PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension

Abstract

Background: Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed.

Methods: Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry.

Results: Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle.

Conclusions: PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

Keywords: cell proliferation; cytokinesis; pulmonary hypertension; rats; smooth muscle cells; vascular remodeling.

Fig. 1.. PBK expression is increased in PH.

A, microarray analysis of PBK mRNA expression in PA from MCT-treated (4wk; 60mg/kg IP) rats. B, RNAseq analysis of PBK expression in lungs from 4wk MCT-treated rats. C, PBK expression in a microarray of healthy and PAH human lungs (GSE113439), D, qRT-PCR analysis of PBK mRNA in healthy and PAH human lung. E, Western Blot (WB) of PBK expression in healthy, iPAH and APAH human lung, F, WB of control (CTRL) and MCT-treated rat lung, G, WB of control (CTRL) and MCT rat PA and H, WB of control (Ctrl) and Sugen/Hypoxia mouse lung (Su/H; 20mg/kg IP 1x wkly; 3wks 10% O₂). I, WB of the time-dependent expression of PBK in lung tissue from CTRL and MCT rats. * p<0.05 versus CTRL (n=3–7 for experimental PH, 4–15 for human PAH). All results are shown as mean±SEM. Parametric Student t tests were used for B,D,F,G,H; Non-parametric Mann-Whitney U test was used for C, Ordinary One-way ANOVA with Tukey multiple comparison test used for E and Two-way ANOVA with Sidak’s multiple comparison test used for I.

Fig. 2.. PBK is elevated in hypertensive PA and found in medial layer smooth muscle cells.

Lung sections from A, rat control, MCT 4wk and Su/H, B, mouse control, Su/H, C, human control and iPAH patients. Sections were immunostained with antibodies to SMA and PBK and DAPI was used to stain nuclei , D, Western Blot (WB) of PBK expression in control and PAH PASMCs. * p<0.05 versus CTRL or Healthy (n=4–10 for experimental PH). All results are shown as mean±SEM. Parametric Student t tests were used for A-D.

Fig. 3.. PBK promotes PASMC proliferation.

A, adenovirus-mediated expression of PBK increases proliferation of HPASMC as quantified by the MTT assay, qRT-PCR analysis of MKI67 mRNA and cell count. Relative expression of PBK was determined by Western blot (right panel). B, siRNA mediated silencing of PBK in HPASMCs decreased cell proliferation as measured using the MTT assay, MKI67 mRNA expression, cell count and C, EdU incorporation. D, inhibition of PBK with Hi-TOPK-032 decreased HPASMC proliferation as quantified by the MTT assay, qRT-PCR analysis of MKI67 mRNA and cell count. E, the kinase activity of PBK is necessary for increased HPASMC proliferation. HPASMC were transduced with adenovirus encoding βgal (Control), wild type (WT), constitutively active (Active) and kinase dead or dominant negative PBK (DN) and cell proliferation measured by MTT Assay and EdU incorporation, F, siRNA mediated silencing of PBK in PAH PASMCs decreased cell proliferation as measured using the WST-1 assay. Relative PBK protein expression was determined by Western blot (bottom panel and quantified on right) in PAH PASMCs, G, inhibition of PBK with OTS-514 decreased PAH PASMC proliferation as quantified by the MTT assay. * p<0.05 vs. control, # vs. WT (n=4–6). All results are shown as mean±SEM. Parametric Student t tests were used A-D,F and G; Ordinary One-way ANOVA with Tukey multiple comparison test used for E.

Fig. 4.. The PBK inhibitor, OTS-514 reverses remodeling of the lung vasculature and right ventricle and decreases PH in MCT rats.

A, experimental design for the treatment of MCT-rats with OTS-514. B, ultrasound measurements (Vevo3100) of VTI and C, Right ventricle thickness in control (Ctrl), MCT and MCT rats treated with OTS-514 (5mg/kg IV) daily after three weeks of MCT treatment. D, postmortem Fulton index at 5wks (n=4). E, H&E staining of lung sections from control (vehicle), MCT and MCT rats administered OTS-514. F, Masson’s Trichrome staining of lung sections from control (vehicle), MCT and MCT rats administered OTS-514. G, quantification of PA vascular remodeling that is shown in panel E and H, quantification of collagen deposition (fibrosis) that is shown in panel F. I, qRT-PCR analysis of the mRNA expression of the marker of cell proliferation, Ki-67 and J-L, qRT-PCR analysis of markers of fibrosis including Periostin (Postn), Col1a1 and Mmp-2 in the lungs of control (vehicle), MCT and MCT rats administered OTS-514. n=4. * p<0.05 versus Veh, # p<0.05 versus MCT. M, experimental design for the treatment of MCT-rats with OTS-514 and sildenafil. N, ultrasound measurements (Vevo3100) of Right Ventricle thickness, O, Velocity Time Integral (VTI), R, Cardiac Output (CO) and Q, Tricuspid Annular Plane Systolic Excursion (TAPSE) in control (Ctrl), MCT, MCT rats treated with OTS-514 and/ or with Sildenafil every other day after two weeks of MCT treatment. P, Right Ventricle Systolic Pressure (RVSP) measurement and S, postmortem Fulton index at 5wks (n=5). * p<0.05 versus Veh, # p<0.05 versus MCT, $ p<0.05 versus MCT, & p<0.05 versus MCT. All results are shown as mean±SEM. Ordinary One-way ANOVA with Tukey multiple comparison test used for B-D, G-I, J-L, N-S.

Fig. 5.. Inhibition of PBK with OTS-514 protects against PH in mice.

