Research ArticleVASCULAR BIOLOGY

The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses and protects against the development of pulmonary arterial hypertension

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Science Signaling  12 Nov 2019:
Vol. 12, Issue 607, eaay4430
DOI: 10.1126/scisignal.aay4430
  • Fig. 1 ATOH8 is a SMAD1/5 target gene that plays important roles in the cardiovascular system.

    (A) Overlap of genes induced by BMP-9/ALK-1 (GSE27661) (5), Notch ligand DLL4 (21), or the NICD (GSE29850) (20) in HUVECs, illustrated by a Venn diagram. (B) List of genes that are induced by ALK-1 activation but not induced by Notch (top 10 genes are presented). (C) Visualization of the ATOH8 locus and the result of SMAD1/5 ChIP-seq analysis. Red peaks represent ChIP regions (top). The conservation plots for mouse/human, frog/human, and zebrafish/human are derived from the VISTA genome browser (middle) (68), which presents the sequence conservation between species. SBR, SMAD1/5-binding region; Control, a negative control region used in ChIP-qPCR experiment. (D) ISH for the expression of mouse Atoh8 mRNA (red dots, indicated by arrows) and control genes in the E17.5 embryo. High-magnification images of the dashed square areas are presented. Images are representative of different experiments (more than n = 3 independent samples). Scale bars, 200 μm (left) and 50 μm (right).

  • Fig. 2 ATOH8 is induced by BMP-9/ALK-1 but not by Notch in ECs.

    (A) qRT-PCR analysis of HUVECs treated for 2 hours with angiogenic cytokines as indicated. GAPDH was used as endogenous control, and data were normalized to the control condition. Results of n ≥ 3 independent experiments are shown by scatter plots with bars representing the means. (B) qRT-PCR analysis of HUVECs treated with BMP-9 (1 ng ml−1) for the indicated time periods. GAPDH was used as endogenous control. Results of n = 3 independent experiments are shown by scatter plots with bars representing the means. n.s., not significant. (C and D) qRT-PCR analysis of HUVECs transfected with siRNA against SMAD4 (siSMAD4) or control (siControl) for 48 hours and treated with BMP-9 (1 ng ml−1) as indicated for 2 hours. Knockdown efficiency of SMAD4 is presented in (D). Results of n = 3 independent experiments are shown by scatter plots with bars presenting the means. (E) qRT-PCR analysis of HUVECs infected with control (Ad-LacZ) or NICD-expressing adenoviruses (Ad-NICD). GAPDH was used as endogenous control, and data were normalized to the control condition. Results of n = 3 independent experiments are shown by scatter plots with bars representing the means. (F) Western blot analysis for endogenous ATOH8 protein in HUVECs, which were transfected with siRNA against ATOH8 (siATOH8) or control (siControl) and treated with BMP-9 (1 ng ml−1) for 24 hours. HDAC1 (nuclear marker) and α-tubulin (αTUB) (cytosol marker) were used as markers for cellular fraction and loading controls. Blots are representative of n = 3 independent experiments. Right: Quantification of each lane of the blots. Results of n = 3 independent experiments are shown by scatter plots with bars representing the means. (G) SMAD1/5 ChIP-qPCR analysis of HUVECs treated with BMP-9 (1 ng ml−1) for 1.5 hours. ChIP was performed using antibody against SMAD1/5. The HPRT1 gene locus was used as a negative control region, and fold enrichment was calculated. Results of n = 3 independent experiments are shown by scatter plots with bars representing the means. (H) Luciferase reporter assay of HUVECs transfected with the indicated reporter constructs using lentiviral vector system and treated with BMP-9 (0.2 or 1 ng ml−1) for 16 hours. Results of n ≥ 3 independent experiments are shown by scatter plots with bars representing the means. Differences between the conditions were analyzed by Welch’s t test, or unequal variances t test, for a single comparison (Fig. 2D) and Tukey’s honestly significant difference (HSD) test for multiple comparisons (A to C and E to H). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Zebrafish atoh8 is expressed in large vessels, but atoh8 knockout fish display normal vascular patterning.

