Nephroblastoma overexpressed (Nov) induces gremlin in ST-2 stromal cell lines by post-transcriptional mechanisms.
Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis in part because it binds bone morphogenetic protein (BMP)-2. In the present study, we investigated whether Nov regulated the expression of the BMP antagonist gremlin. Overexpression of Nov increased gremlin mRNA levels in ST-2 cells, and its downregulation by RNA interference decreased gremlin mRNA. Nov did not affect Grem1 transcription, but prolonged the half-life of gremlin mRNA in ST-2 cells, demonstrating that Nov acts by post-transcriptional mechanisms. This was confirmed by demonstrating that downregulation of Nov destabilizes gremlin transcripts. To assess whether the 3'-untranslated region (UTR) of gremlin mRNA mediated the effect of Nov, the decay of a chimeric cfos gremlin 3'-UTR construct was compared to that of cfos in ST-2 cells. The presence of the gremlin 3'-UTR prolonged the half-life of cfos and was responsible for the effect of Nov. To examine the binding of the gremlin 3'-UTR to ribonucleoproteins, radiolabeled gremlin RNA fragments were incubated with cytosolic extracts from Nov overexpressing and control cells. RNA electrophoretic mobility analysis revealed that Nov enhanced the binding of cytosolic proteins to the fragments spanning the 3'-UTR of gremlin between bases 1,358-1,557 and 1,158-1,357 from the transcriptional start. Mutations of AU-rich elements in these two RNA fragments prevented the formation of RNA-protein complexes induced by Nov. Nov did not alter the binding of cytosolic extracts to sequences present in the 5'-UTR or coding region of gremlin. In conclusion, Nov stabilizes gremlin transcripts, and this effect is possibly mediated by AU-rich elements present in the 3'-UTR of gremlin.
Department of Research, Saint Francis Hospital, Medical Center, Hartford, Connecticut 06105-1299, USA.
Anna
A
Smerdel-Ramoya
Stefano
S
Zanotti
Ernesto
E
Canalis
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D024202:Electrophoretic Mobility Shift Assay; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D055514:Nephroblastoma Overexpressed Protein / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D016207:Cytokines
D020413:3' Untranslated Regions; D036341:Intercellular Signaling Peptides and Proteins; D055514:Nephroblastoma Overexpressed Protein; C113178:Grem1 protein, mouse; D016207:Cytokines
Bone. 1999 Jul;25(1):91-3
10423029
Nucleic Acids Res. 2002 May 1;30(9):2089-195
11972350
J Immunol. 2006 Nov 15;177(10):6999-7006
17082615
Mol Cell. 1998 Apr;1(5):673-83
9660951
Proc Natl Acad Sci U S A. 1988 Apr;85(7):2171-5
3127826
J Clin Invest. 1998 Dec 15;102(12):2106-14
9854046
Genes Dev. 2001 Mar 1;15(5):485-90
11238371
Mol Cell Biol. 1997 Aug;17(8):4801-10
9234736
J Biol Chem. 2007 Oct 26;282(43):31549-57
17785465
J Clin Invest. 2008 Feb;118(2):479-90
18188456
Mol Cell Biol. 2001 Mar;21(6):2070-84
11238942
J Biol Chem. 2007 Jul 6;282(27):19762-72
17500060
Cell. 1992 Sep 4;70(5):829-40
1339313
J Clin Invest. 2009 Oct;119(10):3102-14
19770516
J Biol Chem. 2004 Feb 13;279(7):5397-404
14645243
Endocrinology. 2003 May;144(5):1972-8
12697704
Nucleic Acids Res. 2002 May 1;30(9):e37
11972352
Genes Dev. 1989 Jan;3(1):60-72
2496006
Endocrinology. 1992 Mar;130(3):1318-24
1311236
Mol Cell Biol. 2000 Mar;20(6):1982-92
10688645
Nucleic Acids Res. 1985 Apr 11;13(7):2485-502
2987855
Oncogene. 1987;2(1):79-84
3325886
J Biol Chem. 2006 Mar 10;281(10):6203-10
16407293
Endocrinology. 2008 Sep;149(9):4374-81
18535099
J Biol Chem. 2000 Mar 24;275(12):8785-93
10722723
Mol Cell Biol. 2001 Feb;21(3):721-30
11154260
Mol Endocrinol. 1995 Mar;9(3):375-87
7776983
Endocrinology. 2010 Jan;151(1):221-33
19934377
Nat Cell Biol. 2002 Aug;4(8):599-604
12134160
Nature. 2001 May 24;411(6836):494-8
11373684
Mol Pathol. 2003 Apr;56(2):127-8
12665631
Rev Endocr Metab Disord. 2006 Jun;7(1-2):51-65
17029022
Am J Physiol. 1999 Jul;277(1):C132-8
10409116
J Clin Invest. 1997 Oct 1;100(7):1797-803
9312180
Mol Cell Biol. 1994 Dec;14(12):8471-82
7969180
Bone. 2003 Feb;32(2):111-9
12633782
Development. 2004 May;131(9):2137-47
15105373
Endocr Rev. 2003 Apr;24(2):218-35
12700180
Gene. 2002 Apr 3;287(1-2):39-47
11992721
J Biol Chem. 2008 Aug 15;283(33):22690-9
18583340
Endocrinology. 2005 Feb;146(2):655-65
15539560
J Neurosci. 1995 Sep;15(9):6077-84
7666191
J Endocrinol. 2003 Aug;178(2):169-75
12904165
Endocrinology. 2000 Dec;141(12):4558-63
11108268