Table 1.
Primers used to clone PDI C53, 397S, HIF-1alpha WT, HIF-1alpha K719T, Ero-1 WT, Ero-1 (24–469 aa), Ero-1 C104, 131S, Ero-1 C394, 397S, HIF-1alpha (1–245 aa), HIF-1alpha (245–826 aa), HIF-1alpha C90S (1–245 aa), HIF-1alpha C210S (1–245 aa), and HIF-1alpha C219S (1–245 aa).
Fig 1.
CA9 and Glut1 mRNA levels in PDI-overexpressing cells.
Hep3B cells overexpressing PDI were cultured for 6 h under hypoxic conditions. Total RNA was isolated from cells in three different plates, and the mRNA levels of CA9 or Glut1 were assessed by RT-PCR. The Glut1 mRNA levels of mock cells under normoxic conditions or the CA9 mRNA levels of mock cells under hypoxic conditions were set to 1.0. Proteins (3 μg) were separated by SDS-PAGE and immunoblotting was performed with an anti-PDI or -beta-actin antibody (1:1000 dilution). The PDI protein levels of mock cells under hypoxic conditions were set at 1.0. Values are expressed as the mean ± S.D. (error bars) of three different plates. N, Normoxia; H, Hypoxia. * p<0.05; ** p<0.01, significantly different from mock cells under hypoxic conditions. #, less than 0.05.
Fig 2.
HIF-1alpha protein levels in PDI-overexpressing cells.
(A-F) PDI was overexpressed in Hep3B cells (A and D), HEK293T cells (B and E), and HeLa cells (C and F), and cells were cultured for 6 h under hypoxic conditions. Proteins (3 or 15 μg) were separated by SDS-PAGE and immunoblotting was performed with an anti-PDI, -HIF-1alpha, and -beta-actin antibody (1:1000 dilution). (G) Hep3B cells, HEK293T cells, and HeLa cells were cultured for 6 h under hypoxic conditions. The expression of PDI was evaluated by immunoblotting with an anti-PDI antibody. (H) The PDI/3×FLAG-pcDNA4 vector (6 or 12 μg) was transfected into Hep3B cells, and cells were cultured for 6 h under hypoxic conditions. Proteins (3 μg) were separated by SDS-PAGE, and immunoblotting was performed with an anti-FLAG tag antibody (1:1000 dilution). (I) PDI/pcDNA 3.1 (+) and si-PDI were transfected into Hep3B cells, cells were cultured for 6 h under hypoxic conditions, and immunoblotting was performed. The PDI or HIF-1alpha protein levels of mock cells (A-F and H), or si-ctrl cells (I) under hypoxic conditions were set at 1.0. Values are expressed as the mean ± S.D. (error bars) of three different plates. N, Normoxia. * p<0.05; ** p<0.01, significantly different from mock or ctrl cells (A-G) or si-ctrl cells (I) under hypoxic conditions.
Fig 3.
HIF-1alpha protein levels in catalytic inactive mutant PDI-overexpressing cells.
(A) Hep3B cells overexpressing PDI WT or PDI C53, 397S were cultured for 6 h under hypoxic conditions, and the mRNA levels of CA9 or Glut1 were assessed by RT-PCR. (B) Hep3B cells overexpressing PDI WT or PDI C53, 397S were cultured for 6 h under hypoxic conditions and then subjected to immunoblotting. The overexpression of PDI WT or PDI C53, 397S in Hep3B cells was assessed by Western blotting with an anti-PDI antibody (right panel) (A and B). The HIF-1alpha or PDI protein levels of mock cells under hypoxic conditions were set to 1.0. Values are expressed as the mean ± S.D. (error bars) of three different plates. N, Normoxia; H, Hypoxia. * p<0.05; ** p<0.01, significantly different from mock cells under hypoxic conditions. #, less than 0.05.
Fig 4.
Effects of Ero-1 on HIF-1alpha expression.
