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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 - J. Cell Biol. (2004)

Bottom Line: Coexpression of TRbetaA1 with retinoid X receptor did not enhance regulation.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.We conclude that T3-bound mitochondrial targeted TRs acutely modulate IP3-mediated Ca2+ signaling by increasing mitochondrial metabolism independently of transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 78229, USA.

ABSTRACT
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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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).
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fig1: 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).

Mentions: Acute nongenomic effects of thyroid hormones occur within minutes of ligand treatment (Hummerich and Soboll, 1989). To examine the importance of TRs on the nongenomic modulation of intracellular Ca2+ signaling, stage VI Xenopus oocytes were injected with mRNA encoding the Xenopus thyroid hormone receptor betz subtype A1 (xTRβA1) as described previously (Camacho and Lechleiter, 2000). Expression of xTRβA1 was confirmed by Western blot analysis, 2–3 d after mRNA injection (Fig. 1). The Ca2+ indicator dye was injected into oocytes 30–45 min before confocal imaging. When oocytes were injected with IP3, we observed repetitive Ca2+ wave activity with interwave periods of 6.62 ± 0.20 s (n = 70; Fig. 1). When xTRβA1 expressing oocytes were treated with T3 10 min before IP3 injection, the Ca2+ wave periodicity increased significantly to 8.40 ± 0.30 s (Fig. 1, a and c; n = 24, P < 0.0001 ANOVA single factor). Treatment of oocytes with T3 by itself did not induce Ca2+ release and no detectable changes in basal intracellular Ca2+ concentrations were observed. Application of T3 ligand to nonexpressing control oocytes, had no effect on the Ca2+ interwave period (6.38 ± 0.34 s, n = 27, Fig. 1, a and c). Similarly, xTRβA1 expressing oocytes without T3 treatment exhibited no change in Ca2+ wave periodicity (6.97 ± 0.24 s, n = 35). Peak Ca2+ wave amplitudes (ΔF/F) for xTRβA1-expressing oocytes exposed to T3 (0.69 ± 0.04, n = 29) was also significantly higher than that of control oocytes exposed to T3 (0.55 ± 0.04, n = 20; P < 0.05, t test). We conclude from these data that T3-stimulated xTRβA1 acutely modulates IP3-mediated Ca2+ wave activity and that both thyroid hormone and receptor expression are required for these effects.


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 - J. Cell Biol. (2004)

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).
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fig1: 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).
Mentions: Acute nongenomic effects of thyroid hormones occur within minutes of ligand treatment (Hummerich and Soboll, 1989). To examine the importance of TRs on the nongenomic modulation of intracellular Ca2+ signaling, stage VI Xenopus oocytes were injected with mRNA encoding the Xenopus thyroid hormone receptor betz subtype A1 (xTRβA1) as described previously (Camacho and Lechleiter, 2000). Expression of xTRβA1 was confirmed by Western blot analysis, 2–3 d after mRNA injection (Fig. 1). The Ca2+ indicator dye was injected into oocytes 30–45 min before confocal imaging. When oocytes were injected with IP3, we observed repetitive Ca2+ wave activity with interwave periods of 6.62 ± 0.20 s (n = 70; Fig. 1). When xTRβA1 expressing oocytes were treated with T3 10 min before IP3 injection, the Ca2+ wave periodicity increased significantly to 8.40 ± 0.30 s (Fig. 1, a and c; n = 24, P < 0.0001 ANOVA single factor). Treatment of oocytes with T3 by itself did not induce Ca2+ release and no detectable changes in basal intracellular Ca2+ concentrations were observed. Application of T3 ligand to nonexpressing control oocytes, had no effect on the Ca2+ interwave period (6.38 ± 0.34 s, n = 27, Fig. 1, a and c). Similarly, xTRβA1 expressing oocytes without T3 treatment exhibited no change in Ca2+ wave periodicity (6.97 ± 0.24 s, n = 35). Peak Ca2+ wave amplitudes (ΔF/F) for xTRβA1-expressing oocytes exposed to T3 (0.69 ± 0.04, n = 29) was also significantly higher than that of control oocytes exposed to T3 (0.55 ± 0.04, n = 20; P < 0.05, t test). We conclude from these data that T3-stimulated xTRβA1 acutely modulates IP3-mediated Ca2+ wave activity and that both thyroid hormone and receptor expression are required for these effects.

Bottom Line: Coexpression of TRbetaA1 with retinoid X receptor did not enhance regulation.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.We conclude that T3-bound mitochondrial targeted TRs acutely modulate IP3-mediated Ca2+ signaling by increasing mitochondrial metabolism independently of transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 78229, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus