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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 critically regulates the neonatal vascular permeability to small size molecule via suppressing the Rho signals.(a–d) In vivo leakage experiments show only small-molecular-weight tracers leaked from brain microvessels in Mekk3 iKO and iEC−/− neonatal mice. (a) Sulfo-NHS-biotin (556.6 dalton) was injected into the hearts of the tamoxifen-treated Mekk3 NCL or Mekk3 iKO neonatal pups. Brain sections were analysed for Sulfo-NHS-biotin leakage by staining. Objective lens power: 2.5 × . (b–d) Fluorescent-labelled tracers with different molecular weights were injected into tamoxifen-treated Mekk3 NCL or Mekk3 iEC−/− P7 neonatal pups' hearts. After euthanization, brains were fixed and sectioned and tracer leakage to the brain was determined. (b) Hoechst 33342 (616 dalton; blue) plus Dextran–Rhodamine (2 M Dalton; red), N=7, (c) Dextran–FITC (MW: 4K Dalton; green) plus Dextran–Rhodamine (2 M Dalton)(red), N=4, (d) Dextran–FITC (40 K Dalton; green) plus Dextran–Rhodamine (2 M Dalton; red). Bar graphs on the left of each panel show quantification of the relative leakage of the tracers normalized to the intensity of Dextran–Rhodamine, and shows no leakage in either NCL nor Mekk3 iEC−/− neonatal pups. Error bars indicate s.d. N=4. Objective lens power: 10 × . (e) ROCK inhibitor Y27632 partially rescues survival of Mekk3 iEC−/− neonatal pups. NCL or Mekk3 iEC−/− pups were treated with tamoxifen at P1, and continued daily in the absence (open circle and black diamond, respectively) or presence of Y27632 (cross and black triangle, respectively). Survival of pups was monitored daily until P20. (f) Hoechst 33342 (616 Dalton; blue) plus Dextran K–Rhodamine (2 M Dalton; red) were injected into tamoxifen-treated Mekk3 iEC−/− P7 neonatal pups' hearts fed either with water or Y27632. After euthanization, brains were fixed and sectioned at 30-μm thickness and leakage determined as described above. N=5. Objective lens power: 10 × . (g) Wild-type P7 neonatal pups were treated with cell-permeable wild-type Mekk3-peptide (MEKK3N-peptide) or A6D/L7D mutated Mekk3-peptide (MEKK3mutant-N-peptide). Brain leakage determined using Hoechst 33342 (blue) plus Dextran K–Rhodamine (red). N=5. Objective lens power: 10 × . (h) Proposed signalling pathway. The interactions of CCM2 and MEKK3 are critical for maintenance of vasculature integrity and permeability by control of Rho/ROCK signalling.
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f4: MEKK3 critically regulates the neonatal vascular permeability to small size molecule via suppressing the Rho signals.(a–d) In vivo leakage experiments show only small-molecular-weight tracers leaked from brain microvessels in Mekk3 iKO and iEC−/− neonatal mice. (a) Sulfo-NHS-biotin (556.6 dalton) was injected into the hearts of the tamoxifen-treated Mekk3 NCL or Mekk3 iKO neonatal pups. Brain sections were analysed for Sulfo-NHS-biotin leakage by staining. Objective lens power: 2.5 × . (b–d) Fluorescent-labelled tracers with different molecular weights were injected into tamoxifen-treated Mekk3 NCL or Mekk3 iEC−/− P7 neonatal pups' hearts. After euthanization, brains were fixed and sectioned and tracer leakage to the brain was determined. (b) Hoechst 33342 (616 dalton; blue) plus Dextran–Rhodamine (2 M Dalton; red), N=7, (c) Dextran–FITC (MW: 4K Dalton; green) plus Dextran–Rhodamine (2 M Dalton)(red), N=4, (d) Dextran–FITC (40 K Dalton; green) plus Dextran–Rhodamine (2 M Dalton; red). Bar graphs on the left of each panel show quantification of the relative leakage of the tracers normalized to the intensity of Dextran–Rhodamine, and shows no leakage in either NCL nor Mekk3 iEC−/− neonatal pups. Error bars indicate s.d. N=4. Objective lens power: 10 × . (e) ROCK inhibitor Y27632 partially rescues survival of Mekk3 iEC−/− neonatal pups. NCL or Mekk3 iEC−/− pups were treated with tamoxifen at P1, and continued daily in the absence (open circle and black diamond, respectively) or presence of Y27632 (cross and black triangle, respectively). Survival of pups was monitored daily until P20. (f) Hoechst 33342 (616 Dalton; blue) plus Dextran K–Rhodamine (2 M Dalton; red) were injected into tamoxifen-treated Mekk3 iEC−/− P7 neonatal pups' hearts fed either with water or Y27632. After euthanization, brains were fixed and sectioned at 30-μm thickness and leakage determined as described above. N=5. Objective lens power: 10 × . (g) Wild-type P7 neonatal pups were treated with cell-permeable wild-type Mekk3-peptide (MEKK3N-peptide) or A6D/L7D mutated Mekk3-peptide (MEKK3mutant-N-peptide). Brain leakage determined using Hoechst 33342 (blue) plus Dextran K–Rhodamine (red). N=5. Objective lens power: 10 × . (h) Proposed signalling pathway. The interactions of CCM2 and MEKK3 are critical for maintenance of vasculature integrity and permeability by control of Rho/ROCK signalling.

