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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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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.
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fig6: 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.

Mentions: We reported previously that pyruvate/malate-energized mitochondria increase the amplitude and interwave period of IP3-induced Ca2+ waves in Xenopus oocytes (Jouaville et al., 1995). These effects on Ca2+ wave activity were similar to those observed in TRβA1 overexpressing oocytes with acute T3 incubation (Fig. 1). Sterling and colleagues (Sterling, 1980) initially reported that T3 increases mitochondrial metabolism, particularly oxidative phosphorylation, in less than 30 min. Consequently, we hypothesized that the regulation of Ca2+ signaling by T3-activated xTRβA1 was mediated by its acute modulation of mitochondrial metabolism, which, in turn, increased mitochondrial Ca2+ uptake. Our strategy to test this hypothesis was threefold. First, we examined the effect of T3 on ΔΨ in TRβA1 expressing oocytes using the potential sensitive dye tetramethylrhodamine ethyl ester (TMRE). Oocytes were bathed in 200 nM TMRE for 5 min before imaging fluorescence with two-photon excitation (800 nm). ΔΨ was estimated by monitoring Log(Fmito/Fcyto) where Fmito is the fluorescence intensity of individual mitochondria and Fcyto is the cytosolic fluorescence (Farkas et al., 1989). We found that T3 significantly increased ΔΨ from a resting value of 0.33 ± 0.01 (n = 75) to 0.44 ± 0.01 (n = 92, P < 0.0001) at 5 min and to 0.48 ± 0.02 (n = 48, P < 0.0001) at 25 min (Fig. 5). These data suggest that T3-bound TRβA1 regulates Ca2+ signaling by increasing ΔΨ. Second, we injected a subgroup of the TRβA1-expressing oocytes with ruthenium 360 (Ru360; Calbiochem, ∼1 μM final concentration), a polycation that inhibits the electrogenic mitochondrial Ca2+ uniporter (Ying et al., 1991) ∼60 min before IP3 injection and Ca2+ imaging. A control group of TRβA1-expressing oocytes were injected with buffer only. We found that Ru360 treatment completely inhibited the affect of T3-bound TRβA1 on Ca2+ wave activity (Fig. 6). Untreated TRβA1 expressing oocytes exhibited the expected increase in wave periodicity (7.65 ± 0.4 s, n = 11) when preexposed to T3 for 10 min. However, the average wave period of Ru360-treated TRβA1 expressing oocytes was only 5.75 ± 0.22 s (n = 13) when preexposed to T3. The Ru360-treated average was nearly identical to untreated control oocytes (5.59 ± 0.04 s, n = 4) as well as Ru360-treated nonexpressing oocytes (5.53 ± 0.4 s, n = 3). These data are consistent with the hypothesis that T3-bound TRβA1 regulates Ca2+ signaling by increasing mitochondrial Ca2+ uptake via an increase in ΔΨ.


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)

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.
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Related In: Results  -  Collection

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fig6: 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.
Mentions: We reported previously that pyruvate/malate-energized mitochondria increase the amplitude and interwave period of IP3-induced Ca2+ waves in Xenopus oocytes (Jouaville et al., 1995). These effects on Ca2+ wave activity were similar to those observed in TRβA1 overexpressing oocytes with acute T3 incubation (Fig. 1). Sterling and colleagues (Sterling, 1980) initially reported that T3 increases mitochondrial metabolism, particularly oxidative phosphorylation, in less than 30 min. Consequently, we hypothesized that the regulation of Ca2+ signaling by T3-activated xTRβA1 was mediated by its acute modulation of mitochondrial metabolism, which, in turn, increased mitochondrial Ca2+ uptake. Our strategy to test this hypothesis was threefold. First, we examined the effect of T3 on ΔΨ in TRβA1 expressing oocytes using the potential sensitive dye tetramethylrhodamine ethyl ester (TMRE). Oocytes were bathed in 200 nM TMRE for 5 min before imaging fluorescence with two-photon excitation (800 nm). ΔΨ was estimated by monitoring Log(Fmito/Fcyto) where Fmito is the fluorescence intensity of individual mitochondria and Fcyto is the cytosolic fluorescence (Farkas et al., 1989). We found that T3 significantly increased ΔΨ from a resting value of 0.33 ± 0.01 (n = 75) to 0.44 ± 0.01 (n = 92, P < 0.0001) at 5 min and to 0.48 ± 0.02 (n = 48, P < 0.0001) at 25 min (Fig. 5). These data suggest that T3-bound TRβA1 regulates Ca2+ signaling by increasing ΔΨ. Second, we injected a subgroup of the TRβA1-expressing oocytes with ruthenium 360 (Ru360; Calbiochem, ∼1 μM final concentration), a polycation that inhibits the electrogenic mitochondrial Ca2+ uniporter (Ying et al., 1991) ∼60 min before IP3 injection and Ca2+ imaging. A control group of TRβA1-expressing oocytes were injected with buffer only. We found that Ru360 treatment completely inhibited the affect of T3-bound TRβA1 on Ca2+ wave activity (Fig. 6). Untreated TRβA1 expressing oocytes exhibited the expected increase in wave periodicity (7.65 ± 0.4 s, n = 11) when preexposed to T3 for 10 min. However, the average wave period of Ru360-treated TRβA1 expressing oocytes was only 5.75 ± 0.22 s (n = 13) when preexposed to T3. The Ru360-treated average was nearly identical to untreated control oocytes (5.59 ± 0.04 s, n = 4) as well as Ru360-treated nonexpressing oocytes (5.53 ± 0.4 s, n = 3). These data are consistent with the hypothesis that T3-bound TRβA1 regulates Ca2+ signaling by increasing mitochondrial Ca2+ uptake via an increase in ΔΨ.

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