Specific GPCRs elicit unique extracellular vesicle miRNA array signatures
- Research Service, Veterans Affairs Portland Health Care System, United States
- Department of Behavioral Neuroscience, Oregon Health and Science University, United States
- Biostatistics and Design Program, OHSU-PSU School of Public Health, Oregon Health and Science University, United States
- Department of Psychiatry, Oregon Health and Science University, United States
Figures
Figure 1 with 1 supplement
G protein-coupled receptor (GPCR) activation in U2OS cells by selective agonists.
(A) Dose-dependent cAMP accumulation after ADORA1 activation with agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), or inhibition with antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (5 μM). (B) Dose-dependent IP1 accumulation after 2-pyridylethylamine dihydrochloride (PEA) stimulation of histamine receptor H1 (HRH1) and inhibition by cetirizine (1 μM). (C) Alkaline phosphatase (ALP) activity after stimulation of frizzled class receptor 4 (FZD4) by Wnt3a. (D) The ratio of phosphorylated ERK1/2 (pERK1/2) by total ERK 1/2 (tERK1/2) after stimulation of ACKR3 by SDF-1α. For all graphs, data are shown as mean ± SD, with n=3 independent repeats, each having duplicate determinants, ns = not significant, *p<0.05, **p<0.01, ***p<0.001 vs. vehicle control (VC). Statistical significances were determined by one-way or two-way ANOVA of receptors by agonists or antagonists and post hoc Tukey’s or Sidak’s testing for multiple comparisons.
Figure 1—figure supplement 1
Representative immunoblots show stimulation of ACKR3 with SDF-1α-induced phosphorylation of ERK1/2 (pERK1/2, 44kDa/42kDa) compared to total ERK1/2 (tERK1/2, 44kDa/42kDa).
β-Actin (43kDa) was used as a loading control.
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Figure 1—figure supplement 1—source data 1
Original western blots for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/107865/elife-107865-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file containing the original western blots for Figure 1—figure supplement 1, with relevant bands and experimental conditions labeled.
- https://cdn.elifesciences.org/articles/107865/elife-107865-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
Characterization of extracellular vesicles (EVs) isolated from U2OS cell culture media.
(A) Representative immunoblots of EVs isolated by size exclusion chromatography (SEC) from U2OS cell culture media (U2) but not from media control (MC) show detection of EV markers, including CD9, CD63, CD81, flotillin, and syntenin, in EV fractions 7–9. Endoplasmic reticulum marker, calnexin, was not detected in the EV fractions. (B) Representative images from transmission electron microscopy of isolated EVs (red arrows) from pooled EV-enriched fractions 7–9. (C) Size distribution of pooled EV fractions 7–9 isolated from MC and U2. (D) Quantification of pooled EV fractions 7–9 of MC and U2 measured by fluorescence nanoparticle tracking analysis (f-NTA). Data are shown as mean ± SEM, n=7–8. A Student’s t-test determined significant differences in the EV concentration between the MC and U2, *p<0.05.
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Figure 2—source data 1
Original western blots for Figure 2A.
- https://cdn.elifesciences.org/articles/107865/elife-107865-fig2-data1-v1.zip
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Figure 2—source data 2
PDF file containing the original western blots for Figure 2A, with relevant bands and experimental conditions labeled.
- https://cdn.elifesciences.org/articles/107865/elife-107865-fig2-data2-v1.zip
Figure 2—figure supplement 1
Quantification of extracellular vesicles (EVs) in U2OS media in response to receptor activation.
U2OS cells were incubated with VC or a selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA) (A), 2-pyridylethylamine dihydrochloride (PEA) (B), Wnt3a (C), or SDF-1α (D) for ADORA1, HRH1, FZD4, and ACKR3, respectively. EVs were isolated, and the concentration was normalized as a percentage of the media control (MC). EV size distribution following agonist or VC treatment in ADORA1 (E), HRH1 (F), FZD4 (G), and ACKR3 (H). Data are shown as mean ± SEM, n=3. A t-test determined no significant differences in EV concentration or size distribution between the VC and agonist stimulation in all G protein-coupled receptors (GPCRs), p>0.05.
Figure 3 with 1 supplement
Differentially expressed extracellular vesicle (EV) microRNAs (miRNAs) in response to G protein-coupled receptor (GPCR) activation.
(A) The heatmap shows unsupervised hierarchical clustering of miRNA expression (row) following GPCR stimulation (column). The relative abundance of miRNAs is represented in Z-score value (z-transformed fold changes); blue, below-mean expression; red, above-mean expression. (B–E) Volcano plots display the analysis of EV miRNAs after ADORA1 (B), HRH1 (C), FZD4 (D), and ACKR3 (E) activation. On the x-axis, the dotted line indicates miRNAs that satisfied the |log2 fold change|≥ 1.5 cutoff, while the dotted line on the y-axis indicates miRNA that met Skillings-Mack p-value<0.2. (F) Venn diagram shows the miRNAs of interest with at least 1.5-fold change across four GPCR groups following treatment. n=5–6 replicates per GPCR group.
