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CCM3 signaling through sterile 20–like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations
Xiangjian Zheng, … , William C. Sessa, Mark L. Kahn
Xiangjian Zheng, … , William C. Sessa, Mark L. Kahn
Published July 1, 2010
Citation Information: J Clin Invest. 2010;120(8):2795-2804. https://doi.org/10.1172/JCI39679.
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Research Article Cardiology

CCM3 signaling through sterile 20–like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations

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Abstract

Cerebral cavernous malformation is a common human vascular disease that arises due to loss-of-function mutations in genes encoding three intracellular adaptor proteins, cerebral cavernous malformations 1 protein (CCM1), CCM2, and CCM3. CCM1, CCM2, and CCM3 interact biochemically in a pathway required in endothelial cells during cardiovascular development in mice and zebrafish. The downstream effectors by which this signaling pathway regulates endothelial function have not yet been identified. Here we have shown in zebrafish that expression of mutant ccm3 proteins (ccm3Δ) known to cause cerebral cavernous malformation in humans confers cardiovascular phenotypes identical to those associated with loss of ccm1 and ccm2. CCM3Δ proteins interacted with CCM1 and CCM2, but not with other proteins known to bind wild-type CCM3, serine/threonine protein kinase MST4 (MST4), sterile 20–like serine/threonine kinase 24 (STK24), and STK25, all of which have poorly defined biological functions. Cardiovascular phenotypes characteristic of CCM deficiency arose due to stk deficiency and combined low-level deficiency of stks and ccm3 in zebrafish embryos. In cultured human endothelial cells, CCM3 and STK25 regulated barrier function in a manner similar to CCM2, and STKs negatively regulated Rho by directly activating moesin. These studies identify STKs as essential downstream effectors of CCM signaling in development and disease that may regulate both endothelial and epithelial cell junctions.

Authors

Xiangjian Zheng, Chong Xu, Annarita Di Lorenzo, Benjamin Kleaveland, Zhiying Zou, Christoph Seiler, Mei Chen, Lan Cheng, Jiping Xiao, Jie He, Michael A. Pack, William C. Sessa, Mark L. Kahn

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Figure 2

Expression of ccm3 proteins lacking the 18 amino acids encoded by exon 3 confers cardiovascular phenotypes characteristic of heg, ccm1, and ccm2 deficiency.

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Expression of ccm3 proteins lacking the 18 amino acids encoded by exon 3...
(A) Light images of the hearts of 48-hpf zebrafish control embryos, ccm2 mutant embryos, and embryos injected with morpholinos that block splicing into exon 3 of ccm3a only (ccm3aX3, 3 ng/embryo), ccm3b only (ccm3bX3, 3 ng/embryo), both ccm3a and ccm3b [ccm3(a+b)X3], or exon 2 of ccm3a (ccm3aX2, 3 ng/embryo) are shown. Arrows indicate the embryo hearts. (B) Fluorescence images of the hearts of transgenic embryos in which myocardial cells express GFP following injection of the indicated morpholinos. ccm2MO indicates a morpholino that blocks splicing of the ccm2 gene. (C) Thinned myocardium in ccm3(a+b)X3 morphants is identical to that seen in ccm2 mutants. Shown are hematoxylin/eosin-stained sagittal sections of the indicated 48-hpf embryos. a, atrium; v, ventricle; hw, heart wall. (D) Angiography of 48-hpf control and ccm3(a+b)X3 morphant embryos reveals blocked circulation at the cardiac outflow tract. (E) Vascular endothelial patterning as revealed in Tg (fli1a:EGFP)y1 embryos is undisturbed in ccm3(a+b)X3 morphant embryos. The images are composites of 2–3 images taken of the same embryos. (F) The big heart phenotype conferred by morpholinos that block splicing into exon 3 of ccm3a and ccm3b is rescued by coinjection of cRNAs (100 pg/embryo) encoding either ccm3a or ccm3b (right 2 bars). Shown are mean and SEM. The number of embryos examined is indicated above each bar, and the number of injections performed for each group shown in parentheses. ***P < 0.001 by Student’s t test. Scale bars: 20 μm.

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