Pharmacological studies of the mechanism and function of interleukin-1beta-induced miRNA-146a expression in primary human airway smooth muscle

Abstract

Background: Despite the widespread induction of miR-146a during the innate immune response little is known regarding its biogenesis, function and mechanism. We have therefore examined the role of miR-146a during the interleukin (IL)-1beta-stimulated IL-6 and IL-8 release and proliferation in primary human airway smooth muscle (HASM) cells.

Methods: HASM cells were isolated from human lung re-section, cultured to a maximum of 3 - 6 passages and then exposed to IL-1beta. miR-146a expression were determined by qRT-PCR, IL-6 and IL-8 release by ELISA and proliferation using bromodeoxyuridine incorporation. The role of NF-kappaB and the MAP kinase pathways was assessed using pharmacological inhibitors of IKK2 (TPCA-1), JNK (SP600125), p38 MAP kinase (SB203580) and MEK-1/2 (PD98059). miR-146a function was determined following transfection of HASM with inhibitors and mimics using Amaxa electroporation.

Results: IL-1beta induced a time-dependent and prolonged 100-fold induction in miR-146a expression, which correlated with release of IL-6 and IL-8. Exposure to IL-1beta had no effect upon HASM proliferation. Pharmacological studies showed that expression of primary miR-146a was regulated at the transcriptional levels by NF-kappaB whilst post-transcriptional processing to mature miR-146a was regulated by MEK-1/2 and JNK-1/2. Functional studies indicated that IL-1beta-induced miR-146a expression does not negatively regulate IL-6 and IL-8 release or basal proliferation. However, inhibition of IL-1beta-induced IL-6 and IL-8 release was observed at the super-maximal intracellular miR-146a levels obtained by transfection with miR-146a mimics and indicates that studies using miRNA mimics can produce false positive results. Mechanistic studies showed that in the presence of super-maximal levels, the action of miR-146a mimics was mediated at a step following IL-6 and IL-8 mRNA transcription and not through down-regulation of IL-1 receptor associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6) protein expression, two predicted miR-146a targets involved in IL-1beta signalling.

Conclusions: We have shown that IL-1beta-induced miR-146a expression in HASM and that this was regulated at the transcriptional level by NF-kappaB and at the post-transcriptional level by the MEK-1/2 and JNK-1/2. Unlike previous reports, studies using miRNA inhibitors showed that miR-146a expression did not regulate IL-6 and IL-8 release or proliferation and suggest miR-146a function and mechanism is cell-type dependent.

Figure 1

IL-1β-induced a time- and concentration-dependent increase in miR-146a expression. HASM cells were exposed to either vehicle control or 1 ng/ml IL-1β for the indicated time or to the indicated IL-1β concentration for 24 h. The time dependent expression of miR-146a, miR-146a*, miR-146b and miR-155 (A) or the concentration dependent miR-146a expression (B) was determined by qRT-PCR. The results are expressed as the mean ± SEM of four independent experiments where * p < 0.05 versus time-matched controls.

Figure 2

IL-1β-induced a time- and concentration-dependent increase in primary miR-146a expression. HASM cells were exposed to either vehicle control or 1 ng/ml IL-1β for the indicated time or to the indicated IL-1β concentration for 24 h. The time- (A) and concentration- (B) dependent expression of primary miR-146a was determined by qRT-PCR. The results are expressed as the mean ± SEM of four independent experiments where * p < 0.05 versus time-matched controls.

Figure 3

IL-1β-induced a time- and concentration-dependent release of IL-6 and IL-8. HASM cells were exposed to either vehicle control or 1 ng/ml IL-1β for the indicated time (A) or to the indicated IL-1β concentration for 24 h (B) before measurement of IL-6 and IL-8 release. The results are expressed as the mean ± SEM of four independent experiments where * p < 0.05 versus time-matched controls.

Figure 4

Effect of inhibitors of IKK2 and MAP kinases upon IL-1β-induced IL-6 and IL-8 release. HASM cells were pre-treated for 60 min with the indicated concentrations of the inhibitors of IKK-2 (TPCA-1), MEK-1/2 (PD098059), JNK-1/2 (SP600125) and p38 MAP kinase (SB203580). Following exposure to vehicle control or IL-1β (1 ng/ml) for 24 h the release of IL-6 and IL-8 was determined by ELISA. Results are the mean ± SEM of 3 independent experiments where * p < 0.05 versus IL-1β-stimulated cells.

Figure 5

Effect of inhibitors of IKK2 and MAP kinases upon IL-1β-induced miR-146a and primary miR-146a expression. HASM cells were pre-treated for 60 min with the indicated concentrations of the inhibitors of IKK-2 (TPCA-1), MEK-1/2 (PD098059), JNK-1/2 (SP600125) and p38 MAP kinase (SB203580). Following exposure to vehicle control or IL-1β (1 ng/ml) for 24 h the expression of miR-146a and primary miR-146a was determined by qRT-PCR. Results are the mean ± SEM of 3 independent experiments where * p < 0.05 versus IL-1β-stimulated cells.

Figure 6

Effect of miR-146a inhibitors and mimics upon IL-1β-induced IL-6 and IL-8 release. HASM cells were electroporated in the presence of buffer (A-C), control inhibitor or miR-146a inhibitor (A), control mimic or miR-146a mimic (B) and control siRNA or an siRNA targeted at IL-6 (C). Cells were then exposed to vehicle control or 1 ng/ml IL-1β and the release of IL-6 and IL-8 was measured by ELISA at 24 h. The results are mean ± SEM of three independent experiments where * p < 0.05 versus IL-1β-stimulated cells.

Figure 7

Cellular miR-146a expression levels. HASM cells were electroporated in the presence of buffer, control mimic or miR-146a mimic. Following repeated washes, cells were exposed to vehicle control or 1 ng/ml IL-1β and the levels miR-146a were measured by TaqMan RT-PCR at 24 h. The results are mean ± SEM of three independent experiments.

Figure 8

Effect of miR-146a inhibitors and mimics upon IL-1β-induced proliferation and cell number. HASM cells were exposed to either vehicle control, the indicated FCS concentration or the indicated concentration of IL-1β for 48 h, 72 h or 96 h before measurement of proliferation (A) or cell number (B). In panel C, HASM cells were electroporated in the presence of buffer, control inhibitor or miR-146a inhibitor, control mimic or miR-146a mimic. Cells were then exposed to vehicle control or 1 ng/ml IL-1β and proliferation and cell number was measured at 48 h. These results are mean ± SEM of three independent experiments.

Figure 9

IRAK-1 and TRAF6 mRNA and protein expression following exposure to IL-1β and miR-146a mimic. HASM cells were transfection with either miR-146a mimics or control and then exposed to either vehicle control or 1 ng/ml IL-1β and the expression of IRAK-1 and TRAF6 mRNA (A) and protein (B) were detected at 24 h. The results in panel A are mean ± SEM of three independent experiments where * p < 0.05 versus time-matched controls whilst panel B is representative of three independent experiments.

Figure 10

Effect of miR-146a mimics upon IL-1β-induced IL-6 and IL-8 mRNA expression. HASM cells were electroporated in the presence of buffer, control mimic or miR-146a mimic. Cells were then exposed to vehicle control or 1 ng/ml IL-1β and the expression of IL-6 and IL-8 mRNA was measured by qRT-PCR at 24 h. The results are the mean ± SEM of three independent experiments. Panel A shows the absolute increases in IL-6 and IL-8 mRNA whilst the results in panel B are expressed as a % compared to IL-1β stimulation (100%).