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Non-raft adenylyl cyclase 2 defines a cAMP signaling compartment that selectively regulates IL-6 expression in airway smooth muscle cells: differential regulation of gene expression by AC isoforms

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Abstract

Adenylyl cyclase (AC) isoforms differ in their tissue distribution, cellular localization, regulation, and protein interactions. Most cell types express multiple AC isoforms. We hypothesized that cAMP produced by different AC isoforms regulates unique cellular responses in human bronchial smooth muscle cells (BSMC). Overexpression of AC2, AC3, or AC6 had distinct effects on forskolin (Fsk)-induced expression of a number of known cAMP-responsive genes. These data show that different AC isoforms can differentially regulate gene expression. Most notable, overexpression and activation of AC2 enhanced interleukin 6 (IL-6) expression, but overexpression of AC3 or AC6 had no effect. IL-6 production by BSMC was induced by Fsk and select G protein-coupled receptor (GPCR) agonists, though IL-6 levels did not directly correlate with global cAMP levels. Treatment with PKA selective 6-Bnz-cAMP or Epac selective 8-CPT-2Me-cAMP cAMP analogs revealed a predominant role for PKA in cAMP-mediated induction of IL-6. IL-6 promoter mutations demonstrated that AP-1 and CRE transcription sites were required for Fsk to stimulate IL-6 expression. Our present study defines an AC2 cAMP signaling compartment that specifically regulates IL-6 expression in BSMC via Epac and PKA and demonstrates that other AC isoforms are excluded from this pool.

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Abbreviations

AC:

Adenylyl cyclase

cAMP:

Cyclic 3′,5′ adenosine monophosphate

BSMC:

Bronchial smooth muscle cells

Fsk:

Forskolin

IL-6:

Interleukin 6

GPCR:

G protein-coupled receptor

Epac:

Exchange protein activated by cAMP

PKA:

Protein kinase A

AP-1:

Activator protein 1

CRE:

cAMP response element

βAR:

Beta-adrenergic receptor

PKC:

Protein kinase C

CaM:

Calmodulin

Iso:

Isoproterenol

NECA:

5′-N-ethylcarboxamidoadenosine

AVP:

Arginine vasopressin

α-CGRP:

α-Calcitonin gene related peptide

PGD2:

Prostaglandin D2

IBMX:

3-Isobutyl-1-methylxanthine

CP:

Crossing point

AREG:

Amphiregulin

SCG2:

Secretogranin II

CCND1:

Cyclin D1

SST:

Somatostatin

EP2R:

Prostaglandin E2 receptor

Buta:

Butaprost

8-CPT-2Me-cAMP:

8-(4-Chlorophenylthio)-2′-O-methylad­enosine-3′,5′-cyclic monophosphate sodium salt

8-Br-cAMP:

8-Bromoadenosine 3′,5′-cyclic monophosphate

p38 MAPK:

p38 mitogen-activated protein kinase

PI3K:

Phosphatidylinositol 3-kinase

PGE2:

Prostaglandin E2

qRT-PCR:

Quantitative reverse transcriptase polymerase chain reaction

C/EBP:

CCAAT-enhancer-binding protein

RNAi:

RNA interference

PDE:

Phosphodiesterase

AKAP:

A kinase anchoring protein

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Acknowledgments

This work was supported by the National Institutes of Health National Heart, Lung and Blood Institute [Grant HL079166].

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Authors and Affiliations

  1. Department of Pharmacology, University of Tennessee Health Science Center, 874 Union Ave., Crowe 115, Memphis, TN, 38163, USA

    Amy S. Bogard, Anna V. Birg & Rennolds S. Ostrom

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  1. Amy S. Bogard
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  2. Anna V. Birg
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  3. Rennolds S. Ostrom
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Correspondence to Rennolds S. Ostrom.

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Bogard, A.S., Birg, A.V. & Ostrom, R.S. Non-raft adenylyl cyclase 2 defines a cAMP signaling compartment that selectively regulates IL-6 expression in airway smooth muscle cells: differential regulation of gene expression by AC isoforms. Naunyn-Schmiedeberg's Arch Pharmacol 387, 329–339 (2014). https://doi.org/10.1007/s00210-013-0950-4

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