Fig 1: HINT3 is increased in clinical and experimental PAH samples. (A,B) Fail donor (CON) or idiopathic pulmonary arterial hypertension (IPAH) patient lungs were subjected to qRT-PCR and Western blots. n = 4–5. Densitometry with a representative blot and GAPDH loading control is shown in Figure 2B. (C,D) Whole lung homogenates were collected from normoxic/sugen (NOR/SU) or hypoxic/Sugen (HYP/SU)-treated mice. Densitometry with a representative blot and GAPDH loading control is shown in Figure 2D. All bars represent mean HINT3 mRNA or protein levels ± SEM relative to GAPDH expressed as fold-change vs. control (CON) or vs. NOR/SU. n = 5. * p < 0.05 vs. respective control condition.
Fig 2: HINT3 regulates BCL2 expression in HPAECs in vitro. (A) Human pulmonary artery endothelial cells (HPAECs) were cultured for 48 h. Cell lysates were harvested and subjected to immunoprecipitation (IP) with either anti-IgG antibody or anti-HINT3 antibody, and then immunoblotted with anti-HINT3, anti-BCL2, or anti-β-actin (ACTB) antibody. (B) HPAECs were treated with scrambled (SCR) or HINT3 (20 nM) siRNAs for 6 h, and the media was replaced with an endothelial growth medium (EGM) containing 5% FBS. And then incubated for an additional 72 h. Western blotting was performed for HINT3, BCL2, USP11, or GAPDH protein. (C) HPAECs were transfected with either the control plasmid (Mock) or USP11 (1 μg, oxUSP11) plasmid for 6 h, media were replaced with endothelial growth medium (EGM) containing 5% FBS. And then incubated for an additional 72 h. Cell lysates were collected and immunoblotted with anti-BCL2 or ACTB antibody. (D) Hypothetical schema defining the role of USP11/HINT3/BCL2 signaling in PAH pathogenesis. Hypoxia induces USP11, which stabilizes HINT3 levels through HINT3 deubiquitination. Increases in HINT3 stimulate anti-apoptosis marker, BCL2 expression promoting PAH pathogenesis
Fig 3: HINT3 is degraded by ubiquitination and USP11 deubiquitinates HINT3 in HPAECs in vitro. (A) Human pulmonary artery endothelial cells (HPAECs) were cultured for 48 h. Cell lysates were harvested and subjected to immunoprecipitation (IP) with either anti-IgG antibody or anti-HINT3 antibody and then immunoblotted with anti-USP11, anti-HINT3, or anti-β-actin (ACTB) antibody. (B) HPAECs were transfected for 24 h with HA-ubiquitin (1 μg, HA-Ubi) plasmid or the control plasmid (empty vector), then collected and assayed by immunoblotting for anti-HA, anti-HINT3, and ACTB (loading control) antibody. (C) HPAECs were pretreated with MG132 for 2 h, and then were treated with cycloheximide (CHX, 20 μg/mL) for 0, 2, 4, and 8 h, to inhibit protein synthesis, and harvested for immunoblotting. The level of HINT3 was set as 100% at time 0, and the percent HINT3 protein remaining after CHX treatment at each time point was calculated accordingly. All bars represent mean HINT3 protein levels ± SEM relative to ACTB expressed as fold-change vs. CON * p < 0.05 vs. CON, n = 3. (D) HPAECs were transfected with USP11 (1 μg, oxHUWE1) plasmid or the control plasmid (empty vector) for 6 h. After media replacement, HPAECs were incubated for additional 72 h. Cell lysates were collected and immunoblotted with anti-HINT3 or ACTB antibody. (E) HPAECs were treated with scrambled (SCR) or HINT3 (20 nM) siRNAs for 6 h, media were replaced with endothelial growth medium (EGM) containing 5% FBS. And then incubated for an additional 72 h. Western blotting was performed for HINT3 or GAPDH protein. (F) HPAECs were treated with dimethyl sulfoxide (DMSO) or USP11 inhibitor (mitoxantrone) for 0–4 h. Western blotting was performed for HINT3 or GAPDH protein.
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