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Defining an Olfactory Receptor Function in Airway Smooth Muscle Cells

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Defining an Olfactory Receptor Function in Airway Smooth Muscle Cells

William H Aisenberg et al. Sci Rep.

Abstract

Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma.

Figures

Figure 1
Figure 1. Expression of olfactory receptor mRNA transcripts in human ASM and transfected HEK-293T cells.
(a) Golf and AC3 are both expressed in human ASM cells (sequenced to confirm). (b) The five ORs transcripts identified by degenerate PCR were also confirmed in human ASM, using gene-specific primers to each OR. Mock RT samples (below) serve as negative controls. (c) Confirmed expression of the full-length transcript of OR51E2 in RT, but not in mRT. (d) Three of the five ASM ORs are able to traffic to the surface of HEK-293T cells. The top panel shows cell surface OR population as detected by immunostaining against an N-terminal extracellular FLAG epitope tag with a polyclonal antibody. For detecting the internal OR population, cells were subsequently fixed, permeabilized, and stained again for Flag using a monoclonal antibody (bottom panel). All constructs with the exception of OR51E2 contain a leucine rich N-terminal signal peptide and are co-transfected with chaperon proteins as previously described; “+” and “−” indicate cell surface localization of ORs.
Figure 2
Figure 2. Human body atlas of ASM OR.
(a) Expression of the five de novo identified ASM ORs across 30 human tissue types (GTEx RNA-Seq tissues). Each colored block provides a quantile spread representing expression levels expressed as fragments per kilobase of transcript per million mapped reads (FPKM) across all samples of that tissue type. The color at the middle of the strip represents the median expression, and the color at the end of the strip represents the maximum expression. A complete absence of reads over an OR is represented as grey. The number of human samples in the collection is displayed next to the tissue name. (b) Cultured lung cell expression of the indicated ORs (also see Supplementary Table 1). (c) RT-PCR cross-validation of the most abundant lung-resident OR genes (shown in panel b) in human ASM cells isolated from 6 non-asthmatic lung donors. Results are shown as 2−ΔCt X 10000, where ΔCt = [Ct(target gene) − Ct(gapdh)].
Figure 3
Figure 3. De-orphanization of OR51E2 signaling and function in human ASM.
(a) Agonist profiles of OR51E2 as detected by a luciferase-based reporter assay in transfected HEK-293T cells. For each ligand dosage/condition (n = 3), we used ANOVA followed by the Student-Newman-Keuls method to determine significance. Data are presented as mean ± SE (*P < 0.05 vs. 0 mM dose considered significant). (b) Trajectories of unforced motions of RGD-coated beads functionalized on the living human ASM. Spontaneous bead motions were recorded over the course of 5 min (10X Objective). Cells were treated for 24 h with or without 10 mM of the respective SCFAs (formate, acetate, propionate, and butyrate). For clarity, only a few representative tracings (n = 20) are depicted for each experimental conditions. (c) The trajectories of bead motions in two dimensions were characterized by computed mean square displacement (MSD) of all beads as function of time t. Data are presented as mean ± SE (untreated, n = 15; formate, n = 9; acetate, n = 15; propionate, n = 17; butyrate, n = 5 individual cell-wells, comprising ~442-1874 individual beads measurements). (d,e) For each individual cell-well, the exponent α and the diffusion coefficient (D*) were estimated from a least square fit of a power law. (f) MSD measured at 10 s and 300 s, respectively. *P < 0.05 compared with untreated cells by ANOVA and post hoc t-tests.
Figure 4
Figure 4. Characteristics of the OR51E2 homologue Olfr78.
(a) Cytoskeletal dynamics of ASM cells derived from several inbred mouse strains as measured by SNTM. Data are presented as mean ± SE (AJ, n = 825; BALB/c, n = 599; C57BL/6, n = 805 individual beads measurements). (b) A schematic diagram of murine Olfr78 locus and its exons; three target sites in exon 4 are shown below, indicating the CRISPR N20NGG target sites. (c) Genome editing efficiency of CRISPR-Cas9 in the primary mouse ASM cells as determined by western blot. Full-length gels/blots are presented in Supplementary Figure 7a. (d) Computed MSD at 300 s for Olfr78 WT and Olfr78 KO cells (C57BL/6) in response to 24 h exposures to acetate and propionate (10 mM). Data are presented as geometric mean ± 95% CI (n = 307–379 individual beads measurements for each group). (e) Isolated mouse ASM cells (C57BL/6) were transfected with or without FLAG-tagged full-length construct encoding human OR51E2. OR51E2 protein expression was detected by anti-Flag antibody. Full-length gels/blots are presented in Supplementary Figure 7b. Although the expected band is ~36 kDa, ORs typically also appear as multiple higher molecular weight bands. (f) Computed MSD at 300 s for wild-type (WT) and OR51E2-overexpressing (Flag-OR51E2) mouse ASM cells. Data are presented as geometric mean ± 95% confidence interval (n = 354–364 individual beads measurements for each group).
Figure 5
Figure 5. Activation of OR51E2 inhibits ASM proliferation.
(a) Representative photomicrographs of cells treated for 24 h with or without formate, acetate, and propionate (10 mM) as measured by EdU cell proliferation assay. EdU positive nuclei are labeled with Alexa Fluor 647 (purple), and all nuclei are labeled with DAPI (blue). (b) The ratio of Alexa Fluor 647 positive and DAPI positive nuclei were counted in four non-overlapping fields per sample. Data are presented as mean ± SE (n = 3 independent samples). (c) ASM proliferation was measured by live cell counting over 6 days in culture with SCFAs. Data are presented as mean ± SE (n = 4 independent measurements). (d) The effects of SCFAs on cellular proliferation of WT and OR51E2 KO cells as quantified by Cell Counting Kit-8 (CCK-8, Sigma). For these studies, cells were treated for 24 h with 10 mM SCFAs and then 1 mM SCFAs for the next 5 days in culture. CCK-8 assay was performed on day 6. Data are presented as mean ± SE (n = 3 independent measurements). (e) The relative transcript levels of OR51E2 in isolated human ASM (non-asthmatics, n = 6 lung donors; asthmatics, n = 6 lung donors). (f) The effects of SCFAs, acetate and propionate, on cellular proliferation of asthmatic ASM derived from 6 independent lung donors as measured by live cell counting over 6 days in culture.

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