(A) Hep3B cells overexpressing Ero-1 WT were cultured for 6 h under hypoxic conditions. Cells were harvested with PBS, and proteins were incubated in the absence or presence of 5 mM diamide or 10 mM DTT. Precipitated proteins were incubated with 20 mM NEM. After the removal of NEM, proteins were incubated with 1 mM DTT. Precipitated proteins were incubated with 1 mM 5kDa PEG-maleimide. An up-shift in molecular weight by the binding of PEG-maleimide was detected by immunoblotting with the anti-PDI antibody. To evaluation of the overexpression of Ero-1 (left panel), proteins (3 μg) were separated by SDS-PAGE, and immunoblotting was performed with the anti-Ero-1 antibody (1:1000 dilution). red, Reduced form. (B) Hep3B cells overexpressing Ero-1 WT or Ero-1 C104, 131S, and Ero-1 C394, 397S were cultured for 6 h under hypoxic conditions, and immunoblotting was performed. (C) Ero-1/pcDNA 3.1 (+) and si-PDI were transfected into Hep3B cells, cells were cultured for 6 h under hypoxic conditions, and immunoblotting was performed. The overexpression of Ero-1 or PDI was evaluated by immunoblotting with an anti-Ero-1 or PDI antibody (1:1000 dilution). The HIF-1alpha, Ero-1, or PDI protein levels of mock cells under hypoxic conditions were set to 1.0. Values are expressed as the mean ± S.D. (error bars) of three different plates. N, Normoxia; H, Hypoxia; n. s., not significant. * p<0.05; ** p<0.01, significantly different from mock cells under hypoxic conditions.
Fig 5.
Effects of PHD on HIF-1alpha expression.
(A) Hep3B cells overexpressing PDI were cultured under normoxic conditions, and total RNA was isolated from cells in three different plates. HIF-1alpha mRNA levels were measured by RT-PCR. The HIF-1alpha mRNA levels of mock cells were set to 1.0. (B and C) Hep3B cells overexpressing PDI were cultured for 6 h in the presence of 100 μM dipyridyl (B) or for 16 h in the presence of 0.5 mM DMOG (C) under normoxic conditions, and immunoblotting was performed. The HIF-1alpha protein levels of mock cells in the presence of dipyridyl (B) or DMOG (C) were set to 1.0. (D and E) PDI/pcDNA 3.1 (+) and si-PHD2 (D) or sh-Ref-1 (E) were transfected into Hep3B cells, cells were cultured for 6 h under hypoxic conditions (E). Proteins (3 or 15 μg) were separated by SDS-PAGE, and immunoblotting was performed with the anti-PHD2 or -Ref-1 antibody (1:1000 dilution). The HIF-1alpha protein levels of si-ctrl or sh-ctrl cells under hypoxic or normoxia conditions were set to 1.0. Values were expressed as the mean ± S.D. (error bars) of three different plates. N, Normoxia; ctrl, control; ##, non-specific band. * p<0.05; ** p<0.01, significantly different from mock cells in the presence of dipyridyl or DMOG or si-ctrl cells.
Fig 6.
Effects of HSC70 on HIF-1alpha regulation by PDI.
(A) Hep3B cells overexpressing PDI or HSC70 were cultured in the presence of 10 mM NH4Cl or 10 μM leupeptin for 8 h under hypoxic conditions. Proteins (3 μg) were separated by SDS-PAGE, and immunoblotting was performed with the anti-HSC70 antibody (1:1000 dilution). The overexpression of HSC70 was assessed by immunoblotting with the anti-HSC70 antibody (upper panel). The HIF-1alpha protein levels of mock cells in the absence of NH4Cl under hypoxic conditions were set to 1.0. (B) PDI/pcDNA 3.1 (+) and si-HSC70 or si-PDI and HSC70/pcDNA 3.1 (+) were transfected into Hep3B cells, cells were cultured for 6 h under hypoxic conditions, and immunoblotting was performed. The HIF-1alpha protein levels of si-ctrl cells were set to 1.0. (C) Hep3B cells overexpressing PDI were cultured for 6 h under hypoxic conditions and then subjected to immunoblotting. Values are expressed as the mean ± S.D. (error bars) of three different plates. N, Normoxia. * p<0.05, ** p<0.01, significantly different from mock cells (A and C) or si-ctrl cells (B) under hypoxic conditions. (D) Hep3B cells overexpressing PDI WT or PDI C53, 397S were cultured under hypoxic conditions for 8 h in the presence of NH4Cl. Cell extracts were subjected to immunoprecipitation (IP) using the anti-HIF-1alpha antibody (Ab). Precipitated proteins were separated by SDS-PAGE, and immunoblotting was performed with the anti-HSC70 or -HIF-1alpha Ab, respectively. Arrows indicate bands corresponding to HSC70 or HIF-1alpha. N, Normoxia; H, Hypoxia; ctrl, control.
