Article Figures & Data
Figures
- Figure 1.
Increased nuclear D3 labeling in the ipsilateral hippocampus of rats with MCAO. A, Boxes showing the areas of the hippocampal formation in a Nissl-stained section of an intact animal, which were comparatively investigated on the contra- and ipsilateral sides, respectively (B-box 1 b vs b′ and C-box 2 c vs c′) after MCAO (ipsilateral). Scale bars, 1000 μm. B, D3 immunoreactivity in the perikaryal layers of CA1 (CA1/SP) and the upper blade of the dentate gyrus (box 1). Scale bar, 100 μm. C, D3 immunoreactivity in the CA4/hilus (CA4/H) and the lower blade of dentate gyrus (DG (lb)) (box 2). Scale bar, 100 μm. D, E, High-power light microscopic images of nuclear D3 labeling in the contra- and ipsilateral cells of the DG (lb) (D, d vs d′) and the CA4/H (E, e vs e′) revealed by the α-D3(CT) antibody. Scale bar, 20 μm. F–H, A toluidine blue-stained semithin section (F) and an ultrathin section labeled with silver-gold enhanced nickel-diaminobenzidine (G, H) show D3 in the granular cells of the ipsilateral dentate gyrus. The labeling (black arrowheads) forms clusters in the cell nucleus (N) and the cytoplasm (Cp), as shown at high power. In H, the nuclear membrane is marked by white arrowheads. Scale bars: F, 5 μm; G, 2 μm; H, 400 nm. A, Alveus; CA, cornu Ammonis; H, hilus; HS, hippocampal sulcus; PL, polymorphic layer; SG, stratum granulosum; SL/M, stratum lacunosum/moleculare; SL, stratum lucidum; SM, stratum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.
- Figure 2.
Catalytically active D3 is present in the cell nucleus. A, Immunofluorescence of normoxic SK-N-AS cells stained with α-D3(CT) antibody. B, Same as in A except that α-nuclear lamin antibody was also used for staining and images were overlaid. C, Western blotting with α-D3(NT) antibody or α-BiP antibody. SK-N-AS cells homogenized with the ball homogenizer (Isobiotec). Nuclear pellet was obtained after centrifugation at 800 × g and washing. Supernatant was centrifuged at 200,000 × g to yield the membrane pellet. D, D3 activity in nuclear or membrane fractions shown in C; data are the mean ± SEM; n = 2 per group; experiments were repeated 3 times.
- Figure 3.
Time-dependent hypoxia-induced nuclear D3 import. A, Immunofluorescence of normoxic SK-N-AS cells processed with α-D3(CT) antibody, α-nuclear lamin antibody, and image overlay. Cells were transferred to a hypoxic chamber (1% O2) and processed for immunofluorescence 8, 16, or 24 h later. B, Western blotting of the indicated SK-N-AS cellular fractions using α-HIF1α, α-nuclear lamin, α-Bip, and α-D3(CT) antibodies; hypoxia lasted for 24 h. N is nucleus (800 g pellet), NN is non-nuclear supernatant prepared in 0.5% Triton X-100 after 800 × g centrifugation; N′ is nucleus (800 g pellet), M is membrane pellet prepared by centrifugation only, after homogenization by ball homogenizer, no detergent was used. α-Nuclear lamin, α-Bip antibodies were used as loading control for nuclear (N′) and membrane (M) fraction, respectively. C, D, D3 activity in nuclear (N′) and membrane (M) fractions prepared by centrifugation only; data are the mean ± SEM; n = 2 per group; experiments were repeated 3 times. E, F, Nuclear localization of D3 under hypoxia in primary hippocampal neuronal cells. Quantification of cells that showed nuclear localization of D3 was done by using a confocal scanning microscope.
- Figure 4.
T3/T2 levels in the nucleus during hypoxia and effect of glucose deprivation on D3 under hypoxia. A, SK-N-AS cells were incubated with high glucose (4.5 g/L) versus glucose-free medium for 24 h under hypoxia; nuclear localization of D3 was examined by immunofluorescence using α-D3(CT) antibody. B, C, Nuclear T3 and T2 profiles of normoxic (B) or hypoxic (C) SK-N-AS cells. D, Relative concentrations of T3 and T2 in the nucleus of SK-N-AS under normoxia and hypoxia. E, Immunoprecipitation of D3 was done in HEK-293 cells transiently expressing flag-tagged D3-Cys and TRα. IN, 20% of total input; IgG, immunoprecipitated by IgG; Flag, immunoprecipitated by α-FLAG. The presence of TRα was examined by Western blot with α-TRα antibody.
- Figure 5.
