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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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Overexpression of CK1δ impairs and inhibition of CK1δ by D4476 increases formation of HIF-1α/ARNT complexes.(a) HeLa cells were co-transfected with pcDNA3.1 or pcDNA3.1-CK1δ and pEGFP plasmids. Twenty hours post-transfection cells were incubated under hypoxia (1% O2) for 4 h and HIF-1α/ARNT complexes were detected by in situ PLA. (b) Detection of HIF-1α/ARNT complexes, in the absence or presence of D4476 (10 μΜ) under hypoxia (1% O2) for 4 h, by in situ PLA. For (a) and (b): left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). (c) Determination of HIF-1 transcriptional activity in HeLa cells incubated for 16 h under normoxia or hypoxia (1% O2) in the absence or presence of D4476 (10 μΜ). Results are shown as fold increase in relation to the corresponding normoxic conditions and represent the mean of three independent experiments performed in triplicate ± SEM. (d) Western blot analysis of HeLa cells incubated for 4 h under normoxia or hypoxia (1% O2) in the absence or presence of D4476 (10 μΜ), for detection of HIF-1α and ARNT protein levels. (e) Histograms show the HIF-1α/actin (left) or ARNT/actin (right) protein levels ratio according to quantification of blots from three independent experiments performed as in (d).
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f0010: Overexpression of CK1δ impairs and inhibition of CK1δ by D4476 increases formation of HIF-1α/ARNT complexes.(a) HeLa cells were co-transfected with pcDNA3.1 or pcDNA3.1-CK1δ and pEGFP plasmids. Twenty hours post-transfection cells were incubated under hypoxia (1% O2) for 4 h and HIF-1α/ARNT complexes were detected by in situ PLA. (b) Detection of HIF-1α/ARNT complexes, in the absence or presence of D4476 (10 μΜ) under hypoxia (1% O2) for 4 h, by in situ PLA. For (a) and (b): left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). (c) Determination of HIF-1 transcriptional activity in HeLa cells incubated for 16 h under normoxia or hypoxia (1% O2) in the absence or presence of D4476 (10 μΜ). Results are shown as fold increase in relation to the corresponding normoxic conditions and represent the mean of three independent experiments performed in triplicate ± SEM. (d) Western blot analysis of HeLa cells incubated for 4 h under normoxia or hypoxia (1% O2) in the absence or presence of D4476 (10 μΜ), for detection of HIF-1α and ARNT protein levels. (e) Histograms show the HIF-1α/actin (left) or ARNT/actin (right) protein levels ratio according to quantification of blots from three independent experiments performed as in (d).

Mentions: Having established the suitability of in situ PLA for generating quantifiable data in a cell-based system, we investigated the effect of CK1δ on the formation of HIF-1 complexes. HeLa cells were co-transfected with a CK1δ overexpressing plasmid (pcDNA3.1-CK1δ) or the corresponding empty vector (pcDNA3.1) and a GFP expressing plasmid (pEGFP; to detect transfected cells) and incubated under hypoxic conditions (Supplementary Fig. 1a). Visualization and quantification of the PLA signals show a significant decrease in the number of HIF-1α/ARNT complexes specifically in cells that overexpress CK1δ compared to control cells that are transfected with the empty vector (Fig. 2a) Therefore, CK1δ does indeed inhibit HIF-1 heterodimerization in intact cells in accordance with our previous in vitro experiments [19].


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)

Overexpression of CK1δ impairs and inhibition of CK1δ by D4476 increases formation of HIF-1α/ARNT complexes.(a) HeLa cells were co-transfected with pcDNA3.1 or pcDNA3.1-CK1δ and pEGFP plasmids. Twenty hours post-transfection cells were incubated under hypoxia (1% O2) for 4 h and HIF-1α/ARNT complexes were detected by in situ PLA. (b) Detection of HIF-1α/ARNT complexes, in the absence or presence of D4476 (10 μΜ) under hypoxia (1% O2) for 4 h, by in situ PLA. For (a) and (b): left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). (c) Determination of HIF-1 transcriptional activity in HeLa cells incubated for 16 h under normoxia or hypoxia (1% O2) in the absence or presence of D4476 (10 μΜ). Results are shown as fold increase in relation to the corresponding normoxic conditions and represent the mean of three independent experiments performed in triplicate ± SEM. (d) Western blot analysis of HeLa cells incubated for 4 h under normoxia or hypoxia (1% O2) in the absence or presence of D4476 (10 μΜ), for detection of HIF-1α and ARNT protein levels. (e) Histograms show the HIF-1α/actin (left) or ARNT/actin (right) protein levels ratio according to quantification of blots from three independent experiments performed as in (d).
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f0010: Overexpression of CK1δ impairs and inhibition of CK1δ by D4476 increases formation of HIF-1α/ARNT complexes.(a) HeLa cells were co-transfected with pcDNA3.1 or pcDNA3.1-CK1δ and pEGFP plasmids. Twenty hours post-transfection cells were incubated under hypoxia (1% O2) for 4 h and HIF-1α/ARNT complexes were detected by in situ PLA. (b) Detection of HIF-1α/ARNT complexes, in the absence or presence of D4476 (10 μΜ) under hypoxia (1% O2) for 4 h, by in situ PLA. For (a) and (b): left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). (c) Determination of HIF-1 transcriptional activity in HeLa cells incubated for 16 h under normoxia or hypoxia (1% O2) in the absence or presence of D4476 (10 μΜ). Results are shown as fold increase in relation to the corresponding normoxic conditions and represent the mean of three independent experiments performed in triplicate ± SEM. (d) Western blot analysis of HeLa cells incubated for 4 h under normoxia or hypoxia (1% O2) in the absence or presence of D4476 (10 μΜ), for detection of HIF-1α and ARNT protein levels. (e) Histograms show the HIF-1α/actin (left) or ARNT/actin (right) protein levels ratio according to quantification of blots from three independent experiments performed as in (d).
Mentions: Having established the suitability of in situ PLA for generating quantifiable data in a cell-based system, we investigated the effect of CK1δ on the formation of HIF-1 complexes. HeLa cells were co-transfected with a CK1δ overexpressing plasmid (pcDNA3.1-CK1δ) or the corresponding empty vector (pcDNA3.1) and a GFP expressing plasmid (pEGFP; to detect transfected cells) and incubated under hypoxic conditions (Supplementary Fig. 1a). Visualization and quantification of the PLA signals show a significant decrease in the number of HIF-1α/ARNT complexes specifically in cells that overexpress CK1δ compared to control cells that are transfected with the empty vector (Fig. 2a) Therefore, CK1δ does indeed inhibit HIF-1 heterodimerization in intact cells in accordance with our previous in vitro experiments [19].

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