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Cited by 105Barrier Structure and Function StudiesDrug Transport and Resistance MechanismsWnt/β-catenin signaling in development and cancer

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Modulation of Wnt/β-catenin signaling promotes blood-brain barrier phenotype in cultured brain endothelial cells

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Laksitorini, Marlyn D., Yathindranath, Vinith, Xiong, Wei, Hombach-Klonisch, Sabine, Miller, Donald W.·2019·Scientific Reports
DOI 10.1038/s41598-019-56075-wPMC PMC6928218
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Abstract

Wnt/β-catenin signaling is important for blood-brain barrier (BBB) development and is implicated in BBB breakdown under various pathophysiological conditions. In the present study, a comprehensive characterization of the relevant genes, transport and permeability processes influenced by both the autocrine and external activation of Wnt signaling in human brain endothelial cells was examined using hCMEC/D3 culture model. The hCMEC/D3 expressed a full complement of Wnt ligands and receptors. Preventing Wnt ligand release from hCMEC/D3 produced minimal changes in brain endothelial function, while inhibition of intrinsic/autocrine Wnt/β-catenin activity through blocking β-catenin binding to Wnt transcription factor caused more modest changes. In contrast, activation of Wnt signaling using exogenous Wnt ligand (Wnt3a) or LiCl (GSK3 inhibitor) improved the BBB phenotypes of the hCMEC/D3 culture model, resulting in reduced paracellular permeability, and increased P-glycoprotein (P-gp) and breast cancer resistance associated protein (BCRP) efflux transporter activity. Further, Wnt3a reduced plasmalemma vesicle associated protein (PLVAP) and vesicular transport activity in hCMEC/D3. Our data suggest that this in vitro model of the BBB has a more robust response to exogenous activation of Wnt/β-catenin signaling compared to autocrine activation, suggesting that BBB regulation may be more dependent on external activation of Wnt signaling within the brain microvasculature.

