title: Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments.
abstract: Vascularization is essential in bone tissue engineering and recent research has focused on interactions between osteoblasts (hOBs) and endothelial cells (ECs). It was shown that cocultivation increases the stability of osteoblastic alkaline phosphatase (ALP) mRNA. We investigated the mechanisms behind this observation, focusing on mRNA binding proteins. Using a luciferase reporter assay, we found that the 3'-untranslated region (UTR) of ALP mRNA is necessary for human umbilical vein endothelial cells (HUVEC)-mediated stabilization of osteoblastic ALP mRNA. Using pulldown experiments and nanoflow-HPLC mass spectrometry, vimentin was identified to bind to the 3'-UTR of ALP mRNA. Validation was performed by Western blotting. Functional experiments inhibiting intermediate filaments with iminodipropionitrile and specific inhibition of vimentin by siRNA transfection showed reduced levels of ALP mRNA and protein. Therefore, ALP mRNA binds to and is stabilized by vimentin. This data add to the understanding of intracellular trafficking of ALP mRNA, its function, and have possible implications in tissue engineering applications.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Schmidt
authors.forename: Martin
authors.initials: M
authors.lastname: Biniossek
authors.forename: G Björn
authors.initials: GB
authors.lastname: Stark
authors.forename: Günter
authors.initials: G
authors.lastname: Finkenzeller
authors.forename: Filip
authors.initials: F
authors.lastname: Simunovic
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D057075:Enzyme Assays; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D013058:Mass Spectrometry; D036103:Nanotechnology; D010006:Osteoblasts / Q000201:enzymology*; D011485:Protein Binding; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D034741:RNA, Small Interfering; D014746:Vimentin; D001710:Biotin; D008156:Luciferases; D000469:Alkaline Phosphatase