Endothelin-1 upregulates activin receptor-like kinase-1 expression via Gi/RhoA/Sp-1/Rho kinase pathways at transcriptional and post-transcriptional levels in human pulmonary arterial endothelial cells

Backgrounds induces through activation of RhoA. we previously demonstrated that Gi, a heterotrimeric G protein, functions upstream of RhoA activation. A gene mutation of activin receptor-like kinase (ACVRL)-1 is recognized in idiopathic or heritable PAH patients. However, little is known about the association between ET-1 and ACVRL-1. In the present study, we investigated the effect of ET-1 on expression and aimed to the of the


Introduction
Pulmonary arterial hypertension (PAH) is a disease with poor prognosis that is characterized by pulmonary vasoconstriction and organic stenosis due to abnormal proliferation of pulmonary artery endothelial cells and smooth muscle cells [1,2,3]. It is considered that endothelial dysfunction is associated with these vascular pathologies [4,5]; however, their detailed molecular mechanisms are still unknown.
Endothelin-1 (ET-1) is a major vasoconstrictor derived from endothelial cells [6]. Today, endothelin receptor agonist is widely used for PAH treatment, and has contributed to the improvement of PAH prognosis. This shows that endothelin plays a crucial role in PAH. It has been demonstrated that ET-1 induces pulmonary vasoconstriction through activation of RhoA, which is a small GTP protein [7]. Many studies have reported that the RhoA/Rho-kinase pathways implicate pulmonary hypertension [8,9,10]. Additionally, endothelin receptors are G-protein-coupled receptors [7], and we have previously demonstrated that Gi, which is a heterotrimeric G protein, functions upstream of RhoA activation [11].
Activin receptor-like kinase-1 (ACVRL-1) is one of the type I cell surface receptors for the transforming growth factor-β (TGF-β) family that is mainly expressed in vascular endothelial cells [12]. A gene mutation of ACVRL-1 is recognized in idiopathic or heritable PAH patients [13]. Although both ET-1 and ACVRL-1 are important molecules for the pathogenesis of PAH, little is known about the association between ET-1 and ACVRL-1.
In the present study, we investigated the effect of ET-1 on ACVRL-1 expression, and aimed to delineate the involvement of the Gi/RhoA/Rho kinase pathway in pulmonary arterial endothelial cells.

Materials
Most of the reagents used in this study have been described previously [14,15,16]. Recombinant human ET-1 was obtained from R&D systems (Minneapolis, MN, USA), and cell permeable exoenzyme C3 transferase (C3T) was purchased from Cytoskeleton, Inc. (Denver, CO, USA). Pertussis toxin (PTX) and actinomycin D were purchased from Merck KGaA (Darmstadt, Germany). Y27632 was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Preparation of Endothelial Cells
Human pulmonary arterial endothelial cells (PAECs) were cultured according to the manufacturers' instructions (PromoCell, Heidelberg, Germany) and used for all experiments after 5 to 10 passages.

Western Blotting
Western blotting was performed as described previously [14,15,16]. The lysates of lung tissues were mixed at a ratio of 4:1 with loading buffer (75 mM Tris-HCl, pH 6.8; 10% glycerol; 3% 2-mercaptoethanol, and 2% sodium dodecyl sulfate [SDS]) and heated at 95 °C for 10 minutes. Aliquots containing 20 µg of protein were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were then transferred onto polyvinylidene di uoride membranes (Merck KGaA). After incubation with blocking solution at room temperature for 30 minutes, the membranes were incubated for 1 hour at room temperature with a monoclonal antibodies to RhoA (Santa Cruz Biotechnology, Santa Cruz, CA) and ACVRL-1 (Abcam, Cambridge, UK) at a dilution of 1∶500, and to β-actin (Santa Cruz Biotechnology) diluted 1∶1000 or a rabbit polyclonal antibody to Sp-1 (GeneTex, Inc., Irvine, CA, USA) diluted 1:1000 for immunoblotting. The signals from immunoreactive bands were visualized by a Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The optical densities of the individual bands were analyzed using Image J 1.48. GTP/GDP Exchange of RhoA GTP-bound active forms of RhoA were assessed using a commercially available assay kit (Cytoskeleton Inc.) according to the manufacturer's instructions [17].
Quantitative reverse transcription polymerase chain reaction Total RNA was extracted from PAECs using TRIzol reagent (Invitrogen Carlsbad, CA, USA). The RNA was reverse transcribed into rst-strand cDNA with a ReverseTra Ace qPCR RT kit (Toyobo Co., Ltd, Osaka, Japan), and the cDNA was subjected to quantitative polymerase chain reaction (qPCR) using a Thunderbird SYBR qPCR Mix (Toyobo) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primers were designed on the basis of GenBank sequences (ACVRL-1, NM_009612.3 and GAPDH, NM_001289726.1). qPCR reactions were run in duplicates, and the delta-delta Ct method was applied for quanti cation.

