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. 2026 Jan 29;27:65. doi: 10.1186/s12931-026-03534-4

Activation of PBK by Gal-3 contributes to pulmonary artery hypertension by promoting PRC1 activation

Jia Zhang 1, Jin Liu 1, Yuqian Chen 1, Huan Chen 1, Yihan Meng 1, Yan Wang 1, Yuanjie Qiu 1, Huizhong Hu 1, Wenhua Shi 2, Manxiang Li 1,✉
PMCID: PMC12895640  PMID: 41612370

Abstract

Background

Galactose lectin-3 (Gal-3) has been shown to promote the progression of pulmonary arterial hypertension (PAH), yet the intricate molecular mechanisms are still unclear.

Methods

Primary cultured rat pulmonary arterial smooth muscle cells (PASMCs) and PAH rats were used in this study. Protein phosphorylation and expression levels were identified using Western blotting, while mRNA level was quantified through qRT-PCR. Hemodynamic measurements, hematoxylin-eosin and immunohistochemistry staining were employed to assess the PAH progression.

Results

Yes-associated protein 1 (YAP1) mediated Gal-3/toll-like receptor 4 (TLR4)-induced PDZ-binding kinase (PBK) upregulation. Furthermore, polo-like kinase 1 (PLK1)/cyclin-dependent kinase 1 (CDK1) mediated Gal-3/TLR4-induced PBK phosphorylation. Activated PBK further phosphorylated protein regulator of cytokinesis 1 (PRC1), which ultimately led to PASMC proliferation. In monocrotaline-induced PAH rat models, inhibition of Gal-3, TLR4, YAP1, CDK1 or silencing of PLK1, PBK, PRC1 attenuated PAH progression.

Conclusion

Our study presents novel evidence that Gal-3 promotes PASMC proliferation and pulmonary vascular remodeling by activating PBK/PRC1 through the TLR4/YAP1 and TLR4/PLK1/CDK1 signaling pathways, suggesting that this signaling pathway might be a promising target for the treatment of PAH.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12931-026-03534-4.

Keywords: Gal-3, PBK, PRC1, Pulmonary arterial remodeling, PAH

Introduction

Pulmonary arterial hypertension (PAH) is caused by persistent vasoconstriction, in situ thrombosis, and vascular remodeling, which leads to right heart failure and eventually death [1, 2]. Pulmonary vascular remodeling is considered to be an important pathological change in PAH, driven by the proliferation of pulmonary arterial smooth muscle cells (PASMCs) [3]. Thus, investigating the mechanisms underlying PASMC proliferation and vascular remodeling is vital to identify therapeutic targets for PAH management.

Galactose lectin-3 (Gal-3) is a member of the beta-galactoside-binding lectin family [4]. Gal-3 can be secreted into the extracellular mesenchyme to bind to cell surface glycoconjugates containing β-galactoside, and this interaction is pivotal in numerous physiological and pathophysiological processes [5]. Research indicates a notable elevation of Gal-3 level in PAH patients and animal models [6–8]. Our previous study has also shown that Gal-3 induces PASMC proliferation [9]. Furthermore, Gal-3 contributes to the pathogenesis of several diseases by triggering toll-like receptor 4 (TLR4) activity [10–12]. However, whether Gal-3 can promote PAH progression and the particular molecular mechanisms remains to be elucidated.

Yes-associated protein 1 (YAP1), a pivotal component of the HIPPO signaling cascade, serves as a crucial transcriptional coactivator [13]. Our previous study has shown that Gal-3 upregulates and activates YAP1 in PASMCs [9]. However, the mechanism by which Gal-3 upregulates YAP1 and facilitates PASMC proliferation is unknown.

Both Polo-like kinase 1 (PLK1) and cyclin-dependent kinase 1 (CDK1) are associated with mitosis regulation [14, 15]. PLK1 regulates several cell cycle phases and is expressed at an elevated level in a variety of tumors [14, 16]. Elevated PLK1 in tumor patients predicts poor prognosis, while intervention with PLK1 suppresses tumor cell proliferation and tumor development [17–19]. CDK1 is an important therapeutic target that mediates the growth of a variety of tumors [20–22]. Research has shown that PLK1 activates CDK1, thus promoting the development of PAH [23]. However, whether PLK1 and CDK1 are involved in Gal-3-induced PASMC proliferation remains poorly elucidated.