    (A) Whole-mount ISH of zebrafish atoh8 at 48 hpf (hours after fertilization). (B) Schematic illustration of the CRISPR-Cas9/guide RNA (gRNA)–targeting sites in the exon 1 of the atoh8 gene. The gRNA-targeting sequence is marked by a gray box, and the protospacer-adjacent motif (PAM) sequence is marked by an orange box in the WT sequence. The sequence of two mutant lines is also shown. (C) Results of Sanger sequencing of genomic DNA from the mutants. Dashed lines represent the deletion location. (D) Representative bright-field (left) and fluorescence images (right; dashed square area of the bright-field image) of atoh8WT/WT and atoh8uu3112/uu3112 fishes at 36 hpf are shown. Scale bars, 500 μm. (E) Assessment of ISV formation in atoh8WT/WT, atoh8WT/uu3112, and atoh8uu3112/uu3112 fishes at 36 hpf (n = 8 for atoh8WT/WT, n = 7 for atoh8WT/uu3112, and n = 10 for atoh8uu3112/uu3112). Differences between the conditions were analyzed by Tukey’s HSD test corrected for multiple comparisons. *P < 0.05.

  • Fig. 4 Loss of Atoh8 results in a delay in retinal angiogenesis in Atoh8-deficient mice.

    (A) Schematic presentation of Atoh8 WT allele, the targeting vector, and the Atoh8 mutant allele. (B) Representative images of whole-mount retinas of P5 Atoh8+/− and Atoh8−/− mice. The tiling images were manually merged. Scale bars, 500 μm. (C) Bar graphs show the mean distance of radial expansion, vessel area, and junction density of Atoh8+/− and Atoh8−/− mice. Data are presented as dot plot (n = 5 and 7 mice, respectively), and differences between the conditions were analyzed by Welch’s t test. **P < 0.01. a.u., arbitrary units.

  • Fig. 5 Atoh8-deficient mice exhibit a phenotype resembling human PAH.

    (A) ISH for expression of mouse Atoh8 mRNA (red dots, indicated by arrows) and control genes in the lungs and hearts of aged mice in normoxic condition. High-magnification images of the dashed square areas are presented. Images are representative of different experiments (more than n = 3 independent samples). Scale bars, 500 μm (left) and 50 μm (right three panels). (B and C) Assessment of right ventricular systolic pressure (RVSP) and systemic systolic blood pressure (SBP) of Atoh8−/− mice. RVSP was measured in normoxic (B) and hypoxic (C) conditions. Data are presented as scatter plot with mean (RVSP: n = 6 for each group in normoxia and n = 7 in hypoxia; systemic SBP: n = 12), and differences between the conditions were analyzed by Welch’s t test. **P < 0.01. (D and E) Assessment of right ventricular hypertrophy of Atoh8−/− mice. The hearts of Atoh8+/+ and Atoh8−/− male mice in the normoxic and hypoxic conditions were analyzed (n = 14 Atoh8+/+ mice under normoxia, n = 16 Atoh8−/− mice under normoxia, and n = 15 mice for each genotype under hypoxia). Data are presented as scatter plot with mean (D). Representative images of hearts of mice in normoxia are presented (E). Differences between the conditions were analyzed by Tukey’s HSD test corrected for multiple comparisons. *P < 0.05 and ***P < 0.001. Scale bars, 1 mm. (F and G) Assessment of pulmonary arterial muscularization of Atoh8−/− mice. Nonmuscularized and partially and fully muscularized arteries, as a percentage of total alveolar wall and duct arteries, were scored in Atoh8+/+ (n = 17) and Atoh8−/− (n = 19) male mice. Data are presented as a stacked bar plot [means ± SEM (F)], and representative images of immunohistochemical staining for smooth muscle α-actin are shown (G). Scale bars, 50 μm. Welch’s t test for fully muscularized vessels, ***P < 0.001. (H) Volcano plot of differentially expressed genes from patients with PAH (n = 12) and healthy control participants (n = 11) (GSE53408) (34).

