Chemotactic G protein-coupled receptors control cell migration by repressing autophagosome biogenesis

<p>Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in physiological processes such as organogenesis and angiogenesis, as well as in pathological processes such as tumor metastasis. The majority of chemotactic stimuli activate cell surface receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCRs remains largely unexplored. We found that ligand-induced activation of 2 chemotactic GPCRs, the chemokine receptor CXCR4 and the urotensin 2 receptor UTS2R, triggers a marked reduction in the biogenesis of autophagosomes, in both HEK-293 and U87 glioblastoma cells. Chemotactic GPCRs exert their anti-autophagic effects through the activation of CAPNs, which prevent the formation of pre-autophagosomal vesicles from the plasma membrane. We further demonstrated that CXCR4- or UTS2R-induced inhibition of autophagy favors the formation of adhesion complexes to the extracellular matrix and is required for chemotactic migration. Altogether, our data reveal a new link between GPCR signaling and the autophagy machinery, and may help to envisage therapeutic strategies in pathological processes such as cancer cell invasion.</p

P2X7 purinoceptor involvement in the dystrophic pathology.

<p>Absence of dystrophin and resulting loss of the DAP complex lead to myofiber damage. Degenerating/dying muscle releases large quantities of DAMPs, including ATP, which trigger chronic inflammation. P2RX7 activation on dystrophic myofibers exacerbates injury by promoting intracellular Ca²⁺ build-up and autophagic cell death. Infiltrating macrophages (Mφ), T-cells, and granulocytes (GrC) cause further myofiber damage, while chronically elevated levels of inflammatory mediators disturb normal brain and bone functions. Chronic inflammation also reduces repair by altering satellite cell (SC) activation and muscle precursor cell differentiation, while high eATP levels combined with P2RX7 overexpression contribute to their death and thus reduce muscle regeneration further still.</p

<i>P2RX7</i> ablation reduces inflammation and fibrosis in 20-mo-old tibialis anterior muscles.

<p>(A) Representative immunofluorescence micrographs (left) and enumeration of CD11b-expressing cells in 20-mo-old TA showing no significant difference in the numbers of infiltrating leukocytes in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> muscles (<i>t</i>-test, <i>t</i> = 1.82, df = 8, <i>p</i> = 0.107). (B) qPCR gene expression analyses: relative expression levels (2^−ΔΔCT) in muscle-derived mRNAs from <i>mdx</i> and <i>mdx</i>/P2X7^−/− TA demonstrate significant decreases in P2RX4 (<i>t</i>-test, <i>t</i> = 4.04, df = 17, <i>p</i> = 0.001) and TNFα (<i>t</i>-test, <i>t</i> = 3.07, df = 18, <i>p</i> = 0.006), with concomitant increases in expression levels of IL12α (<i>t</i>-test, <i>t</i> = 2.56, df = 18, <i>p</i> = 0.020) and Foxp3 (<i>t</i>-test, <i>t</i> = 6.8, df = 17, <i>p <</i> 0.001) in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> muscles. (C) Representative images of trichrome staining (left) and trichrome average intensities in 20-mo-old TA muscles demonstrating a significant decrease in fibrosis in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> mice (ANOVA, <i>F</i> = 6.18, df = 2, <i>n</i> = 3, 5, 4, <i>p</i> = 0.020; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.038). *<i>p <</i> 0.05, **<i>p <</i> 0.005, ***<i>p <</i> 0.001.</p

Generation and characterization of <i>mdx</i>/P2X7^−/− mice.

<p>(A) Schematics of mouse breeding. (B) Representative Western blots showing increased expression of P2RX7 in 4-wk-old <i>mdx</i> compared to wild-type (WT) gastrocnemius and its absence in <i>mdx</i>/P2X7^−/−. Use of separate Western blots is indicated by solid black lines. (C) Micrographs of P2RX7 immunofluorescence localization (green signal) in 4-wk-old tibialis anterior (TA) muscle from WT, <i>mdx</i>, and Pf-<i>mdx</i>/P2X7^−/− mice showing expression in areas rich with infiltrating cells, and negative control using no primary antibody and with a blue signal denoting nuclear counterstaining.</p

Expression of genes associated with fibrosis as measured by RNA-Seq.

