Saelim N et al. (2004), Nontranscriptional modulation of intracellular ...

XB-ART-2670

J Cell Biol 2004 Dec 06;1675:915-24. doi: 10.1083/jcb.200409011.

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Nontranscriptional modulation of intracellular Ca2+ signaling by ligand stimulated thyroid hormone receptor.

Saelim N , John LM , Wu J , Park JS , Bai Y , Camacho P , Lechleiter JD .

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Thyroid hormone 3,5,3'-tri-iodothyronine (T3) binds and activates thyroid hormone receptors (TRs). Here, we present evidence for a nontranscriptional regulation of Ca2+ signaling by T3-bound TRs. Treatment of Xenopus thyroid hormone receptor beta subtype A1 (xTRbetaA1) expressing oocytes with T3 for 10 min increased inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ wave periodicity. Coexpression of TRbetaA1 with retinoid X receptor did not enhance regulation. Deletion of the DNA binding domain and the nuclear localization signal of the TRbetaA1 eliminated transcriptional activity but did not affect the ability to regulate Ca2+ signaling. T3-bound TRbetaA1 regulation of Ca2+ signaling could be inhibited by ruthenium red treatment, suggesting that mitochondrial Ca2+ uptake was required for the mechanism of action. Both xTRbetaA1 and the homologous shortened form of rat TRalpha1 (rTRalphaDeltaF1) localized to the mitochondria and increased O2 consumption, whereas the full-length rat TRalpha1 did neither. Furthermore, only T3-bound xTRbetaA1 and rTRalphaDeltaF1 affected Ca2+ wave activity. We conclude that T3-bound mitochondrial targeted TRs acutely modulate IP3-mediated Ca2+ signaling by increasing mitochondrial metabolism independently of transcriptional activity.

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Species referenced: Xenopus laevis
Genes referenced: pnma1