PDZ-binding kinase (PBK) is a serine/threonine protein kinase that regulates cell proliferation, apoptosis, and inflammatory [24, 25]. PBK is activated and upregulated in various tumor cells and mediates cell proliferation [26–29]. A recent study has shown that PBK is upregulated in PASMCs, and the active form of PBK increases proliferation of PASMCs, whereas inhibition of PBK decreases proliferation [30]. It has been demonstrated that PBK is regulated by YAP1 in breast cancer cells [26]. In addition, studies have shown that PBK-Thr9 is activated by CDK1/cyclin B, and activated PBK has the ability to bind with CDK1/cyclin B and microtubules and accumulate on the mitotic spindle at the late stage of division, regulating spindle formation and chromosome segregation during mitosis [27, 31, 32]. PBK significantly influences tumor cell proliferation as a crossroads of multiple signaling pathways, but the role of PBK in PAH is currently uncertain.

Protein regulator of cytokinesis 1 (PRC1) has been demonstrated to contribute to spindle formation, especially the central spindle [33, 34]. PBK has been found to act as a carrier by simultaneously binding the CDK1/cyclinB1 complex and PRC1 in breast cancer cells, phosphorylating PRC1-Thr481 and linking it to microtubule proteins, and ultimately promoting cytoplasmic division [32]. A recent study has demonstrated that PBK promotes PRC1 activation and consequently facilitates cytokinesis in PASMCs [30]. Further investigation is needed to elucidate the regulatory role of PBK on PRC1 during PAH development.

Taken together, we propose the hypothesis that Gal-3 induces PBK expression and phosphorylation via the TLR4/YAP1 and TLR4/PLK1/CDK1 axis, respectively, which further activates PRC1, ultimately promoting PAH development.

Materials and methods

Cell culture and reagents

Primary PASMCs from male Sprague-Dawley (SD) rats were extracted using established methods [35]. PASMCs were maintained in high glucose Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin-streptomycin, incubated in a 37 °C, 5% CO2 incubator, and passaged using 0.25% trypsin. PASMCs within six passages were utilized, and immunofluorescence staining with α-SMA (Proteintech, Wuhan, China, 14395-1-AP) was used to verify the cells contained over 95% PASMCs. PASMCs were incubated in serum-free DMEM overnight before each experiment. Gal-3 (Peprotech, NJ, USA, 450 − 38) and TAK-242 (1 µM) (MedChemExpress, NJ, USA) were used in the experiment.

Cell proliferation assessment

After different treatments, cells in each well were treated with CCK-8 reagent (diluted 1:10, GlpBio, CA, USA) for 4 h, and the absorbance was measured at 450 nm by using a microplate reader (Bio-Rad, USA). The EdU incorporation rate was determined using the EdU-488 Kit (Beyotime, Wuxi, China) following the provided protocol. Images were observed with an inverted fluorescence microscope and quantified utilizing Image-J (NIH, USA).

SiRNA transfection

At 30%-50% confluency, PASMCs were transfected with Lipofectamine ™ 3000 reagent (Invitrogen, USA), with knockdown efficiency evaluated via immunoblotting.

siRNA was synthesized by GenePharma: PLK1 siRNA, sense 5’-GUGUGGGAGUGCUCUUUAATT-3’, antisense 5’-UUAAAGAGCACUCCCACACTT-3’; CDK1 siRNA, sense 5’-GCCAAACGAAUCUCUGGAATT-3’, antisense 5’-UUCCAGAGAUUCGUUUGGCTT-3’; YAP1 siRNA, sense 5’-CUGCCACCAAGCUAGAUAATT-3’, antisense 5’-UUAUCUAGCUUGGUGGCAGTT-3’; PBK siRNA sense 5’-GGAGACAUAAAGUCUUCAATT-3’, antisense 5’-UUGAAGACUUUAUGUCUCCTT-3’; PRC1 siRNA, sense 5’-GCUCCGUGAAAUUCUGUGUTT-3’, antisense 5’-ACACAGAAUUUCACGGAGCTT-3’; NC siRNA, sense 5’-UUCUCCGAACGUGUCACGUTT-3’, antisense 5’-ACGUGACACGUUCGGAGAATT -3’.

qRT-PCR

The RNA isolation and reverse transcription were processed using RNA fast200 Kit (Feijie, Shanghai, China) and PrimeScript RT Reagent Kit (TaKaRa, Japan, RR037A). qRT-PCR was performed with TB Green Premix Ex Taq II (TaKaRa, Japan, RR820A). The following primers were provided by TSINGKE for qRT-PCR: YAP1, Forward: 5’-AGACCACAGGCAATACGGAATAT-3’, Reverse: 5’-TGCCACTGTTAAGAAAGGGATCT-3’; PBK, Forward: 5’-AGTGTGTATGGTGGTCAGAAGAT-3’, Reverse: 5’-GCATAAATGGAGAGGCAGGGATA-3’; β-Actin, Forward: 5’-CTGAGAGGGAAATCGTGCGTGAC-3’, Reverse: 5’-AGGAAGAGGATGCGGCAGTGG-3’.

