Fig 1: Linalool blocks the MAPK signaling pathway via downregulating the expression of CHRM3. (A) Western blotting was applied to detect the expression of proteins related to the MAPK signaling pathway in A7r5 and transfected cells. (B) The semi-quantitative graphs of western blotting. ***P<0.001 vs. control; ###P<0.001 vs. Ang II; &&&P<0.01 vs. Ang II+linalool+pcDNA3.1-NC (n =3). Ang II, angiotensin II; CHRM3, cholinergic receptor muscarinic 3; NC, negative control; p-, phosphorylated.
Fig 2: mMSCs express M3R in the cytoplasmic compartment co-localized to the endoplasmic reticulum (ER). (A) Generalized expression of M3R (green) localized to the cytoplasmic space of mMSCs, as shown in the top panel. Magnification of × 20; scale bar, 20 µm. (B) To confirm M3R localization to the ER, cells were co-stained with antibodies specific to M3R (green) and ER (red). At the single cell level, the merged image confirms the localization of M3R in the ER by the observed orange color (scale bar, 2 µm). DAPI was used for nuclear staining. (C) Furthermore, WB was performed to confirm M3R protein expression in transfected and untransfected MSCs. (D) RT-PCR was also performed to confirm M3R mRNA expression of MSCs. (E) Additionally, nuclear and cytoplasmic protein fractions from mMSCs analyzed by WB confirmed the confinement of M3R to the cytoplasmic compartment. (F) Knocking down M3R with a sequence-specific siRNA led to a significant reduction of the M3R level. ** p < 0.01 by Student’s t-test. (G) At 24-h post-transfection, TCF3 expressing mMSCs demonstrated reduced expression of M3R observed in the cytoplasm (white arrows). Cells that have low or no expression of TCF3 maintained M3R expression (green arrows). Magnification of × 40; scale bar, 5 µm.
Fig 3: Pharmacological inhibition of MRTF-SRF signaling in organ culture, and knockout of Srf in vivo, reduces M3 receptor expression. To examine if MRTF-SRF signaling regulates muscarinic M3 receptor expression in situ, we first isolated organs from wild-type C57Bl/6 mice. Organs were split in half and maintained for 96h in organ culture with vehicle (DMSO) or CCG-1423 (10µM). A clear reduction of Chrm3 relative to the house-keeping gene 18s was seen in the esophagus (A), but in the remainder of the organs, the house-keeping genes examined declined (not shown). We also found that organ culture of the mouse caudal artery in the presence of CCG-1423 eliminated force development on stimulation with the a1-adrenergic agonist cirazoline (B), suggesting that this experimental paradigm is unsuitable for studying effects on endothelium-dependent dilatation. Mice with SMC-specific knockout of Srf were next obtained by injecting Srffl/fl mice harboring the Myh11-Cre/ERT2 transgene with tamoxifen for 5 consecutive days (knockout: KO). Cre-negative Srffl/fl mice injected with tamoxifen were used as controls (wild-type: WT). Organs were harvested and frozen 10days after the first injection and transcript levels were determined by RT-qPCR. At this time, body weights were unchanged, but Srf depletion was seen in some organs (C). (D) Shows that Chrm3 was reduced in parallel with Srf in the bladder, but this was not seen elsewhere (not shown). We therefore next used mice at 21days post tamoxifen. Two control groups were included in this second experiment along with the tamoxifen-treated knockouts (TKO): vehicle-treated Cre-positive mice (VC) and tamoxifen-treated Cre-negative mice (TC). At 21days, mobility on provocation was reduced, the intestines had started to swell [(E), white arrows], and the urinary bladders were often enlarged [(E), black arrows]. Both Chrm3 and Srf were reduced in the bladder (F) and ileum (G). For the ileum, the two knockouts with the most modest Srf depletion (TKO 173 and TKO 193) are highlighted. No change of Chrm3 was seen in the aorta (H), despite significant Srf depletion. These findings show that MRTF-SRF signaling is critical for Chrm3 expression in gastrointestinal and urogenital organs in vivo. ***p<0.001, **p<0.01, and *p<0.05.
Fig 4: Linalool inhibits the proliferation of Ang II-induced vascular smooth muscle cells via downregulating CHRM3 expression. (A) Reverse transcription-quantitative PCR and (B)western blotting were employed to detect the expression level of CHRM3 in transfected cells. ***P<0.001 vs. pcDNA3.1-NC (n=3). (C) MTT assay was used to detect cell proliferation. *P<0.05, **P<0.01 vs. Ang II; #P<0.05, ##P<0.01 vs. Ang II+linalool+pcDNA3.1-NC (n =3). (D) The expression of PCNA, which is relevant to cell proliferation, was detected via western blotting. ***P<0.001 vs. control; ###P<0.001 vs. Ang II; &&&P<0.001 vs. Ang II+linalool+pcDNA3.1-NC (n =3). Ang II, angiotensin II; CHRM3, cholinergic receptor muscarinic 3; NC, negative control; PCNA, proliferating cell nuclear antigen; NC, negative control.
Fig 5: Linalool downregulates CHRM3 expression in Ang II-induced vascular smooth muscle cells. (A) Reverse transcription-quantitative PCR and (B) western blotting were used to detect CHRM3 expression in cells treated with Ang II and different concentrations of linalool. ***P<0.001 vs. control; ##P<0.01, ###P<0.001 vs. Ang II (n =3). Ang II, angiotensin II; CHRM3, cholinergic receptor muscarinic 3.
Supplier Page from Abcam for Anti-Muscarinic Acetylcholine Receptor M3/CHRM3 antibody