DNA transfection and luciferase assay
We cloned the re y luciferase reporter construct ACVRL-1/Luc containing the human putative promoter region of ACVRL-1 (GeneBank: NC_000012.12, position 51906383 to 51907627) into pGL3-Basic vector [18]. pNL1.1.TK [NLuc/TK] was used as a control vector. The constructs were co-transfected into 70% con uent PAECs using a Screenfect A (FUJIFILM Wako Pure Chemical Corporation). Forty-eight hours after transfection, cell lysates were assayed for luciferase activity using a Nano-Glo Dual Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer's instructions. Cell culture experiments were performed in triplicate.

Determination of eNOS mRNA stability
To analyze the effect of ET-1 on ACVRL-1 mRNA stability, PAECs were stimulated by ET-1 for 6 hours with or without Y27632, then 5 µg/mL actinomycin D was added. ACVRL-1 mRNA levels were determined by qPCR.

Statistical analysis
Statistical analyses were performed using ANOVA with Tukey's post hoc test or Student's t-test where appropriate. A value of P < 0.05 was considered signi cant. Data are expressed as means ± standard errors (SE).

Results
Western blotting and qPCR demonstrated that protein and mRNA expressions of ACVRL-1 were increased by ET-1 stimulation in the PAECs (Figs. 1A and B). Further, these results indicate that the upregulations of ACVRL-1 were not necessarily dependent on the dose of ET-1.
Pull-down assay revealed that ET-1 rapidly induced a GTP-loading of RhoA, which is active form of RhoA in the PAECs, whereas the levels of RhoA in whole cell lysates were not changed. C3T prevented ET-1induced RhoA activation ( Fig. 2A). Figure 2B shows that the ET-1-induced RhoA activation was suppressed by pretreatment with PTX, suggesting that Gi is upstream of RhoA activation.
To elucidate whether ET-1-induced ACVRL-1 upregulation occurs at the transcriptional or posttranscriptional level, we evaluated the promoter activity of ACVRL-1 and the stability of mRNA of ACVRL-1. Figure 4A shows that transcriptional activity of the ACVRL-1 promotor was increased by ET-1. In addition, the lifespan of mRNA of ACVRL-1 was sustained in ET-1-stimulated PAECs, and Rho kinase inhibition by Y27632 reversed the stabilization of ACVRL-1 mRNA by ET-1 to the control level (Fig. 4B).
Further, we investigated the role of Sp-1, which is one of the transcriptional factors for ACVRL-1, in ET-1stimulated PAECs. Figure 5A shows that, after adding ET-1, the level of Sp-1 was increased within 10 min, and peaked after 15 min. Both Gi inhibition by PTX and RhoA inhibition by C3T prevented the increase of Sp-1 level in response to ET-1 (Fig. 5B, C). These data indicate that rapid RhoA activation via Gi affects Sp-1 regulation.

Discussion
We for the rst time demonstrated that ET-1 upregulated ACVRL-1 expression at the transcriptional and the post-transcriptional levels via Gi/RhoA/Rho kinase pathway in PEACs. Our results suggest that Sp-1 is associated with the promoter activity of ACVRL-1 via RhoA activation.
In patients with idiopathic PAH, an ET-1 plasma level is correlated with mean pulmonary arterial pressure and pulmonary vascular resistance [19]. Further, it has been reported that ACVRL-1 expression is increased in the PAECs of patients with idiopathic PAH [20]. Taking these clinical ndings and our in vitro data together, it is highly possible that elevated ET-1 levels actually increase ACVRL-1 expression in PAH patients. In addition, a previous study has shown that ACVRL-1 de ciency induced the synthesis and release of ET-1 [21]. This phenomenon might have been caused to maintain the levels of ACVRL-1 expression by increasing ET-1 stimulation.
However, in mice models, there have been opposing results of the effect of TGF-β receptors de ciency on PAH. While Jerkic et al. reported that adult ACVRL-1 heterozygous mice spontaneously develop PAH [22], Gore et al. demonstrated that hypoxia-induced pulmonary hypertension was ameliorated by de ciency of endoglin, which is a TGF-β receptor. Similar to ACVRL-1, gene mutation in endoglin is implicated to PAH [20]. Therefore, the signi cance of increases in ACVRL-1 due to ET-1 in PAH patients remains unclear, and needs to be resolved in future studies.
Previous studies have shown that whether the modi cation of RNA expression by RhoA is upregulated or downregulated depends on the type of molecule [23,24]. Marshall et al. demonstrated that ET-1 upregulated RNA expression of some molecules (e.g. Abra, Srf and Egr2), which were inhibited by C3T in rat cardiomyocytes [23]. On the other hand, Laufs et al. reported that RhoA activation downregulated the expression of endothelial nitric oxide synthase (eNOS) at the post-transcriptional level in endothelial cells. They showed that actin stress ber reorganization by RhoA activation was associated with posttranscriptional regulation of eNOSmRNA [24].
Murthy et al. reported that Rac1, which is a small GTP protein similar to RhoA, stabilized Sp-1 expression in macrophages [25]. Interestingly, in the current study, ET-1-triggered RhoA activation has been increased the Sp-1 protein within 15 min, whereas the stabilization of Sp-1 by Rac1 was shown to take several hours.
Several studies have reported on the association between ET-1 and PTX-sensitive G protein [26,27].