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Normal autophagic activity in macrophages from mice lacking Gαi3, AGS3, or RGS19.

Vural A, McQuiston TJ, Blumer JB, Park C, Hwang IY, Williams-Bey Y, Shi CS, Ma DZ, Kehrl JH - PLoS ONE (2013)

Bottom Line: In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination.As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy.These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

View Article: PubMed Central - PubMed

Affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-), Gpsm1(-/-), or Rgs19(-/-) mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-), and Gpsm1(-/-) macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

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The lack of Gαi3 or PTX treatment does not affect autophagy induction in primary mouse macrophages.(A) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3-/- mice; or wild type mice pre-exposed to PTX (200 ng/ml for 2h), or not, to assess the steady state levels of Gαi3, Gαi2, p62 and ubiquitin proteins. (B) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3-/- mice; or wild type mice pre-exposed to PTX as above, or not, either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios. (C) Endogenous LC3 immunocytochemistry of BMDM from wild type mice, Gnai3-/- mice, or from wild type mice pre-exposed to PTX (200 ng/ml for 2h) either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h). Representative images are shown for each condition. (D) Quantification of endogenous LC3 dots performed by fluorescence microscopy for at least 70-100 cells from experiment shown in part C. Data represents the mean LC3 puncta per cytosol ± SEM for three independent experiments for each condition. (E) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7-/- mice to assess the steady state levels of p62 and ubiquitin proteins. (F) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7-/- deficient mice, either not further manipulated, or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios.
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pone-0081886-g001: The lack of Gαi3 or PTX treatment does not affect autophagy induction in primary mouse macrophages.(A) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3-/- mice; or wild type mice pre-exposed to PTX (200 ng/ml for 2h), or not, to assess the steady state levels of Gαi3, Gαi2, p62 and ubiquitin proteins. (B) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3-/- mice; or wild type mice pre-exposed to PTX as above, or not, either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios. (C) Endogenous LC3 immunocytochemistry of BMDM from wild type mice, Gnai3-/- mice, or from wild type mice pre-exposed to PTX (200 ng/ml for 2h) either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h). Representative images are shown for each condition. (D) Quantification of endogenous LC3 dots performed by fluorescence microscopy for at least 70-100 cells from experiment shown in part C. Data represents the mean LC3 puncta per cytosol ± SEM for three independent experiments for each condition. (E) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7-/- mice to assess the steady state levels of p62 and ubiquitin proteins. (F) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7-/- deficient mice, either not further manipulated, or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios.

Mentions: We first verified that bone marrow derived macrophages (BMDM) from the Gnai3-/- mice lacked Gαi3 and assessed the impact of the loss on the expression of Gαi2 as well as the effect of PTX treatment on the expression of both isoforms of Gαi. Like other hematopoietic cells macrophages express little or no Gαi1. As expected the Gnai3-/- macrophages lacked Gαi3 and Gαi2 levels were not appreciably altered (Figure 1A). PTX treatment resulted in a modest increase in Gαi3, but not Gαi2 (Figure 1A). To check basal autophagy levels in BMDM we measured steady-state levels of autophagy marker protein SQSTM1/p62 and of ubiquitinated proteins since they increase following the suppression of basal autophagy [20]. We found similar levels of p62 expression and of ubiquitinated proteins in cell lysates prepared from wild type versus Gαi3-deficient BMDM; and in PTX treated versus control BMDM (Figure 1A). These results indicate that neither Gαi3 deficiency nor the inhibition of Gαi nucleotide exchange with PTX results in a significant difference in steady-state autophagy levels in primary mouse BMDM.


Normal autophagic activity in macrophages from mice lacking Gαi3, AGS3, or RGS19.

Vural A, McQuiston TJ, Blumer JB, Park C, Hwang IY, Williams-Bey Y, Shi CS, Ma DZ, Kehrl JH - PLoS ONE (2013)

The lack of Gαi3 or PTX treatment does not affect autophagy induction in primary mouse macrophages.(A) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3-/- mice; or wild type mice pre-exposed to PTX (200 ng/ml for 2h), or not, to assess the steady state levels of Gαi3, Gαi2, p62 and ubiquitin proteins. (B) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3-/- mice; or wild type mice pre-exposed to PTX as above, or not, either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios. (C) Endogenous LC3 immunocytochemistry of BMDM from wild type mice, Gnai3-/- mice, or from wild type mice pre-exposed to PTX (200 ng/ml for 2h) either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h). Representative images are shown for each condition. (D) Quantification of endogenous LC3 dots performed by fluorescence microscopy for at least 70-100 cells from experiment shown in part C. Data represents the mean LC3 puncta per cytosol ± SEM for three independent experiments for each condition. (E) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7-/- mice to assess the steady state levels of p62 and ubiquitin proteins. (F) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7-/- deficient mice, either not further manipulated, or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842979&req=5

pone-0081886-g001: The lack of Gαi3 or PTX treatment does not affect autophagy induction in primary mouse macrophages.(A) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3-/- mice; or wild type mice pre-exposed to PTX (200 ng/ml for 2h), or not, to assess the steady state levels of Gαi3, Gαi2, p62 and ubiquitin proteins. (B) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice; Gnai3-/- mice; or wild type mice pre-exposed to PTX as above, or not, either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios. (C) Endogenous LC3 immunocytochemistry of BMDM from wild type mice, Gnai3-/- mice, or from wild type mice pre-exposed to PTX (200 ng/ml for 2h) either not further manipulated, starved (HBSS for 1h with 100 nM Bafilomycin A1), or treated with nigericin (4 µM for 4h). Representative images are shown for each condition. (D) Quantification of endogenous LC3 dots performed by fluorescence microscopy for at least 70-100 cells from experiment shown in part C. Data represents the mean LC3 puncta per cytosol ± SEM for three independent experiments for each condition. (E) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7-/- mice to assess the steady state levels of p62 and ubiquitin proteins. (F) Immunoblot analysis of cell lysates from BMDM prepared from wild type mice and Atg7-/- deficient mice, either not further manipulated, or treated with nigericin (4 µM for 4h) to assess LC3-II/actin band ratios.
Mentions: We first verified that bone marrow derived macrophages (BMDM) from the Gnai3-/- mice lacked Gαi3 and assessed the impact of the loss on the expression of Gαi2 as well as the effect of PTX treatment on the expression of both isoforms of Gαi. Like other hematopoietic cells macrophages express little or no Gαi1. As expected the Gnai3-/- macrophages lacked Gαi3 and Gαi2 levels were not appreciably altered (Figure 1A). PTX treatment resulted in a modest increase in Gαi3, but not Gαi2 (Figure 1A). To check basal autophagy levels in BMDM we measured steady-state levels of autophagy marker protein SQSTM1/p62 and of ubiquitinated proteins since they increase following the suppression of basal autophagy [20]. We found similar levels of p62 expression and of ubiquitinated proteins in cell lysates prepared from wild type versus Gαi3-deficient BMDM; and in PTX treated versus control BMDM (Figure 1A). These results indicate that neither Gαi3 deficiency nor the inhibition of Gαi nucleotide exchange with PTX results in a significant difference in steady-state autophagy levels in primary mouse BMDM.

Bottom Line: In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination.As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy.These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

View Article: PubMed Central - PubMed

Affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-), Gpsm1(-/-), or Rgs19(-/-) mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-), and Gpsm1(-/-) macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

Show MeSH
Related in: MedlinePlus