Figure 3—figure supplement 1
Sensitivity analysis across alternative estimates of treatment effects.
(A) Scatterplot matrix showing pairwise concordance of alternative treatment effect estimators, including all per-microRNA (miRNA) estimates from all receptors combined, with text labels (in quotes) indexed to the table below; note that the perfect correlation in the ‘individual’ and ‘matched’ approaches is expected, and the ‘pooled’ and ‘batched’ approaches only apply to the receptors ADORA1 and H1R. All estimators not based on matched pairs demonstrate bias in the form of exaggeration of effect size on average, and estimators employing a higher degree of aggregation (either at the treatment group or batch level) show worse correlation with the unbiased matched-pairs estimator (an example of ecological bias); it is clear that process variance brings considerable risk of bias in experiments of this type, and that great care should be taken to provide adequate representation of process variance via controls across experiments. (B) Comparison table detailing the differences between alternative estimators, including advantages and disadvantages of each approach. Our preferred approach is ‘matched’, on both theoretical and empirical grounds. Briefly, the six combinations were: (1) treatment effect estimated as difference of group medians with permutation-based p-value; (2) difference of group medians with p-value from Welch’s robust t-test; (3) (applies to batched receptors only) treatment coefficient from median regression of pooled-batch data with p-value from the rank-based Mann-Whitney test; (4) (applies to batched receptors only) sample-weighted average of treatment coefficients from separate median regressions of data from each batch with p-value as the harmonic mean of Mann-Whitney p-values calculated separately on each batch; (5) mean matched-pairs estimator (simple average of all within-pair differences between agonist and vehicle control) with permutation-based p-value; and (6) regression matched-pairs estimator (treatment coefficient from a within-pairs linear regression) with p-value from the unbalanced-robust rank-based Skillings-Mack test. Note that (1) was the only approach where we applied group-based normalization (for all others we used run-based), and that the treatment effects in (5) and (6) are not strictly the same except in the absence of missing data, but they are operationally 100% correlated.
Figure 4 with 2 supplements
Pathway analysis of the differentially expressed extracellular vesicle (EV) microRNA (miRNA) after G protein-coupled receptor (GPCR) activation.
The bubble plots show the top 25 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for the miRNAs (≥1.5 fold change) of individual GPCR. (A) ADORA1, (B) HRH1, (C) FZD4, (D) ACKR3. The dot size represents the number of enriched gene targets, and the color shows the p-value of the enrichment. For the enrichment analysis, cutoff criteria were p-value (FDR)<0.05 and gene count >2. KEGG terms: * Endocrine and other factor-regulated calcium reabsorption, ^ Progesterone-mediated oocyte maturation.
Figure 4—figure supplement 1
The top 10 hub genes identified in microRNA (miRNA) targets (≥1.5 fold change) PPI network.
Figure 4—figure supplement 2
Pathway analysis of miR-502-3p and miR-137 targets.
The bubble plots show the top 25 significantly enriched KEGG pathways for the targets of (A) miR-502-3p and (B) miR-137. The dot size represents the number of enriched gene targets, and the color shows the p-value of the enrichment. For the enrichment analysis, the cutoff criteria were p-value (FDR)<0.05 and gene count >2. KEGG term abbreviations: ** Aldosterone-regulated sodium reabsorption.
Figure 5
Stimulation of G protein-coupled receptors (GPCRs) activates extracellular vesicle (EV) microRNA signatures.
Tables
Table 1
Extracellular vesicle (EV) microRNAs (miRNAs) differentially expressed in four G protein-coupled receptor (GPCR) groups.
Differentially expressed miRNAs (p<0.05) were identified in each treatment group compared to its vehicle control, n=5–6 samples of each group.
