Pulmonary arterial hypertension (PAH) is a progressive and fatal disease clinically defined by abnormally high mean pulmonary arterial pressure (> 25 mmHg at rest or > 30 mmHg during exercise), leading to heart failure and ultimately death, if not treated. The increase in mean pulmonary arterial pressure is a consequence of pulmonary vascular remodeling that involves a phenotypic switch of pulmonary arterial smooth muscle cells (PASMCs) from a quiescent state to a synthetic state (1). While PASMCs in healthy vasculature do not proliferate and express high levels of contractile marker genes, those in PAH have elevated proliferation rates and reduced expression of contractile marker genes in response to genetic and environmental cues. This phenotype switching leads to hypertrophy and PASMC overgrowth, increasing pulmonary vascular resistance and elevating pulmonary arterial pressure.
A comprehensive understanding of the molecular etiology of PAH has been achieved by identifying genetic mutations in genes such as bone morphogenetic protein receptor 2 (
Although these studies clearly identified the individual roles of BMP signaling and KCNK3 during the establishment and progression of PAH, little attention has been given to the possible crosstalk between BMP signaling and KCNK3 in PAH pathogenesis. In the present study, we aimed to explore how BMP/BMPR2 signaling and KCNK3 work together to regulate the PASMC phenotypic switch. We found that canonical BMP- BMPR2-Smad1/5 signaling upregulates KCNK3 in PASMCs and that BMP-induced expression of KCNK3 is required for BMP- mediated maintenance of the contractile and quiescent PASMC phenotype, suggesting a connection between BMP signaling and KCNK3 in the regulation of PASMC phenotypic switching.
We first measured KCNK3 and BMPR2 expression levels in lung tissues from PAH animal models and normal controls. We found that KCNK3 and BMPR2 protein levels were substantially lower in lung tissues of rats with MCT-induced PAH compared with those in the normal controls (Fig. 1A). We then conducted immunohistochemical staining to identify correlations between KCNK3 and BMPR2 expression patterns in pulmonary vasculature. While KCNK3 and BMPR2 were highly co-expressed in the α-SMA+ PASMC layer of normal pulmonary arterioles, their expression was simultaneously downregulated in the thickened medial layer of pulmonary arterioles associated with PAH (Fig. 1B, Supplementary Fig. 1). These data imply that impaired BMP/BMPR2 signaling due to reduced BMPR2 expression may be associated with downregulation of KCNK3 in pulmonary vasculature of the PAH animal model.
Assuming a potential association between BMP/BMPR2 signaling and KCNK3 expression in PASMCs, we investigated whether BMPs can regulate KCNK3 expression in HPASMCs. Previous studies have shown that BMP2, BMP4, and BMP7, regulate cell proliferation, apoptosis, and phenotypic changes in PASMCs (7-9). We found that BMP2, BMP4, and BMP7 treatment significantly increased KCNK3 expression in HPASMCs, as observed using RT-qPCR and western blot analyses (Fig. 2A, B). To investigate whether the BMP-induced increase in KCNK3 expression is mediated by BMPR2, we silenced
Next, we explored whether KCNK3, whose expression is upregulated by BMPs, might be involved in the BMP-induced switching of PASMC phenotypes. HPASMCs cultured in growth media containing growth factors and fetal bovine serum exhibit proliferative or synthetic phenotype characterized by high cell proliferation rates and decreased expression of VSMC contractile marker genes (11). In contrast, BMPs enhance the expression of contractile marker genes and suppress cell proliferation, inhibiting VSMCs from changing into proliferative or synthetic phenotypes in response to growth factors produced following vascular injury (7-9). We found that BMP2, BMP4, and BMP7 treatment substantially inhibited cell proliferation, as illustrated using the anti-Ki67 staining assay (Fig. 3A, B). This anti-proliferative effect of BMPs was abrogated by siRNA-mediated knockdown of
PASMCs undergo a phenotypic switch during PAH development, displaying high proliferation rates and reduced expression of contractile marker genes, which is one of central events in pulmonary vascular remodeling. Recent studies of patients with PAH have identified BMP/BMPR2 signaling and KCNK3 as key factors regulating the phenotypic switching of PASMCs in PAH. Although the individual roles of BMP/BMPR2 signaling and KCNK3 are well defined, it is not known whether a crosstalk between these two factors occur during PAMSC phenotype switching. In this study, we demonstrated that canonical BMP/BMPR2 signaling induces KCNK3 expression in PASMCs, and KCNK3 mediates BMP-induced phenotypic switching of PASMCs. This crosstalk between BMP/BMPR2 signaling and KCNK3 that is involved in the regulation of PASMC phenotype may provide insights into the complex molecular pathogenesis of PAH.