Western blotting

Proteins were extracted using RIPA lysis buffer, subsequently separated by 10% SDS-PAGE gel, and transferred to a polyvinylidene fluoride membrane. The membranes received an overnight incubation with primary antibodies at 4 °C and then were probed with secondary antibodies at room temperature for 1 h. Primary antibodies against p-PLK1 (Immunoway, TX, USA, YP0964, 1:500), t-PLK1 (Proteintech, Wuhan, China, 10305-1-AP, 1:500), p-CDK1 (SAB, MD, USA, 12493, 1:500), t-CDK1 (Proteintech, Wuhan, China, 19532-1-AP, 1:2000), p-PBK (abcam, Cambs, UK, ab236872, 1:1000), t-PBK (Proteintech, Wuhan, China, 16110-1-AP, 1:3000), p-PRC1 (Affinity, CA, USA, AF8394, 1:1000), t-PRC1 (Proteintech, Wuhan, China, 10110-2-AP, 1:2000), YAP1 (Proteintech, Wuhan, China, 66900-1-Ig, 1:5000), β-actin (Immunoway, TX, USA, YM3028, 1:5000), secondary antibodies anti-mouse/anti-rabbit (ZhuangzhiBio, Xi’an, China, 1:8000 dilution) were used according to the instructions. Finally, chemiluminescence was performed using the ChemiDoc XRS system and measured by Image-J. Detailed images of the uncropped blots are provided in Supplementary material.

Animal experiments

Animal experiments were performed according to the Institutional Animal Ethics Committee of Xi’an Jiaotong University (Permit No. 2021 − 667). Male SD rats (200–220 g) were kept on a 12-h light/dark cycle at 18–22 °C and 40%-60% humidity in the SPF animal laboratory and divided into 11 groups, with 6 rats in each group: Control group: administered intraperitoneal vehicle solution (60 mg/kg) on day 1, intraperitoneal injection of saline for 28 days; MCT group: administered intraperitoneal monocrotaline (MCT) (60 mg/kg, Must Bio-Technology, China) on day 1; MCT + DMSO group: administered 28 days of DMSO intraperitoneal injection after MCT administration on day 1; MCT + MCP group: received 28 days of Gal-3 inhibitor PectaSol modified citrus pectin (MCP) (100 mg/kg/d, orally, Econugenics, Santa Rosa, CA, USA) after MCT administration [36]; MCT + TAK-242 group: received TLR4 receptor inhibitor TAK-242 (3 mg/kg/d, intraperitoneal injection, Aladdin, T125887) for 28 days after MCT administration [37]; MCT + VP group: received YAP1 inhibitor verteporfin (VP) (4.5 mg/kg, every 2 days, intraperitoneal injection, Aladdin, V129759) for 28 days after MCT administration [38]; MCT + NC siRNA group: received NC siRNA (0.2 mg/kg/w tail-vein injection, GenePharma, China) for 28 days after MCT administration; MCT + PLK1 siRNA-targeted group: received PLK1 siRNA molecule for 28 days after the injection of MCT; MCT + RO-3306 group: received CDK1 inhibitor RO-3306 (4 mg/kg, every 2 days, orally, MedChemExpress, USA, HY-12529) for 28 days after the injection of MCT [39]; MCT + PBK-targeted siRNA group: received PBK siRNA molecule for 28 days after the injection of MCT; MCT + PRC1-targeted siRNA group: received PRC1 siRNA molecule for 28 days after the injection of MCT. The PLK1, PBK, and PRC1 siRNA sequences were consistent with those used in the cell experiments, and the administered dosage and technique are the same as those of NC siRNA.

Measurement of hemodynamic and the RVHI

After 28 days, all rats were anesthetized to perform hemodynamic measurements. Right heart catheterization was employed to evaluate right ventricle systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP). The right ventricle (RV)/ (left ventricle (LV) plus ventricular septum (S)) ratio was measured to assess the right ventricle hypertrophy index (RVHI).

Histological and immunohistochemistry staining

After hemodynamic study, the rats were euthanized by cervical dislocation. After fixing the right upper pulmonary lobes and heart tissues in 4% paraformaldehyde for 48 h, the samples were embedded in paraffin wax and subsequently sectioned at 5 μm thickness. These sections were then stained with Hematoxylin-eosin (HE), Elastin Van Gieson (EVG) and α-SMA antibody. Image Pro Plus software was used for image analysis. Medial wall thickness was assessed by EVG staining using the following formula: (vessel outer diameter -vessel inner diameter) / vessel outer diameter × 100% (n = 8 per rat). The degree of muscularization was determined by the percentage of α-SMA-positive cellular infiltration (n = 10–12 per rat). Non muscular, partially muscular and fully muscular were defined as having α-SMA-positive cellular infiltration of < 25%, between 25% and 75%, and > 75%, respectively.