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Figures

Figure 1 Expression of Wnt/β-catenin components in hCMEC/D3 cells. Expression of Wnt receptors and co-receptors (Panel a), Wnt ligands (Panel b) and Wnt modulators (Panel c) were examined by RT-PCR in confluent hCMEC/D3 monolayers. Total RNA were isolated for further PCR studies. Human Fetal Brain RNA were used as a control positive. Asterisk (*) is the correct PCR product in the primer that shown multiple bands.
Figure 1 Expression of Wnt/β-catenin components in hCMEC/D3 cells. Expression of Wnt receptors and co-receptors (Panel a), Wnt ligands (Panel b) and Wnt modulators (Panel c) were examined by RT-PCR in confluent hCMEC/D3 monolayers. Total RNA were isolated for further PCR studies. Human Fetal Brain RNA were used as a control positive. Asterisk (*) is the correct PCR product in the primer that shown multiple bands.CC BY · PubMed Central
Figure 2 Evidence for autocrine activation of Wnt/β-catenin signaling in hCMEC/D3 monolayers. WntC59 1 µM treatment for 15–20 hour reduced β-catenin abundance (Panel a), downregulated Axin-2 and APCDD1 mRNA expression (Panel b), diminished Claudin-5 mRNA expression (Panel c) and reduced Claudin-5 protein expression (Panel d). The decreased Claudin-5 expression associated with reductions in paracellular barrier were examined using real time cell analyzer (Panel e). hCMEC/D3 cell viability post 24 hours exposure to various concentrations of WntC59 (Panel f). Panel a and d show representative Western blotting of each protein. The cropped blot are used in the figure and full length blot are available at Supplementary Figs. S5 and S6. a and d One tail t-test; n: 3–4. (b,c) multitple t-test n: 4. (e): n of 4. (f) n of 8, One way ANOVA followed by LSD Fisher’s test. (a–f) *p < 0.05. All values represent the mean ± SEM except (panel e): value is mean ± SD.
Figure 2 Evidence for autocrine activation of Wnt/β-catenin signaling in hCMEC/D3 monolayers. WntC59 1 µM treatment for 15–20 hour reduced β-catenin abundance (Panel a), downregulated Axin-2 and APCDD1 mRNA expression (Panel b), diminished Claudin-5 mRNA expression (Panel c) and reduced Claudin-5 protein expression (Panel d). The decreased Claudin-5 expression associated with reductions in paracellular barrier were examined using real time cell analyzer (Panel e). hCMEC/D3 cell viability post 24 hours exposure to various concentrations of WntC59 (Panel f). Panel a and d show representative Western blotting of each protein. The cropped blot are used in the figure and full length blot are available at Supplementary Figs. S5 and S6. a and d One tail t-test; n: 3–4. (b,c) multitple t-test n: 4. (e): n of 4. (f) n of 8, One way ANOVA followed by LSD Fisher’s test. (a–f) *p < 0.05. All values represent the mean ± SEM except (panel e): value is mean ± SD.CC BY · PubMed Central
Figure 3 Functional impact of Wnt/β-catenin pathway modulation in hCMEC/D3 monolayers. Effects of Wnt3a exposure on electrical impedance (Panel a); transcellular permeability to small hydrophilic probe sodium fluorescein (Panel b), large hydrophilic probe IRdye 800 PEG (Panel c) and P-glycoprotein permeability probe, Rhodamine 800 (Panel d); and intracellular accumulation of P-glycoprotein and Breast Cancer Resistance Protein dependent fluorescent dyes (Panel e and f, respectively). Electrical impedance changes in response to Wnt3a were measured in real time and expressed as Cell index (CI). Influence of Wnt/β-catenin on transcellular permeability and transport were examined 15-hours following exposure to Wnt3a or WntC59. Values represent the mean ± SEM of 3–4 monolayers for the impendance and permeability studies and 8 monolayers for transporter studies. *p < 0.05; **p < 0.01; ***p < 0.001 as determined using one way ANOVA and LSD Fisher’s test.
Figure 3 Functional impact of Wnt/β-catenin pathway modulation in hCMEC/D3 monolayers. Effects of Wnt3a exposure on electrical impedance (Panel a); transcellular permeability to small hydrophilic probe sodium fluorescein (Panel b), large hydrophilic probe IRdye 800 PEG (Panel c) and P-glycoprotein permeability probe, Rhodamine 800 (Panel d); and intracellular accumulation of P-glycoprotein and Breast Cancer Resistance Protein dependent fluorescent dyes (Panel e and f, respectively). Electrical impedance changes in response to Wnt3a were measured in real time and expressed as Cell index (CI). Influence of Wnt/β-catenin on transcellular permeability and transport were examined 15-hours following exposure to Wnt3a or WntC59. Values represent the mean ± SEM of 3–4 monolayers for the impendance and permeability studies and 8 monolayers for transporter studies. *p < 0.05; **p < 0.01; ***p < 0.001 as determined using one way ANOVA and LSD Fisher’s test.CC BY · PubMed Central
Figure 4 Modulation of Wnt/β-catenin response in hCMEC/D3 using TCF inhibitor ICRT-3. Effects of ICRT-3 treatment on select gene (Panel a) and protein expression (Panels b–d) and endothelial cell function (Panels e and g). Viability of hCMEC/D3 after being exposed to various ICRT-3 concentrations for 24 hours (Panel f). The blots in the middle show representative Westerns for each protein. The cropped blots are used in the figure and full length blots are available in Supplementary Fig. S7. For gene and protein expression and functional studies, cells were exposed to ICRT-3 (20 µM) for 15-hrs. For the electrical impedance studies readings were taken in real-time and expressed as the cell index (CI) normalized to control wells receiving no ICRT-3 (Panel e). Values in all panels represent the mean ± SEM except panel e which is mean ± SD. *p < 0.05 **p < 0.01 ***p < 0.001. Panel a was determined by multiple t-test; (Panel b–d) one tail t-test; (Panel f,g): One way ANOVA followed by LSD Fisher’s test.
Figure 4 Modulation of Wnt/β-catenin response in hCMEC/D3 using TCF inhibitor ICRT-3. Effects of ICRT-3 treatment on select gene (Panel a) and protein expression (Panels b–d) and endothelial cell function (Panels e and g). Viability of hCMEC/D3 after being exposed to various ICRT-3 concentrations for 24 hours (Panel f). The blots in the middle show representative Westerns for each protein. The cropped blots are used in the figure and full length blots are available in Supplementary Fig. S7. For gene and protein expression and functional studies, cells were exposed to ICRT-3 (20 µM) for 15-hrs. For the electrical impedance studies readings were taken in real-time and expressed as the cell index (CI) normalized to control wells receiving no ICRT-3 (Panel e). Values in all panels represent the mean ± SEM except panel e which is mean ± SD. *p < 0.05 **p < 0.01 ***p < 0.001. Panel a was determined by multiple t-test; (Panel b–d) one tail t-test; (Panel f,g): One way ANOVA followed by LSD Fisher’s test.CC BY · PubMed Central
Figure 5 Modulation on Wnt/β-catenin signaling in hCMEC/D3. Natural ligand Wnt3a was a stronger Wnt/β-catenin activator compared to LiCl 10 mM (Panel a). Wnt 3a and LiCl treatment increased β-catenin abundance in whole cell lysate (Panel b,c). The activation of Wnt/β-catenin signaling alters select transporters and intercellular junction proteins (Panel d and e). Wnt3a increased protein expression of Pgp and claudin-1 (Panel f and g). Panels b, c, f, g are representative Western blots of each protein. The cropped blots are used in the figure and full length blots are available in Supplementary Figs. S8–S11: One way ANOVA followed by LSD Fisher’s test.*p < 0.05; ***p < 0.001. (b,c,f,g,h): One tail t test. P < 0.05. n: 3–4. Mean ± SEM. (d,e): Two way ANOVA followed by LSD Fisher’s test. n: 4. *p < 0.05.Mean ± SEM.
Figure 5 Modulation on Wnt/β-catenin signaling in hCMEC/D3. Natural ligand Wnt3a was a stronger Wnt/β-catenin activator compared to LiCl 10 mM (Panel a). Wnt 3a and LiCl treatment increased β-catenin abundance in whole cell lysate (Panel b,c). The activation of Wnt/β-catenin signaling alters select transporters and intercellular junction proteins (Panel d and e). Wnt3a increased protein expression of Pgp and claudin-1 (Panel f and g). Panels b, c, f, g are representative Western blots of each protein. The cropped blots are used in the figure and full length blots are available in Supplementary Figs. S8–S11: One way ANOVA followed by LSD Fisher’s test.*p < 0.05; ***p < 0.001. (b,c,f,g,h): One tail t test. P < 0.05. n: 3–4. Mean ± SEM. (d,e): Two way ANOVA followed by LSD Fisher’s test. n: 4. *p < 0.05.Mean ± SEM.CC BY · PubMed Central
Figure 6 Wnt/β-catenin dependent responses to LiCl treatment in hCMEC/D3 monolayers. ICRT-3 inhibited the effects of LiCl (10 mM) on gene expression (Panel a); paracellular permeability (Panel b) and drug efflux transporter activity (Panels c and d). Values represent the mean ± SEM of n of 4–6 monolayers per treatment group. *p < 0.05; **p < 0.01; ***p < 0.001 as determined using two way ANOVA and LSD Fisher’s test for (Panel a) and one way ANOVA and LSD Fisher’s test for (Panel c,d).
Figure 6 Wnt/β-catenin dependent responses to LiCl treatment in hCMEC/D3 monolayers. ICRT-3 inhibited the effects of LiCl (10 mM) on gene expression (Panel a); paracellular permeability (Panel b) and drug efflux transporter activity (Panels c and d). Values represent the mean ± SEM of n of 4–6 monolayers per treatment group. *p < 0.05; **p < 0.01; ***p < 0.001 as determined using two way ANOVA and LSD Fisher’s test for (Panel a) and one way ANOVA and LSD Fisher’s test for (Panel c,d).CC BY · PubMed Central
Figure 7 Modulation of Wnt/β-catenin in hCMEC/D3 cells alters endocytic activity. Altered PLVAP gene expression with pharmacological modulation of Wnt activity (Panel a). The reduction of PLVAP gene expression by Wnt3a is associated with less vesicular transport activity (Panel b). a and b, n: 4–6; value was Mean ± SEM. *p < 0.05; **p < 0.005 and ***p < 0.001 determined by One Way ANOVA followed by LSD Fisher’s test
Figure 7 Modulation of Wnt/β-catenin in hCMEC/D3 cells alters endocytic activity. Altered PLVAP gene expression with pharmacological modulation of Wnt activity (Panel a). The reduction of PLVAP gene expression by Wnt3a is associated with less vesicular transport activity (Panel b). a and b, n: 4–6; value was Mean ± SEM. *p < 0.05; **p < 0.005 and ***p < 0.001 determined by One Way ANOVA followed by LSD Fisher’s testCC BY · PubMed Central

