Cytokines regulate beta-2-adrenergic receptor responsiveness in airway smooth muscle via multiple PKA- and EP2 receptor-dependent mechanisms.
Beta2AR desensitization in airway smooth muscle (ASM) mediated by airway inflammation has been proposed to contribute to asthma pathogenesis and diminished efficacy of beta-agonist therapy. Mechanistic insight into this phenomenon is largely conceptual and lacks direct empirical evidence. Here, we employ molecular and genetic strategies to reveal mechanisms mediating cytokine effects on ASM beta2AR responsiveness. Ectopic expression of inhibitory peptide (PKI-GFP) or a mutant regulatory subunit of PKA (RevAB-GFP) effectively inhibited intracellular PKA activity in cultured human ASM cells and enhanced beta2AR responsiveness by mitigating both agonist-specific (beta-agonist-mediated) desensitization and cytokine (IL-1beta and TNF-alpha)-induced heterologous desensitization via actions on multiple targets. In the absence of cytokine treatment, PKA inhibition increased beta2AR-mediated signaling by increasing both beta2AR-G protein coupling and intrinsic adenylyl cyclase activity. PKI-GFP and RevAB-GFP expression also conferred resistance to cytokine-promoted beta2AR-G protein uncoupling and disrupted feed-forward mechanisms of PKA activation by attenuating the induction of COX-2 and PGE2. Cytokine treatment of tracheal ring preparations from wild-type mice resulted in a profound loss of beta-agonist-mediated relaxation of methacholine-contracted rings, whereas rings from EP2 receptor knockout mice were largely resistant to cytokine-mediated beta2AR desensitization. These findings identify EP2 receptor- and PKA-dependent mechanisms as the principal effectors of cytokine-mediated beta2AR desensitization in ASM.
Guo
Manhong
M
Department of Internal Medicine and Center for Human Genomics, Wake Forest University Health Sciences Center, Winston-Salem, NC 27157, USA.
Pascual
Rodolfo M
RM
Wang
Siwei
S
Fontana
Mary F
MF
Valancius
Cathryn A
CA
Panettieri
Reynold A
RA
Tilley
Stephen L
SL
Penn
Raymond B
RB
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018341:Receptors, Adrenergic, alpha-2 / Q000502:physiology*; D018078:Receptors, Prostaglandin E / Q000378:metabolism*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D014132:Trachea / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism
D016207:Cytokines; C545644:PTGER2 protein, human; C545645:Ptger2 protein, mouse; D018341:Receptors, Adrenergic, alpha-2; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C