Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium–Sensing Receptor
Qinghua Hu，Liping Zhu
All studies using Sprague‐Dawley rats were approved by the institutional animal care and use committee of Tongji Medical College, Huazhong University of Science and Technology, and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.To determine whether monocrotaline was capable of binding to CaSR, this study used 3 divergent techniques well‐suited to detect any ligation or combining of a chemical compound with a protein molecule in different types of sample preparations.To explore whether monocrotaline was able to bind to CaSR in vitro, we conducted WaterLOGSY NMR screening with purified protein preparation of CaSR ECD. As shown in Figure 2A, 5 μmol/L CaSR ECD and 1 mmol/L monocrotaline exhibited a distinct proton spectrum with weak signals in 10 mmol/L PBS containing 10% D2O on NMR (marked “a1” and “a2,” respectively). The concomitant presence of CaSR ECD and monocrotaline resulted in enormous enhancement of proton signals in several down fields of the monocrotaline spectrum, at 1.1, 1.3, 1.4, 2.2, 3.0, 3.6, 3.8, and 6.2 ppm on NMR (marked “a3” in Figure 2A), and the enhancement effect was dose‐dependent on monocrotaline (marked “a4” through “a7” in Figure 2A), strongly suggesting the possible binding of monocrotaline to CaSR ECD. Furthermore, we carried out saturation transfer difference NMR analysis with the purified CaSR ECD protein. As shown in Figure 2B, 5 μmol/L CaSR ECD and 1 mmol/L monocrotaline exhibited different proton spectrums in 10 mmol/L Tris in D2O on NMR (marked “b1” and “b2,” respectively). When CaSR‐ECD was irradiated in the presence of 1 mmol/L monocrotaline on NMR for 3 seconds at 2.0 and 30 ppm for saturation and nonsaturation, respectively, of CaSR‐ECD, the resulting saturation transfer difference (STD) clearly identified several increased proton signals at the down fields around 0.7, 0.9, 1.0, 1.7, 2.5, 3.0, and 6.5 ppm (marked “b3” in Figure 2B), which completely coincided with the corresponding down fields of monocrotaline proton spectrum (marked “b2” in Figure 2B) and thus confirmed the binding of monocrotaline to CaSR ECD. It is noted that N,N‐dimethylformamide, the solvent of monocrotaline pyrrole (MCTP) prevented us from comparing any potentially enhanced or altered binding capacity of MCTP to CaSR versus monocrotaline, since N,N‐dimethylformamide usually induces denaturalization of purified protein in vitro. In addition, N,N‐dimethylformamide contains the intrastructures of methyl groups, which most likely interfere with the proton NMR spectrum by giving rise to 2 singlets of 3 protons by itself38 and thus is treated as an inappropriate component in the sample preparations for NMR monitoring, even at a trace amount.To examine whether monocrotaline was capable of ligating the native CaSR, we performed immunocytochemical staining of cultured PAECs. For this purpose, we labeled monocrotaline with FITC and then incubated FITC‐MCT with PAECs with or without coimmunostaining of CaSR. As shown in Figure 3, FITC‐MCT incubation clearly showed localization of monocrotaline on the cell membrane and, to some extent, in the cytosol of PAECs (shown in green in Figure 3A and
3C, left), not in FITC‐incubated control PAECs, ensuring the specificity of this staining (Figure 3B, left). Also shown in Figure 3, indirect immunostaining showed the localization of CaSR on the cell membrane and, to some extent, in the cytosol of PAECs (shown in red in Figure 3A and
3B, middle). The expression of CaSR on the cell membrane and in the cytosol of PAECs was completely consistent with previous reports on other types of vascular endothelial cells including human aortic endothelial cells.39 The coimmunostaining of CaSR in FITC‐MCT–incubated PAECs further showed the yellow fluorescence resulting from the merging of the green fluorescence of monocrotaline and the red fluorescence of CaSR, indicating colocalization of monocrotaline together CaSR on the cell membrane and, to a lesser extent, in the cytosol of PAECs (Figure 3A, right). It is noted that the structure of monocrotaline pyrrole (MCTP) and its relatively unstable property prevented us from labeling it with FITC or other types of fluorescent tracer. Considering the conversion