A, experimental design for the OTS-514 treatment of Sugen/Hypoxia (Su/H) mice. Su/H mice were administered 20mg/kg of SU5416 IP 1x wkly for 3wks, housed in 10% O₂ for 4wks and administered vehicle or OTS-514 1.25mg/kg IP every other day starting at the end of week 1 for 3 weeks in total. B, right ventricular systolic pressure (RVSP). C, right ventricle thickness (Vevo3100). D, postmortem analysis of Fulton index at Wk4 (n=4). E, H&E staining of lung sections and F, Masson’s Trichrome staining of lung sections from control vehicle treated (normoxia), and Su/H mice administered OTS-514. G, quantification of PA vascular remodeling. H, quantification of collagen accumulation. I, qRT-PCR analysis of Ki-67 mRNA levels and J, qRT-PCR analysis of Periostin (Postn), K, Col1a1, and L, Mmp-2 in the lungs of vehicle (normoxia), Su/H and Su/H mice administered OTS-514 (n=4).* p<0.05 versus Veh, # p<0.05 versus Su/H. All results are shown as mean±SEM. Ordinary One-way ANOVA with Tukey multiple comparison test used for B-D, G-I, J-L.

Fig. 6.. Genetic deletion of PBK protects against PH.

Using a guide RNA targeting exon4 of the rat PBK gene, knockout rats were generated harboring a single T insertion resulting in a frame shift that was expressed in the germline. A, Genotyping strategy for PBK KO mice using PCR of genomic DNA and restriction digestion with BsaJI. B, Western blots of lung segments from WT and PBK^−/− mice. PH was analyzed in control (SD) and PBK KO rats administered MCT or vehicle. C, RVSP. D, RV thickness and E, VTI using digital ultrasound (Vevo3100) and F, Fulton index (n=5). G, H&E staining of lung sections from littermate control (SD), MCT (SD+MCT), PBK KO and PBK+ MCT treated rats and H, quantification of PA vascular remodeling. I, Masson’s Trichrome staining of lung sections from littermate control (SD), MCT (SD+MCT), PBK KO and PBK+ MCT treated rats. J, quantification of fibrosis. K, qRT-PCR analysis of Ki-67 mRNA levels and L, qRT-PCR analysis of Periostin (Postn), M, Col1a1, N, Mmp-2 and in lungs of (Ctrl), MCT, PBK KO and PBK+ MCT treated rats (n=4). * p<0.05 versus Veh, # p<0.05 versus MCT. All results are shown as mean±SEM. Ordinary One-way ANOVA with Tukey multiple comparison test used for C-F, H, J-N.

Fig. 7.. Molecular regulation of PBK expression.

A, qRT-PCR analysis of PBK mRNA levels in control HPASMCs or iPAH HPASMCs treated with or without actinomycin D (1µg/ml) and cells harvested in Trizol at the times indicated for qRT-PCR analysis (n=3) *p<0.05 versus CTRL HPASMC. B, WB of PBK expression in control HPASMCs and iPAH HPASMCs treated with vehicle (Veh.) or cyclohexamide (CHX) (1µg/ml) for 24 hours, *p<0.05 versus HPASMC Veh. (n=3). C, Western Blot (WB) of relative YAP expression in control (CTRL) and MCT-treated rat lung (n=3), D-E, Expression of YAP1 and TEAD1 mRNA in healthy and PAH lungs (n=11–15). F, WB of relative PBK and YAP expression in YAP silenced iPAH HPASMCs. G, WB of PBK and YAP expression in HPASMCs transduced with control (GFP) or active YAP1 adenovirus. H, HEK293 cells were transfected with ctrl plasmid and plasmid encoding active YAP1 and PBK proximal promoter activity was measured by luciferase activity assay I, PBK was overexpressed in YAP silenced PASMCs transduced with adenovirus encoding CTRL (GFP) or WT or active PBK and proliferation was assessed by the WST-1 assay (n=9) *p<0.05 versus control, # p<0.05 versus YAP silenced cells. All results are shown as mean±SEM. Two-way ANOVA with Tukey’s multiple comparisons test for A, One-way ANOVA with Dunnett’s multiple comparisons test was used for B, Parametric Student t test was used for D and E; Ordinary One-way ANOVA with Tukey multiple comparison test were used for H and I.

Fig. 8.. PBK binds Protein Regulator of Cytokinesis1 (PRC1), a controller of cytokinesis, which is upregulated in PH.

Evidence of altered cytokinesis in PA from MCT-treated rats and human PAH. A, Prc1 mRNA expression levels from microarray analysis of PA(upper) and RNAseq of total lung (lower) from vehicle and MCT rats. B, PRC1 mRNA is elevated in the lungs of PAH humans and C, markers of cytokinesis are elevated in human PAH. D, Co-IP assays in HEK293 cells showing that IP of HA-PBK co-precipitates PRC1 and the reciprocal IP of PRC1-MYC, co-precipitates PBK. E, Phosphoproteins were isolated using affinity columns and levels of P-PRC1 were determined by Western blotting. F-G, PRC1 was silenced in HPASMC using siRNA and effects on proliferation determined by cell number and WST-1 assay(n=5–8). H, HPASMC were transduced with active and inactive PBK adenovirus and 48h later cells were fixed with 4% PFA, stained with DAPI, and the stages of mitosis assessed using fluorescence microscopy (n=10). I, q-RT-PCR analysis of Prc1 in the lungs of SD and PBK KO rat treated with vehicle or MCT , J, Cell cycle analysis in HPASMC transduced with I, control (β-gal), active PBK and dominant negative PBK (DN) or K, HPASMC transfected with non-targeting (CTRL) or PBK siRNA (n=4). * p<0.05 versus CTRL/ β-gal. All results are shown as mean±SEM. Parametric Student t test was used for A; Non-parametric Student t test with Mann-Whitney test was used for B; One-way ANOVA with Tukey’s multiple comparisons test for I-K.