AU-Rich Elements in the Collagenase 3 mRNA Mediate Stabilization of the Transcript by Cortisol in Osteoblasts
| Autor: | Sheila Rydziel, Ernesto Canalis, Anne M. Delany |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Untranslated region
Time Factors Hydrocortisone MMP1 Recombinant Fusion Proteins Biology Transfection Biochemistry Mass Spectrometry Rats Sprague-Dawley Cytosol Matrix Metalloproteinase 13 medicine Animals Electrophoretic mobility shift assay Collagenases RNA Messenger 3' Untranslated Regions Molecular Biology Polyacrylamide gel electrophoresis Ions AU-rich element Messenger RNA Osteoblasts Models Genetic RNA Cell Biology Molecular biology Vinculin Rats Mutation Collagenase Electrophoresis Polyacrylamide Gel Female RNA Interference 5' Untranslated Regions Proto-Oncogene Proteins c-fos Ultracentrifugation Plasmids medicine.drug |
| Zdroj: | Journal of Biological Chemistry. 279:5397-5404 |
| ISSN: | 0021-9258 |
| Popis: | Collagenase 3 degrades collagen fibrils and is necessary for bone resorption. Cortisol increases collagenase 3 mRNA in osteoblasts by stabilizing collagenase 3 transcripts. To understand mechanisms involved, we used RNA electrophoretic mobility shift assay and RNA turnover studies. Cortisol increased the binding of Ob cell cytosolic extracts to AU-rich sequences in the collagenase 3 3'-untranslated region (UTR). No cortisol-dependent protein complexes were formed with the coding region or the 5'-UTR. Functional assays, using transient transfections of CMV-driven c-fos collagenase 3'-UTR chimeric constructs, demonstrated that the 3'-UTR of collagenase 3 stabilizes c-fos mRNA in transcriptionally arrested Ob cells, cortisol prolongs the transcript half-life, and mutations of AU-rich sequences destabilize c-fos transcripts precluding the cortisol effect. Purification of osteoblast cytosolic extracts by ultracentrifugation, ion exchange, and RNA affinity chromatography, and polyacrylamide gel electrophoresis followed by mass spectroscopy identified specific proteins. RNA gel mobility supershift assays demonstrated that vinculin and far upstream element (FUSE)-binding protein 2 interacted with collagenase 3 3'-UTR sequences, and RNA interference demonstrated these proteins altered collagenase mRNA stability. In conclusion, AU-rich sequences of the 3'-UTR of collagenase 3 and vinculin and FUSE-binding protein 2 regulate collagenase mRNA stability in osteoblasts. |
| Databáze: | OpenAIRE |
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