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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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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).
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fig2: 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).

Mentions: Classically, activated thyroid hormone receptors heterodimerize to initiate transcription responses. Retinoid X receptor (RXR) is the most common dimerization partner that binds to the thyroid hormone response element (TRE; Leid et al., 1992; Bhat et al., 1994; Wong and Shi, 1995). To investigate the transcriptional activity of xTRβA1, we coinjected oocytes with xTRβA1 mRNA and a plasmid reporting vector containing a TRE system with two direct repeats (DR4) upstream of the secreted placental alkaline phosphatase (SEAP) gene (p-TRE-SEAP; CLONTECH Laboratories, Inc.). If the hormone receptor dimerizes and binds to the TRE enhancer, the oocyte expresses SEAP, which is secreted into the medium. mRNA-injected oocytes were continuously bathed in T3 (100 nM) for 3 d and the presence of SEAP was subsequently quantified by Western blot analysis and used as a marker for transcriptional activity. Using this TRE-reporting system, we observed no transcriptional activity in oocytes expressing the xTRβA1 protein by itself (Fig. 2 a, lane 3). However, when we coexpressed xRXRα with xTRβA1 and oocytes were incubated with T3 (100 nM) and 9-cis retinoic acid (RA; 100 nM) for 3 d, SEAP expression was significantly increased (Fig. 2 a, lane 5). Note that xTRβA1/xRXRα-mediated transcription requires both ligands, T3 and RA (Fig. 2 a, lanes 4 and 5). These data indicate that stimulation of xTRβA1 by T3 does not initiate detectable transcription in Xenopus oocytes.


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)

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).
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fig2: 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).
Mentions: Classically, activated thyroid hormone receptors heterodimerize to initiate transcription responses. Retinoid X receptor (RXR) is the most common dimerization partner that binds to the thyroid hormone response element (TRE; Leid et al., 1992; Bhat et al., 1994; Wong and Shi, 1995). To investigate the transcriptional activity of xTRβA1, we coinjected oocytes with xTRβA1 mRNA and a plasmid reporting vector containing a TRE system with two direct repeats (DR4) upstream of the secreted placental alkaline phosphatase (SEAP) gene (p-TRE-SEAP; CLONTECH Laboratories, Inc.). If the hormone receptor dimerizes and binds to the TRE enhancer, the oocyte expresses SEAP, which is secreted into the medium. mRNA-injected oocytes were continuously bathed in T3 (100 nM) for 3 d and the presence of SEAP was subsequently quantified by Western blot analysis and used as a marker for transcriptional activity. Using this TRE-reporting system, we observed no transcriptional activity in oocytes expressing the xTRβA1 protein by itself (Fig. 2 a, lane 3). However, when we coexpressed xRXRα with xTRβA1 and oocytes were incubated with T3 (100 nM) and 9-cis retinoic acid (RA; 100 nM) for 3 d, SEAP expression was significantly increased (Fig. 2 a, lane 5). Note that xTRβA1/xRXRα-mediated transcription requires both ligands, T3 and RA (Fig. 2 a, lanes 4 and 5). These data indicate that stimulation of xTRβA1 by T3 does not initiate detectable transcription in Xenopus oocytes.

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