Fig 7.
Interaction between PDI and HIF-1alpha.
(A and B) Hep3B cells were cultured under hypoxic conditions for 6 h. Cell extracts were subjected to immunoprecipitation using the anti-HIF-1alpha antibody. Precipitated proteins were separated by SDS-PAGE, and immunoblotting was performed with the anti-HIF-1alpha, -PDI (A), or -Ero-1 antibody (B). Arrows indicate bands corresponding to HIF-1alpha, PDI (A and B), or Ero-1 (B). ctrl, control. (C) HEK293 cells overexpressing HIF-1alpha WT or HIF-1alpha K719T were cultured under hypoxic conditions for 6 h. Cells were stained with the anti-FLAG tag and Hsp90 antibody and analyzed under a confocal microscope. (D) HEK293 cells overexpressing HIF-1alpha WT or HIF-1alpha K719T were cultured under hypoxic conditions for 6 h, and immunoblotting was performed. Proteins (15 μg) were separated by SDS-PAGE, and immunoblotting was performed with the anti-FLAG antibody. (E) Hep3B cells were cultured under hypoxic conditions for 6 h or 12 h, and the cytosol or microsomal fraction was extracted (6 h). Proteins was separated by SDS-PAGE, and immunoblotting was performed with the anti-PDI, -NPR, or -Hsp90 antibody. (F and G) Hep3B cells were cultured under hypoxic conditions for 4 h (F) or 6 h (F and G). Cells were treated with 20 mM NEM for 15 min (G). The cytosol (F and G) or microsomal fraction (F) was extracted. Proteins was separated by SDS-PAGE, and immunoblotting was performed with the anti-PDI (F), -NPR (F), or -Hsp90 (F and G) antibody. Immunoprecipitation was performed with the anti-HIF-1alpha antibody (F). The redox states of proteins were detected using the same method as in Fig 4A (G). An up-shift in molecular weight by the binding of PEG-maleimide was detected by immunoblotting with the anti-PDI (G). (H) Hep3B cells overexpressing PDI were cultured under hypoxic conditions for 6 h, and the cytosol fraction was extracted. Proteins was separated by SDS-PAGE, and immunoblotting was performed with the anti-PDI, or -Hsp90 antibody. The PDI protein levels of ctrl, or mock cells under normoxic or hypoxic conditions were set to 1.0. Values are expressed as the mean ± S.D. (error bars) of three different plates. ctrl, control. * p<0.05; ** p<0.01, significantly different from ctrl, mock, or si-ctrl cells.
Fig 8.
Detection of the HIF-1alpha redox state by PEG-maleimide.