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Metadata

Authors
Laksitorini, M. D., Yathindranath, V., Xiong, W., Hombach-Klonisch, S., & Miller, D. W.
Affiliations
  • Universitas Gadjah Mada (ID)
  • University of Manitoba (CA)
  • Health Sciences Centre (CA)
Corresponding author
Miller, Donald W.
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2019-12-23
Volume
9
Issue
1
Pages
19718-19718
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-019-56075-w
PMC
PMC6928218
OpenAlex
W2997752650
Cited by
105
References
63
Citations by year
2026: 13 · 2025: 9 · 2024: 21 · 2023: 12 · 2022: 21 · 2021: 19 · 2020: 10
FWCI
5.50
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Manitoba Health Research Council
  • University of Manitoba
  • Canadian Breast Cancer Foundation
  • Cancer Research Society
Topics
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
Keywords
Wnt/β-catenin signaling, blood-brain barrier, brain endothelial cells, hCMEC/D3, Wnt3a, paracellular permeability, P-glycoprotein, ABCG2, efflux pumps, PLVAP, vesicle trafficking, GSK3 inhibition, blood-brain barrier disruption
MeSH terms
Blood-Brain Barrier, Cells, Cultured, Endocytosis, Gene Expression Regulation, Humans, Ligands, Membrane Proteins, Oxazoles, Phenotype, Receptors, Cell Surface, Lithium Chloride, Autocrine Communication, Endothelial Cells, Wnt Signaling Pathway

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