Elisa

The lung tissues were fully ground and centrifuged, and the supernatant was analyzed for Gal-3 concentration using commercial ELISA kits (Westang, Shanghai, China).

Statistical analysis

Statistical analyses were conducted with Prism version 8.0 software, defining significance at P < 0.05. Data were expressed as mean ± standard deviation and analyzed using a t-test or one-way ANOVA with multiple comparisons.

Results

Gal-3 induces PASMC proliferation

To assess the effect of Gal-3 on PASMC proliferation, cells were treated with Gal-3 for different concentrations or times, and PASMC proliferation was assessed. Figure 1A demonstrated that Gal-3 stimulated cell proliferation concentration-dependently, with 30 nM inducing a significant increase. As shown in Fig. 1B, Gal-3 triggered cell proliferation over time. Based on these results, 30 nM of Gal-3 was employed in the following experiments.

Fig. 1.

Fig. 1

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Gal-3 induces PASMC proliferation. A PASMCs were subjected to various concentrations of Gal-3 for 48 h, with proliferation assessed via CCK-8 assay (n = 4). B PASMCs were subjected to Gal-3 for the indicated time, with proliferation assessed via CCK-8 assay (n = 4). * P < 0.05 versus control, **** P < 0.0001 versus control.

TLR4/YAP1 mediates Gal-3-induced PBK upregulation

We next investigated the effect of Gal-3 on the expression of YAP1 and PBK. Figure 2A showed a 1.50 ± 0.07-fold increase in YAP1 protein level and a 1.46 ± 0.11-fold rise in PBK protein level following Gal-3 stimulation for 48 h compared to controls.

Fig. 2.

Fig. 2

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TLR4/YAP1 mediates Gal-3-induced PBK upregulation. A PASMCs were subjected to Gal-3 stimulation, YAP1 and PBK protein levels were examined. B and C PASMCs were pretreated with TAK-242 and then incubated with Gal-3. YAP1 and PBK mRNA levels were measured. D PASMCs were pretreated with TAK-242 and then incubated with Gal-3. YAP1 and PBK protein levels were detected. E PASMCs were transfected with YAP1 siRNA. YAP1 protein level was measured. F and G PASMCs were transfected with YAP1 siRNA and then incubated with Gal-3. PBK mRNA and protein levels were measured. Protein levels were examined by immunoblotting, and mRNA levels were measured by qRT-PCR (n = 3). * P < 0.05, ** P < 0.01, **** P < 0.0001.

To explore whether Gal-3 promotes the upregulation of YAP1 and PBK mRNA levels and the underlying mechanism, PASMCs were pretreated with TLR4 inhibitor TAK-242 (1 µM) for 30 min before stimulation with Gal-3 for 48 h. As shown in Fig. 2B and C, Gal-3 upregulated YAP1 and PBK mRNA levels. Meanwhile, pretreatment with TAK-242 reversed the Gal-3-induced increase in YAP1 and PBK mRNA levels. TAK-242 also reduced Gal-3-induced elevation of protein levels of YAP1 and PBK in PASMCs (Fig. 2D). These results suggested that TLR4 mediated Gal-3 upregulation of YAP1 and PBK at both mRNA and protein levels in PASMCs.

To further investigate whether elevation of YAP1 contributes to PBK upregulation in Gal-3-stimulated PASMCs, YAP1 was silenced with sequence-specific siRNA. As depicted in Fig. 2E, transfection of YAP1 siRNA (150 nM for 48 h) effectively reduced YAP1 protein expression. As shown in Fig. 2F and G, pre-silencing of YAP1 (YAP1 siRNA 150 nM for 48 h) before stimulation with Gal-3 for 48 h reversed the Gal-3-induced increase of PBK mRNA and protein levels, whereas NC siRNA did not show any effects. These results indicated that YAP1 lying downstream of TLR4 mediated Gal-3-induced PBK transcription and translation in PASMCs.

TLR4/PLK1/CDK1 mediates Gal-3-induced PBK phosphorylation

To investigate the role of Gal-3 in the activation of PLK1, CDK1 and PBK, PASMCs were stimulated with Gal-3 for different times. Figure 3A and B showed that Gal-3 induced a time-dependent increase in PLK1 and CDK1 phosphorylation in PASMCs, with the maximal effect at 15 min. As shown in Fig. 3C, Gal-3 phosphorylated PBK over time in PASMCs, with the maximal effect at 6 h.

Fig. 3.

