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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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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).
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fig9: 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).

Mentions: T3 treatment has previously been reported to increase mitochondrial metabolism (Sterling et al., 1980; Soboll, 1993a). Our data suggest that the acute effects of T3 on mitochondrial metabolism are likely to be mediated by T3-activated thyroid hormone receptors. A truncated form of rat TRα1 (rTRα1ΔF) has been shown to localize to mitochondria matrix (Ardail et al., 1993; Wrutniak et al., 1995; Casas et al., 1999). Furthermore, the NH2 terminus of the Xenopus TRβA1 that we used throughout this work has a high homology to the NH2 terminus of rTRα1ΔF (Fig. 8 a). Our strategy in this experiment was to test whether mitochondrial targeting of TRs was necessary to modulate Ca2+ signaling. First, we examined the cellular targeting of xTRβA1, rTRα1, and rTRα1ΔF by injecting Xenopus oocytes with their respective mRNAs. After 3 d of expression, mitochondria were isolated by centrifugation. Whole oocyte extract (minus mitochondria) and mitochondrial extract from each group were subjected to immunoprecipitation using a TR antibody (MA1-215; Affinity BioReagents, Inc.). The immunocomplexes (TRs/MA1-215) were loaded onto a 10% SDS-PAGE gel for Western blot analysis. As shown in Fig. 8 c, only xTRβA1 and rTRα1ΔF were detected in the mitochondria extracts. Full-length rTRα1 did not localize to mitochondria. These results are consistent with previous reports (Ardail et al., 1993; Wrutniak et al., 1995; Casas et al., 1999). Our next step was to compare the rate of O2 consumption for oocytes expressing either rTRα1 or rTRα1ΔF (Fig. 9). Consistent with its mitochondrial targeting, the rate of O2 consumption in rTRα1ΔF expressing oocytes after T3 exposure was significantly increased 1.88 ± 0.35 nmol/min (n = 3, P < 0.05). In contrast, the rate of O2 consumption in oocytes expressing the full-length rTRα1 was not significantly affected by T3 exposure (−0.16 ± 0.55, n = 3). We conclude that mitochondrial targeting of TRs is required for a T3 mediated increase in mitochondrial respiration.


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)

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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172460&req=5

fig9: 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).
Mentions: T3 treatment has previously been reported to increase mitochondrial metabolism (Sterling et al., 1980; Soboll, 1993a). Our data suggest that the acute effects of T3 on mitochondrial metabolism are likely to be mediated by T3-activated thyroid hormone receptors. A truncated form of rat TRα1 (rTRα1ΔF) has been shown to localize to mitochondria matrix (Ardail et al., 1993; Wrutniak et al., 1995; Casas et al., 1999). Furthermore, the NH2 terminus of the Xenopus TRβA1 that we used throughout this work has a high homology to the NH2 terminus of rTRα1ΔF (Fig. 8 a). Our strategy in this experiment was to test whether mitochondrial targeting of TRs was necessary to modulate Ca2+ signaling. First, we examined the cellular targeting of xTRβA1, rTRα1, and rTRα1ΔF by injecting Xenopus oocytes with their respective mRNAs. After 3 d of expression, mitochondria were isolated by centrifugation. Whole oocyte extract (minus mitochondria) and mitochondrial extract from each group were subjected to immunoprecipitation using a TR antibody (MA1-215; Affinity BioReagents, Inc.). The immunocomplexes (TRs/MA1-215) were loaded onto a 10% SDS-PAGE gel for Western blot analysis. As shown in Fig. 8 c, only xTRβA1 and rTRα1ΔF were detected in the mitochondria extracts. Full-length rTRα1 did not localize to mitochondria. These results are consistent with previous reports (Ardail et al., 1993; Wrutniak et al., 1995; Casas et al., 1999). Our next step was to compare the rate of O2 consumption for oocytes expressing either rTRα1 or rTRα1ΔF (Fig. 9). Consistent with its mitochondrial targeting, the rate of O2 consumption in rTRα1ΔF expressing oocytes after T3 exposure was significantly increased 1.88 ± 0.35 nmol/min (n = 3, P < 0.05). In contrast, the rate of O2 consumption in oocytes expressing the full-length rTRα1 was not significantly affected by T3 exposure (−0.16 ± 0.55, n = 3). We conclude that mitochondrial targeting of TRs is required for a T3 mediated increase in mitochondrial respiration.

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