Heat shock enhances CMV-IE promoter-driven metabotropic glutamate receptor expression and toxicity in transfected cells.
In CHO-K1 cells, heat shock strongly activated reporter-gene expression driven by the cytomegalovirus immediate-early (CMV-IE) promoter from adenoviral and plasmid vectors. Heat shock treatment (2h at 42.5 °C) significantly enhanced the promoter DNA-binding activity in nuclear extracts. In CHO cells expressing mGluR1a and mGluR5a receptors under the control of the CMV promoter, heat shock increased receptor protein expression, mRNA levels and receptor function estimated by measurement of PI hydrolysis, intracellular Ca²+ and cAMP. Hyperthermia increased average amplitudes of Ca²+ responses, the number of responding cells, and revealed the toxic properties of mGluR1a receptor. Heat shock also effectively increased the expression of EGFP. Hence, heat shock effects on mGluR expression and function in CHO cells may be attributed to the activation of the CMV promoter. Moreover, this effect was not limited to CHO cells as heat shock also increased EGFP expression in PC-12 and HEK293 cells. Heat shock treatment may be a useful tool to study the function of proteins expressed in heterologous systems under control of the CMV promoter. It may be especially valuable for increasing protein expression in transient transfections, for enhancing receptor expression in drug screening applications and to control the expression of proteins endowed with toxic properties. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.
Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20057, USA.
Sergey
S
Pshenichkin
Alexander
A
Surin
Elena
E
Surina
Małgorzata
M
Klauzińska
Ewa
E
Grajkowska
Victoria
V
Luchenko
Monika
M
Dolińska
Barbara
B
Wroblewska
Jarda T
JT
Wroblewski
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors / Q000235:genetics; D018869:Heat-Shock Response / Q000502:physiology*; D006358:Hot Temperature; D017874:Immediate-Early Proteins / Q000819:agonists / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D018675:Toxicity Tests; D014162:Transfection
C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; C108822:metabotropic glutamate receptor 2; C104077:metabotropic glutamate receptor type 1
J Mol Cell Cardiol. 2003 Jul;35(7):823-31
12818573
Biochem Pharmacol. 2000 Jan 1;59(1):55-63
10605935
Nucleic Acids Res. 1991 Jul 11;19(13):3715-21
1649457
J Immunol Methods. 1983 Dec 16;65(1-2):55-63
6606682
Mol Cell Biol. 2002 Jan;22(1):343-56
11739746
Exp Lung Res. 1998 Jan-Feb;24(1):3-14
9457465
J Biol Chem. 1992 Jul 5;267(19):13361-8
1320017
Virology. 1995 Apr 1;208(1):197-206
11831701
Am J Physiol. 1999 Nov;277(5):L1051-6
10564192
J Virol. 1997 Sep;71(9):6683-91
9261391
Nitric Oxide. 2003 Jun;8(4):262-5
12895436
Br J Pharmacol. 1993 Dec;110(4):1305-10
8306069
Annu Rev Pharmacol Toxicol. 1997;37:205-37
9131252
Biochem Biophys Res Commun. 2001 Aug 31;286(4):807-14
11520069
J Am Coll Surg. 2002 Nov;195(5):619-26
12437247
Cancer Gene Ther. 2002 Mar;9(3):267-74
11896443
Am J Physiol Gastrointest Liver Physiol. 2003 Feb;284(2):G255-62
12388181
J Gen Virol. 1988 Jun;69 ( Pt 6):1179-93
2838570
Biochem Biophys Res Commun. 1993 Dec 15;197(2):709-15
8267607
J Gen Virol. 1987 Nov;68 ( Pt 11):2925-31
2824669
Neuropharmacology. 2007 Mar;52(3):744-54
17095021
Mol Endocrinol. 1996 Jul;10(7):781-93
8813719
Int J Hyperthermia. 1997 Sep-Oct;13(5):547-57
9354939
J Neurochem. 1997 Jul;69(1):174-81
9202308
J Virol. 1996 Dec;70(12):8590-605
8970984
J Cell Biochem. 2001;83(4):563-73
11746500
J Biol Chem. 2000 Dec 8;275(49):38213-20
10982802
Biochem Biophys Res Commun. 2005 Jul 1;332(2):441-9
15894289
Differentiation. 1994 Apr;56(1-2):119-29
8026641
J Cell Physiol. 1999 Aug;180(2):255-62
10395295
J Neurochem. 2001 Jun;77(6):1664-7
11413250
Trends Neurosci. 1996 Jul;19(7):267-71
8799968
J Gen Virol. 1990 Jan;71 ( Pt 1):105-13
2154528
Neuron. 1992 Apr;8(4):757-65
1314623
Neuropharmacology. 2008 Sep;55(4):500-8
18619982
Eur J Biochem. 2004 Mar;271(6):1094-105
15009188