<p>Fragments per kilobase per million fragments mapped (FPKM) values for each sample were obtained using Cuffnorm (Tuxedo suite), log-transformed, and normalized to zero mean and unit standard deviation (rows with missing expression values were removed). Hierarchical clustering was performed using the Ward’s method [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1001888#pmed.1001888.ref042" target="_blank">42</a>]. KO, knockout.</p

Short-term P2RX7 antagonist administration reduces severity of muscle pathology.

<p>(A) Comparison of serum CK levels in <i>mdx</i> mice (control) and <i>mdx</i> mice injected with the P2RX7 antagonist CBB. Note the high variability of CK levels in the dystrophic sera. Daily administration of CBB over the 4-wk period reduced CK levels (<i>t-</i>test, <i>t</i> = 2.3, df = 26, <i>p</i> = 0.030), in line with the effects of P2RX7 ablation in 4-wk-old Pf-<i>mdx</i>/P2X7^−/− mice. (B) Representative Western blots (left) and average value plots (right) demonstrating significantly decreased levels of the CD11b leukocyte marker in gastrocnemius muscles of <i>mdx</i> mice injected with ox-ATP compared to <i>mdx</i> saline-injected controls (<i>t-</i>test, <i>t</i> = 2.84, df = 4, <i>p</i> = 0.047). Use of separate Western blots is indicated by solid black lines. (C) Comparisons of serum CK levels (left) and F4/80⁺ macrophage loads in <i>mdx</i> mice showing significant decreases (CK <i>t-</i>test, <i>t</i> = 2.26, df = 9, <i>p</i> = 0.050; F4/80 <i>t-</i>test, <i>t</i> = 4.34, df = 4, <i>n</i> = 3, <i>p</i> = 0.012) following 14 daily administrations of the competitive P2RX7 antagonist A-438079. *<i>p <</i> 0.05.</p

<i>P2RX7</i> ablation improves <i>mdx</i> mouse muscles.

<p>(A) Autophagy induction (LC3I to LC3II shift in representative Western blots) found in <i>mdx</i> muscles is blocked in Pf-<i>mdx</i>/P2X7^−/− muscles, with average values shown in (B) (ANOVA, <i>F</i> = 11.57, df = 2, <i>n</i> = 4, <i>p</i> = 0.003; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.008). Note: use of separate Western blots is indicated by solid black lines. (C) Greater average diaphragm isometric tetanic forces at 4 mo in both Pf-<i>mdx</i>/P2X7^−/− and G-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> mice (ANOVA, <i>F</i> = 37.97, df = 2, <i>n</i> = 4, 4, 5, <i>p <</i> 0.001; Tukey’s test, G-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.010; Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p <</i> 0.001). *<i>p <</i> 0.05, ***<i>p <</i> 0.001.</p

<i>P2RX7</i> ablation continues to reduce dystrophic pathology in 20-mo-old diaphragm and heart.

