The kinetics of fibronectin synthesis and release in normal and tumor promoter-treated human lung fibroblasts |
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Authors: | Beth A Burrous George Wolf |
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Institution: | (1) Department of Applied Biological Sciences, Massachusetts Institute of Technology, MA 02139 Cambridge, USA;(2) Present address: U. S. Patent and Trade Mark Office, 22202 Arlington, VA;(3) Present address: Department of Nutritional Sciences, University of California, 94720 Berkeley, CA, USA |
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Abstract: | Summary The present study is a detailed kinetic analysis of the synthesis, release and multimerization of fibronectin (FN) in normal and tumor promoter-treated human lung fibroblasts. Pulse/chase and surface labeling experiments were performed to follow the fate of both newly synthesized and pre-existing cell-surface FN over time. The majority of FN (80%) left the intracellular compartment within one hour of synthesis. However, the rate of direct secretion was very low and after one hour, 70% of newly synthesized FN was still at the cell surface. This material was primarily dimeric. Dimeric and multimeric (very high molecular weight) FN was detectable at the cell surface and in the medium 4 hours after synthesis. Pulse-labeled FN multimer levels peaked at 12 hours and declined thereafter. After 24 hours, 85% of pulse-labeled FN had been shed into the medium and the labeled FN remaining at the cell surface was primarily multimeric. Surface labeling experiments confirmed that the majority of FN resides at the cell surface prior to release into the medium.One hour treatment with the phorbol ester tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), stimulated a nine-fold increase in release of preexisting, dimeric cell-surface FN (125I-labeled). The major effect of longer term TPA treatment up to nine hours was continued depletion of dimeric cell-surface FN. Increased release of cell-surface multimeric FN was also stimulated by TPA, but to a much lesser extent. Release of newly synthesized (pulse-labeled) dimeric FN was also stimulated by TPA though much less than pre-existing FN, and TPA treatment produced a small decrease in the steady-state level of multimeric FN. Thus, preexisting cell-surface IN and newly synthesized FN differ dramatically in their susceptibility to TPA treatment.Abbreviations FN
fibronectin
- HLF
human lung fibroblasts
- PMSF
phenylmethylsulfonylfluoride
- TPA
12-O-tetradecanoyl phorbol-13-acetate
- DMSO
dimethylsulfoxide
- MEM
minimal essential medium
- ELISA
enzyme linked immunosorbent assay
- PBS
phosphate-buffered saline
- BSA
bovine serum albumin
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis
- DOC
deoxycholate
- DTT
dithiotreitol |
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Keywords: | fibronectin tumor promoter 12-O-tetradecanoyl phorbol-13-acetate TPA cell surface |
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