Regulation of m2 Muscarinic Receptor Gene Expression by Platelet-Derived Growth Factor: Involvement of Extracellular Signal-Regulated Protein Kinases in the Down-Regulation Process
- Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College, London SW3 6LY, United Kingdom
Abstract
To study the role of mitogen-activated protein kinase in the regulation of M2 receptors, we studied the effect of platelet-derived growth factor (PDGF) on M2 receptor gene expression. PDGF (4 ng/ml) caused a time-dependent decrease in M2 receptor number and in m2 receptor mRNA levels in HEL 299 cells. The PDGF-induced loss in m2 mRNA required de novo protein synthesis and occurred through a decrease in the rate of transcription of the m2 receptor gene. The down-regulation of M2receptors was not accompanied by an uncoupling of the remaining receptors, indicating a large receptor reserve in these cells. Preincubations with the phosphatidylinositol 3-kinase inhibitor wortmannin, the protein kinase C inhibitor GF 109203X and the cAMP-dependent protein kinase inhibitor H-8 did not attenuate PDGF-induced down-regulation, indicating a lack of involvement of these enzymes in the down-regulation process. Activation of the extracellular signal-regulated protein kinase (ERK) 1 and 2 proteins was measured by an “in gel” phosphorylation assay. Carbachol did not activate ERK1 or 2, whereas PDGF and 4β-phorbol 13,14-dibutyrate resulted in a large increase in ERK1 and 2 activity along with a decrease in m2 mRNA. Preincubation with PD 098059, an inhibitor of mitogen-activated protein kinase kinase, inhibited PDGF- and 4β-phorbol 13,14-dibutyrate-mediated activation of ERK 1 and 2 in a concentration-dependent manner. The inhibitory action of PD 098059 was reflected at the mRNA level attenuating both PDGF- and 4β-phorbol 13,14-dibutyrate-mediated decreases in m2 mRNA. These results suggest a role of ERK1 and 2 in the regulation of muscarinic m2 receptor gene expression.
Footnotes
-
Send reprint requests to: Peter J. Barnes, Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College, Dovehouse Street, London SW3 6LY, UK. E-mail: p.j.barnes{at}ic.ac.uk
-
This work was funded by the Medical Research Council, UK, and by a European Union postdoctoral fellowship fund (E.-B.H.).
- Abbreviations:
- PKC
- protein kinase C
- ERK extracellular-signal regulated protein kinase
- GAPDH, glyceraldehyde-3-phosphate dehydrogenase
- GF 109203X
- 2-[1-(3-dimethylaminopropyl)-inol-3-yl]-3-(indol-3-yl)maleimide
- H-8
- N-[2-(methylamino)ethyl]-5′-isoquinoline-sulfonamide
- HBSS
- Hanks’ balanced salt solution
- MAPK
- mitogen-activated protein kinase
- NMS
- N-methyl-scopolamine
- PD 098059
- 2-(2-amino-3-methoxyphenyl)4H-1-benzopyran-4-one
- PI
- phosphatydylinositol
- PDGF
- platelet-derived growth factor BB chain
- PDBu
- 4β-phorbol 12,13-dibutyrate
- PKA
- cAMP-dependent protein kinase, TGF-β1, transforming growth factor β1
- SDS
- sodium dodecyl sulfate
- EGF
- epidermal growth factor
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
-
- Received March 26, 1997.
- Accepted August 21, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