D3 with glycosylation motifs and its glycosylation. A, D3 constructs fused with FLAG epitope or glycosylation sequences as indicated. B, Constructs containing N-glycosylation site with different orientation (a1, a2) were transfected into CHO cells. The cell lysates were analyzed by Western blot using α-FLAG antibody; GFP was used as a control for transfection. C, The cell lysates used in B were immunoprecipitated by α-FLAG antibody. The immunoprecipitate from a2 was treated with EndoH. D, D3 constructs without (b1) and with (b2) O-glycosylation motif as indicated. E, The constructs b1 and b2 were transfected into CHO cells and total cell lysates were analyzed by Western blot using α-FLAG antibody. F, For N-glycosylation, constructs a0 and a2 were transfected into CHO cells. After 24 h of hypoxia treatment, cells were lysed in 0.5% Triton X-100; after 800 × g centrifugation, resulting nuclear (N) and non-nuclear (NN) supernatant fractions were analyzed by Western blot using α-FLAG antibody. G, For O-glycosylation, constructs b1 and b2 were transfected into CHO cells, treated with 24 h of hypoxia, and lysed in 0.5% Triton X-100. After 800 × g centrifugation, nuclear (N) and non-nuclear (NN) supernatant fractions were analyzed by Western blot using α-FLAG antibody as in F.
- Figure 6.
Hsp40 interacts with and colocalizes with D3. A, HEK-293 cells transiently expressing FLAG-tagged D3 were lysed with Triton X-100; samples were immunoprecipitated with IgG or α-FLAG antibody and analyzed by Western blotting with different antibodies as indicated; IN is 10% of input, IP is immunoprecipitate pellet. B, Western blotting of SK-N-AS cell sonicates with α-Hsp40 antibody; hypoxia lasted for 24 h. C, Immunofluorescence of normoxic SK-N-AS cells processed with α-D3(CT) and α-Hsp40 antibodies and image overlay with two different magnifications. D, Same as in C, except that cells were treated with 24 h of hypoxia instead of 24 h of normoxia. E, Quantification of D3 or Hsp40 colocalization with the DAPI signal in the nucleus; also shown in the far right column, colocalization of D3 and Hsp40, p < 0.001. F, Colocalization of D3 with Hsp40 in cell nucleus of neuron in the rat hippocampal CA1 region. D3, Green; Hsp40, red; nucleus, blue (Hoechst). Scale bar, 5 μm.
- Figure 7.
Hsp40 mediates hypoxia-induced D3 nuclear import. A, SK-N-AS cells electroporated with a GFP-Hsp40 expression plasmid (Addgene 19495) were stained with both α-D3(CT) antibody and DAPI. Overlay is shown on the third row. Small panel on lower right shows GFP-Hsp40-expressing cells. B, Colocalization between D3 and DAPI signal from GFP-Hsp40-expressing cells was analyzed by Pearson's coefficients using NIS-AR software (Nikon Instruments); data are presented as mean ± SEM, n = 20. C, SK-N-AS cells stably expressing either a scrambled (left) or a shRNA plasmid targeting endogenous Hsp40 mRNA molecules (right). KD, Knockdown. D, Hsp40 protein levels as analyzed by Western blot of SK-N-AS cells from C. shRNA construct is specific for DNAJB1 and DNAJB1 (Hsp40) antibody used in this experiment (ab78437) does not cross-react with other J-domain proteins such as YDJ2. In E and F, SK-N-AS cells stably expressing the Hsp40 shRNA were exposed for 24 h to normoxic (E) or hypoxic (F) condition and then stained with DAPI and α-D3(CT) antibody as in A. For C–F, stable SK-N-AS cell lines expressing a control or an Hsp40 shRNA plasmid were generated by lentiviral transduction coupled with 2 weeks of puromycin (1 μg/ml) selection.
- Figure 8.
Nuclear D3 import dampens thyroid hormone signaling and slows down cellular metabolism. A, Effect of Hsp40 knockdown (KD) on the T3 inducibility of ENPP2 gene expression in hypoxic SK-N-AS cells; hypoxia and 50 nm T3 treatments were simultaneous and lasted for 24 h. Results are presented as a ratio between the T3 induction during hypoxia and the T3 induction during normoxia. B, OCR and ECAR (glycolysis) of SK-N-AS cells transiently expressing a control or an Hsp40 shRNA; 15,000 cells/well were plated and baseline values for both OCR and ECAR were measured as previously described (Simonides et al., 2008). Results are expressed as the OCR or ECAR ratio between Hsp40 KD and the control constructs at two different times: baseline and after treatment with 50 nm T3 for 24 h; data are presented as mean ± SEM; n = 30 per group; *p < 0.01 versus baseline reading by Student's t test. C, Same as in B except that studies were performed in cells transiently expressing a GFP-Hsp40. Both in B and C, D3 protein level did not change. D, Model explaining how O2 availability and Hsp40 regulate D3 subcellular localization. When O2 is available, ER-born D3 reaches the Golgi system and plasma membrane (PM), where it is internalized and recycled. During hypoxia, there is Hsp40-mediated nuclear import of D3, thus placing D3 physically closer to the thyroid hormone receptor.
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