<p>(A) Representative immunofluorescence micrographs (left) and enumeration of CD11b- and CD68-expressing cells in 20-mo-old diaphragms showing reduced numbers of infiltrating leukocytes (CD11b <i>t-</i>test, <i>t</i> = 3.68, df = 6, <i>p</i> = 0.015) and macrophages (CD68⁺<i>t</i>-test, <i>t</i> = 4.73, df = 4, <i>p</i> = 0.009) in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> diaphragms. (B) Trichrome staining (left) and its average intensity in 20-mo-old diaphragms demonstrating no increase in fibrosis in Pf-<i>mdx</i>/P2X7^−/− over <i>mdx</i> mice (ANOVA, <i>F</i> = 60.32, df = 2, <i>n</i> = 5, 5, 3, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.095; permutation analysis, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>F</i> = 4.47, <i>p</i> = 0.095). (C) Representative ImageJ output masks from morphometric analyses of diaphragm fibers (left) demonstrating the increased average minimum Feret diameter in <i>mdx</i>/P2X7^−/− compared to <i>mdx</i> diaphragms (ANOVA, <i>F</i> = 75.17, df = 2, <i>n</i> = 3, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.006; permutation analysis, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>F</i> = 20.01, <i>p</i> = 0.099). (D) Graphs showing a lower total number of diaphragm fibers per unit area in Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i> mice (left; ANOVA, <i>F</i> = 52.77, df = 2, <i>n</i> = 3, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.001; Pf-<i>mdx</i>/P2X7^−/− versus WT, <i>p</i> = 0.051) and the increased proportion of C/N fibers (right; <i>t-</i>test, <i>t</i> = 5, df = 4, <i>p</i> = 0.008). (E) Dystrophin immunofluorescence in representative transverse sections of 20-mo-old diaphragms showing the typical staining (green signal) in dystrophin-positive muscles and clusters of revertant dystrophin-positive fibers in dystrophic samples. The data show significantly fewer revertant fibers in 20-mo-old Pf-<i>mdx</i>/P2X7^−/− than in <i>mdx</i> diaphragms (<i>t-</i>test, <i>t</i> = 5.12, df = 4, <i>p</i> = 0.007). (F) Representative trichrome staining (left) of whole hearts from 20-mo-old mice showing a significant decrease in cardiac muscle damage (histological lesions) and average area of fibrosis (blue signal in trichrome staining) in Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i> mice (ANOVA, <i>F</i> = 166.29, df = 2, <i>n</i> = 4, 3, 3, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p <</i> 0.001). (G) Representative examples of CD11b⁺ leukocyte marker staining (left; red immunofluorescence) and infiltrating cell counts demonstrating fewer infiltrations in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> hearts (ANOVA, <i>F</i> = 19.65, df = 2, <i>n</i> = 3, <i>p</i> = 0.002; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.005). *<i>p <</i> 0.05, **<i>p <</i> 0.005, ***<i>p <</i> 0.001.</p

P2RX7 ablation reduces <i>mdx</i> mouse muscle pathology.

<p>The color-coding legend applies to all graphs in the figure. (A) Collagen type-IV (green) and nuclei (blue) immunofluorescence with an accompanying frequency histogram (B) of the minimum Feret diameter of C/N fibers from 4-wk-old <i>mdx</i> and Pf-<i>mdx</i>/P2X7^−/− mice showing the right shift in TA muscle fiber size corresponding with the greater average Feret diameter of Pf-<i>mdx</i>/P2X7^−/− fibers (<i>t</i>-test, <i>t</i> = 6.99, df = 6, <i>p</i> < 0.001). (C) Elevated myogenin levels (average Western blot values) in both Pf-<i>mdx</i>/P2X7^−/− and G-<i>mdx</i>/P2X7^−/− muscle (ANOVA, <i>F</i> = 33.38, df = 2, <i>n</i> = 4, 3, 4, <i>p <</i> 0.001; Tukey’s test, G-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> < 0.001; G-<i>mdx</i>/P2X7^−/− versus Pf-<i>mdx</i>/P2X7^−/−, <i>p</i> = 0.516; Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> < 0.001) and (D) significantly lower average serum CK levels in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> muscle (Mann-Whitney U test, <i>W</i> = 388, <i>n</i> = 18, 16, <i>p</i> = 0.012; permutation analysis, <i>F</i> = 7.07, <i>p</i> = 0.013; log₁₀ serum CK ANOVA, <i>F</i> = 3.76, df = 2, <i>n</i> = 19, 18, 16, <i>p</i> = 0.030; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.025). (E) Example immunofluorescence micrographs of IgG penetration into muscle fibers and (F) chart showing reduced average IgG influx into Pf-<i>mdx</i>/P2X7^−/− muscle (ANOVA, <i>F</i> = 5.52, df = 2, <i>n</i> = 3, 5, 3, <i>p</i> = 0.031; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.032). *<i>p <</i> 0.05, ***<i>p <</i> 0.001.</p

<i>P2RX7</i> ablation improves <i>mdx</i> muscle strength and endurance and object recognition memory and decreases anxiety in vivo.