Fig. 3

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TLR4/PLK1/CDK1 mediates Gal-3-induced PBK phosphorylation. A-C Gal-3 increased PLK1, CDK1 and PBK phosphorylation time-dependently in PASMCs. p-PLK1, t-PLK1, p-CDK1, t-CDK1, p-PBK and t-PBK levels were examined. D PASMCs were pretreated with TAK-242 and then incubated with Gal-3 for 15 min. p-PLK1, t-PLK1, p-CDK1 and t-CDK1 protein levels were detected. E PASMCs were pretreated with TAK-242 and then incubated with Gal-3 for 6 h. p-PBK and t-PBK protein levels were detected. F PASMCs were transfected with PLK1 siRNA. PLK1 protein level was measured. G and H PASMCs were transfected with PLK1 siRNA, followed by Gal-3 stimulation for 15 min or 6 h. p-CDK1, t-CDK1, p-PBK and t-PBK protein levels were measured. I PASMCs were transfected with CDK1 siRNA. CDK1 protein level was measured. J PASMCs were transfected with CDK1 siRNA, followed by Gal-3 for 6 h. p-PBK and t-PBK protein levels were measured. Protein levels were examined by immunoblotting (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

We then examined the role of TLR4 in the Gal-3-induced phosphorylation of PLK1, CDK1 and PBK. PASMCs were pre-treated with TLR4 inhibitor TAK-242 (1 µM for 30 min) and then stimulated with Gal-3 for 15 min. Figure 3D showed that inhibition of TLR4 significantly suppressed Gal-3-induced PLK1 and CDK1 phosphorylation. In addition, Gal-3-induced PBK phosphorylation was also blocked by pretreatment with TAK-242 (Fig. 3E). These results indicated that TLR4 mediated Gal-3 induction of PLK1, CDK1 and PBK phosphorylation in PASMCs.

To explore whether PLK1 plays a role in Gal-3-induced CDK1 and PBK phosphorylation, siRNA was employed to knock down PLK1 expression. As shown in Fig. 3F, transfection of PLK1 siRNA (150 nM for 48 h) reduced the PLK1 protein level. Figure 3G showed that stimulation with Gal-3 for 15 min significantly increased the phosphorylation level of CDK1, whereas loss of PLK1 (PLK1 siRNA 150 nM for 48 h) significantly attenuated Gal-3-induced CDK1 phosphorylation. Furthermore, stimulation with Gal-3 for 6 h increased the phosphorylation of PBK, whereas knockdown of PLK1 (PLK1 siRNA 150 nM for 48 h) reduced Gal-3-induced PBK phosphorylation (Fig. 3H). These results suggested that activation of PLK1 mediated Gal-3-induced phosphorylation of CDK1 and PBK.

We further determined whether CDK1 mediates Gal-3-induced PBK phosphorylation. CDK1 siRNA (150 nM for 48 h) was transfected and showed a dramatic reduction of CDK1 protein level in PASMCs (Fig. 3I). Figure 3J demonstrated that lacking of CDK1 (CDK1 siRNA 150 nM for 48 h) blunted Gal-3-induced PBK phosphorylation.

Taken together, the above results demonstrated that TLR4/PLK1/CDK1 axis mediated Gal-3-induced PBK phosphorylation.

PBK mediates Gal-3/TLR4-induced PRC1 expression and phosphorylation

It has been reported that mRNA level of PRC1 is upregulated in pulmonary arteries from the MCT-induced PAH rats and facilitates cytokinesis in PASMCs [30]. To determine whether PRC1 is upregulated by Gal-3, PASMCs were treated with Gal-3. Figure 4A showed an elevated PRC1 expression following Gal-3 treatment for 48 h. We further examined whether Gal-3 promotes PRC1 phosphorylation. Figure 4B demonstrated that Gal-3 time-dependently phosphorylated PRC1 in PASMCs with peak time at 6 h.

Fig. 4.

Fig. 4

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PBK mediates Gal-3/TLR4-induced PRC1 expression and phosphorylation. A PASMCs were incubated with Gal-3, PRC1 protein level was examined. B Gal-3 increased PRC1 phosphorylation time-dependently in PASMCs, p-PRC1 and t-PRC1 protein levels were examined. C PASMCs were pre-treated with TAK-242 and then stimulated with Gal-3 for 48 h. PRC1 protein level was detected. D PASMCs were pre-treated with TAK-242 and then stimulated with Gal-3 for 6 h. p-PRC1 and t-PRC1 protein levels were detected. E PASMCs were transfected with PBK siRNA. PBK protein level was measured. F PASMCs were transfected with PBK siRNA and then stimulated with Gal-3 for 48 h. PRC1 protein level was measured. G PASMCs were transfected with PBK siRNA and then stimulated with Gal-3 for 6 h. p-PRC1 and t-PRC1 protein levels were measured. H PASMCs were transfected with YAP1 siRNA and then followed with Gal-3 for 48 h. PRC1 protein level was measured. I and J PASMCs were transfected with PLK1 siRNA or CDK1 siRNA and then followed with Gal-3 for 6 h. p-PRC1 and t-PRC1 protein levels were measured. Protein levels were examined by immunoblotting (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

