10016, USA
³ Pulmonary and Critical Care Medicine, Harborview Medical Center, University of Washington, Seattle, WA 98104, USA

View larger version (27K): [in a new window]   Fig. 1. (A) 8ß1 adhesion to LAP-TGFß1. Mock transfected or 8-transfected cells (50,000/well) were allowed to attach to wells precoated with 5 µg/ml of recombinant LAP-TGFß1. In some cases, cells were incubated with v integrin antibody (L230) or ß1 integrin antibody (5D1) prior to adhesion assay. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD595. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (B) Adhesion of SW4808 or SW480ß6-transfected cells to increasing concentrations of LAP-TGFß1 with or without 1mM Mn2+. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD595. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (C) Adhesion of 8 or ß6-transfected cells to fibronectin. AtT208, SW4808 or SW480ß6 cells were allowed to attach to wells precoated with the indicated concentration of fibronectin. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD595. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (D) Mutation of RGD site in LAP TGFß1 eliminates 8ß1 adhesion. 8-transfected cells were allowed to attach to wells coated with 5 µg/ml of authentic recombinant LAP-TGFß1 (RGD-LAP-TGFß1) or recombinant LAP-TGFß1 containing a single glutamic acid for aspartic acid substitution mutation in the RGD site (RGE-LAP-TGFß1) for 1 hour; adhesion was then assessed by absorbance. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of BSA-coated well. (E) Adhesion of AtT208 cells to LAP-TGFß3 is similar to adhesion to LAP-TGFß1. AtT20 or AtT208 cells were allowed to adhere to increasing concentrations of recombinant LAP-TGFß1 or LAP-TGFß3 for 1 hour; adhesion was then assessed by absorbance. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well.

View larger version (83K): [in a new window]   Fig. 2. (i) 8ß1 mediates spreading on LAP-TGFß1. 8-transfected or mock-transfected AtT20 cells were plated on 5 µg/ml of recombinant LAP-TGFß1 in serum-free media for 24 or 48 hours. At 24 hours, mock transfected AtT20 cells remain rounded and unattached on LAP-TGFß1 (A), whereas 8-transfected cells attach and spread on LAP-TGFß1 (B). Further spreading is seen at 48 hours (C). (ii) Adhesion of 8ß1 to LAP-TGFß1 results in FAK phosphorylation. 8-transfected or mock-transfected AtT20 cells were plated on 0.01% poly-L-lysine (PLL), 5 µg/ml fibronectin (FN), 5 µg/ml LAP-TGFß1 (LAP1) or 5 µg/ml RGE-LAP-TGFß1 (RGE) for 30 minutes in serum-free media. Cells were lysed in buffer containing phosphatase inhibitors, immunoprecipitated with anti-FAK antibody followed by western blotting with either anti-phosphotyrosine antibody PY20 (top panel) or anti-FAK antibody (bottom panel).

View larger version (36K): [in a new window]   Fig. 3. (A) Proliferation of cells adherent to LAP-TGFß1. Equal numbers of 8-transfected or mock-transfected AtT20 cells were plated on 5 µg/ml LAP-TGFß1 or 5 µg/ml fibronectin (FN) in serum-free media. After 3 days, proliferation was assayed using the Roche Cell Proliferation kit (MTT). Results for four independent clones of AtT208 cells are shown. Data is reported as the mean absorbance of triplicate wells±s.d. (B) ERK phosphorylation on LAP-TGFß1. 8-transfected or mock-transfected AtT20 cells were plated on 0.01% poly-L-lysine (PLL) 5 µg/ml fibronectin (FN), 5 µg/ml LAP-TGFß1 (LAP1), or 5 µg/ml RGE-LAP-TGFß1 (RGE) for 30 minutes in serum-free media and then lysed in buffer containing phosphatase inhibitors. Equal amounts of protein were loaded and probed with an antibody to phospho-ERK (top) or ERK (bottom).

View larger version (16K): [in a new window]   Fig. 4. (A) Adhesion of LAP-TGFß1 to 8ß1 does not activate TGFß1. Equal number of Mv1Lu reporter cells and test cells (SW4808 or SW480ß6) were co-cultured for 16 hours with indicated concentrations of the integrin-activating antibody 8A2 and lysed for measurement of luciferase activity. Results are the means of at least two experiments done in duplicate. (B) 8-transfected cells do not affect TGF-ß1 activation by SW480ß6 cells. Mv1Lu reporter cells were cultured with SW4808 cells and SW480ß6 cells, or mock-transfected SW480 cells and SW480ß6 cells, for 16 hours and lysed for measurement of luciferase activity. Results are the means of duplicate experiments±s.d. (C) Adhesion of LAP-TGFß3 to 8ß1 does not activate TGFß3. CHO or CHO8 cells were transiently transfected with full-length expression construct of TGFß3. After 16 hours, transfected cells were cultured for 24 hours with Mv1Lu reporter cells. Cells were lysed and luciferase activity was measured. Results are the mean of triplicate experiments±s.d.

View larger version (147K): [in a new window]   Fig. 5. LAP-TGFß1 localization in adult lung tissue. Paraffin-embedded, formalin fixed tissues were stained with an antibody specific for LAP-TGFß1 and counterstained with hematoxylin. Immunoreactivity for LAP-TGFß1 is seen on a macrophage (arrowhead) and interstitial cells (arrows). Magnification: A, 4x; B,C, 20x; D, 40x.