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. 2019 Nov 12;12(607):eaay4430.
doi: 10.1126/scisignal.aay4430.

The ALK-1/SMAD/ATOH8 Axis Attenuates Hypoxic Responses and Protects Against the Development of Pulmonary Arterial Hypertension

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Free PMC article

The ALK-1/SMAD/ATOH8 Axis Attenuates Hypoxic Responses and Protects Against the Development of Pulmonary Arterial Hypertension

Masato Morikawa et al. Sci Signal. .
Free PMC article

Abstract

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) is implicated in vascular diseases such as pulmonary arterial hypertension (PAH). Here, we showed that the transcription factor ATOH8 was a direct target of SMAD1/5 and was induced in a manner dependent on BMP but independent of Notch, another critical signaling pathway in ECs. In zebrafish and mice, inactivation of Atoh8 did not cause an arteriovenous malformation-like phenotype, which may arise because of dysregulated Notch signaling. In contrast, Atoh8-deficient mice exhibited a phenotype mimicking PAH, which included increased pulmonary arterial pressure and right ventricular hypertrophy. Moreover, ATOH8 expression was decreased in PAH patient lungs. We showed that in cells, ATOH8 interacted with hypoxia-inducible factor 2α (HIF-2α) and decreased its abundance, leading to reduced induction of HIF-2α target genes in response to hypoxia. Together, these findings suggest that the BMP receptor type II/ALK-1/SMAD/ATOH8 axis may attenuate hypoxic responses in ECs in the pulmonary circulation and may help prevent the development of PAH.

Conflict of interest statement

Competing Interests

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. ATOH8 is a SMAD1/5 target gene which 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 Notch intracellular domain (NICD) (GSE29850) (20) in human umbilical vein endothelial cells (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 panel). The conservation plots for mouse/human, frog/human and zebrafish/human are derived from the VISTA genome browser (middle panel) (68), which presents the sequence conservation between species. SBR, Smad 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 bar: 200 µm (left) and 50 µm (right).
Figure 2
Figure 2. ATOH8 is induced by BMP-9/ALK-1 but not by Notch in ECs
(A) qRT-PCR analysis of HUVECs treated for 2 h 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. (C and D) qRT-PCR analysis of HUVECs transfected with siRNA against SMAD4 (siSMAD4) or control (siControl) for 48 h, and treated with BMP-9 (1 ng ml-1) as indicated for 2 h. Knock-down 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 Notch intracellular domain (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 h. 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 h. 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 h. 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 (Fig. 2A-C, 2E-H); *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3. Zebrafish atoh8 is expressed in large vessels but atoh8 knockout fish display normal vascular patterning
(A) Whole-mount in situ hybridization 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 grey box and the protospacer-adjacent motif (PAM) sequence by an orange box in the wild-type sequence. The sequence of 2 mutant lines is also shown. (C) Results of Sanger sequencing of genomic DNA from the mutants are presented. Dashed lines represent the deletion location. (D) Representative bright field (left) and fluorescence images (right, the dashed-square area of the bright-field image) of atoh8WT/WT and atoh8uu3112/uu3112 fish at 36 hpf are shown. Scale bar: 500 µm. (E) Assessment of ISV formation in atoh8WT/WT, and atoh8WT/uu3112 and atoh8uu3112/uu3112 fish at 36 hpf (n=8 for atoh8WT/WT, n=7 for atoh8WT/uu3112, n=10 for atoh8uu3112/uu3112). Differences between the conditions were analyzed by Tukey's HSD test corrected for multiple comparisons; *p < 0.05.
Figure 4
Figure 4. Loss of Atoh8 results in a delay in retinal angiogenesis in Atoh8-deficient mice
(A) Schematic presentation of Atoh8 wild-type (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 bar: 500 µm. (C) Bar graphs show the mean distance of radial expansion, vessel area, and junction density of Atoh8+/− and Atoh8−/− mice. The 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.
Figure 5
Figure 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 bar: 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. The 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, n=15 mice for each genotype under hypoxia). The 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, ***p < 0.001. Scale bar: 1 mm. (F and G) Assessment of pulmonary arterial muscularization of Atoh8/− mice. Non-, 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. The data is presented as a stacked bar plot, mean ± 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 PAH patients (n=12) and healthy control subjects (n=11) (GSE53408) (34).
Figure 6
Figure 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 panel). (C) SMAD1/5 ChIP-qPCR analysis of HPAECs treated with BMP-9 (1 ng ml-1) for 1.5 h. 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 < 0.05. (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 h and then treated with 100 μg/ml cycloheximide (CHX) for indicated time periods. αTUB 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 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 h. Cells were then lysed and subjected to SDS-PAGE and Western blotting. αTUB was used as a loading control. Blots are representative of n=3 independent experiments. FL, full-length.
Figure 7
Figure 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. αTUB 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 proteasomal inhibition modulates the effect of ATOH8 coexpression on HIF-2α abundance. αTUB 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. αTUB 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 h. 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.
Figure 8
Figure 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 h, then transferred to hypoxic (≤1% O2) or normal (21% O2) conditions for an additional 24 h. Cell lysates were analysed by Western blotting for endogenous HIF proteins. αTUB 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 bar presenting the means. Differences between the lanes were analyzed by Tukey's HSD test corrected for multiple comparisons; **p < 0.01, ***p < 0.001. (B) qRT-PCR analysis of HPAECs infected with either Ad-ATOH8 or control (Ad-LacZ) for 24 h, then transferred to hypoxic (≤1% O2) or normal (21% O2) conditions for an additional 24 h. 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 bar representing the means. exo: exogenous. Differences between the conditions were analyzed by Tukey's HSD test corrected for multiple comparisons; **p < 0.01, ***p < 0.001. (C) qRT-PCR analysis of HPAECs transfected with either siRNAs specific for ATOH8 or control siRNA for 60 h. 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 bar representing the means. Differences between the conditions were analyzed by Tukey's HSD test corrected for multiple comparisons; *p < 0.05, ***p < 0.001. (D) HPAECs were seeded 24 h before treatment. Cells were treated with BMP-9 (5 ng ml-1) or infected with either Ad-ATOH8 or control (Ad-LacZ) for 24 h, then transferred to hypoxic (≤1% O2) or normal (21% O2) conditions for an additional 48 h. 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 bar representing the means. Differences between the conditions were analyzed by Tukey's HSD test corrected for multiple comparisons; *p < 0.05.

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