  • Fig. 6 ATOH8 does not function as a DNA binding factor in HPAECs.

    (A) A Venn diagram indicating overlap of SMAD1/5-binding sites of HUVECs (5) and HPAECs treated with BMP-9. The numbers of overlapping regions are not identical because some of the peaks do not show a one-by-one correspondence. (B) Visualization of the ATOH8 gene locus and the result of SMAD1/5 (n = 2) and FLAG-ATOH8 (n = 2) ChIP-seq in HPAECs. Colored peaks represent ChIP regions (top). (C) SMAD1/5 ChIP-qPCR analysis of HPAECs treated with BMP-9 (1 ng ml−1) for 1.5 hours. ChIP was performed using antibody against SMAD1/5. The HPRT1 gene locus was used as a negative control region, and fold enrichment was calculated. Results of n = 5 independent experiments are shown by scatter plots with bars representing the means. Differences between the conditions were analyzed by Tukey’s HSD test corrected for multiple comparisons. **P < 0.01. (D) De novo motif prediction analysis of the FLAG-ATOH8–binding sites. (E) Western blot analysis of FLAG-tagged ATOH8 protein expression in HEK293T cells cultured with or without 10 μM MG132 for 16 hours and then treated with cycloheximide (CHX) (100 μg/ml) for the indicated time periods. α-Tubulin was used as a loading control. Blots are representative of n = 3 independent experiments. (F) Western blot analysis of FLAG-tagged ATOH8 protein in HEK293T cells. Cells were transfected with the indicated ATOH8 mutants (0.3 μg for Δ231-286 and 1 μg for the others). Twenty-four hours after transfection, cells were incubated with or without 10 μM MG132 for 16 hours. Cells were then lysed and subjected to SDS-PAGE and Western blotting. α-Tubulin was used as a loading control. Blots are representative of n = 3 independent experiments. FL, full length.

  • Fig. 7 ATOH8 physically interacts with HIF-2α and decreases its abundance.

    (A) HEK293T cells were transiently transfected with the indicated plasmids. FLAG immunoprecipitates (IP) were subjected to Western blotting for FLAG (to detect HIF-1α or HIF-2α) or Myc (to detect ATOH8). Blots are representative of n = 3 independent experiments. (B) HEK293T cells were transfected with the indicated plasmids and subjected to Western blotting to assess the effect of ATOH8 coexpression on HIF-2α abundance. α-Tubulin was used as a loading control. Blots are representative of n = 4 independent experiments. Right: Quantification of each lane of the blots. Results of n = 4 independent experiments are shown by scatter plots with bars representing the means. Differences between the conditions were analyzed by Tukey’s HSD test corrected for multiple comparisons. *P < 0.05. (C) HEK293T cells were transfected with the indicated plasmids, treated with MG132, and subjected to Western blotting to assess that proteasomal inhibition modulates the effect of ATOH8 coexpression on HIF-2α abundance. α-Tubulin was used as a loading control. Representative images are taken from n ≥ 3 experiments. (D) HEK293T cells were transfected with the indicated plasmids and subjected to Western blotting to determine the binding site of HIF-2α on ATOH8. α-Tubulin was used as a loading control. Blots are representative of n = 3 independent experiments (see also fig. S5A). (E) FLAG immunoprecipitates were subjected to Western blotting to assess the requirement for the bHLH domain of ATOH8 on the ATOH8/HIF-2α interaction. Blots are representative of n = 3 independent experiments. (F) HPAECs stably expressing FLAG-ATOH8 were maintained under hypoxic conditions (≤1% O2) for 24 hours. FLAG immunoprecipitates were subjected to Western blotting for FLAG (to detect HIF-1α or HIF-2α) or Myc (to detect ATOH8). Blots are representative of n = 3 independent experiments.

  • Fig. 8 ATOH8 attenuates hypoxia-induced HIF-2α activation and target gene expression.