We next examined whether TLR4 mediates the upregulation and phosphorylation of PRC1 by Gal-3 in PASMCs. As shown in Fig. 4C, Gal-3 stimulation for 48 h increased PRC1 protein expression, whereas pretreatment with TAK-242 (1 µM for 30 min) significantly reduced this upregulation. Figure 4D indicated that Gal-3 stimulation increased PRC1 phosphorylation at 6 h, whereas pretreatment with TAK-242 reversed Gal-3-induced PRC1 phosphorylation.

To determine whether PBK mediates Gal-3-induced PRC1 expression and phosphorylation in PASMCs, PBK was silenced with PBK siRNA (150 nM for 48 h). As shown in Fig. 4E, transfection of PBK siRNA dramatically reduced PBK protein level. Figure 4F showed that Gal-3 stimulation for 48 h upregulated the protein expression of PRC1, whereas knockdown of PBK (PBK siRNA 150 nM for 48 h) suppressed this effect. Figure 4G showed that incubation with Gal-3 for 6 h significantly increased the phosphorylation of PRC1 protein, while prior silencing of PBK (PBK siRNA 150 nM for 48 h) significantly blocked this phosphorylation. These results suggested that PBK mediated the Gal-3-induced expression and phosphorylation of PRC1 in PASMCs.

In addition, we investigated whether YAP1, PLK1, and CDK1 are involved in Gal-3-induced PRC1 activation in PASMCs. YAP1, PLK1 and CDK1 were silenced using sequence-specific siRNA. As shown in Fig. 4H, I and J, silencing of YAP1 (YAP1 siRNA 150 nM for 48 h) suppressed Gal-3-induced PRC1 upregulation, and prior transfection with PLK1 siRNA or CDK1 siRNA (PLK1 or CDK1 siRNA 150 nM for 48 h) suppressed Gal-3-induced PRC1 phosphorylation.

Interfering PBK or PRC1 inhibits Gal-3-induced proliferation of PASMCs

To determine whether targeting PBK/PRC1 suppresses Gal-3-triggered PASMC proliferation, PASMCs were transfected with PBK siRNA or PRC1 siRNA (150 nM for 48 h) and then stimulated with Gal-3 for 48 h. Figure 5 indicated that Gal-3 significantly promoted PASMC proliferation, while prior knockdown PBK or PRC1 reduced Gal-3-induced cell proliferation. Fig. S1 depicted the transfection efficiency of PRC1 siRNA. These results indicated that PBK/PRC1 mediated Gal-3-induced PASMC proliferation.

Fig. 5.

Fig. 5

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Interfering PBK or PRC1 inhibits Gal-3-induced proliferation of PASMCs. PASMCs were transfected with PBK siRNA or PRC1 siRNA and then incubated with Gal-3 for 48 h. Cell proliferation was detected by EdU assay (scale bar = 100 μm, n = 3). *** P < 0.001.

Inhibition of Gal-3/TLR4/PBK/PRC1 axis attenuates the development of PAH in MCT-induced PAH rats

To determine the involvement of the Gal-3/TLR4/PBK/PRC1 axis in vascular remodeling and PAH progression, MCT-induced PAH rat models were established. Compared with controls, a significantly higher level of Gal-3 was observed in the lung tissue of MCT-induced PAH rats (Fig. 7A). The Gal-3 inhibitor MCP reduced the elevation of mPAP and RVSP in the PAH rats (Fig. 6A and B). Furthermore, MCP treatment attenuated the elevations in RV/LV + S, medial wall thickness, and muscularized arteries (Fig. 6C-F). We also showed that the expression of YAP1, phosphorylation of PLK1 and CDK1, and activation of PBK and PRC1 were significantly increased in lung tissues of PAH rat models (Fig. 7B). However, administration of MCP reversed all these changes (Fig. 7C).

Fig. 7.

Fig. 7

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Relevant interventions regulate their downstream molecules phosphorylation or expressions in MCT-induced PAH rats. A Concentration of Gal-3 was measured by Elisa (n = 6). B-D Protein levels of YAP1, p/t-PLK1, p/t-CDK1, p/t-PBK, and p/t-PRC1 were measured. E Protein levels of PBK and PRC1 were measured. F Protein levels of p/t-CDK1, p/t-PBK, and p/t-PRC1 were measured. G Protein levels of p/t-PBK and p/t-PRC1 were measured. H Protein levels of p/t-PRC1 were measured. Protein levels in lung tissues were examined by immunoblotting (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 6.

