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CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation.

Kourti M, Ikonomou G, Giakoumakis NN, Rapsomaniki MA, Landegren U, Siniossoglou S, Lygerou Z, Simos G, Mylonis I - Cell. Signal. (2015)

Bottom Line: We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT.This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation.These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, Larissa, Greece.

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HIF-1α/ARNT complexes can be specifically detected and quantified by in situ PLA in HeLa cells.(a) Cells were incubated at normoxia or hypoxia (1% O2) for 4 h and processed for the detection of HIF-1α/ARNT interaction using simultaneous incubation with both primary anti-HIF-1 and anti-ARNT antibodies and the in situ PLA method (panels i and ii). Treatment of cells with a single primary antibody (panels iii–vi) or no primary antibodies (panels vii and viii) was used as negative controls. Panels i–viii show microscopic images of the PLA signal as nuclear dots while the corresponding panels i′–viii′ show the cell nuclei stained with DAPI. (b) Detection of HIF-1α/ARNT complexes by in situ PLA in HeLa cells incubated under hypoxia (1% Ο2) for 4 h in the absence or presence of kaempferol (50–100 μΜ). (c) Twenty hours post-transfection, HeLa cells expressing GFP or GFP-HIF-1α 1-347 were incubated for 4 h in hypoxia (1% O2) and HIF-1α/ARNT complexes were detected by in situ PLA. Arrows point to transfected cells.Left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). Inset in (c): immunobloting analysis of cell lysates with an anti-GFP antibody to show expression of GFP alone or GFP-HIF-1α 1-347.
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f0005: HIF-1α/ARNT complexes can be specifically detected and quantified by in situ PLA in HeLa cells.(a) Cells were incubated at normoxia or hypoxia (1% O2) for 4 h and processed for the detection of HIF-1α/ARNT interaction using simultaneous incubation with both primary anti-HIF-1 and anti-ARNT antibodies and the in situ PLA method (panels i and ii). Treatment of cells with a single primary antibody (panels iii–vi) or no primary antibodies (panels vii and viii) was used as negative controls. Panels i–viii show microscopic images of the PLA signal as nuclear dots while the corresponding panels i′–viii′ show the cell nuclei stained with DAPI. (b) Detection of HIF-1α/ARNT complexes by in situ PLA in HeLa cells incubated under hypoxia (1% Ο2) for 4 h in the absence or presence of kaempferol (50–100 μΜ). (c) Twenty hours post-transfection, HeLa cells expressing GFP or GFP-HIF-1α 1-347 were incubated for 4 h in hypoxia (1% O2) and HIF-1α/ARNT complexes were detected by in situ PLA. Arrows point to transfected cells.Left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). Inset in (c): immunobloting analysis of cell lysates with an anti-GFP antibody to show expression of GFP alone or GFP-HIF-1α 1-347.

Mentions: In order to study the regulation of complex formation of endogenous HIF-1 in intact cells, we applied the in situ proximity ligation assay (PLA). Using this method, the HIF-1α/ARNT interaction was monitored in HeLa cells that were incubated under normoxic or hypoxic conditions. Following treatment with both anti-HIF-1α and anti-ARNT primary antibodies and analysis by in situ PLA, a very weak signal could be detected in cells incubated under normoxia while, in contrast, the signal was drastically amplified in cells grown under hypoxic conditions (Fig. 1a, panels i and ii and chart). The detected signals were specific for the HIF-1α/ARNT complex as no signal was obtained when one or both of the primary antibodies were omitted (Fig. 1a, panels iii–viii). We, therefore, concluded that in situ PLA could be used for specific detection of HIF-1α/ARNT heterodimerization in intact cells.


CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation.

Kourti M, Ikonomou G, Giakoumakis NN, Rapsomaniki MA, Landegren U, Siniossoglou S, Lygerou Z, Simos G, Mylonis I - Cell. Signal. (2015)

HIF-1α/ARNT complexes can be specifically detected and quantified by in situ PLA in HeLa cells.(a) Cells were incubated at normoxia or hypoxia (1% O2) for 4 h and processed for the detection of HIF-1α/ARNT interaction using simultaneous incubation with both primary anti-HIF-1 and anti-ARNT antibodies and the in situ PLA method (panels i and ii). Treatment of cells with a single primary antibody (panels iii–vi) or no primary antibodies (panels vii and viii) was used as negative controls. Panels i–viii show microscopic images of the PLA signal as nuclear dots while the corresponding panels i′–viii′ show the cell nuclei stained with DAPI. (b) Detection of HIF-1α/ARNT complexes by in situ PLA in HeLa cells incubated under hypoxia (1% Ο2) for 4 h in the absence or presence of kaempferol (50–100 μΜ). (c) Twenty hours post-transfection, HeLa cells expressing GFP or GFP-HIF-1α 1-347 were incubated for 4 h in hypoxia (1% O2) and HIF-1α/ARNT complexes were detected by in situ PLA. Arrows point to transfected cells.Left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). Inset in (c): immunobloting analysis of cell lysates with an anti-GFP antibody to show expression of GFP alone or GFP-HIF-1α 1-347.
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f0005: HIF-1α/ARNT complexes can be specifically detected and quantified by in situ PLA in HeLa cells.(a) Cells were incubated at normoxia or hypoxia (1% O2) for 4 h and processed for the detection of HIF-1α/ARNT interaction using simultaneous incubation with both primary anti-HIF-1 and anti-ARNT antibodies and the in situ PLA method (panels i and ii). Treatment of cells with a single primary antibody (panels iii–vi) or no primary antibodies (panels vii and viii) was used as negative controls. Panels i–viii show microscopic images of the PLA signal as nuclear dots while the corresponding panels i′–viii′ show the cell nuclei stained with DAPI. (b) Detection of HIF-1α/ARNT complexes by in situ PLA in HeLa cells incubated under hypoxia (1% Ο2) for 4 h in the absence or presence of kaempferol (50–100 μΜ). (c) Twenty hours post-transfection, HeLa cells expressing GFP or GFP-HIF-1α 1-347 were incubated for 4 h in hypoxia (1% O2) and HIF-1α/ARNT complexes were detected by in situ PLA. Arrows point to transfected cells.Left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). Inset in (c): immunobloting analysis of cell lysates with an anti-GFP antibody to show expression of GFP alone or GFP-HIF-1α 1-347.
Mentions: In order to study the regulation of complex formation of endogenous HIF-1 in intact cells, we applied the in situ proximity ligation assay (PLA). Using this method, the HIF-1α/ARNT interaction was monitored in HeLa cells that were incubated under normoxic or hypoxic conditions. Following treatment with both anti-HIF-1α and anti-ARNT primary antibodies and analysis by in situ PLA, a very weak signal could be detected in cells incubated under normoxia while, in contrast, the signal was drastically amplified in cells grown under hypoxic conditions (Fig. 1a, panels i and ii and chart). The detected signals were specific for the HIF-1α/ARNT complex as no signal was obtained when one or both of the primary antibodies were omitted (Fig. 1a, panels iii–viii). We, therefore, concluded that in situ PLA could be used for specific detection of HIF-1α/ARNT heterodimerization in intact cells.

Bottom Line: We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT.This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation.These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, Larissa, Greece.

Show MeSH
Related in: MedlinePlus