An essential role of intestinal cell kinase in lung development is linked to the perinatal lethality of human ECO syndrome

Abstract

Human endocrine-cerebro-osteodysplasia (ECO) syndrome, caused by the loss-of-function mutation R272Q in the intestinal cell kinase (ICK) gene, is a neonatal-lethal developmental disorder. To elucidate the molecular basis of ECO syndrome, we constructed an Ick R272Q knock-in mouse model that recapitulates ECO pathological phenotypes. Newborns bearing Ick R272Q homozygous mutations die at birth due to respiratory distress. Ick mutant lungs exhibit not only impaired branching morphogenesis associated with reduced mesenchymal proliferation but also significant airspace deficiency in primitive alveoli concomitant with abnormal interstitial mesenchymal differentiation. ICK dysfunction induces elongated primary cilia and perturbs ciliary Hedgehog signaling and autophagy during lung sacculation. Our study identifies an essential role for ICK in lung development and advances the mechanistic understanding of ECO syndrome.

Keywords: Hedgehog signaling; autophagy; endocrine-cerebro-osteodysplasia syndrome; intestinal cell kinase; lung development; primary cilium.

Fig. 1

Ick R272Q knock-in mouse model for human ECO syndrome displayed a hypoplastic lung with abnormal air saccular structures. (A) Diagram of the targeting strategy used to generate the Ick R272Q knock-in allele and the genotyping strategy used to determine the wild-type and the targeted Ick alleles. (B) E18.5 embryos of three genotypes based on tail DNA PCR results. Scale bar, 1cm. (C) Genomic DNA sequencing data confirming the presence of the R272Q mutation in mouse Ick gene. (D) Gross view of neonatal lungs at P0 and Hematoxylin & Eosin-stained lung tissue sections. Scale bar, 100 μm. (E) Quantification of lung saccular areas in E18.5 lungs (Mean ± SD, n=4, *P<0.01). (F–K) E18.5 lung tissue sections immuno-stained with alveolar type 1 and type 2 cell markers. Scale bar, 20 μm. (L) Assessing the ICK catalytic activity using its specific in vivo target, the phospho-Scythe^T1080 signal. Top blot: GST-ICK wild type (WT) or kinase dead (KD) and HA-Scythe wild type (WT) or T1080A mutant were co-expressed in HEK293T cells. HA-Scythe was purified and analyzed for p-Scythe^T1080 signal. Bottom blot: Equal amount of total proteins extracted from E18.5 lungs were blotted for p-T1080 specific and total Scythe signals.

Fig. 2

Ick^R272Q/R272Q mutant lung exhibited impaired branching associated with reduced distal mesenchymal proliferation. (A–B) Whole-mount lungs isolated from E13.5 embryos. (A'–B') Blow-up images illustrating lung buds, as denoted by the asterisk symbol. (C) Quantification of the number of lung buds per area indicates about 50% reduction in the number of branching points in Ick mutant lungs. Shown are representative images and data from five pairs of E12.5–13.5 wild type and Ick mutant lungs. Scare bars: 500 μm (A–B), 250 μm (A'–B'). (D–E) E12.5 lung tissue sections immuno-stained with proliferation marker Ki67. Scale bar, 50 μm. (F) Quantification of Ki67 and phospho-Histone H3 positive cells in E12.5 lung epithelium and mesenchyme indicates a significant decrease in the number of proliferating mesenchymal cells in Ick mutant lungs during branching morphogenesis (Mean ± SD, n=3, *P<0.05, **P<0.01).

Fig. 3

Ick^R272Q/R272Q mutant lung displayed an excessive amount of smooth muscle actin (SMA)-positive mesenchymal cells in the hypercellular interstitium. (A–B) E18.5 lung tissue sections immuno-stained for α-smooth muscle actin (α-SMA). (A´–B´) High magnification images showing an excess of SMA-positive cells in Ick mutant lung interstitium. Scale bars, 20 μm. (C) Western blots of α-SMA, E-cadherin (CDH1), β-Actin, SFTPC, total and phospho-Smad2/3 signals in E18.5 lung tissue extracts. Quantification data of α-SMA signals against β-Actin and phospho-Smad2 against total Smad2 indicate up-regulated expression of α-SMA proteins and down-regulated phospho-Smad2 signals in Ick mutant lungs (Mean ± SD, n=6, *P<0.01, **P<0.001).

Fig. 4

Loss of ICK function induced elongated primary cilia. (A, B) Lung tissue sections from Ick^+/+ and Ick^R272Q/R272Q E18.5 embryos stained for the primary cilium marker Arl13B. (A´, B´) Blow up images of a primary cilium. Scale bar, 10 μm. (C) Ciliary length in E18.5 Ick^R272Q/R272Q mutant (2.53 ± 0.55 μm, n=64 cilia) and Ick^+/+ wild type (1.53 ± 0.32 μm, n=87 cilia) lungs were measured using Image J. **P<0.001. (D) Representative scanning electron micrographs showing elongated primary cilium in E18.5 Ick ^R272Q/^R272Q mutant lung. Scale bar, 500 nm. (E–L) Immunofluorescence images of primary cilium marker (Acetylated tubulin and Arl13B) staining on mouse embryonic fibroblast (MEF) cells isolated from Ick mutant and wild type littermate embryos. Scale bar, 50 μm. (M) Ciliary length in Ick^R272Q/R272Q mutant MEF cells (3.56 ± 0.97 μm, n=30 cells) and wild type MEF cells (2.55 ± 0.6 μm, n=30 cells) were measured using Image J. *P < 0.01.

Fig. 5

ICK dysfunction altered ciliary Hedgehog signaling and autophagy. (A–D) E18.5 lung tissue sections immuno-stained for Gli2 and Ptch2. (A´–D´) High magnification images showing elevated expression of Gli2 and Ptch2 in a subset of cells in Ick R272Q homozygous mutant lungs. Scale bars: 20 μm (A–D), 10 μm (A´–D´). (E) Relative mRNA levels of the Sonic Hedgehog pathway components in E18.5 Ick R272Q homozygous mutant versus wild type lungs (Mean ± SD, n=6, *P<0.05). (F) Western blots of autophagy markers LC3 and SQSTM1 in E18.5 lung tissue extracts. Quantification of SQSTM1 against β-Tubulin signals indicates a significant decrease in SQSTM1 signals in Ick R272Q homozygous mutant lungs (Mean ± SD, n=6, **P<0.001). (G) Western blots of autophagy markers LC3 and SQSTM1 in mouse embryonic fibroblasts (MEFs) grown in either normal medium (control) or starved in Hank’s Balanced Salt Solution (HBSS) for 2 hours. Quantification of LC3 and SQSTM1 against β-Actin signals indicates a significant decrease in autophagy markers in Ick R272Q homozygous mutant MEFs under the steady-state or starvation-induced autophagic flux (Mean ± SD, n=3, *P<0.05, **P<0.01).