Disruption of DLL4/NOTCH1 Causes Dysregulated PPARγ/AKT Signaling in Pulmonary Arterial Hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2-silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3Kδ/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2-silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Keywords: AKT; BMPR2; DLL4; HPAH; IPAH; N1ICD; PPARγ; endothelial dysfunction; signal transduction; vascular remodeling.

Figure 1.. BMPR2 knockdown activates AKT and protects the cells against apoptosis.

(A) Human primary pulmonary artery endothelial cells were transfected with control (siCTRL) or BMPR2 siRNA (siBMPR2) for 48 h and total protein lysates were collected. Western blots of BMPR2, phosphorylation of AKT at S473 and T308, total AKT, phosphorylation of BCL2-associated agonist of cell death (BAD) at S136 and total BAD were detected. Densitometric analysis of each protein relative to total β-actin, total AKT or total BAD and normalized to its corresponding siCTRL. Representative Western blots are shown (n=5). (B-C) Immunofluorescence of pAKT (S473) (red), CD31 (green) antibodies and nuclear staining with Hoechst 33342 (blue) in HPAH, IPAH and control (CTRL) lung. Scale bar, 20 μm. Insert scale bar, 5 μm. (D) Caspase 3/7 activity was measured after 24 h withdrawal of serum and growth media (SFM-GF; n=5). Data presented as mean ± SEM; (A) paired t-test, *P < 0.05; **P < 0.01; ****P < 0.001; (C) 2-way ANOVA with Tukey HSD ^###P < 0.005 (siBMPR2-complete vs siBMPR2-SFM-GF)

Figure 2.. Loss of activated JNK1 contributes to apoptosis resistance as measured by reduced caspase 3 cleavage and flow cytometry analysis.

Human primary pulmonary artery endothelial cells (PAECs) were transfected with control (siCTRL), BMPR2 (siBMPR2), or JNK1 (siJNK1) siRNA for 48 h and total protein lysates were collected. (A) phosphorylated JNK1 (pJNK), (B) cleaved caspase 3, (C) BIM and (D) phosphorylated ERK (pERK) protein levels were measured and densitometric analysis was performed relative to total JNK2, β-actin or total ERK and normalized to its corresponding siCTRL. Representative Western blots are shown (n=5). (E) After siRNA transfection (48 h), cells were cultured in either complete media or serum-free media without growth factors (SFM-GF) for 24 h followed by staining with annexin and propidium iodide (PI) for 15 min and data acquired on MACSquant. (F) Column scatter plot representing the percentage of apoptotic cells (cells stained with annexin V not PI, quadrant 3). Data presented as mean ± SEM; (A-D) 1-way ANOVA with Tukey HSD, *P < 0.05; ****P < 0.001; (F) 2-way ANOVA with Tukey HSD; ^##P < 0.01 (siCTRL-SFM-GF vs siBMPR2-SFM-GF)

Figure 3.. DLL4, a target of JNK1, is decreased in BMPR2- or JNK1-silenced PAECs and in lung tissue from patients with pulmonary arterial hypertension (PAH).

Human primary pulmonary artery endothelial cells (PAECs) were transfected with control (siCTRL), BMPR2 (siBMPR2), or JNK1 (siJNK1) siRNA for 48 h and total protein lysates were collected for Western blotting of (A) DLL4, (B) N1ICD, (C) NOTCH1, (D) N2ICD and (E) N4ICD. (F) DLL4 protein was also analyzed in PAECs transfected with either control (siCTRL) or BMPR2 (siBMPR2), CAV1 (siCAV1) or SMAD9 (siSMAD9) gene-specific siRNA pools. CAV1 and SMAD9 loss-of-function mutations, like BMPR2, have been associated with the development of PAH. Densitometric analysis relative to β-actin and normalized to its corresponding siCTRL. Representative Western blots are shown (n=5). (G) Immunohistochemical staining of DLL4 in paraffin embedded lung of failed donor controls (CTRL; n=5), HPAH (n=3) and IPAH (n=2). Scale bar, 50μm and 20μm. Data are presented as mean ± SEM; 1-way ANOVA with Tukey HSD *P < 0.05; ***P < 0.005; ****P < 0.001

Figure 4.. Immobilized DLL4 activates NOTCH1 signaling and increases BMPR2 transcription.

(A) Human primary pulmonary artery endothelial cells (PAECs) were grown on BSA or DLL4-coated plates and the following day transfected with either non-targeting control (siCTRL) or BMPR2 (siBMPR2) gene-specific siRNA pools. After BMPR2 knockdown (48 h), total protein lysates were collected and analyzed by Western blotting for expression of DLL4, N1ICD, NOTCH1, N2ICD and N4ICD. Representative Western blots are shown (n=5). Densitometric analysis (mean±SEM) relative to β-actin and normalized to its corresponding siCTRL-BSA. (B) The schematic of BMPR2 promoter with putative RBPJ (recombinant signal binding protein) binding sites indicated. (C) BMPR2 promoter activity in PAECs grown on BSA or DLL4 coated plates and transfected with the promoter driven luciferase reporter. (D) BMPR2 promoter activity in PAECs transfected with the promoter driven luciferase reporter plus either control (CTRL) or DLL4 over-expression plasmid (DLL4-OE). (E and F) Quantitative RT-PCR of BMPR2 mRNA from (E) PAECs or from (F) healthy or IPAH PAECs grown on BSA or DLL4 coated plates. For quantitative RT-PCR, data are presented as the geometric mean±SD (n=5). 2-way ANOVA with Tukey HSD; *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001 (siCTRL-BSA versus siBMPR2-BSA); ^##P < 0.01; ^###P < 0.005; ^####P < 0.001 (siBMPR2-BSA versus siBMPR2-DLL4)

Figure 5.. DLL4-induced NOTCH1 activation blocks AKT activation, inhibits proliferation and reverses EndoMT.