(A) Hep3B cells overexpressing PDI or PDI C53, 397S were cultured under hypoxic conditions for 8 h in the presence of NH4Cl. (B) Hep3B cells were cultured under hypoxic conditions for 6 h and harvested with 50 mM Hepes, pH 7.5, containing 0.5% Nonidet P-40. The lysate from Hep3B cells was then treated with 17.5 nM purified His-tagged PDI WT or PDI C53, 397S. The redox states of proteins were detected using the same method as in Fig 4A. An up-shift in molecular weight by the binding of PEG-maleimide was detected by immunoblotting with the anti-PDI (A), -HIF-1alpha (A and B), or -His tag antibody (B). (C and D) PDI-overexpressing Hep3B cells were cultured for 8 h under hypoxic conditions in the presence of NH4Cl (C). PDI/pcDNA 3.1 (+) and si-HSC70 were transfected into Hep3B cells, and cells were cultured for 6 h under hypoxic conditions (D). Cells were harvested with PBS. The redox states of proteins were detected using the same method as that in Fig 4A. Since PDI decreased HIF-1alpha expression levels in the absence of NH4Cl (C) or in si-ctrl cells (D), 1.5-fold amount of protein was applied in Lane 4 in order to achieve an equal intensity in the reduced form of HIF-1alpha in other lanes. The knockdown of HSC70 was checked by immunoblotting with the anti-HSC70 antibody (upper panel).
Fig 9.
Effects of PDI on HIF-1alpha (1–245 aa) expression.
(A) Hep3B cells overexpressing PDI WT or PDI C53, 397S were cultured under hypoxic conditions for 8 h in the presence of NH4Cl. The biotin-switch method was performed, and HIF-1alpha was detected by immunoblotting with the anti-HIF-1alpha antibody. (B) Hep3B cells overexpressing HIF-1alpha 1–245 aa or PDI were cultured for 8 h in the presence of NH4Cl. Cell extracts were subjected to immunoprecipitation using the anti-Myc antibody (1:1000 dilution). Precipitated proteins (15 or 3 μg) were separated by SDS-PAGE, and immunoblotting was performed with the anti-Myc or -HSC70 antibody. (C) HIF-1alpha full length or 1–245 aa/3×FLAG-pcDNA4 vector, or PDI WT, or PDI C53, 397S / pcDNA 3.1 (+) was transfected into Hep3B cells, and immunoblotting was performed with the anti-FLAG, -PDI, or-beta-actin antibody. The HIF-1alpha protein levels of mock cells under normoxic conditions were set to 1.0. Values are expressed as the mean ± S.D. (error bars) of three different plates. ctrl, control. ** p<0.01, significantly different from mock cells.
Fig 10.
Detection of the HIF-1alpha (1–245 aa) redox state.
(A) Hep3B cells overexpressing HIF-1alpha 1–245 aa or HIF-1alpha 245–826 aa were cultured under hypoxic conditions for 6 h, and 17.5 nM purified His-tagged PDI WT or PDI C53, 397S was added to the lysate from Hep3B cells. The redox states of proteins were detected using the same method as in Fig 4A. An up-shift in molecular weight by the binding of PEG-maleimide was detected by immunoblotting with the anti-Myc antibody. (B) 350 nM purified His-tagged PDI was added to 350 nM His-tagged HIF-1alpha 1–245 aa, and proteins were incubated in the presence or absence of 10 mM DTT. Precipitated proteins were incubated with 50 mM NEM, and immunoblotting was performed with the anti-HIF-1alpha antibody. (C) 350 nM purified His-tagged PDI WT or PDI C53, 397S was added to 350 nM His-tagged HIF-1alpha 1–245 aa, and proteins were incubated with 20 mM NEM. After the removal of NEM, proteins were incubated with 1 mM DTT. Precipitated proteins were incubated with HRP Maleimide Conjugate. An up-shift in molecular weight by the binding of HRP was detected by immunoblotting with or without the anti-HIF-1alpha antibody. ##, non-specific band.
Fig 11.
Identification of critical cysteine residue in HIF-1alpha regulation by PDI.
HIF-1alpha C90S, HIF-1alpha C210S, or HIF-1alpha C219S/3×FLAG-pcDNA4 vector, or PDI WT, or PDI C53, 397S / pcDNA 3.1 (+) vector was transfected into Hep3B cells, and immunoblotting was performed with the anti-FLAG, -PDI, or-beta-actin antibody. The HIF-1alpha protein levels of mock cells under normoxic conditions were set to 1.0. Values are expressed as the mean ± S.D. (error bars) of three different plates. ctrl, control. * p<0.05; ** p<0.01, significantly different from mock cells.