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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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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.
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fig3: 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.

Mentions: To test whether heterodimerization of xTRβA1 with xRXRα affects the acute modulation of Ca2+ activity, we coinjected oocytes with both xRXRα and xTRβA1 mRNA and confirmed protein expression levels using Western analysis 2–3 d after injection of mRNA (Fig. 3 b). Oocytes were loaded with Ca2+ indicator dye and confocally imaged. Oocytes coexpressing xTRβA1 and xRXRα were initially exposed to both T3 (100 nM) and RA (100 nM) 10 min before injection with IP3 (∼300 nM). The average Ca2+ interwave period for xTRβA1-expressing oocytes was 6.58 ± 0.26 s (n = 67), whereas that of xRXRα/xTRβA1 coexpressing oocytes was 6.72 ± 0.31 s (n = 82; Fig. 3, a and c). These values were not significantly different from each other (P = 0.22), but were both significantly larger than values in the control oocytes that exhibited an average Ca2+ interwave period of 5.90 ± 0.43 s (n = 55, ANOVA single factor, P < 0.0001; Fig. 3, a and c). We conclude that the xRXRα coexpression does not affect the ability of T3-bound xTRβA1 to modulate Ca2+ signaling.


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)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: To test whether heterodimerization of xTRβA1 with xRXRα affects the acute modulation of Ca2+ activity, we coinjected oocytes with both xRXRα and xTRβA1 mRNA and confirmed protein expression levels using Western analysis 2–3 d after injection of mRNA (Fig. 3 b). Oocytes were loaded with Ca2+ indicator dye and confocally imaged. Oocytes coexpressing xTRβA1 and xRXRα were initially exposed to both T3 (100 nM) and RA (100 nM) 10 min before injection with IP3 (∼300 nM). The average Ca2+ interwave period for xTRβA1-expressing oocytes was 6.58 ± 0.26 s (n = 67), whereas that of xRXRα/xTRβA1 coexpressing oocytes was 6.72 ± 0.31 s (n = 82; Fig. 3, a and c). These values were not significantly different from each other (P = 0.22), but were both significantly larger than values in the control oocytes that exhibited an average Ca2+ interwave period of 5.90 ± 0.43 s (n = 55, ANOVA single factor, P < 0.0001; Fig. 3, a and c). We conclude that the xRXRα coexpression does not affect the ability of T3-bound xTRβA1 to modulate Ca2+ signaling.

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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