Microtubule involvement in translational regulation of fibronectin expression by light chain 3 of microtubule-associated protein 1 in vascular smooth muscle cells.
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.
Zhou
B
B
Hospital for Sick Children, Department of Pediatrics, University of Toronto, Ontario, Canada.
Rabinovitch
M
M
D000818:Animals; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics*; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000502:physiology*; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000502:physiology*; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D012756:Sheep; D013347:Subcellular Fractions / Q000378:metabolism
D005353:Fibronectins; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D003078:Colchicine