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	Figure 1. T3-bound TRβA1 increases IP3-induced Ca2+ wave period. (a) Spatial-temporal stacks of IP3 (∼300 nM)-induced Ca2+ wave activity in a representative control (water injected) oocyte, a T3-treated (100 nM) oocyte expressing TRβA1 and a T3 (100 nM) treated oocyte. Each image is 745 × 745 μm. (b) Western blot showing expression of TRβA1. Protein extracts from all groups were collected and loaded at 0.5 oocytes per lane onto 10% SDS-PAGE. The membrane was probed with a monoclonal mouse anti–human TRs antibody (MA1-215) and labeled with an HRP-conjugated secondary antibody. (c) Histogram of average interwave period for each group of oocytes. n values in parentheses represent the total number of oocytes pooled from at least two frogs. Error bars correspond to the mean ± SEM. The asterisks (**) denote a statistic significant difference (ANOVA single factor, P < 0.0001).
	Figure 2. Transcriptional activity of TRβA1 requires xRXRα and both cognate ligands. Transcriptional activity was monitored with the TRE-reporter vector, pSEAP (TRE). (a) Lanes 1 and 2 are negative (pSEAP(−ve)) and positive (pSEAP(+ve)) vector controls. Oocytes expressing TRβA1 or TRβA1 plus xRXRα were incubated with 100 nM T3 (lanes 3–5) plus 100 nM RA (lane 5) for 3 d. Cytosolic extracts from each group of oocytes was prepared and loaded onto a 10% SDS-PAGE at 2.5 oocytes equivalents per lane. SEAP was detected with the polyclonal rabbit anti–human SEAP antibody and an HRP-conjugated secondary antibody. The SP labeled arrow indicates SEAP immunoreactivity, which was present only in oocytes expressing TRβA1 and xRXRα exposed to both T3 and RA. (b) Transcriptional activity of TRβA1 requires the pBOX within the DBD and the NLS. Oocytes expressing xTRβA1ΔpBox-NLS and xRXRα show no SEAP immunoreactivity when incubated with T3 (lane 6) or T3 plus RA (lane 7). Western blot analysis shows that xRXRα, TRβA1, and xTRβA1ΔpBox-NLS are expressed at comparable levels (Western blots below lanes 4–7). TRβA1 and xTRβA1ΔpBox-NLS were detected with the monoclonal mouse anti–human TRs antibody (MA1-215). xRXRα was detected with a polyclonal rabbit anti–human RXR antibody (Sc-774).
	Figure 3. Acute modulation of Ca2+ signaling does not require heterodimerization of TRβA1 with xRXRα. (a) Spatial-temporal stacks of IP3-induced Ca2+ wave activity in control oocytes compared with oocytes expressing TRβA1 or TRβA1 with xRXRα. T3 (100 nM) and RA (100 nM) were added as indicated 10–15 min before injection with IP3 (∼300 nM). Scale is the same as Fig. 1. (b) Western blots of oocytes expressing TRβA1 and xRXRα. Primary and secondary antibodies were identical to those used in Figs. 1 and 2. (c). Histogram of average interwave period (second) of each group of oocytes. The asterisks (**) denote a statistic significance using ANOVA single factor (P < 0.0001). Values in parentheses represent the number of oocytes.
	Figure 4. The pBOX and NLSs of TRβA1 are not required for the acute regulation of Ca2+ signaling. (a) Schematic figure depicting the position of the pBOX deletion in the DBD and the NLS modification within TRβA1. (b) Spatial-temporal stack of IP3-induced Ca2+ wave activity in control oocytes compared with oocytes expressing TR mutants ΔpBox-NLS and ΔNLS. Oocytes expressing the TR mutants were incubated with T3 (100 nM) 10–15 min before IP3 (∼300 nM) injections. (c) Western blot analysis confirming comparable levels of protein expression for both wild-type and mutant TRβA1. (d) Histogram of the average Ca2+ wave periods for each group of oocytes (n values are in parentheses). Statistic significance over control oocytes is indicated by the asterisks (**; ANOVA single factor, P < 0.0001).
	Figure 5. T3 stimulation of oocytes expressing TRβA1 increases the ΔΨ. (a) Images of mitochondria labeled with the potential sensitive dye TMRE. The oocytes are expressing TRβA1 and have been exposed to T3 for the indicated amount of time. Images are 50 × 100 μm. (b) Histogram of the log of mitochondrial TMRE fluorescence (Fmito) divided by the cytosolic fluorescence (Fcyto) at the indicated times of T3 exposure. Values in parentheses refers to the number of mitochondrion analyzed. Statistical significance is indicated by the asterisks (**; ANOVA single factor, P < 0.001).
	Figure 6. Ru360 blocks T3-bound TRβA1 increases in IP3-induced Ca2+ wave period. (a) Spatial-temporal stacks of the effect of Ru360 treatment on Ca2+ wave activity in control oocytes compared with oocytes expressing TRβA1as labeled. (b) Histogram of average interwave period (seconds) of each group of oocytes shown in a. The asterisk (*) denotes a statistic significance using ANOVA single factor (P < 0.01). Values in parentheses represent the number of oocytes.
	Figure 7. T3 stimulation of oocytes expressing TRβA1 increases O2 consumption. (a) Plots of O2 levels in oocytes as labeled (n = 200 oocytes per group). (b) Histogram represents average change of O2 consumption rates (before and after T3 exposure) in control and xTRβA1 groups. Statistical significance is indicated by the asterisk (*; t test, P < 0.05).
	Figure 8. Xenopus TRβA1 and NH2-terminal truncated rat TRα1 (rTRα1ΔF) localize to mitochondria. (a) Schematic diagram of TRs showing that rTRα1ΔF and xTRβA1 have a similar NH2 terminus. (b and c) Western blots of TRα1, rTRα1ΔF, and xTRβA1 expression in whole oocytes and mitochondrial extracts respectively. FL, full-length receptor; SH, shortened form of the receptor. Extracts were prepared from 300 oocytes in each group. All oocytes were exposed to 100 nM T3 for at least 15 min before organelle extraction. TRs were immunoprecipitated with a monoclonal mouse anti–human TRs antibody (MA1-215), captured with immobilized protein G, concentrated, and loaded onto a 10% SDS-PAGE. An HRP-conjugated secondary antibody was used for visualization.
	Figure 9. NH2-terminal truncated rat TRα1 (rTRα1ΔF) stimulates O2 consumption. (a) Plots of O2 levels for oocytes expressing full-length rTRα1 with and without T3 compared with oocytes expressing the NH2-terminal truncated rTRα1ΔF with or without T3. Protocols used were identical to those described in Fig. 7. (b) Histogram of the average change of the O2 consumption rates after T3 exposure in rTRα1 versus rTRα1ΔF groups. The asterisk (*) indicates statistical significance (t test, P < 0.05).
	Figure 10. The truncated rTRα1ΔF regulates intracellular Ca2+ release. (a) Spatio-temporal stacks of IP3-induced Ca2+ wave activity in control oocytes compared with oocytes expressing rTRα1ΔF or rTRα1ΔF. TR expressing oocytes were treated with 100 nM T3 10–15 min before IP3 (∼300 nM) injections and confocal imaging. (b) Western blots of rTRα1 and rTRα1ΔF expression levels in experimental oocytes. (c) Histogram of the average interwave periods for each group (n values in parentheses). Note that rTRα1ΔF has significantly longer periods even though its expression levels are lower than those of full-length rTRα1. The asterisks (**) indicate statistical significance with P < 0.01 using ANOVA single factor.

References [+] :

Abbaticchio, Hormones in the seminal fluid. The transport proteins of the thyroid hormones. 1981, Pubmed