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Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.

Fisher OS, Deng H, Liu D, Zhang Y, Wei R, Deng Y, Zhang F, Louvi A, Turk BE, Boggon TJ, Su B - Nat Commun (2015)

Bottom Line: Here we report that Mekk3 plays an intrinsic role in embryonic vascular development.We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage.We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, USA.

ABSTRACT
Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development. Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage. We discover direct interaction between CCM2 harmonin homology domain (HHD) and the N terminus of MEKK3, and determine a 2.35 Å cocrystal structure. We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage. We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

No MeSH data available.


Related in: MedlinePlus

MEKK3 is essential for embryonic vascular development and neonatal brain vascular integrity.(a) Survival lines of neonatal pups of normal control littermate (NCL) Cad5-ERCre/Mekk3fl/+ (open circles, n=17) or Cad5-ERCre/Mekk3fl/− (black diamonds, n=16) with no tamoxifen treatment or treated with 5 μl of tamoxifen (10 mg ml−1) orally at day-1 after birth and continuously daily (Cad5-ERCre/Mekk3fl/+, black crosses, n=17; Cad5-ERCre/Mekk3fl/−, black triangles, n=10). The pups were observed for 20 days for survival. (b–g) Organ examples collected from neonatal littermates of tamoxifen-treated Cad5-ERCre/Mekk3fl/+ (Mekk3NCL) pups or EC-specific Mekk3 deletion Cad5-ERCre/Mekk3fl/− (Mekk3 iEC−/−) pups as indicated: (b) kidneys of postnatal day (P)10 littermate pups, (c) small intestines from P10 littermate pups, (d) lungs from P6 littermate pups, (e) colons from P10 littermate pups, (f) brains from P7 littermate pups. (g) Light microscopic pictures showing brain surface of P7 littermate pups. Scale bar, 5 mm. Arrowheads in f show bleeding spots observed from the surface of Mekk3 iEC−/− brain. (h) Haematoxylin and eosin staining of paraffin-embedded sections of P7 neonatal Mekk3NCL and Mekk3 iEC−/− brains visualized by a light microscope under a 2.5 × objective lens. Blue arrowheads indicate the haemorrhage sites inside the brains. (i) Immunoblots show expression of VEGFR2, MEKK3 and T18/S19 phosphorylated MLC2 (MLC2pT18/S19) in freshly isolated BMEC from three Mekk3NCL and three Mekk3 iEC−/− littermate pups as indicated. GAPDH expression is shown as loading controls. Elevated MLC2pT18/S19 was observed in three independently isolated pairs. (j) Confocal microscopy to visualize the expression of VE-Cadherin (green) and MLC2pT18/S19 (red) in primary BMEC from P7 Mekk3NCL, or Mekk3 iEC−/−, or Mekk3 iEC−/− pups treated with Y27632. Cell nuclei were stained with DAPI (blue). MLC2pT18/S19 was elevated in Mekk3 iEC−/−BMEC, which was suppressed by addition of ROCK inhibitor Y27632. Scale bar, 100 μm.
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f1: MEKK3 is essential for embryonic vascular development and neonatal brain vascular integrity.(a) Survival lines of neonatal pups of normal control littermate (NCL) Cad5-ERCre/Mekk3fl/+ (open circles, n=17) or Cad5-ERCre/Mekk3fl/− (black diamonds, n=16) with no tamoxifen treatment or treated with 5 μl of tamoxifen (10 mg ml−1) orally at day-1 after birth and continuously daily (Cad5-ERCre/Mekk3fl/+, black crosses, n=17; Cad5-ERCre/Mekk3fl/−, black triangles, n=10). The pups were observed for 20 days for survival. (b–g) Organ examples collected from neonatal littermates of tamoxifen-treated Cad5-ERCre/Mekk3fl/+ (Mekk3NCL) pups or EC-specific Mekk3 deletion Cad5-ERCre/Mekk3fl/− (Mekk3 iEC−/−) pups as indicated: (b) kidneys of postnatal day (P)10 littermate pups, (c) small intestines from P10 littermate pups, (d) lungs from P6 littermate pups, (e) colons from P10 littermate pups, (f) brains from P7 littermate pups. (g) Light microscopic pictures showing brain surface of P7 littermate pups. Scale bar, 5 mm. Arrowheads in f show bleeding spots observed from the surface of Mekk3 iEC−/− brain. (h) Haematoxylin and eosin staining of paraffin-embedded sections of P7 neonatal Mekk3NCL and Mekk3 iEC−/− brains visualized by a light microscope under a 2.5 × objective lens. Blue arrowheads indicate the haemorrhage sites inside the brains. (i) Immunoblots show expression of VEGFR2, MEKK3 and T18/S19 phosphorylated MLC2 (MLC2pT18/S19) in freshly isolated BMEC from three Mekk3NCL and three Mekk3 iEC−/− littermate pups as indicated. GAPDH expression is shown as loading controls. Elevated MLC2pT18/S19 was observed in three independently isolated pairs. (j) Confocal microscopy to visualize the expression of VE-Cadherin (green) and MLC2pT18/S19 (red) in primary BMEC from P7 Mekk3NCL, or Mekk3 iEC−/−, or Mekk3 iEC−/− pups treated with Y27632. Cell nuclei were stained with DAPI (blue). MLC2pT18/S19 was elevated in Mekk3 iEC−/−BMEC, which was suppressed by addition of ROCK inhibitor Y27632. Scale bar, 100 μm.

