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

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.

Fig. 1

cAMP accumulation in control and AC overexpressing BSMC. cAMP EIA’s were used to measure cAMP following 10 min exposure to 1 μM Fsk or vehicle in the presence of broad-spectrum PDE inhibitor IBMX. mean ± SEM, n=3. * indicates p<.05 compared to basal.

Fig. 2

a: Fsk-induced gene regulation in AC overexpressing BSMC compared to control BSMC. RT² q PCR arrays (SA Biosciences) were used to measure mRNA levels following 24 h treatment with 1μM Fsk. mRNA levels of AC overexpressing, Fsk-treated cells are expressed as fold change with respect to lacZ (control), Fsk- treated. Amphiregulin (AREG), Secretogranin II (SCG2), Cyclin D1 (CCND1), Interleukin 6 (IL-6), Somatostatin (SST). Data are presented as fold change over lacZ, mean ± SEM, n=3. b: IL-6 protein production in AC2 or AC6 overexpressing BSMC. IL-6 protein in cell culture media was measured by ELISA following 24 h treatment with 1 μM of the indicated drug. Data are presented as % change with respect to lacZ, mean ± SEM, n=3. * indicates p<.05 compared to lacZ

Fig. 2

a: Fsk-induced gene regulation in AC overexpressing BSMC compared to control BSMC. RT² q PCR arrays (SA Biosciences) were used to measure mRNA levels following 24 h treatment with 1μM Fsk. mRNA levels of AC overexpressing, Fsk-treated cells are expressed as fold change with respect to lacZ (control), Fsk- treated. Amphiregulin (AREG), Secretogranin II (SCG2), Cyclin D1 (CCND1), Interleukin 6 (IL-6), Somatostatin (SST). Data are presented as fold change over lacZ, mean ± SEM, n=3. b: IL-6 protein production in AC2 or AC6 overexpressing BSMC. IL-6 protein in cell culture media was measured by ELISA following 24 h treatment with 1 μM of the indicated drug. Data are presented as % change with respect to lacZ, mean ± SEM, n=3. * indicates p<.05 compared to lacZ

Fig. 3

a: IL-6 protein production in response to Gα_s-coupled receptor agonists. BSMC were treated with the indicated drug for 24 h, and IL-6 secreted into culture media was measured by ELISA. 1μM Fsk, AVP, CGRP, glucagon, PGD₂, substance P; 10 μM ATPƔS, BRL 37344, NECA. cAMP measured by EIA. b: cAMP production in response to Gα_s – coupled receptor agonists. BSMC were pretreated with 0.2 mM IBMX prior to 10 min agonist exposure: 1μM Fsk, AVP, CGRP, glucagon, PGD₂, substance P; or 10 μM ATPƔS, BRL 37344, NECA. cAMP was measured by EIA. Dashed line represents basal level. Mean ± SEM, n=3–4. * indicates p<.05 compared to vehicle

Fig. 3

a: IL-6 protein production in response to Gα_s-coupled receptor agonists. BSMC were treated with the indicated drug for 24 h, and IL-6 secreted into culture media was measured by ELISA. 1μM Fsk, AVP, CGRP, glucagon, PGD₂, substance P; 10 μM ATPƔS, BRL 37344, NECA. cAMP measured by EIA. b: cAMP production in response to Gα_s – coupled receptor agonists. BSMC were pretreated with 0.2 mM IBMX prior to 10 min agonist exposure: 1μM Fsk, AVP, CGRP, glucagon, PGD₂, substance P; or 10 μM ATPƔS, BRL 37344, NECA. cAMP was measured by EIA. Dashed line represents basal level. Mean ± SEM, n=3–4. * indicates p<.05 compared to vehicle

Fig. 4

cAMP and IL-6 production in BSMC by GPCR agonists alone or with concurrent Fsk treatment. a: Cells were treated with 0.1 μM of the indicated drug with or without concurrent treatment with 0.1 μM Fsk for 10 min in the presence of 0.2 mM IBMX. cAMP was measured by EIA.. b: Cells were treated with 0.1 μM of the indicated drug with or without concurrent treatment with 0.1 μM Fsk. IL-6 in culture media was measured by ELISA following 24 h drug treatment. Mean ± SEM, n=3 * indicates p<.05 compared to single drug

Fig. 4

cAMP and IL-6 production in BSMC by GPCR agonists alone or with concurrent Fsk treatment. a: Cells were treated with 0.1 μM of the indicated drug with or without concurrent treatment with 0.1 μM Fsk for 10 min in the presence of 0.2 mM IBMX. cAMP was measured by EIA.. b: Cells were treated with 0.1 μM of the indicated drug with or without concurrent treatment with 0.1 μM Fsk. IL-6 in culture media was measured by ELISA following 24 h drug treatment. Mean ± SEM, n=3 * indicates p<.05 compared to single drug

Fig. 5

IL-6 protein production in response to non-selective, Epac selective, or PKA selective cAMP analogs. BSMC were treated with the indicated concentration of either 8-Br-cAMP (non-selective), 8-CPT-2Me-cAMP (Epac selective), or 6-Bnz-cAMP (PKA selective) for 24 h and IL-6 in culture supernatant was measured by ELISA. Dashed line represents vehicle. Mean ± SEM, n=3–4 * indicates p<.05 compared to vehicle

Fig. 6

Fsk-induced IL-6 production with inhibition of PKC (GF 109203X), p38 MAPK (SB 202190) or PI3K (wortmanin). BSMC were pretreated with 10 μM of the indicated inhibitor for 1 h prior to addition of 1 μM Fsk. RNA was isolated after 1 h Fsk treatment and measured by qRT-PCR. Data presented as fold over vehicle, mean ± SEM, n=3

Fig. 7

Fsk-induced promoter activation of IL-6 promoter mutants. IL-6 promoter activity was measured by luciferase assay in BSMC that were transfected with 1168 bp wild type or mutated human IL-6 promoters driving expression of luciferase. Luciferase activity in cell lysate was measured following 6 h treatment with 1 μM Fsk. Data is presented as fold over the basal wild-type promoter activity, mean ± SEM, n=11. * indicates p<0.05 compared to Fsk wild-type promoter