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      Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signaling

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          Abstract

          Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause stroke and seizures in younger individuals 1 . CCMs arise from endothelial cell loss of KRIT1, CCM2, or PDCD10, non-homologous proteins that form an adaptor complex 2 . How disruption of the CCM complex results in disease remains controversial, with numerous signaling pathways (including Rho 3, 4 , SMAD 5 and Wnt/β-catenin 6 ) and processes such as endothelial-mesenchymal transition (EndMT) 5 proposed to play causal roles. CCM2 binds MEKK3 711 , and we have recently demonstrated that CCM complex regulation of MEKK3 is essential during vertebrate heart development 12 . Here, we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we find that expression of the MEKK3 target genes KLF2 and KLF4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. In contrast, we find no evidence of EndMT or increased SMAD or Wnt signaling during early CCM formation. Endothelial-specific loss of Mekk3, Klf2, or Klf4 dramatically prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we demonstrate that endothelial expression of KLF2 and KLF4 is elevated in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signaling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics.

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          Most cited references 26

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          The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon.

          Klf4 (formerly GKLF) is a zinc-finger transcription factor expressed in the epithelia of the skin, lungs, gastrointestinal tract and several other organs. In vitro studies have suggested that Klf4 plays an important role in cell proliferation and/or differentiation. Mice homozygous for a null mutation in Klf4 die within 15 hours of birth and show selective perturbation of late-stage differentiation structures in the epidermis, but the function of Klf4 in the gastrointestinal tract has not been investigated. To address this issue, we have generated Klf4(-/-) mice by homologous recombination in embryonic stem cells. In this study, we provide the first in vivo evidence that Klf4 is a goblet cell-specific differentiation factor in the colon. Klf4(-/-) mice exhibit normal cell proliferation and cell death rates in the colon on postnatal day 1. However, Klf4(-/-) mice demonstrate a 90% decrease in the number of goblet cells in the colon, show abnormal expression of the goblet cell-specific marker Muc2 by in situ hybridization, have abnormal staining of the colonic epithelium with Alcian Blue for acidic mucins, and lack normal goblet cell morphology by ultrastructural analysis. All other epithelial cell types are present in the colon of Klf4(-/-) mice. In summary, Klf4 plays a crucial role in colonic epithelial cell differentiation in vivo.
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            Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity

            Endothelial cell–cell junctions regulate vascular permeability, vasculogenesis, and angiogenesis. Familial cerebral cavernous malformations (CCMs) in humans result from mutations of CCM2 (malcavernin, OSM, MGC4607), PDCD10 (CCM3), or KRIT1 (CCM1), a Rap1 effector which stabilizes endothelial cell–cell junctions. Homozygous loss of KRIT1 or CCM2 produces lethal vascular phenotypes in mice and zebrafish. We report that the physical interaction of KRIT1 and CCM2 proteins is required for endothelial cell–cell junctional localization, and lack of either protein destabilizes barrier function by sustaining activity of RhoA and its effector Rho kinase (ROCK). Protein haploinsufficient Krit1 +/− or Ccm2 +/− mouse endothelial cells manifested increased monolayer permeability in vitro, and both Krit1 +/− and Ccm2 +/− mice exhibited increased vascular leak in vivo, reversible by fasudil, a ROCK inhibitor. Furthermore, we show that ROCK hyperactivity occurs in sporadic and familial human CCM endothelium as judged by increased phosphorylation of myosin light chain. These data establish that KRIT1–CCM2 interaction regulates vascular barrier function by suppressing Rho/ROCK signaling and that this pathway is dysregulated in human CCM endothelium, and they suggest that fasudil could ameliorate both CCM disease and vascular leak.
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              The Cerebral Cavernous Malformation signaling pathway promotes vascular integrity via Rho GTPases

              SUMMARY Cerebral cavernous malformation (CCM) is a common vascular dysplasia that affects both systemic and CNS blood vessels. Loss of function mutations in the CCM2 gene cause CCM. Here we show that targeted disruption of Ccm2 in mice results in failed lumen formation and early embryonic death through an endothelial cell autonomous mechanism. We demonstrate that CCM2 regulates endothelial cytoskeletal architecture, cell-cell interactions and lumen formation. Heterozygosity at Ccm2, a genotype equivalent to human CCM, results in impaired endothelial barrier function. Because our biochemical studies indicate that loss of CCM2 results in activation of RHOA GTPase, we rescued the cellular phenotype and barrier function in heterozygous mice using simvastatin, a drug known to inhibit Rho GTPases. These data offer the prospect for pharmacologic treatment of a human vascular dysplasia using a widely available and safe drug.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                2 February 2016
                30 March 2016
                7 April 2016
                30 September 2016
                : 532
                : 7597
                : 122-126
                Affiliations
                [1 ]Department of Medicine and Cardiovascular Institute, University of Pennsylvania, 3400 Civic Center Blvd, Philadelphia PA 19104
                [2 ]Lab of Cardiovascular Signaling, Centenary Institute, Sydney NSW 2050, Australia
                [3 ]Neurovascular Surgery Program, Section of Neurosurgery, Department of Surgery, The University of Chicago Medicine and Biological Sciences, Chicago, Illinois, USA
                [4 ]Department of Radiology, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia PA 19104
                [5 ]Sydney Microscopy & Microanalysis, University of Sydney, Sydney, NSW 2050, Australia
                [6 ]Division of Cell Signaling and Immunology, University of Dundee, Dundee, United Kingdom, DD1 5EH
                [7 ]Division of Cardiovascular Medicine and the Program in Molecular Medicine, University of Utah, Salt Lake City, UT 84112
                [8 ]Faculty of Medicine, Sydney Medical School, University of Sydney, Sydney, NSW 2050, Australia
                Author notes
                Correspondence should be addressed to: M.L.K. ( markkahn@ 123456mail.med.upenn.edu ), Telephone: 215-898-9007 FAX: 215-573-2094
                [*]

                These authors contributed equally

                Article
                NIHMS755502
                10.1038/nature17178
                4864035
                27027284

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