Mentions: To understand the impact of Mekk3 loss at a later stage of development and its role in normal physiological and pathological settings, we generated EC-specific tamoxifen-inducible Mekk3 iKO mice (Mekk3 iEC−/−). Mekk3 deletion was induced at postnatal day (P) 1 by oral feeding of tamoxifen daily. Mekk3 iEC−/− pups became obviously sick 2–3 days after the initiation of tamoxifen administration, and most died within 15 days (Fig. 1a). In contrast, control Mekk3 iEC+/− pups, although only having one copy of the Mekk3 gene in EC, survived at similar doses of tamoxifen (Fig. 1a), as did the untreated pups (Fig. 1a). The Mekk3 iEC−/− pups showed haemorrhage in various organs, including the kidney, small intestine, lung and colon (Fig. 1b–e). Although neonatal loss of MEKK3 could result in haemorrhages of multiple organs, the brain is the most affected organ, which is also likely the main cause of lethality (Fig. 1). In fact, almost all the Mekk3 iEC−/− pups showed brain haemorrhage, but only a fraction of other organs were affected. This may be due to fact that brain vascular MEKK3 is more efficiently deleted by tamoxifen administration. Interestingly, we observed that about 50% of Mekk3 iEC−/− pups developed signs of paralysis before death. Indeed, after 5–7 days of tamoxifen treatment, the Mekk3 iEC−/− pups harboured multiple focal haemorrhages on the surface and inside of the brain, but this was not observed in control brains (Mekk3 iEC+/− treated with tamoxifen termed Mekk3NCL; Fig. 1f). The neonatal vasculature was also malformed when Mekk3 was deleted (Fig. 1g, Supplementary Fig. 2) and haematoxylin and eosin staining of the neonatal brain confirmed bleeding in the cerebella and brain surface areas (Fig. 1h, Supplementary Fig. 3). The postnatal loss of Mekk3 in the endothelium is therefore associated with severe haemorrhage in the brain vasculature.


Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.

Fisher OS, Deng H, Liu D, Zhang Y, Wei R, Deng Y, Zhang F, Louvi A, Turk BE, Boggon TJ, Su B - Nat Commun (2015)

MEKK3 is essential for embryonic vascular development and neonatal brain vascular integrity.(a) Survival lines of neonatal pups of normal control littermate (NCL) Cad5-ERCre/Mekk3fl/+ (open circles, n=17) or Cad5-ERCre/Mekk3fl/− (black diamonds, n=16) with no tamoxifen treatment or treated with 5 μl of tamoxifen (10 mg ml−1) orally at day-1 after birth and continuously daily (Cad5-ERCre/Mekk3fl/+, black crosses, n=17; Cad5-ERCre/Mekk3fl/−, black triangles, n=10). The pups were observed for 20 days for survival. (b–g) Organ examples collected from neonatal littermates of tamoxifen-treated Cad5-ERCre/Mekk3fl/+ (Mekk3NCL) pups or EC-specific Mekk3 deletion Cad5-ERCre/Mekk3fl/− (Mekk3 iEC−/−) pups as indicated: (b) kidneys of postnatal day (P)10 littermate pups, (c) small intestines from P10 littermate pups, (d) lungs from P6 littermate pups, (e) colons from P10 littermate pups, (f) brains from P7 littermate pups. (g) Light microscopic pictures showing brain surface of P7 littermate pups. Scale bar, 5 mm. Arrowheads in f show bleeding spots observed from the surface of Mekk3 iEC−/− brain. (h) Haematoxylin and eosin staining of paraffin-embedded sections of P7 neonatal Mekk3NCL and Mekk3 iEC−/− brains visualized by a light microscope under a 2.5 × objective lens. Blue arrowheads indicate the haemorrhage sites inside the brains. (i) Immunoblots show expression of VEGFR2, MEKK3 and T18/S19 phosphorylated MLC2 (MLC2pT18/S19) in freshly isolated BMEC from three Mekk3NCL and three Mekk3 iEC−/− littermate pups as indicated. GAPDH expression is shown as loading controls. Elevated MLC2pT18/S19 was observed in three independently isolated pairs. (j) Confocal microscopy to visualize the expression of VE-Cadherin (green) and MLC2pT18/S19 (red) in primary BMEC from P7 Mekk3NCL, or Mekk3 iEC−/−, or Mekk3 iEC−/− pups treated with Y27632. Cell nuclei were stained with DAPI (blue). MLC2pT18/S19 was elevated in Mekk3 iEC−/−BMEC, which was suppressed by addition of ROCK inhibitor Y27632. Scale bar, 100 μm.
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Related In: Results  -  Collection

