A Shh/miR-206/BDNF Cascade Coordinates Innervation and Formation of Airway Smooth Muscle

Abstract

Dysfunctional neural control of airway smooth muscle (ASM) is involved in inflammatory diseases, such as asthma. However, neurogenesis in the lung is poorly understood. This study uses mouse models to investigate developmental mechanisms of ASM innervation, a process that is highly coordinated with ASM formation during lung branching morphogenesis. We show that brain-derived neurotrophic factor (BDNF) is an essential ASM-derived signal for innervation. Although BDNF mRNA expression is temporally dissociated with ASM formation and innervation, BDNF protein is coordinately produced through post-transcriptional suppression by miR-206. Using a combination of chemical and genetic approaches to modulate sonic hedgehog (Shh) signaling, a pathway essential for lung branching and ASM formation, we show that Shh signaling blocks miR-206 expression, which in turn increases BDNF protein expression. Together, our work uncovers a functional cascade that involves Shh, miR-206 and BDNF to coordinate ASM formation and innervation.

miR-326 Is Downstream of Sonic Hedgehog Signaling and Regulates the Expression of Gli2 and Smoothened

“…The importance of the crosstalk between miRNAs and the Shh signaling pathway during embryonic development is increasingly recognized (25,26). We reported that brain-derived neurotrophic factor is a target of miR-206.…”

“…We reported that brain-derived neurotrophic factor is a target of miR-206. Shh induces the expression of brain-derived neurotrophic factor by suppression of miR-206 in the formation and innervation of airway smooth muscle cells (26). miR-214 is expressed in somites of Zebrafish during early stages of segmentation and plays a role in muscle cell fate specification by down-regulating the expression of Sufu, a negative regulator of Hedgehog signaling (25).…”

“…Embryonic lung cultures were performed as described previously (26,30). For the various purposes, BGjb medium was supplemented with 5 mM cyclopamine (Calbiochem, Darmstadt, Germany), 1 mg/ml Smo agonist (SAG) (Calbiochem), or the TGF-b receptor 1 antagonist SB431542 (10 mM) (Sigma-Aldrich, St. Louis, MO).…”

“…In situ hybridization (ISH) of freshly isolated embryonic lungs, cultured lungs, or frozen sections of embryonic lungs were performed as described previously (26,31,33,34) by using DIG-labeled sense and antisense RNA probes or 59-DIG-labeled LNA miRNA probes.…”

Airway Contractility in the Precision-Cut Lung Slice after Cryopreservation

“…The PCLSs were then embedded in optical cutting temperature compound and sectioned to 10-mm-thick slices. Using a previously established protocol (28), the slices were immunostained with antibodies against the lung epithelium marker CC10 (1:500, T-18; Santa Cruz Biotechnology, Santa Cruz, CA) and the ASM marker a-smooth muscle actin (1:500, MS-113; Thermo Fisher Scientific). Viability is preserved in frozen-thawed precision-cut lung slice (PCLS).…”