Notch transcriptional target tmtc1 maintains vascular homeostasis

Abstract

Proper lung function requires the maintenance of a tight endothelial barrier while simultaneously permitting the exchange of macromolecules and fluids to underlying tissue. Disruption of this barrier results in an increased vascular permeability in the lungs, leading to acute lung injury. In this study, we set out to determine whether transcriptional targets of Notch signaling function to preserve vascular integrity. We tested the in vivo requirement for Notch transcriptional signaling in maintaining the pulmonary endothelial barrier by using two complementary endothelial-specific Notch loss-of-function murine transgenic models. Notch signaling was blocked using endothelial-specific activation of an inhibitor of Notch transcriptional activation, Dominant Negative Mastermindlike (DNMAML; CDH5Cre^ERT2), or endothelial-specific loss of Notch1 (Notch1^f/f; CDH5Cre^ERT2). Both Notch mutants increased vascular permeability with pan-Notch inhibition by DNMAML showing a more severe phenotype in the lungs and in purified endothelial cells. RNA sequencing of primary lung endothelial cells (ECs) identified novel Notch targets, one of which was transmembrane O-mannosyltransferase targeting cadherins 1 (tmtc1). We show that tmtc1 interacts with vascular endothelial cadherin (VE-cadherin) and regulates VE-cadherin egress from the endoplasmic reticulum through direct interaction. Our findings demonstrate that Notch signaling maintains endothelial adherens junctions and vascular homeostasis by a transcriptional mechanism that drives expression of critical factors important for processing and transport of VE-cadherin.

Keywords: Endothelial cells; Notch signaling; Vascular permeability.

Fig. 1

Comparative inducible transgenic model systems for in vivo tissue specific inhibition of Notch pathway in lung endothelial cells (ECs). A (Top) experimental design of WT, DNMAML CDH5Cre^ERT2 (DNMAML), Notch1^f/f CDH5Cre^ERT2 (Notch1^f/f) mice, and (bottom) illustration of the mechanism of pathway inhibition for each mutant model. Mice were injected with tamoxifen for 5 consecutive days to induce Cre activity in CDH5+ endothelial cells. Mice were rested for 2-weeks before used for experiments. B Representative gating strategy for FACS-purified ECs in tamoxifen-treated WT, DNMAML and Notch1^f/f mice. Cells were sorted for DAPI⁻ CD45⁻ CD31⁺. C mRNA expression of canonical Notch targets Hes1, Hey1, and EphrinB2 in WT, DNMAML, and Notch1^f/f mice. Hes1: n (individual mouse replicates) = 5, 3, and 3 from left to right for each genotype. Hey1: n (individual mouse replicates) = 5, 4, and 3 for each genotype. Ephrin B2: n (individual mouse replicates) = 5, 3, and 3 for each genotype. Statistical analysis: one-way ANOVA. D Representative Western blot of cleaved-Notch1, Notch4, and VE-Cadherin in FACS-sorted lung ECs. GAPDH was used as a loading control. E Quantification of Western blot protein levels for cleaved-Notch1: n (individual mouse replicates) = 3, 2, 3 for each genotype. VE-Cadherin: n (individual mouse replicates) = 3, 3, 2 or each genotype. Notch4: n (individual mouse replicates) = 3, 2, 3 for each genotype

Fig. 2

Loss of Notch signaling disrupts pulmonary vascular integrity and inhibition of Notch signaling increases vascular permeability. A (Top) Representative images and (bottom) quantification of EBA extravasation in perfused lungs. n (individual mouse replicates) = 12, 16, 12 from left to right for each genotype. Statistical analysis: one-way ANOVA. B Wet to dry ratio of lungs. n (individual mouse replicates) = 6, 4, 7 for each genotype. Statistical analysis: one-way ANOVA. C Capillary filtration coefficient (Kfc) in lungs. n (individual mouse replicates) = 12, 4, 7 for each genotype. Statistical analysis: one-way ANOVA. D H&E staining of section of WT, DNMAML, and Notch1^f/f lungs. E IF staining of lung sections of WT, DNMAML and Notch1 for Ki67 (top) and cleaved Caspase3 (bottom). scale bar 50um. Quantification of Ki67 positive cells (%) in indicated genotypes (right). Statistical analysis: one-way ANOVA. F Representative flow cytometry histogram of ECs in tamoxifen-treated WT, DNMAML and Notch1^f/f mice gated for DAPI⁻ CD45⁻ CD31⁺ and analyzed for Ki67 (left) and AnnexinV (right). G Quantification of lung inflammation score. n (individual mouse replicates) = 3 for all conditions. Statistical analysis: one-way ANOVA. H Absolute granulocyte (CD45⁺ CD31⁻ CD11b⁺ Gr1⁺) numbers in WT and Notch1^f/f lungs. n (individual mouse replicates) = 3. Statistical analysis: Welch’s t-test. I Experimental design. WT mice were injected with 2 consecutive injections of DMSO or GSI (10 mg/kg total). Lungs ECs were FACS-purified for further experiments. J mRNA expression of Notch targets Hes1, Hey1, and EphrinB2 in FACS-sorted lung ECs from DMSO- or GSI-treated mice. Expression was normalized to GAPDH. n (individual mouse replicates) = 4 for both conditions. Statistical analysis: Welch’s t-test. K Wet/Dry ratio. n (individual mouse replicates) = 4 for both conditions. Statistical analysis: Welch’s t-test. L EBA extravasation of perfused lungs. n (individual mouse replicates) = 4 for both conditions. Statistical analysis: Welch’s t-test. M (left) TEER of HLMVECs transfected with empty vector (EV) or DNMAML-encoding plasmids. Resistance was measured 48 h post-transfection. n (biological replicates) = 3 and 4. (right) quantification n (biological replicates) = 3, 3, 4, 4 from left to right. Statistical analysis: one-way ANOVA. Scale bar for D and E 100 μm and 25 μm, respectively