We found that KCNK3 and BMPR2 expression was simultaneously reduced in PASMCs of rats with PAH, allowing us to postulate that impaired BMP/BMPR2 signaling might be associated with reduced KCNK3 expression in PASMCs. In support of this postulation, BMP2, BMP4, and BMP7 significantly enhanced the expression of KCNK3 in HPASMCs via BMPR2-Smad1/5 signaling pathway, which was confirmed using
We also showed that KCNK3 expression and its ion channel activity are required for BMP-induced PASMC phenotype switching. As TASK-1, encoded by KCNK3, is a major K+ channel/outward rectifier regulating the resting membrane potential in PASMCs, siRNA-mediated knockdown of
In summary, we addressed a crosstalk between BMP signaling and KCNK3 expression during the PASMC phenotype switching process, a hallmark of PAH pathogenesis. We showed that the dysfunction and/or downregulation of BMPR2 and KCNK3 observed in patients with PAH may work together to induce aberrant switching of PASMC phenotypes. Our findings provide novel mechanistic insights into the multifactorial pathogenesis of pulmonary vascular remodeling in PAH.
All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Chung-Ang University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the United States National Institutes of Health. Seven-week-old male Sprague Dawley rats (Orient, Seoul, Korea) received either a single subcutaneous injection of monocrotaline (MCT; 60 mg/kg, Sigma, St. Louis, MO) or saline solution. Five weeks later, the rats were anesthetized using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg). The animals were then euthanized by cervical dislocation, and lung tissues were extracted for further analyses.
Primary human pulmonary artery smooth muscle cells (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA) were seeded and cultured at 37°C in smooth muscle cell growth medium (ScienCell Research Laboratories) from passages five to seven in a humidified atmosphere containing 5% CO2. Cells were treated with or without recombinant human bone morphogenetic proteins (BMPs; BMP2, BMP4, and BMP7, R&D Systems, Minneapolis, MN), LDN193189 (Cayman Chemical, Ann Arbor, MI), and A293 (Selleck Chemicals, Houston, TX) and cultured in growth media for 48-72 h.
Lung tissue sections and HPASMCs were stained with primary immunoglobulins (IgGs) and then incubated with fluorescent secondary IgGs. Nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Tissue sections and cells were visualized using a confocal laser scanning microscope (Carl Zeiss, Jena, Germany). All images presented are representative of more than three independent experiments. The IgGs used in the experiment are listed in Supplementery Table 1.
Tissue and cell lysates were sonicated, centrifuged, and the insoluble fraction were discarded. The concentration of the protein in the supernatant was determined using the bicinchoninic acid protein assay. Protein samples were then boiled in sample buffer and size-fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were hybridized with the appropriate primary IgG and then incubated with horseradish peroxidase-conjugated secondary IgG. Bound IgGs were detected using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). Images captured using a western blot imaging system (Vilber, Marne-la-Valleé, France) were analyzed using ImageJ software (National Institute of Health, Bethesda, MD). The IgGs used in the experiment are listed in Supplementery Table 1.
To silence the expression of
Total RNA was extracted using TRIzol reagent (Invitrogen). Complementary DNA was synthesized using the Superscript first-strand synthesis kit (Invitrogen), and conventional PCR was performed using Bio-Rad PCR system (Hercules, CA). Real-time quantitative reverse transcription-PCR (RT-qPCR) was performed on the StepOnePlusTM Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using SYBR Green PCR Master Mix (Applied Biosystems), following the manufacturer’s instructions. Data were analyzed using the ΔΔCt method. Glyceraldehyde-3-phhosphate dehydrogenase (
GraphPad prism software (GraphPad Software Inc, San Diego, CA) was used to analyze the data. Statistical significance was evaluated using an unpaired Student’s
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) [2020R1A2C1012930] [2020R1A4A4079817] [2018M3A9H2019 045] and basic science research program through the NRF funded by the Ministry of Education [NRF-2020R1I1A1A0106 6886].
The authors have no conflicting interests.