title: Effects of A-CREB, a dominant negative inhibitor of CREB, on the expression of c-fos and other immediate early genes in the rat SON during hyperosmotic stimulation in vivo.
abstract: Intraperitoneal administration of hypertonic saline to the rat supraoptic nucleus (SON) increases the expression of several immediate early genes (IEG) and the vasopressin gene. These increases have usually been attributed to action of the cyclic-AMP Response Element Binding Protein (CREB). In this paper, we study the role of CREB in these events in vivo by delivering a potent dominant-negative form of CREB, known as A-CREB, to the rat SON through the use of an adeno-associated viral (AAV) vector. Preliminary experiments on HEK 293 cells in vitro showed that the A-CREB vector that we used completely eliminated CREB-induced c-fos expression. We stereotaxically injected this AAV-A-CREB into one SON and a control AAV into the contralateral SON of the same rat. Two weeks following these injections we injected hypertonic saline intraperitoneally into the rat. Using this paradigm, we could measure the relative effects of inhibiting CREB on the induced expression of c-fos, ngfi-a, ngfi-b, and vasopressin genes in the A-CREB AAV injected SON versus the control AAV injected SON in the same rat. We found only a small (20%) decrease of c-fos expression and a 30% decrease of ngfi-b expression in the presence of the A-CREB. There were no significant changes in expression found in the other IEGs nor in vasopressin that were produced by the A-CREB. This suggests that CREB may play only a minor role in the expression of IEGs and vasopressin in the osmotically activated SON in vivo.
authors.affiliation: Laboratory of Neurochemistry, Molecular Neuroscience Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. DLubelski@gmail.com
authors.forename: Daniel
authors.initials: D
authors.lastname: Lubelski
authors.forename: Todd A
authors.initials: TA
authors.lastname: Ponzio
authors.forename: Harold
authors.initials: H
authors.lastname: Gainer
mesh_terms: D000818:Animals; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D017781:Genes, Immediate-Early / Q000187:drug effects; D016762:Genes, fos / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012462:Saline Solution, Hypertonic / Q000494:pharmacology; D013495:Supraoptic Nucleus / Q000187:drug effects / Q000378:metabolism*; D014667:Vasopressins / Q000096:biosynthesis
chemical_list: C508138:A-CREB protein; D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012462:Saline Solution, Hypertonic; D014667:Vasopressins
references.citation: J Neurosci. 2004 Jul 21;24(29):6553-62
references.pmid: 15269267
references.citation: Nucleic Acids Res. 2008 May;36(8):2594-607
references.pmid: 18334532
references.citation: Nat Rev Mol Cell Biol. 2001 Aug;2(8):599-609
references.pmid: 11483993
references.citation: Nat Protoc. 2006;1(6):3166-73
references.pmid: 17406580
references.citation: J Neurosci Methods. 1980 Dec;3(2):129-49
references.pmid: 6110810
references.citation: J Biol Chem. 1992 Nov 25;267(33):23520-6
references.pmid: 1331087
references.citation: Nature. 2001 Sep 13;413(6852):179-83
references.pmid: 11557984
references.citation: EMBO J. 1995 Oct 16;14(20):4905-13
references.pmid: 7588619
references.citation: J Neurosci. 2003 Jun 1;23(11):4760-5
references.pmid: 12805315
references.citation: Mol Cell Biol. 1993 Oct;13(10):6260-73
references.pmid: 8413226
references.citation: Mol Cell Biol. 1998 Feb;18(2):967-77
references.pmid: 9447994
references.citation: Cell. 1994 Jun 3;77(5):713-25