Mentions: The above biochemical, biophysical and in vitro studies provided the first direct evidence to link the physiological function of an interaction between MEKK3 and CCM2 in brain vasculature. Since ample evidence has suggested the importance of CCM proteins in endothelial cell junctions15161718, to better understand the impact of Mekk3 deletion in brain vasculature development, we tested if there were any EC cell permeability alterations in the neurovasculature of developing mice. To understand why MEKK3 deficiency causes brain haemorrhage, we examined the integrity of brain blood vessels in Mekk3 iEC−/− and control mice. We used Sulfo-NHS-biotin to visualize the brain vessels and detect possible leaks. Normal control littermate pups or Mekk3 iKO pups were fed tamoxifen from day P1 and analysed at P6, a time-point chosen because it is before the onset of massive cerebral haemorrhage. In contrast to the control brains, the Mekk3 iKO brains showed massive leakage of Sulfo-NHS-biotinylated materials, which stained blue (Fig. 4a). The leakage appeared throughout much of the brain in the cerebral cortex, thalamus and hypothalamus areas indicating a defective brain blood barrier. The hippocampus area of the Mekk3 iKO brains showed significant blue staining compared to the control hippocampus, strongly indicating that the defect was due to blood vessel leakage.


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 critically regulates the neonatal vascular permeability to small size molecule via suppressing the Rho signals.(a–d) In vivo leakage experiments show only small-molecular-weight tracers leaked from brain microvessels in Mekk3 iKO and iEC−/− neonatal mice. (a) Sulfo-NHS-biotin (556.6 dalton) was injected into the hearts of the tamoxifen-treated Mekk3 NCL or Mekk3 iKO neonatal pups. Brain sections were analysed for Sulfo-NHS-biotin leakage by staining. Objective lens power: 2.5 × . (b–d) Fluorescent-labelled tracers with different molecular weights were injected into tamoxifen-treated Mekk3 NCL or Mekk3 iEC−/− P7 neonatal pups' hearts. After euthanization, brains were fixed and sectioned and tracer leakage to the brain was determined. (b) Hoechst 33342 (616 dalton; blue) plus Dextran–Rhodamine (2 M Dalton; red), N=7, (c) Dextran–FITC (MW: 4K Dalton; green) plus Dextran–Rhodamine (2 M Dalton)(red), N=4, (d) Dextran–FITC (40 K Dalton; green) plus Dextran–Rhodamine (2 M Dalton; red). Bar graphs on the left of each panel show quantification of the relative leakage of the tracers normalized to the intensity of Dextran–Rhodamine, and shows no leakage in either NCL nor Mekk3 iEC−/− neonatal pups. Error bars indicate s.d. N=4. Objective lens power: 10 × . (e) ROCK inhibitor Y27632 partially rescues survival of Mekk3 iEC−/− neonatal pups. NCL or Mekk3 iEC−/− pups were treated with tamoxifen at P1, and continued daily in the absence (open circle and black diamond, respectively) or presence of Y27632 (cross and black triangle, respectively). Survival of pups was monitored daily until P20. (f) Hoechst 33342 (616 Dalton; blue) plus Dextran K–Rhodamine (2 M Dalton; red) were injected into tamoxifen-treated Mekk3 iEC−/− P7 neonatal pups' hearts fed either with water or Y27632. After euthanization, brains were fixed and sectioned at 30-μm thickness and leakage determined as described above. N=5. Objective lens power: 10 × . (g) Wild-type P7 neonatal pups were treated with cell-permeable wild-type Mekk3-peptide (MEKK3N-peptide) or A6D/L7D mutated Mekk3-peptide (MEKK3mutant-N-peptide). Brain leakage determined using Hoechst 33342 (blue) plus Dextran K–Rhodamine (red). N=5. Objective lens power: 10 × . (h) Proposed signalling pathway. The interactions of CCM2 and MEKK3 are critical for maintenance of vasculature integrity and permeability by control of Rho/ROCK signalling.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526114&req=5

