ABSTRACT
BACKGROUND: Cavernous cerebral malformations can arise because of mutations in the CCM1, CCM2, or CCM3 genes, and lack of Cdc42 has also been reported to induce these malformations in mice. However, the role of the CCM3 (cerebral cavernous malformation 3)-associated kinases in cavernoma development is not known, and we, therefore, have investigated their role in the process. METHODS: We used a combination of an in vivo approach, using mice genetically modified to be deficient in the CCM3-associated kinases STK24 and STK25 (serine/threonine kinases 24 and 25), and the in vitro model of human endothelial cells in which expression of STK24 and STK25 was inhibited by RNA interference. RESULTS: Mice deficient for both Stk24 and Stk25, but not for either of them individually, developed aggressive vascular lesions with the characteristics of cavernomas at an early age. Stk25 deficiency also gave rise to vascular anomalies in the context of Stk24 heterozygosity. Human endothelial cells deficient for both kinases phenocopied several of the consequences of CCM3 loss, and single STK25 deficiency also induced KLF2 expression, Golgi dispersion, altered distribution of ß-catenin, and appearance of stress fibers. CONCLUSIONS: The CCM3-associated kinases STK24 and STK25 play a major role in the inhibition of cavernoma development.
Subject(s)
Central Nervous System Neoplasms/genetics , Germinal Center Kinases/genetics , Hemangioma, Cavernous, Central Nervous System/genetics , Human Umbilical Vein Endothelial Cells/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Animals , Central Nervous System Neoplasms/metabolism , Germinal Center Kinases/metabolism , Hemangioma, Cavernous, Central Nervous System/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Knockout , Phosphorylation , Protein Serine-Threonine Kinases/metabolismABSTRACT
Cerebral cavernous malformations (CCMs) are vascular malformations that can be the result of the deficiency of one of the CCM genes. Their only present treatment is surgical removal, which is not always possible, and an alternative pharmacological strategy to eliminate them is actively sought. We have studied the effect of the lack of one of the CCM genes, CCM3, in endothelial and non-endothelial cells. By comparing protein expression in control and CCM3-silenced cells, we found that the levels of the Epidermal Growth Factor Receptor (EGFR) are higher in CCM3-deficient cells, which adds to the known upregulation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in these cells. Whereas VEGFR2 is upregulated at the mRNA level, EGFR has a prolonged half-life. Inhibition of EGFR family members in CCM3-deficient cells does not revert the known cellular effects of lack of CCM genes, but it induces significantly more apoptosis in CCM3-deficient cells than in control cells. We propose that the susceptibility to tyrosine kinase inhibitors of CCM3-deficient cells can be harnessed to kill the abnormal cells of these lesions and thus treat CCMs pharmacologically.
ABSTRACT
One of the CCM genes, CCM3/PDCD10, binds to the protein kinase family GCKIII, which comprises MST3/STK24, SOK1/STK25, and MST4/STK26. These proteins have been shown to have the same effect as CCM3, both in endothelial cells and in animal models such as zebrafish and are most likely involved in CCM pathogenesis. We describe here an in vitro kinase assay of GCKIII proteins which can be used to study their regulation in endothelial and other cells under different circumstances.