Fig. 3

Tmtc1 is a novel Notch transcriptional target in lung ECs. A Heat map of WT, DNMAML, and Notch1^f/f lung bulk RNA-sequencing of sorted lung EC gene expression. Heat map shows the 15 highest upregulated and downregulated genes for each genotype. n (individual mouse replicates) = 3 for all conditions. B Volcano plot from RNAseq data of WT, DNMAML, and Notch1^f/f. C Enrichment plot for S phase genes in DNMAML lung ECs. D Venn diagram of DNMAML and Notch1^f/f showing number of genes that were (top) downregulated or (down) upregulated from both RNAseq datasets compared to WT. E Representative Notch1 and RBPJ predicted regulatory binding regions identified by ChIP-seq for tmtc1 (top) and slc6a2 (bottom) in HUVECs. F Tmtc1 mRNA expression of FACS-sorted lung endothelial cells from mutant mice. Expression was normalized to GAPDH. n (individual mouse replicates) = 4, 4, and 3. Statistical analysis: one-way ANOVA. G Representative Western blot of Tmtc1.  H Quantification of western blot of FACS-sorted lung ECs from WT, DNMAML, and Notch1^f/f mice. GAPDH was used as a loading control. n (mouse biological replicates) = 3. Statistical analysis one-way ANOVA. I (left) Tmtc1 and (right) Hes1 mRNA expression of HLMVECs treated with DMSO, GSI, and 4-h washout. Expression was normalized to GAPDH. n (biological replicates) = 4, 4, and 3. Statistical analysis: one-way ANOVA. J Chromatin Immunoprecipitation of the TMTC1 promoter using Notch1 Val1744 antibody in HLMVECs treated with DMSO or GSI. n (technical replicates) = 5. J (left) Tmtc1 and (right) Hes1 mRNA expression of HLMVECs treated with DMSO, GSI, and 4-h washout. Expression was normalized to GAPDH. n (biological replicates) = 4, 4, and 3. Statistical analysis: one-way ANOVA. K Tmtc1 mRNA expression of WT mice treated with DMSO or GSI. Expression was normalized relative to GAPDH. n (individual mouse replicates) = 4 for both conditions. Statistical analysis: Welch’s t-test

Fig. 4

Loss of tmtc1 negatively impacts endothelial junctional integrity. A Tmtc1 mRNA expression of HLMVECs transfected with siRNA Scrambled (siSc) or siTmtc1 at 24 h, 48 h, and 72 h. Expression was normalized relative to GAPDH. n (biological replicates) = 5, 4, 3, 5, 5 and 6. from left to right. Statistical analysis: one-way ANOVA. B (left) TEER of control siRNA (siSc) or tmtc1 siRNA treated cells (siTmtc1) and (right) quantification of TEER. n (biological replicates) = 3 for all conditions. Statistical analysis: one-way ANOVA. C Immunofluorescent staining of siSc- and siTmtc1-treated HLMVECs and D Quantification of junctional VE-Cadherin of siSc- and siTmtc1-treated HLMVECs n (biological replicates) = 3 and 4, respectively. Quantification determined by averaging VE-cadherin junctional fluorescent intensity per field of view. Statistical analysis: Welch’s t-test. Cells were stained with VE-Cadherin (green) and Hoechst 33342 (blue)

Fig. 5

Accumulation of VE-Cadherin in the endoplasmic reticulum after tmtc1 knockdown. A Immunofluorescent imaging of siSc-, siTmtc1-, EV-, and DNMAML-treated HLMVECs. Cells were stained with SERCA2 (red), VE-Cadherin (magenta) and DAPI (blue). B Quantification of colocalization of SERCA2 and VE-Cadherin from (left) siSc- and siTmtc1-, and (right) EV- and DNMAML-treated HLMVECs is represented by individual experiments. n (biological replicates) = 6 for all conditions. Statistical analysis: Welch’s t-test. C RT-qPCR of Tmtc1 in EV, DNMAML, and DNMAML+TMTC1 transfected HLMVECs, n (technical replicates) = 3. D Surface VE-Cadherin expression detected by flow cytometry of HLMVECs transfected with EV, DNMAML, or DNMAML+TMTC1. n (biological replicates) = 5 for all conditions. Statistical analysis one-way ANOVA. E (Top) Co-Immunoprecipitation analysis of Tmtc1 and VE-Cadherin. HLMVECs were transfected with siSc, siTmtc1, and DNMAML. The cell lysates were immunoprecipitated with anti-Tmtc1 and were probed for VE-Cadherin. Representative (bottom) loading Western blot lysate for VE-Cadherin, Tmtc1, and GAPDH from total HLMVECs transfected with siSc, siTmtc1, and DNMAML. n (biological replicates) = 2