references.pmid: 8205620
references.citation: J Neurosci Res. 2010 May 1;88(6):1364-73
references.pmid: 19938175
references.citation: PLoS One. 2010 Jun 21;5(6):e11235
references.pmid: 20574536
references.citation: Nat Biotechnol. 2004 May;22(5):589-94
references.pmid: 15064769
references.citation: J Neurochem. 2009 Mar;108(5):1251-65
references.pmid: 19141071
references.citation: J Neurosci. 2003 Nov 12;23(32):10231-7
references.pmid: 14614081
references.citation: Proc Natl Acad Sci U S A. 2005 Jun 7;102(23):8357-62
references.pmid: 15923261
references.citation: Cell Biochem Biophys. 2005;42(3):263-76
references.pmid: 15976459
references.citation: Genes Dev. 1988 Dec;2(12A):1529-38
references.pmid: 2850967
references.citation: Endocrinology. 1997 Dec;138(12):5266-74
references.pmid: 9389510
references.citation: Anal Biochem. 2010 Apr 1;399(1):144-6
references.pmid: 19951694
references.citation: Mol Reprod Dev. 2008 May;75(5):931-9
references.pmid: 18163434
references.citation: Science. 1998 Dec 18;282(5397):2272-5
references.pmid: 9856954
references.citation: J Neurosurg. 2007 Sep;107(3):560-7
references.pmid: 17886556
references.citation: Curr Biol. 1995 Oct 1;5(10):1191-200
references.pmid: 8548291
references.citation: Science. 1991 Jun 7;252(5011):1427-30
references.pmid: 1646483
references.citation: Cereb Cortex. 2009 Nov;19(11):2535-47
references.pmid: 19213815
references.citation: Nature. 1984 Oct 4-10;311(5985):433-8
references.pmid: 6090941
references.citation: J Neuroendocrinol. 2005 Apr;17(4):227-37
references.pmid: 15842234
references.citation: J Neurosci Methods. 2011 Apr 30;197(2):238-48
references.pmid: 21392530
references.citation: Endocrinology. 2006 Jun;147(6):2850-63
references.pmid: 16543367
references.citation: EMBO J. 1995 Oct 16;14(20):5037-47
references.pmid: 7588632
references.citation: Exp Neurol. 2009 Sep;219(1):212-22
references.pmid: 19463813
references.citation: J Neurosci Methods. 2007 Aug 15;164(1):149-54
references.pmid: 17540451
references.citation: Genes Dev. 1989 Feb;3(2):198-211
references.pmid: 2541049
references.citation: Neuron. 1990 Apr;4(4):477-85
references.pmid: 1969743
references.citation: Am J Physiol Cell Physiol. 2008 Apr;294(4):C1046-55
references.pmid: 18272822
references.citation: Genes Dev. 1990 Feb;4(2):255-68
references.pmid: 2110922
references.citation: Cell. 1989 Nov 17;59(4):675-80
references.pmid: 2573431
references.citation: Nucleic Acids Res. 2001 May 1;29(9):e45
references.pmid: 11328886
references.citation: J Biol Chem. 2011 Mar 11;286(10):8094-8105
references.pmid: 21209091
references.citation: J Biol Chem. 2003 Oct 31;278(44):43014-9
references.pmid: 12939274
references.citation: J Neurosci. 2005 Jul 6;25(27):6296-303
references.pmid: 16000619
references.citation: Virol J. 2006 Mar 15;3:14
references.pmid: 16539700
references.citation: Brain Res. 1990 Oct 29;531(1-2):299-303
references.pmid: 2126973
references.citation: Neuron. 1990 Apr;4(4):571-82
references.pmid: 2157471
references.citation: Hum Gene Ther. 2002 Nov 1;13(16):1935-43
references.pmid: 12427305
references.citation: Neuron. 2002 Aug 15;35(4):605-23
references.pmid: 12194863
references.citation: Mol Cell Biochem. 2010 Jun;339(1-2):119-25
references.pmid: 20066477
references.citation: Mol Endocrinol. 1993 Mar;7(3):365-79
references.pmid: 8483478
references.citation: Neurosci Lett. 2010 Dec 3;486(1):5-9
references.pmid: 20850504
references.citation: Curr Biol. 1996 Jan 1;6(1):16-9
references.pmid: 8805215
references.citation: Mol Cell Biol. 1999 Feb;19(2):1313-24
references.pmid: 9891065