| GPCRs | miRNAs | log2 (FC) | p-Value | SD of log2(FC) |
|---|---|---|---|---|
| ADORA1 | hsa-miR-550a-5p | 1.70 | 0.0455 | 0.22 |
| hsa-miR-1227-3p | 1.49 | 0.0253 | 0.16 | |
| hsa-miR-454-5p | 1.17 | 0.0143 | 0.27 | |
| hsa-miR-31-5p | 0.58 | 0.0143 | 0.07 | |
| hsa-miR-135a-5p | –0.79 | 0.0143 | 0.18 | |
| HRH1 | hsa-miR-502-3p | 1.57 | 0.025 | 0.26 |
| hsa-miR-423-5p | 0.59 | 0.025 | 0.16 | |
| hsa-miR-744-5p | –0.62 | 0.025 | 0.14 | |
| FZD4 | hsa-miR-203a-3p | –0.90 | 0.0143 | 0.16 |
| ACKR3 | hsa-miR-135b-3p | 0.67 | 0.0253 | 0.11 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | U2OS (epithelial, osteosarcoma) | ATCC | HTB-96 RRID:CVCL_0042 | |
| Antibody | Rabbit polyclonal anti-Flotillin1 | Abcam | Ab41927 RRID:AB_941621 | 1:1000 |
| Antibody | Rabbit monoclonal anti-Syntenin | Abcam | Ab133267 RRID:AB_11160262 | 1:1000 |
| Antibody | Mouse monoclonal anti-CD63 | Santa Cruz Biotechnology | sc-5275 RRID:AB_627877 | 1:1000 |
| Antibody | Mouse monoclonal anti-CD81 | Santa Cruz Biotechnology | sc-166029 RRID:AB_2275892 | 1:500 |
| Antibody | Rabbit polyclonal anti-CD9 | Abcam | ab223052 RRID:AB_2922392 | 1:1000 |
| Antibody | Rabbit polyclonal anti-calnexin | Abcam | ab22595 RRID:AB_2069006 | 1:1000 |
| Antibody | Rabbit monoclonal anti-phospho-ERK1/2 (pERK1/2) | Millipore | 05-797R RRID:AB_1587016 | 1:1000 |
| Antibody | Rabbit polyclonal anti-p44/42 MAPK (total ERK1/2) | Cell Signaling Technology | 9102 RRID:AB_330744 | 1:1000 |
| Antibody | Mouse monoclonal anti-β-actin | Santa Cruz Biotechnology | sc-69879 RRID:AB_1119529 | 1:1000 |
| Sequence-based reagent | Megaplex RT primer human pool set v3.0 | Thermo Fisher Scientific | 4444750 | |
| Sequence-based reagent | Megaplex PreAmp primers human pool set v3.0 | Thermo Fisher Scientific | 4444748 | |
| Sequence-based reagent | TaqMan Array Human MicroRNA A+B Card Set v3.0 | Thermo Fisher Scientific | 4444913 | |
| Peptide, recombinant protein | Human recombinant SDF-1α | MilliporeSigma | GF344 | |
| Peptide, recombinant protein | Human recombinant Wnt3a | MilliporeSigma | H17001 | |
| Commercial assay or kit | cAMP EIA kit | Cayman Chemical | 581001 RRID:AB_3095671 | |
| Commercial assay or kit | ALP assay kit | Abcam | ab83369 | |
| Commercial assay or kit | Cisbio IP-1 Elisa kit | Revvity | 72IP1PEA RRID:AB_2904131 | |
| Commercial assay or kit | MagMax mirVana total RNA isolation kit | Applied Biosystems, Thermo Fisher Scientific | A27828 | |
| Commercial assay or kit | Qubit miRNA assay kit | Thermo Fisher Scientific | Q32880 | |
| Chemical compound, drug | Di8-ANEPPS | Thermo Fisher Scientific | D3167 | |
| Chemical compound, drug | Fluronic F-127 | Thermo Fisher Scientific | P3000MP | |
| Chemical compound, drug | 2-Chloro-N6-cyclopentyladenosine (CCPA) | Tocris Bioscience | 1705 CAS:37739-05-2 | |
| Chemical compound, drug | 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) | Tocris Bioscience | 0439 CAS:102146-07-6 | |
| Chemical compound, drug | 2-Pyridylethylamine dihydrochloride | Tocris Bioscience | 2478 CAS:3343-39-3 | |
| Chemical compound, drug | Cetirizine dihydrochloride | Tocris Bioscience | 2577 CAS:83881-52-1 | |
| Software, algorithm | GraphPad Prism software version 10 | GraphPad Software Inc | RRID:SCR_002798 | |
| Software, algorithm | String (v12.0) | http://string.embl.de/ | RRID:SCR_005223 | |
| Software, algorithm | Gene Ontology | http://www.geneontology.org/ | RRID:SCR_002811 | |
| Software, algorithm | SR plot web service | https://www.bioinformatics.com.cn/srplot | RRID:SCR_025904 | |
| Software, algorithm | miRNet online web service v2.0 | http://www.mirnet.ca | RRID:SCR_024567 | |
| Software, algorithm | CytoScape software (v3.10.1) | https://cytoscape.org | RRID:SCR_003032 |
Additional files
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Supplementary file 1
The analysis of G protein-coupled receptor (GPCR) gene expression across various cell lines, as sourced from the Human Protein Atlas.
- https://cdn.elifesciences.org/articles/107865/elife-107865-supp1-v1.docx
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Supplementary file 2
microRNAs (miRNAs) detected in media control.
- https://cdn.elifesciences.org/articles/107865/elife-107865-supp2-v1.docx
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Supplementary file 3
Differentially expressed microRNAs (miRNAs) in the isolated extracellular vesicles (EVs) following G protein-coupled receptor (GPCR) activation.
All differentially expressed miRNAs (meeting p<0.2) after stimulation were listed.
- https://cdn.elifesciences.org/articles/107865/elife-107865-supp3-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107865/elife-107865-mdarchecklist1-v1.docx
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Specific GPCRs elicit unique extracellular vesicle miRNA array signatures
eLife 14:RP107865.
https://doi.org/10.7554/eLife.107865.3
