Abstract
Cyclic AMP Response Element-Binding Protein 1 (Creb1) is a transcription factor that mediates cyclic adenosine 3′, 5′-monophosphate (cAMP) signalling in many tissues. Creb1−/− mice die at birth due to respiratory failure and previous genome-wide microarray analysis of E17.5 Creb1−/− fetal mouse lung identified important Creb1-regulated gene targets during lung development. The lipogenic enzymes stearoyl-CoA desaturase 1 (Scd1) and fatty acid synthase (Fasn) showed highly reduced gene expression in Creb1−/− lungs. We therefore hypothesized that Creb1 plays a crucial role in the transcriptional regulation of genes involved in pulmonary lipid biosynthetic pathways during lung development. In this study we confirmed that Scd1 and Fasn mRNA levels were down regulated in the E17.5 Creb1−/− mouse lung while the lipogenic-associated transcription factors SrebpF1, C/ebpα and Pparγ were increased. In vivo studies using germline (Creb1−/−) and lung epithelial-specific (Creb1EpiΔ/Δ) Creb1 knockout mice showed strongly reduced Scd1, but not Fasn gene expression and protein levels in lung epithelial cells. In vitro studies using mouse MLE-15 epithelial cells showed that forskolin-mediated activation of Creb1 increased both Scd1 gene expression and protein synthesis. Additionally, MLE15 cells transfected with a dominant-negative ACreb vector blocked forskolin-mediated stimulation of Scd1 gene expression. Lipid profiling in MLE15 cells showed that dominant-negative ACreb suppressed forskolin-induced desaturation of ether linked lipids to produce plasmalogens, as well as levels of phosphatidylethanolamine, ceramide and lysophosphatidylcholine. Taken together these results demonstrate that Creb1 is essential for the induction and maintenance of Scd1 in developing fetal mouse lung epithelial cells.
Highlights
Prematurity is a major complication for newborn infants and accounts for,60% of the perinatal morbidity and mortality associated with birth
Microarray analysis of the E17.5 Creb12/2 mouse lung and littermate controls identified two lipogenic gene targets: stearoyl-CoA desaturase 1 (Scd1) (7.8 fold, p,0.05) and fatty acid synthase (Fasn) (1.9 fold, p,0.05) to be significantly downregulated in Creb12/2 fetal mice (Table 1), that was subsequently verified by qPCR (Figure 1A)
Gene expression levels of these lipogenic genes and transcription factors were found to be developmentally regulated during normal lung development, with very low expression before E16.5, but thereafter there is a marked increase in mRNA levels until after birth (Figure 1C, D, E, F, G)
Summary
Prematurity is a major complication for newborn infants and accounts for ,60% of the perinatal morbidity and mortality associated with birth. This is primarily due to an incomplete development of the lung which cannot adequately fulfil the demands of oxygenation for the body. A key event of late lung development is the differentiation and maturation of the type II alveolar epithelial cell (AEC) in the distal lung, which primarily functions to synthesize and secrete surfactant into the airways. Lung surfactant is composed of approximately 90% phospholipids, and 10% surfactant associated proteins [1,2] This complex mixture reduces the surface tension at the air-liquid interface after birth that prevents alveolar collapse and allows normal lung function. The genetic programs which drive pulmonary morphogenesis, and in particular stimulate epithelial cell differentiation and surfactant production in the lung are controlled by the actions of specific transcription factors, which regulate a complex array of gene expression networks
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