of monocrotaline to MCTP in the liver after their injection into animals and the subsequent targeting of MCTP on PAECs,5, 6, 7 the potential binding of MCTP to CaSR was explored in monocrotaline‐injected rats as follows.To finally verify whether MCTP was able to combine with native CaSR in vivo, we conducted the cellular thermal shift assay on pulmonary arteries isolated from monocrotaline‐injected rats. The cellular thermal shift assay is a recently established approach to reveal whether a chemical compound or drug can bind to a protein.40, 41 The new technique methodologically exploits the chemical property of enhanced thermal stability of a protein on specific binding of a compound.40, 41 The protein lysates of pulmonary arteries isolated from rats administered control (the vehicle DMSO), the chemical inhibitor of CaSR ([1S,2S,1′R]‐N1‐[4‐chlorobenzoyl]‐N2‐[1‐(1‐naphthyl)ethyl]‐1,2‐diaminocyclo‐hexane, Calhex 231), or monocrotaline were heated at a series of temperatures from 42 to 72°C for 3 minutes and then subjected to immunoblotting analysis. In control‐ or DMSO‐treated animals, the CaSR was stable below or at 48°C, was slightly degraded from 51 to 54°C, was significantly degraded at 57°C, and was almost completely degraded at 60°C (Figure 3D). In Calhex 231‐treated animals, the CaSR was stable below or at 57°C, was slightly or significantly degraded from 60 to 63°C, and was almost completely degraded at 69 to 72°C (Figure 3D). In monocrotaline‐treated animals, the CaSR was stable below or at 63°C, was slightly or significantly degraded from 66 to 69°C, and was not completely degraded even at 72°C (Figure 3D). As summarized in Figure 3E, the statistical regression analysis showed the significant right shift of thermal stability curves of CaSR in pulmonary arteries from animals administered monocrotaline or Calhex 231 compared with control or vehicle, clearly indicating the specific binding of MCTP to CaSR in vivo.These results consistently demonstrated the binding of monocrotaline or MCTP to CaSR.Figure S1. The systemic and lung knockdown of CaSR. Representative immunoblots and statistical summaries of CaSR expression levels in lung (A), liver (B), kidney (C), brain (D), and heart (E) as well as serum levels of phosphate (F), calcium (G), and parathyroid hormone (H) in blank control rats and rats intravenously or intratracheally transduced with CaSR shRNA or control shRNA for systemic and lung CaSR knockdown, respectively. *P<0.05 vs control, n=3 for each group. CaSR indicates extracellular calcium–sensing receptor; shRNA, short hairpin RNA.Figure S2. The map of the pRP[CRISPR]‐hCas9_D10A‐U6 vector.Figure S3. Genome editing via the Cas9/gRNA system in Sprague‐Dawley rats. A, Constructs and schematic illustration of the Cas9/gRNA system used in this experiment. The U6 promoter drives transcription of the gRNA, which consists of a target sequence and a scaffold sequence. The CBh promoter drives the expression of Cas9 nuclease. BGH polyA facilitates transcriptional termination of the upstream ORF. B, Target sequence of Casr and parathyroid hormone. The rat Casr gene is located on chromosome 11, and 7 exons have been identified. E3 was selected as the Cas9 targeting region, and gRNA targeting sequences were labeled above in the E3. The rPth gene is located on chromosome 1, and 3 exons have been identified. E2 was selected as the Cas9 targeting region, and gRNA targeting sequences were labeled above in E2. PAM (which is indispensable for Cas9 binding and cleavage) sequences are highlighted in pink. BGH polyA indicates bovine growth hormone polyadenylation signal; CaSR indicates extracellular calcium–sensing receptor; E2, exon 2; E3, exon 3; gRNA, guide RNA; NLS, nuclear localization signal; PAM, protospacer adjacent motif.Figure S4. Generation of CaSR and PTH double‐knockout rats. A, Detailed mutations of CaSR and PTH genes in F0 KO rats. Deletions are indicated by dashes, insertions are indicated in blue, and substitutions are indicated in red. Deletions (−) and insertions (+) are shown to the right of each allele. B, Detailed mutations of CaSR and PTH genes in F1 KO rats. C, Detailed mutations of CaSR and PTH genes in F2 KO rats. CaSR indicates extracellular calcium–sensing receptor; KO, knockout; PTH, parathyroid hormone.