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f1: MEKK3 is essential for embryonic vascular development and neonatal brain vascular integrity.(a) Survival lines of neonatal pups of normal control littermate (NCL) Cad5-ERCre/Mekk3fl/+ (open circles, n=17) or Cad5-ERCre/Mekk3fl/− (black diamonds, n=16) with no tamoxifen treatment or treated with 5 μl of tamoxifen (10 mg ml−1) orally at day-1 after birth and continuously daily (Cad5-ERCre/Mekk3fl/+, black crosses, n=17; Cad5-ERCre/Mekk3fl/−, black triangles, n=10). The pups were observed for 20 days for survival. (b–g) Organ examples collected from neonatal littermates of tamoxifen-treated Cad5-ERCre/Mekk3fl/+ (Mekk3NCL) pups or EC-specific Mekk3 deletion Cad5-ERCre/Mekk3fl/− (Mekk3 iEC−/−) pups as indicated: (b) kidneys of postnatal day (P)10 littermate pups, (c) small intestines from P10 littermate pups, (d) lungs from P6 littermate pups, (e) colons from P10 littermate pups, (f) brains from P7 littermate pups. (g) Light microscopic pictures showing brain surface of P7 littermate pups. Scale bar, 5 mm. Arrowheads in f show bleeding spots observed from the surface of Mekk3 iEC−/− brain. (h) Haematoxylin and eosin staining of paraffin-embedded sections of P7 neonatal Mekk3NCL and Mekk3 iEC−/− brains visualized by a light microscope under a 2.5 × objective lens. Blue arrowheads indicate the haemorrhage sites inside the brains. (i) Immunoblots show expression of VEGFR2, MEKK3 and T18/S19 phosphorylated MLC2 (MLC2pT18/S19) in freshly isolated BMEC from three Mekk3NCL and three Mekk3 iEC−/− littermate pups as indicated. GAPDH expression is shown as loading controls. Elevated MLC2pT18/S19 was observed in three independently isolated pairs. (j) Confocal microscopy to visualize the expression of VE-Cadherin (green) and MLC2pT18/S19 (red) in primary BMEC from P7 Mekk3NCL, or Mekk3 iEC−/−, or Mekk3 iEC−/− pups treated with Y27632. Cell nuclei were stained with DAPI (blue). MLC2pT18/S19 was elevated in Mekk3 iEC−/−BMEC, which was suppressed by addition of ROCK inhibitor Y27632. Scale bar, 100 μm.
Mentions: To understand the impact of Mekk3 loss at a later stage of development and its role in normal physiological and pathological settings, we generated EC-specific tamoxifen-inducible Mekk3 iKO mice (Mekk3 iEC−/−). Mekk3 deletion was induced at postnatal day (P) 1 by oral feeding of tamoxifen daily. Mekk3 iEC−/− pups became obviously sick 2–3 days after the initiation of tamoxifen administration, and most died within 15 days (Fig. 1a). In contrast, control Mekk3 iEC+/− pups, although only having one copy of the Mekk3 gene in EC, survived at similar doses of tamoxifen (Fig. 1a), as did the untreated pups (Fig. 1a). The Mekk3 iEC−/− pups showed haemorrhage in various organs, including the kidney, small intestine, lung and colon (Fig. 1b–e). Although neonatal loss of MEKK3 could result in haemorrhages of multiple organs, the brain is the most affected organ, which is also likely the main cause of lethality (Fig. 1). In fact, almost all the Mekk3 iEC−/− pups showed brain haemorrhage, but only a fraction of other organs were affected. This may be due to fact that brain vascular MEKK3 is more efficiently deleted by tamoxifen administration. Interestingly, we observed that about 50% of Mekk3 iEC−/− pups developed signs of paralysis before death. Indeed, after 5–7 days of tamoxifen treatment, the Mekk3 iEC−/− pups harboured multiple focal haemorrhages on the surface and inside of the brain, but this was not observed in control brains (Mekk3 iEC+/− treated with tamoxifen termed Mekk3NCL; Fig. 1f). The neonatal vasculature was also malformed when Mekk3 was deleted (Fig. 1g, Supplementary Fig. 2) and haematoxylin and eosin staining of the neonatal brain confirmed bleeding in the cerebella and brain surface areas (Fig. 1h, Supplementary Fig. 3). The postnatal loss of Mekk3 in the endothelium is therefore associated with severe haemorrhage in the brain vasculature.

Bottom Line: Here we report that Mekk3 plays an intrinsic role in embryonic vascular development.We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage.We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, USA.

ABSTRACT
Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development. Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage. We discover direct interaction between CCM2 harmonin homology domain (HHD) and the N terminus of MEKK3, and determine a 2.35 Å cocrystal structure. We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage. We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

No MeSH data available.


Related in: MedlinePlus