<p>Forelimb grip strength (A) and voluntary wheel run distance (B) were significantly greater in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> mice (grip strength ANOVA, <i>F</i> = 22.99, df = 2, <i>n</i> = 9, 9, 10, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.0118; run distance ANOVA, <i>F</i> = 12.73, df = 2, <i>n</i> = 9, 9, 10, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.016). The rotarod test (C) showed no difference for total average run time and average speed (run time ANOVA, <i>F</i> = 1.23, df = 2, <i>n</i> = 9, 9, 10, <i>p</i> = 0.310; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.300; speed ANOVA, <i>F</i> = 2.23, df = 2, <i>n</i> = 9, 9, 10, <i>p</i> = 0.129; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.109), and the parallel rod floor test (D) showed no significant differences in the average number of activations over several run time-spans between WT, <i>mdx</i>, and Pf-<i>mdx</i>/P2X7^−/− mice (ANOVA, <i>F</i> = 2.6, 0.62, 1.34; df = 2; <i>n</i> = 9, 9, 10; <i>p</i> = 0.094, 0.545, 0.212; at 0–5, 5–10, and 0–10 min, respectively). In the object recognition test (E) there was no significant difference between genotypes at 10-min retention delay, but at 24 h and 48 h, both duration and contact discrimination were significantly different from the 50% chance level for the Pf-<i>mdx</i>/P2X7^−/− mice, but not for <i>mdx</i> mice. Memory retention in Pf-<i>mdx</i>/P2X7^−/− mice was equal to that in WT mice, while <i>mdx</i> mice performed at a lower level than WT (contact discrimination 10 min, 24 h, 48 h ANOVA; <i>F</i> = 0.08, 7.37, 6.83; df = 2; <i>n</i> = 10, 9, 10; <i>p</i> = 0.922, 0.003, 0.004; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.984, 0.004, 0.006; Pf-<i>mdx</i>/P2X7^−/− versus WT, <i>p</i> = 0.970, 0.908, 0.890; <i>mdx</i> versus WT, <i>p</i> = 0.916, 0.012, 0.017; duration discrimination 10 min, 24 h, 48 h ANOVA; <i>F</i> = 0.09, 16.17, 5.1; df = 2; <i>n</i> = 10, 9, 10; <i>p</i> = 0.913, < 0.001, = 0.013; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.905, < 0.001, = 0.031; Pf-<i>mdx</i>/P2X7^−/− versus WT, <i>p</i> = 0.969, 0.994, 0.985; <i>mdx</i> versus WT, <i>p</i> = 0.980, < 0.001, = 0.021,). (F) In the elevated zero maze anxiety test, both the duration and the distance travelled by Pf-<i>mdx</i>/P2X7^−/− mice within the open arm of the maze were greater than those of <i>mdx</i> mice, and <i>mdx</i> mice performed at a lower level than WT (distance and duration ANOVA, <i>F</i> = 10.51, 11.76; df = 2; <i>n</i> = 10, 9, 10; <i>p <</i> 0.001, < 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.045, 0.047; Pf-<i>mdx</i>/P2X7^−/− versus WT, <i>p</i> = 0.109, 0.060; <i>mdx</i> versus WT, <i>p</i> < 0.001, < 0.001). In the closed arm (F, bottom), there was no difference in distance travelled, but Pf-<i>mdx</i>/P2X7^−/− mice spent less time in this arm than <i>mdx</i> mice, and <i>mdx</i> mice more time than WT (distance and duration ANOVA, <i>F</i> = 1.42, 11.76; df = 2; <i>n</i> = 10, 9, 10; <i>p</i> = 0.259, < 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.687, 0.047; Pf-<i>mdx</i>/P2X7^−/− versus WT, <i>p</i> = 0.659, 0.060; <i>mdx</i> versus WT, <i>p</i> = 0.230, < 0.001). *<i>p <</i> 0.05, **<i>p <</i> 0.005, ***<i>p <</i> 0.001.</p