Abstract
Muscarinic type 3 receptor (M3R) plays a pivotal role in the induction of glandular fluid secretions. Although M3R is often the target of autoantibodies in Sjögren's syndrome (SjS), chemical agonists for M3R are clinically used to stimulate saliva secretion in patients with SjS. Aside from its activity in promoting glandular fluid secretion, however, it is unclear whether activation of M3R is related to other biological events in SjS. This study aimed to investigate the cytoprotective effect of chemical agonist-mediated M3R activation on apoptosis induced in human salivary gland (HSG) cells. Carbachol (CCh), a muscarinic receptor-specific agonist, abrogated tumor necrosis factor α/interferon γ-induced apoptosis through pathways involving caspase 3/7, but its cytoprotective effect was decreased by a M3R antagonist, a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) inhibitor, a phosphatidylinositol 3-kinase/Akt inhibitor, or an epidermal growth factor receptor (EGFR) inhibitor. Ligation of M3R with CCh transactivated EGFR and phosphorylated ERK and Akt, the downstream targets of EGFR. Inhibition of intracellular calcium release or protein kinase C δ, both of which are involved in the cell signaling of M3R-mediated fluid secretion, did not affect CCh-induced ERK or Akt phosphorylation. CCh stimulated Src phosphorylation and binding to EGFR. A Src inhibitor attenuated the CCh/M3R-induced cytoprotective effect and EGFR transactivation cascades. Overall, these results indicated that CCh/M3R induced transactivation of EGFR through Src activation leading to ERK and Akt phosphorylation, which in turn suppressed caspase 3/7-mediated apoptotic signals in HSG cells. This study, for the first time, proposes that CCh-mediated M3R activation can promote not only fluid secretion but also survival of salivary gland cells in the inflammatory context of SjS.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This study was supported by the National Institutes of Health National Institute of Dental and Craniofacial Research [Grant DE019644].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- SjS
- Sjögren's syndrome
- HSG
- human salivary gland
- M3R
- muscarinic type 3 receptor
- CCh
- carbachol
- ERK
- extracellular signal-regulated kinase
- EGFR
- epidermal growth factor receptor
- EGF
- epidermal growth factor
- siRNA
- small interfering RNA
- MEK
- mitogen-activated protein kinase kinase
- NC
- nitrocellulose
- TNFα
- tumor necrosis factor α
- IFNγ
- interferon γ
- AMPK
- AMP-activated protein kinase
- HB
- heparin-binding
- TUNEL
- terminal deoxynucleotidyl transferase dUTP nick end labeling
- GPCR
- G protein-coupled receptor
- PKC
- protein kinase C
- PI3K
- phosphatidylinositol 3-kinase
- 4-DAMP
- 4-diphenylacetoxy-N-methylpiperidine methiodide
- BAPTA-AM
- 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester
- LY294002
- 2-morpholin-4-yl-8-phenylchromen-4-one
- AG1478
- N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinamine
- PP2
- 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine
- GF109203X
- 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide
- MTS
- 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene.
- Received December 21, 2011.
- Accepted April 17, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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