Microtubule involvement in translational regulation of fibronectin expression by light chain 3 of microtubule-associated protein 1 in vascular smooth muscle cells

Bin Zhou, Marlene Rabinovitch

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule- associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.

Original languageEnglish (US)
Pages (from-to)481-489
Number of pages9
JournalCirculation Research
Volume83
Issue number5
StatePublished - Sep 7 1998
Externally publishedYes

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Microtubule-Associated Proteins
Vascular Smooth Muscle
Fibronectins
Microtubules
Smooth Muscle Myocytes
Polyribosomes
Light
Messenger RNA
Ductus Arteriosus
Rough Endoplasmic Reticulum
Colchicine
Protein Biosynthesis
Membranes
Sucrose
Eukaryotic Large Ribosome Subunits
AU Rich Elements
RNA Transport
Proteins
3' Untranslated Regions
Ribonucleases

Keywords

  • Fibronectin
  • Light chain 3
  • Microtubule
  • Smooth muscle cell
  • Translation

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

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abstract = "Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule- associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.",
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T1 - Microtubule involvement in translational regulation of fibronectin expression by light chain 3 of microtubule-associated protein 1 in vascular smooth muscle cells

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AU - Rabinovitch, Marlene

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N2 - Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule- associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.

AB - Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule- associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.

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