f4: MEKK3 critically regulates the neonatal vascular permeability to small size molecule via suppressing the Rho signals.(a–d) In vivo leakage experiments show only small-molecular-weight tracers leaked from brain microvessels in Mekk3 iKO and iEC−/− neonatal mice. (a) Sulfo-NHS-biotin (556.6 dalton) was injected into the hearts of the tamoxifen-treated Mekk3 NCL or Mekk3 iKO neonatal pups. Brain sections were analysed for Sulfo-NHS-biotin leakage by staining. Objective lens power: 2.5 × . (b–d) Fluorescent-labelled tracers with different molecular weights were injected into tamoxifen-treated Mekk3 NCL or Mekk3 iEC−/− P7 neonatal pups' hearts. After euthanization, brains were fixed and sectioned and tracer leakage to the brain was determined. (b) Hoechst 33342 (616 dalton; blue) plus Dextran–Rhodamine (2 M Dalton; red), N=7, (c) Dextran–FITC (MW: 4K Dalton; green) plus Dextran–Rhodamine (2 M Dalton)(red), N=4, (d) Dextran–FITC (40 K Dalton; green) plus Dextran–Rhodamine (2 M Dalton; red). Bar graphs on the left of each panel show quantification of the relative leakage of the tracers normalized to the intensity of Dextran–Rhodamine, and shows no leakage in either NCL nor Mekk3 iEC−/− neonatal pups. Error bars indicate s.d. N=4. Objective lens power: 10 × . (e) ROCK inhibitor Y27632 partially rescues survival of Mekk3 iEC−/− neonatal pups. NCL or Mekk3 iEC−/− pups were treated with tamoxifen at P1, and continued daily in the absence (open circle and black diamond, respectively) or presence of Y27632 (cross and black triangle, respectively). Survival of pups was monitored daily until P20. (f) Hoechst 33342 (616 Dalton; blue) plus Dextran K–Rhodamine (2 M Dalton; red) were injected into tamoxifen-treated Mekk3 iEC−/− P7 neonatal pups' hearts fed either with water or Y27632. After euthanization, brains were fixed and sectioned at 30-μm thickness and leakage determined as described above. N=5. Objective lens power: 10 × . (g) Wild-type P7 neonatal pups were treated with cell-permeable wild-type Mekk3-peptide (MEKK3N-peptide) or A6D/L7D mutated Mekk3-peptide (MEKK3mutant-N-peptide). Brain leakage determined using Hoechst 33342 (blue) plus Dextran K–Rhodamine (red). N=5. Objective lens power: 10 × . (h) Proposed signalling pathway. The interactions of CCM2 and MEKK3 are critical for maintenance of vasculature integrity and permeability by control of Rho/ROCK signalling.
Mentions: The above biochemical, biophysical and in vitro studies provided the first direct evidence to link the physiological function of an interaction between MEKK3 and CCM2 in brain vasculature. Since ample evidence has suggested the importance of CCM proteins in endothelial cell junctions15161718, to better understand the impact of Mekk3 deletion in brain vasculature development, we tested if there were any EC cell permeability alterations in the neurovasculature of developing mice. To understand why MEKK3 deficiency causes brain haemorrhage, we examined the integrity of brain blood vessels in Mekk3 iEC−/− and control mice. We used Sulfo-NHS-biotin to visualize the brain vessels and detect possible leaks. Normal control littermate pups or Mekk3 iKO pups were fed tamoxifen from day P1 and analysed at P6, a time-point chosen because it is before the onset of massive cerebral haemorrhage. In contrast to the control brains, the Mekk3 iKO brains showed massive leakage of Sulfo-NHS-biotinylated materials, which stained blue (Fig. 4a). The leakage appeared throughout much of the brain in the cerebral cortex, thalamus and hypothalamus areas indicating a defective brain blood barrier. The hippocampus area of the Mekk3 iKO brains showed significant blue staining compared to the control hippocampus, strongly indicating that the defect was due to blood vessel leakage.

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