A CREB-mediated increase in miRNA let-7f during prolonged β-agonist exposure: a novel mechanism of β2-adrenergic receptor down-regulation in airway smooth muscle

Abstract

β₂-Adrenergic receptors (β₂ARs) desensitize during continuous agonist activation, which manifests clinically as tachyphylaxis. β-Agonist desensitization of β₂ARs in human airway smooth muscle (HASM) cells is recognized in the treatment of asthma and may be related to poor outcomes. Rapid events in desensitization include receptor phosphorylation and internalization, but mechanisms responsible for the decrease in receptor protein after prolonged agonist exposure (down-regulation) are ill defined. The microRNA (miRNA) let-7f regulates β₂AR expression by translational repression. In cultured HASM cells from nonasthmatic and asthmatic lungs, 18 h of β-agonist exposure increased let-7f by 2-3-fold, concomitant with a ∼90% decrease in β₂ARs. Inhibition of let-7f attenuated this down-regulation response by ∼50%. The let-7f increase was found to be cAMP/PKA-dependent. The mechanism of the let-7f increase was found by chromatin immunoprecipitation to be from activated cAMP response element-binding protein (CREB) binding to the let-7f promoter, thereby increasing let-7f expression. Knockdown of CREB attenuated agonist-promoted β₂AR down-regulation by ∼50%. Thus, β₂AR down-regulation occurs as a result of not only internalized receptor degradation but also a novel cAMP/PKA/CREB-mediated increase in let-7f, which causes enhanced repression of the β₂AR gene, adrenoreceptor β₂ ( ADRB2) translation and represents ∼50% of the net loss of receptors observed after prolonged agonist exposure. This mechanism is apparent in asthmatic HASM cells, indicating relevance in a disease model.-Kim, D., Cho, S., Woo, J. A., Liggett, S. B. A CREB-mediated increase in miRNA let-7f during prolonged β-agonist exposure: a novel mechanism of β₂-adrenergic receptor down-regulation in airway smooth muscle.

Keywords: G protein; asthma; microRNA; tachyphylaxis.

Figure 1

Agonist regulation of β₂AR and let-7f in HASM cells. A, B) HASM cells from nonasthmatic or asthmatic lung donors were maintained in culture and treated with 100 µM ascorbic acid (control) or ascorbic acid with 30 µM ISO for 18 h at 37°C. RNA and protein were derived from these cells and let-7f miRNA and β₂AR protein expression were determined as described in Materials and Methods (n = 4 experiments each from a different donor). C) The 2 greatest and 2 least changes in β₂AR expression found in experiments from panels A and B are indicated with the changes in let-7f levels. D) ADRB2 mRNA is not altered in HASM cells by 18 h ISO treatment (n = 4 experiments from the D9 line). E) Knockdown of let-7f attenuates ISO-promoted down-regulation of β₂AR in HASM cells (n = 6 experiments) *P < 0.01.

Figure 2

cAMP/PKA-dependent agonist regulation of let-7f in HASM cells. A, B) Cultured HASM cells derived from asthmatic (A) and nonasthmatic (B) donor lungs were treated with 10 µM H89, 30 µM ISO, H89 + ISO, 10 µM forskolin, or H89 + forskolin for 18 h at 37°C and let-7f miRNA expression was determined by quantitative RT-PCR. The expression of let-7f miRNA was increased by ISO and forskolin, which was blocked by the PKA inhibitor H89 (n = 4 experiments, each from a different donor). C, D) The same cells in panels A and B were treated with 100 µM cell-permeable cAMP analog dibutyryl cAMP (dB-cAMP) and let-7f miRNA expression was determined by quantitative RT-PCR. dB-cAMP increased let-7f expression, which was blocked by H89. (n = 4 experiments, each from a different donor) *P < 0.01, ^#P > 0.05.

Figure 3

CREB modulates let-7f expression and long-term agonist-promoted down-regulation of β₂AR in HASM cells. A) Transfection of CREB increases let-7f expression in HASM cells (n = 3). B) Knockdown of CREB by siCREB ablates agonist-promoted increases in let-7f (n = 4). C) Knockdown of CREB impairs agonist-promoted down-regulation of β₂AR. A representative experiment (1 of 4) shows the silencing of CREB expression by siCREB, and the lack of ISO-promoted down-regulation under these conditions. D) Knockdown of CREB attenuates ISO-promoted down-regulation of β₂AR (n = 5). All experiments performed with ISO used a concentration of 30 µM with an exposure period of 18 h at 37°C. *P < 0.01.

Figure 4

Localization of CREB binding sites in the let-7f pri-miRNA promoter. A) Potential CREB binding sites in the 5′-upstream region of pri-let-7f as identified by JASPER. B) Chromatin immunoprecipitation results using a CREB antibody and pri-let-7f primers for PCR (see Materials and Methods). Sites 1, 2, and 3 were identified as CREB binding sites based on the presence of a PCR product. Results are representative of 4 experiments performed. IgG was utilized as a negative control in the immunoprecipitation step. The input refers to cell extracts prior to immunoprecipitation and the primers were validated by the presence of PCR products.

Figure 5

Summary of the mechanisms responsible for β₂AR down-regulation by CREB-mediated/let-7f processes in HASM cells. HASM cells express β₂AR at a stable level when receptor production and degradation are at an equilibrium. Upon agonist activation of β₂AR, intracellular cAMP levels increase due to receptor-G_s coupling. cAMP activates PKA, which in turn activates the CREB transcription factor. The transcription factor subsequently binds to the promoter of pri-let-7f, increasing expression of let-7f. Then let-7f binds to the 3′UTR of ADRB2 and represses translation, contributing to agonist-promoted long-term down-regulation of β₂AR. Another mechanism of down-regulation that is apparent in HASM cells is the degradation of β-arrestin–mediated internalized receptors.