Fig. 6

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Relevant interventions attenuate the development of MCT-induced PAH rat. A Comparison of mPAP (n = 6). B Comparison of RVSP (n = 6). C Comparison of RV/ (LV + S) (n = 4). D Quantification of wall thickness (n = 4). E Quantification of muscularization (n = 4). F HE staining reveals right ventricular hypertrophy, scale bar = 5000 μm; HE staining and EVG staining reflect medial wall thickness, scale bar = 50 μm; muscularization revealed by immunohistochemical staining of α-SMA, scale bar = 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

We next investigated the effects of targeting TLR4, YAP1, PLK1, CDK, PBK and PRC1 in PAH rat models. As shown in Fig. 6A-F, inhibition of each target effectively reduced mPAP and RVSP, attenuated right ventricular hypertrophy, medial wall thickness and muscularized arteries. In addition, TAK-242 treatment reduced YAP1 protein expression, blocked PLK1 and CDK1 phosphorylation, and attenuated PBK1 and PRC1 activation in PAH rats (Fig. 7D). Treatment with YAP1 inhibitor VP reduced MCT-induced increase in PBK1 and PRC1 protein expression (Fig. 7E). Administration of PLK1 siRNA inhibited CDK1, PBK and PRC1 phosphorylation in PAH rat models, and CDK1 inhibitor RO-3306 treatment also attenuated PBK and PRC1 phosphorylation in PAH rats (Fig. 7F and G). Targeting PBK by siRNA suppressed the expression and phosphorylation of PRC1 in PAH rat models as shown in Fig. 7H.

Discussion

The present study demonstrates that Gal-3 activated PBK/PRC1 through TLR4/YAP1 and TLR4/PLK1/CDK1 signaling pathways, thereby promoting PASMC proliferation and pulmonary arterial remodeling (Fig. 8).

Fig. 8.

Fig. 8

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The molecular mechanisms of PBK mediation of Gal-3-induced PASMC proliferation and pulmonary arterial remodeling. Gal-3 induces PBK expression and phosphorylation via TLR4/YAP1 and TLR4/PLK1/CDK1 axis, which further activates PRC1, ultimately promoting PASMC proliferation and pulmonary arterial remodeling, therefore PAH development.

Gal-3 is a regulatory factor in PAH development [7, 40]. It has been found that pre-silencing Gal-3 or pre-inhibiting its function blocked PASMC proliferation and attenuated the development of PAH in animal model [40–42]. In alignment with these studies, our study confirmed Gal-3’s role of stimulating PASMC proliferation and pulmonary arterial remodeling. Several reports have demonstrated that Gal-3 upregulates the expression of TLR4 as well as its adaptor protein myeloid differentiation factor-88 adaptor protein and activates its downstream signaling pathway [11, 43]. Our previous study has indicated that Gal-3 induces the upregulation, dephosphorylation and nuclear translocation of YAP1, thereby promoting the proliferation of PASMCs [9]. In this study, we demonstrated that TLR4 mediated the upregulation of YAP1 transcription and translation by Gal-3 in PASMCs. In addition, our study showed that TLR4 mediated Gal-3-induced phosphorylation of PLK1, thus promoting PASMC proliferation and pulmonary arterial remodeling.

CDK1 serves as a crucial regulator of the cell cycle, forming a complex with cyclin B1 that allows the cell to enter M-phase and initiate cell division [44]. CDK1 can phosphorylate and activate a variety of substrates, with its dysregulation closely associated with tumorigenesis [45]. There is evidence that CDK1 phosphorylation is elevated in G1/S phase in PASMCs derived from PAH patient [23]. In addition, it has been found that PLK1 plays a role upstream of CDK1, and PLK1 inhibitor reduces CDK1 activation [15, 46]. In alignment with prior studies, our study demonstrated a marked increase in the phosphorylation level of CDK1 in Gal-3-treated PASMCs and in the lung tissue of PAH rats, and PLK1 mediated the Gal-3-induced CDK1 phosphorylation.

