A critical role for miR-142 in alveolar epithelial lineage formation

Abstract

The development of a functional lung, capable of gas exchange, requires proper alveologenesis. Mechanisms regulating AT1 and AT2 cell maturation are poorly defined. We report the activation of the glucocorticoid pathway in an in vitro alveolar epithelial lineage differentiation assay led to increased AT2 marker Sftpc and decreased miR-142 expression.Using a constitutive KO mouse model, we further demonstrate a relative increase of AT2 and a decrease in AT1 cell number with a global decrease of AT2 gene profile signature in miR-142 KO AT2 cells. Over-expression of miR-142 in alveolar progenitor cells in vivo led to an opposite effect. Examination of the KO lungs at E18.5, revealed enhanced expression miR-142 targets like Apc, Ep300 and Kras associated with increased Ctnnb1 and p-Erk signaling. Pharmacological inhibition of Ep300-Ctnnb1 in vitro prevented an increase in Sftpc expression triggered by loss of miR-142. These results together suggest glucocorticoid-miR-142-p300 signaling axis controls pneumocyte maturation. 5 Material and Methods MiceThe miR-142 KO mice on a pure C57BL6 background were previously generated (Shrestha et al., 2015). miR-142 heterozygous males and females were crossed to generate KO and WT littermate embryos at different stages. We also generated a knockin of miR-142 in the Rosa26R locus. Rosa26-LoxP-STOP-loxP-miR-142 (aka Rosa26R miR-142/miR-142 ) mice were crossed with Sftpc CreERT2/+ mice (kind gift from Dr. Chapman) to generate control (Sftpc +/+ ; Rosa26 miR-142/+ ) and Experimental (Sftpc CreERT2/+ ; Rosa26R miR-142/+ ) embryos. Pregnant females were injected with tamoxifen IP (0.1 mg/ g of mouse) at E14.5 and E15.5 and collected at E18.5. Animal experiments were approved by the Federal Authorities of Animal Research of the Regierungspräsidium Giessen, Hessen, Germany with approved protocol numbers 405_M, 423_M, G 47/2017 and G54/2017 Quantitative Real-time PCR Analysis Briefly, total RNA from embryonic lungs were extracted and used for cDNA synthesis. RT-PCR for mRNA was carried out using Quantitative Reverse Transcription kit (205314; Qiagen) and Taqman MicroRNA Reverse Transcription kit (4366597; Applied Biosystem)was used in RT-PCR for miRNA. In both cases, reactions were assembled following the manufacturer's recommendations. qPCR was performed on a Light Cycler 480 system (Roche Applied Science). The TaqMan microRNA assay (Applied Biosystem) was used for screening the differential expression of miRNAs whereas SYBR Green (Platinum SYBR Green qPCR SuperMix-UDG Invitrogen) was used for the analysis of mRNA expression. U6and Hprt (Hypoxanthine phosphporibosyl transferase1) were used as a reference for normalization of miRNA and mRNA respectively. Results were collected from at least three lung samples and each reaction was run in triplicate. Primers for miR142-3p, miR142-5p and U6 were obtained from Applied Biosystems. In-situ hybridization and Immunofluorescence stainingFreshly isolated lungs were washed in PBS, then fixed in 4% PFA, gradually dehydrated in ethanol, impregnated...