Intestinal cell kinase, a protein associated with endocrine-cerebro-osteodysplasia syndrome, is a key regulator of cilia length and Hedgehog signaling

Abstract

Endocrine-cerebro-osteodysplasia (ECO) syndrome is a recessive genetic disorder associated with multiple congenital defects in endocrine, cerebral, and skeletal systems that is caused by a missense mutation in the mitogen-activated protein kinase-like intestinal cell kinase (ICK) gene. In algae and invertebrates, ICK homologs are involved in flagellar formation and ciliogenesis, respectively. However, it is not clear whether this role of ICK is conserved in mammals and how a lack of functional ICK results in the characteristic phenotypes of human ECO syndrome. Here, we generated Ick knockout mice to elucidate the precise role of ICK in mammalian development and to examine the pathological mechanisms of ECO syndrome. Ick null mouse embryos displayed cleft palate, hydrocephalus, polydactyly, and delayed skeletal development, closely resembling ECO syndrome phenotypes. In cultured cells, down-regulation of Ick or overexpression of kinase-dead or ECO syndrome mutant ICK resulted in an elongation of primary cilia and abnormal Sonic hedgehog (Shh) signaling. Wild-type ICK proteins were generally localized in the proximal region of cilia near the basal bodies, whereas kinase-dead ICK mutant proteins accumulated in the distal part of bulged ciliary tips. Consistent with these observations in cultured cells, Ick knockout mouse embryos displayed elongated cilia and reduced Shh signaling during limb digit patterning. Taken together, these results indicate that ICK plays a crucial role in controlling ciliary length and that ciliary defects caused by a lack of functional ICK leads to abnormal Shh signaling, resulting in congenital disorders such as ECO syndrome.

Keywords: Gli2; LF4; MRK; Smoothened; ciliopathy.

Fig. 1.

Ick mutant mice have ECO syndrome-like features. (A) RT-PCR analyses with RNAs from embryonic day (E) 10.5 embryos confirmed that, whereas normal transcripts spliced between exons 5 and 6 of the Ick gene were expressed in wild type, truncated Ick transcripts, fused with LacZ, were expressed in Ick^tm1a/tm1a homozygotes. (B) E15.5 Ick^tm1a/tm1a embryos showed extensive edema at the head and back (red arrowhead). (Scale bars, 1.5 mm.) (C) Ick^tm1a/tm1a mutants exhibited cleft palates in the roof of the oral cavity (Left, asterisk). (Scale bars, 1 mm.) Coronal sections further demonstrate the cleft palate in mutant embryos (Right). (Scale bars, 300 µm.) (D) Developing telencephalon in Ick^tm1a/tm1a embryos showed severely dilated ventricles. (Scale bars, 300 µm.) (E) E15.5 Ick^tm1a/tm1a forelimbs displayed polydactyly (Upper). (Scale bars, 200 µm.) Additionally, alcian blue/alizarin red staining in E18.5 Ick^tm1a/tm1a embryos revealed delayed ossification in the digits and severely distorted and shortened long bones (Lower). (Scale bars, 500 µm.)

Fig. 2.

ICK kinase activity levels determine the length of primary cilia. (A and B) FLAG-tagged wild-type and kinase-inactive forms of ICK (K33R and TDY mut) were transfected into cultured cells. (A) Cilia and transfected ICK were visualized by immunofluorescent staining with acetylated tubulin (acTub, green) and FLAG (red) antibodies, respectively. Overexpression of wild-type ICK either suppressed cilia formation or shortened ciliary length. By contrast, overexpression of inactive ICK increased ciliary length. (B) Quantification of ciliary length in CTRL (n = 56), K33R (n = 56), or TDY mut (n = 58) ICK overexpressing cells. (C) Western blot analysis showed that ICK protein levels were significantly decreased by Ick siRNA 2 compared with control siRNA or siRNA 4. (D and E) Primary cilia were stained by Arl13b antibody (green) to analyze cilia length (n = 14) (D) and morphology (E). Knockdown of ICK by siRNA 2 resulted in longer primary cilia than those observed after control siRNA or siRNA 4 treatment. Error bars reflect standard SEM and asterisk denotes statistical significance according to Student’s t tests in B and D (*P < 0.01). (Scale bars in A and E, 5 µm.)