    (A) HPAECs were infected with either Ad-ATOH8 or control (Ad-LacZ) for 24 hours and then transferred to hypoxic (≤1% O2) or normal (21% O2) conditions for an additional 24 hours. Cell lysates were analyzed by Western blotting for endogenous HIF proteins. α-Tubulin was used as a loading control. Blots are representative of n = 3 independent experiments. Right: Quantification of each lane of the blots. Results of n = 3 independent experiments are shown by a scatter plot with bars presenting the means. Differences between the lanes were analyzed by Tukey’s HSD test corrected for multiple comparisons. **P < 0.01 and ***P < 0.001. (B) qRT-PCR analysis of HPAECs infected with either Ad-ATOH8 or control (Ad-LacZ) for 24 hours and then transferred to hypoxic (≤1% O2) or normal (21% O2) conditions for an additional 24 hours. HPRT1 was used as endogenous control, and data were normalized to the control condition. Results of n = 3 independent experiments are shown by a scatter plot with bars representing the means. exo, exogenous. Differences between the conditions were analyzed by Tukey’s HSD test corrected for multiple comparisons. **P < 0.01 and ***P < 0.001. (C) qRT-PCR analysis of HPAECs transfected with either siRNAs specific for ATOH8 or control siRNA for 60 hours. HPRT1 was used as endogenous control, and data were normalized to the control condition. Results of n = 5 independent experiments are shown by a scatter plot with bars representing the means. Differences between the conditions were analyzed by Tukey’s HSD test corrected for multiple comparisons. *P < 0.05 and ***P < 0.001. (D) HPAECs were seeded 24 hours before treatment. Cells were treated with BMP-9 (5 ng ml−1) or infected with either Ad-ATOH8 or control (Ad-LacZ) for 24 hours and then transferred to hypoxic (≤1% O2) or normal (21% O2) conditions for an additional 48 hours. The ratio of the number of the cells under hypoxia to that under normoxia was calculated. Results of n ≥ 3 independent experiments are shown by a scatter plot with bars representing the means. Differences between the conditions were analyzed by Tukey’s HSD test corrected for multiple comparisons. *P < 0.05.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/607/eaay4430/DC1

    Fig. S1. ATOH8 is induced by BMP-9/ALK-1 but not by Notch in ECs.

    Fig. S2. Zebrafish atoh8 is expressed in large vessels, and atoh8 morphants have slight delay in ISV formation.

    Fig. S3. Atoh8−/− mice are small in body size but are viable and fertile.

    Fig. S4. Atoh8 is expressed in ECs in the lung, and Atoh8−/− mice exhibit PAH-like phenotype.

    Fig. S5. ATOH8 interacts with HIF-1α and decreases its protein abundance.

    Data file S1. Gene regulation of 70 putative direct target genes of BMP-9/ALK-1.

    Data file S2. SMAD1/5-binding sites in HUVECs that are related to blood vessels or the heart.

    Data file S3. Genomic regions commonly bound by FLAG-ATOH8 in HPAEC-FLAG-ATOH8 cells.

    Data file S4. Primer sets used for qRT-PCR and ChIP-qPCR.

  • The PDF file includes:

    • Fig. S1. ATOH8 is induced by BMP-9/ALK-1 but not by Notch in ECs.
    • Fig. S2. Zebrafish atoh8 is expressed in large vessels, and atoh8 morphants have slight delay in ISV formation.
    • Fig. S3. Atoh8−/− mice are small in body size but are viable and fertile.
    • Fig. S4. Atoh8 is expressed in ECs in the lung, and Atoh8−/− mice exhibit PAH-like phenotype.
    • Fig. S5. ATOH8 interacts with HIF-1α and decreases its protein abundance.
    • Legends for data files S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Gene regulation of 70 putative direct target genes of BMP-9/ALK-1.
    • Data file S2 (Microsoft Excel format). SMAD1/5-binding sites in HUVECs that are related to blood vessels or the heart.
    • Data file S3 (Microsoft Excel format). Genomic regions commonly bound by FLAG-ATOH8 in HPAEC-FLAG-ATOH8 cells.
    • Data file S4 (Microsoft Excel format). Primer sets used for qRT-PCR and ChIP-qPCR.

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