(A) Human primary pulmonary artery endothelial cells (PAECs) were grown on BSA or DLL4-coated plates and the following day, cells were transfected with either non-targeting control (siCTRL) or BMPR2 (siBMPR2) gene-specific siRNA pools. After BMPR2 knockdown (48 h), total protein lysates were collected and analyzed by Western blotting for expression of phosphorylated AKT (pAKT S473 and pAKT T308) or phosphorylated ERK (pERK). Representative Western blots are shown (n=4–6, as indicated). Densitometric analysis (mean ± SEM) relative to total AKT or total ERK and normalized to its corresponding siCTRL. (B) BrdU cell proliferation of PAECs transfected with control or BMPR2 siRNA for 48 h and then replated onto BSA or DLL4-coated plates for an additional 48 h (n=5). (C) BrdU cell proliferation of healthy and IPAH ECs were grown on either BSA or DLL4 for 48 h (n=5). (D and E) Immunofluorescence staining of (D) α-SMA (ACTA2) and (E) VE-Cadherin (CDH5) in ECs from healthy and IPAH grown on either BSA or DLL4. Scale bar, 100μm. Data are presented as mean±SEM, 2-way ANOVA with Tukey HSD; *P < 0.05; ****P < 0.001 (siCTRL-BSA versus siBMPR2-BSA); ^#P < 0.05; ^##P < 0.01; ^###P < 0.005; ^####P < 0.001 (siBMPR2-BSA versus siBMPR2-DLL4)

Figure 6.. DLL4-induced NOTCH1 activation rescues PPARγ expression.

(A) PPARγ immunofluorescence staining of PAECs endothelial cells from healthy or IPAH patients grown on either BSA or DLL4 (n=5). Scale bar, 100 μm. (B) Human primary pulmonary artery endothelial cells (PAECs) were transfected with empty vector (CTRL) or DLL4 overexpression plasmid (DLL4-OE) for 24 h and PPAR-driven reporter activity assessed (n=5). (C) mRNA levels from healthy (CTRL) or IPAH ECs grown on BSA or DLL4 for 48 h were analyzed for PPARγ expression and PPARγ target genes. (D) mRNA from PAECs transfected with control (siCTRL) or BMPR2 (siBMPR2) siRNA and grown on BSA or DLL4 for 48 h were analyzed for PPARγ expression and PPARγ target genes. (E) After 24 h of gene silencing with either control or BMPR2 siRNAs, PAECs were then transfected with empty vector (CTRL) or PPARγ overexpression plasmid for 24 h and expression of PPARγ and phosphorylated AKT (pAKT T308) protein was analyzed by Western blotting. Densitometric quantification (mean±SEM) relative to β-actin or total AKT and normalized to its corresponding siControl. Representative Western blots are shown (n=5 or 6). The mRNA levels were measured by quantitative RT-PCR and presented as the geometric mean ± SD (n=5). 2-way ANOVA with Tukey HSD; *P < 0.05; ***P < 0.005 (siCTRL-BSA versus siBMPR2-BSA); ^#P < 0.05; ^###P < 0.005; ^####P < 0.001 (siBMPR2-BSA versus siBMPR2-DLL4)

Figure 7.. PI3Kδ inhibitor, leniolisib, decreases AKT activation, proliferation, and EndoMT while increasing apoptosis in BMPR2-silenced PAECs.

(A) Human primary pulmonary artery endothelial cells (PAECs) were transfected with control (siCTRL) or BMPR2 (siBMPR2) siRNA for 48 h and then exposed to leniolisib for 4 h at the indicated concentrations. (B) Effect of 10 μM leniolisib on phosphorylation of AKT (S473 and T308) in BMPR2- and CAV1-silenced PAECs. Representative Western blots are shown (n=5) and densitometric analysis (mean±SEM) relative to β-actin or total AKT and normalized to its corresponding siCTRL. (C) BrdU cell proliferation of PAECs transfected with control (siCTRL), BMPR2 (siBMPR2) or CAV1 (siCAV1) siRNA for 48 h then replated and treated with vehicle (DMSO), 1 μM or 10 μM leniolisib for an additional 72 h. (D) Representative immunofluorescence of endothelial markers (VE-cadherin, VWF and CD31) and mesenchymal markers (α-SMA, SNAIL/SLUG and CD44) in PAECs transfected with control, BMPR2, or CAV1 siRNA for 48 h then treated with 10 μM leniolisib for 24 h (n=5). Scale bar, 100 μm. (E) Leniolisib reactivated apoptosis as assessed by annexin and PI staining. *P < 0.05 (siCTRL versus siBMPR2), # P < 0.05 (vehicle versus treatment)

Figure 8.. Graphical Abstract.

Crosstalk among DLL4/NOTCH1, JNK1, AKT with BMPR2 and PPARγ signaling pathways. BMPR2 loss or Mutation culminates in the hyperactivation of AKT and ERK along with loss of DLL4/NOTCH1, JNK and PPARγ leading to increased proliferation, endothelial to mesenchymal transition (EndoMT) and an apoptosis resistant phenotype. DLL4/N1ICD Activation increases BMPR2/PPARγ expression blocking AKT and ERK activation leading to a decrease in proliferation and EndoMT.