Fig. 3.

ICK activity is required for its ciliary localization. To determine the subcellular localization of WT or mutant (K33R or TDY mut) ICK in primary cilia, cultured cells were transfected with appropriate plasmids and immunostained with Arl13b (blue) and γ-tubulin (γ-Tub, red) antibodies for cilia and basal bodies, respectively. Wild-type ICK was localized near the basal body of shortened cilia. By contrast, inactive ICK mutants were localized at the distal tip of cilia, which displayed swollen morphology. (Scale bars: 5 µm, Left; 1 µm, Right.)

Fig. 4.

ICK regulates Smo and Gli2 localization in primary cilia. (A) Responsiveness to Shh was assayed in cultured cells using a 8 × 3′ Gli-BS luciferase reporter construct. Responsiveness to Shh was decreased in cells overexpressing any types of ICK proteins compared with nontransfected (CTRL) cells. Error bars = SEM. *P < 0.01 (Student’s t tests). (B) In cells treated with control siRNA (CTRL), Smo-EGFP fluorescence was evenly distributed across entire cilia, which were stained with acetylated tubulin (acTub) antibody. By contrast, Smo-EGFP was observed in a punctate pattern in elongated cilia in ICK siRNA 2 transfected cells (Right). In CTRL cells, endogenous GLI2 proteins were localized at the tip of primary cilia, whereas in Ick siRNA 2 transfected cells, GLI2 proteins showed additional sites of accumulation in the axoneme of elongated cilia (Right). (Scale bars, 2.5 µm.)

Fig. 5.

Ick mutant embryos have defects in HH signaling and ciliogenesis. (A) Expressions of Gli1, Ptch1, and Hoxd13, which were normally restricted to posterior regions of E10.5 wild-type limb buds, were decreased in the posterior regions and ectopically increased in the anterior regions of limb buds of Ick^tm1a/tm1a mutants (arrowheads). (B and C) Frozen sections of E11.5 limb buds were stained with Arl13b (green) and γ-Tub (red) antibodies. Ciliary lengths in Ick^tm1a/tm1a mutants (3.35 ± 0.10 µm, n = 96) were ∼2.5-fold longer than those in wild type (1.38 ± 0.06 µm, n = 54). *P < 0.01 (unpaired Student’s t tests). (D) Gli3 processing was analyzed by Western blotting with anti-GLI3 antibody for cell lysates from E9.5 wild-type (WT) or Ick mutant (m) whole embryos or limb buds. The antibody recognized full-length [GLI3(FL)] and processed [GLI3(R)] forms of GLI3 protein. Extracts from Gli3 knockout embryos were used as a control. (E) Scanning electron microscopy (EM) showed that primary cilia in the limb ectoderm of Ick^tm1a/tm1a mutants were significantly longer compare with Ick^+/+ controls. (Scale bars: Left, 500 µm; Right, 300 µm.) Transmission EM showed that ultrastructures of primary cilia in the limb mesenchyme of Ick^tm1a/tm1a mutants were indistinguishable from those of controls in both longitudinal and cross-section views. (Scale bars: Left, 2 µm; Right, 0.5 µm.)

Fig. 6.

The ICK mutation identified in human ECO syndrome causes defects in ciliogenesis and HH signaling. ICK-3FLAG mutant (R272Q and R272A) plasmids were transfected into cultured cells. After transfection, cells were stained with fluorescent Arl13b (green) and FLAG (red) antibodies. (A and B) Ciliary length was increased by approximately twofold by overexpression of ICK mutant proteins. Transfected ICK mutant proteins were localized at the bulged tips of primary cilia. (Scale bars, 5 µm.) (C) SHH responsiveness was severely compromised by overexpression of wild-type or mutant forms of ICK in cultured cells treated with SHHN-conditioned media. Error bars in B and C represent SEM. *P < 0.01; **P < 0.05 (Student’s t tests).