Figure 1.
Relative expression of Scap and Insig1 in ScapΔ/Δ and Insig1/2Δ/Δ mice.
Scap (A) and Insig1 (B) mRNA were measured by mRNA microarray analysis and qPCR. Total lung RNA was isolated from ScapΔ/Δ, Insig1/2Δ/Δ and littermate controls at E17.5, E18.5 and PN1. Data were normalized to 18S and presented as mean ± S.E.; n = 2–3/group.
Figure 2.
Altered lung surfactant SatPC content in ScapΔ/Δ mice.
SatPC (A), total cholesterol (C) and triglycerides (E) were measured using whole lung samples from control (open bars) and ScapΔ/Δ mice (closed bars) at E17.5, E18.5 and PN1. *p<0.05. n = 5–8/group. Correlation coefficients between Srebf-1c mRNA and SatPC (B), cholesterol (D) and triglyceride levels (F) were measured at age PN1, n = 5–8/group.
Figure 3.
Genes and biological processes affected by Scap deletion in the developing lung.
A) Differentially expressed genes in ScapΔ/Δ lungs were identified by mRNA microarray analysis. Heatmap analysis shows the two-dimensional clustering of 1079 mRNAs that were significantly altered in ScapΔ/Δ mice compared with controls. The intensity in the red and green color ranges indicates increased versus decreased mRNAs, respectively. Each row represents a single mRNA, and each column represents an average fold change between knockout and control mice at the specified gestational age. B and C) Biological processes significantly influenced by epithelial Scap deletion were identified using ToppGene Suite (http://toppgene.cchmc.org). Enriched bioprocesses in genes induced (B) or suppressed (C) in ScapΔ/Δ lungs are represented as red bars and green bars respectively. Statistical significance of each bioprocess was presented using negative log2 transformation of P value. D and E) qPCR analysis confirmed RNA microarray data demonstrating altered expression of genes involved in lipid biosynthesis/transport (D) and in lung development and maturation (E) in ScapΔ/Δ mice (filled bars) compared with control (open bars), n = 3/group, *p<0.05.
Table 1.
Enriched functions altered by Scap deletion in perinatal lung.
Table 2.
Enriched canonical pathways altered by Scap deletion in perinatal lung.
Figure 4.
SREBP transcriptional network regulates lipogenic pathways.
A) Clustering analysis of genes differentially expressed in ScapΔ/Δ mice vs. control at E17.5 and PN1 using a Self-organizing map. Genes from Clusters 5 and 7 were functionally enriched in lipid biosynthesis and metabolism. B) Upstream network analysis of Cluster 5 genes. Genes down-regulated in response to Scap deletion are shown as green nodes, genes that were unchanged but predicted to be involved in the process are shown in orange (activation) and blue (inhibition) respectively. C) Upstream network analysis of Cluster 7 genes. In addition to Srebf1/2, Cebpa, Foxa2 and Foxo1 were decreased in response to Scap deletion (shown as green nodes). Genes that were unaltered but predicted to be involved in the regulation of Cluster 7 genes, including Por and Pten (orange nodes indicate activation), Tgfb1, Nkx2-1, Ifng, Pparg, Ctnnb1 and Hif1a (blue indicates inhibition), are shown.
Figure 5.
Putative SREBP co-factors mediating perinatal lung lipid homeostasis.
A SREBP related transcriptional network was constructed based on the promoter analysis of genes within Clusters 5 and 7. Each connection between a matrix family (white rectangle) and a target gene (blue oval) represents at least one predicted binding site for a given transcription factor on the promoter region of that target gene.
Table 3.
Potential upstream regulators of SCAP/SREBP pathway (ranked by P-value).
Table 4.
Potential upstream regulators of SCAP/SREBP pathway (ranked by Z-score).
Figure 6.
Epithelial Scap deletion in the perinatal lung suppresses Wnt/β-catenin signaling.
(A) A schematic representation of mRNAs changed in the canonical Wnt/β-catenin pathway in ScapΔ/Δ perinatal lung (E17.5) is shown. Green nodes indicate mRNAs that were decreased in ScapΔ/Δ lungs with respect to control lungs. Wnt pathway genes that were not changed in response to Scap deletion are depicted as empty nodes. (B) Heatmap demonstrates decreased expression of genes encoding a number of key components in the Wnt/β-catenin pathway in ScapΔ/Δ vs. control mice, n = 3/group.
Figure 7.
Scap modulates expression of Wnt pathway components.
A and B) qPCR analysis demonstrates altered expression of Wnt signaling genes in ScapΔ/Δ E17.5 (A) and PN1 (B) whole lung tissue compared to littermate controls. Results were normalized to 18 s RNA, n = 3–4/group, *p<0.05 vs. control at the respective time point. C and D) qPCR analysis demonstrates decreased expression of Scap (C) and of Wnt pathway (D) genes in MLE15 cells following transfection with negative control (neg con) and two distinct Scap siRNAs (si1 and si2). Results were normalized to 18 s, n = 6 samples/group representing 3 independent experiments, *p<0.05 vs negative control. ND = non-detectable.
Table 5.
Glucocorticoid receptor signaling was suppressed in ScapΔ/Δ mice in perinatal lung at E17.5.
Table 6.
Differentially expressed miRNA in E18.5 ScapΔ/Δ vs. control mouse lungs.