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

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 (ccm3Delta) known to cause cerebral cavernous malformation in humans confers cardiovascular phenotypes identical to those associated with loss of ccm1 and ccm2. CCM3Delta 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.

SUBMITTER: Zheng X

PROVIDER: S-EPMC2912181 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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

Zheng Xiangjian X Xu Chong C Di Lorenzo Annarita A Kleaveland Benjamin B Zou Zhiying Z Seiler Christoph C Chen Mei M Cheng Lan L Xiao Jiping J He Jie J Pack Michael A MA Sessa William C WC Kahn Mark L ML

The Journal of clinical investigation 20100701 8

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. He ...[more]

PMID: 20592472

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Project description:Cerebral cavernous malformations (CCMs) are dilated capillaries causing epilepsy and stroke. Inheritance of a heterozygous mutation in CCM3/PDCD10 is responsible for the most aggressive familial form of the disease. Here we studied the differences and commonalities between the transcriptomes of microdissected lesional neurovascular units (NVUs) from acute and chronic in vivo Ccm3/Pdcd10ECKO mice, and cultured brain microvascular endothelial cells (BMECs) Ccm3/Pdcd10ECKO.We identified 2409 differentially expressed genes (DEGs) in acute and 2962 in chronic in vivo NVUs compared to microdissected brain capillaries, as well as 121 in in vitro BMECs with and without Ccm3/Pdcd10 loss (fold change ≥ |2.0|; p < 0.05, false discovery rate corrected). A functional clustered dendrogram generated using the Euclidean distance showed that the DEGs identified only in acute in vivo NVUs were clustered in cellular proliferation gene ontology functions. The DEGs only identified in chronic in vivo NVUs were clustered in inflammation and immune response, permeability, and adhesion functions. In addition, 1225 DEGs were only identified in the in vivo NVUs but not in vitro BMECs, and these clustered within neuronal and glial functions. One miRNA mmu-miR-3472a was differentially expressed (FC = - 5.98; p = 0.07, FDR corrected) in the serum of Ccm3/Pdcd10+/- when compared to wild type mice, and this was functionally related as a putative target to Cand2 (cullin associated and neddylation dissociated 2), a DEG in acute and chronic lesional NVUs and in vitro BMECs. Our results suggest that the acute model is characterized by cell proliferation, while the chronic model showed inflammatory, adhesion and permeability processes. In addition, we highlight the importance of extra-endothelial structures in CCM disease, and potential role of circulating miRNAs as biomarkers of disease, interacting with DEGs. The extensive DEGs library of each model will serve as a validation tool for potential mechanistic, biomarker, and therapeutic targets.

Project description:BackgroundCerebral cavernous malformations (CCMs) are vascular anomalies of the nervous system mostly located in the brain presenting sporadically or familial. Causes of familial forms are mutations in CCM1 (Krit1), CCM2 (MGC4607) and CCM3 (PDCD10) genes. Sporadic forms with no affected relative most often have only one lesion and no germ line mutations. However, a number of sporadic cases with multiple lesions have been reported and are indeed genetic cases with a de novo mutation or a mutation inherited from an asymptomatic parent.MethodsHere, we performed an analysis of regulatory region of CCM genes in 60 sporadic patients, negative for mutations in coding region and intron-exon boundaries and large deletion/duplications in CCM genes by direct sequencing and MLPA. Among 5 variants identified in 851-bp region shared by CCM3 and SERPINI1 genes and acting as asymmetric bidirectional promoter, two polymorphisms c.-639 T > C/rs9853967 and c.-591 T > C/rs11714980 were selected. A case-control study was performed to analyze their possible relationships with sporadic CCMs. Promoter haplotypes activities on CCM3/SERPINI1 genes expression were tested by dual-luciferase assay.ResultsNo variants were identified in CCM1 and CCM2 regulatory regions. In CCM3/SERPINI1 asymmetric bidirectional promoter 5 variants, 2 of them unknown and 3 corresponding to polymorphisms c.-639 T > C/rs9853967, c.-591 T > C/rs11714980 and c.-359G > A/rs9834676 were detected. While rs9853967 and rs11714980 polymorphisms fall in a critical regulatory fragment outside the minimal promoter in intergenic region, other variants had no effects on transcription factor binding according to RegRNA tool. Case-control study performed on 60 patients and 350 healthy controls showed frequencies of the mutated alleles significantly higher in the control group than in patients. Furthermore, the functional assay showed a significant reduction of CCM3 expression for C-C haplotype even more than for T-C and C-T haplotypes. In SERPINI1 direction, the reduction was not statistically significant.ConclusionsOur data indicated that rs9853967 and rs11714980 polymorphisms could be associated with a protective role in CCM disease.

Project description:Cerebral cavernous malformations (CCMs) are dilated capillaries causing epilepsy and stroke. Inheritance of a heterozygous mutation in CCM3/PDCD10 is responsible for the most aggressive familial form of the disease. Here we studied the differences and commonalities between the transcriptomes of microdissected lesional neurovascular units (NVUs) from acute and chronic in vivo Ccm3/Pdcd10ECKO mice, and cultured brain microvascular endothelial cells (BMECs) Ccm3/Pdcd10ECKO. We identified 2409 differentially expressed genes (DEGs) in acute and 2962 in chronic in vivo NVUs compared to microdissected brain capillaries, as well as 121 in in vitro BMECs with and without Ccm3/Pdcd10 loss (fold change ≥ |2.0|; p < 0.05, false discovery rate corrected). A functional clustered dendrogram generated using the Euclidean distance showed that the DEGs identified only in acute in vivo NVUs were clustered in cellular proliferation gene ontology functions. The DEGs only identified in chronic in vivo NVUs were clustered in inflammation and immune response, permeability, and adhesion functions. In addition, 1225 DEGs were only identified in the in vivo NVUs but not in vitro BMECs, and these clustered within neuronal and glial functions. One miRNA mmu-miR-3472a was differentially expressed (FC = -5.98; p = 0.07, FDR corrected) in the serum of Ccm3/Pdcd10+/- when compared to wild type mice, and this was functionally related as a putative target to Cand2 (cullin associated and neddylation dissociated 2), a DEG in acute and chronic lesional NVUs and in vitro BMECs. Our results suggest that the acute model is characterized by cell proliferation, while the chronic model showed inflammatory, adhesion and permeability processes. In addition, we highlight the importance of extra-endothelial structures in CCM disease, and potential role of circulating miRNAs as biomarkers of disease, interacting with DEGs. The extensive DEGs library of each model will serve as a validation tool for potential mechanistic, biomarker, and therapeutic targets.

Dataset Information

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

Publications

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

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