AU-rich elements in the collagenase 3 mRNA mediate stabilization of the transcript by cortisol in osteoblasts.
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.
Rydziel
Sheila
S
Department of Research, Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105, USA.
Delany
Anne M
AM
Canalis
Ernesto
E
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D017364:Collagenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006854:Hydrocortisone / Q000378:metabolism*; D007477:Ions; D013058:Mass Spectrometry; D053509:Matrix Metalloproteinase 13; D008957:Models, Genetic; D009154:Mutation; D010006:Osteoblasts / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014461:Ultracentrifugation; D016596:Vinculin / Q000737:chemistry / Q000378:metabolism
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D007477:Ions; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D012313:RNA; D017364:Collagenases; D053509:Matrix Metalloproteinase 13; C507083:Mmp13 protein, rat; D006854:Hydrocortisone