PBK exhibits overexpression in several cancers and shows a correlation with disease prognosis [27, 28]. In our study, we demonstrated that YAP1 mediated the Gal-3/TLR4-induced upregulation of PBK in PASMCs, which further promoted PASMC proliferation and pulmonary arterial remodeling. The phosphorylation of PBK is cell cycle dependent in mitosis, with PBK not being phosphorylated in asynchronous cells [47]. The intracellular localization of PBK throughout the cell cycle is very similar to that of CDK1, and Thr9 phosphorylation of PBK enhances its ability to bind to CDK1 [31]. In addition, in vitro kinase assays have demonstrated that PBK can be phosphorylated by CDK1/cyclin B [47]. Our study demonstrated that TLR4/PLK1/CDK1 axis mediated Gal-3 activation of PBK. These results suggest that Gal-3 can induce the expression and phosphorylation of PBK through the TLR4/YAP1 and TLR4/PLK1/CDK1 axis, respectively.

PRC1 is involved in the development of many malignant tumors, such as colon, lung and breast cancer [48–50]. Previous studies have shown that PRC1 is concentrated in the central spindle during mitotic metaphase and is involved in the microtubule formation, chromosome alignment, chromosome segregation and cytokinesis, and loss of PRC1 leads to disruption of the central spindle, abnormalities of chromosome segregation and incomplete cell divisions [51–54]. Several studies have demonstrated that PRC1 can be phosphorylated by PBK and thereby promotes cytokinesis [32]. In this study, we demonstrated that PRC1 could be time-dependently phosphorylated by Gal-3 to promote PASMC proliferation, and pre-silencing PBK or its upstream regulators reversed this process. Furthermore, we found that PBK also mediated the Gal-3-induced upregulation of PRC1. The exact regulatory mechanisms are not clear, but as PBK has been shown to be essential for transcriptional activity of several promoters, it may involve upregulating PRC1 expression by activating downstream transcription factors [55].

Although current treatment strategies targeting endothelin, nitric oxide and prostacyclin signaling pathway have significantly improved PAH patients outcome, the disease remains refractory and warrants further investigations [56]. The proliferation of PASMCs is a critical pathological mechanism in PAH, whereas current targeted medicines primarily reduce pulmonary circulation pressure through vasodilation [57]. As PBK/PRC1 plays a regulatory role in PASMC proliferation and pulmonary arterial remodeling, targeting this signaling pathway may have significant potential value in the treatment of PAH.

However, this study also has several limitations. In in vitro experiments, we used primary cultured rat PASMCs to construct the cellular model, but human PASMCs, especially those derived from lung tissue of PAH patients better reflect the clinical pathological state. Moreover, this study only employed the MCT-induced PAH rat animal models. To validate the universality of the results, future studies using human PASMCs and other PAH models, such as Sugen 5416/hypoxia-induced PAH models, are needed to investigate the role of this signaling pathway in PAH development.

Conclusion

In conclusion, we have demonstrated that Gal-3 induces PBK expression and activation through TLR4/YAP1 and TLR4/PLK1/CDK1 axis, thus promoting upregulation and phosphorylation of PRC1 and ultimately contributing to PASMC proliferation and pulmonary arterial remodeling. Our results provide novel insights, and targeting this signaling pathway might have potential value in the management of PAH.

Supplementary Information

Supplementary Material 1. (73KB, pdf)
Supplementary Material 2. (704.9KB, pdf)

Acknowledgements

We thank the Center for Translational Medicine of the First Affiliated Hospital of Xi’an Jiaotong University for the laboratory and experimental platform.

Abbreviations

Gal-3

Galactose lectin-3

TLR4

Toll-like receptor 4

PAH

Pulmonary arterial hypertension

PASMCs

Pulmonary arterial smooth muscle cells

MCT

Monocrotaline

YAP1

Yes-associated protein 1

PLK1

Polo-like kinase 1

CDK1

Cyclin-dependent kinase 1

PBK

PDZ-binding kinase

PRC1

Protein regulator of cytokinesis 1

SD

Sprague-Dawley

DMEM

Dulbecco’s modified eagle medium

FBS

Fetal bovine serum

MCP

PectaSol modified citrus pectin

VP

Verteporfin

RVSP

Right ventricle systolic pressure

mPAP

Mean pulmonary arterial pressure

RV

Right ventricle

LV

Left ventricle

S

Septum

RVHI

Right ventricle hypertrophy index

H&E

Hematoxylin and eosin

EVG

Elastin Van Gieson

Authors’ contributions

JZ: Conceptualization, Data curation, Formal analysis, Validation, Methodology, Visualization, Writing – original draft. JL: Validation, Methodology. YQ C: Validation, Methodology. HC: Methodology. YH M: Methodology. YW: Formal analysis. YJ Q: Formal analysis. HZ H: Formal analysis. WH S: Supervision, Funding acquisition. MX L: Writing, Conceptualization, Administration, Funding acquisition. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82270056) and the Natural Science Foundation of Shaanxi Province (2024JC-YBQN-0833).

Data availability

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (73KB, pdf)
Supplementary Material 2. (704.9KB, pdf)

Data Availability Statement

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

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